Forestry, Faculty of
DSpace
UBCV
McMahen, Katie (Kathleen)
Doctor of Philosophy - PhD
University of British Columbia
Primary successional post-mining landscapes present physical, chemical and biological barriers to terrestrial ecosystem recovery. I investigated if legacies of the pre-disturbance ecosystem can be applied to help overcome these barriers, leveraging the processes that guide succession following natural disturbances. In a three-year field experiment at the Mount Polley Mine, Canada and in a greenhouse experiment using tailings, overburden and forest soil from the mine, I tested if (1) adding small volumes of forest soil to the seedling rooting zone during planting and (2) spatial connectivity with undisturbed forest would improve soil food web recovery and plant establishment. Soil additions improved seedling growth and/or nutrition in the greenhouse and field, corresponding with greater root colonization by microbial mutualists. In the field, soil additions increased soil faunal diversity and caused bacterial community shifts consistent with advancing succession, with greater benefits in lower quality soils. Sterilized greenhouse controls revealed that a later successional conifer benefited primarily from the biological inoculum, while an early successional shrub benefited more from the improved soil physiochemical conditions. Greater proximity to undisturbed forest increased plant diversity and shifted soil microbial communities, showing that retained patches of intact ecosystems can counteract dispersal limitations. Short-distance belowground connectivity with undisturbed forest improved plant diversity and mycorrhizal fungal community establishment but decreased seedling growth compared to areas “disconnected” with trenches. Field-testing of soil additions and spatial connectivity in conjunction with site preparation showed that application of ecosystem legacies can have additive benefits with reclamation methods targeting soil physical improvements. In a second greenhouse experiment, I tested if plants establishing in mine reclamation materials regenerate soil microbial legacies that facilitate successional advancement. I found negative feedbacks due to fungal pathogen accumulation promoted turnover of an early successional shrub, while formation of distinct mycorrhizal fungal communities selectively promoted establishment and stability of compatible later successional tree species. This reinforces that reclamation plant species selection can influence long-term plant community assembly. Ecosystem legacy-based mine reclamation methods could be used operationally to simultaneously enhance dispersal and establishment of native plants and soil organisms, which, over the long-term, is anticipated to have positive feedbacks on ecosystem recovery.
INVESTIGATION OF ECOSYSTEM LEGACY-BASED MINE RECLAMATION TECHNIQUES: EFFECTS OF SOIL TRANSFERS, FOREST CONNECTIVITY AND PLANT–SOIL FEEDBACKS ON SOIL FOOD WEB RECOVERY AND PLANT ESTABLISHMENT by Katie (Kathleen) McMahen B.Sc., The University of British Columbia, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2020 © Katie (Kathleen) McMahen, 2020 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Investigation of ecosystem legacy-based mine reclamation techniques: effects of soil transfers, forest connectivity and plant–soil feedbacks on soil food web recovery and plant establishment submitted by Katie McMahen in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Forestry Examining Committee: Dr. Suzanne Simard (Faculty of Forestry, UBC) Supervisor Dr. Leslie Lavkulich (Faculty of Land and Food Systems, UBC) Supervisory Committee Member Dr. Susan Grayston (Faculty of Forestry, UBC) Supervisory Committee Member Dr. Cindy Prescott (Faculty of Forestry, UBC) University Examiner Dr. Sean Smukler (Faculty of Land and Food Systems, UBC) University Examiner Additional Supervisory Committee Members: Dr. Carolyn Anglin (Anglin and Associates) Supervisory Committee Member iii Abstract Primary successional post-mining landscapes present physical, chemical and biological barriers to terrestrial ecosystem recovery. I investigated if legacies of the pre-disturbance ecosystem can be applied to help overcome these barriers, leveraging the processes that guide succession following natural disturbances. In a three-year field experiment at the Mount Polley Mine, Canada and in a greenhouse experiment using tailings, overburden and forest soil from the mine, I tested if (1) adding small volumes of forest soil to the seedling rooting zone during planting and (2) spatial connectivity with undisturbed forest would improve soil food web recovery and plant establishment. Soil additions improved seedling growth and/or nutrition in the greenhouse and field, corresponding with greater root colonization by microbial mutualists. In the field, soil additions increased soil faunal diversity and caused bacterial community shifts consistent with advancing succession, with greater benefits in lower quality soils. Sterilized greenhouse controls revealed that a later successional conifer benefited primarily from the biological inoculum, while an early successional shrub benefited more from the improved soil physiochemical conditions. Greater proximity to undisturbed forest increased plant diversity and shifted soil microbial communities, showing that retained patches of intact ecosystems can counteract dispersal limitations. Short-distance belowground connectivity with undisturbed forest improved plant diversity and mycorrhizal fungal community establishment but decreased seedling growth compared to areas “disconnected” with trenches. Field-testing of soil additions and spatial connectivity in conjunction with site preparation showed that application of ecosystem legacies can have additive benefits with reclamation methods targeting soil physical improvements. iv In a second greenhouse experiment, I tested if plants establishing in mine reclamation materials regenerate soil microbial legacies that facilitate successional advancement. I found negative feedbacks due to fungal pathogen accumulation promoted turnover of an early successional shrub, while formation of distinct mycorrhizal fungal communities selectively promoted establishment and stability of compatible later successional tree species. This reinforces that reclamation plant species selection can influence long-term plant community assembly. Ecosystem legacy-based mine reclamation methods could be used operationally to simultaneously enhance dispersal and establishment of native plants and soil organisms, which, over the long-term, is anticipated to have positive feedbacks on ecosystem recovery. v Lay Summary Mining operations severely disturb ecosystems and create challenging conditions for forest restoration. I demonstrated that remnants of the pre-existing forest can be used in mine reclamation to help plants reestablish. Firstly, adding small volumes of fresh forest soil to the rooting zone of seedlings during planting improved seedling establishment and increased colonization of seedling roots by mycorrhizal fungi (beneficial microbes that improve plant nutrient acquisition). Secondly, greater proximity to undisturbed forest enhanced dispersal and recovery of plant communities and soil microbes. I also found that plants growing in mining waste formed distinct communities of mycorrhizal fungi. This selectively promoted establishment of tree species that are compatible with those mycorrhizal fungi, highlighting the long-term implications of plant species selection in reclamation. My research advanced our understanding of the role of soil microbes in mine reclamation and identified reclamation methods that can improve plant establishment by promoting recovery of beneficial soil microbes. vi Preface This dissertation is an original, unpublished product of the author, Katie McMahen, with guidance from Suzanne Simard. The initial research partnership was formed when Katie McMahen was an employee of the Mount Polley Mine and Suzanne Simard was retained as a contractor to support the ecological risk assessment of the effects of the 2014 Mount Polley Mine tailings dam failure. This thesis is based on a successful Natural Sciences and Engineering Research Council (NSERC) Engage grant proposal prepared by Suzanne Simard, which identified the research direction. Katie McMahen developed the specific research questions and designed the experiments with input from Suzanne Simard and supervisory committee members Susan Grayston, Les Lavkulich and ‘Lyn (Carolyn) Anglin. Importantly, ‘Lyn Anglin was the Chief Scientific Officer for Imperial Metals Corporation (Mount Polley Mining Corporation’s parent company) during the period when the experiments were designed, and she provided insight on behalf of the industry partner. Katie McMahen carried out all fieldwork, laboratory work and data analysis, with support from field and laboratory assistants and volunteers. Shannon Guichon provided technical laboratory support and assistance with high-throughput sequencing data processing. Valerie LeMay, Susan Dudley and STAT 551 students Miguel Biron and Shuxian Fan provided support with statistical analyses. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................ xxii List of Abbreviations ............................................................................................................. xxxvi Acknowledgements ............................................................................................................... xxxvii Chapter 1: Introduction ................................................................................................................1 1.1 The role of soil biological communities in mine reclamation ........................................ 2 1.2 Ecosystem legacies as sources of soil biological communities ...................................... 5 1.2.1 Spatial ecosystem legacies .......................................................................................... 5 1.2.2 Topsoil ecosystem legacies ......................................................................................... 8 1.2.3 Ecosystem legacy effects on succession ..................................................................... 9 1.3 Study site ....................................................................................................................... 11 1.4 Thesis objectives ........................................................................................................... 13 Chapter 2: General methodology ...............................................................................................17 2.1 Mount Polley Mine site description .............................................................................. 17 2.1.1 Climate ...................................................................................................................... 17 2.1.2 Geology and soils ...................................................................................................... 17 2.1.3 Vegetation ................................................................................................................. 19 viii 2.2 Plant species selection................................................................................................... 20 2.3 Greenhouse experiments ............................................................................................... 21 2.4 Soil physiochemical analyses ....................................................................................... 23 2.5 Analysis of root mycorrhizal fungi communities ......................................................... 23 2.6 Microbial high-throughput sequencing bioinformatics ................................................ 25 Chapter 3: Forest soil transfers increase root symbiont colonization and seedling establishment in tailings and overburden ..................................................................................27 3.1 Introduction ................................................................................................................... 27 3.2 Methods......................................................................................................................... 29 3.2.1 Experimental design.................................................................................................. 29 3.2.2 Data collection and laboratory analyses ................................................................... 30 3.2.3 Statistical analyses .................................................................................................... 32 3.3 Results ........................................................................................................................... 33 3.3.1 Forest soil transfer effects on seedling establishment ............................................... 33 3.3.2 Root colonization by microbial symbionts ............................................................... 40 3.3.3 Soil Sterilization........................................................................................................ 42 3.4 Discussion ..................................................................................................................... 42 3.5 Conclusion .................................................................................................................... 47 Chapter 4: Soil and spatial ecosystem legacies promote plant and soil food web dispersal and establishment at the Mount Polley Mine ............................................................................49 4.1 Introduction ................................................................................................................... 49 4.2 Methods......................................................................................................................... 54 4.2.1 Experimental design.................................................................................................. 54 ix 4.2.2 Baseline soil data collection...................................................................................... 57 4.2.3 Vegetation monitoring .............................................................................................. 57 4.2.4 Seedling destructive harvest ..................................................................................... 58 4.2.5 Microbial analyses .................................................................................................... 60 4.2.6 Statistical analyses .................................................................................................... 61 4.3 Results ........................................................................................................................... 65 4.3.1 Spruce seedling establishment .................................................................................. 65 4.3.2 Spruce seedling destructive harvest .......................................................................... 69 4.3.2.1 Spruce seedling growth and physiology ........................................................... 69 4.3.2.2 Spruce root EM fungal colonization ................................................................. 72 4.3.2.3 Rooting zone soil fungal communities ............................................................. 77 4.3.2.4 Rooting zone soil bacterial communities .......................................................... 82 4.3.2.5 Rooting zone soil faunal communities .............................................................. 85 4.3.3 Plant ingress .............................................................................................................. 88 4.3.4 Soil physiochemical properties ................................................................................. 91 4.3.4.1 Baseline ............................................................................................................. 91 4.3.4.2 Spruce seedling rooting zone soil after three years .......................................... 92 4.4 Discussion ..................................................................................................................... 93 4.4.1 Site preparation ......................................................................................................... 97 4.4.2 Soil transfers ............................................................................................................. 99 4.4.3 Forest connectivity .................................................................................................. 103 4.5 Conclusion .................................................................................................................. 106 x Chapter 5: Plant–soil feedbacks promote forest succession through plant species turnover and mycorrhizal fungal historical contingencies ....................................................................109 5.1 Introduction ................................................................................................................. 109 5.2 Methods....................................................................................................................... 113 5.2.1 Experimental design................................................................................................ 113 5.2.2 Data collection ........................................................................................................ 117 5.2.3 Root fungal community assessment........................................................................ 117 5.2.4 Statistical analyses .................................................................................................. 119 5.3 Results ......................................................................................................................... 121 5.3.1 Plant–soil feedback effects on seedling growth ...................................................... 121 5.3.2 Historical contingency effects on root fungal symbionts ....................................... 124 5.3.1 Plant–soil feedback effects on root morphology .................................................... 130 5.4 Discussion ................................................................................................................... 131 5.4.1 Plant–soil feedbacks promote plant species turnover and persistence in forest succession ........................................................................................................................... 131 5.4.2 Mycorrhizal fungal community compatibility mediates historical contingencies .. 135 5.4.3 Plant–soil feedbacks are consistent in primary successional soils.......................... 138 5.5 Conclusion .................................................................................................................. 141 Chapter 6: Discussion and synthesis ........................................................................................143 6.1 Overview ..................................................................................................................... 143 6.2 Operational applications of key findings .................................................................... 146 6.3 Implications for Mount Polley Mine........................................................................... 152 6.4 Limitations and future research .................................................................................. 155 xi 6.5 Conclusion .................................................................................................................. 159 References ...................................................................................................................................161 Appendices ..................................................................................................................................189 Appendix A Chapter 1 supplementary material ...................................................................... 189 Appendix B Chapter 3 supplementary material ...................................................................... 192 Appendix C Chapter 4 supplementary material ...................................................................... 204 C.1 Supplemental baseline soil data .............................................................................. 236 Appendix D Chapter 5 PLFA analyses ................................................................................... 241 D.1 Chapter 5 PLFA analyses........................................................................................ 241 Appendix E Chapter 5 supplementary material ...................................................................... 243 xii List of Tables Table 2-1 Properties of deposited tailings, glacial parent material exposed by erosion and reference forest mineral soil in Hazeltine Creek following the Mount Polley Mine tailings dam embankment breach. Bold values indicate an exceedance of BC Contaminated Sites Regulation Standards (parkland guidelines). Table modified from Miller et al. (2015). ................................ 19 Table 2-3 Plant species used in the greenhouse and field experiments presented in Chapter 3, Chapter 4 and Chapter 5. .............................................................................................................. 21 Table 2-2 Chemical and physical properties of the tailings, overburden and forest soil collected at the Mount Polley Mine, BC. Forest soil includes the fermentation layer, humic layer and upper mineral horizons. Cations and effective cation exchange capacity (CEC) = Barium Chloride exchangeable cations. EC = electrical conductivity. .................................................................... 22 Table 3-1 Linear model and generalized linear model results of soil type x soil transfer volume ( 0%, 5% or 25% forest soil addition) effects on spruce, willow and alder seedling establishment with soil transfer volume as a continuous variable. Soil type = tailings, overburden or tailings + overburden. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Df = 2 for soil type, df = 1 for transplant volume, and df = 2 for soil type x soil transfer volume........................................................................................................................................... 36 Table 4-1 Field experiment hypotheses for the effects of site preparation, soil transfer and forest connectivity (both aerial and belowground connectivity) on plant establishment, soil food web recovery and soil physiochemical properties. Site preparation refers to mounding in the till zone, and mounding and tailings removal in the tailings zone. Increasing bacterial succession is based xiii on comparison of phyla relative abundances with forest soil and successional chronosequences. NA = hypothesis not tested or not applicable. OM = organic matter; EM = ectomycorrhizal. .... 53 Table 4-2 Height, root collar diameter (RCD) and volume (mean ± 1 SE) of spruce seedlings in the till zone and tailings zone after three growing seasons. The tailings zone is separated by no site preparation (undisturbed tailings) vs. site preparation (mounding and tailings removal) because site preparation had a major effect. ................................................................................. 67 Table 4-3 Generalized linear models (Poisson, link = logit) of soil faunal abundance and linear models of soil faunal broad group richness in rooting zone soil of harvested spruce seedlings against soil physiochemical parameters across all treatments (site preparation, soil transfer and forest belowground connectivity). Outliers above the 75th percentile or below the 25th percentile by greater than 1.5 x the interquartile range were removed prior to regressions. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). .......................................... 87 Table 4-4 Plant richness and cover (mean ± 1 SE) in the till zone, tailings zone and reference forest. The tailings zone is separated by no site preparation (undisturbed tailing) vs. site preparation (mounding and tailings removal) because site preparation had a major effect on the plant community. .......................................................................................................................... 90 Table 4-5 Field experiment results for site preparation, soil transfer and forest connectivity effects on plant establishment, soil food web recovery and soil physiochemical properties. Only main effects are presented (interactions are discussed in-text). Site preparation refers to mounding in the till zone, and mounding and tailings removal in the tailings zone. Increasing bacterial succession is based on comparison of phyla relative abundances with forest soil and successional chronosequences. Colours indicate positive (dark green), weakly positive (light green), neutral (white) or negative (orange) effects. NA = hypothesis not tested or not applicable. xiv N = total nitrogen, P = available phosphorus, CEC = effective cation exchange capacity; OM = organic matter; EM = ectomycorrhizal. ........................................................................................ 96 Table 5-1 Combinations of conditioning and feedback phase plant species tested in the two-phase plant–soil feedback experiment. AMF = arbuscular mycorrhizal fungi; EMF = ectomycorrhizal fungi. ................................................................................................................ 115 Table 6-1 Barriers to ecosystem recovery following mining disturbance and key findings of this thesis on the role of ecosystem legacy-based reclamation techniques (soil transfers, proximity to undisturbed forest and plant–soil feedbacks) as well as site preparation (a common reclamation technique for improving soil physical properties) in overcoming these barriers. Barriers adapted from (Bradshaw, 2000). .............................................................................................................. 151 Table A-1 Tailings, overburden and forest soil collection locations at the Mount Polley Mine, British Columbia for the greenhouse experiments in Chapter 3 and Chapter 5. ........................ 190 Table B-1 Post hoc soil type contrasts of linear model and generalized linear model results of soil type x soil transfer volume (0%, 5% or 25% forest soil addition) effects on spruce, willow and alder seedling establishment with soil transfer volume as a continuous variable. Soil type = tailings, overburden or tailings + overburden. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Df = 2 for soil type, df = 1 for transplant volume, and df = 2 for soil type x soil transfer volume. ...................................................................................... 192 Table B-2 Results of spruce ectomycorrhizal (EM) fungal morphotype identification with high-throughput sequencing. Grey shaded rows indicate confirmation of non-mycorrhizal morphotype vouchers. ..................................................................................................................................... 193 xv Table B-3 Spruce, willow and alder seedling establishment (mean ± 1 SE). Significant differences of soil treatments from 100% forest soil are indicated by *** (P < 0.001), ** (0.001 ≤ P < 0.01), * (0.01 ≤ P < 0.05) and . (0.05 ≤ P < 0.1). Df = 9 for soil treatment. Growth parameters were relativized per growing day to account for growth period differences due to replanting. ................................................................................................................................... 194 Table B-4 Results from linear models for unsterilized soil treatments with symbiont colonization as the explanatory variable and soil treatment (soil type (tailings, overburden or tailings + overburden) and transplant volume (0%, 5% or 25%) combined) as a covariate. Shoot biomass, root:shoot ratio and specific root length were tested as response variables for each plant species. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). .......... 196 Table B-5 Change in slope of effect of increasing forest soil transfer volume on seedling establishment with soil sterilization. Bold = significant P-value (P< 0.05); italics = marginally significant (0.05 < P < 0.1). ........................................................................................................ 197 Table C-1 Basal area of trees species at forest soil transfer collection sites in the undisturbed forest adjacent to each trial block in the till and tailings zones (mean of two 50 m2 plots). ...... 204 Table C-2 Spruce seedling vigour assessment criteria ............................................................... 204 Table C-3 Till zone post hoc contrasts of site preparation, soil transfer and forest belowground connectivity effects on spruce seedling survival, growth, vigour and chlorosis. Contrasts were run on binomial generalized mixed models (survival and chlorosis), Poisson generalized mixed models (vigour) and linear mixed models (height, root collar diameter and volume growth). Interactions only shown where significant. Survival is cumulative (i.e., total survival after second and third growing seasons). On the vigour scale, 1 = highest and 4 = lowest, thus negative vigour xvi model results indicate improved vigour. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). RCD = root collar diameter. ........................................ 205 Table C-4 Tailings zone post hoc contrasts of soil transfer, site preparation and forest belowground connectivity effects on spruce seedling survival, growth, vigour and chlorosis. Contrasts were run on binomial generalized mixed models (survival and chlorosis), Poisson generalized mixed models (vigour) and linear mixed models (height, root collar diameter and volume growth). Interactions only shown where significant. Survival is cumulative (i.e., total survival after the second and third growing seasons). On the vigour scale, 1 = highest and 4 = lowest, thus negative vigour model results indicate improved vigour. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). RCD = root collar diameter. ........ 207 Table C-5 Post hoc contrasts of soil transfer, site preparation and forest belowground connectivity effects on spruce seedling biomass, foliar nitrogen (N) ratio and foliar δ13C. Contrasts were run on linear mixed models. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). δ13C data were not available for four of eight treatments in one till trial block. ................................................................................................................... 209 Table C-6 Sanger sequencing results for ectomycorrhizal fungal morphotype vouchers from the roots of harvested spruce seedlings. Fungal taxa were assigned using a 97% sequence similarity threshold. ..................................................................................................................................... 210 Table C-7 High-throughput sequencing results for ectomycorrhizal (EM) fungal morphotype vouchers from the roots of harvested spruce seedlings. Samples with poor amplification or no dominant EM taxa (> 10% of reads) were assigned as non-mycorrhizal (grey highlighting). Samples with poor amplification but a dominant EM taxon that aligned with morphology from morphotyping were retained. Blue highlighting denotes secondary EM taxa (not the most xvii abundant) also present in vouchers i.e., mixed vouchers. Sample F148 was assigned to Russula nauseosa despite it being the second most abundant EM taxa based on morphology recorded during morphotyping. ................................................................................................................. 211 Table C-8 Post hoc contrasts of site preparation, soil transfer and forest belowground connectivity effects on spruce seedling ectomycorrhizal fungal percent colonization, richness and Shannon diversity. Contrasts were run on binomial generalized linear mixed models (colonization) and linear mixed models (richness and diversity). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ............................................................. 213 Table C-9 Relative abundance and taxa richness of saprotrophic, ectomycorrhizal (EM), and plant pathogenic fungi amplicon sequence variants (ASVs) in spruce seedling rooting zone soil in the tailings and till zones compared to the reference forest. ................................................... 213 Table C-10 PERMANOVA results of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of spruce seedling rooting zone soil fungal communities (Hellinger transformed) with distance from forest edge as a covariate. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ........................................................................ 214 Table C-11 Post hoc contrasts of mounding, soil transfer and forest belowground connectivity effects on richness, diversity and abundance of spruce seedling rooting zone soil fungal amplicon sequencing variants (ASVs) and guilds in the till zone. Contrasts were run on linear mixed models (richness and diversity) and binomial generalized linear mixed models (abundance). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ................... 215 Table C-12 Post hoc contrasts of distance covariate effects on spruce seedling rooting zone soil fungi, bacteria and fauna in the till and tailings zones. Contrasts were run on linear mixed models xviii (richness and diversity), binomial generalized linear mixed models (relative abundance) and Poisson generalized linear mixed models (faunal abundance). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ASV = amplicon sequencing variant. . 216 Table C-13 Post hoc contrasts of site preparation, soil transfer and forest belowground connectivity effects on richness, diversity and abundance of spruce seedling rooting zone soil amplicon sequencing variants (ASVs) and guilds in the tailings zone. Contrasts were run on linear mixed models (richness and diversity) and binomial generalized linear mixed models (abundance). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ............................................................................................................................................. 217 Table C-14 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity combined), distance from forest edge, soil properties (pH, organic matter, moisture and electrical conductivity) and naturally ingressing plant community on spruce seedling rooting zone soil fungal and bacterial communities (Bray Curtis distances of Hellinger-transformed community data). Marginal significance was tested with db-RDAs for each factor conditioned on trial block. Conditional significance was tested with RDAs of each factor conditioned on the other factors and trial block. Significance was tested with ANOVA run on 999 permutations restricted within levels of block. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ........................................................................ 218 Table C-15 PERMANOVA results of site preparation (mounding in the till zone, tailings removal and mounding in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of spruce seedling rooting zone soil bacterial communities (Hellinger transformed) with distance from forest edge as a covariate. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ..................................................... 219 xix Table C-16 Post hoc contrasts of linear mixed models of site preparation, soil transfer and forest belowground connectivity effects on richness, diversity and abundance of spruce seedling rooting zone soil bacterial amplicon sequencing variant (ASV) richness and Shannon diversity. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). .......... 220 Table C-17 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity combined), distance from forest edge and soil properties (pH, organic matter, moisture and electrical conductivity) and naturally ingressing plant community effects on spruce seedling rooting zone soil faunal communities (Bray Curtis distances). Marginal significance was tested with db-RDAs for each factor conditioned on trial block. Conditional significance was tested with RDAs of each factor conditioned on the other factors and trial block. Soil pH had a significant marginal effect in the tailings zone (P = 0.045) and was used to assess conditional effects. Significance was tested with ANOVA run on 999 permutations restricted within levels of block. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ...................................................................................... 221 Table C-18 PERMANOVA results of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of the log-transformed naturally ingressing plant community with distance from forest edge as a covariate. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ........................................................................................................ 222 Table C-19 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity combined), distance from forest edge and soil properties (pH, organic matter, moisture and electrical conductivity) on log-transformed naturally ingressing plant communities (Bray Curtis distances). Marginal significance was tested with db-RDAs for xx each factor conditioned on trial block. Conditional significance was tested with RDAs of each factor conditioned on the other factors and trial block. Significance was tested with ANOVA run on 999 permutations restricted within levels of block. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ........................................................................ 223 Table C-20 PERMANOVA results of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of baseline soil chemical parameters with distance from forest edge as a covariate. Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ........................................................................ 224 Table C-21 Soil chemical properties of forest soil used in transfers for block and mean baseline soil properties of the till and tailings zones by site preparation type. Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. CEC = cation exchange capacity; EC = electrical conductivity. ............................. 239 Table E-1 Next-generation sequencing identification of spruce ectomycorrhizal (EM) fungi morphotype vouchers. Grey cells indicate non-mycorrhizal samples. ....................................... 243 Table E-2 Conditioning species post hoc comparisons of shoot biomass linear models by feedback species (willow, spruce and redcedar). Bold values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). ...................................................................... 244 Table E-3 Shoot biomass linear model results by feedback phase species (willow, spruce and redcedar) with soil type (forest, overburden, tailings) and forest soil addition (yes, no) treatments combined into the soil treatment model term. Foliar carbon to nitrogen (C:N) ratio, soil depth xxi (settling in pot) and drought damage (willow only) were included as covariates. Bold values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). .................. 244 Table E-4 Shoot biomass linear model results by feedback phase species (willow, spruce and redcedar) with conditioning species, soil type (overburden, tailings; forest soil not analyzed here) and inoculation (yes, no) treatments as explanatory variables. Foliar carbon to nitrogen (C:N) ratio, soil depth (settling in pot) were included as covariates. Bold italic values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). ......................................... 245 Table E-5 Indicator species analysis of willow and redcedar root fungal communities from next-generation sequencing for a subset of soil treatments (forest soil, overburden and tailings only i.e., no forest soil additions) and spruce ectomycorrhizal fungi communities based on morphotyping (combined across all soil types). ......................................................................... 246 Table E-6 Sanger sequencing identification of ectomycorrhizal fungi vouchers from morphotyping of spruce seedling roots. ...................................................................................... 248 Table E-7 Conditioning species post hoc comparisons of shoot biomass linear models by feedback species (willow, spruce and redcedar) with no foliar nutrient covariate in the model. Bold values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). ........................................................................................................................................... 248 xxii List of Figures Figure 1-1 Location of the Mount Polley Mine, British Columbia, Canada (A) and overview of the 2014 Mount Polley Mine tailings dam failure flow path from the Tailings Storage Facility into Polley Lake, then down Hazeltine Creek into Quesnel Lake (B). ......................................... 13 Figure 3-1 Spruce seedling linear model (A, B) and binomial generalized linear model (C) results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling shoot biomass (A), foliar nitrogen (B) and ectomycorrhizal fungal (EMF) percent colonization (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Log transformation = loge. Shoot biomass was relativized per growing day to account for growth period differences due to replanting. .................................................. 37 Figure 3-2 Willow seedling linear model (A, B) and binomial generalized linear model (C, D) results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling shoot biomass (A), foliar nitrogen (B), ectomycorrhizal fungal (EMF) colonization (C), and fungal endophyte colonization (D) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Shoot biomass was relativized per growing day to account for growth period differences due to replanting. ............................................................. 38 Figure 3-3 Alder seedling linear model (A–C) and binomial generalized linear model (D) results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling shoot biomass (A), foliar nitrogen (B), nitrogen-fixing nodule wet weight (C) and fungal endophyte xxiii percent colonization (D) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Log transformation = loge. Shoot biomass was relativized per growing day to account for growth period differences due to replanting ............. 39 Figure 4-1 Depiction of the field trial experimental design in one till zone block (A) with a 2x2x2 factorial design (n = 4 spruce seedlings x 15 distances, totalling 60 seedlings per treatment unit). Factors were site preparation (excavator mounding), soil transfers and belowground connectivity to the adjacent forest (trench vs. no trench). Three blocks with randomized treatments were installed. The same design was applied to three blocks in the tailings zone (B), except three site preparation treatments were tested: no site preparation (undisturbed tailings), mounding and tailings removal. Due to space restrictions, the no site preparation treatment (undisturbed tailings) was tested with and without forest transfers, but only with belowground connectivity (i.e., 2 x 2 x 2 full factorial design plus two additional no site preparation treatments, totaling ten treatment units per tailings zone block). ................................................. 56 Figure 4-2 Till zone: third growing season effects of mounding (A, C), soil transfers (A, C) and forest belowground connectivity (B, D) on spruce seedling shoot volume growth (A, B) and chlorosis (C, D). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from linear mixed models (A, B) and binomial generalized linear mixed models (C, D). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). ................................................................................................................................... 68 Figure 4-3 Tailings zone: third growing season effects of site preparation (mounding and tailings removal; A, C), soil transfers (B, D) and forest belowground connectivity (B, D) on spruce seedling shoot volume growth (A, B) and chlorosis (C, D). Bar plots represent main effects and xxiv line plots represent interactions. Soil transfers significantly reduced seedling chlorosis with no connectivity interaction (D). Bars/points are the back-transformed means ± 1 SE from linear mixed models (A, B) and binomial generalized linear mixed models (C, D). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). ................................................................................................... 69 Figure 4-4 Till zone: effects of mounding, soil transfers and forest belowground connectivity on spruce seedling foliar N (A) and foliar δ13C (‰) (B). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). .......................................... 71 Figure 4-5 Tailings zone: effects of site preparation (mounding and tailings removal; A, C), soil transfers (A, C) and forest belowground connectivity (B, D) on spruce seedling shoot biomass (A, B) and foliar nitrogen (C, D). Models for the no site preparation treatment in A and C exclude outlier seedlings with access to organic matter deposits. Bar plots represent main effects and line plots represent interactions. Mounding and tailings removal increased shoot biomass with no soil transfer interaction (A) and tailings removal increased foliar nitrogen with no interaction (C). Bars/points are the back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). ....................................................................... 72 Figure 4-6 A: Relative abundance of ectomycorrhizal fungal taxa on spruce seedling roots. Baseline nursery seedling root fungal communities were assessed with high-throughput sequencing prior to planting. Spruce seedling roots following three growing seasons in the till and tailings zones were assessed through morphotyping and DNA sequencing of morphotype xxv vouchers. B: Relative abundance of dominant fungal genera (> 2% of total sequencing reads) in spruce seedling rooting zone soil in the till zone, tailings zone and reference forest. Fungal guilds are indicated in brackets. EM = ectomycorrhizal; endo = endophyte; path = pathogen; sap = saprotroph. .................................................................................................................................... 75 Figure 4-7 Ectomycorrhizal fungi colonizing spruce seedlings roots after three growing seasons in areas of till and tailings at the Mount Polley Mine, British Columbia as identified through morphotyping and DNA sequencing of morphotype vouchers. Dominant taxa were Amphinema byssoides (A), Wilcoxina rehmii (B), Thelephora terrestris (C), Pseudotomentella sp. (D), Inocybe rufoalba (E), Cenococcum sp. (F), and Tuber annaie (G). ............................................. 76 Figure 4-8 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity on spruce seedling root ectomycorrhizal fungal percent colonization (A, C), morphotype Shannon diversity (B) and morphotype richness (D) in the till zone (A, B) and tailings zone (C, D). Bar plots represent main effects and line plots represent interactions. Mounding and tailings removal significantly increased EM fungal colonization without a soil transfer interaction (C). Bars/points are the back-transformed means ± 1 SE from binomial generalized linear mixed models (A, C) and linear mixed models (B, D). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant)...................... 77 Figure 4-9 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity (and their interactions) on ectomycorrhizal (EM), plant pathogen and saprotroph fungal amplicon sequence variant (ASV) richness in rooting zone soil of spruce seedlings in the till zone (A, B) and tailings zone (C, D). Bar plots represent main effects and line plots represent interactions. Soil transfers xxvi significantly increased EM and saprotroph fungal richness in the tailings zone with no connectivity interaction (D). Bars/points are back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). ....................................................... 81 Figure 4-10 db-RDA of soil transfer and site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone) effects on Bray Curtis distances of Hellinger-transformed bacterial amplicon sequence variant (ASV) communities in the till zone (A) and tailings zone (B). Full models included forest belowground connectivity and treatment interaction terms, and a distance from forest edge covariate (Table C-15). PERMANOVA analysis found significant effects of site preparation (till: P = 0.001; tailings: P = 0.002) and soil transfers (till: P = 0.002; tailings: P = 0.031). .............................................................................. 84 Figure 4-11 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity on soil faunal abundance (A, D), broad group richness (B, E) and broad group Shannon diversity (C, F) in the top 6 cm of rooting zone soil of spruce seedlings in the till zone (A–C) and tailings zone (D–F; site preparation effects only). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from binomial generalized linear mixed models (abundance) and linear mixed models (diversity and richness). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). ................................................................................................... 88 Figure 4-12 Representative examples of plant diversity and percent cover in the tailings zone (A, B) and till zone (C, D) with no site preparation (A, C) and mounding (B, C). All plots are at the 1–5m from forest edge. Photo credit: Katie McMahen. ............................................................... 91 xxvii Figure 5-1 Shoot biomass loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to control unconditioned soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ................................................................................................................................... 123 Figure 5-2 Shoot biomass loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned reclamation soils (tailings, overburden and tailings + overburden) compared to control unconditioned soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ............................................................................................................................. 124 Figure 5-3 Percent colonization of ectomycorrhizal fungi (EMF; A, D), arbuscular mycorrhizal fungi (AMF; B, E), and non-AMF fungal endophytes (C, F) on willow (A, B, C), spruce (D) and redcedar (E, F) seedling roots grown in control, willow-conditioned and spruce-conditioned soils (forest, overburden, tailings and tailings + overburden). “+ forest soil” treatments were mixed with 25% v/v forest soil. Bars represent mean ± 1 SE. Significant differences between conditioning species in each soil treatment are indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05) or NS (not significant) in A–D, or by different letters in C–F (P < 0.05). ........................................................................................................................................... 129 Figure 5-4 Willow root fungal pathogen amplicon sequence variant (ASV) abundance (A) and fungal symbiont ASV richness (B) in forest soil, overburden and tailings treatments without forest soil additions. Bars represent mean ± 1 SE. Pathogen abundance increased (Z = 3.18, P = 0.002) and symbiont richness decreased (T25 = -2.47, P = 0.021) in willow-conditioned soil compared to unconditioned control soil. ..................................................................................... 130 xxviii Figure 5-5 Summary of successional plant–soil feedback results in early successional mine reclamation soils (A) and mid–late successional forest soil (B). Arrow colour indicates the feedback direction and symbols indicate the feedback mechanism. These results were integrated with available literature to develop a conceptual model for plant–soil feedbacks effects on forest succession (C). ............................................................................................................................ 134 Figure 6-1 Representative photos of till zone (A, B) and tailings zone (C, D) treatment units without mounding (A, C) and with mounding (B, D) at the Mount Polley Mine, British Columbia. Photo credit: Katie McMahen. .................................................................................. 154 Figure A-1 Locations at the Mount Polley Mine, British Columbia where forest soil, overburden (glacial till) and mine tailings were collected for the greenhouse experiments in Chapter 3 and Chapter 5. .................................................................................................................................... 191 Figure B-1 Diffrences in total Phospholipid fatty acid (PLFA) biomass (A) and PLFA biomass of microbial broad groups (B) between freshly harvested forest soil and potted forest soil maintained under greenhouse conditions for the time period (6 months) required to acquire more viable alder seed (n = 3). Total biomass did not signficantly change with storage (P = 0.622). There were no significant differences between baseline and stored (greenhouse) microbial group biomasses except for protozoa, which increased with storage (P = 0.006). ............................... 198 Figure B-2 Spruce seedling linear model results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling root biomass (A), root:shoot ratio (B) and chlorophyll fluorescence (C) in sterilized and unsterilized soils. The dashed line is the mean value for xxix seedlings grown in unsterilized forest soil. Growth parameters were relativized per growing day to account for growth period differences due to replanting. ....................................................... 199 Figure B-3 Willow seedling linear model results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% addition) effects on seedling root biomass (A), root:shoot ratio (B) and chlorophyll fluorescence (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Growth parameters were relativized per growing day to account for growth period differences due to replanting. ....................................................... 200 Figure B-4 Alder seedling linear model results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% addition) effects on seedling root biomass (A), root:shoot ratio (B) and chlorophyll fluorescence (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Log transformation = loge. Growth parameters were relativized per growing day to account for growth period differences due to replanting. .......... 201 Figure B-5 Soil sterilization effects on soil root symbiont colonization. Post-hoc comparisons were conducted on linear models (E) and generalized linear models (A–D) with soil treatment (soil type and forest soil transfer (addition) volume combined) and sterilization as the explanatory variables. Significant differences are indicated by *** (P < 0.001), ** (0.001 ≤ P < 0.01), * (0.01 ≤ P < 0.05) and . (0.05 ≤ P < 0.1). ....................................................................... 202 Figure B-6 Representative spruce, willow and alder seedlings (median biomass from replicates in treatment unit) growing in Mount Polley Mine tailings with 0%, 5% or 25% forest soil additions. 100% forest soil treatments are shown for compression. Photo credit: Katie McMahen. .......... 203 xxx Figure C-1 Field trial block locations in areas impacted by the Tailings Storage Facility embankment failure at the Mount Polley Mine, British Columbia, Canada. Note that some trial blocks locations are in the forest; these areas were logged after aerial imagery was taken due to forest disturbance to facilitate recontouring of steep, eroded banks (till zone) or tree die-off from anaerobic soil conditions created by saturated tailings deposition (tailings zone). .................... 225 Figure C-2 db-RDA of spruce seedling rooting zone soil fungal community (Hellinger-transformed) Bray Curtis dissimilarities between the reference forest, tailings zone and till zone site. The reference forest soil fungal community was highly dissimilar from the tailings zone (Bray Curtis dissimilarity = 0.92) and till zone (Bray Curtis dissimilarity = 0.92) soil, and the till and tailings zones were dissimilar from each other (Bray Curtis dissimilarity = 0.89). ............ 226 Figure C-3 Relative abundance of dominant bacterial phyla (> 1.5% of total sequencing reads) in spruce seedling rooting zone soil in the till zone, tailings zone and reference forest. ................ 227 Figure C-4 Binomial generalized linear mixed model T-value results of effects of site preparation, soil transfers and forest belowground connectivity on bacterial phyla abundance in spruce seedling rooting zone soil. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). OM = organic matter; EC = electrical conductivity. ............................................................................. 228 Figure C-5 Relative abundance of soil faunal broad groups in spruce seedling rooting zone soil in the till zone, tailings zone and reference forest. All broad groups are at the order level except Chilopoda is a class, and Acari and Collembola are subclasses. ................................................ 229 Figure C-6 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity) and soil properties (pH) on soil faunal community composition Bray Curtis dissimilarities in the tailings zone. .......................................................................... 230 xxxi Figure C-7 db-RDA of natural ingressing plant community (log-transformed) Bray Curtis dissimilarities between the reference forest, tailings zone and till zone site. Plant community composition was highly dissimilar between till and tailings zones (Bray Curtis dissimilarity = 0.78) and from the reference forest (till zone Bray Curtis dissimilarity = 0.93; tailings zone Bray Curtis dissimilarity = 0.92). ........................................................................................................ 231 Figure C-8 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity on plant percent cover (A, C), non-native plant percent cover (B) and Shannon diversity (D) of naturally ingressing plants in the till zone (A, B) and tailings zone (C, D). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from binomial generalized linear mixed models (cover) and linear mixed models (diversity). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). OM = organic matter; EC = electrical conductivity. .............. 232 Figure C-9 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity), distance from forest edge and soil properties (pH, organic matter, moisture and electrical conductivity) on Bray Curtis distances of log-transformed naturally ingressing plant community in the till zone (A) and tailings zone (B). communities. Only marginally significant factors, as tested by db-RDAs for each factor conditioned on trial block, were included in variation partitioning. ........................................................................... 233 Figure C-10 Effects of site preparation (mounding and tailings removal) on bulk density in the till and tailings zones, with comparison to reference forest soil. Box centerlines = mean, hinges = 25th and 75th percentiles, whiskers = 1.58 x interquartile range (≈ 95% confidence interval) and points = outliers. ......................................................................................................................... 234 xxxii Figure C-11 Correlation matrices of soil baseline chemical parameter Pearson correlation coefficients in the till zone (A) and tailings zone (B). Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. EC = electrical conductivity (μS/cm); CEC = effective cation exchange capacity (cmol+/kg); N_tot = total nitrogen (%); C_tot = total carbon (%); P = PO4-P (mg/kg). All metals = exchangeable ions (cmol+/kg). ................................................................................................ 235 Figure C-12 Effects of site preparation (mounding in the till zone; mounding and tailings removal in the tailings zone; C–H) and mounding x soil transfer interactions (A, B) on spruce seedling rooting zone soil parameters in the till zone (A–D) and tailings zone (E–H). Bar plots represent main effects and line plots represent interactions. Bars/points are back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). OM = organic matter; EC = electrical conductivity. .......................................................................... 236 Figure C-13 db-RDA of site preparation effects on Bray Curtis distances of baseline soil chemical properties. Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. Full models included soil transfer, forest belowground connectivity and treatment interactions, and a distance from forest edge covariate (Table C-20). PERMANOVA analysis found significant effects of site preparation in the till zone (P = 0.048) and the tailings zone (P = 0.003). ................................. 240 Figure D-1 Phospholipid fatty acid (PLFA) total biomass (A) and microbial broad group biomass (B) results for control forest soil before (= Baseline) and after (= Storage) storage at -4 °C during the conditioning phase of the experiment. .................................................................................. 242 xxxiii Figure E-1 Seedling height loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ................................................................................................................................... 249 Figure E-2 Seedling root collar diameter loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ........................................................................................................ 250 Figure E-3 Seedling root biomass loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ............................................................................................................................. 251 Figure E-4 Regressions of shoot biomass feedbacks against ectomycorrhizal fungi (EMF; A, C), arbuscular mycorrhizal fungi (AMF; E) and root fungal endophyte (B, F) percent colonization feedbacks, and shoot biomass feedbacks against EMF species richness (E) for willow (A, B), spruce (C, D) and redcedar (E, F) seedling roots. Each data point represents loge response ratios of conditioning treatment vs. control for one soil type x forest soil amendment treatment. ...... 252 Figure E-5 Representative willow, spruce and redcedar seedlings (replicate with median shoot biomass in treatment) grown in control, willow-conditioned or spruce-conditioned forest soil for six months in a greenhouse. ........................................................................................................ 253 xxxiv Figure E-6 NMDS analysis of high-throughput sequencing fungal (ITS2) community data for willow (A) and redcedar (B) in willow- and spruce-conditioned soils (forest soil, overburden and tailings) compared to unconditioned control soils. Ellipses could not be draw for willow and control (none) conditioning treatments in redcedar due to poor survival. .................................. 254 Figure E-7 Amplicon sequence variant (ASV) richness (A, C, E) and relative abundance (B, D, E) of pathogenic (A, B), symbiotic (C, D) and saprotrophic (E, F) fungi on roots of willow seedlings grown in unconditioned control and willow-conditioned soils (tailings, overburden and tailings + overburden). “+ forest soil” treatments were mixed with 25% v/v forest soil. Bars represent mean ± 1 SE. ............................................................................................................... 255 Figure E-8 Spruce seedling ectomycorrhizal fungal (EMF) species richness loge response ratios in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ............................................................................................................................. 256 Figure E-9 Spruce seedling ectomycorrhizal fungal (EMF) species richness loge response ratios of in willow- and spruce-conditioned reclamation soils (tailings, overburden and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ............................................................................................................................. 256 Figure E-10 Amplicon sequence variant (ASV) richness (A, C, E) and relative abundance (B, D, E) of pathogenic (A, B), symbiotic (C, D) and saprotrophic (E, F) fungi on roots of redcedar seedlings grown in unconditioned control, willow-conditioned and spruce-conditioned soils xxxv (tailings, overburden and tailings + overburden). “+ forest soil” treatments were mixed with 25% v/v forest soil. Bars represent ± 1SE. .......................................................................................... 257 Figure E-11 Spruce (A) and redcedar (B) specific root length loge response ratios of in willow- and spruce-conditioned reclamation soils (tailings, overburden and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. ................................................................................................................................... 258 Figure E-12 Regressions of shoot biomass (A, C) and mycorrhizal fungal percent colonization (B, D) feedbacks against specific root length feedbacks for spruce (A, B) and redcedar (C, D) seedlings. Each data point represents loge response ratios of conditioning treatment vs. control for one soil type x forest soil addition treatment. EMF = ectomycorrhizal fungi; AMF = arbuscular mycorrhizal fungi. ..................................................................................................... 259 xxxvi List of Abbreviations AM Arbuscular mycorrhizal ANOVA Analysis of variance ASV Amplicon sequence variant BC British Columbia C Carbon CEC Cation exchange capacity DBH Diameter at breast height EM Ectomycorrhizal GDP Gross domestic product ITS Internal transcribed spacer MPMC Mount Polley Mining Corporation NSERC Natural Sciences and Engineering Research Council N Nitrogen P Phosphorus PCR Polymerase chain reaction PERMANOVA Permutational multivariate analysis of variance PLFA Phospholipid fatty acid UBC University of British Columbia xxxvii Acknowledgements I first have to thank Trish Miller, ‘Lyn Anglin and Suzanne Simard who were inspired to carry out research following the tailings dam failure at the Mount Polley Mine, generating positive outcomes from the otherwise devastating incident by advancing reclamation science. I am grateful to have been a part of this team and thank my colleagues at the Mount Polley Mine for supporting a smooth transition to graduate school. I am incredibly fortunate to have been supervised by Suzanne Simard whose positivity, big ideas, passion and impressive ability to share that passion are a constant source of inspiration. I am grateful for the encouragement, insight and helpful feedback of my supervisory committee members Susan Grayston, Les Lavkulich and ‘Lyn Anglin, and for their support of my transition from a MSc to a PhD program. I would like to thank Mount Polley Mining Corporation (MPMC) staff including Art Frye, Colleen Hughes, Gabriel Holmes, Shauna Litke and Valerie Holweck for always accommodating my field visits and on-site needs. Thank you to my undergraduate research assistants Maria Continentino, Alyx Hough, Jordan Carter, Erica Xu and Tong Ye for their laboratory and greenhouse help. Thank you to Jean Roach, Eva Snyder, Daniel Melvin, Sophie Vanderbanck, Rachel Green and Abbey Clancy for help with annual seedling measurements. A special thank you to Maria Continentino for her positivity and hard work during seedling harvesting in the field. Thanks also to undergraduate laboratory and greenhouse volunteers Cassandra Harper, Aman Kandola, Jenny Mackay, Hilary Clark, Levi Robson, Thomson Harris, Lukas Charron, Chany Lapierre, Priya Joshi, Mariella Becu, Curtis Logan and Jennifer Ahn. I am grateful for the camaraderie and guidance of friends and colleagues at UBC, including Eva Snyder, Allen Laroque, Amanda Asay, Monika Gorzelak, Camille Defrenne, xxxviii Gabriel Orrego, Dixi Modo, Elana Evans, Laura Super, Dylan Mendenhall, Catch Catomeris. Devin de Zwaan, Cora Skaien and Hannah Visty. A thank you to Melina Biron, the Greenhouse Research Manager, and Belowground Ecosystem Group Lab Mangers Shannon Guichon, Mina Momayyezi and Alice Chang for their logistical, technical and administrative assistance. I am extremely grateful to my partner, Luke Moger, my parents, Ann Carter and Ivor McMahen, and my siblings, Marshall and Monica McMahen, for their constant encouragement and support throughout the PhD process and in all that I do. I am especially indebted to my parents for housing and feeding my field assistants and me throughout three field seasons. On top of this, my mom helped manage the microarthropod “lab” and my dad helped with a couple long field days of harvesting seedlings; they are an amazing support system. Financial support for this research was from an NSERC Engage grant and Mitacs Accelerate internship, both in partnership with MPMC, as well as a Puget Sound Mycological Society Ben Woo grant. MPMC provided in-kind support for the field trial, including excavator assistance for trial installation. In-kind sequencing support was provided by Agriculture and Agrifoods Canada. I was supported by an NSERC Canada Graduate Scholarship, a Killam Graduate Memorial Doctoral Scholarship and a UBC Four Year Doctoral Fellowship. Additional financial support came from the Edward W. Bassett Memorial Scholarship in Reforestation, Adrian Weber Memorial Scholarship in Forest Ecology, Peter Rennie Memorial Award, David L. Oswald Forestry Award, VanDusen Graduate Fellowship in Forestry, BC Technical and Research Committee on Reclamation Jake McDonald Memorial Scholarship, BC Contaminated Sites Approved Professionals Mike Macfarlane Memorial Scholarship, BC Institute of Agrologists Scholarship and Metro Women’s Soccer League Scholarship. I am incredibly honoured to have received these awards and greatly appreciate the financial support. 1 Chapter 1: Introduction Surface mining operations severely disturb terrestrial ecosystems, removing vegetation and soil, and creating landscape-level changes that result in conditions comparable to the early stages of primary succession (Bradshaw, 1984). The main materials requiring reclamation following mining are waste rock (blasted rock removed to access ore) and tailings (finely ground rock that is a waste product from ore processing; Tordoff et al., 2000). Both have poor physical structure and limited hydraulic capacity, are low in nutrients and organic matter, and host minimal soil biological activity (Bois et al., 2005; Huang et al., 2012; Sheoran et al., 2010). Although not the focus of this thesis, tailings and waste rock can also leach metals and other elements at varying rates depending on their geochemistry (Jamieson et al., 2015). Topsoil and overburden (subsoil) are typically salvaged prior to mining disturbance and stored during operations to use as supplementary growing mediums in reclamation following mine closure (Tordoff et al., 2000). However, overburden lacks the organic matter and active biological communities of topsoil, and salvaged topsoil can be of variable quality due to degradation during long-term storage in stockpiles (Gorzelak et al., 2020; Paterson et al., 2019). Further, many mine sites do not have sufficient topsoil or overburden for use in reclamation due to a lack of historic salvaging practices and/or due to naturally thin soil types that are challenging to salvage and redistribute (Sheoran et al., 2010). Consequently, the physical, chemical and biological constraints of mining waste materials combined with the limited availability of high-quality reclamation soils and the harsh, exposed nature of many post-mining landscapes present limitations for plant community recovery. Given that an estimated 30–88 million (M) hectares (ha) of land have been disturbed by mining and quarrying activities globally (Cherlet et al., 2018), implementation of reclamation 2 practices that address these barriers to revegetation are critical for restoring terrestrial ecosystem functions. Soil organisms carry out many of the ecosystem processes necessary to ameliorate degraded soil conditions and support reestablishment of plant communities, such as litter decomposition, formation of soil structure and enhancement of plant nutrient uptake. This thesis investigated if legacies of the pre-disturbance ecosystem, including proximity to undisturbed forest and fresh topsoil, can be applied to promote recovery of soil organisms, and assessed if soil biological community recovery corresponded to improved plant establishment. I also examined if topsoil additions and plant establishment in mine reclamation materials regenerate soil ecosystem legacies, and if these newly formed legacies influence vegetation successional trajectories. The following section outlines of the role of soil biological communities in mine reclamation. I then present a literature review describing current knowledge and research gaps in (1) application of spatial and topsoil ecosystem legacies in mine reclamation, and (2) the role of ecosystem legacies in long-term plant community assembly. Finally, I introduce the study site and provide an outline of the thesis chapters and hypotheses. 1.1 The role of soil biological communities in mine reclamation Soil biological communities (the soil food web) are key drivers of soil development following ecosystem disturbance, including formation of soil structure, decomposition and nutrient cycling, and development of soil organic matter (Haynes, 2014). These processes are mediated by complex interactions of the microbial and faunal assemblages that comprise the trophic levels of the soil food web, including photosynthesizers, decomposers, mutualists, pathogens, parasites, shredders, grazers and predators (Coleman et al., 2018; Ingham et al., 1985). Soil structure is 3 created by macrofauna burrowing and mixing soil (bioturbation), while smaller fauna contributes to pore formation (Coleman, 2008). In mine reclamation, increased macrofaunal density and activity have been linked with improved soil microstructure and increased organic matter translocation within the soil profile, both of which can help ameliorate compaction and increase water and air flow (Frouz et al., 2006, 2007). Microbes further contribute to development of soil structure and aggregate development through physical binding (e.g., by fungal hyphae) and production of metabolites that promote binding and formation of organic colloids (Chotte, 2005). The breakdown and transformation of plant litter by soil fauna through ingestion combined with bioturbation also supports nutrient cycling. These processes redistribute nutrients and increase soil organic carbon, stimulating colonization and activity of microbes that mineralize nutrients into plant-available forms (Drenovsky et al., 2004). This process of nutrients being transformed into available forms for the subsequent trophic level has been shown in mine reclamation soils, supporting the need to rehabilitate all functional groups (taxa groupings based on morphological traits and life history) in reclamation. For example, higher densities of soil fauna have been shown to increase microbial activity and indirectly increase nutrient availability (Topp et al., 2001). In contrast, a lack of macrofauna at reclaimed mine sites can result in increased accumulation of litter fragments, forming a recalcitrant fermentation layer on the surface instead of promoting decomposition and humus formation (Frouz et al., 2007). The importance of these biological-mediated processes is underscored by research showing that belowground biodiversity is positively related to forest ecosystem function (Bakker et al., 2019). Soil microbes, in particular mycorrhizal fungi, play an additional critical role in supporting plant establishment by forming mutualisms with plant roots. Mycorrhizal fungi provide plants with soil nutrients and resources in exchange for photosynthates (Smith & Read, 4 2008). Plants benefit from improved nutrition, as mycorrhizal fungal hyphae access a greater volume of soil than plant roots and take up nutrients, such as phosphorus, in forms that are not available to plants (Bolduc, 2011; Johnson et al., 2016). It is estimated that 92% of plant families form mycorrhizae (Wang & Qiu, 2006), with the arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi being the two major classes. AM fungi, mainly Glomeromycota, are obligate symbionts that colonize approximately 85% of plant species and are dominant in temperate grasslands, tropical forests and agricultural systems (Brundrett, 2009). AM fungi are characterized by arbuscules (and sometimes vesicles) that form in the cortical cells of plant roots (Smith & Read, 2008). EM fungi, typically Basidiomycetes and Ascomycetes, colonize approximately 10% of plant species and generally associate with woody perennial species (Brundrett, 2009). In temperate forest soils, EM fungi can constitute up to 80% of the fungal community and 30% of total microbial biomass (Högberg & Högberg, 2002). EM are characterized by a fungal sheath or mantle around a root from which hyphae grow inwards between the epidermal and cortical cells forming the Hartig net (there is no fungal penetration of the root cells), as well as outwards as extrametrical hyphae into the soil (Smith & Read, 2008). Formation of mycorrhizae has been shown to increase plant resilience to environmental stressors such as drought, herbivory, pathogens, nutrient deficiencies and elevated levels of metals or salt (Bennett & Bever, 2007; Brundrett & Tedersoo, 2018; Crossay et al., 2019; Evelin et al., 2009; Johnson et al., 2016; Lehto & Zwiazek, 2011), making mycorrhizal relationships of particular importance for seedling establishment in harsh, low nutrient reclamation environments. The roots of certain plant species, including legumes (Fabaceae spp.) and alder (Alnus spp.), similarly form mutualistic relationships with nitrogen-fixing bacteria, exchanging photosynthates for nitrogen. Nitrogen is commonly a limiting nutrient for plant establishment in 5 mine reclamation and biological nitrogen fixation is the primary natural pathway for low-nutrient reclamation soils to accumulate nitrogen (Bradshaw, 1984; Handley et al., 1978). Planted or naturally ingressing nitrogen-fixing species have been shown to promote early successional plant establishment in diverse ecosystems by increasing microbial activity and organic matter accumulation through litter inputs and root exudate, i.e., through positive feedbacks (Franco & De Faria, 1997; Lefrançois et al., 2010; Skeffington & Bradshaw, 1980). A lack of mycorrhizal fungi and N-fixing bacteria can limit ecosystem recovery following disturbance (Mueller et al., 2019; Nara, 2006; Seeds & Bishop, 2009), which suggests that revegetating without reestablishing the soil microbial community may hinder restoration success (Harris, 2009; Koziol et al., 2018). Thus, mine reclamation methods that promote recolonization of soil organisms in substrates with low biological activity can potentially bootstrap ecosystem recovery (Perry et al., 1989). 1.2 Ecosystem legacies as sources of soil biological communities Soil biota naturally recolonize mine reclamation areas over time from surviving components of the pre-disturbance ecosystem termed “ecosystem legacies” (Dangi et al., 2012; Ezeokoli et al., 2019). Ecosystem legacies, for example patches of trees, seeds and spores, act as “memories” or “threads of continuity” that guide recovery of disturbed ecosystems towards the pre-disturbance state and promote resilience of natural systems (Franklin et al., 2000; Johnstone et al., 2016). 1.2.1 Spatial ecosystem legacies Nearby undisturbed ecosystems can act as ecosystem legacies, but the large size of many industrial disturbances may present limitations for natural dispersal of soil biota. For example, 6 microarthropods were found to ingress only 30 m in 30 years in a birch forest (Ojala & Huhta, 2001). While the spores of many EM fungal species have been shown to disperse well up to 1 kilometre (km; Peay et al., 2012), some EM taxa and the majority of AM fungi rely on more limited mechanisms such as root to root contact, erosion and transport by other organisms (Camargo Ricalde, 2002; Egan et al., 2014). A review by Hanson et al. (2012) found significant correlations between distance and microbial composition in fifteen studies, with seven studies showing spatial effects at the < 1 km scale, highlighting the importance of dispersal for creating and maintaining microbial biogeographic patterns. Similarly, at a small scale (metres), microbe dispersal has been shown to have a stronger influence on microbial community assembly than environmental filtering (Chen et al., 2020). This variation in community assembly of soil organisms can affect plant fitness and scale up to community- and ecosystem-level effects (Gorzelak et al., 2015; Klironomos, 2002). Mine planning approaches that retain patches of undisturbed native ecosystems may preserve spatial ecosystem legacies from which soil biota can recolonize disturbed areas, similar to retention patches or trees in silviculture (MacArthur & Wilson, 1967; Timpane-Padgham et al., 2017). In addition to improved dispersal, short-distance connectivity with the mycorrhizal fungal hyphae of established trees can facilitate increased mycorrhizal colonization: retention trees and patches in silviculture have been found to increase EM colonization and diversity approximately 10 m into forestry cutblocks (Jones et al., 2008; Luoma et al., 2006). Further, colonization of seedling roots by mycorrhizal fungal hyphae from established trees enables formation of mycorrhizal networks, in which the mycelium of a fungal individual or genet forms a physical connection between the roots of at least two plants (Beiler et al., 2010; van der Heijden & Horton, 2009). Mycorrhizal networks can facilitate plant–fungal–plant relationships, 7 including plant to plant (interspecific and intraspecific) transfers of carbon, nitrogen, water, and defense or stress signals (Simard et al., 2015). These transfers have been linked with improved plant survival and growth, highlighting the potential role of mycorrhizal network connectivity in supporting plant establishment during ecosystem restoration (Bingham & Simard, 2011; Teste et al., 2010). Limited research on mycorrhizal colonization of seedlings through connectivity with fungal networks has been conducted in primary successional environments. Based on studies of plant establishment in volcanic outwash (scoria), Nara (2015) suggests that mycorrhizal networks may benefit establishing plants not only by facilitating faster mycorrhizal colonization of seedlings so that plants benefit from the mutualism at an earlier life stage, but also by transferring nutrients from richer to poorer nutrient areas (e.g., from patches of established vegetation) and following sources-sink gradients between plants and fungi, with greater transfer occurring where gradients are stronger (e.g., seedlings have accumulated relatively less nitrogen through their shorter lifespan than more established trees, thus forming a steeper nutrient gradient with nitrogen-rich fungi). These mechanisms are not, however, well understood, and it is likely that multiple mechanisms are involved and play varying roles depending on the environmental conditions and primary constraints to plant establishment (Horton, 2015). In summary, spatial ecosystem legacies may benefit plant survival and growth in early successional conditions by facilitating dispersal of soil biota and mycorrhizal network formation. Yet, these processes have not been tested in a mine reclamation setting and further studies are required to assess if they can be scaled up into practical mine reclamation techniques. 8 1.2.2 Topsoil ecosystem legacies Topsoil, another form of ecosystem legacy, is commonly spread on mine reclamation areas to create more hospitable soil conditions and inoculate plant propagules and soil organisms, ultimately improving soil food web recolonization and plant establishment (Boldt-Burisch & Naeth, 2017; Cristescu et al., 2012; Mackenzie & Naeth, 2010). However, as described above, topsoil availably is often limited due to a lack of historic salvaging practices and/or topsoil degradation during long-term storage (Abdul-Kareem & McRae, 1984; Harris et al., 1989). A potential alternative for sites with minimal or no fresh topsoil is to inoculate the rooting zone of seedlings during planting with small volumes of fresh topsoil from recently disturbed or undisturbed local ecosystems (herein referred to as “soil transfers”; Amaranthus & Perry, 1988; Borchers & Perry, 1990). Inoculation with native microbes has been shown to benefit plant growth more than foreign or commercial inoculants (Ji et al., 2010; Maltz & Treseder, 2015). Generalist species used in commercial inocula do not account for local adaptations, which can support a home-field advantage for native plant–soil–microbe relationships (Rúa et al., 2016). Further, commercial inoculations may have low viability or contain fertilizer, which can suppress mycorrhizal fungi colonization (Chaudhary et al., 2020; Rowe et al., 2007). Use of native inoculum also avoids the unstudied risk that microbes introduced in commercial inoculums could become invasive or pathogenic (Thomsen & Hart, 2018). Recognizing that soil microbes function as part of a complex and interconnected network (Bonfante & Anca, 2009), another benefit of using whole soil amendments is that they contain the full soil biological community (not just one or a few species; Hoeksema et al., 2010), including many species that are unculturable and/or disperse by root or hyphae fragments instead 9 of spores. For example, Shishido et al. (1996) found that plant growth-promoting bacteria and mycorrhizal fungi enhanced conifer growth through unrelated mechanisms, with the potential for additive benefits if both are present in soil. Even in cases where most of the growth benefit is from, for example, a single mycorrhizal fungal species (e.g., Cheeke et al., 2019), inoculation of diverse species increases the probability of a beneficial species being present (Koziol et al., 2018). The diversity of soil microbes contained in whole soil can be of particular benefit when plants are exposed to different environmental stressors (Bradáčová et al., 2019; Crossay et al., 2019). Limited testing of soil transfers has been done in mine reclamation settings, but Helm & Carling (1993a, 1993b) found transfers to improve balsam poplar (Populus balsamifera) seedling growth, physiology and root mycorrhizal fungal colonization in overburden. Greenhouse experiments have shown unsterilized soil transfers to benefit seedlings more than sterilized soil, highlighting the biological effectiveness of inoculations beyond the localized improvement in soil physiochemical conditions (Emam, 2016; Saxerud & Funke, 1991). However, further research is necessary to refine recommended transfer volumes as well as to investigate soil transfer effects in mine tailings, effects on different plant functional groups, and effects on soil organisms other than mycorrhizal fungi. 1.2.3 Ecosystem legacy effects on succession Plant litter inputs, root development and rhizodeposition influence soil chemical and physical properties and shape the soil food web, including communities of litter decomposers, belowground herbivores, pathogens and mutualists (van der Putten et al., 2013). These abiotic and biotic soil changes affect the fitness and niche space of successive plants in a process termed 10 “plant–soil feedbacks” (Bever et al., 1997; Ke & Wan, 2019). Plant–soil feedbacks are the net outcome of negative (e.g., root herbivory, plant root competition, pathogens) and positive (e.g., plant–microbe mutualisms) belowground interactions and are a central factor in structuring plant populations and communities (Eisenhauer, 2012). Soil biota are key drivers of plant–soil feedbacks and have been shown to promote community stability or instability, plant species invasions and plant species coexistence (Chung et al., 2019; Crawford et al., 2019; Klironomos, 2002; Pickett et al., 2019). The ability of soil biota to influence plant community composition has the potential to shape temporal trajectories (Chung et al., 2019; Duhamel et al., 2019), challenging the view that ecosystem succession is solely a function of abiotic and stochastic factors (Connell & Slatyer, 1977). There is accumulating evidence that biological soil ecosystem legacies associated with plant communities promote species turnover in early succession due to pathogen accumulation and selectively promote late-successional species through positive plant–soil feedbacks associated with mycorrhizal fungi (van der Putten et al., 2013). Successional plant–soil feedback processes have important implications for ecological restoration, as highlighted by studies showing that spreading of topsoil can guide vegetation recovery in the direction of the plant community present at the topsoil source (Wubs et al., 2016), as well as studies showing that soil microbial legacies of invasive plants can hinder native plant establishment (Dierks et al., 2019; Pickett et al., 2019). Consequently, without restoration methods that promote establishment of native soil biota and plants, there is potential that non-native species may establish and shift the successional trajectory to an unhealthy alternative stable state in which the sequence of plant species colonization results in a different final community composition (Chase, 2003; Fukami, 2015; Johnstone et al., 2016). Research on the ecological theory underpinning plant–soil feedback effects on succession and implications for 11 restoration ecology have, however, primarily been conducted in secondary successional and non-forested ecosystems (e.g., grasslands, old-field agricultural sites). Given that plant–soil feedback effects can vary with plant functional group (Cortois et al., 2016), further studies are necessary to investigate plant–soil feedback effects on succession in primary successional forest ecosystems. This information will refine reclamation best practices for soil/microbial inoculation and plant species selection to set ecosystems on the desired successional trajectory (Kardol & Wardle, 2010). 1.3 Study site My research was conducted in partnership with the Mount Polley Mine, an open pit copper and gold mine located in the Quesnel River watershed of the Cariboo region in interior British Columbia (BC), Canada (52°32'42.35" N, 121°37'58.67" W; Figure 1-1). The mine site is approximately 57 km northeast of Williams Lake, near the community of Likely, which is located at the outlet of Quesnel Lake into Quesnel River. Operations at Mount Polley started in 1997 and the current mine life extends to 2026, although at the time of writing the site was in care and maintenance (i.e., not operating). The mill facilities have a capacity to process approximately 16,800 tonnes of ore per day. The tailings produced by the mill are stored in a 2.4 km2 Tailings Storage Facility southeast of the mine facilities and adjacent to (west of) Polley Lake and Hazeltine Creek, which flow into Quesnel Lake (Figure 1-1). I carried out my experiments on materials requiring reclamation following the August 2014 tailings dam embankment failure. The tailings dam embankment failure released 25M m3 of water, mine tailings and dam construction materials to the downstream environment (Miller et al., 2015). The released material flowed into the adjacent Polley Lake, then down the 9 km 12 Hazeltine Creek corridor into Quesnel Lake (Figure 1-1; Miller et al., 2015). The debris flow removed vegetation and eroded an estimated 1.2M m3 of native material, exposing the underlying glacial sediments and till in a 136 ha area (Miller et al., 2015). Tailings were deposited in portions of this eroded area, as well as on top of the intact forest floor in a 99 ha area along the edges of the disturbed corridor (Miller et al., 2015). Where tailings were deposited on the forest floor, vegetation remained in place, but ultimately died due to the saturated tailings creating prolonged anaerobic conditions in the rooting zone (Miller & Simard, 2015). The Mount Polley Mine tailings are non-acid generating and thus less likely to leach dissolved metals, which tend to be more bioavailable than those that remain bound in mineral lattices (Van Geest et al., 2017). While levels of copper and vanadium are elevated in the tailings compared to reference soils, ecological risk assessments have found no negative toxicological effects of these metals on soil fauna and plants (Van Geest et al., 2017). As such, my research focused on limitations to plant establishment associated with the severe physical disturbance and poor soil quality, not metal toxicity. 13 Figure 1-1 Location of the Mount Polley Mine, British Columbia, Canada (A) and overview of the 2014 Mount Polley Mine tailings dam failure flow path from the Tailings Storage Facility into Polley Lake, then down Hazeltine Creek into Quesnel Lake (B). 1.4 Thesis objectives The objective of this thesis was to investigate if ecosystem legacy-based mine reclamation methods can enhance soil food web recovery and if this translates into improved plant establishment. Plant establishment, in this thesis, is defined as plant growth, nutrition and physiology. The thesis contains three data chapters that test the following overarching hypotheses: Hypothesis 1 (Chapter 3, Chapter 4): forest soil transfers (small volume additions to the seedling rooting zone) in mine reclamation soils would increase soil food web diversity and 14 abundance, increase seedling root colonization by microbial mutualists (mycorrhizal fungi and endophytic N-fixing bacteria), and improve seedling establishment. Hypothesis 2 (Chapter 4): proximity to undisturbed forest (a spatial ecosystem legacy) would enhance recovery of the soil food web and improve plant establishment in mine reclamation areas through aerial propagule dispersal and belowground connectivity. Hypothesis 3 (Chapter 5): plant establishment in mine reclamation materials would generate soil microbial ecosystem legacies that promote turnover of early successional plant species and selectively influence establishment of next stage successional plant species, with positive feedbacks occurring where plant species and mycorrhizal fungi communities are compatible. Chapter 2 describes the study ecosystem and general methodologies used in multiple data chapters, including greenhouse methods, test plant species selection, soil physiochemical analyses, and analysis of root and soil microbial communities. In Chapter 3, I conducted a greenhouse experiment to investigate the effects of forest soil transfers (small volume additions of fresh forest soil) on seedling establishment in overburden and mine tailings. I tested plant species representing a cross section of functional groups, successional stages and plant–microbe associations (Section 2.2). Two soil transfer volumes were tested and compared with tailings/overburden controls and reference forest soil to determine the effectiveness of different transfer volumes. Seedling root colonization by mycorrhizal fungi was assessed through microscopy and DNA sequencing, and nitrogen-fixing bacteria colonization was measured through nodule biomass measurements. The experiment was replicated with sterilized soil to isolate the biological effects of the soil transfers from the physiochemical effects. 15 In Chapter 4, a three-year field trial was carried out at the Mount Polley Mine to test the effects of: (1) forest soil transfers, (2) belowground connectivity with undisturbed forest, (3) distance from undisturbed forest edge, and (4) site preparation (a common mine reclamation technique; Polster, 2015) on seedling establishment, natural plant ingress and soil food web recovery (bacteria, fungi and fauna). The trial included annual growth monitoring of > 3,200 spruce (Picea engelmannii x glauca) seedlings planted in six trial blocks, three in areas of overburden (glacial till) and three in areas of tailings. The soil food web was assessed in the rooting zone of > 200 destructively harvested spruce seedlings through high-throughput sequencing of soil fungal and bacterial communities, morphotyping of EM fungi colonizing spruce seedling roots, and faunal community characterization through morphological identification and microscopy. This experiment enabled testing of the soil transfers investigated in Chapter 3 at an operational scale under field conditions. In Chapter 5, I investigated if plants growing in primary successional mine reclamation soils generate soil microbial ecosystem legacies that promote forest succession. Specifically, I tested if: (1) negative intraspecific plant–soil feedbacks promote turnover of early successional plant species, (2) if compatibility vs. mis-matches in plant species mycorrhizal guild (i.e., EM vs. AM fungi) exerts historical contingencies on establishment of next stage successional species, and (3) if plant–soil feedback strength and direction are consistent in soils of different successional stages. To address these questions, I conduced a two-phase greenhouse experiment in which I conditioned soil with early and mid–late successional plant species. The same plant species (intraspecific) and later stage successional plants species (interspecific), including plants from the same or different mycorrhizal guild, were then grown as feedback-phase seedlings in the conditioned soil and unconditioned control soil. These treatments were tested in forest soil 16 and primary successional mine reclamation substrates (overburden and tailings) with or without forest soil amendment. Root fungal communities, including mycorrhizal fungi and pathogens, were characterized through microscopy and high-throughput sequencing to link seedling responses with soil microbial mechanisms. In Chapter 6, findings from the experiments in Chapters 3, 4 and 5 are integrated in the context of my overarching thesis objective and hypotheses, summarizing how ecosystem legacies can be applied in mine reclamation to support soil food web recovery and plant establishment. Opportunities and approaches for application of my findings in operational-scale mine reclamation, as well as specific findings relevant to the Mount Polley Mine, are presented. Strengths and limitations of this thesis are discussed, along with avenues for future research to support further incorporation of above ground–belowground linkages and natural ecosystem recovery processes into mine reclamation best practices. 17 Chapter 2: General methodology This chapter includes a description of the Mount Polley Mine site as well as methods common to more than one of the experiments presented in Chapter 3, Chapter 4 and Chapter 5. 2.1 Mount Polley Mine site description 2.1.1 Climate Average annual precipitation at the Mount Polley Mine is 670 mm and average monthly temperatures range from -6.0 °C in January to 15.3 °C in July and August (Irving et al., 2015). Precipitation typically occurs as snowfall from November through March, with an average maximum snowpack of 178 mm (snow water equivalent) occurring at the end of March (Irving et al., 2015). 2.1.2 Geology and soils The Mount Polley Mine is located on an alkalic porphyry copper gold deposit within the Quesnel Terrane, a belt of mafic to intermediate volcanic rocks (BC Ministry of Energy and Mines, 2013). Geochemical characterization of the tailings produced during ore processing has found a negligible potential for acid rock drainage due to the low sulphur content (0.1 to 0.3%) and high carbonate buffering capacity (Van Geest et al., 2017). Copper, the primary parameter of concern associated with the tailings, has low leaching potential under both subaerial and subaqueous conditions because the copper is primarily associated with the non-sulphide minerals, which are relatively insoluble (Van Geest et al., 2017). Properties of the tailings spilled in the 2014 tailings dam embankment breach are presented in Table 2-1. 18 The Mount Polley site is covered by blankets and veneers of glacial tills with small inclusions of alluvial, lacustrine, colluvial, and organic landforms (Lord, 1984). Morainal tills deposited by glacial ice with little or no re-working by water are the dominant material. Consequently, the tills are massive or poorly sorted with a wide range of particle sizes (Lord, 1984). Local soils have developed from the loamy to clay loam morainal parent material and are classified as Podzolic to Luvisolic soils, or Brunisolic soils where less developed (Lord, 1984). Properties of the glacial deposits exposed by erosion during the 2014 tailings dam embankment breach and reference forest mineral soil are presented in Table 2-1. 19 Table 2-1 Properties of deposited tailings, glacial parent material exposed by erosion and reference forest mineral soil in Hazeltine Creek following the Mount Polley Mine tailings dam embankment breach. Bold values indicate an exceedance of BC Contaminated Sites Regulation Standards (parkland guidelines). Table modified from Miller et al. (2015). 2.1.3 Vegetation The Mount Polley Mine is located in a temperate coniferous forest ecosystem classified as the Interior Cedar Hemlock biogeoclimatic zone (Steen and Coupé, 1997). The mine site is in the Till units Lacustrine unitChemical - pH and metals n = 71 n = 23pH8.5 (7.51 - 9.09)5.62(4.69 - 7.13)Copper (mg/kg)869 (185 - 1560)35.6 (5.98 - 135)Vanadium (mg/kg)187 (106 - 289)70 (40.2 - 133)Chemical - Nutrients n = 27 n = 18Total carbon (%)0.5(0.3 - 0.9)2.0 (0.3 - 10.5)Total nitrogen (%)0.011(<0.02 - 0.028)0.117 (<0.02 - 0.667)Mineralizable nitrogen (μg/g)9(3 - 86)26(8 - 103)CEC (meq/100g)7.28(2.55 - 30.4)18.9(6.1 - 81.3)Avalable phosphate (μg/g)1.3(<2 - 3.9)41.17(1 - 293)Available potassium (μg/g)148.3(36 - 418)84.27(32 - 232)Physical - Texture n = 20 n = 9 n = 1 n = 18Sand (%)49 (0.6 - 92.7)42.4(25.8 - 61.7)0.8 -Silt (%)41.4(6.8 - 80.2)42.4(31.5 - 58.1)66.7 -Clay (%)9.6(0.5 - 31.5)15.4(6.3 - 27.1)32.5 -Gravel (%)1.7(0.1 - 17.1)9.6(0 - 32.4)0 -Physical - Bulk density n = 9 n = 5 n = 3 n = 4Bulk density 1.54 1.74 1.77ParameterReference forest mineral soiln = 1763.15(32 - 113)7.3(5.95 - 8.48)36.1 (6.28 - 86.7)59.7 (2.04 - 100)Glacial parent mateiralTailingsn = 131.2(0.1 - 4.6)0.061(<0.02 - 0.261)21(1.3 - 95)12.2(4.21 - 28.3)2.31(<2 - 9.4)20 moist cool subzone (Horsefly variant; ICHmk3) and the area disturbed by the tailings dam failure extends into the wet cool subzone (Quesnel variant, ICHwk2; Steen and Coupé, 1997). Dominant climax tree species in the ICHmk3 variant are western redcedar (Thuja plicata) with less frequent hybrid white spruce (Picea engelmannii x glauca) and subalpine fir (Abies lasiocarpa). The ICHwk2 occurs in wetter, milder areas than the ICHmk3, and can be differentiated by the presence of western hemlock (Tsuga heterophylla) and less abundant subalpine fir in mature stands (Steen and Coupé, 1997). 2.2 Plant species selection Plant species used for the experiments in all three data chapters are native to the Mount Polley Mine area (Table 2-2). The selected species represent a range of functional groups, successional stages and plant–microbe associations. Willow and alder were chosen because they are commonly used in reclamation at the Mount Polley Mine, consistent with best practices for revegetation with plant species adapted to early successional conditions (Groninger et al., 2017). Hybrid white spruce is the primary silvicultural species planted in the region and representative of a tree species that may be included in revegetation species mixtures to initiate conifer establishment (BC Ministry of Forests, 2002). Redcedar is a climax tree species and was used as a late successional species in investigations of successional processes (Chapter 5). For the greenhouse experiments (Chapter 3 and Chapter 5), willow and alder seeds were collected from the Mount Polley Mine area, however, due to low willow seed viability, non-regional willow seed was purchased from Tipi Mountain Native Plants, Cranbrook, BC. Spruce and redcedar seeds of appropriate provenance for the Mount Polley site were obtained from the BC Tree Seed Centre, Surrey, BC. 21 Table 2-2 Plant species used in the greenhouse and field experiments presented in Chapter 3, Chapter 4 and Chapter 5. Species Functional group Successional stage Plant – microbe association(s) Experiments Willow (Salix scouleriana Barratt ex Hook.) Deciduous shrub Early EM (AM) Chapter 3 Chapter 5 Sitka alder (Alnus viridis (Chaix.) D.C.) Deciduous shrub Early EM Nitrogen-fixing bacteria Chapter 3 Hybrid white spruce (Picea engelmannii Parry ex Engelm. x glauca (Moench) Coniferous tree Mid–late EM Chapter 3 Chapter 4 Chapter 5 Western redcedar (Thuja plicata Donn ex D.Don) Coniferous tree Late AM Chapter 5 2.3 Greenhouse experiments Soils used in the greenhouse experiments in Chapter 3 and Chapter 5 were collected from the Mount Polley Mine in the area disturbed by the 2014 tailings dam failure using shovels and trowels. Collection tools were cleaned with deionized water between sites. 440 L of deposited mine tailings and 460 L of overburden (native glacial sediments and till) exposed by erosion were collected (tailings: one collection location; overburden: composite from two collection locations; Figure A-1). 300 L of forest soil were collected from two locations in the adjacent undisturbed mid- and late seral ICHmk3 forests and composited (Figure A-1). The forests at these sites were dominated by western redcedar (65.32 m2/ha basal area), subalpine fir (9.65 m2/ha basal area) and hybrid white spruce (6.17 m2/ha basal area; Table A-1). At each reference site, litter was removed and the top 15–20 cm of soil was collected (fermentation layer, humic layer and upper mineral soil horizons). Soils were stored in plastic pails or totes at 4 °C until use (approximately 8 weeks), when they were homogenized and mixed with 20% v/v perlite to 22 compensate for structural losses due to handling. Average physical and chemical properties of each soil type are presented in Table 2-3. In both greenhouse experiments, for each replicate of each plant species (Scouler’s willow, Sitka alder, hybrid white spruce and/or redcedar; see Section 2.2), five sterilized seeds were planted in each container and thinned to one seedling following germination. Some re-planting and transplanting within treatment groups was necessary early in the growing period to achieve the desired replication. Pots were randomly arranged and grown in the University of British Columbia (UBC) Horticulture Greenhouse, Vancouver, BC under 600 watt high pressure sodium lights (17 h photoperiod) and hand-watered as-needed. To avoid nutrient enrichment effects on root-microbe associations and mimic mine site conditions, seedlings were not fertilized. Table 2-3 Chemical and physical properties of the tailings, overburden and forest soil collected at the Mount Polley Mine, BC. Forest soil includes the fermentation layer, humic layer and upper mineral horizons. Cations and effective cation exchange capacity (CEC) = Barium Chloride exchangeable cations. EC = electrical conductivity. Soil parameter Tailings Overburden Forest soil Sand (%) 62.07 48.12 44.46 Silt (%) 29.08 36.69 34.71 Clay (%) 8.85 15.18 20.83 Texture Sandy loam Loam Loam pH 8.23 8.09 5.2 EC 543 230 417 Total N (%) 0.013 0.025 0.605 Total C (%) 0.48 0.82 14.28 PO4-P (mg/kg) 1.21 1.42 41.98 Al (cmol+/kg) 0.026 0.010 0.186 Ca (cmol+/kg) 5.333 7.532 28.305 Fe (cmol+/kg) < 0.001 < 0.001 0.005 K (cmol+/kg) 0.279 0.140 0.761 Mg (cmol+/kg) 0.757 1.163 5.366 Mn (cmol+/kg) < 0.001 0.003 0.394 Na (cmol+/kg) 0.347 0.081 0.123 CEC (cmol+/kg) 6.742 8.929 35.139 23 2.4 Soil physiochemical analyses Soil physiochemical analyses were conducted to characterize the tailings, overburden and forest soil used in greenhouse experiments (Chapter 3 and Chapter 5) and to characterize baseline conditions and forest soil used for soil transfers in the field experiment (Chapter 4). Soil samples were air dried and sieved to < 2 mm. pH and electrical conductivity (2:1 w/v deionized water:soil solution) were measured with a Seven Compact PH/Ion meter S220 and S230 Conductivity meter, respectively. The following parameters were analyzed by the BC Analytical Laboratory, Victoria, BC: • Exchangeable cations (Al, Ca, Fe, K, Mg, Mn, Na) and effective cation exchange capacity (CEC) through Barium chloride extraction (0.1 N) and ICP-OES analysis; • Total nitrogen (N) and total carbon (C) through combustion and analysis with an elemental analyzer; and • Available phosphorus (PO4-P; P) by Bray P-1 extraction (1 minute shake) and UV-Vis analysis. 2.5 Analysis of root mycorrhizal fungi communities To assess mycorrhizal fungal percent colonization of willow, alder and redcedar seedling roots (Chapter 3 and Chapter 5), approximately 1 cm root segments were cleared in 10% KOH and stained in an ink and vinegar solution (Vierheilig et al., 2005). AM and non-AM fungal endophyte percent colonization were evaluated at 100 intersections under a compound microscope as per McGonigle et al. (1990). The proportion of tips colonized by EM fungi was evaluated for the stained willow and alder root segments using the gridline intersect method (Giovannetti & Mosse, 1980). 24 Morphotyping of spruce seedling roots to assess EM fungal percent colonization and diversity was conducted under a dissecting microscope for all three experiments (Chapter 3, Chapter 4 and Chapter 5) as per Goodman et al. (1996). Vouchers of each morphotype (one or a few root tips from an individual root system) were collected, rinsed with distilled water and stored at -20 °C. Individual voucher specimens (minimum three per morphotype except for rare morphotypes observed less than three times) were ground with a sterile micropestle and sterile microsand followed by DNA extraction with PowerPlant Pro kits (Qiagen, 2017; Chapter 3, Chapter 5) and DNeasy PowerSoil Pro kits (Qiagen, 2019; Chapter 4), both as per the manufacturer’s instructions. The ribosomal internal transcribed spacer (ITS) regions ITS1 and ITS2 were amplified using polymerase chain reaction (PCR) with the fungal-specific primers ITS4 (White et al., 1990) and ITS1F (Gardes & Bruns, 1993) using GE illustra PuReTaq Ready-To-Go PCR Beads, following the manufacturer’s protocol. Thermocycler parameters for PCR involved an initial denaturation of 5 minutes at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds and elongation at 72 °C for 30 seconds, followed by 7 minutes at 72 °C for a final elongation. Bidirectional Sanger sequencing of PCR products was performed at Macrogen, Korea. Bidirectional reads were trimmed (error probability limit = 0.01) and aligned into a consensus contig using Geneious Prime (version 2019.2.3). The resulting sequences were identified using the Basic Local Alignment Search Tool (BLAST) against the National Center for Biotechnology Information (NCBI) GenBank database and the UNITE database (Abarenkov et al., 2010) using a 97% sequence similarity threshold. Where PCR products had more than one band in gel electrophoresis or Sanger sequencing failed, and for all Chapter 3 morphotype vouchers, genomic DNA extracts were sent to the Integrated Microbiome Resource at Dalhousie University, Halifax, Nova Scotia for high-25 throughput Illumina MiSeq sequencing using the fungal-specific ITS2 region primers ITS86 and ITS4 (Turenne et al., 1999; Vancov & Keen, 2009; White et al., 1990). Following processing of high-throughput sequencing data (Section 2.6) each morphotype voucher was assigned to the EM fungal taxon with the highest number of reads. The assigned EM fungal taxa were cross-referenced with documented morphology and photos from morphotyping and the Ectomycorrhizae Descriptions Database (Armstrong et al., 2007) and Determination of Ectomycorrhizae database (Agerer & Rambold, 2020). Non-mycorrhizal root tips were confirmed by poor PCR amplification and low EM fungal abundance. 2.6 Microbial high-throughput sequencing bioinformatics Data from high-throughput sequencing of seedling roots (fungal ITS2 region) and soil (fungal ITS2 region and bacterial 16S region) were processed as follows. Sequences were initially trimmed for quality using the sliding window function in Trimmomatic (version 0.39; Bolger et al., 2014) where the average quality per base within the four base pair window fell below fifteen (ITS2 root samples only). Subsequent sequence processing and analyses were conducted using QIIME2 (version 2019.7 for root data, version 2020.2 for soil data; Caporaso et al., 2010). Sequences were imported into a QIIME2 artifact and trimmed using Cutadapt to remove primers and read through (ITS2 root samples and soil 16S samples only; Martin, 2011). Following, for fungal samples, the ITS2 region was extracted and chimeras and non-ITS2 sequences were removed using the ITSxpress QIIME2 plugin (version 1.8.0; Rivers et al., 2018). Bidirectional reads from ITS2 and 16S samples were assembled, denoised and assigned to amplicon sequence variants (ASVs), dereplicated, and filtered for chimeras using the DADA2 pipeline (Callahan et al., 2016). Taxonomic identities of the ASVs were determined using a Naïve Bayes classifier 26 trained on the UNITE database for ITS2 sequences (Abarenkov et al., 2010) and SILVA database for 16S sequences (Glöckner et al., 2017; Quast et al., 2012). ITS2 ASVs were assigned to fungal guilds using the FUNGuild database (Nguyen et al., 2016). 27 Chapter 3: Forest soil transfers increase root symbiont colonization and seedling establishment in tailings and overburden 3.1 Introduction Topsoil is applied in mine reclamation to improve the physical and chemical properties of reclamation materials, including increasing organic matter and nutrient availability (Tordoff et al., 2000). A benefit of topsoil over other soil amendments is that it contains ecosystem legacies, such as native plant and soil microbe propagules, which can support rapid establishment of diverse native plant species and increase microbial diversity, abundance and activity (Macdonald et al., 2015; McMillan et al., 2007; Moynahan et al., 2002; Rivera et al., 2014). Topsoil applications promote colonization of plant roots by microbial mutualists, such as mycorrhizal fungi and N-fixing bacteria, which can improve plant establishment in harsh reclamation conditions (Hankin et al., 2015; Saxerud & Funke, 1991; Zong et al., 2015). At reclamation sites where fresh topsoil availability is limited or topsoil quality has degraded during long-term storage in stockpiles (Gorzelak et al., 2020; Paterson et al., 2019), a potential method of inoculating plants with beneficial soil biota is to mix small volumes of fresh topsoil, either salvaged from recently disturbed native ecosystems or from undisturbed areas, into the rooting zone soil of seedlings during planting (termed “soil transfers”). This approach maximizes use of limited quantities of fresh topsoil (i.e., the upper soil layers, which have the greatest inoculum potential; Macdonald et al., 2015; Rivera et al., 2012). Testing of soil transfers in silvicultural and ex-agricultural sites has had mixed results (Amaranthus & Perry, 1987; Pareliussen et al., 2006; St-Denis et al., 2017). However, this technique may be of greater benefit at mine sites because: (1) limited soil microbial community 28 establishment has occurred, thus minimal competition from established microbes is expected, and (2) reclamation materials are low in nutrients and microbial symbionts can be critical for nutrient acquisition in low nutrient environments (Carbajo et al., 2011; Getachew et al., 2019). Limited soil transfer research has been conducted at mine reclamation sites, but Helm and Carling (1993a; 1993b) showed soil transfers from a balsam poplar (Populus balsamifera) stand to the rooting zone of balsam poplar seedlings growing in overburden increased seedling growth, nutrient status and EM fungal colonization. Two other studies found amendment with unsterilized soil to have a greater positive effect on plant growth and mycorrhizal fungal colonization than sterilized soil, showing the effectiveness of the biological inoculation (Emam, 2016; Saxerud & Funke, 1991). While having less impact than a layer of topsoil, soil amendment of seedling rooting zones may also cause localized improvements in soil physiochemical conditions for initial establishment of roots and soil organisms, especially compared to commercial microbial inoculants. The objective of this study was to assess the effects of forest soil transfers on seedling establishment in the dominant materials requiring reclamation at the Mount Polley Mine following the 2014 tailings dam failure (Section 1.3). I conducted a greenhouse study in which seedlings representing different functional groups (including conifers and EM plant species that have been less commonly studied) were grown in tailings and overburden with different forest soil transfer volumes. Two soil transfer volumes were tested and compared with tailings/overburden controls and reference forest soil to determine the effectiveness of different transfer volumes. The experiment was replicated with sterilized soil to isolate the biological effects of the transfers from the physiochemical effects. Treatment effects were evaluated by assessing seedling growth, foliar nutrition, physiology (chlorophyll fluorescence) and root 29 colonization by microbial mutualists. I hypothesized that: (1) seedling growth, nutrition and root symbiont colonization would increase with forest soil transfer volume for all plant species tested, (2) the biological component of the added forest soil would be partially responsible for positive effects on seedling establishment, (3) forest soil transfers would have the greatest benefit in tailings, where seedling establishment was expected to be poorest, and (4) seedling establishment comparable to forest soil controls could be achieved with soil transfers. 3.2 Methods 3.2.1 Experimental design A 3 x 3 x 2 factorial set of treatments applied to greenhouse-grown seedlings was replicated five times in a completely randomized design using soils collected from the Mount Polley Mine as described in Section 2.3. Individual seedlings were grown in 750 mL Ray Leach tubes and treated with one of three forest soil transfer rates (0% (0 mL), 5% (38 mL), or 25% (188 mL)) mixed with one of three reclamation substrates: tailings, overburden and a 1:1 mixture of tailings and overburden by volume (herein referred to as “tailings + overburden”). These treatments targeted lower ratios of forest soil addition where the greatest relative improvements in seedling establishment were expected. Further, when scaled up, these soil transfer volumes would equate to lower soil quantities than are typically spread at mine sites that are not topsoil-limited (e.g., Bendfeldt et al., 2001; Rokich, 2016). One additional treatment of 100% forest soil (n = 5) was added for comparison to reference conditions. The third factor tested was sterilization, where all soils were autoclaved for 1 hr at 121°C (Trevors, 1996) to differentiate between the physiochemical and biological effects of the soil transfers. 30 The soil factors were tested on three species native to the Mount Polley Mine site: hybrid white spruce (Picea engelmannii x glauca), willow (Salix scouleriana) and Sitka alder (Alnus viridis; Section 2.2). These species represent a cross section of functional groups, plant–microbe associations and successional stages: spruce is a mid–late seral conifer forming EM, while willow and alder are early successional stage deciduous shrubs that form EM and possibly AM (Molina et al., 1992). Alder roots also associate with N-fixing bacteria (Frankia spp.). Delays were experienced in initiating the experiment for alder due to poor viability of the seeds collected at the Mount Polley Mine (Section 2.3). Phospholipid fatty acid (PLFA) analysis, which provides an estimate of the living biomass of broad microbial groups (Quideau et al., 2016), was conducted at Ward Lab, Kearny, Nebraska to assess potential changes in the soil microbial community of potted forest soil stored in the greenhouse for 6 months (the length of time required to obtain new alder seed). There were no changes in total biomass or biomass of any microbial groups, except protozoa, which make up < 0.4% of the total community (Figure B-1). These results indicate negligible effects on the soil microbial community and are not discussed further. 3.2.2 Data collection and laboratory analyses Light-adapted chlorophyll fluorescence is a measure of plant photosynthetic efficiency (specifically, photosystem II capacity) and is sensitive to environmental stressors, with plants showing reduced chlorophyll fluorescence when exposed to stressors, such as drought (Murchie & Lawson, 2013). Light-adapted chlorophyll fluorescence was assessed prior to harvest with an Opti-Sciences OS-30p handheld meter. Shoots were dried for 72 h at 70°C and weighed. Samples of the youngest foliage were analyzed for total (%) N and C using an Elemetar vario EL 31 Cube elemental analyzer at the UBC Stable Isotope Facility, Vancouver, BC. Washed roots were scanned for morphological analysis using WinRhizo software. As an indicator of soil microbial activity and the biological effects of the soil transfers, symbiotic relationships with soil microbes were evaluated. The number of N-fixing nodules on each alder root system was counted and the wet weight of nodules was measured. For analysis of mycorrhizal fungi, 50 random spruce root tips and 50 random 1 cm willow and alder root tip segments (or 50% of available tips/segments for small root systems) were collected from each root system and rinsed with distilled water. Remaining roots were dried for 72 h at 70°C and weighed. Willow and alder root segments were assessed for percent colonization by AM fungi, non-AM fungal endophytes and EM fungi as described in Section 2.5. AM fungal colonization data were not used in statistical analyses, as colonization was not detected on alder roots and was negligible on willow roots. Spruce root tips were morphotyped as per Section 2.5 to assess EM fungal percent colonization and diversity. Morphotypes were identified through high-throughput sequencing (Section 2.5, Section 2.6). For all but two morphotype voucher samples, the assigned EM fungal taxa corresponded to the ASV with the most reads (Table B-2). Results indicated two vouchers were mixed (>1 EM fungal taxa constituting > 10% of reads; Table B-2), whereas the rest of the voucher samples each had a single EM dominant fungal species present, but also had some saprophytic, parasitic and/or low abundance EM (< 10% of total reads) fungal DNA present. Where pairwise identity was < 97%, the taxa was recorded as “[taxon]-like”, e.g., Pseudotomentella rhizopunctata-like. 32 3.2.3 Statistical analyses Growth parameters were relativized per day to account for growth period differences due to replanting. Analyses were conducted in R (version 3.5.3; R Core Team, 2019). Linear models were used except for EM and root endophytic fungal percent colonization, where binomial generalized linear models (link = logit) were used. Models were run with an interaction term and significance of linear models assessed with Type III analysis of variance (ANOVA). Models were re-run without the interaction term if no significant or marginally significant interaction occurred, and linear model significance tested with Type II ANOVA. For all analyses, significance was evaluated as P ≤ 0.05 and marginal significance as 0.05 < P < 0.1. Data for linear models were transformed, as required, to meet model assumptions of normality and equal variance (Figure 3-1–Figure 3-3; Figure B-2–Figure B-4). Separate models for each plant species (spruce, willow and alder) were run for each plant establishment parameter (response variable) with soil type (overburden, tailings and tailings + overburden) x soil transfer volume (0%, 5% and 25%; continuous variable) as the explanatory variables. Post hoc contrasts of soil type (valid when no significant soil type x soil transfer volume interaction was present) were done using the emmeans package (Lenth, 2020) with the Tukey adjustment for multiple comparisons. Models were also run separately for each soil type to provide additional insight into soil type x transfer volume interactions. Models were run for sterilized and unsterilized soil and the slopes (i.e., effect of increased transfer volume) of a combined model with sterilization as an explanatory variable were compared using the emtrends function in the emmeans package (Lenth, 2020). Pairwise post hoc comparisons of root symbiont colonization in unsterilized vs. sterilized soil were done with models using soil treatment (soil type and transfer volume combined) and sterilization treatment as the explanatory variables. 33 Comparison of the seedlings grown in different soil treatments vs. 100% forest soil was done through post hoc contrasts (emmeans; Lenth, 2020) applied to models with unsterilized soil treatment (soil type and transfer volume combined) as the explanatory variable. To provide insight into the effects of root symbiont colonization, linear models were run for unsterilized soil treatments with symbiont colonization as the explanatory variable and shoot biomass, root:shoot ratio or specific root length (root length / biomass) as the response variable. Soil treatment (soil type and transfer volume combined) was used as a covariate to account for differential effects of soil treatments on these relationships. 3.3 Results 3.3.1 Forest soil transfer effects on seedling establishment Spruce seedling shoot biomass, foliar N and chlorophyll fluorescence increased with increasing soil transfer volume (Table 3-1). While there were no significant soil transfer volume x soil type interactions for these parameters, separate models for each soil type (overburden, tailings, and tailings + overburden) showed significant shoot biomass increases in tailings and tailings + overburden, but not overburden (Figure 3-1). Soil transfers also had no effect on the foliar N in tailings (Figure 3-1). Root biomass increased with soil transfer volume in tailings and tailings + overburden (Figure B-2). In overburden, there was no effect of increasing soil transfer volume on root biomass but root:shoot ratio significantly decreased (Figure B-2). Spruce seedling biomass did not differ with soil type, but seedlings had higher chlorophyll fluorescence and foliar N in overburden compared to tailings (Table B-1). Spruce seedlings grown in tailings + overburden had higher chlorophyll fluorescence than in tailings but did not significantly differ from seedlings grown in overburden for any parameters (Table B-1). Compared to the 100% forest 34 soil treatment, spruce seedling biomass was significantly lower in tailings with 0% or 5% forest soil but not with 25% forest soil (Figure 3-1; Table B-3). Chlorophyll florescence and foliar N were significantly lower in all tailings treatments compared to 100% forest soil (except foliar N in tailings with 5% forest soil; Figure 3-1; Figure B-2; Table B-3). In overburden and tailings + overburden treatments, spruce seedling biomass was similar to 100% forest soil, however 25% forest soil additions were necessary for comparable foliar nutrition and chlorophyll fluorescence (Figure 3-1; Figure B-2; Table B-3). Willow shoot and root biomass increased with soil transfer volume with no soil type interaction (Table 3-1). Trends were not always significant within each soil type (Figure 3-2; Figure B-3), but low survival in some treatments hindered statistical analysis at this resolution (Table B-3). Root:shoot ratio increased with soil transfer volume in overburden but not the other soil treatments (marginally significant soil type interaction; Figure B-3; Table 3-1). Chlorophyll fluorescence and foliar N were not affected by increasing soil transfer volume (Figure B-3), although foliar N decreased with increasing soil transfer volume in the tailings + overburden treatment (Figure 3-2; Table 3-1). There were no differences in willow seedling biomass and foliar nutrition among soil types, however, seedling chlorophyll fluorescence was significantly lower in tailings + overburden than overburden (Figure B-3). Compared to 100% forest soil, willow seedlings had significantly poorer growth in 100% overburden, tailings and tailings + overburden (Figure 3-2; Table B-3). These differences were not present with 5% and 25% forest soil additions to tailings and overburden or 25% forest soil additions to tailings + overburden (Figure 3-2; Table B-3). Willow chlorophyll fluorescence and foliar nutrition did not differ from 100% forest soil in any soil treatments (Figure 3-2; Figure B-3; Table B-3). 35 In alder, soil transfers had no effect on seedling biomass but chlorophyll fluorescence tended to increase with increasing soil transfer volume (Table 3-1), although this was primarily driven by the tailings + overburden treatment (Figure B-4). Foliar N also tended to increase with increasing soil transfer volume in tailings (Figure 3-3). Seedling establishment did not vary among soil types, but biomass tended to be greater in treatments containing tailings (Figure 3-3). In fact, alder had greater shoot biomass in the 100% tailings treatment than any other soil treatment except 100% forest soil, although variance was high (Table B-3). Alder shoot biomass was significantly lower than in the forest soil control in all treatments except 100% tailings and tailings + overburden with 25% forest soil (Figure 3-3; Table B-3). Alder chlorophyll fluorescence and foliar nutrition did not differ from 100% forest soil in any soil treatments (Figure 3-3; Figure B-4; Table B-3). 36 Table 3-1 Linear model and generalized linear model results of soil type x soil transfer volume ( 0%, 5% or 25% forest soil addition) effects on spruce, willow and alder seedling establishment with soil transfer volume as a continuous variable. Soil type = tailings, overburden or tailings + overburden. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Df = 2 for soil type, df = 1 for transplant volume, and df = 2 for soil type x soil transfer volume. Model termF P F P F P F P F P ꭕ2P ꭕ2P F PSpruceSoil type 1.49 0.238 3.35 0.047 1.69 0.198 8.21 0.001 4.14 0.234 100.11 <0.001 - - - -Transfer volume 12.82 0.001 1.30 0.261 5.16 0.029 7.70 0.008 8.26 0.007 50.37 <0.001 - - - -Interaction 2.37 0.107 4.34 0.020 2.71 0.080 0.10 0.901 1.83 0.175 33.79 <0.001 - - - -WillowSoil type 0.73 0.492 0.69 0.508 1.99 0.156 3.74 0.037 0.93 0.406 43.78 <0.001 40.76 <0.001 - -Transfer volume 8.30 0.007 16.22 <0.001 6.25 0.019 2.31 0.141 2.84 0.104 93.47 <0.001 161.32 <0.001 - -Interaction 0.09 0.917 0.28 0.762 3.19 0.057 0.64 0.536 0.86 0.435 4.59 0.101 93.07 <0.001 - -AlderSoil type 1.05 0.360 1.25 0.299 0.35 0.710 1.20 0.313 1.02 0.372 - - 1.42 0.492 1.99 0.151Transfer volume 0.00 0.950 0.00 0.975 0.01 0.930 3.21 0.082 2.68 0.111 - - 23.22 <0.001 1.52 0.226Interaction 0.08 0.927 0.07 0.934 0.62 0.542 2.24 0.122 0.407 0.669 - - 62.12 <0.001 1.18 0.320Shoot biomass (mg/day)Root biomass (mg/day)Root:shoot biomassChlorophyll fluorescence (Fv/Fm) Foliar N (%)EMF colonization (%)Root SymbiontsPhysiology and NutritionGrowthEndophyte colonization (%)N-fixing nodule wet weight (mg)37 Figure 3-1 Spruce seedling linear model (A, B) and binomial generalized linear model (C) results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling shoot biomass (A), foliar nitrogen (B) and ectomycorrhizal fungal (EMF) percent colonization (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Log transformation = loge. Shoot biomass was relativized per growing day to account for growth period differences due to replanting. 38 Figure 3-2 Willow seedling linear model (A, B) and binomial generalized linear model (C, D) results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling shoot biomass (A), foliar nitrogen (B), ectomycorrhizal fungal (EMF) colonization (C), and fungal endophyte colonization (D) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Shoot biomass was relativized per growing day to account for growth period differences due to replanting. 39 Figure 3-3 Alder seedling linear model (A–C) and binomial generalized linear model (D) results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling shoot biomass (A), foliar nitrogen (B), nitrogen-fixing nodule wet weight (C) and fungal endophyte percent colonization (D) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Log transformation = loge. Shoot biomass was relativized per growing day to account for growth period differences due to replanting 40 3.3.2 Root colonization by microbial symbionts Spruce root EM fungal colonization significantly increased with forest soil transfer volume, however a soil type x soil transfer volume interaction occurred where the trend of increased EM fungal colonization was weak in tailings + overburden (Figure 3-1; Table 3-1). Soil type had a significant effect on EM fungal colonization, with lower rates in tailings compared to overburden and tailings + overburden (Figure 3-1; Table B-1). Compared to forest soil, EM fungal colonization rates were significantly lower in tailings and tailings + overburden, even when amended with forest soil (Figure 3-1; Table B-3). In contrast, soil transfers in overburden resulted in colonization rates comparable to 100% forest soil (Figure 3-1; Table B-3). Overall EM fungal morphotype richness on spruce seedlings was low (maximum three species on an individual). Increased soil transfer volume did not affect EM fungal richness (F1 = 2.43, P = 0.923), but soil type did (F2 = 7.49, P = 0.009); richness was lower in tailings than in overburden and tailings + overburden treatments (Table B-1). Additionally, EM fungal species richness was lower in all tailings and tailings + overburden treatments than in forest soil but was comparable to forest soil in overburden with or without soil transfers (0% forest soil: T35 = 2.21, P = 0.401; 5% forest soil: T35 = 1.85, P = 0.702; 25% forest soil: T35 = 2.61, P = 0.250). Willow root colonization by EM fungi and non-AM fungal endophytes increased with forest soil transfer volume across all soil types, except there was no effect on endophyte colonization evident in tailings + overburden or EM fungal colonization in tailings due to poor seedling survival (Figure 3-2; Table 3-1). Soil type significantly affected colonization, with lower EM fungal colonization in tailings than overburden and tailings + overburden (Figure 3-2; Table B-1). Endophyte colonization was lowest in tailings and highest in overburden, although a significant soil type x transfer volume interaction was present (Figure 3-2; Table B-1). Compared 41 to forest soil, willow seedlings had lower EM and endophyte fungal colonization in 100% overburden and tailings (Table B-3). These differences were not present in overburden with 5% and 25% forest soil, tailings with 25% forest soil or any tailings + overburden treatments (Table B-3). Soil transfers had no overall effect on alder nodulation, but nodulation tended to increase with soil transfer volume in tailings (Figure 3-3; Table 3-1). Non-AM fungal endophyte colonization of alder roots increased with forest soil transfers in overburden and tailings but not tailings + overburden (Figure 3-3). Endophyte colonization was lower in tailings compared to overburden and tailings + overburden (Figure 3-3; Table B-1), although a significant soil type x transfer volume interaction was present (Table 3-1). Compared to the forest soil control, endophyte colonization and N-fixing nodule biomass were lower in all soil treatments (Table B-3). Shoot biomass was positively related to EM fungal colonization in spruce (P < 0.001) and to nodule biomass in alder (P < 0.001) in models across all unsterilized treatments where soil treatment was included as a covariate (Table B-4). EM fungal colonization did not, however, relate to shoot biomass in willow (P = 0.225; Table B-4). Symbiont colonization also correlated with seedling morphology: root:shoot ratio was positively related to willow EM (P = 0.025) and endophyte (P = 0.032) fungal colonization, but tended to negatively relate to spruce EM fungal colonization (p = 0.074; Table B-4). Root:shoot ratio was not affected in alder, but specific root length was negatively related to nodulation (P = 0.009; Table B-4). Aside from willow root:shoot ratio, non-AM fungal endophyte colonization did not relate to willow or alder shoot biomass or seedling morphology measures (Table B-4). 42 3.3.3 Soil Sterilization Soil sterilization significantly reduced colonization of root symbionts in all three species except where colonization was already low in unsterilized treatments (e.g., 100% tailings) and/or where low levels of colonization occurred in sterilized soil due to imperfect sterilization or colonization by greenhouse species (Figure B-5). Increases in seedling shoot growth and/or foliar N with forest soil transfers to spruce and alder seedlings were reduced or lost with sterilization (Figure 3-1; Figure 3-3). This aligned with significantly more positive slopes in the soil transfer volume models for unsterilized soil compared to sterilized soil (Table B-5). In contrast, greater willow seedling shoot growth with increased soil transfer volume was maintained in sterilized soil (Figure 3-2). For willow, slopes of the soil transfer volume effect models did not differ significantly between unsterilized and sterilized soil, except for tailings where root:shoot ratio foliar N decreased at greater rates in sterilized tailings compared to unsterilized tailings (Table B-5). 3.4 Discussion This study showed that forest soil transfers can provide microbial root symbionts and improve seedling growth and foliar nutrition in overburden and tailings. Spruce and willow seedling growth and EM fungal colonization increased with forest soil transfer volume, supporting my first hypothesis. These results are consistent with Helm and Carling (1993a; 1993b) who found soil transfers to balsam poplar seedlings at a mine reclamation site increased seedling height, foliar N and EM fungal colonization after two years. Their findings suggest that the greenhouse effects observed in this study may translate to field conditions. Willow seedlings did, however, experience lower foliar N levels with increased soil transfer volume in tailings + overburden, but 43 this may be due to growth dilution in the larger seedlings. Unlike spruce and willow, soil transfers did not affect alder seedlings growth. Soil transfers did, however, increase non-AM root fungal endophyte colonization across all soils and N-fixing nodule biomass in tailings. This corresponded with increased foliar N in tailings, while soil transfers increased chlorophyll fluorescence in tailings + overburden. The variation in species-specific responses may relate to mismatches between the test species and the plant community of the soil donor site (Taheri & Bever, 2011; van der Putten et al., 2013). Amaranthus and Perry (1988) and Amaranthus et al. (1990) found that soil transfers to Douglas-fir (Psuedotsuga menziesii) seedlings with soil from an arbutus (Arbutus menziesii) stand had limited benefits, whereas Amaranthus and Perry (1987) found regeneration of Douglas-fir at a forest site was promoted by soil transfers from older Douglas-fir plantations. Similarly, Guichon (2015) found soil transfers of western redcedar (Thuja plicata) soil to western hemlock (Tsuga heterophylla) seedlings had an inhibitory effect on their growth and mycorrhizal colonization. In this study, soil was collected from mid to late seral forests where spruce was present but not alder or willow and it is possible that limited compatible microbial inoculum was available. PLFA tests showed that N-fixing bacteria were absent from fresh forest soil, underscoring the low inoculum levels in the forest soil, even though nodulation did occur in the 100% forest soil treatment. Further, alder associate with a high proportion of genus-specific EM fungi, an exception to the low host specificity of EM taxa that are typically dominant on other trees and shrubs (Molina, 1979; Tedersoo et al., 2009), potentially explaining the negligible EM fungal colonization I observed. This may have been compounded by the fact that alder generally does not form symbioses with EM fungi until roots have been colonized by N-fixing bacteria (Koo et al., 1995; Miller et al., 1992). 44 Plant species successional stage has also been shown to influence the effectiveness of microbial inoculation, with late successional species benefiting most (Cheeke et al., 2019). Improvements in spruce foliar nutrition and growth with soil transfers declined or disappeared with soil sterilization, indicating that EM fungi play a key role in plant establishment. This supports my second hypothesis that the biological component of forest soil would be partially responsible for soil transfer benefits. Correspondingly, increased EM fungal colonization altered spruce seedling morphology, increasing shoot biomass and decreasing root allocation. These results for spruce align with research from grassland ecosystems where soil and native mycorrhizal fungal inoculations were more beneficial for late successional species, which have higher mycorrhizal dependence (Cheeke et al., 2019; Koziol & Bever, 2015). Negative effects of soil sterilization on spruce establishment are also consistent with Saxerud and Funke (1991) and Emam (2016) who found increased biomass, foliar nutrition and AM fungal colonization of grasses with unsterilized versus sterilized soil transfers to mine reclamation soils. In contrast to spruce, soil transfer benefits to willow, an early successional species, were maintained in sterilized soil, indicating that willow primarily benefited from the physiochemical components of the soil transfer. This is supported by the lack of relationship between shoot biomass and mycorrhizal colonization. These results contradict my second hypothesis that the biological component of soil transfers would benefit seedlings, but may be explained by the lower mycorrhizal dependence of early successional species (i.e., these plants are adapted to soils with low biological activity; Cheeke et al., 2019; Koziol & Bever, 2015). Willow has fibrous fine roots for nutrient acquisition without relying on mycorrhizal fungi. In fact, root:shoot ratio increased with endophyte and EM fungal colonization suggesting that the fungal symbionts did not improve willow uptake of nutrients and shoot growth was not improved with greater 45 mycorrhizal fungal colonization. This finding does not mean use of willow or similar early successional species should be avoided in restoration—these plants play an important role in soil development. This result simply helps inform practitioners of when and how soil transfers are anticipated to be of greater benefit to seedlings. It is possible, however, that willow would show a biological benefit with compatible inoculum (i.e., not soil from late-seral stands where willow was not present). Additionally, greater positive effects of mycorrhizal colonization may occur on willow under field conditions compared the controlled greenhouse environment in this experiment, because seedlings were not exposed to environmental stressors, such as drought, which are known to enhance the benefits of microbial symbioses (Lehto & Zwiazek, 2011). While alder is also an early successional species, the few parameters that did improve with soil transfers were lost with sterilization. Further, increased nodulation positively related to shoot biomass and negatively related to specific root length (i.e., root systems became thinner). This indicates that N-fixing bacteria may be an exception to the low microbial symbiont dependence of early successional species, highlighting that soil transfers may be of particular benefit for trees and shrubs that associate with N-fixing bacteria (Seeds & Bishop, 2009). Soil transfers benefited alder and spruce more in tailings than overburden (Figure B-6), supporting my third hypothesis, but overall there were limited significant interactions between soil transfer volume and soil type, indicating that transfers were beneficial in all substrates tested. Consistent with other mine reclamation studies, both the overburden and tailings had low nutrient levels and inoculum potential (Bois et al., 2005; Moynahan et al., 2002; Zong et al., 2015), supporting the idea that facilitation of plant establishment by soil microbes is expected to be more effective in low fertility soils (Carbajo et al., 2011; Schultz et al., 2001). Despite similar soil transfer responses (slopes), general seedling establishment did vary among the reclamation 46 soil types with the poorest growth occurring in tailings, supporting my third hypothesis. This aligns with the lower nutrient levels and inoculum potential of tailings compared to overburden. Results from the tailings + overburden treatment were comparable to the overburden treatment, suggesting that mixing the substrates can alleviate some of the negative effects of tailings on plant establishment and reduce the volume of topsoil needed for reclamation. The one exception is alder, which grew relatively well in tailings, demonstrating its usefulness as a species for tailings reclamation and aligning with research showing broad-leaved trees establish better in tailings (Zong et al., 2015). In support of my fourth hypothesis, the larger soil transfer volume (188 mL; 25% of the greenhouse pot) was generally sufficient for improving seedling growth, physiology and root symbiont colonization to levels comparable with seedlings grown in 100% forest soil. The larger volume (compared to the 38 mL or 5% treatment) may not only provide a higher inoculum load, but may alter rhizosphere conditions such as pH to be more amenable to root development and/or symbiont colonization (a potential benefit over commercial inoculants; Dumbrell et al., 2010). This volume is comparable to Helm and Carling (1993a; 1993b; 250 mL), Amaranthus and Perry (1989;150 mL), Querejeta et al. (1998; 150 mL) and Berman and Bledsoe (1998; 250 mL) who saw positive effects of soil transfers on plant growth and/or mycorrhizal fungal colonization across different disturbance types. These results suggest diminishing returns of soil volumes greater than approximately 200 mL, although this will be influenced by the native soil inoculum density (Koziol et al., 2018). Two exceptions to the 188 mL soil transfer volume as ideal were observed. Firstly, willow in overburden only required the smaller soil transfer volume (38 mL) for growth, nutrition and EM fungal colonization comparable to willow seedlings grown in forest soil. This indicates that lower soil transfer volumes may be appropriate for early successional 47 species in higher quality reclamation soils (i.e., overburden, not tailings). Secondly, even with the larger soil transfer volume, spruce seedlings in tailings had lower foliar nutrition levels, chlorophyll fluorescence and EM fungal colonization than in 100% forest soil, while spruce in tailings + overburden had lower EM fungal colonization. This may indicate that growth will lag over the longer-term. Further research is required to assess if soil transfers will have lasting effects on seedling growth and nutrition as roots extend beyond the treated rooting zone, as the findings from this experiment only apply to initial seeding establishment. Middleton & Bever (2012) found AM fungal inoculation benefits to spread to neighouring plants 2 m away within a couple of growing seasons, suggesting that although growth may lag compared to seedlings growing in reference forested areas, over time established plants will maintain and further develop communities of microbial mutualists through plant–soil feedbacks (Krüger et al., 2017). Alternatively, if site-wide topsoil application occurs at a rate of 25% of the growing medium volume (the more effective ratio found in this experiment) as, for example, a layer of topsoil, my findings suggest that plant establishment benefits may persist (Ryszka & Turnau, 2007, but see Bendfeldt et al., 2001). 3.5 Conclusion Application of forest soil transfers to mine reclamation materials (overburden and tailings) increased seedling growth, chlorophyll fluorescence and foliar N under greenhouse conditions. These improvements generally correlated with increased colonization by mycorrhizal fungi and N-fixing bacteria. Differential results among plant species in sterilized soil indicated that the later successional species, spruce, was more mycorrhizal-dependent and benefited from the biological component of the forest soil, while willow, an early successional species, benefited 48 more from the improved physiochemical properties of the forest soil. The limited benefit of soil transfers to alder seedlings was likely due to a lack of compatible soil microbes at the donor site, highlighting the importance of matching the donor site with the target tree species. Soil transfers had greater benefit in mine tailings (the lowest quality substrate) but were effective in both overburden and tailings, with approximately 200 mL forest soil additions generally supporting initial seedling establishment comparable to forest soil controls. While long-term field validation of these findings in mine reclamation settings is necessary, including investigation of the best methods for large-scale application, this study shows that small volume transfers of topsoil from native ecosystems are a promising method for promoting recovery of beneficial root microbial symbionts and improving seedling establishment at highly disturbed sites. 49 Chapter 4: Soil and spatial ecosystem legacies promote plant and soil food web dispersal and establishment at the Mount Polley Mine 4.1 Introduction Mine reclamation materials such as tailings and overburden typically have limited biological activity (Bois et al., 2005; Moynahan et al., 2002) and even stockpiled topsoil is subject to loss of vegetative propagules and beneficial microbes, including mycorrhizal fungi and N-fixing bacteria (Dhar et al., 2019; Gorzelak et al., 2017; Harris et al., 1987). Ecosystem legacies are a source of biological propagules that can be leveraged in mine reclamation to support ecosystem recovery, emulating the processes through which ecosystems recover from natural disturbances (Johnstone et al., 2016). While manipulation of the biotic component of ecosystems does not always accelerate succession (Kardol et al., 2009), research in primary successional systems has shown that lack of plant propagules or soil microbes can act as biotic barriers to plant survival or growth, and even arrest succession (McClanahan, 1986; Nara, 2006; Seeds & Bishop, 2009). Additionally, early colonization of mutualistic soil microbes may support seedling survival in stressful primary successional conditions during the critical establishment stage (Nara, 2015). Spatial ecosystem legacies such as undisturbed patches of forest can be a low-cost source of aerially dispersed seeds and microbes, with seeds traveling up to hundreds of meters and some fungal spores traveling kilometers (Peay et al., 2012; Vittoz & Engler, 2007). Additional benefits may be associated with short-distance connectivity due to increased seed rain (Gorchov et al., 1993) and ingress of species including soil fauna and many mycorrhizal fungi that spread by root contact (Egan et al., 2014; Ojala & Huhta, 2001). Short-distance connectivity with established forest can also allow for formation of mycorrhizal networks that have been shown to transfer 50 nutrients from established trees to seedlings 2.5–10 m away (Churchland & Grayston, 2014; Teste & Simard, 2008). Studies on volcanic outwash suggest seedling spatial connectivity with established plants in primary successional soils is a critical source of nutrients (accessed via mycorrhizal networks) and microbial inoculum (Nara, 2006; Seeds & Bishop, 2009). Yet, ingress of soil biota and facilitation through belowground connectivity with nearby forest have not been tested in a mine reclamation setting. Fresh topsoil is another ecological legacy that, when in limited supply at a mine site, could be applied as soil transfers (targeted small volume additions to the seedling rooting zone). Soil transfers have been shown to benefit seedling and mycorrhizal fungal establishment in other restoration applications (Cortese & Bunn, 2017; Lance et al., 2019; Middleton & Bever, 2012; Querejeta et al., 1998; Urgiles et al., 2014). However, further research is necessary to test the transferability of this technique to mine reclamation conditions (but see Helm and Carling, 1993), with field trials being critical for validating greenhouse results and assessing operational scalability. Field trials also enable longer-term testing to address outstanding uncertainties regarding persistence of inoculated microbes and long-term effects on plant establishment and/or community composition (Hart et al., 2018). Site preparation, such as ripping, blading or mounding, is commonly applied in mine reclamation prior to revegetation to reduce compaction and improve the soil moisture and temperature conditions of planting microsites (Burton et al., 2000; Skousen et al., 2009). Soil compaction restricts water as well as dissolved nutrient and oxygen flow due to loss of pore space and can have long-term negative effects on plant growth and soil microbial communities (Ezzati et al., 2012; Hartmann et al., 2012). Fungal communities, in particular mycorrhizal fungi, have been shown to be negatively impacted by compaction, potentially due to hindered hyphal 51 penetration of soil pores (Amaranthus et al., 1996; Hartmann et al., 2012; Skinner & Bowen, 1974). Site preparation can improve woody seedling survival and growth in mine reclamation (Ashby, 1997; Burger & Evans, 2010) and Bauman et al. (2013) found EM colonization and richness to increase along with improved seedling establishment. Limited mine reclamation research has been conducted on other soil taxa, but Topp et al. (2001) found improved soil fauna colonization and increased microbial activity in troughs compared to crests, leading to improved soil quality. In contrast, a silviculture study found arthropods to be resilient to site preparation, but not benefit from it (Bird et al., 2004). Incorporation of site preparation into this study enabled investigation of its effects on diverse soil organisms and whether its effectiveness was influenced by application of ecosystem legacies. I conducted a field experiment to test the effects of the following three ecosystem legacy-based reclamation techniques on plant establishment, soil food web recovery and soil physiochemical properties at the Mount Polley Mine in areas of till and tailings: (1) site preparation, (2) small volume forest soil transfers, and (3) connectivity with the undisturbed forest. Beyond to the typical motivations for site preparation described above, in the areas with relatively thin layers of spilled tailings (≤ 1m) at the Mount Polley Mine, site preparation also resurfaced the underlying forest floor material, giving seedlings access to this previously buried ecological legacy. Treatment effects on plant establishment were evaluated through growth and nutrition of planted spruce seedlings, and diversity and abundance of naturally ingressing plants. Soil food web recovery was assessed through high-throughput sequencing of soil fungal and bacterial communities, morphotyping of EM fungi colonizing spruce seedling roots, and faunal community characterization through morphological identification and microscopy. Treatment effects on soil physiochemical properties were assessed by measuring bulk density, nutrient 52 levels, organic matter content, pH and electrical conductivity. My four associated hypotheses for the effects of each treatment factor on spruce seedling establishment, naturally ingressing plants, soil food web recovery and soil physiochemical properties are presented in Table 4-1. I additionally expected to find interactions between distance from forest edge and forest belowground connectivity, with greater benefit of forest belowground connectivity closer to the forest edge.53 Table 4-1 Field experiment hypotheses for the effects of site preparation, soil transfer and forest connectivity (both aerial and belowground connectivity) on plant establishment, soil food web recovery and soil physiochemical properties. Site preparation refers to mounding in the till zone, and mounding and tailings removal in the tailings zone. Increasing bacterial succession is based on comparison of phyla relative abundances with forest soil and successional chronosequences. NA = hypothesis not tested or not applicable. OM = organic matter; EM = ectomycorrhizal. No. Factor Spruce seedlings Plant ingress Spruce root EM fungi Soil microbes (fungi, bacteria) Soil fauna Soil properties 1 Site preparation ↑ establishment ↑ diversity ↑ abundance ↑ diversity ↑ colonization ↑ diversity ↑ EM fungi relative abundance ↑ bacteria succession ↑ diversity ↑ abundance ↓ bulk density ↑ OM (tailings) 2 Soil transfers ↑ establishment No effect ↑ diversity ↑ colonization ↑ diversity ↑ EM fungi relative abundance ↑ bacteria succession ↑ diversity ↑ abundance No effect 3 Forest belowground connectivity ↑ establishment ↑ diversity ↑ abundance ↑ diversity ↑ colonization ↑ diversity ↑ EM fungi relative abundance ↑ bacteria succession ↑ diversity ↑ abundance No effect 4 Increasing distance from forest edge NA ↓ diversity ↓ abundance NA ↓ diversity ↓ abundance ↓ diversity ↓ abundance NA 54 4.2 Methods 4.2.1 Experimental design The field experiment was established in May 2016 at the Mount Polley Mine in areas of exposed native glacial till (primarily basil till; the “till zone”) and areas of tailings overlying intact forest floor (the “tailings zone”) located within the tract of land disturbed by the 2014 tailings dam failure. In the till zone, a factorial 2 x 2 x 2 randomized complete block design was established in three trial blocks along the disturbed corridor with approximately 5 m buffers separating treatment units in each block (Figure 4-1; Figure C-1). The first factor was site preparation: sites were excavator mounded (i.e., underlying soil overturned) vs. no mounding. The second factor was forest soil transfers: 750 mL of forest soil was mixed into the seedling rooting zone during planting vs. no soil transfers. Soil was collected from the undisturbed forest adjacent to each block by removing the litter layer and collecting the top 20 cm of soil (fermentation layer, humic layer and upper mineral soil horizons). Treeplanters added the forest soil from one of their planting bags into the shovel hole opened for each seedling, and the forest soil was lightly mixed into the surrounding soil with the planting shovel. EM conifers—spruce (Picea glauca x engelmannii) and/or firs (Abies lasiocarpa and Pseudotsuga menziesii)—were present at forest soil collection sites adjacent to all trial blocks (Table C-1). Spruce trees were present adjacent to all trial blocks except one in the till zone, although the proportion of spruce ranged from 2 to 98% of basal area (Table C-1). Forest soil at each block was homogenized before use in transfers. The third factor was belowground connectivity with the undisturbed forest: seedlings out-planted from the forest edge were either isolated (“disconnected”) using linear trenches excavated along the forest edge or remained connected to the adjacent forest (i.e. no trenches were dug). The ≥ 1 m deep trenches extended below the forest rooting zone. 55 The same experimental design was applied to three trial blocks in the tailings zone (Figure C-1), but three site preparation treatments were tested: undisturbed tailings (no site preparation), mounding, and removal of tailings in strips down to forest floor, with seedlings planted in these strips. Mounding and tailings removal treatments were installed in the 2 x 2 x 2 full factorial design described above, but due to space restrictions, the no-site preparation treatment (undisturbed tailings) was tested with and without forest transfers, but only with belowground connectivity (i.e., 2 x 2 x 2 design plus two additional no site preparation treatments, totaling ten treatment units per block; Figure 4-1). Prior to installation of the experiment, the eroded banks in the till zone were re-sloped with an excavator. In the tailings zone, dead trees (killed by anerobic soil conditions created by the saturated tailings deposited on the forest floor; Miller & Simard, 2015) were logged and excess tailings were removed to ≤ 1m depth. Depth of tailings was measured at 2, 8 and 14 m from the forest edge in each tailings zone treatment prior to site preparation to confirm that tailings depths were similar across treatment units. Tailings depths increased with distance from forest edge, but increases were consistent among treatment units and this was accounted for by using distance as a random factor during statistical analyses (Section 4.2.6). Trial blocks represented the variation present within the disturbed corridor, including different slope aspects and, in the till zone, a range of slope gradients, with two having low slope angles (~1–2 °) and one having a greater slope angle (~ 9 °). The tailings zone blocks were relatively flat, as these were depositional areas. Following site preparation, in each treatment unit, four lines of hybrid white spruce seedlings (Picea engelmannii x glauca) were out-planted perpendicular to the forest edge into the disturbed area every 1m for 15m, with 1 m spacing between lines, totaling 60 seedlings per 56 treatment unit (Figure 4-1). The planting layout was designed to enable assessment of short-distance spatial ingress and facilitation from the forest edge. For general comparison of the treatment areas to the reference forest, a seedling was planted at 1 m and 10 m into the forest adjacent to each treatment unit. Spruce seedlings were 1-year-old nursery container stock of appropriate stock type and provenance for the region (PRT Red Rock Nursery, Prince George, BC). Figure 4-1 Depiction of the field trial experimental design in one till zone block (A) with a 2x2x2 factorial design (n = 4 spruce seedlings x 15 distances, totalling 60 seedlings per treatment unit). Factors were site preparation (excavator mounding), soil transfers and belowground connectivity to the adjacent forest (trench vs. no trench). Three blocks with randomized treatments were installed. The same design was applied to three blocks in the tailings zone (B), except three site preparation treatments were tested: no site preparation (undisturbed tailings), mounding and tailings removal. Due to space restrictions, the no site preparation treatment (undisturbed tailings) was tested with and without forest transfers, but only with belowground connectivity (i.e., 2 x 2 x 2 full factorial design plus two additional no site preparation treatments, totaling ten treatment units per tailings zone block). 57 4.2.2 Baseline soil data collection At the time of planting, bulk density samples were collected using the excavation method (10 cm deep x 10.5 cm diameter excavation) as per Krzic et al. (2010). One sample per site preparation treatment type and one sample of the adjacent reference forest was taken per block. For chemical analyses, a 15 cm deep core was collected adjacent to a planted spruce seedling at each distance from forest edge (one of four replicate seedlings at each distance was randomly selected) and cores composited for 1–5 m, 6–10 m and 11–15 m in each treatment unit. Samples of the homogenized forest soil used for transfers in each block were also collected. Soil samples were processed and analyzed for pH, electrical conductivity, total C, total N, available P and exchangeable cations as described in Section 2.4. 4.2.3 Vegetation monitoring Spruce seedling survival, height and root collar diameter were measured immediately following planting and at the end of the growing season (August) for the first three years. Vigour was qualitatively assessed where 1 = excellent and 4 = poor (detailed criteria are provided in Table C-2). Seedling microsite (water-receiving, neutral or water-shedding) and damage, including browsing, flooding (defined as evidence of standing water at the base of the seedling) and chlorosis, were noted. In the third growing season, naturally ingressing plants were surveyed in 1 m2 quadrats at 3 m, 7.5 m and 13 m from the forest edge in each treatment unit, representing “close” (1–5 m), “mid” (6–10 m) and “far” (11–15 m) distances. Ocular estimates of percent cover of each plant species (excluding moss) was recorded along with percent cover of the tree, shrub, herb and moss layers and total non-moss plant cover. Reference forest shrubs and herbs were surveyed in 58 1 m2 quadrats in two plots 10 m into the adjacent forest at each block (12 plots total). Additionally, percent cover and diameter at breast height (DBH) of tree species were measured in 3.99 m radius (50 m2) plots at the reference forest sites. Basal area of each tree species was calculated from DBH. 4.2.4 Seedling destructive harvest At the end of the third growing season, a subset of spruce seedlings was destructively harvested, including excavation of the root systems. One representative seedling was harvested at 1, 3, 8 and 15 m from forest edge (approximately following a logarithmic scale) in each treatment unit, totaling 207 seedlings. No seedling was available for harvest at 1 m in one of the tailings zone treatments due to herbivory, or at 15 m in ten tailings zone treatments due to poor survival associated with flooding. In six instances, all seedlings were dead at the desired distance and a seedling 1 m away was harvested instead. At harvest, a 20 cm wide x 20 cm long x 6 cm deep soil monolith centered on each seedling and its root system was excavated and the monolith soil was homogenized in a clean plastic container. First, an approximately 100 mL sample was collected for microbial analysis in a Nasco Whirl-Pak bag and a 230 mL sample collected for microarthropod analysis in a VWR HDPE plastic container. The remaining monolith soil was then hand-sorted in the field for soil macrofauna, which were identified to broad group (class for Chilopoda, subclass for Acari and Collembola, order for the remaining taxa). Finally, a soil sample was collected for chemical and physical analyses. The full root system was then excavated. Roots were washed and all root and soil samples were stored at 4 °C until analysis, except the soil microbial samples were frozen at -59 20 °C. For general comparison of the treatment areas to the reference forest, soil samples were also collected from two spruce seedlings per block that were planted in the reference forest. Microarthropods were extracted using modified Burlese-Tullgren funnels constructed out of plastic drink bottles cut in half and placed upside down with 2 mm mesh over the mouth. Bottles of 70% ethanol were placed at the base of each funnel to preserve specimens. Soil samples were placed in the funnels under 7 watt incandescent bulbs within 14 days of collection and left in place until no signs of soil moisture were present (up to 26 days). Samples were transferred to a petri dish and abundance and diversity of microarthropod morphospecies assessed under a dissecting microscope (40x). Small specimens were mounted on slides and viewed under a compound microscope (40–400x). A 10 g sample of bulk soil was dried for 48 h at 105 °C and weighed for gravimetric water content. The remaining sample was air-dried and sieved to < 2 mm. Soil organic matter was measured through loss on ignition at 450 °C for 4 h. pH and electrical conductivity were measured as described in Section 2.4. Spruce shoots were dried at 70 °C for 72 h and weighed. Samples of the youngest foliage were analyzed for total (%) N and C, as well as δ13C with an Elementar Vario EL Cube Elemental Analyzer and an Isoprime Isotope Ratio Mass Spectrometer (GV Instruments) at the UBC Stable Isotope Facility, Vancouver, BC. δ13C is a measure of the ratio of stable isotopes 13C:12C and 13C isotope discrimination can be used as an indicator of greater water-use and photosynthetic efficiency in C3 plants, with higher δ13C corresponding to increased efficiency (Arslan et al., 1999; Kranabetter et al., 2010; Zou et al., 2019). 100 random root tips were collected from each spruce seedling for morphotyping of EM fungi and the remaining root systems were dried at 70 °C for 72 h and weighed. 60 4.2.5 Microbial analyses 50 root tips were collected from five of the nursery-grown spruce seedlings at the time of planting to characterize their baseline EM fungal communities. The root tips were rinsed with distilled water and DNA was extracted using PowerSoil DNA Isolation kits (MoBio, 2015) as per the manufacturer’s instructions. Extracts underwent high-throughput sequencing and bioinformatics as described in Sections 2.5 and 2.6. Root tips collected from spruce seedlings harvested after three growing seasons (Section 4.2.4) were morphotyped to assess EM fungal percent colonization and diversity as described in Section 2.5. Morphotypes were identified through Sanger sequencing; where Sanger sequencing failed or PCR products had more than one band in gel electrophoresis, high-throughput sequencing was used (Section 2.5, Section 2.6). DNA was extracted from 0.25 g of the spruce seedling rooting zone soil microbe samples (collected from the 20 cm wide x 20 cm long x 6 cm deep monolith centered on each seedling at harvest; Section 4.2.4) using DNeasy PowerLyzer PowerSoil kits (Qiagen, 2017) as per the manufacturer’s instructions. Fungal (ITS2) and bacterial (16S) marker genes were PCR amplified using ITS7f/ITS4 (Ihrmark et al., 2012; White et al., 1990) and 515f/806r (Apprill et al., 2015; Parada et al., 2016) primer combinations, respectively, followed by Illumina Miseq sequencing of the amplicons at the Génome Québec Centre d’expertise et de services, Montréal, Quebec. High-throughput sequence data were processed as described in Section 2.6. Following, non-fungal and non-bacterial ASVs were removed from fungal (ITS2) and bacterial (16S) sequence data, respectively. ASVs < 0.1% of mean sample depth (23, 060 for fungi, 32,328 for bacteria) were removed to remove potential Mi-Seq bleedthrough. Samples were rarified to the minimum sample depth of 5,423 for fungi (resulted in loss of one ASV) and 7,884 for bacteria (no ASVs lost). Twenty bacteria samples did not amplify during PCR and were not sequenced 61 (four from the till zone, fifteen from the tailings zone, one from the reference forest). FunGuild assignments were made for 77% of fungal ASVs, with 62% of fungal ASVs used in analyses following removal of mixed trophic mode taxa. 4.2.6 Statistical analyses Data analyses were carried out in R (version 3.6.1; R Core Team, 2020). Models were run separately for the till and tailings zones. Treatment effects were assessed with linear mixed models (LMMs) using the lmer function (lme4 package) and generalized linear mixed models GLMMs using the glmer function (lme4 package; Bates et al., 2019). Till zone models had treatment factors as the fixed effects with distance from forest edge as a covariate (site preparation x soil transfer x forest belowground connectivity + distance) and block as the random effect. Tailings zone model fixed effects were adjusted to account for the added site preparation treatment: preparation x soil transfer + soil transfer x forest belowground connectivity + distance. For LMMs, data were transformed to meet assumptions of normality and equal variance, as required. If binomial GLMMs were overdispersed, betabinomial models were run using the glmmTMB function (glmmTMB package; Brooks et al., 2017). Pre-planned post hoc contrasts were used to test the effects of treatment factors and their interactions using the emmeans function (emmeans package; Lenth, 2020) with the Tukey adjustment for multiple comparisons. Secondary mixed models were run to test distance x forest belowground connectivity interactions (i.e., changes in effects of connectivity with distance from forest edge) with distance x connectivity + site preparation + transfer as the fixed effects. For the tailings zone, the no-site preparation treatment was removed from these models, as no equivalent 62 treatment without forest belowground connectivity was included in the design. For all analyses, significance was evaluated as P ≤ 0.05 and marginal significance as 0.05 < P < 0.1. Effects of experimental treatments on baseline soil chemistry (samples collected after site preparation) were tested with permutational multivariate analysis of variance (PERMANOVA) using the adonis function (999 permutations, strata = block, distance from forest edge = covariate) in the vegan package on Bray Curtis distances (Oksanen et al., 2019). Correlation matrices of soil parameter Pearson correlation coefficients and P-values were constructed with the cor function and rcorr function (Hmisc package; Harrell & Dupont, 2020), respectively, to identify key soil parameters. Treatment effects on key parameters were then assessed with LMMs. For bulk density, LMMs deviated from the structure described above and included site preparation x zone (till, tailings, reference forest) as the fixed effects and block as the random effect. Treatment effects on the richness and Shannon diversity of naturally ingressing plants were tested (LMMs) as well as effects on percent cover of all plants, trees, shrubs and herbs, and proportion of cover from non-native plants (binomial GLMMs). Effects on Bray Curtis distances of log-transformed plant community composition were tested with PERMANOVA using the adonis function (999 permutations, strata = block, distance from forest edge = covariate; Oksanen et al., 2019). Effects of baseline soil chemistry and distance from forest edge on plant community composition were tested with db-RDAs conditioned on block using the capscale function in the vegan package (ANOVA run with 999 permutations restricted within levels of block; Oksanen et al., 2019). Variation partitioning between treatment effects (site preparation, soil transfers and forest belowground connectivity combined), distance from forest edge, and soil properties was conducted using the varpart function (vegan package) and conditional 63 significance of the components was assessed with RDAs (vegan package) using the Condition argument (ANOVA run on 999 permutations restricted within levels of block; Oksanen et al., 2019). Bray Curtis dissimilarities between till zone, tailings zone and reference forest plant communities were compared, with differences visualized using db-RDA (vegan package, Oksanen et al., 2019). Treatment effects on spruce seedling size and condition were assessed using binomial GLMMs (survival, proportion of chlorotic or flooded seedlings seedling), Poisson GLMMs (vigour) and LMMs (volume growth, microsite). Seedling volume was calculated as the volume of a cone from height and root collar diameter (Aphalo & Rikala, 2003) and used as the primary growth measure, as volume had a stronger relationship with shoot biomass (R2adj = 0.71) than height (R2adj = 0.63) or root collar diameter (R2adj = 0.63) based on harvested seedlings. Similarly, volume had the strongest relationship with root biomass of harvested seedlings (volume: R2adj = 0.56; height: R2adj = 0.38; root collar diameter: R2adj = 0.54). Seedlings that were browsed by wildlife were removed from the dataset prior to analysis. Measures were averaged among distance replicates (the four seedlings planted at each distance within each treatment unit) and distance groupings of 1–5 m, 6–10 m, and 11–15 m to allow identification of more generalizable distance effects. Data were analyzed for each of the three growing seasons, as well as total growth over the time period (three-year time period not applicable for vigour, chlorosis or survival endpoints; survival was assessed cumulatively, i.e., total survival at the end of each year). Model interpretation focused on the third growing season, as first year growth was primarily reflective of nursery conditions and second year growth was overall poor due to drought and severe wildfire smoke (Abbott & Chapman, 2018), while third year growth was reflective of any lasting treatment effects. Temperature was approximately average during the 64 third growing season (2018), but only 117.3 mm of rain fell compared to approximately 243 mm on average (MPMC, 2019). Treatment effects on harvested spruce seedling biomass, foliar nutrition and EM fungal communities were tested with LMMs (shoot biomass, root biomass, root:shoot ratio, foliar N, foliar δ13C, EM fungal morphotype richness and Shannon diversity) and binomial GLMMs (EM fungal percent colonization). For soil samples collected from the rooting zone of harvested seedlings, treatment effects on soil physiochemical properties were tested with LMMs. Fungal high-throughput data were assessed at two levels: ASVs, and key guilds (saprotrophs, plant pathogens and EM fungi). Treatment effects on fungal richness and Shannon diversity (ASVs only) were tested using LMMs, while effects on guild relative abundance were tested with GLMMs. Bacterial ASV richness and Shannon diversity were similarly tested with LMMs. Relative abundance of dominant phyla (> 1.5% of reads) was tested with GLMMs. For soil fauna, treatment effects on richness of broad groups (LMMs) and total abundance (Poisson GLMMs) were tested, and richness and abundance data were regressed using linear models against rooting zone soil properties (outliers above the 75th percentile or below the 25th percentile by greater than 1.5 x the interquartile range were removed for regressions). Treatment effects on the community composition Bray Curtis distances of soil fungi (Hellinger transformed), bacteria (Hellinger transformed) and fauna were tested with PERMANOVA using the adonis function (999 permutations, strata = block, distance from forest edge = covariate; Oksanen et al., 2019). Effects of soil physiochemical properties, distance from forest edge and the naturally ingressing plant community (scores from the first two PCA axes) on belowground community composition were assessed with db-RDA as described above. Where one or more of these factors (experimental treatments, soil physiochemical properties, distance 65 from forest edge and plant community) were significant, conditional significance was assessed with RDAs using the Condition argument as described above. For fauna, varpart was used, as fewer than four factors were significant. Bray Curtis dissimilarities between till zone, tailings zone and reference forest fungal communities were compared, with differences visualized using db-RDA (vegan package, Oksanen et al., 2019). 4.3 Results 4.3.1 Spruce seedling establishment In the till zone, survival in the first growing season was 98.3%, then dropped to 91.3% in the second growing season with minor losses in the third growing season (91.0% survival). In the third growing season, mounding reduced the proportion of chlorotic seedlings in treatments without soil transfers but decreased survival and shoot volume growth in treatments with soil transfers (Figure 4-2; Table C-3). Soil transfer interactions with mounding similarly showed that transfers tended to reduce seedling chlorosis in treatments with no mounding, while transfers had negative effects on survival, shoot volume growth and chlorosis in treatments with mounding (Figure 4-2; Table C-3). Forest belowground connectivity negatively affected all seedling establishment measures in the third growing season (Figure 4-2; Table C-3), although a significant interaction showed vigour only decreased in treatments without soil transfers (T76.8 = -2.73, P = 0.008). Distance from forest edge and connectivity significantly interacted: year three shoot volume growth was lower with connectivity at 6–10 m (T76.8 = -2.05, P = 0.044) and 11–15 m (T76.8 = -1.95, P = 0.055) but not at 1–5 m (T76.8 = -1.17, P = 0.246). Similarly, survival was lower with connectivity at 6–10 m (Z = -1.93, P = 0.054) and 11–15 m (Z = -2.44, P = 0.015) but not 1–5 m (Z = -1.03, P = 0.304). 66 The proportion of seedlings planted in water-receiving microsites in the till zone (i.e., low microsites or in small depressions) was higher with mounding (Z = 4.10, P < 0.001), soil transfers (Z = 10.95, P < 0.001) and forest belowground connectivity (Z = 4.33, P < 0.001). This corresponded to a greater proportion of seedlings affected by flooding with mounding (T60 = 5.18, P < 0.001), soil transfers (only in mounded treatments; T60 = 2.87, P = 0.006), and connectivity (only in non-mounded treatments: T60 = 3.78, P < 0.001). Flooding was negatively related to seedling shoot volume (R2adj = 0.19, P < 0.001). In the tailings zone, overall survival was high in the first growing season (98.4%) but dropped to 68.8% after the second growing season (due to flooding in two blocks) with minor losses in the third growing season (66.9% survival). Site preparation (mounding and tailings removal) increased spruce seedling survival, volume growth and vigour, and reduced chlorosis in the second and third growing seasons (Figure 4-3; Table C-4). The only difference between tailings removal and mounding was that seedlings in mounded treatments tended to be more chlorotic in the third growing season (Figure 4-3; Table C-4). Soil transfers increased vigour and reduced the proportion of chlorotic seedlings in the second and third growing seasons (Figure 4-3; Table C-4). Transfers also increased seedling volume growth in the mounding treatment (all three growing seasons), but not the undisturbed tailings or tailings removal treatments (Table C-4). Forest soil connectivity decreased survival and interacted with soil transfers, having negative effects on seedling growth, vigour and chlorosis in treatments with no soil transfers (Figure 4-3; Table C-4). Interactions between distance from forest edge and forest belowground connectivity revealed lower third year seedling volume growth adjacent to the forest (1–5 m) where there was connectivity compared to without connectivity (T74.8 = -2.13, P = 0.037), but no difference between connected and unconnected treatments at 6–10 m or 11–15 m. The proportion 67 of seedlings affected by flooding decreased with soil transfers in undisturbed tailings (i.e., no site preparation; Z76 = -2.21, P = 0.030) and tended to decrease in mounded treatments (Z76 = -1.84, P = 0.070). Incidence of seedling flooding damage was weakly negatively related to seedling shoot volume (R2adj = 0.02, P = 0.083). Treatment effects on seedling size over the full three years were reflective of year three results for both the tailings zone (Table C-3) and till zone (Table C-4). Overall, seedlings in undisturbed tailings (no site preparation) were the smallest, while seedlings in the tailings zone site preparation treatments (mounding or tailings removal) were largest; till zone seedlings were intermediate (Table 4-2). Table 4-2 Height, root collar diameter (RCD) and volume (mean ± 1 SE) of spruce seedlings in the till zone and tailings zone after three growing seasons. The tailings zone is separated by no site preparation (undisturbed tailings) vs. site preparation (mounding and tailings removal) because site preparation had a major effect. Zone Height (cm) RCD (mm) Volume (cm3) Till 34.1 ± 0.3 7.9 ± 0.1 20.6 ± 0.5 Tailings (no site preparation) 28.9 ± 0.5 6.0 ± 0.1 10.3 ± 0.7 Tailings (site preparation) 44.5 ± 0.6 8.3 ± 0.1 32.5 ± 1.1 68 Figure 4-2 Till zone: third growing season effects of mounding (A, C), soil transfers (A, C) and forest belowground connectivity (B, D) on spruce seedling shoot volume growth (A, B) and chlorosis (C, D). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from linear mixed models (A, B) and binomial generalized linear mixed models (C, D). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 69 Figure 4-3 Tailings zone: third growing season effects of site preparation (mounding and tailings removal; A, C), soil transfers (B, D) and forest belowground connectivity (B, D) on spruce seedling shoot volume growth (A, B) and chlorosis (C, D). Bar plots represent main effects and line plots represent interactions. Soil transfers significantly reduced seedling chlorosis with no connectivity interaction (D). Bars/points are the back-transformed means ± 1 SE from linear mixed models (A, B) and binomial generalized linear mixed models (C, D). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 4.3.2 Spruce seedling destructive harvest 4.3.2.1 Spruce seedling growth and physiology After three growing seasons, till zone spruce seedlings that received soil transfers tended to have higher foliar N and foliar δ13C (i.e., increased water-use and photosynthetic efficiency; Arslan et al., 1999; Zou et al., 2019; Figure 4-4). Transfers had no effect on root biomass or root:shoot ratio (Table C-5) but decreased shoot biomass in treatments with mounding (T107 = -2.96, P = 70 0.004). Contrary to soil transfers, forest belowground connectivity decreased foliar N (Figure 4-4). Significant interactions showed connectivity decreased δ13C in treatments without mounding, while mounding decreased foliar δ13C in treatments without connectivity (Figure 4-4). Mounding and forest belowground connectivity had minimal effects on root and shoot growth (Table C-5). No interactions between distance from forest edge and forest belowground connectivity occurred. In the tailings zone, site preparation (tailings removal and mounding) resulted in greater spruce shoot biomass and tailings removal increased foliar N (Figure 4-5; Table C-5. Conversely, site preparation (tailings removal and mounding) had limited effects on root biomass (only marginally significant increases with mounding), leading to significant decreases in root:shoot ratio (Table C-5). Soil transfers increased root and shoot biomass in the mounding treatment, with no effect on root:shoot ratio (Figure 4-5; Table C-5). While soil transfers had no significant effects in undisturbed tailings (i.e., no site preparation), trends of increased shoot biomass, root biomass and foliar N and foliar δ13C were evident (Table C-5). Removal of known outliers (seedlings with access to pockets of organic matter or forest soil as observed during sampling and verified against rooting zone soil organic matter data) increased the strength of these trends and showed transfer tended to increase foliar N (T21 = -1.99, P = 0.060; Figure 4-5). In contrast to these positive effects, soil transfers decreased δ13C in the tailings removal treatment and forest soil connectivity decreased root and shoot biomass, with no effects on root:shoot ratio (Figure 4-5; Table C-5). No interactions between distance from forest edge and forest belowground connectivity occurred adjacent to the forest edge (at 1 m or 3 m). 71 Spruce seedling foliar N levels were within the range of moderate to severely deficient (1.05–1.25%), indicating that foliar N is a limiting nutrient and increases are expected to be beneficial for plant growth (i.e., not simply indicative of growth dilution; Carter, 1992). Figure 4-4 Till zone: effects of mounding, soil transfers and forest belowground connectivity on spruce seedling foliar N (A) and foliar δ13C (‰) (B). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 72 Figure 4-5 Tailings zone: effects of site preparation (mounding and tailings removal; A, C), soil transfers (A, C) and forest belowground connectivity (B, D) on spruce seedling shoot biomass (A, B) and foliar nitrogen (C, D). Models for the no site preparation treatment in A and C exclude outlier seedlings with access to organic matter deposits. Bar plots represent main effects and line plots represent interactions. Mounding and tailings removal increased shoot biomass with no soil transfer interaction (A) and tailings removal increased foliar nitrogen with no interaction (C). Bars/points are the back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 4.3.2.2 Spruce root EM fungal colonization Seventeen EM fungal taxa were identified during morphotyping of spruce seedling root systems following three growing seasons through Sanger sequencing (Table C-6) and high-throughput sequencing (Table C-7). The dominant morphotypes (present on > 5% of seedlings) were Wilcoxina rehmii, Amphinema spp., Thelephora terrestris, Cenococcum sp. Pseudotomentella sp. and Tuber annaie in both the till and tailings zones, plus Inocybe rufoalba in the till zone only 73 (Figure 4-6, Figure 4-7). Of the thirteen EM fungal taxa on nursery seedlings prior to planting (baseline), only T. terrestris, W. rehmii and Amphinema sp. were observed on seedlings harvested from the till and tailings zones after three years (Figure 4-6). Pezoloma ciliifera was the most abundant taxon on nursery seedlings (43% of reads on average) but was not observed on harvested seedlings or detected in high-throughput sequencing of the rooting zone soil samples. T. terrestris was present on all five baseline nursery seedlings tested (9% of reads on average) and was the third most common morphotype at harvest (39% of seedlings, 8% of all EM root tips). The two most abundant taxa on harvested seedlings were Amphinema spp. (95% of seedlings, 32% of EM all root tips) and W. rehmii (68% of seedlings, 35% of all EM root tips), however, were both only present in low abundances on one of five baseline nursery seedlings sampled (< 0.1% and 4% of reads, respectively). In the till zone, mean EM fungal colonization (± 1 SE) was 82.5 ± 0.7%. Soil transfers tended to increase spruce root EM fungal colonization and Shannon diversity, while mounding increased Shannon diversity (Figure 4-8; Table C-8). Forest belowground connectivity had no effect on EM fungal colonization or diversity and no treatments affected EM morphotype richness, which was low overall (3.1 taxa per root system on average; Figure 4-8; Table C-8). No interactions between distance from forest edge and forest belowground connectivity occurred. In the tailings zone, mean EM fungal colonization (± 1 SE) was 80.6 ± 1.1%. Site preparation increased spruce root EM fungal colonization from 69.8 ± 2.7% to 83.5 ± 1.0% and increased taxa richness, with no difference between mounding and removal treatments (Figure 4-8; Table C-8). Soil transfers increased taxa richness and tended to increase Shannon diversity (Figure 4-8; Table C-8). Soil transfers also increased EM fungal colonization in undisturbed tailings (i.e., no site preparation treatment; Figure 4-8) to colonization levels equivalent with the 74 mounding treatment (T96 = -1.42, P = 0.336). Forest belowground connectivity increased EM fungal colonization in treatments with no transfers, while transfers only increased colonization in treatments with no connectivity (Figure 4-8; Table C-8). Analysis of distance from forest edge x connectivity interactions showed greater EM fungal colonization at 1, 3 and 8 m but not 15 m with connectivity (1 m: Z = 2.40, P = 0.017; 3 m: Z = 4.43 , P < 0.001; 8 m: Z = 1.99, P = 0.047). 75 Figure 4-6 A: Relative abundance of ectomycorrhizal fungal taxa on spruce seedling roots. Baseline nursery seedling root fungal communities were assessed with high-throughput sequencing prior to planting. Spruce seedling roots following three growing seasons in the till and tailings zones were assessed through morphotyping and DNA sequencing of morphotype vouchers. B: Relative abundance of dominant fungal genera (> 2% of total sequencing reads) in spruce seedling rooting zone soil in the till zone, tailings zone and reference forest. Fungal guilds are indicated in brackets. EM = ectomycorrhizal; endo = endophyte; path = pathogen; sap = saprotroph. 76 Figure 4-7 Ectomycorrhizal fungi colonizing spruce seedlings roots after three growing seasons in areas of till and tailings at the Mount Polley Mine, British Columbia as identified through morphotyping and DNA sequencing of morphotype vouchers. Dominant taxa were Amphinema byssoides (A), Wilcoxina rehmii (B), Thelephora terrestris (C), Pseudotomentella sp. (D), Inocybe rufoalba (E), Cenococcum sp. (F), and Tuber annaie (G). 77 Figure 4-8 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity on spruce seedling root ectomycorrhizal fungal percent colonization (A, C), morphotype Shannon diversity (B) and morphotype richness (D) in the till zone (A, B) and tailings zone (C, D). Bar plots represent main effects and line plots represent interactions. Mounding and tailings removal significantly increased EM fungal colonization without a soil transfer interaction (C). Bars/points are the back-transformed means ± 1 SE from binomial generalized linear mixed models (A, C) and linear mixed models (B, D). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 4.3.2.3 Rooting zone soil fungal communities High-throughput sequencing of fungi in spruce seedling rooting zone soil after three growing seasons generated 1,198,483 sequences assigned to 4,819 ASVs, of which 3,233 were identified to genus. In the till zone, dominant rooting zone soil fungal genera (each > 2% of reads, 51% of reads combined) were, in descending order, Amphinema, Thelephora, Inocybe, Tetracladium, Wilcoxina, Oidiodendron, Hygrophorus, Paraphoma, Trichophaea, Plenodomus and Venturia 78 (Figure 4-6). In the tailings zone, the dominant fungal genera (> 2% of reads each, 38% of reads combined) were, in descending order, Oidiodendron, Wilcoxina, Amphinema, Tetracladium, Trichophaea, Thelephora, Pseudogymnoascus, Cladosporium, Vishniacozyma and Mortierella (Figure 4-6). Consistent with the dominant genera being EM in both zones, 39.2% of reads were assigned to EM fungi, followed by saprotrophs (19.7%) and plant pathogens (10.7%). The dominant EM genera align with morphotyping results of Wilcoxina, Amphinema, Thelephora and Inocybe (till only) being the most abundant taxa (Figure 4-6). While Wilcoxina, Amphinema, and Thelephora were all detected in baseline nursery seedling roots, these genera were also abundant (> 2% of reads) in reference forest soil (Figure 4-6). Despite similarities in dominant EM fungal genera, soil fungal communities in the reference forest were highly dissimilar from the tailings and till zones, and both zones were dissimilar from each other (Figure C-2). Forest soil tended to have greater EM fungal richness and relative abundance, lower plant pathogen richness and relative abundance, and lower saprotroph richness (Table C-9). After three growing seasons, site preparation, soil transfers and belowground forest connectivity had lasting effects on spruce seedling rooting zone soil fungal community composition in both the till and tailings zones (PERMANOVA P = 0.001; Table C-10). In the till zone, soil transfers increased soil fungal ASV richness compared with no transfers (Table C-11). At the guild level, this translated to soil transfers increasing EM fungal ASV richness and marginally decreasing saprotroph relative abundance with no significant effects on plant pathogen ASV richness or relative abundance (Figure 4-9; Table C-11). While no mounding or forest belowground connectivity main effects occurred, the two treatment factors consistently interacted (Table C-11). At sites with forest belowground connectivity, mounding decreased total and EM fungal ASV richness, tended to decrease plant pathogen and saprotroph richness, and 79 marginally decreased EM fungal relative abundance while plant pathogen relative abundance increased (Figure 4-9; Table C-11). Forest belowground connectivity increased total fungal and saprotroph ASV richness only in treatments with no mounding (Table C-11). No interactions between distance from forest edge and forest belowground connectivity occurred, but across connected and unconnected treatments, plant pathogen abundance was greater closer to forest edge (Table C-12). In the tailings zone, site preparation (with no difference between mounding and tailings removal) increased spruce seedling rooting zone soil fungal ASV richness and Shannon diversity and ASV richness of all guilds assessed when no soil transfers were applied (Table C-13). In treatments without site preparation (i.e., undisturbed tailing) and in treatments without forest belowground connectivity, soil transfers increased spruce seedling rooting zone soil fungal ASV richness and Shannon diversity, and ASV richness of all guilds assessed (Figure 4-9; Table C-13). Soil transfers also increased plant pathogen relative abundance, while decreasing saprotroph relative abundance in undisturbed tailings (Table C-13). Distance from forest edge x belowground forest connectivity interactions showed greater EM fungal richness (T93 = 2.12, P = 0.037) and relative abundance (T74 = 2.042, P = 0.045) with connectivity than without at 1m, but no significant differences at 3, 8 or 15 m. Saprotroph relative abundance was lower at 1 m with forest belowground connectivity (T74 = -2.42, P = 0.018). Across connected and unconnected treatments, fungal ASV richness was greater at 1 m and 3 m than 15 m (Table C-12). Accordingly, EM and saprotroph fungal richness tended to be higher at 1 m and 3 m, respectively, compared to at 15 m (Table C-12). Spruce seedling rooting zone soil fungal community composition significantly varied with soil physiochemical properties (pH, organic matter, moisture, and electrical conductivity), 80 distance from forest edge and naturally ingressing plant community composition in both zones (plant community was only marginally significant in till; Table C-14). Variation partitioning of fungal community composition among treatment variables (site preparation, soil transfers and forest belowground connectivity combined), soil physiochemical properties, naturally ingressing plant community (PCA scores) and distance from forest edge showed that in the till zone, while overall variation explained was low (18%), treatment, distance and soil properties but not plant community had significant conditional effects (Table C-14). In the tailings zone, fungal community variation explained by the model was low (16%), but all four components had a significant conditional effect (Table C-14). 81 Figure 4-9 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity (and their interactions) on ectomycorrhizal (EM), plant pathogen and saprotroph fungal amplicon sequence variant (ASV) richness in rooting zone soil of spruce seedlings in the till zone (A, B) and tailings zone (C, D). Bar plots represent main effects and line plots represent interactions. Soil transfers significantly increased EM and saprotroph fungal richness in the tailings zone with no connectivity interaction (D). Bars/points are back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 82 4.3.2.4 Rooting zone soil bacterial communities High-throughput sequencing of bacteria in spruce seedling rooting zone soil after three growing seasons generated 1,584,684 sequences that were assigned to 12,695 ASVs. The most abundant phyla (> 1.5% of reads) across both zones were, in descending order of abundance, Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, Verrucomicrobia, Planctomycetes, Gemmatimonadetes, Firmicutes, Bacteroidetes and Rokubacteria (Figure C-3). In the till zone, mounding, soil transfers, and mounding x forest belowground connectivity were significant terms in explaining bacterial community composition (Figure 4-10; Table C-15). This corresponded to soil transfers tending to increase ASV Shannon diversity (Table C-16), while increasing relative abundances of Proteobacteria and Gemmatimonadetes (marginally significant) and decreasing Chloroflexi relative abundance (Figure C-4). Aligning with the significant mounding x connectivity interaction, mounding decreased Acidobacteria (T82 = -3.75, P = 0.003) and increased Bacteroidetes (T82 = 3.40, P = 0.001) relative abundances in treatments with connectivity (Figure C-4). In treatments with no mounding, connectivity tended to increase ASV Shannon diversity (Table C-15) and increased Acidobacteria relative abundance (T82 = 1.83, P = 0.013), while tending to decrease Actinobacteria (T82 = -1.88, P = 0.064) and Bacteroidetes (T82 = -1.74, P = 0.086) relative abundances. In the tailings zone, site preparation and soil transfer main effects, plus site preparation x transfer and transfer x forest belowground connectivity interactions were significant terms explaining bacterial community composition (Figure 4-10; Table C-15). The db-RDA visualization clearly showed separation of site preparation treatments (Figure 4-10). This aligned with site preparation (mounding and tailings removal) increasing bacterial ASV Shannon diversity, with greater increases in tailings removal than mounding treatments (Table C-16). Site 83 preparation increased relative abundances of Acidobacteria, Rokubacteria, Verrucomicrobia and decreased relative abundances of Bacteroidetes and Chloroflexi (Figure C-4). Relative abundances of Firmicutes and Planctomycetes were higher and lower, respectively, in the mounding treatment compared to no site preparation and tailings removal (Figure C-4). The significant PERMANOVA site preparation x soil transfer interaction and separation of the soil transfer treatments within the undisturbed tailings treatment in the db-RDA visualization (Figure 4-10) align with soil transfers increasing bacterial ASV richness and Shannon diversity only in undisturbed tailings (Table C-16). Transfers decreased Firmicutes relative abundance (T81 = -2.13, P = 0.036) and increased Planctomycetes relative abundance (T81 = 2.93, P = 0.085) only in undisturbed tailings. Similarly, site preparation increased bacterial ASV richness and Shannon diversity (and shifted Acidobacteria and Bacteriodete relative abundances) but only when no soil transfers occurred (Table C-16). Soil transfers also interacted with forest belowground connectivity, increasing Actinobacteria relative abundance (T81 = 2.37, P = 0.021) and decreasing Acidobacteria relative abundance (T81 = -2.53, P = 0.013) only in treatments without connectivity. Forest belowground connectivity decreased Acidobacteria relative abundance (Figure C-4). The bacterial community composition of spruce seedling rooting zone soil was significantly related to soil physiochemical properties (pH, organic matter, moisture, and electrical conductivity), naturally ingressing plant community composition and distance from forest edge in both zones (plant community was only marginally significant in till; Table C-14). Variation partitioning of bacterial community composition among treatment variables (site preparation, soil transfers and forest belowground connectivity combined), significant soil physiochemical properties, the ingressing plant community (PCA scores) and distance from 84 forest edge showed that, in the till zone, model variables accounted for 26% of community variability and all four components had significant conditional effects (Table C-14). In the tailings zone, the model accounted for 23% of bacterial community variability and while treatment, soil properties and plant community had significant conditional effects, the distance conditional effect was only marginally significant (Table C-14). Despite conditional distance effects, no interactions between distance from forest edge and forest belowground connectivity occurred and but minor shifts in phyla relative abundances with distance from forest edge occurred (lower Firmicutes closer to forest edge in the till zone and higher acidobacteria at 15 m in the tailings zone; Table C-12). Figure 4-10 db-RDA of soil transfer and site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone) effects on Bray Curtis distances of Hellinger-transformed bacterial amplicon sequence variant (ASV) communities in the till zone (A) and tailings zone (B). Full models included forest belowground connectivity and treatment interaction terms, and a distance from forest edge covariate (Table C-15). PERMANOVA analysis found significant effects of site preparation (till: P = 0.001; tailings: P = 0.002) and soil transfers (till: P = 0.002; tailings: P = 0.031). 85 4.3.2.5 Rooting zone soil faunal communities Thirteen broad groups of soil fauna were observed in spruce seedling rooting zone soil including spiders (Aranaea; 36%), beetles (Coleoptera; 18%), mites (Acari; 8%), true bugs (Hemiptera; 7%), termites (Blattodea; 6%), fleas (Siphonaptera; 4%) and worms (Haplotaxid; 3%; Figure C-5). Broad group richness and total faunal abundance were low, with a maximum of eight individuals and three broad groups per sample, including in the reference forest samples (samples = the 2000 mL monolith macrofauna sample and 230 mL microarthropod sample data combined). Mean abundances (± 1 SE) by zone were 3.9 ± 0.4 in the till zone, 6.4 ± 0.5 in the tailings zone and 5 ± 1.1 in the reference forest soil. In the till zone, an interaction between mounding and forest belowground connectivity was the only significant treatment factor explaining soil faunal community composition (PERMANOVA R2 = 0.03, P = 0.036). This corresponded to forest belowground connectivity decreasing faunal richness and abundance only in treatments with mounding (Figure 4-11), and mounding decreasing faunal abundance and tending to decrease Shannon diversity only in treatments with connectivity (Figure 4-11). Soil transfers increased faunal broad group richness in treatments without mounding, but decreased faunal broad group richness, abundance, and Shannon diversity in treatments with mounding, (Figure 4-11). Mounding decreased faunal abundance (Figure 4-11) in treatments with soil transfers. In the tailings zone, site preparation was the only significant treatment factor explaining soil faunal community composition (PERMANOVA R2 = 0.06, P = 0.001). This aligned with site preparation (mounding and tailings removal) increasing faunal richness, abundance and diversity, with tailings removal increasing abundance more than mounding (Figure 4-11). Soil 86 transfers increased faunal abundance in the undisturbed tailings (no site preparation) treatment (Z = 2.02, P = 0.043). Faunal community composition was not related to distance from forest edge in either zone (Table C-17) and no interactions between distance from forest edge and forest belowground connectivity occurred. Yet, both zones had lower faunal abundance at 15 m compared to 1 m, 3 m, and 8 m (Table C-12). Faunal community composition was not significantly related to the naturally ingressing plant community in the till zone but was in the tailings zone (Table C-17). While faunal community composition was also not related to soil physiochemical properties (pH, specific conductance, moisture and organic matter) in the till zone, pH was a significant term in the tailings zone (F4 = 1.84, P = 0.045). Variation partitioning between soil pH, ingressing plant community (PCA scores) and treatment effects (site preparation, soil transfers and forest belowground connectivity combined) in the tailings zone showed a conditional effect of plant community, but no conditional soil or treatment effects (Figure C-6; Table C-17). Unlike community composition, richness and abundance were significantly related to soil physiochemical parameters; abundance and richness increased with organic matter content and soil moisture in both zones and decreased with pH and electrical conductivity in the tailings zone (Table 4-3). 87 Table 4-3 Generalized linear models (Poisson, link = logit) of soil faunal abundance and linear models of soil faunal broad group richness in rooting zone soil of harvested spruce seedlings against soil physiochemical parameters across all treatments (site preparation, soil transfer and forest belowground connectivity). Outliers above the 75th percentile or below the 25th percentile by greater than 1.5 x the interquartile range were removed prior to regressions. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Soil parameter Estimate SE Z P Relation Df SS Mean Sq F P Adj. R2RelationpH -0.0467 0.044 -1.07 0.284 NA 1 1.25 1.25 0.53 0.470 0.00 NAElectrical conductivity (µS/cm) -0.0003 0.001 -0.22 0.829 NA 1 0.17 0.17 0.07 0.789 -0.01 NAOrganic matter (%) 0.1815 0.054 3.38 < 0.001 + 1 15.62 15.62 6.83 0.010 0.06 +Gravimetric water content (%) 0.0381 0.014 2.71 0.007 + 1 6.83 6.83 2.92 0.091 0.02 +Tailings zonepH -0.3679 0.059 -6.23 < 0.001 - 1 29.82 29.82 9.55 0.003 0.07 -Electrical conductivity (µS/cm) -0.0024 0.001 -3.87 < 0.001 - 1 15.61 15.61 4.91 0.029 0.04 -Organic matter (%) 0.0465 0.007 6.72 < 0.001 + 1 45.03 45.03 14.82 < 0.001 0.12 +Gravimetric water content (%) 0.0318 0.004 7.19 < 0.001 + 1 70.83 70.83 26.06 < 0.001 0.20 +Soil faunal abundance Soil faunal richnessTill zone88 Figure 4-11 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity on soil faunal abundance (A, D), broad group richness (B, E) and broad group Shannon diversity (C, F) in the top 6 cm of rooting zone soil of spruce seedlings in the till zone (A–C) and tailings zone (D–F; site preparation effects only). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from binomial generalized linear mixed models (abundance) and linear mixed models (diversity and richness). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). 4.3.3 Plant ingress Tailings zone treatments with tailings removal and mounding had greater plant species richness and cover than the till zone including total, tree, shrub and herb percent cover (Figure 4-12; Table 4-4). The till zone had lower vegetative cover than undisturbed tailings (no site preparation), but similar species richness (Table 4-4). The plant communities were highly 89 dissimilar between till and tailings and from the reference forest (Figure C-7). Overall the non-native plant cover across both zones was low (on average ≤ 15%), but higher than in the reference forest where almost no non-native plants were present (0.1% cover; Table 4-4). In the till zone, mounding and an interaction between mounding and forest belowground connectivity were significant parameters in explaining naturally ingressing plant community composition (Table C-18). Aligning with this interaction, mounding decreased total plant cover (Figure C-8) and non-planted tree seedling cover (T60 = -2.34, P = 0.023) only in treatments with connectivity. Mounding decreased native plant cover while forest belowground connectivity increased native plant cover (Figure C-8). Distance from forest edge did not affect till zone plant community composition (Table C-19) and no interactions between distance from forest edge and forest belowground connectivity occurred. Plant community composition was significantly related to baseline soil properties (Table C-19). Variation partitioning between treatment effects (mounding, soil transfers and connectivity combined) and soil effects found a significant conditional effect of soil properties and a marginally significant conditional treatment effect (Figure C-9; Table C-19), with the model explaining 24% of plant community composition variation. In the tailings zone, site preparation and soil transfers significantly and marginally significantly explained plant community composition variation, respectively (Table C-18). This aligned with tailings removal and mounding increasing in plant species richness, Shannon diversity, total cover, tree seedling cover (tailings removal only) and shrub cover (P ≤ 0.006; Figure 4-12; Figure C-8). Significant interactions found soil transfers decreased plant community Shannon diversity (Figure C-8), species richness (T97 = -2.49, P = 0.015), and shrub percent 90 cover (T78 = -2.05, P = 0.044) only in treatments with connectivity. No treatment variables affected the relative abundance of native species. Tailings zone plant community composition was significantly structured by baseline soil parameters and distance from forest edge (Table C-19). Consistent with a distance effect, species richness was greater at 1–5 m from forest edge than 6–10 m (T97 = 3.4, P = 0.003) and 11–15 m (T97 = 5.30, P < 0.001), and vegetative cover and Shannon diversity were greater at 1–5 m than 11–15 m (cover: T78 = 2.24, P = 0.071; diversity T97 = 4.29, P < 0.001). Distance from forest edge x connectivity interactions showed that plant species richness and Shannon diversity were higher at 1–5m from forest edge with connectivity than without (richness: T95.8 = 2.32, P = 0.23; diversity; T95.8 = 1.97, P = 0.058). Variation partitioning among effects of treatment (site preparation, soil transfers and connectivity combined), distance from forest edge and soil properties found soil properties and distance had significant conditional effects, while the experimental treatments did not (Figure C-9; Table C-19). The model explained 22% of plant community variation (Figure C-9). Table 4-4 Plant richness and cover (mean ± 1 SE) in the till zone, tailings zone and reference forest. The tailings zone is separated by no site preparation (undisturbed tailing) vs. site preparation (mounding and tailings removal) because site preparation had a major effect on the plant community. ZoneForest 23.8 ± 1.5 84.9 ± 3.1 52.9 ± 4.8 14.5 ± 2.4 19.1 ± 3.3 0.1 ± 0.1Till 8.5 ± 0.5 26.9 ± 2.0 5.7 ± 0.4 3.7 ± 0.8 18.5 ± 1.6 15.2 ± 1.5Tailings (no site prep.) 7.9 ± 1.1 39.4 ± 7.6 2.5 ± 1.1 2.8 ± 0.7 37.1 ± 7.4 13.3 ± 4.4Tailings (site prep.) 12.1 ± 0.4 63.8 ± 2.3 16.7 ± 1.7 8.3 ± 0.6 46.1 ± 2.7 10.6 ± 1.5Non-native (%)Richness Total cover (%) Tree cover (%) Shrub cover (%) Herb cover (%)91 Figure 4-12 Representative examples of plant diversity and percent cover in the tailings zone (A, B) and till zone (C, D) with no site preparation (A, C) and mounding (B, C). All plots are at the 1–5m from forest edge. Photo credit: Katie McMahen. 4.3.4 Soil physiochemical properties 4.3.4.1 Baseline Analysis of baseline soil results is presented in Appendix C.1. Key findings are that mounding tended to reduce bulk density across both zones (P = 0.095; Figure C-10). In the tailings zone, tailings removal also reduced bulk density (P = 0.04) and there was a non-significant trend towards lower bulk density with tailings removal than mounding (Figure C-10). Baseline soil chemistry varied significantly with distance from forest edge in both zones (till: P = 0.031; 92 tailings: P = 0.031), supporting the inclusion of distance as a covariate in statistical analysis of treatment effects. In the till zone, pH was higher with mounding (P = 0.004), forest belowground connectivity (P = 0.033) and increasing distance from forest edge (P ≤ 0.005). Higher pH corresponded to higher electrical conductivity, Ca, K, Mg Na and effective CEC, but lower Al, Fe, Mn, total N and available P (Figure C-11). Total soil C did not change with distance from forest edge but mounding decreased total C in treatments with connectivity (P = 0.002). Similarly, treatments with connectivity tended to have lower total C with mounding (P = 0.079), but higher total C in treatments without mounding (P = 0.075). In the tailings zone, pH was not affected by distance from forest edge but was lower in mounding and tailings removal treatments than undisturbed tailings (P < 0.001), with lower pH following tailings removal than mounding (P < 0.001). All other soil parameters were significantly negatively correlated with pH (P < 0.001; Figure C-11) except electrical conductivity, which was not affected by site preparation but was higher at 11–15 m from forest edge compared to 1–5 m (P = 0.004). Forest soil used for transfers generally had lower pH and higher total N, total C, available P and effective CEC compared to the treatment zones (Table C-21). 4.3.4.2 Spruce seedling rooting zone soil after three years Analysis of spruce seedling rooting zone soil after three growing seasons in the till zone found that mounding increased soil moisture (gravimetric water content) and, in treatments with belowground connectivity, increased pH and decreased organic matter (Figure C-12). Treatments with connectivity had higher organic matter levels when no mounding occurred but lower 93 organic matter with mounding (Figure C-12). Forest belowground connectivity treatments also had higher pH (T107 = 2.68, P = 0.042) and electrical conductivity (T107 = 2.77, P = 0.025) with no interactions. Soil transfer treatments tended to have lower pH than where no soil transfer occurred (T107 = -1.94, P = 0.055). In the tailings zone, site preparation (tailings removal and mounding) decreased pH and increased soil organic matter content and moisture, with tailings removal resulting in higher organic matter and moisture levels compared to mounding (Figure C-12). No main effects of soil transfers or forest belowground connectivity occurred, but significant interactions showed that soil transfers decreased organic matter (T117 = -3.56, P < 0.001) and moisture (T117 = -2.64, P = 0.009) in the tailings removal site preparation treatment. Soil transfers increased pH (T117 = 2.83, P =0.005) and decreased organic matter (T117 = -4.46, P < 0.001) and moisture (T117 = -2.83, P = 0.006) but only in treatments with forest belowground connectivity. Similarly, treatments with forest belowground connectivity increased organic matter content when no transfers were applied (T117 = 2.85, P =0.005), but decreased organic matter content when transfers were applied (T117 = -2.22, P =0.023). 4.4 Discussion After three growing seasons, the ecosystem legacy-based reclamation treatments had significant lasting effects on plant and soil biological communities as summarized in Table 4-5. Where the experimental treatments improved spruce seedling establishment, EM fungal community diversity and/or abundance on their roots and in the rooting soils also increased, and the relative abundance of bacterial phyla increased in similarity with the reference forest (Figure C-3) and more advanced successional soils (based on post-glacial and sand dune succession studies; 94 Haynes, 2014), indicating positive feedbacks between the soil and plant communities. Spruce rooting zone soil fungal and bacterial communities were influenced by soil physiochemical properties, consistent with the research showing the importance of soil properties, particularly pH, in structuring soil microbial communities (Uroz et al., 2016). Yet, the experimental treatments and distance from forest edge also had significant conditional effects on spruce rooting zone soil microbial communities beyond variation accounted for by soil physiochemical properties, indicating that that the treatments had a lasting biological effect. In contrast to microbes, experimental treatments had no conditional effect on soil faunal community composition beyond the variation accounted for by soil physiochemical properties and naturally ingressing plant community composition, although soil transfers did increase abundance and/or diversity in the lowest quality soils. Faunal richness and abundance were positively related to soil organic matter and moisture in both zones and negatively related to soil pH and electrical conductivity ⁠—properties associated with tailings ⁠—in the tailings zone. This suggests that soil physiochemical properties inhibited fauna recovery more than dispersal limitations and reclamation treatments that promote soil organic matter accumulation are necessary for faunal community recovery (Kielhorn et al., 1999). Naturally ingressing plant communities were primarily structured by soil properties (both zones) and distance from the forest edge (tailings zone). This is expected given that viable plant propagules would have been scarce in treatment areas and thus not affected by site preparation, aerial seed dispersal would not be affected by the trenching (belowground connectivity) treatment, and soil transfers were only applied in a localized manner. While benefits of microbial inoculations on seedlings have been shown to spread to neighbouring plants 2 m away within a couple of years (Middleton & Bever, 2012), my findings indicate that under the conditions at 95 Mount Polley Mine, larger soil transfer volumes, alternative soil application methods or longer passage of time may be necessary for applied nucleation to occur (Santala et al., 2016; Wubs et al., 2016). 96 Table 4-5 Field experiment results for site preparation, soil transfer and forest connectivity effects on plant establishment, soil food web recovery and soil physiochemical properties. Only main effects are presented (interactions are discussed in-text). Site preparation refers to mounding in the till zone, and mounding and tailings removal in the tailings zone. Increasing bacterial succession is based on comparison of phyla relative abundances with forest soil and successional chronosequences. Colours indicate positive (dark green), weakly positive (light green), neutral (white) or negative (orange) effects. NA = hypothesis not tested or not applicable. N = total nitrogen, P = available phosphorus, CEC = effective cation exchange capacity; OM = organic matter; EM = ectomycorrhizal. Factor Zone Spruce seedlingsSpruce root EM fungiSpruce rooting zone soil fungiSpruce rooting zone soil bacteria Soil fauna Plant ingress Soil propertiesSite preparation Till No effect ↑ diversity Community shift Community shift No effect ↑ non-native plants ↓ bulk density, N, P↑ pH, CECTailings ↑ establishment ↑ diversity↑ colonizationNo effect ↑ bacteria succession Community shift↑ richness↑ abundance↑ diversity ↑ abundance↓ bulk density, pH↑ OM, nutrientsSoil transfers Till ↑ nutrition and physiology↑ diversity↑ colonization ↑ diversityCommunity shift↑ diversityCommunity shiftNo effect No effect ↓ pH, ECTailings ↑ nutrition and physiology↑ diversity No effect Community shift No effect No effect No effectForest belowground connectivityTill ↓ establishment* but not close to treeline No effect No effect No effect No effect ↓ non-native plants No effectTailings ↓ establishment↓ biomass close to treeline↑ colonization close to treelineNo effect ↑ succession No effect ↑ diversity close to treelineNo effectCloser to forest Till NA NA Community shift†↑ plant pathogen relative abundanceCommunity shift†↑ successionNo community shift† ↑ abundanceNo effect NATailings NA NA Community shift†↑ total, EM and saprotroph richness Community shift†↑ successionNo community shift† ↑ abundanceCommunity shift†↑ diversity NA97 4.4.1 Site preparation Resurfacing of buried forest soil in the tailings zone either through tailings removal or mounding decreased bulk density and increased soil organic matter, nutrients and CEC. As hypothesized, this had positive effects on the soil food web including increased faunal richness and abundance, increased richness of all fungal guilds, and greater bacterial diversity. Tailings removal and mounding also shifted bacterial phyla relative abundances: Acidobacteria and Verrucomicrobia increased, while Bacteroidetes and Chloroflexi decreased. This aligns with changes in bacterial community composition documented to occur with advancing succession and pedogenesis (Haynes, 2014) and is likely reflective of pH declining during succession (or, in this study, with site preparation; Williams et al. 2013). Site preparation effects on the soil food web corresponded with improved spruce seedling survival, growth and foliar N, greater spruce root EM fungal colonization and richness, and greater plant cover and diversity. An interaction showed soil fungal and bacterial community improvements primarily happened when soil transfers weren’t applied, indicating comparable benefits of soil transfers for the microbial community, but additive site preparation and transfer benefits for soil fauna and seedling establishment (including root EM fungal colonization and diversity). Recovery of soil faunal communities with tailings removal and mounding suggests that fauna may have survived the 1.5-year burial period, which would also explain the lack if dispersal (i.e., distance from forest edge) effects. However, it is likely that only certain taxa would have survived, given topsoil storage studies that have found negative effects of burial on earthworms (Abdul-Kareem & McRae, 1984). Tailings removal and mounding treatments yielded similar results for all ecosystem attributes assessed except there was greater faunal abundance, marginally significantly reduced spruce seedling chlorosis and greater bacterial ASV diversity (as well as higher Planctomycetes 98 and lower Firmicutes relative abundance, respectively) where tailings were removed, probably due to greater soil organic matter content (i.e., forest soil was not diluted by tailings). Given these limited differences, leaving tailings in place and mounding appears to be a reasonable treatment for areas of tailings overlying forest soil at Mount Polley. In addition, plant ingress and spruce seedling establishment in the tailings zone where tailings removal or mounding was applied were better than in the till zone. While additional challenges including tailings depth and hydraulic limitations are associated with reclamation of tailings within storage facilities, these findings show that with appropriate amendments and structural improvements (organic matter likely being of critical importance), mine tailings with neutral drainage (i.e., not acidic) can support plant community establishment and a recovering soil food web (Huang et al., 2012). The mounding treatment in the till zone reduced bulk density and increased soil moisture, which corresponded to decreased spruce seedling chlorosis (when no soil transfers were added) and increased spruce root EM fungal morphotype diversity, supporting my first hypothesis. This aligns with previous mine reclamation studies that found site preparation to improve seedling establishment and increase root EM fungal colonization and richness (Ashby, 1997; Bauman et al., 2013; Burger & Evans, 2010). Despite these benefits, a greater proportion of spruce seedlings were exposed to standing water in mounding treatments. Mounding created depressions that held water in the poorly draining, highly compacted glacial till (basal till in some locations). In two blocks, this was compounded by the shallow slope gradients. Seedlings were planted lower on the mounds (not on the hinge or top) because in temperate climates, moisture is typically a limiting factor for plants in reclamation materials that lack organic matter. Yet, this meant that seedlings were more exposed to the standing water. Further, baseline soil results of decreased total N and available P with mounding suggest mounding may have reduced soil nutrient 99 availability by exposing lower quality underlying soil. This suggests that there were unintended side-effects of mounding that served to counteract some of the soil physical improvements, and benefits could possibly be enhanced with adjusted seedling microsite selection and careful management of varying soil quality with depth (Schmidt et al., 1996; Sutton, 1993). Mounding also increased the proportion of non-native species likely as a result of the resilience of many weedy plants to physical land disturbance, highlighting the need for immediate seeding and planting following site preparation (Bauman et al., 2015). Mounding also consistently masked any positive effects of belowground connectivity to the surrounding forest in the till zone, causing lower spruce seedling performance, reduced plant cover and decreased abundance and diversity of beneficial soil organisms. Soil sampling showed a significant interaction where soil organic matter was lower in mounded treatments with (but not without) forest belowground connectivity at baseline and this difference persisted through three growing seasons. This was reflected in the bacteria communities, where Acidobacteria were less abundant and Bacteriodetes were more abundant, which is characteristic of disturbed or reconstructed soils, rather than forest soil (Masse et al. 2017). Given the overall absence of connectivity x distance from forest edge interactions that would be expected with spatial connectivity effects, this negative interaction is likely due to baseline soil quality differences. 4.4.2 Soil transfers In support of my second hypothesis, after three years, soil transfers had lasting positive effects on spruce seedling nutrition and physiology, with higher foliar N and δ13C in the till zone, and increased vigour and reduced incidence of chlorosis in the tailings zone. This aligned with increased EM fungal morphotype colonization and diversity in the till zone, and increased EM 100 fungal morphotype diversity and richness in the tailings zone. In the till zone, transfers also increased soil faunal broad group richness (when no mounding occurred) and increased bacterial ASV diversity. Soil transfer improvements to rooting zone soil conditions in the till zone were reflected by a decrease in the relative abundance of Chloroflexi: Anaerolineae was the dominant Chloroflexi class and been linked to compact, anaerobic soils (Masse et al., 2017). These results suggest that positive effects of soil transfers on balsam poplar (Populus balsamifera) in overburden found by Helm and Carling (1993a, 1993b) are transferable to other plant functional groups and mine tailings. These findings also indicate that soil transfers are suitable for nursery-grown seedlings, not just seedlings grown from seed (Chapter 3). There was a clear shift away from the EM fungal taxa colonizing the nursery spruce seedling roots at time of planting, although the main taxa colonizing spruce roots after three years (Amphinema spp., Wilcoxina rehmii and Thelephora terrestris) were dominant in all treatments as well as forest soil, making treatment-specific trends difficult to identify (aligning with other studies showing that these early successional fungi persist through late successional stages; Dickie et al., 2013). Soil transfers had no lasting effect on rooting zone soil chemistry in the tailings zone, which indicates their effectiveness can be attributed mainly to their biological rather than physiochemical properties. This finding aligns with the rapid mineralization of organic matter amendments commonly observed in mine reclamation (Bendfeldt et al., 2001) and is consistent with greenhouse studies comparing sterilized vs. unsterilized topsoil additions (Emam, 2016; Saxerud & Funke, 1991). In the till zone, transfers increased rooting zone soil electrical conductivity and decreased pH, corresponding with the transferred forest soil having pH of approximately 5.5 vs. the till pH of 7. These chemical changes may have supported improved EM colonization by creating more amenable rhizosphere conditions (Dumbrell et al., 2010). This 101 indicates that select physiochemical benefits of the soil transfers may occur in the short-term and work in concert with effects of the biological inoculum (Lance et al., 2019). Increased EM fungal morphotype diversity and improved spruce seedling nutrition and physiology did not consistently translate into increased seedling growth. This may be because other soil factors continued to limit seedling establishment (Kardol et al., 2009). Other studies with this finding have also suggested that a short-term cost associated with increased EM fungal colonization may occur (Lance et al., 2019) and that whole soil transfers can increase the presence of antagonistic organisms as well as beneficial soil microbes (St-Denis et al., 2017). I saw some evidence of this with soil transfers increasing fungal pathogen ASV richness and relative abundance in undisturbed tailings, but effects on EM fungi were more pronounced and occurred across all treatment units. In the till zone, application of soil transfers created more water-receiving seedling microsites (small depressions) and the negative effects on seedling growth associated with increased incidence of standing water may have outweighed transfer benefits to seedling growth. Despite a lack of consistent growth benefits, improved seedling nutrition and physiology (Salifu et al., 2009) and increased EM fungal colonization have been shown to correspond with increased seedling resilience to environmental stressors (Ortega et al., 2004; Rasmann et al., 2017). Thus, positive growth effects may materialize over time. Soil transfers negatively interacted with site preparation in both zones. In the till zone, transfers decreased incidence of spruce seedling chlorosis and increased faunal richness and abundance in treatments without mounding but had negative effects on faunal communities and spruce seedling chlorosis and shoot volume growth in treatments with mounding. I expect this is because mounding and soil transfers increased the proportion of seedlings growing in water-receiving microsites (application of soil transfers in high bulk density till was observed to create 102 small depressions), which consequently increased incidence of standing water in the poorly draining till material. In the tailings zone, the positive effects of site preparation and resurfacing of buried forest soil (an ecosystem legacy) reduced benefits of soil transfers. Transfers increased spruce seedling growth in the mounding treatment (forest soil mixed with tailings), but not in the removal treatment. Transfers had the greatest benefit in undisturbed tailings where no site preparation occurred, increasing EM fungal colonization, bacterial ASV richness and richness of all fungal guilds, consistent with the low inoculum potential of tailings (Moynahan et al., 2002; Zong et al., 2015). Soil transfers decreased Firmicutes relative abundance in undisturbed tailings and increased Planctomycetes relative abundance, indicating a transition from oligotrophic to copiotroph bacteria, potentially due to greater carbon availability from root exudates or the forest soil itself (Masse et al. 2017).These soil biological community shifts in undisturbed tailings corresponded to improved spruce seedling nutrition and a trend of increased shoot biomass, but overall growth was poor, indicating that soil transfers improve growing conditions, but ideally tailings can be amended with greater soil volumes. These findings align with studies showing the reduced effectiveness of soil transfers during restoration of higher fertility agricultural and tropical soils (Pareliussen et al., 2006; St-Denis et al., 2017). Soil transfers interacted with forest belowground connectivity in the tailings zone, with transfers increasing spruce root EM fungal colonization and richness of all fungal guilds and only in treatments with no connectivity. Similarly, actinobacteria relative abundance increased and Acidobacteria decreased with transfers only in treatments without connectivity, shifting towards relative abundances more representative of forest soil, specifically the FH layers as compared to mineral soils (Modi et al., 2020). Given that connectivity increased the abundance of soil EM fungi and EM fungal morphotypes colonizing spruce seedlings adjacent to the forest 103 edge, this suggests that transfers have stronger effects on soil microbial community recolonization at sites without adjacent undisturbed forest. Soil transfers also decreased spruce seedling volume growth in treatments with forest belowground connectivity and increased growth in treatments without connectivity. These differential effects may be due to soil physiochemical conditions, because rooting zone soil organic matter was lower in soil transfer treatments with belowground connectivity than without connectivity. 4.4.3 Forest connectivity Contrary to my third hypothesis, forest belowground connectivity had limited effects, increasing the proportion of native plant cover in the till zone, but having no significant main effects on fungal, bacterial or faunal community composition in either zone. In the till zone, connectivity had negative effects on spruce seedling establishment (except where soil transfers ameliorated negative effects on vigour); however, this is likely associated with the increased proportion of seedlings being located in water-receiving microsites (subject to standing water during wet periods). This may be a legacy of the experimental design: all of the till zone blocks sloped away from the forest (although gradually in two blocks, ~1–2 °) and the trenches may have diverted runoff water from the disconnected blocks, reducing the risk of water accumulating in the poorly draining glacial till treatment areas, particularly where mounding impeded overland flow. Neutral spruce seedling survival and growth adjacent to the forest edge in treatments with connectivity, but lower survival and growth farther from the forest edge (6–15 m) may, however, indicate some forest edge connectivity benefit counteracting these negative side effects. Forest belowground connectivity in the till zone also interacted with mounding, with connectivity decreasing faunal richness and abundance, and shifting bacteria phyla relative 104 abundance only in treatments with mounding. However, this is likely due to lower baseline total soil carbon in the mounding treatment given the significant relationships of fauna and bacteria with soil organic matter and a lack of effects in connectivity x distance from forest edge models. In treatments without mounding, connectivity increased saprotroph fungal ASV richness and decreased spruce seedling foliar δ13C, which were likely due to the increased incidence of standing water in seedling microsites in this treatment during wet periods, as opposed to a connectivity effect. Yet, acidobacteria relative abundance increased, while Actinobacteria were less abundant, which aligns with increasing similarity to the undisturbed forest soil. In the tailings zone, stronger connectivity effects on spruce seedlings were observed, potentially due to a greater proportion of spruce trees in the adjacent forests (Table C-1) and the absence of negative treatment effects associated with standing water in seedling microsites during wet periods. Forest belowground connectivity decreased spruce seedling survival and biomass, suggesting increased root competition may have reduced seedling establishment, which is supported by results of greater diversity of naturally ingressing plants and reduced seedling volume growth adjacent to the forest edge (1–5 m) in treatments with connectivity. Conversely, EM fungal colonization was greater adjacent to connected forest edges compared to disconnected forest edges, potentially indicating an initial seedling cost due to EM formation (Lance et al., 2019). Increased EM fungal diversity and abundance at this scale are consistent with silviculture studies finding increased EM fungal colonization and richness 2–10 m from tree retention patches (Jones et al., 2008; Luoma et al., 2006). It is possible that greater effects on mycorrhizal fungal communities may have occur if more long-distance exploration type EM fungi were present. In this experiment, the dominant EM taxa present were contact (Wilcoxina and Tuber) and short-distance (Cenococcum and Inocybe) exploration types, with only Thelephora, 105 Pseudotomentella and Amphinema being medium-distance explorers (Agerer, 2001). No long-distance explorers were dominant. Given the mixed positive and negative effects of forest belowground connectivity and the large-scale disturbance of mining operations, other ecosystem legacy reclamation approaches are expected to provide greater benefit. In the tailings zone, forest belowground connectivity interacted with soil transfers, with connectivity increasing spruce root EM fungal colonization and plant community cover but decreasing spruce seedling growth only in treatments without soil transfers. These results are consistent with connectivity x distance from forest edge models showing greater plant diversity, higher soil EM fungal richness and relative abundance, and higher spruce root EM fungal colonization adjacent to forest edges (1–5 m and 1–8 m, respectively). These effects may have been limited to treatments without soil transfers because transfers also improved EM fungal community establishment. In support of soil organism dispersal occurring through aerial dispersal and mechanisms other than movement through soil (my fourth hypothesis), variation partitioning that controlled for treatment, plant community and soil physiochemical effects showed that short-scale (15 m) distance from forest edge had a significant conditional effect on fungal and bacterial communities in both zones. Bacterial communities closer to the forest edge were more reflective of the adjacent forest soil (lower Firmicutes and Acidobacteria relative abundances in the till zone and tailings zone, respectively) and EM and saprotroph richness were greater with increased proximity to the forest edge in the tailings zone. This supports research findings that stochastic processes can govern microbial colonization in restoration, with dispersal effects explaining a significant portion of variation in microbial community composition at spatial scales ranging from meters to hundreds of kilometers (Chen et al., 2020; Hanson et al., 2012). Distance 106 from forest edge had a significant conditional effect on plant community composition in the tailings zone (but not the till zone), aligning with greater plant species richness and diversity closer to the forest edge. These results are supported by a seed rain study showing reduced species richness and abundance of seed at 15 m from forest edge at the Mount Polley Mine (Dan Deseniuk, unpublished data). This suggests that undisturbed patches of forest may be important sources of, or refuges for, plant propagules in mine reclamation. Access to seed sources can influence plant colonization as much as soil properties (Alday et al., 2011) and a lack of seed sources can even arrest succession (McClanahan, 1986). The relative importance of dispersal over habitat filtering in early successional plant community assembly highlights the need to consider dispersal processes in reclamation and take actions to establish the desired plant communities from the start so that plant–soil feedbacks promote the desired successional trajectories (Fukami, 2015; Ulrich et al., 2016). In contrast to plant ingress, distance had no significant effect on soil faunal community composition (although abundance was lower at 15 m than 1 m). This may be due to the ability of many arthropods to aerially disperse (Wanner & Dunger, 2002), but short-distance dispersal limitations found in other studies (Ojala & Huhta, 2001; Rantalainen et al., 2006) suggest soil conditions were a greater limiting factor in this experiment, outweighing any dispersal effects. 4.5 Conclusion This experiment showed that ecosystem legacy-based reclamation methods can support soil food web recovery and plant community establishment in mine reclamation. Soil transfers improved EM fungal community development and spruce seedling nutrition and physiology across all site preparation and connectivity treatments in areas of till and tailings, with the greatest benefits in 107 lower quality soils. This technique is anticipated to be of use at topsoil-limited mines, at sites where topsoil biological communities have diminished during long-term storage (similar to the forest soil that was buried by tailings then resurfaced with site preparation), and at remote mine sites, where access to soil amendments is costly and challenging. Forest belowground connectivity improved EM fungal community establishment and naturally ingressing plant diversity in the tailings zone and increased the proportion of native plants while counteracting negative side effects of other treatments on seedling growth in the till zone. Yet, the spatial scale of effects (generally ≤ 8 m) likely limits the applicability of this practice for mining-scale disturbances. In addition, interaction effects showed that comparable benefits can be achieved at a broader spatial scale using soil transfers. Proximity to undisturbed forest had significant aerial dispersal benefits for plants and microbes. While further research on longer distance plant and microbe propagule dispersal is necessary, preservation of refuge sites has the potential to be more broadly applied in mine reclamation. Mounding improved soil physical properties and increased spruce root EM fungal diversity, although results from the till zone highlight that careful consideration of drainage pathways and planting microsites is necessary to maximize plant establishment benefits. In the tailings zone, site preparation (mounding and tailings removal) positively affected ecosystem recovery by resurfacing buried forest floor material, improving soil physicochemical conditions. The additive benefit of soil transfers and spatial legacies on microbial and plant community establishment in these treatments suggests that ecosystem legacy-based reclamation methods can be combined with techniques that improve soil physical conditions for enhanced reclamation outcomes (Querejeta et al., 1998). 108 Future research should explore the effectiveness of ecosystem legacy-based reclamation techniques on other plant species and in other ecosystems and to verify transferability of these findings to overburden-covered waste rock dumps and tailings contained in storage facilities. Long-term monitoring of this trial will be necessary to assess if treatments continue to provide lasting benefits to soil food web and vegetation recovery, and to investigate long-term community assembly implications. In particular, litter from treatments resulting in greater plant cover and diversity may support faster amelioration of soil physiochemical limitations, and greater spruce and EM fungal community establishment may have positive feedbacks on ingressing EM conifers (Lefrançois et al., 2010; Wulantuya et al., 2020). While the soil food web largely reflects the physiochemical environment (Harris, 2009), this experiment showed that ecosystem legacy-based mine reclamation approaches can enhance dispersal and establishment of plant and soil biological communities, which over the long-term is anticipated to have positive feedbacks on ecosystem recovery. 109 Chapter 5: Plant–soil feedbacks promote forest succession through plant species turnover and mycorrhizal fungal historical contingencies 5.1 Introduction Plants influence the physical, chemical and biological properties of the soil they grow in through root development, root exudate and litter inputs, and creation of habitats for soil organisms (Bardgett & Wardle, 2010; van der Putten et al., 2013). These soil property changes subsequently influence plant growth in a process referred to as “plant–soil feedbacks” (the term “feedback” used herein refers to plant–soil feedbacks; Bever et al., 1997). Intraspecific plant–soil feedbacks are typically negative⁠—likely due to density-dependent pathogen accumulation—which promotes plant community diversity and coexistence (Crawford et al., 2019). Evidence is accumulating that plant–soil feedbacks can support directional plant community shifts towards late successional species (Bauer et al., 2015). In early secondary succession, negative intraspecific feedbacks due to soil-borne pathogen accumulation can promote species turnover by breaking plant species dominance (Kardol et al., 2007; Klironomos, 2002; van der Putten et al., 1993). Early successional plants generally invest more in growth than defense, making them susceptible to pathogen–mediated feedbacks (Koziol & Bever, 2015; Lemmermeyer et al., 2015). Early successional species also typically have lower mycorrhizal dependence and more explorative root systems, traits that have been shown to correspond to stronger negative feedbacks (Cheeke et al., 2019; Cortois et al., 2016; Koziol & Bever, 2015). As these negative feedbacks reduce vitality and abundance of the early successional plants, niche space opens for the next successional stage plant species. 110 As succession proceeds, neutral feedbacks have been found in mid-successional plants and positive feedbacks in late successional plants, suggesting that positive plant–soil feedbacks can stabilize climax communities (Carbajo et al., 2011; Kardol et al., 2006). This corresponds to a shift in functional traits where later successional plants have slower growth but increased pathogen resistance and higher dependence on mycorrhizal fungi (Cheeke et al., 2019; Koziol & Bever, 2015). Positive feedbacks on late successional grassland species have been shown to be dependent on the presence of beneficial mycorrhizal fungi (Koziol & Bever, 2019). Similarly, empirical studies have shown the importance of mycorrhizal fungi in maintaining stability of climax tree species (Booth, 2004; Dickie et al., 2002; McGuire, 2007). Historical contingencies, “the effect[s] of the order and timing of past events on community assembly”, occur when new plant species colonize the niche space created through negative intraspecific plant–soil feedbacks of previous plants (Fukami, 2015). The plant community that subsequently develops is a function of abiotic conditions and stochastic community-level processes, such as dispersal, as well as interspecific plant–soil feedbacks associated with plant–microbe interactions (Dumbrell et al., 2010). The latter concept has been illustrated through research on EM and AM fungi. Plants typically only associate with one of these guilds (EM or AM; Brundrett & Tedersoo, 2018) and Horton et al. (1999) found EM Douglas-fir (Pseudotsuga menziesii) to establish only near EM manzanita (Arctostaphylos spp.) not AM chamise (Adenostoma fasciculatum). These findings are consistent with studies reporting overlap in the EM mycorrhizal fungal taxa colonizing understorey and overstorey vegetation (Kennedy et al., 2003) and of mid-seral and late seral tree species (Kennedy et al., 2012). Together, this research indicates that interspecific feedbacks are influenced by mycorrhizal relationships and plant species forming compatible mycorrhizae may be promoted during forest 111 succession. Yet, aside from these few examples, the majority of the research to date on plant–soil feedbacks has been in grasslands and non-forested ecosystems, which are dominated by AM plant species (Crawford et al., 2019). Plant–soil feedback effects can vary with plant functional group (Cortois et al., 2016) and limited research has been conducted on conifers and EM plants that dominate temperate forests and tend to display more positive feedbacks than AM plants (Bennett et al., 2017; Seiwa et al., 2020; Wulantuya et al., 2020). The corresponding mechanistic understanding of the relationship between mycorrhizal fungi and plant–soil feedbacks promoting forest succession requires further investigation (Twieg et al., 2007). The plant–soil feedback successional theory described above was mainly developed through research on secondary successional systems. To inform application of plant–soil feedback theory into restoration of highly disturbed sites, which are characterized by low nutrient levels and minimal soil biological activity, more research on primary successional systems is necessary (Castle et al., 2016; Kuťáková et al., 2020). Although early successional plant species are typically less dependent on mutualists (Koziol et al., 2018), colonization of N-fixing bacteria and EM fungi have been shown to be critical for plant establishment in the severely nutrient-limiting conditions of primary succession (Nara & Hogetsu, 2004; Seeds & Bishop, 2009). This indicates some level of reliance on microbial mutualisms under low nutrient conditions that may be compromised in reclamation materials lacking microbial activity (Bois et al., 2005). Thus, in contrast to the negative intraspecific feedbacks observed in early secondary succession, during early primary succession, the high importance of microbial symbionts may result in initial intraspecific plant–soil feedbacks being neutral to positive (Kuťáková et al., 2020; Semchenko et al., 2018). 112 In this study, I investigated if plant–soil feedbacks promote forest succession and compared feedback strength and direction (positive or negative) in forest soil and primary successional mine reclamation soils with or without forest soil additions. My three research objectives and hypotheses were as follows. Objective 1: to test if intraspecific plant–soil feedbacks promote plant species turnover in the early stages of forest succession. Consistent with the non-forested ecosystem literature, I hypothesized that an early successional shrub (willow) would have negative intraspecific feedbacks corresponding to root fungal pathogen accumulation, while a later seral stage conifer (spruce) would have neutral to positive intraspecific feedbacks associated with colonization by mycorrhizal fungi. Objective 2: to test if historical contingencies due to formation of plant species-specific mycorrhizal fungal communities contribute to plant–soil feedback responses of next successional stage species. I hypothesized that next successional stage species associating with compatible mycorrhizal fungal guilds would benefit, while lack of compatible mycorrhizal fungal guilds would correspond with neutral feedbacks (i.e., EM willow and spruce would have positive feedbacks on EM spruce but neutral feedbacks on AM redcedar). This is of particular interest in the study ecosystem at the Mount Polley Mine, where EM tree species dominate but one of the major climax community tree species (redcedar) forms AM. Objective 3: to investigate if plant–soil feedbacks are consistent in primary successional soils. I hypothesized that intraspecific feedbacks of an early successional plant species (willow) in primary successional soils would initially be neutral due to lower microbial abundance (i.e., fewer pathogens) and increased importance of any mutualists present. I predicted that the directions of interspecific feedbacks (willow-redcedar and spruce-redcedar) and late successional species intraspecific feedbacks (spruce-spruce) would be maintained but the strength would be decreased due to lower microbial abundance. I expected 113 that amending reclamation soils with forest soil would transition feedbacks towards the models described in the hypotheses for objectives one and two. To address test these hypotheses, I conducted a two-phase greenhouse experiment in which I conditioned soil with early and mid–late successional plants. The same plant species (intraspecific) and later stage successional plant species (interspecific), including plant species from the same or different mycorrhizal guild, were then grown as feedback-phase seedlings in the conditioned soil and unconditioned control soil. These treatments were tested in forest soil and primary successional mine reclamation substrates (overburden and tailings) with or without forest soil amendment. Root fungal communities, including mycorrhizal fungi and pathogens, were characterized through microscopy and high-throughput sequencing to link seedling growth and nutrition responses with soil microbial mechanisms. 5.2 Methods 5.2.1 Experimental design A two-phase greenhouse experiment was conducted with tailings, overburden and forest soil collected from the Mount Polley Mine (Section 2.3). Plant species of different successional stages were grown to condition the soil microbiome (conditioning phase) followed by a second growing period (feedback phase) in which conditioning phase soil was used to grow the same species (intraspecific) or later successional stage species (interspecific). I tested three factors in a completely randomized experimental design. (1) Soil type: four soil types were tested including forest soil and three reclamation “soils” (mine tailings, overburden and a 1:1 v/v mixture of mine tailings and overburden, herein referred to as “tailings +overburden”). (2) Forest soil addition: reclamation soils (but not forest soil) were mixed with 25% v/v forest soil vs. left unamended. 114 (3) Plant–soil feedback: eight combinations of conditioning phase–feedback phase plant species were tested on each soil treatment, amounting to a total of 56 treatments (Table 5-1). Specifically, soil conditioned by willow (Salix scouleriana), an early successional species, was used to grow willow, hybrid white spruce (Picea glauca x engelmannii; a climax species that is present through mid to late successional stages) and western redcedar (Thuja plicata; a late successional species; Section 2.2). Spruce-conditioned soil was used to grow spruce and redcedar. Willow, spruce and redcedar were also grown in unconditioned control soil during the feedback phase for comparison (Table 5-1). The successional transitions also represent changes in plant functional group and mycorrhizal guild (Table 5-1). 115 Table 5-1 Combinations of conditioning and feedback phase plant species tested in the two-phase plant–soil feedback experiment. AMF = arbuscular mycorrhizal fungi; EMF = ectomycorrhizal fungi. SpeciesSuccessional stageFunctionalgroupMycorrhizal fungalassociationSpeciesSuccessional stageFunctionalgroupMycorrhizal fungalassociationWillow Intraspecific Early Deciduous shrub EMF (AMF)Hybrid white spruce Interspecific Mid-late1EMFWestern redcedar Interspecific Late AMFHybrid white spruce Intraspecific Mid-late1EMFWestern redcedar Interspecific Late AMFWillow Intraspecific Early Deciduous shrub EMF (AMF)Hybrid white spruce Interspecific Mid-late1EMFWestern redcedar Interspecific Late AMF1. Climax species but is present through mid to late successional stagesWillow EarlyHybrid white spruceMid-late1Conditioning phaseEMF (AMF)EMFDeciduous shrubControl (soil stored at 4 °C)- - -Coniferous treeConiferous treeConiferous treeConiferous treeFeedback phase116 In the conditioning phase, seedlings were grown in 3 L pots in the greenhouse for six months, then harvested and the soil and seedlings retained. Because majority of the spruce seedlings did not grow enough to extend roots into the entire pot, only soil and roots from 6 cm diameter cores centered on the seedlings were retained. The roots of the conditioning phase seedling were cut into approximately 1 cm pieces and mixed back into their respective soils. Soil was not homogenized among replicates within treatments between phases; soil from each individual conditioning phase seedling formed an individual feedback phase treatment. However, due to poor survival in some conditioning phase treatments, soil from an individual conditioning phase seedling was subdivided into two or three feedback phase replicates to attain the desired sample size. Furthermore, due to substantially higher mortality of willow seedlings than spruce seedlings in the conditioning phase, fewer replicates of feedback phase seedlings could be grown in willow-conditioned soil (n = 6) than in spruce-conditioned and control soil (n = 7). In the feedback phase, willow, spruce and redcedar seedlings were grown in 250 mL Ray Leach cones containing conditioned soil. As a control, seedlings were also planted in untreated soil that was stored at 4 °C during the conditioning phase. This control soil approach was selected because it allowed investigation of changes in specific microbial functional groups, as opposed to the net effect of the full soil biological community, which many studies test by comparing seedlings grown in sterilized vs. unsterilized conditioning phase soil (Brinkman et al., 2010). Nutrient release from decomposing organisms caused by sterilization treatments was also avoided (Brinkman et al., 2010). By using only field soil in this study, I emulated conditions that were more representative of field conditions and better preserved the original density of microorganisms present within the soil. 117 Phospholipid fatty acid (PLFA) analysis, which provides an estimate of living biomass of broad microbial groups (Quideau et al., 2016), was conducted by Wards Lab, Kearney, Nebraska as per Hamel et al. (2006) to assess potential changes in the soil microbial community of the control soil stored at 4 °C during the conditioning phase. PLFAs were run on forest soil samples from each reference site (n = 3; Table A-1) following soil collection and after the storage period. Results indicated no inoculum load losses and are not discussed further (Appendix D.1). 5.2.2 Data collection For each feedback phase seedling, initial survival prior to thinning was documented. At harvest, height and root collar diameter were measured, then harvested shoots were dried for 72 h at 70 °C and weighed. Samples of the youngest foliage were analyzed for total (%) N and C using an Elemetar vario EL Cube elemental analyzer at the UBC Stable Isotope Facility, Vancouver, BC. Washed roots of spruce and redcedar were scanned for morphological analysis using WinRhizo software; willow roots could not be accurately scanned due to their dense, fibrous nature. Root tips/segments for mycorrhizal fungi analysis were collected immediately post-harvest and the remaining roots were dried for 72 h at 70 °C then weighed. Due to the small size of many redcedar root systems, a wet weight to dry weight ratio was used to account for root segments retained for microbial analysis. 5.2.3 Root fungal community assessment For spruce, 50 random root tips were collected from each root system (or all tips for systems with < 50 tips) and morphotyped as per Section 2.5 to assess EM fungal percent colonization and diversity. Morphotypes were identified through Sanger sequencing; where Sanger sequencing 118 failed or PCR products had more than one band in gel electrophoresis, high-throughput sequencing was conducted (Section 2.5, Section 2.6). For all but two vouchers analyzed with high-throughput sequencing, the ASV with the most reads corresponded to an EM fungal sequence (Table E-1). Results indicated that four vouchers contained mixed EM taxa (>1 dominant EM taxon; Table E-1), whereas the rest of the vouchers each had a single dominant EM fungal species present, but also had some saprophytic, parasitic and/or low abundance EM (0–4% of total reads) fungal DNA contaminating the voucher specimen. For willow and redcedar, 40 x 1 cm randomly selected root segments per sample were collected, rinsed with distilled water and frozen at -20 °C until analysis. 20 segments per sample were assessed for percent colonization by AM fungi, non-AM fungal endophytes and EM fungi (willow only) as described in Section 2.5. To identify and assess the diversity of root-associated fungi, DNA was extracted from the remaining 20 willow and redcedar root segments per sample using DNeasy Plant Pro kits (Qiagen, 2017), followed by high-throughput Illumina MiSeq sequencing at the Integrated Microbiome Resource at Dalhousie University, Halifax, Nova Scotia. Only the root samples from the 100% forest soil, tailings and overburden treatments underwent DNA characterization given that the forest soil addition treatments represented a mixture of the analyzed soils, and therefore likely contained the same fungal species. Bioinformatic processing of high-throughput sequencing results was carried out separately for redcedar and willow as described in Section 2.6. Non-fungal sequences and singletons were removed, then data were rarified to the lowest number of reads. 119 5.2.4 Statistical analyses All statistical analyses were conducted in R (version 3.6.1; R Core Team, 2019). Separate linear models for each feedback phase species (willow, spruce and redcedar) were constructed with soil treatment (soil type x forest soil addition treatments combined) x conditioning species as the explanatory variables with plant growth measures as the response variables. Response variables were natural log (loge) transformed and checked for normality and equal variance. Growth parameters were relativized per growing day to account for growth period differences due to replanting. Foliar C:N ratio was added as a covariate to account for potential soil nutrient depletion during the conditioning phase. Depth to soil from the top of the pot was also included as a covariate to account for variation in soil settling and potential effects on light availability. Model term significance was tested with Type III ANOVA and the overall effect of each conditioning phase treatment compared with pairwise post hoc contrasts using the emmeans function with the Tukey adjustment for multiple comparisons (Lenth, 2020). Loge response ratios and confidence intervals of emmeans outputs contrasting conditioned vs. control soil were constructed, allowing comparison across soil treatments. Secondary models were analyzed for reclamation soils only (i.e., excluding forest soil) with soil type x forest soil addition x conditioning species as the explanatory variables. Response ratios of these models combined plant–soil feedback effects across all reclamation soil types to assess overall effects of forest soil addition. Custom contrasts using the emmeans package (Lenth, 2020) were used to test for differences in feedback effects with and without forest soil. Percent colonization by root symbionts was assessed using binomial generalized linear models (link = logit) with the same explanatory variables as the linear models described above. Post hoc testing with the emmeans function was used to compare conditioning treatment effects 120 in each soil treatment. The relationships between feedback effects (shoot biomass and specific root length; shoot biomass and symbiont percent colonization; specific root length and symbiont percent colonization) were assessed with linear models of the feedback response ratios. For willow and redcedar high-throughput sequencing data, conditioning treatment effects on root fungal community composition for each soil type was visualized with non-metric multidimensional scaling (NMDS) of the Bray-Curtis distances of the fourth root-transformed data using the vegdist and metaMDS functions in the vegan package (Oksanen et al., 2019). Differences between conditioning treatments were assessed through PERMANOVA with the adonis function in vegan (999 permutations). FUNGuild assignments were filtered to remove ASVs with multiple functional guild assignments, i.e., fungi that have varying life histories (Nguyen et al., 2016). FUNGuild provides useful insight into potential shifts in microbial community structure; however, it is important to note that only 50% of willow ASVs and 42% of redcedar ASVs remained after filtering unassigned and multi-guild fungi. Linear models of functional guild ASV richness and quasibinomial (link = logit) generalized linear models of functional guild ASV relative abundance were constructed with soil type x conditioning species as the explanatory variables along with the covariates described above. Post hoc pairwise testing with emmeans was used to test effects of conditioning treatments. Indicator species analysis of spruce EM fungal morphotyping data and willow and redcedar high-throughput sequencing data was done using the multiplatt function of the indicspecies package (de Caceres & Legendre, 2009). FUNGuild assignments were used to support interpretations (multi-guild taxa were excluded from interpretations due to their unknown functional role in the system). For all analyses, significance was evaluated as P ≤ 0.05 and marginal significance as 0.05 < P < 0.1. 121 5.3 Results 5.3.1 Plant–soil feedback effects on seedling growth Willow had significant negative intraspecific feedbacks on shoot biomass (P < 0.001; Figure 5-1; Table E-2). No soil type x conditioning treatment (i.e., conditioning phase species) interaction occurred (P = 0.128; Table E-3), although negative feedback trends were not significant in all individual soil treatments (Figure 5-1). The lack of a soil type interaction is consistent with overall negative feedbacks occurring in reclamation soils (tailings, overburden and tailings + overburden) with and without forest soil addition, and no significant effect of forest soil addition (T58 = 1.47, P = 0.681; Figure 5-2; Table E-4). Willow-conditioned soil had a negative effect on willow germinant survival (Z = -2.55, P = 0.011). Spruce seedling shoot biomass showed negative feedbacks in willow-conditioned soil (P < 0.001) but neutral feedbacks in spruce-conditioned soil (P = 0.211; Figure 5-1; Table E-2; Table E-3). Negative willow-conditioned soil feedbacks and neutral spruce-conditioned soil feedbacks were consistent across reclamation soils with and without forest soil additions, with no significant differences in feedback strength (willow: T97 = -0.25, P = 1.000; spruce: T97 = -1.66, P = 0.939; Figure 5-2; Table E-4). There was, however, a trend of stronger negative feedbacks on spruce in willow-conditioned soils with forest soil additions than without forest soil (Figure 5-2). This trend aligned with willow-conditioned soil feedbacks being neutral in unamended tailings and barely negative in unamended overburden, whereas all the treatments with forest soil additions and 100% forest soil had significantly negative feedbacks (Figure 5-1). Conditioning treatment had no effect on spruce germinant survival (willow: Z = -0.97, P = 0.332; spruce: Z = 0.004, P = 0.997), although there was a tendency for willow-conditioning to decrease survival (from 88% to 76%). 122 Redcedar shoot biomass showed negative feedbacks in willow-conditioned soil (P < 0.001) and spruce-conditioned soil (P = 0.007), with willow-conditioned soil feedbacks being significantly more negative (P < 0.001; Figure 5-1; Table E-2). A marginally significant soil type x conditioning species interaction was present (P = 0.078; Table E-3) and corresponded with spruce-conditioned soil having neutral feedbacks on redcedar in unamended reclamation soils but negative feedbacks with forest soil addition (Figure 5-2; Table E-4). Stronger negative feedbacks in spruce-conditioned reclamation soils with forest soil addition compared to no forest soil addition were consistent with the negative feedback in 100% forest soil (Figure 5-1). Willow-conditioned soil had negative feedbacks on redcedar in reclamation soils with and without forest soil additions, with no significant effect of forest soil additions (T87 = -2.66, P = 0.345; Figure 5-2; Table E-4). Willow-conditioned soil had negative effect on redcedar germinant survival across all soil types (Z = -2.41, P = 0.042), while spruce-conditioned soil had no effect (Z = 0.005, P = 0.996). Height (Figure E-1), root collar diameter (Figure E-2) and root biomass (Figure E-3) results were consistent with shoot biomass results for all test species. Photos of representative seedlings are presented in Figure E-5. 123 Figure 5-1 Shoot biomass loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to control unconditioned soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 124 Figure 5-2 Shoot biomass loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned reclamation soils (tailings, overburden and tailings + overburden) compared to control unconditioned soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 5.3.2 Historical contingency effects on root fungal symbionts Willow EM fungal percent colonization was significantly lower in willow-conditioned forest soil and forest soil-amended reclamation soils (except overburden) compared to controls (Figure 5-3). EM fungal percent colonization was low in unamended reclamation soils and did not significantly vary with conditioning treatment except for tailings where it increased in willow-conditioned soil (Figure 5-3). There was no relationship between the willow-conditioning feedback effects on willow shoot biomass and EM fungal percent colonization (R2adj = -0.17, P = 0.743; Figure E-4). Percent colonization of willow by AM fungi was low (≤2%) and did not differ among treatments (Figure 5-3). Conditioning treatment effects on willow root percent colonization by non-AM fungal endophytes were inconsistent, ranging from negative to positive (Figure 5-3). Yet, a marginally significant negative relationship was present between feedback effects on shoot biomass and fungal endophyte percent colonization (R2adj = 0.48, P = 0.052; Figure E-4). 125 Analysis of willow root high-throughput sequencing results (forest soil, tailings and overburden treatments only) showed that willow conditioning significantly shifted root fungal community composition in forest soil (P = 0.001) and tended to change root fungal community composition in overburden (P = 0.093) and tailings (P = 0.074) compared to control soil (Figure E-6). Relative abundance of pathogens increased in willow-conditioned soils compared to controls (Z = 3.18, P = 0.002; Figure 5-4) with no soil type x conditioning species interaction. Willow-conditioning had no effect on pathogen species richness (T25 = 0.71, P = 0.487), although it tended to cause an increase in richness in forest soil and tailings (Figure E-7). Symbiont richness decreased in willow-conditioned soil compared with control soil (T25 = -2.47, P = 0.021) and a significant soil x conditioning species interaction occurred (P = 0.003) corresponding to greater decreases in forest soil (Figure 5-4). There was no effect of willow conditioning on symbiont relative abundance (Z = -0.10, P = 0.924), although relative abundances in tailings and overburden were low (≤ 3%; Figure E-7). Willow-conditioning decreased soil saprophyte richness overall (Z = -2.46, P = 0.014; Figure E-7) but the direction of effect varied with soil type and there was no effect on relative abundance (T25 = 0.71, P = 0.487; Figure E-7). Indicator species analysis showed the plant pathogen Plectosphaerella cucumerina was present in control tailings and overburden, and maintained in willow-conditioned overburden and tailings (Table E-5). Control forest soil was characterized by the pathogen Neonectria candida, while the pathogens Moesziomyces aphidis and Plectosphaerella cucumerina were indicative of willow-conditioned forest soil. The symbiotrophs Cadophora finlandica and Cadophora sp. were present in control and willow-conditioned forest soil but Tomentella sublilacina, Meliniomyces bicolor and Phialocephala sp. were indicative of control soil only (Table E-5). Saprotrophic taxa also tended to be indicative of willow-conditioned, but 126 not control soil (Tetracladium breve and Hyphopichia burtonii in willow-conditioned overburden; Tetracladium sp. in willow-conditioned forest soil; Table E-5). Spruce EM fungal percent colonization was significantly lower in willow-conditioned soils compared to controls for all soil types except tailings and tailings + overburden without forest soil additions, which both had low EM fungal colonization (Figure 5-3). In contrast, spruce seedlings in spruce-conditioned soil had equivalent or higher EM fungal percent colonization compared to controls (Figure 5-3). Spruce EM fungal percent colonization feedbacks had a positive relationship with shoot biomass feedbacks (R2adj = 0.50, P = 0.003; Figure E-4). Spruce EM fungal species richness (based on sequencing of morphotyping vouchers; Table E-1; Table E-6) had negative feedbacks in willow-conditioned soil (T112 = -2.64 , P = 0.026) and tended to have positive feedbacks in spruce-conditioned soil (T112 = 2.26 , P = 0.066), although a significant soil type x conditioning treatment interaction occurred, with feedbacks in spruce-conditioned soil ranging from neutral to positive (P < 0.001; Figure E-8). Spruce EM fungal species richness feedbacks had a positive relationship with shoot biomass feedbacks (R2adj = 0.33, P = 0.020; Figure E-4). EM fungal species richness feedbacks in willow-conditioned reclamation soils did not differ with forest soil additions, while feedbacks shifted from neutral to positive with forest soil addition in spruce-conditioned reclamation soils (Figure E-9). Indicator species analysis across all soil types showed Wilcoxina rehmii to be dominant in control soil and maintained in spruce- but not willow-conditioned soil. Thelephora terrestris was characteristic of spruce-conditioned soils (Table E-5). A Wilcoxina sp. was characteristic of only control soil, while a Trichophaea sp. was an indicator species for both conditioning treatments (Table E-5). 127 Redcedar AM fungal percent colonization was significantly lower in spruce- and willow-conditioned forest soil and forest soil-amended reclamation soils (except spruce-conditioned overburden amended with forest soil) compared to controls (Figure 5-3). AM fungal percent colonization was low in unamended reclamation soils (≤4%) and did not significantly differ with conditioning treatment (Figure 5-3). There was a positive but non-significant relationship between redcedar AM fungal percent colonization and shoot biomass feedbacks (R2adj = 0.03, P = 0.253; Figure E-4). Redcedar root colonization by non-AM fungal endophytes was significantly lower in all spruce-conditioned soil treatments except forest soil and forest soil-amended tailings + overburden (Figure 5-3). Effects of willow-conditioned soil on endophyte colonization were inconsistent, ranging from negative to positive (Figure 5-3). No significant relationship was present between feedback effects on shoot biomass and endophyte percent colonization (R2adj = -0.06, P = 0.594; Figure E-4). Analysis of redcedar root high-throughput sequencing results (forest soil, tailings and overburden treatments only) showed that the fungal community composition differed with conditioning treatment in forest soil (P = 0.023) and overburden (P = 0.001), but not tailings (P = 0.101; Figure E-6). Despite these shifts, there were no significant changes in relative abundance or species richness of fungal pathogens, symbionts or saprotrophs (Figure E-10). Indicator species analysis reflected this lack of clear shifts: symbiotrophs tended to be present in control and conditioned soil (Cadophora luteo-olivacea in control and spruce-conditioned tailings; Cadophora sp. in control, willow-conditioned and spruce-conditioned forest soil; Phalocephala spp. in control and willow-conditioned forest soil; Table E-5). The saprotroph Thelonectria rubrococca was indicative of all forest soil and overburden treatments except spruce-conditioned forest soil, which had two different saprophytic indicator species (Ilyonectria 128 liriodendra, Thielaviopsis basicola; Table E-5). The only pathogenic indicator species was Olpidium brassicae in control overburden and tailings; it was not indicative of any conditioned soils (Table E-5). 129 Figure 5-3 Percent colonization of ectomycorrhizal fungi (EMF; A, D), arbuscular mycorrhizal fungi (AMF; B, E), and non-AMF fungal endophytes (C, F) on willow (A, B, C), spruce (D) and redcedar (E, F) seedling roots grown in control, willow-conditioned and spruce-conditioned soils (forest, overburden, tailings and tailings + overburden). “+ forest soil” treatments were mixed with 25% v/v forest soil. Bars represent mean ± 1 SE. Significant differences between conditioning species in each soil treatment are indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05) or NS (not significant) in A–D, or by different letters in C–F (P < 0.05). 130 Figure 5-4 Willow root fungal pathogen amplicon sequence variant (ASV) abundance (A) and fungal symbiont ASV richness (B) in forest soil, overburden and tailings treatments without forest soil additions. Bars represent mean ± 1 SE. Pathogen abundance increased (Z = 3.18, P = 0.002) and symbiont richness decreased (T25 = -2.47, P = 0.021) in willow-conditioned soil compared to unconditioned control soil. 5.3.1 Plant–soil feedback effects on root morphology Conditioning species had no overall effect on spruce specific root length (willow: T112 = 0.99, P = 0.587; spruce: T112= -0.64, P = 0.801) or redcedar specific root length (willow: T112 = -1.28, P = 0.4082; spruce: T112= 0.95, P = 0.611). Yet, for both spruce and redcedar there was a trend of specific root length feedbacks being more positive (i.e., feedbacks causing roots to become thinner) in reclamation soils with forest soil additions compared to unamended reclamation soils (Figure E-11). Notably, redcedar specific root length feedbacks were neutral in unamended spruce-conditioned reclamation soils but positive in spruce-conditioned reclamation soils amended with forest soil. This aligns with the trend towards positive feedbacks in forest soil treatments (Figure E-11). Redcedar specific root length feedbacks were not, however, related to shoot biomass or AM fungal percent colonization feedbacks (Figure E-12). In spruce, specific 131 root length feedback tended to decrease (become less acquisitive) with increasing shoot biomass feedback (R2adj = 0.16, P = 0.090; Figure E-12) and decreased with increasing EM fungal colonization feedback (R2adj = 0.39, P = 0.010; Figure E-12). Specific root length data were not available for willow. 5.4 Discussion 5.4.1 Plant–soil feedbacks promote plant species turnover and persistence in forest succession Intraspecific feedbacks were negative in willow, an early successional shrub, and neutral in spruce, a later successional conifer, supporting my first hypothesis (Figure 5-5). Negative growth feedbacks in willow corresponded to an increase in root fungal pathogen relative abundance. The trend of more negative shoot biomass feedbacks with increased non-AM fungal endophyte colonization also suggests negative effects of fungal pathogens. In willow-conditioned soil, pathogenic indicator species present in control soil were maintained or increased, with the root rot pathogen Plectosphaerella cucumerina being dominant across soil types. This supports my prediction that pathogens would promote species turnover in early succession through negative feedbacks (Bever, 1994). While I found evidence of root fungal pathogens, other biota such as root-feeding nematodes may have also played a role as they have been associated with negative feedbacks in other studies (van der Putten et al., 1993; Wilschut et al., 2019). Bacteria and protists are unlikely to have caused the observed feedbacks, however, as their community composition is typically primarily structured by soil abiotic conditions (Semchenko et al., 2018). Negative intraspecific feedbacks in willow corresponded to decreased EM fungal colonization 132 (except where inoculum potential was already low) and loss of EM fungal indicator species, but no relationship between EM fungal colonization and shoot biomass feedbacks was present. The overall low mycorrhizal colonization rates (EM fungi: ≤17%; AM fungi: ≤2%) are consistent with willow being an explorative early successional plant with low mycorrhizal dependency (i.e., adapted to a low abundance of mutualistic microbes; Cheeke et al., 2019). Early successional species are also characterized by higher susceptibility to negative plant–soil feedbacks due to growth defense tradeoffs, supporting pathogen accumulation as the dominant successional process (Cortois et al., 2016; Koziol & Bever, 2015). In contrast to willow, spruce seedlings had equivalent or higher shoot biomass and EM fungal colonization and species richness in intraspecific-conditioned soil compared to controls. A positive relationship between spruce EM fungal colonization and shoot biomass feedbacks was present across all treatments, supporting my hypothesis that this later successional tree species would have higher mycorrhizal dependency and facilitation by mycorrhizal fungi would support persistence of spruce, not plant species turnover (Figure 5-5; Bauer et al., 2015; Kardol et al., 2006). Higher EM fungal colonization of spruce roots corresponded to lower specific root length, showing spruce morphology shifts with the soil microbiome, as has been documented with plant–soil feedback effects on AM plant species (Cortois et al., 2016) and EM Douglas-fir (Psuedotsuga menziesii; Defrenne et al., 2019). Additionally, indicator species analysis showed that dominant EM fungal species were maintained (Wilcoxina rehmii) or gained (a Wilcoxina sp. was replaced with Thelephora terrestris and a Trichophaea sp.) in spruce-conditioned soil. It has been suggested that these ruderal mycorrhizal species colonizing greenhouse seedlings are representative of early colonizers post-disturbance in the field (consistent with Chapter 4 results) 133 and are “keystone mutualists” that enable alternative successional trajectories (Duhamel et al., 2019; Visser, 1995). 134 Figure 5-5 Summary of successional plant–soil feedback results in early successional mine reclamation soils (A) and mid–late successional forest soil (B). Arrow colour indicates the feedback direction and symbols indicate the feedback mechanism. These results were integrated with available literature to develop a conceptual model for plant–soil feedbacks effects on forest succession (C). 135 5.4.2 Mycorrhizal fungal community compatibility mediates historical contingencies Spruce and redcedar seedlings showed negative growth feedbacks in willow-conditioned soil (Figure 5-5). In spruce, negative feedbacks corresponded with decreased EM fungal richness and colonization. A net loss of EM fungal indicator species also occurred in willow-conditioned soil, suggesting low compatibility between willow and spruce EM fungal communities. This leads me to reject my second hypothesis that positive feedbacks would occur where mycorrhizal guilds overlap, highlighting the importance of within-guild plant–mycorrhizal species compatibility (Ke & Wan, 2019); while most EM fungi are generalists, plant taxa-specific EM fungi do occur (Massicotte et al., 1999) and a higher proportion of host-specific EM fungi have been reported in early successional temperate forests (Twieg et al., 2007). Negative feedbacks of willow-conditioned soil on redcedar growth corresponded to decreased AM fungal colonization, except in reclamation soils where inoculation potential was already low. Yet, AM fungal colonization and shoot biomass feedbacks were not significantly related, although it is possible this relationship was masked by low colonization in reclamation soil treatments (this may also have been the reason for no observed AM fungal colonization–specific root length feedback relationship). There was no consistent willow-conditioned soil effect on redcedar fungal endophyte colonization or relative abundance of pathogens or symbiotic endophytes, indicating non-AM fungi were not feedback drivers. However, a significant fungal community shift did occur in willow-conditioned soil, so unclassified fungal taxa may have played a role (Merges et al., 2020). While neutral feedbacks on growth were expected when mycorrhizal communities were not compatible, the observed negative feedbacks may have been due to EM willow not maintaining the AM fungal inoculum potential of forest 136 soil (collected from forests containing spruce and redcedar trees) and, consequently, mycorrhizal fungal colonization declining in redcedar. Yet, negative growth feedbacks also occurred in reclamation soils without forest soil additions where mycorrhizal fungal inoculum potential was low and colonization was not affected by conditioning treatment. Consistent negative willow-conditioned soil feedbacks on growth across all plant species and soil types (even where microbial abundance was low) suggests an alternate or additional plant–soil feedback mechanism unrelated to species-specific fungal pathogens and mutualists is occurring (Figure 5-5). Nutrient depletion in the conditioning phase is an unlikely explanation, given foliar C:N ratio was incorporated into the models as a covariate (Cesarano et al., 2017). It is possible that willow decreased pH—a common occurrence in primary succession—but this typically corresponds to increased nutrient availability (Kuťáková et al., 2020). Accumulation of a generalist non-fungal pathogen, for example root-feeding nematodes, may also have occurred, although Wilschut et al. (2019) found a positive relationship between fungal pathogen and root-feeding nematode presence in their system. Finally, willow is known to produce salicylic acid, a phenolic compound that is involved in plant defense responses, and salicylic acid has been shown to have allelopathic properties, reducing shoot growth in a variety of crop and weed species (Raskin, 1992). Allelopathy effects on next-stage successional species may be a consequence of willow initiating defense responses to pathogen accumulation and/or an adaptation to delay willow’s replacement in succession. I suspect that the interspecific negative feedbacks on seedling growth associated with willow may be species-specific, especially if allelopathy is occurring, as other studies have found early successional species to have neutral to positive interspecific feedbacks on seedling growth (Castle et al., 2016; Kuťáková et al., 2020; van de Voorde et al., 2011). Variability in the strength and direction of interspecific plant–soil 137 feedbacks of early successional species due to mechanisms including allelopathy, varying plant microbial dependencies (e.g., N-fixing bacteria; Seeds and Bishop 2009), specific vs. generalist pathogen accumulation and effects on abiotic soil properties is supported by a study finding neighbourhood effects of woody plants to range from negative to positive in mine reclamation (Ibáñez & Rodríguez, 2020). Spruce-conditioned soils also had negative feedbacks on redcedar growth. Yet, feedbacks were only negative in treatments containing forest soil in which spruce-conditioning decreased AM fungal colonization (Figure 5-5). In contrast, feedbacks on growth were neutral in reclamation soils where AM fungal colonization was not affected (Figure 5-5). This suggests that negative feedbacks occurred due to spruce reducing the AM fungal inoculum potential of forest soil (collected from spruce and redcedar forests). That AM fungi are drivers of the observed feedbacks is further supported by the lack of spruce-conditioned soil effects on redcedar root fungal pathogen and non-AM fungal endophyte communities. My results indicate that interspecific neutral or positive feedbacks on late successional conifer growth are contingent on plant taxa having compatible mycorrhizal fungal communities (Figure 5-5). This is supported by research showing established EM trees to have negative effects on AM seedling establishment (Booth, 2004; Dickie et al., 2002; Guichon, 2015) and vice versa (Seiwa et al., 2020). Similarly, Koziol and Bever (2019) found positive feedbacks in late successional grassland species only occurred in the presence of beneficial AM fungi. However, these findings contrast with a meta-analysis by Crawford et al. (2019) who found that feedbacks tend to be neutral to positive when mycorrhizal guilds differ. Their finding may have been influenced by the fact that plants forming AM, in contrast to plants forming EM, tend to show negative conspecific density dependence, possibly due to less pathogen protection associated 138 with AM fungi as they lack a protective mantle (Bennett et al., 2017). I suggest that the processes maintaining inter-mycorrhizal guild coexistence do not necessarily promote inter-guild establishment or ingress during succession; it is likely that redcedar establishment during succession is facilitated by other species that obligately or facultatively form AM, such as black cottonwood (Populus trichocarpa; Kennedy et al., 2003). Yet, once established in a community, complementarity effects and resource partitioning (as well as potential negative intraspecific feedbacks in AM redcedar; Bennett et al., 2017), may explain coexistence of EM spruce and AM redcedar in the study ecosystem (Veresoglou et al., 2018). Negative feedbacks of spruce-conditioned soil on redcedar growth were not as pronounced as the negative effects on AM fungal colonization (i.e., a positive relationship between redcedar AM fungal colonization and growth feedbacks was not present), suggesting that an alternative plant–soil feedback mechanism not accounted for in this study promotes some level of coexistence (e.g., lower levels of redcedar-specific non-fungal pathogens). 5.4.3 Plant–soil feedbacks are consistent in primary successional soils Plant–soil feedbacks in forest soil-amended reclamation soils were comparable with those in forest soil for all treatments, indicating that the forest soil addition treatment successfully “advanced” the soil successional stage. Willow-conditioned soil feedbacks on willow, spruce and redcedar growth were consistently negative in reclamation soils with and without forest soil addition, leading me to reject my third hypothesis that negative feedbacks would be neutralized in primary successional conditions (Figure 5-5). For all three species, willow conditioned soil decreased mycorrhizal fungal percent colonization in forest soil-amended reclamation soils compared to controls but did not affect mycorrhizal fungal colonization in reclamation soils 139 without forest soil additions where inoculum potential was low. Despite these differential effects on mycorrhizal colonization, no change in growth feedbacks was observed (except a trend towards more negative feedbacks on spruce with forest soil additions), indicating that negative plant–soil feedback mechanisms outweighed effects of mycorrhization irrespective of soil successional stage. For example, willow pathogen accumulation still occurred, potentially eliciting an allelopathic response that subsequently caused negative interspecific feedbacks. This aligns with Castle et al. (2016) who suggested pathogen accumulation may occur faster than mutualist accumulation in primary successional soils. The negative interspecific feedbacks of willow on spruce and redcedar growth in reclamation soils contrasts with findings from (Kuťáková et al., 2020), who found early successional species to have neutral to positive interspecific feedbacks on seedling growth in primary successional soils. However, they attributed neutral to positive feedbacks to plants improving soil nutrient levels. While this study focused on soil microbes and controlled for soil nutrient changes, results of the foliar nutrient covariate indicate abiotic soil nutrient improvements may have occurred (Table E-3) but did not shift net feedback effects (Table E-7). The variable results between studies suggests that neutral to positive interspecific feedbacks of early successional species associated with improved abiotic conditions can be modified by generalist pathogen or allelopathy effects to give species-specific responses (Figure 5-5; Castle et al., 2016; Kuťáková et al., 2020; van de Voorde et al., 2011). More research is needed to parse out the relative roles of biotic and abiotic mechanisms in generating plant–soil feedbacks in primary successional soils. Despite negative intraspecific feedbacks in willow across all successional stage soils, in mine tailings there was an increase in willow EM fungal percent colonization in willow-140 conditioned soil vs. control soil. As tailings was the lowest quality soil tested (Table 2-3), this indicates that some level of intraspecific facilitation may occur in very poor soils, even if a net negative feedback is occurring. Nara and Hogetsu (2004) found EM fungal colonization to benefit Salix reinii establishment in volcanic scoria, yet benefits were attributed to connectivity with larger plants via a common mycorrhizal network, a facilitation method not examined in this greenhouse experiment, suggesting spatial and community-level feedbacks should be explored in future studies (Nara, 2006). Neutral intraspecific spruce feedbacks on growth and EM fungal percent colonization in primary successional soils were consistent in reclamation soils with and without forest soil addition, also contradicting my third hypothesis (Figure 5-5). While positive feedbacks on EM fungal species richness occurred in reclamation soils with forest soil additions but not without forest soil, the lack of growth response to this difference may be explained by plant–soil feedbacks being more dependent on mycorrhizal fungal community species composition than diversity (Crossay et al., 2019; Koziol & Bever, 2019). Spruce feedbacks on redcedar, however, showed evidence of neutralized feedbacks in primary successional soils, as hypothesized. Neutral feedbacks of spruce-conditioned soil on redcedar growth occurred in reclamation soils where the conditioning treatment had no effect on AM fungal percent colonization (Figure 5-5). In contrast, in forest soil-amended reclamation soils, spruce conditioning decreased AMF fungal colonization, which corresponded to negative growth feedbacks and increased specific root length (i.e., a shift to thinner, more acquisitive root systems). Neutralized feedbacks align with the weaker shifts I observed in the soil fungal community in tailings and overburden compared to forest soil with conditioning. This is likely 141 due to the mycorrhizal dependence of this late successional species and underscores the increasing importance of mutualists over the course of succession (Cheeke et al., 2019). 5.5 Conclusion I found negative intraspecific plant–soil feedbacks in an early successional shrub (associated with pathogen accumulation) and neutral intraspecific plant–soil feedbacks in a later successional conifer (associated with mycorrhizal fungal facilitation), indicating a shift from plant–soil feedbacks promoting plant species turnover to stability as succession progresses. These findings highlight the increasing importance of mycorrhizal fungi with successional time and are consistent with successional plant–soil feedback theory developed in grassland ecosystems (van der Putten et al., 2013). In contrast to studies in grassland ecosystems (Kardol et al., 2006; Koziol & Bever, 2019), interspecific feedbacks were generally negative. Negative feedbacks of willow-conditioned soil due to mechanisms not detected here (potentially allelopathy) contrast with neutral to positive interspecific feedbacks of primary successional plants found in other studies associated with microbial mutualists and abiotic soil improvements (Kuťáková et al., 2020; Nara, 2006; Seeds & Bishop, 2009). My proposed conceptual model of the role of plant–soil feedbacks in forest succession (Figure 5-5) reflects these varying interspecific feedback mechanisms, highlighting the complex, species-specific nature of plant–soil feedbacks (Bennett & Klironomos, 2019). In later succession, I found evidence that neutral and positive interspecific feedbacks rely on plant species having compatible mycorrhizal fungal communities. Thus, while species associating with different mycorrhizal guilds may better coexist, plant–soil feedbacks during succession may promote late successional conifers with compatible mycorrhizal fungal guilds (Figure 5-5). I found plant–soil feedbacks to be generally 142 consistent in early and late successional soils, although feedbacks on late successional plant species growth associated with mycorrhizal fungi were weakened where abundance of mutualistic microbes in early successional soils was low. Plant community recovery after a disturbance can follow alternative successional trajectories that are reinforced by plant–soil feedbacks, which can ultimately influence ecosystem functions such as nutrient cycling (Duhamel et al., 2019). The occurrence of plant–soil feedbacks in primary successional soils underscores the long-term implications of revegetation plant species selection, and shows the risks associated with “anything green is good” planting strategies or allowing invasive species to establish (Dierks et al., 2019; Pickett et al., 2019). Instead, early successional native plant species should be used in reclamation to simultaneously build soil and generate soil microbial legacies that selectively support establishment of next stage successional plant that associate with compatible microbial mutualists. Together, my findings indicate that plant species selection in reclamation can influence long-term community composition, highlighting the importance of accounting for plant–microbe relationships in ecosystem restoration and management. 143 Chapter 6: Discussion and synthesis 6.1 Overview Rehabilitation of functional terrestrial ecosystems on barren, primary successional, post-mining landscapes requires implementation of carefully planned reclamation actions that address the numerous and interacting barriers to ecosystem recovery (Bradshaw, 1984; Bradshaw, 2000). In this thesis, I demonstrate that ecosystem legacies can be applied in mine reclamation to help overcome these barriers, emulating and leveraging the ecosystem “memories” (e.g., seeds, spores, surviving patches of trees) that guide succession of terrestrial ecosystems following natural disturbances, such as wildfire or landslides (Johnstone et al., 2016). The thesis objective was to investigate if ecosystem-legacy based mine reclamation techniques can improve soil food web and plant recovery. My overarching hypotheses and evidence generated in support of—or contrary to—them are summarized below. Hypothesis 1: forest soil transfers (small volume additions to the seedling rooting zone) in mine reclamation soils would increase soil food web diversity and abundance, increase seedling root colonization by microbial mutualists (mycorrhizal fungi and endophytic N-fixing bacteria), and improve seedling establishment. In Chapter 3, I found that greenhouse-grown willow (Salix scouleriana) and spruce (Picea engelmannii x glauca) seedlings showed improved growth, physiology and nutrition with forest soil transfers, which corresponded with increased mycorrhizal fungal colonization. Soil transfers were beneficial in overburden (glacial till) but tended to have greater benefit in tailings, the lower quality substrate. However, the plant response mechanism varied with successional stage: sterilized soil controls revealed that spruce, a later successional species, benefited primarily from the biological component of the forest soil amendment, while willow, an early 144 successional species, benefited more from the improved physiochemical soil conditions (due to low reliance on mutualistic microbes and/or a lack of compatible inoculum in the late-seral stage forest soil that was used for the transfers). Alder (Alnus viridis) showed limited response to soil transfers, likely due to a lack of N-fixing bacteria in the added forest soil, although where establishment improvements did occur, they corresponded to increased N-fixing bacteria nodulation. Chapter 4 showed that these findings are applicable under field conditions where, after three growing seasons, soil transfers resulted in improved spruce seedling nutrition and physiology and increased EM fungal diversity and colonization on seedling roots. Transfers also shifted rooting zone soil bacterial communities toward increasing similarity with forest soil and, in the lowest quality overburden and tailings, increased soil faunal diversity and/or abundance. Soil transfer benefits occurred when transfers were applied in conjunction with site preparation (except where site preparation caused flooding), demonstrating the additive benefits of the two methods. In summary, with careful consideration of donor site plant community compatibility, soil transfers can be a beneficial technique for promoting seedling establishment in mine reclamation, with the greatest benefit in low quality soils and for microbial mutualist-dependent plant species (i.e., mycorrhizal fungi- or N-fixing bacteria-dependent). Hypothesis 2: proximity to undisturbed forest (a spatial ecosystem legacy) would enhance recovery of the soil food web and improve plant establishment in mine reclamation areas through aerial propagule dispersal and belowground connectivity. In Chapter 4, I found belowground connectivity with the adjacent forest in the tailings zone increased EM fungal community establishment and spruce seedling EM fungal colonization, although connectivity was not associated with seedling growth benefits and in some cases had negative effects. Belowground connectivity benefitted naturally ingressing 145 plants, increasing the proportion of native plants in the till zone and diversity of plants adjacent to the forest edge in the tailings zone. Distance effects were not observed for soil fauna and it is likely that constraints of soil physiochemical properties, which significantly related to faunal diversity and abundance, outweighed dispersal limitations, however dispersal limitations may occur over time as plant communities ameliorate soil conditions (Rantalainen et al., 2006). Greater aboveground proximity to forest edge increased plant community diversity in the tailings zone (but not the till zone). Fungal and bacterial communities varied significantly with distance from undisturbed forest. The soil microbial community closer to the forest edge was more similar to reference forest soil, with greater EM and saprotroph fungal richness adjacent to forest edge in the tailings zone. Hypothesis 3: plant establishment in mine reclamation materials would generate soil microbial ecosystem legacies that promote turnover of early successional plant species and selectively influence establishment of next stage successional plant species, with positive feedbacks occurring where plant species and mycorrhizal fungi communities are compatible. In Chapter 5, I found negative intraspecific plant–soil feedbacks occurred for an early successional shrub (willow) corresponding to accumulation of fungal pathogens, but a shift to stabilizing neutral intraspecific feedbacks associated with EM fungal facilitation in a mid–late successional conifer species (spruce). Interspecific feedbacks were negative and provided evidence that neutral and positive feedbacks promoting late successional conifers are contingent on species having compatible mycorrhizal fungi communities. Contrasting with other studies on early successional plant species (Castle et al., 2016; Kuťáková et al., 2020; van de Voorde et al., 2011), willow had strong negative interspecific feedbacks. These feedbacks did not correspond with root fungal guild shifts, suggesting another mechanism such as allelopathy may have 146 occurred, highlighting the species-specific nature of plant–soil feedbacks (Bennett & Klironomos, 2019). The strength and direction of plant–soil feedbacks in early successional reclamation soils were generally consistent feedbacks in forest soils, highlighting the long-term implications of initial reclamation actions, such as plant species selections and soil additions, on plant community assembly (Kardol & Wardle, 2010). This provides support for ecosystem legacy-based reclamation methods that, by promoting native plant and soil organism recovery, can initiate a recovery trajectory towards the pre-disturbance ecosystem. 6.2 Operational applications of key findings The ecosystem legacy-based reclamation techniques tested in this thesis have the potential to be scaled up for operational mine reclamation. To summarize, Table 6-1 provides a high-level summary of barriers to ecosystem recovery following mining and the role that forest soil transfers (Chapter 3, Chapter 4), proximity to undisturbed forest (spatial legacies; Chapter 4) and successional plant–soil feedbacks (Chapter 5) can play in addressing these constraints, as found in this thesis. The effects of site preparation (Chapter 4) are also included, demonstrating that additive benefits can occur when living ecosystem legacies are combined with standard mine reclamation techniques aimed at improving soil physical conditions. Chapters 3 and Chapter 4 showed that forest soil transfers can improve establishment of seedlings grown from seed or nursery stock in tailings and overburden. Practitioners should consider the site conditions, as transfers showed greater benefit in reclamation materials with low inoculum potential. The planted species should also be present in the donor site plant community, to ensure abundant compatible soil microbes in the donor soil (e.g., alder in Chapter 3; Amaranthus & Perry, 1988). This may be better achieved with collection of soil from areas of 147 mid-successional plant communities as opposed to climax ecosystems (Taheri & Bever, 2011). Additionally, the species-specific results in Chapter 3 indicate that transfers are more effective for later successional plants that are more mycorrhizal-dependent (Cheeke et al., 2019), as well as plants that rely on a specific mutualism such as alder and N-fixing bacteria (Seeds & Bishop, 2009) or, potentially, pine and N-fixing bacteria (Bal & Chanway, 2012). Early successional species that are less microbe-dependent, e.g., willow, can benefit from the physiochemical properties of the added topsoil, but the field experiment showed that organic matter mineralized over three growing seasons indicating that lasting benefits may only occur if initial seedling survival is increased. Fresh topsoil has higher inoculum potential than stored topsoil (Dhar et al., 2019; Rives et al., 1980) and ideally a thick layer can be applied to inoculate plant propagules and soil organisms while creating widespread improvements in soil physiochemical properties (Boldt-Burisch & Naeth, 2017; Dangi et al., 2012). When fresh topsoil availability is limited, soil transfers are an efficient method for applying small volumes of topsoil across a larger area, potentially in combination with application of larger volumes of low inoculum potential soils (e.g., overburden, degraded stored topsoil), similar to my inoculation of forest soil previously buried by tailings. While I am not advocating for disturbing new areas to source soil⁠—especially sensitive or at-risk ecosystems—if topsoil is not available or has deteriorated in quality from long-term storage in stockpiles, collecting soil from dispersed areas of adjacent undisturbed ecosystems may be a minimally invasive option, depending on the size of the reclamation area. For example, collecting the top 10 cm of soil from 1 m2 area would provide sufficient soil for 500 x 200 mL transfers. 148 Consistent with studies across other restoration sectors, in the greenhouse (Chapter 3) I found 200 mL transfers to generally have sufficient inoculum potential, resulting in initial mycorrhizal fungal colonization comparable to seedlings growing in 100% forest soil (Amaranthus & Perry, 1989; Berman & Bledsoe, 1998; Helm & Carling, 1993a; Querejeta et al., 1998). This indicates that smaller soil transfer volumes than the 750 mL used in the field trial (Chapter 4) may still be effective, however further field experiments are necessary to refine recommended transfer volumes. If greater quantities of topsoil are available, the soil transfer application approach could be adapted by, for example, spreading the topsoil as a thin layer. Unlike soil transfers this could not be carried out by tree planters and would require compaction-causing (and costly) heavy equipment, however, spreading topsoil would likely increase plant establishment from propagules compared to topsoil application exclusively to the rooting zone of seedlings (Mackenzie & Naeth, 2010). For example, application of a thin (< 0.5 cm thick) layer of topsoil at an old agricultural site steered the plant community in the direction of the plant community of the donor soil site (Wubs et al., 2016). Similarly, Wubs et al. (2019) found long-term (20 year) soil food web and plant community recovery benefits following tilling of < 0.5 cm of topsoil into an old agricultural site. Spatial legacies as assessed in Chapter 4 could be incorporated into the mine planning process by prioritizing retention of intact ecosystems patches. The size of mine facilities precludes this in many cases, but any patches that can be retained will be sources of seeds and soil biota that can disperse and support passive recovery in reclamation. It is anticipated that these patches will likely have additional habitat and ecosystem service benefits beyond the scope of this thesis (Moorcroft, 2009). Further, undisturbed patches within the permitted mine 149 disturbance footprint could be sources of fresh topsoil that could be collected and used for soil transfers at closure. The experiment in Chapter 5 found that plant–soil feedback strength and direction were generally consistent in primary successional reclamation soils, topsoil-amended reclamation soils and forest soils. Accordingly, initial reclamation plant species selection can have long-term implications for ecosystem succession, influencing ecosystem composition and function. This supports best practices of planting native species and not allowing invasive plants to establish, which likely avoids negative plant–soil feedbacks on target reclamation species (Dierks et al., 2019; Pickett et al., 2019). Selection of species that have compatible mycorrhizal fungal guilds with the target ecosystem can reinforce succession along the desired trajectory, especially for EM fungal species, which typically exhibit positive conspecific density dependence (Bennett et al., 2017; Wulantuya et al., 2020). The ubiquitous interspecific negative feedbacks of willow support prescribing diverse plant species mixtures in reclamation. Other studies have found early successional plants to have neutral to positive feedbacks (Castle et al., 2016; Kuťáková et al., 2020; van de Voorde et al., 2011), thus a diverse mixture would dilute effects of individual species with negative feedbacks and enable coexistence mechanisms that can promote biodiversity and ecosystem stability (Chung et al., 2019; Crawford et al., 2019). Finally, throughout the research conducted for this thesis, a common theme emerged that plant physiology (e.g., chlorophyll fluorescence) and nutrition (e.g., foliar N) are more sensitive indicators of seedling establishment. For example, in Chapter 3, reduced seedling foliar N and chlorophyll fluorescence were noted, but no differences in seedling growth were detected. In Chapter 4, seedlings showed nutritional and physiological benefits that were not reflected in growth measurements. Measures such as foliar nutrition, may be better predictors of future plant 150 performance than traditional growth measures, as has been shown in silviculture (Kranabetter et al., 2010). Plant pathogens could be used as an additional indicator of plant stress. For example, during the second growing season of the field trial when severe regional wildfires occurred (Abbott & Chapman, 2018), many seedlings were noted to be infected with aphids. Future research and analyses could be applied to link the presence and severity of pathogen infection with seedling growth, nutrition and physiology responses and to investigate how these responses relate to (1) reclamation treatment factors and (2) climatic factors—especially those associated with climate change. 151 Table 6-1 Barriers to ecosystem recovery following mining disturbance and key findings of this thesis on the role of ecosystem legacy-based reclamation techniques (soil transfers, proximity to undisturbed forest and plant–soil feedbacks) as well as site preparation (a common reclamation technique for improving soil physical properties) in overcoming these barriers. Barriers adapted from (Bradshaw, 2000). Barrier to recovery Problem(s) Role of ecosystem legacies and site preparation found in this thesis Soil Soil structure • Compaction • Lack of aggregate structure Site preparation: decreased bulk density (Chapter 4). Soil transfers: provided short-term improved physiochemical soil conditions (Chapter 3). Soil transfers and spatial legacies: improved seedling establishment and plant dispersal, respectively (Chapter 3, Chapter 4), which can ameliorate soil conditions over time through litter inputs and root development (Haynes, 2014). Soil nutrients • Lack of nutrients • Nutrients in inaccessible form • Lack of soil fauna and microbes that mediate nutrient cycling processes Soil transfers: increased root colonization by mutualistic microbes that improved seedling nutrient acquisition (Chapter 3, Chapter 4). Microbial mutualists can provide access to nutrients unavailable to plant roots (e.g., bacteria fix atmospheric nitrogen, mycorrhizal fungi access rock phosphate; Coleman, 2008). Transfers advanced relative abundance of later successional bacterial phyla and increased fauna recolonization in low quality reclamation soils (Chapter 4). Spatial legacies: microbial communities changed significantly with distance from undisturbed forest and, in the tailings zone, forest belowground connectivity improved ectomycorrhizal (EM) fungal community establishment immediately adjacent to the forest edge (Chapter 4). Soil fauna likely require improved physiochemical properties before dispersal limitations would occur, however, as plants improve soil structure over time (see above), spatial legacies are anticipated to support dispersal of fauna (Chapter 4; Rantalainen et al., 2006). Site preparation: increased diversity of EM fungi colonizing spruce roots and any negative interactions of site preparation with soil transfers or spatial legacies were likely due to effects of standing water that could be mitigated with appropriate drainage installation and planting microsite selection (Chapter 4). Plant–soil feedbacks: Plants create microbial community legacies that can promote establishment of successive plants e.g., spruce created EM fungal legacies that benefited successive spruce, but not AM redcedar, seedlings (Chapter 5). Vegetation Plant propagules • Lack of seeds or other propagules in reclamation materials Spatial legacies: increased plant community diversity closer to undisturbed forest; richness and diversity further increased immediately adjacent to the forest edge in treatments with belowground connectivity (tailings zone; Chapter 4). Belowground connectivity with the undisturbed forest increased the proportion of native plants (till zone; Chapter 4). Succession • Arrested succession • Undesired successional pathway Spatial legacies: increased plant community richness and diversity closer to undisturbed forest; richness and diversity further increased immediately adjacent to the forest edge with belowground connectivity (tailings zone; Chapter 4). Spatial legacies may also be a source of next stage successional species seed (and soil organisms) over time when conditions are appropriate (McClanahan, 1986). Plant–soil feedbacks: plant species can influence plant community composition through plant–soil feedbacks (Chapter 5). Plants generate soil microbial legacies that selectively support establishment of next stage successional plant that associate with compatible microbial mutualists (Chapter 5). 152 6.3 Implications for Mount Polley Mine The field trial conducted at the Mount Polley Mine and greenhouse trials carried out with soil collected from the site have further specific implications for the mine’s reclamation program. Alder showed better growth in tailings than willow and spruce in a six-month greenhouse experiment (Chapter 3). While longer-term field trials are recommended, alder will likely be a useful species for reclamation of the Tailings Storage Facility at closure. The plant–soil feedback experiment in Chapter 5 supports Mount Polley Mine’s focus on planting early successional native plants (MPMC, 2015), consistent with best practices (Tordoff et al., 2000), as this is expected to promote the successional trajectory of the local forest ecosystem. However negative feedbacks of willow may merit further investigation or, at minimum, willow should be planted in a diverse mixture of species as described above. In the area impacted by the 2014 tailings dam failure, results from Chapter 4 suggest that soil transfers are a beneficial technique that can be applied across areas of till and tailings to improve seedling EM fungal colonization and nutrition. Soil transfers would be an efficient use of the minimal topsoil available, as topsoil was not salvaged in advance given the unplanned nature of the incident. In the areas where tailings were deposited over forest floor, mounding was a successful reclamation treatment, with limited differences between mounding and tailings removal (Figure 6-1). This suggests the tailings can be effectively remediated in place, which would avoid compaction, road construction and cost associated with heavy equipment removal of tailings. Interestingly, establishment of planted seedlings and naturally ingressing plants was better in the tailings zone (with mounding or tailings removal) than in exposed glacial till areas (Figure 6-1). While environmental impact assessments have focused on the effects of the spilled tailings (Miller et al., 2015), the till areas may recover more slowly and suffer more erosion due 153 to the lower vegetative cover (Runolfsson, 1987). Ongoing monitoring will be necessary to assess if plant community recovery stagnates, necessitating further supplemental reclamation interventions. Additionally, the poor drainage capabilities of both the glacial till and tailings presented challenges for seedling survival and growth in the field experiment (Chapter 4). Incorporation of hydrological planning into re-contouring and selection of high planting microsites following site preparation are likely to improve revegetation outcomes. The linear configuration of the tailings dam failure disturbance (the maximum distance from the undisturbed forest is < 500 m with majority of the disturbed area within 50 m of the forest edge; Miller et al., 2015) positions the recovering areas well to benefit from soil organism and plant dispersal from the spatial ecosystem legacy of the adjacent undisturbed forest. As this research only extended to 15 m from the forest edge, further monitoring will be necessary to identify any dispersal limitations over greater spatial extents. 154 Figure 6-1 Representative photos of till zone (A, B) and tailings zone (C, D) treatment units without mounding (A, C) and with mounding (B, D) at the Mount Polley Mine, British Columbia. Photo credit: Katie McMahen. 155 6.4 Limitations and future research A strength of this thesis was validation of the soil transfer method tested under greenhouse conditions (Chapter 3) over a longer period in the field and on nursery seedlings, which are typically used in reclamation over direct seedling (Chapter 4). Chapter 4, however, only tested spruce seedlings. While Helm & Carling (1993a, 1993b) found soil transfers to benefit balsam poplar (Populus balsamifera), an early successional shrub, over two years in the field, given the differential responses of willow, alder and spruce under greenhouse conditions, long-term field testing with additional species representing diverse functional groups is recommended. Additionally, while three years is longer than other field studies testing soil transfers in ecological restoration (e.g., Amaranthus et al., 1990; Amaranthus & Perry, 1988; Helm & Carling, 1993a; Lance et al., 2019; Querejeta et al., 1998; St-Denis et al., 2017), trees are long-lived and ongoing monitoring of this experiment will be necessary to understand if soil transfer effects continue to persist and scale up to affecting ecosystem community composition or ecosystem function. Longer-term testing would additionally enable comparing the effectiveness of this method without outcomes from longer-term trials and chronosequence studies where soil was applied with different methods and in greater quantities, such as spreading a layer of topsoil (e.g., Boldt-Burisch & Naeth, 2017; Dangi et al., 2012). Chapter 4 of this thesis investigated short-distance (15 m) dispersal and facilitation associated with spatial connectivity to undisturbed forest. Forest belowground connectivity had minor benefits adjacent to the forest edge including improved establishment of EM fungal communities and naturally ingressing plants (primarily in the tailings zone), but these findings likely don’t translate into a scalable mine reclamation technique. Many of the belowground forest connectivity benefits were alternatively achievable through soil transfers. The benefits of 156 spatial proximity to forest for aerial dispersal of plant seeds and microbes may be more broadly applicable. Future research over spatial scales appropriate for large mining disturbances will be necessary to refine the role of spatial ecosystem legacies and if a minimum threshold for retention patch size exists (Kranabetter et al., 2013; Lee et al., 2015). Of particular use in addressing this knowledge gap on community assembly (Dhar et al., 2018) would be studies investigating long-term effects of dispersal on ecosystem community composition and identifying if improved dispersal alleviates biotic barriers such as lack of mycorrhizal fungi and N-fixing bacteria or stagnated succession due to a lack of late successional plant species seed sources. Additionally, while the ecosystem legacy-based mine reclamation techniques tested in Chapter 4 elicited significant ecological responses despite the trial being established in a complex reclamation environment with many interacting factors, the generalizable effectiveness of these methods beyond the unique disturbed area resulting from the tailings dam embankment breach should also be confirmed through studies in traditional mine reclamation settings (e.g., overburden-covered waste rock dumps and tailings storage facilities) and across different ecosystem types. Research at additional mine sites and across different ecosystem types is required to address concerns over pseudoreplication due to the greenhouse and field experiments in this thesis being conducted at only one mine site. This would help address potential influence of climate on my field experiment results. The second and third growing seasons of the field experiment were both much drier than average (Abbott and Chapman, 2018; MPMC, 2019). This had the potential to increase the magnitude of plant responses to ecosystem legacies (e.g., mycorrhizal fungi have been shown to increase seedling resilience to drought; Lehto & Zwiazek, 2011) or decrease the magnitude of plant responses due to overall less growth occurring. 157 Chapter 5 addressed knowledge gaps regarding the role of plant–soil feedbacks in ecosystem succession, including testing early successional (mine reclamation) soils and forest ecosystem plant species. To build on my short-term greenhouse study findings, next steps (as detailed in the subsequent paragraphs) should include research to understand: (1) effects when seedlings are grown as part of a community, not individually; (2) how greenhouse results translate to field settings; and (3) long-term effects. Such research will improve our understanding of complex plant–soil feedback processes involving interacting abiotic and biotic ecosystem components (Bennett & Klironomos, 2019), ultimately informing our ability to restore and manage ecosystems. Firstly, I grew seedlings in individual pots, which does not incorporate plant-plant competition (Ke & Wan, 2019) or conspecific density dependence (Bennett et al., 2017), potentially overestimating or underestimating the plant–soil feedback strength of plants grown in a community, as competitors may have varied sensitivities and dependencies (De Long et al., 2019). For example, in groups, competition may act as a stabilizing mechanism to positive feedbacks associated with mycorrhizal fungi colonization (Veresoglou et al., 2018). Conversely, individual growth excludes the potential for facilitation through mycorrhizal networks (Nara, 2006). Future research should also investigate additional plant species and functional groups given the lack of research on plant–soil feedbacks in temperate forests and my differential species-specific results. Secondly, like Chapter 5, the majority of plant–soil feedback studies are conducted in greenhouses (Crawford et al., 2019), which may affect plant–soil feedbacks through development of different biological communities than in nature (Kivlin et al., 2018). For example, soil collection and storage may exclude some microarthropods or larger soil biota, 158 which can differentially affect plant–soil feedbacks, while warmer climatic conditions in greenhouses may promote faster pathogen reproduction and accumulation (De Long et al., 2019; Kuťáková et al., 2018). Further, it is possible that some positive effects of mycorrhizal colonization were eliminated under the controlled greenhouse conditions where seedlings were not exposed to abiotic stressors (e.g., drought), which have been shown to enhance the benefits of microbial symbionts (e.g, Lehto & Zwiazek, 2011). While my statistical models accounted for nutrient depletion and mycorrhizal colonization was generally higher in controls (suggesting propagules were not lost during storage; Stenberg et al., 1998), the storage of control soil during the conditioning phase may have affected my results. Despite these challenges of greenhouse studies, Duhamel et al. (2019) found greenhouse EM fungal species to be representative of early successional fungal communities in the field and other studies have found consistency between field and greenhouse results (e.g., Bauer et al., 2015; van de Voorde et al., 2011). Future experiments conducted in the field with plants grown in communities will be important for further elucidating the relative importance of plant–soil feedbacks in ecosystem assembly. Thirdly, given the time scale over which forest succession occurs, the role of plant–soil feedbacks should be tested over the long-term, including testing assessing the relative importance of plant–soil feedbacks in the context of other abiotic and stochastic successional drivers, as well as interactions with these factors. For example, gap dynamics and light availability have been shown to shift conspecific plant–soil feedback direction by changing the relative influence of pathogens and mutualists (Wulantuya et al., 2020). Plant–soil feedback effects through the life cycle of long-lived trees should similarly be explored, as feedbacks can change over time with plant age and life stage (Bezemer et al., 2018; Dudenhöffer et al., 2018), 159 even extending to effects on the seed microbiome and seed germination (Kong et al., 2019; Miller et al., 2019). 6.5 Conclusion The mining industry directly employs 40 million people worldwide (Cherlet et al., 2018) and over 400,000 people here in Canada, contributing $60 billion (USD) to our gross domestic product (GDP; Natural Resources Canada, 2019). Mining is anticipated to continue to be economically important as the demand for metals increases with the uptake of electric vehicles and other green technologies that rely on metal components such as batteries (European Commission, 2014). However, the economic benefits of mining are paired with severe ecosystem disturbances, and continued refinement and implementation of mine reclamation best practices is critical to reduce the environmental impacts of the industry and maintain the sector’s social license to operate (Owen & Kemp, 2013). Concepts incorporating linkages between aboveground plant establishment and belowground ecosystems, including the soil food web, are not widely applied in ecological restoration (Kardol & Wardle, 2010). This thesis advanced research on understanding the role of plant–soil microbe interactions in mine reclamation and how these interactions can be incorporated into scalable reclamation techniques. Specifically, I found that small volume forest soil additions to the seedling rooting zone can effectively inoculate mycorrhizal fungi and improve establishment of mycorrhizal-dependent seedlings. Spatial ecosystem legacies (undisturbed forest) can also be a source from which plants and soil organisms disperse to recolonize disturbed areas. Mine reclamation techniques incorporating living ecosystem legacies can have additive benefits with reclamation methods, such as site preparation, which focus on 160 improving soil physical properties. Furthermore, reestablishment of plants in early successional mine reclamation soils creates plant species-specific ecosystem legacies, which can influence successional trajectories through plant–soil feedbacks. Taken together, these findings provide evidence that ecosystem legacies can simultaneously support recovery of the soil food web and plant communities, rebuilding the complex ecological communities characteristic of functional and resilient forests. 161 References Abarenkov, K., Nilsson, H. R., Larsson, K. H., Alexander, I. J., Eberhardt, U., Erland, S., Høiland, K., . . . Kõljalg, U. (2010). 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Vegetation Soil Latitude Longitude Collection Date Proportion Collected Dominant Tree Species Basal Area (m2/ha) Structural Stage Tailings1 52°31'36.76" N 121°35'18.28" W 23-Nov-15 45% - - No vegetation 29-Jun-16 55% Overburden 52°30'47.06" N 121°34'17.22" W 25-Nov-15 22% - - No vegetation 52°31'09.76" N 121°35'04.29" W 28-Jun-16 78% - - No vegetation Forest Soil 52°31'09.94" N 121°35'14.10" W 03-Apr-16 80% Thuja plicata 81.65 Old growth Abies lasiocarpai 11.69 52°30'53.66" N 121°34'44.28" W 28-Jun-16 20% Picea glauca x engelmannii 30.86 Young forest - stem exclusion Abies lasiocarpai 1.49 191 Figure A-1 Locations at the Mount Polley Mine, British Columbia where forest soil, overburden (glacial till) and mine tailings were collected for the greenhouse experiments in Chapter 3 and Chapter 5. 192 Appendix B Chapter 3 supplementary material Table B-1 Post hoc soil type contrasts of linear model and generalized linear model results of soil type x soil transfer volume (0%, 5% or 25% forest soil addition) effects on spruce, willow and alder seedling establishment with soil transfer volume as a continuous variable. Soil type = tailings, overburden or tailings + overburden. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Df = 2 for soil type, df = 1 for transplant volume, and df = 2 for soil type x soil transfer volume. contrastP -value contrastP -value contrastP -value contrastP -value contrastP -value contrastP -value contrastP -value contrastP -value contrastP -valueGrowthShoot biomass (mg/day) 0.564 0.313 -0.013 0.999 -0.577 0.297 1.116 0.764 -0.797 0.850 -1.913 0.460 -0.931 0.442 -0.939 0.409 -0.009 1.000Root biomass (mg/day) 0.079 0.387 -0.018 0.953 -0.097 0.269 0.272 0.602 -0.038 0.988 -0.310 0.518 -1.037 0.342 -0.938 0.387 0.099 0.989Root:shoot biomass 0.060 0.841 0.030 0.960 -0.030 0.959 -0.021 0.973 0.011 0.991 0.031 0.939 -0.028 0.807 0.006 0.989 0.034 0.710Physiology and NutritionChlorophyll fluorescence (Fv/Fm) 0.252 0.004 -0.002 0.999 -0.254 0.004 0.051 0.132 0.055 0.045 0.004 0.985 -0.009 0.281 -0.005 0.705 0.004 0.718Foliar N (%) 0.152 0.017 0.067 0.424 -0.086 0.259 -0.085 0.960 -0.323 0.412 -0.238 0.724 0.073 0.785 -0.072 0.780 -0.145 0.361Root SymbiontsEMF colonization (%) 1.529 <0.001 0.201 0.1878 -1.328 <0.001 1.003 <0.001 -0.228 0.192 -1.231 <0.001 0.828 0.002 - - - -EMF species richness 0.653 0.002 0.171 0.593 -0.482 0.026 - - - - - - - - - - - -Endophyte colonization (%) - - - - - - 1.329 <0.001 0.327 <0.001 -1.002 <0.001 0.828 0.002 0.146 0.682 -0.682 0.014N-fixing nodule wet weight (mg) - - - - - - - - - - - - -3.130 0.804 -9.280 0.141 -6.150 0.410AlderOverburden - TailingsOverburden - Tailings + OverburdenTailings - Tailings + OverburdenSpruceOverburden - TailingsOverburden - Tailings + OverburdenTailings - Tailings + OverburdenWillowOverburden - TailingsOverburden - Tailings + OverburdenTailings - Tailings + Overburden193 Table B-2 Results of spruce ectomycorrhizal (EM) fungal morphotype identification with high-throughput sequencing. Grey shaded rows indicate confirmation of non-mycorrhizal morphotype vouchers. Voucher ID EM fungal taxa Proportion of reads (%) Abundance rank Pairwise identity (%) Total reads A3 Thelephoraceae sp. 1 4.1 Non-mycorrhizal 1,523 D1 Thelephoraceae sp. 1 1.7 Non-mycorrhizal 1,813 D4 Thelephoraceae sp. 1 2.9 Non-mycorrhizal 1,676 G3 Thelephoraceae sp. 1 1.0 Non-mycorrhizal 8,027 M1 Thelephoraceae sp. 2 1.2 Non-mycorrhizal 1,445 M3 Meliniomyces bicolor 86.4 1 100.00 79,346 M4 Meliniomyces bicolor-like 6.1 4 78.00 12,725 P1 Tuber sp. 73.9 1 99.75 29,281 T3 Entoloma sp. 1.7 Non-mycorrhizal 2,297 U3 Meliniomyces bicolor-like 94.2 1 78.00 65,877 W2 Wilcoxina rehmii 40.3 1 100.00 57,084 Piloderma sp. 14.4 3 100.00 57,084 Z3 Thelephoraceae sp. 3 0.4 Non-mycorrhizal 9,647 AA1 Wilcoxina mikolae 54.3 1 98.96 31,583 AB1 Wilcoxina mikolae 67.0 1 98.96 81,940 AB2 Russula sp. 1.8 Non-mycorrhizal 4,564 AB3 Pyronemataceae sp. 80.2 1 94.04 40,278 AC2 No EMF taxa present 0.0 Non-mycorrhizal 4,736 AE1 Wilcoxina rehmii 87.7 1 99.99 70,898 AE3 Pseudotomentella rhizopunctata-like 47.8 1 95.78 76,451 AE4 Wilcoxina rehmii 28.2 2 99.99 19,363 Tuber sp. 10.0 3 99.75 19,363 AG1 Hymenoscyphus kathiae 0.8 Non-mycorrhizal 1,399 AH1 Wilcoxina mikolae 86.3 1 98.96 17,208 AK1 Amphinema byssoides 62.2 1 100.00 29,763 194 Table B-3 Spruce, willow and alder seedling establishment (mean ± 1 SE). Significant differences of soil treatments from 100% forest soil are indicated by *** (P < 0.001), ** (0.001 ≤ P < 0.01), * (0.01 ≤ P < 0.05) and . (0.05 ≤ P < 0.1). Df = 9 for soil treatment. Growth parameters were relativized per growing day to account for growth period differences due to replanting. Forest soil addition (%) nSpruceForest 100 5 0.38 ± 0.18 0.17 ± 0.07 0.52 ± 0.04 0.857 ± 0.028 3.4 ± 0.4 67.7 ± 6.8 - -Overburden 0 5 0.26 ± 0.19 0.15 ± 0.09 0.76 ± 0.11 0.566 * ± 0.114 2.2 . ± 0.2 20.8 *** ± 5.8 - -5 5 0.24 ± 0.11 0.17 ± 0.10 0.71 ± 0.09 0.574 ± 0.157 2.2 . ± 0.1 60.9 ± 16.0 - -25 5 0.32 ± 0.23 0.10 ± 0.07 0.38 ± 0.12 0.703 ± 0.028 2.7 ± 0.3 50.0 * ± 11.6 - -Tailings 0 5 0.05 * ± 0.01 0.03 ± 0.01 0.61 ± 0.10 0.192 *** ± 0.096 1.7 ** ± 0.1 4.3 *** ± 4.3 - -5 5 0.06 * ± 0.03 0.04 ± 0.02 0.59 ± 0.15 0.430 ** ± 0.084 2.3 ± 0.1 14.5 *** ± 14.5 - -25 5 0.39 ± 0.23 0.15 ± 0.08 0.46 ± 0.11 0.466 ** ± 0.092 1.9 ** ± 0.1 32.1 *** ± 18.7 - -0 5 0.09 ± 0.04 0.04 ± 0.01 0.34 ± 0.14 0.493 ** ± 0.076 1.9 ** ± 0.3 43.9 ± 25.3 - -5 5 0.09 ± 0.02 0.06 ± 0.02 0.65 ± 0.13 0.621 . ± 0.065 2.1 * ± 0.2 28.9 *** ± 11.5 - -25 5 0.61 ± 0.22 0.33 ± 0.12 0.67 ± 0.21 0.735 ± 0.009 2.6 ± 0.1 44.7 ** ± 8.1 - -WillowForest 100 5 7.55 ± 0.52 3.42 ± 0.32 0.37 ± 0.10 0.688 ± 0.016 2.6 ± 0.2 21.2 ± 3.7 31.40 ± 4.68 -Overburden 0 3 2.48 . ± 2.00 0.10 ** ± 0.07 0.20 ± 0.10 0.703 ± 0.007 3.3 ± 0.6 2.7 ** ± 0.9 19.68 . ± 6.97 -5 4 4.39 ± 1.76 2.11 ± 0.88 0.40 ± 0.09 0.658 ± 0.014 2.4 ± 0.3 17.8 ± 8.4 35.99 ± 8.08 -25 5 6.53 ± 0.49 3.93 ± 0.45 0.60 . ± 0.04 0.667 ± 0.014 2.5 ± 0.2 22.6 ± 2.9 63.00 *** ± 4.18 -Tailings 0 2 1.00 ** ± 1.00 0.29 ** ± 0.29 0.65 ± 0.35 0.710 ± NA 2.8 ± NA 3.0 * ± 0.0 4.43 *** ± 1.57 -5 2 3.74 ± 3.73 2.73 ± 2.73 0.41 ± 0.32 0.605 ± NA 2.4 ± NA 0.0 * ± 0.0 10.75 ** ± 1.75 -25 5 5.60 ± 1.87 2.61 ± 1.18 0.39 ± 0.06 0.596 ± 0.025 2.8 ± 0.3 15.6 ± 6.6 40.40 ± 5.12 -0 2 5.77 * ± 5.38 2.67 ** ± 2.63 0.30 ± 0.18 0.641 ± 0.028 3.3 ± 0.2 12.0 ± 12.0 43.50 ± 36.50 -5 5 2.92 * ± 1.28 1.00 ** ± 0.49 0.41 ± 0.12 0.619 ± 0.039 3.2 ± 0.2 7.4 *** ± 1.8 31.40 ± 7.93 -25 5 8.38 ± 2.08 4.07 ± 0.82 0.51 ± 0.03 0.605 ± 0.032 2.6 ± 0.2 27.6 ± 4.8 38.40 ± 6.96 -Physiology and Nutrition Root SymbiontsGrowthEMF (% colonization)Endophytes (% colonization)N-fixing nodules wet weight (mg)Tailings + OverburdenTailings + OverburdenChlorophyll fluoresence (Fv/Fm) Foliar N (%)Shoot biomass (mg/day)Root biomass (mg/day)Root:shoot biomass195 Table B-3 cont’d Forest soil addition (%) nAlderForest 100 5 42.79 ± 7.84 14.10 ± 3.09 0.32 ± 0.02 0.689 ± 0.004 3.0 ± 0.2 21.6 ± 1.9 0.48 ± 0.04 187.1 ± 1729.4Overburden 0 5 0.53 ** ± 0.23 0.20 ** ± 0.06 0.51 ± 0.12 0.650 ± 0.033 2.0 ± 0.3 0.0 *** ± 0.0 0.08 *** ± 0.02 113.3 *** ± 211.05 4 0.74 *** ± 0.38 0.25 *** ± 0.14 0.31 ± 0.03 0.689 ± 0.008 1.6 ± 0.3 0.0 *** ± 0.0 0.02 *** ± 0.02 140.4 *** ± 140.425 4 0.40 ** ± 0.09 0.20 ** ± 0.05 0.53 ± 0.08 0.692 ± 0.012 2.1 ± 0.2 0.0 *** ± 0.0 0.16 *** ± 0.02 121.9 *** ± 121.9Tailings 0 4 13.14 ± 12.53 5.44 ± 5.16 0.43 ± 0.05 0.705 ± 0.002 1.6 ± 0.2 0.0 *** ± 0.0 0.01 *** ± 0.00 0.0 *** ± 0.05 4 3.25 ** ± 2.70 1.11 ** ± 0.83 0.57 ± 0.11 0.705 ± 0.010 1.6 ± 0.3 0.0 *** ± 0.0 0.03 *** ± 0.01 274.5 *** ± 410.725 5 1.79 ** ± 0.87 0.83 * ± 0.39 0.46 ± 0.03 0.698 ± 0.008 2.1 ± 0.2 0.0 *** ± 0.0 0.13 *** ± 0.04 103.6 ** ± 407.40 4 6.28 ** ± 5.06 3.11 ** ± 2.69 0.48 ± 0.06 0.552 ± 0.138 1.9 ± 0.3 0.2 *** ± 0.2 0.05 *** ± 0.03 264.4 *** ± 388.15 5 10.14 . ± 8.31 5.49 . ± 4.82 0.43 ± 0.06 0.638 ± 0.039 2.2 ± 0.3 0.8 *** ± 0.6 0.09 *** ± 0.03 247.1 ** ± 597.025 5 2.13 ± 1.09 0.65 ± 0.24 0.41 ± 0.06 0.711 ± 0.022 2.1 ± 0.2 0.8 *** ± 0.6 0.03 *** ± 0.01 165.0 ** ± 548.3Physiology and Nutrition Root SymbiontsGrowthTailings + OverburdenEMF (% colonization)Endophytes (% colonization)N-fixing nodules wet weight (mg)Chlorophyll fluoresence (Fv/Fm) Foliar N (%)Shoot biomass (mg/day)Root biomass (mg/day)Root:shoot biomass196 Table B-4 Results from linear models for unsterilized soil treatments with symbiont colonization as the explanatory variable and soil treatment (soil type (tailings, overburden or tailings + overburden) and transplant volume (0%, 5% or 25%) combined) as a covariate. Shoot biomass, root:shoot ratio and specific root length were tested as response variables for each plant species. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Parameter Species Estimate SE T P Estimate SE T P Estimate SE T PShoot biomass Spruce 0.024 0.005 4.647 <0.001 - - - - - - - -Willow 0.048 0.042 1.162 0.255 0.008 0.042 0.193 0.849 - - - -Alder - - - - 0.063 0.055 1.149 0.258 0.003 0.001 4.355 <0.001Root:shoot ratio Spruce -0.005 0.003 -1.842 0.074 - - - - - - - -Willow 0.005 0.002 2.371 0.025 0.005 0.002 2.256 0.032 - - - -Alder - - - - 0.008 0.009 0.942 0.352 -0.0001 0.0001 -0.435 0.666Specific root length Spruce -0.001 0.002 -0.317 0.753 - - - - - - - -Willow -0.003 0.002 -1.308 0.202 0.001 0.002 0.218 0.829 - - - -Alder - - - - -0.130 0.114 -1.142 0.261 -0.001 0.0003 -2.789 0.009EM fungi Endophytes Nodules197 Table B-5 Change in slope of effect of increasing forest soil transfer volume on seedling establishment with soil sterilization. Bold = significant P-value (P< 0.05); italics = marginally significant (0.05 < P < 0.1). Slope Δ p -value Slope Δ p -value Slope Δ p -value Slope Δ p -value Slope Δ p -valueSpruceOverburden -0.007 0.792 -0.001 0.798 0.011 0.160 -0.0015 0.817 0.0099 0.088Tailings -0.061 0.027 -0.011 0.037 0.004 0.618 -0.0013 0.841 -0.0004 0.942Tailngs + overburden -0.077 0.004 -0.015 0.003 0.011 0.164 -0.0033 0.600 0.0124 0.034WillowOverburden 0.048 0.688 -0.013 0.564 -0.004 0.517 0.0002 0.913 0.0093 0.649Tailings 0.060 0.639 0.020 0.415 0.020 0.005 0.0042 0.072 0.0497 0.049Tailngs + overburden 0.125 0.304 0.020 0.389 0.005 0.419 0.0007 0.738 -0.0093 0.649AlderOverburden 0.041 0.535 0.027 0.681 -0.005 0.214 -0.0002 0.593 0.0128 0.207Tailings -0.019 0.769 -0.023 0.719 -0.001 0.738 0.0001 0.809 0.0165 0.089Tailngs + overburden 0.050 0.426 0.061 0.337 0.003 0.349 -0.0011 0.011 0.0014 0.885Shoot biomass Root biomass Root:shoot biomass Chlorophyll fluorescence Foliar N198 Figure B-1 Diffrences in total Phospholipid fatty acid (PLFA) biomass (A) and PLFA biomass of microbial broad groups (B) between freshly harvested forest soil and potted forest soil maintained under greenhouse conditions for the time period (6 months) required to acquire more viable alder seed (n = 3). Total biomass did not signficantly change with storage (P = 0.622). There were no significant differences between baseline and stored (greenhouse) microbial group biomasses except for protozoa, which increased with storage (P = 0.006). 199 Figure B-2 Spruce seedling linear model results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% additions) effects on seedling root biomass (A), root:shoot ratio (B) and chlorophyll fluorescence (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Growth parameters were relativized per growing day to account for growth period differences due to replanting. 200 Figure B-3 Willow seedling linear model results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% addition) effects on seedling root biomass (A), root:shoot ratio (B) and chlorophyll fluorescence (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Growth parameters were relativized per growing day to account for growth period differences due to replanting. 201 Figure B-4 Alder seedling linear model results with 95% model confidence interval bands of soil type (overburden, tailings and tailings + overburden) and forest soil transfer volume (0, 5% or 25% addition) effects on seedling root biomass (A), root:shoot ratio (B) and chlorophyll fluorescence (C) in sterilized and unsterilized soils. The dashed line is the mean value for seedlings grown in unsterilized forest soil. Log transformation = loge. Growth parameters were relativized per growing day to account for growth period differences due to replanting. 202 Figure B-5 Soil sterilization effects on soil root symbiont colonization. Post-hoc comparisons were conducted on linear models (E) and generalized linear models (A–D) with soil treatment (soil type and forest soil transfer (addition) volume combined) and sterilization as the explanatory variables. Significant differences are indicated by *** (P < 0.001), ** (0.001 ≤ P < 0.01), * (0.01 ≤ P < 0.05) and . (0.05 ≤ P < 0.1). 203 Figure B-6 Representative spruce, willow and alder seedlings (median biomass from replicates in treatment unit) growing in Mount Polley Mine tailings with 0%, 5% or 25% forest soil additions. 100% forest soil treatments are shown for compression. Photo credit: Katie McMahen. 204 Appendix C Chapter 4 supplementary material Table C-1 Basal area of trees species at forest soil transfer collection sites in the undisturbed forest adjacent to each trial block in the till and tailings zones (mean of two 50 m2 plots). Basal area (m2/ha) Block Picea glauca x engelmannii Abies lasiocarpa Pseudotsuga menziesii Thuja plicata Populus trichocarpa Betula papyrifera Till zone 1 - 1.74 - 2.24 11.57 - 2 25.68 13.23 - 0.03 - - 3 11.92 10.90 - 2.49 - 6.49 Tailings zone 1 11.45 2.02 - 10.52 0.52 - 2 24.90 <0.01 - <0.01 0.40 - 3 1.10 6.26 9.26 31.22 - 1.80 Table C-2 Spruce seedling vigour assessment criteria Vigour no. Vigour class Criteria1 Excellent Healthy tree; no chlorosis; good leader growth; may have a defect (e.g., fork or past browsing) if still meets remaining criteria because producing trees for commercial forestry is not the objective2 Good Reasonably healthy, but has one or more minor problems: slightly chlorotic, needles somewhat stunted or sparse, moderate leader growth3 Fair Poor condition, but death is not inevitable; has one or more serious problems: chlorotic, stunted or sparse needles, stem may be leaning heavily or very bent, poor leader growth4 Poor Is almost dead or expected to die; very few green needles5 Dead No live needles6 Missing Can't find205 Table C-3 Till zone post hoc contrasts of site preparation, soil transfer and forest belowground connectivity effects on spruce seedling survival, growth, vigour and chlorosis. Contrasts were run on binomial generalized mixed models (survival and chlorosis), Poisson generalized mixed models (vigour) and linear mixed models (height, root collar diameter and volume growth). Interactions only shown where significant. Survival is cumulative (i.e., total survival after second and third growing seasons). On the vigour scale, 1 = highest and 4 = lowest, thus negative vigour model results indicate improved vigour. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). RCD = root collar diameter. Factor Contrast Z P P P P P PYear 1Site preparation None - mounding 0.26 , 0.798 -0.41 80.9 , 0.681 -1.68 79.3 , 0.097 -1.24 79.3 , 0.219 -0.05 61 , 0.965 0.00 60 , 0.999Soil transfer No - yes 0.44 , 0.662 0.73 80.9 , 0.468 -0.62 79.3 , 0.539 -0.81 79.3 , 0.423 -0.61 61 , 0.546 0.00 60 , 0.999Connectivity No - yes 0.18 , 0.861 -0.49 80.9 , 0.628 -0.13 79.3 , 0.897 0.60 79.3 , 0.553 -0.20 61 , 0.844 0.00 60 , 0.999Interactions:Site prep. = none Transfer: no - yes - - - - -2.25 79.3 , 0.027 -2.11 79.3 , 0.038 - - - -Site prep. = mounding Transfer: no - yes - - - - 1.38 79.3 , 0.172 0.97 79.3 , 0.336 - - - -Connectivity = no Transfer: no - yes - - - - 1.19 79.3 , 0.239 0.94 79.3 , 0.349 - - - -Connectivity = yes Transfer: no - yes - - - - -2.06 79.3 , 0.043 -2.08 79.3 , 0.041 - - - -Year 2Site preparation None - mounding 2.16 , 0.031 0.02 79.3 , 0.988 -0.22 79.3 , 0.829 -0.12 79.3 , 0.907 -1.76 61 , 0.083 -2.81 , 0.005Soil transfer No - yes 3.36 , <0.001 0.44 79.3 , 0.663 1.15 79.3 , 0.255 0.98 79.3 , 0.331 -0.03 61 , 0.975 -1.70 , 0.089Connectivity No - yes 1.29 , 0.197 0.74 79.3 , 0.462 0.32 79.3 , 0.747 0.52 79.3 , 0.606 -0.51 61 , 0.614 -3.17 , 0.002Interactions:Site prep. = none Transfer: no - yes 1.38 , 0.168 - - - - - - - - 0.29 , 0.769Site prep. = mounding Transfer: no - yes 3.57 , <0.001 - - - - - - - - -2.97 , 0.003Transfer = no Site prep. = none - mounding 0.55 , 0.579 - - - - - - - - -0.50 , 0.620Transfer = yes Site prep. = none - mounding 2.98 , 0.003 - - - - - - - - 4.50 , <0.001Connectivity = no Site prep. = none - mounding -0.71 , 0.479 - - - - - - - - -3.42 , 0.001Connectivity = yes Site prep. = none - mounding 3.71 , <0.001 - - - - - - - - -0.35 , 0.727T df T dfChlorisisZ /T dfSurvival Height growth RCD growth Volume growth VigourT df T df206 Table C-3 cont’d Factor Contrast Z P P P P P PYear 3Site preparation None - mounding 1.92 , 0.055 -1.16 79.3 , 0.248 5.91 79.3 , <0.001 2.96 79.3 , 0.004 0.56 , 0.576 2.98 , 0.003Soil transfer No - yes 2.81 , 0.005 0.46 79.3 , 0.644 4.47 79.3 , <0.001 3.20 79.3 , 0.002 0.32 , 0.747 -1.17 , 0.241Connectivity No - yes 2.19 , 0.029 2.59 79.3 , 0.012 5.33 79.3 , <0.001 3.68 79.3 , <0.001 -2.22 , 0.027 -5.30 , <0.001Interactions:Site prep. = none Transfer: no - yes 0.10 0.917 -2.23 79.3 0.029 - - 0.12 79.3 0.905 - - 4.50 <0.001Site prep. = mounding Transfer: no - yes 2.96 0.003 0.58 79.3 0.562 - - 4.06 79.3 <0.001 - - -0.50 0.620Transfer = no Site prep. = none - mounding 0.72 0.474 -1.08 79.3 0.285 1.10 79.3 0.274 0.29 79.3 0.773 - - 1.89 0.058Transfer = yes Site prep. = none - mounding 3.38 0.001 1.73 79.3 0.087 5.22 79.3 <0.001 4.23 79.3 <0.001 - - -3.19 0.001Year 1-3Site preparation None - mounding - - 0.14 79.3 , 0.893 1.92 79.3 , 0.058 1.73 79.3 , 0.088 - - - -Soil transfer No - yes - - 1.44 79.3 , 0.153 2.31 79.3 , 0.024 2.18 79.3 , 0.033 - - - -Connectivity No - yes - - 1.81 79.3 , 0.075 2.31 79.3 , 0.024 2.44 79.3 , 0.017 - - - -Interactions:Site prep. = none Transfer: no - yes - - -0.40 79.3 , 0.692 0.73 79.3 , 0.466 0.21 79.3 , 0.832 - - - -Site prep. = mounding Transfer: no - yes - - 2.44 79.3 , 0.017 2.53 79.3 , 0.013 2.87 79.3 , 0.005 - - - -Transfer = no Site prep. = none - mounding - - - - 0.46 79.3 , 0.648 -0.11 79.3 , 0.916 - - - -Transfer = yes Site prep. = none - mounding - - - - 2.26 79.3 , 0.027 2.55 79.3 , 0.013 - - - -ZT df T df T df ZSurvival Height growth RCD growth Volume growth Vigour Chlorisis207 Table C-4 Tailings zone post hoc contrasts of soil transfer, site preparation and forest belowground connectivity effects on spruce seedling survival, growth, vigour and chlorosis. Contrasts were run on binomial generalized mixed models (survival and chlorosis), Poisson generalized mixed models (vigour) and linear mixed models (height, root collar diameter and volume growth). Interactions only shown where significant. Survival is cumulative (i.e., total survival after the second and third growing seasons). On the vigour scale, 1 = highest and 4 = lowest, thus negative vigour model results indicate improved vigour. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). RCD = root collar diameter. Factor Contrast Z P P P P P PYear 1Site preparation None - tailings removal -1.25 , 0.426 -0.88 97 , 0.658 3.21 97 , 0.005 1.74 97 , 0.194 -1.88 79 , 0.151 8.80 78 , <0.001None - mounding -0.99 , 0.586 -1.13 97 , 0.496 2.21 97 , 0.075 0.45 97 , 0.893 -0.41 79 , 0.913 8.86 78 , <0.001Tailings removal - mounding 0.41 , 0.912 -0.34 97 , 0.938 -1.32 97 , 0.386 -1.71 97 , 0.207 2.02 79 , 0.115 1.56 78 , 0.270Soil transfer No - yes -1.34 , 0.182 -0.41 97 , 0.685 -1.69 97 , 0.093 -1.09 97 , 0.281 0.77 79 , 0.443 -0.71 78 , 0.478Connectivity No - yes -1.56 , 0.119 0.47 97 , 0.637 1.78 97 , 0.078 1.33 97 , 0.186 -1.97 79 , 0.052 3.16 78 , 0.002Interactions:Site prep. = none Transfer: no - yes -2.47 , 0.013 - - - - 0.31 97 , 0.761 - - - -Site prep. = removal Transfer: no - yes 0.54 , 0.590 - - - - -0.74 97 , 0.459 - - - -Site prep. = mounding Transfer: no - yes 0.74 , 0.460 - - - - -2.04 97 , 0.044 - - - -Year 2Site preparation None - tailings removal 9.72 , <0.001 -4.35 93.1 , <0.001 -2.15 93.1 , 0.086 -2.36 93.1 , 0.053 4.70 75 , <0.001 8.36 , <0.001None - mounding 8.64 , <0.001 -4.13 93.1 , <0.001 -2.31 93.1 , 0.059 -3.04 93.1 , 0.008 4.91 75 , <0.001 8.48 , <0.001Tailings removal - mounding -1.82 , 0.162 0.40 93.2 , 0.914 -0.16 93.2 , 0.987 -0.84 93.2 , 0.676 0.08 75 , 0.997 -0.97 , 0.595Soil transfer No - yes 0.08 , 0.933 0.41 93.1 , 0.685 1.08 93.1 , 0.285 0.97 93.1 , 0.332 3.42 75 , 0.001 2.12 , 0.034Connectivity No - yes 4.46 , <0.001 1.51 93.1 , 0.134 3.03 93.1 , 0.003 2.76 93.1 , 0.007 -0.83 75 , 0.410 -0.79 , 0.428Interactions:Site prep. = none Transfer: no - yes -0.79 , 0.432 0.77 93.1 , 0.442 0.96 93.1 , 0.342 1.08 93.1 , 0.285 - - - -Site prep. = removal Transfer: no - yes 2.16 , 0.031 1.53 93.1 , 0.129 3.18 93.1 , 0.002 2.74 93.1 , 0.007 - - - -Site prep. = mounding Transfer: no - yes -0.03 , 0.978 -1.99 93.1 , 0.050 -2.57 93.1 , 0.012 -2.52 93.1 , 0.013 - - - -Transfer = no Connectivity = no - yes - - 2.53 93.1 , 0.013 5.03 93.1 , <0.001 4.29 93.1 , <0.001 - - -2.25 , 0.025Transfer = yes Connectivity = no - yes - - -0.47 93.1 , 0.642 -0.87 93.1 , 0.387 -0.49 93.1 , 0.627 - - 0.66 , 0.510Connectivity = no Transfer: no - yes - - - - 3.27 93.1 , 0.002 2.71 93.1 , 0.008 - - -0.09 , 0.928Connectivity = yes Transfer: no - yes - - - - -2.71 93.1 , 0.008 -2.11 93.1 , 0.037 - - 3.34 , 0.001T df T df T df T df Z /T dfChlorisisSurvival Height growth RCD growth Volume growth Vigour208 Table C-4 cont’d Factor Contrast Z P P P P P PYear 3Site preparation None - tailings removal 9.06 , <0.001 -4.48 90.2 , <0.001 -2.96 90.2 , 0.011 -3.11 90.2 , 0.007 3.95 72 , 0.001 5.66 , <0.001None - mounding 7.76 , <0.001 -5.46 90.1 , <0.001 -4.16 90 , <0.001 -4.25 90.1 , <0.001 4.69 72 , <0.001 5.57 , <0.001Tailings removal - mounding -1.83 , 0.159 -1.08 90.2 , 0.529 -1.42 90.2 , 0.333 -1.35 90.2 , 0.374 0.73 72 , 0.748 -2.12 , 0.086Soil transfer No - yes -0.19 , 0.852 0.56 90.1 , 0.576 -0.60 90.1 , 0.547 0.81 90.1 , 0.420 2.59 72 , 0.012 3.48 , 0.001Connectivity No - yes 5.98 , <0.001 0.52 90.2 , 0.602 0.45 90.3 , 0.654 1.39 90.2 , 0.168 -1.13 72 , 0.260 -0.76 , 0.451Interactions:Site prep. = none Transfer: no - yes -0.88 , 0.379 1.34 90.3 , 0.182 0.50 90.3 , 0.615 1.77 90.2 , 0.080 - - - -Site prep. = removal Transfer: no - yes 2.24 , 0.025 0.88 90.8 , 0.380 0.28 91 , 0.781 1.60 90.5 , 0.114 - - - -Site prep. = mounding Transfer: no - yes -0.96 , 0.337 -1.93 90.1 , 0.057 -2.41 90.1 , 0.018 -2.85 90.1 , 0.005 - - - -Transfer = no Connectivity = no - yes - - 2.24 90.4 , 0.027 5.03 93.1 , <0.001 3.08 90.3 , 0.003 -2.14 72 , 0.036 -3.01 , 0.003Transfer = yes Connectivity = no - yes - - -1.64 90.1 , 0.103 -0.87 93.1 , 0.387 -1.27 90.1 , 0.208 0.42 72 , 0.678 0.71 , 0.477Connectivity = no Transfer: no - yes - - 2.09 90.1 0.040 0.87 90.1 , 0.386 2.46 90.1 , 0.016 - - - -Connectivity = yes Transfer: no - yes - - -1.88 90.4 0.063 -2.18 90.5 , 0.032 -1.98 90.3 , 0.051 - - - -Year 1-3Site preparation None - tailings removal - - -3.81 90.2 , 0.001 -3.62 94.1 , 0.001 -4.84 95.1 , <0.001 - - - -None - mounding - - -4.76 90.1 , <0.001 -4.10 94.1 , <0.001 -5.69 95.1 , <0.001 - - - -Tailings removal - mounding - - -1.08 90.2 , 0.530 -0.54 94.1 , 0.854 -1.04 95.1 , 0.552 - - - -Soil transfer No - yes - - 0.64 90.1 , 0.527 0.35 94.1 , 0.729 1.38 95.1 , 0.171 - - - -Connectivity No - yes - - 0.41 90.2 , 0.685 2.20 94.1 , 0.030 1.84 95.1 , 0.069 - - - -Interactions:Site prep. = none Transfer: no - yes - - 1.34 90.3 , 0.182 1.19 94.1 , 0.237 1.96 95.1 , 0.053 - - - -Site prep. = removal Transfer: no - yes - - 0.88 90.8 , 0.380 1.66 94.2 , 0.099 1.18 95.1 , 0.243 - - - -Site prep. = mounding Transfer: no - yes - - -1.93 90.1 , 0.057 -2.89 94.1 , 0.005 -1.38 95.1 , 0.172 - - - -Transfer = no Connectivity = no - yes - - 2.72 90.3 , 0.008 3.63 94.1 , 0.001 3.09 95.1 , 0.003 - - - -Transfer = yes Connectivity = no - yes - - -2.33 90.1 , 0.022 -0.54 94.1 , 0.588 -0.54 95.1 , 0.591 - - - -Connectivity = no Transfer: no - yes - - 2.65 90 0.010 2.04 94.1 , 0.044 2.43 95.1 , 0.017 - - - -Connectivity = yes Transfer: no - yes - - -2.52 90 0.014 -2.27 94.1 , 0.025 -1.06 95.1 , 0.291 - - - -ChlorisisT df T df T df Z ZSurvival Height growth RCD growth Volume growth Vigour209 Table C-5 Post hoc contrasts of soil transfer, site preparation and forest belowground connectivity effects on spruce seedling biomass, foliar nitrogen (N) ratio and foliar δ13C. Contrasts were run on linear mixed models. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). δ13C data were not available for four of eight treatments in one till trial block. Factor Contrast P P P P PTill ZoneSite preparation None - mounding 0.76 107 , 0.451 107 , 107 , 0.49 108 , 0.625 3.07 92.7 , 0.003Soil transfer No - yes 2.06 107 , 0.042 107 , 107 , -1.86 107 , 0.066 -2.42 92.7 , 0.017Connectivity No - yes 1.27 107 , 0.207 107 , 107 , 2.84 107 , 0.005 1.89 92.7 , 0.062Tailings zoneSite preparation None - tailings removal -4.27 117 , <0.001 -0.96 117 , 0.603 6.88 119 , <0.001 -3.05 119 , 0.008 0.34 117 , 0.940None - mounding -4.43 117 , <0.001 -2.19 117 , 0.078 6.05 119 , <0.001 -1.66 119 , 0.225 -0.37 117 , 0.926Tailings removal - mounding -0.11 117 , 0.994 -1.55 117 , 0.273 -1.24 118 , 0.433 1.86 119 , 0.156 -0.92 117 , 0.630Soil transfer No - yes 0.28 117 , 0.780 0.49 117 , 0.624 0.71 118 , 0.477 -0.48 119 , 0.664 0.47 117 , 0.641Connectivity No - yes 3.52 117 , 0.001 3.33 117 , 0.001 -0.24 118 , 0.810 0.84 119 , 0.404 0.88 117 , 0.383Interactions:Site prep. = yes Transfer: no - yes 0.73 117 , 0.469 1.09 117 , 0.276 0.81 118 , 0.419 -0.45 119 , 0.651 -1.17 117 , 0.245Site prep. = removal Transfer: no - yes 1.67 117 , 0.098 1.62 117 , 0.109 0.25 118 , 0.806 -0.20 119 , 0.844 2.29 117 , 0.024Site prep. = mounding Transfer: no - yes -2.29 117 , 0.024 -2.36 117 , 0.020 -0.01 118 , 0.996 -0.11 119 , 0.917 0.45 117 , 0.651Foliar NT dfFoliar δ13CT dfShoot biomass Root biomass Root:shootT df T df T df210 Table C-6 Sanger sequencing results for ectomycorrhizal fungal morphotype vouchers from the roots of harvested spruce seedlings. Fungal taxa were assigned using a 97% sequence similarity threshold. EM fungal taxa Seedling ID Zone Site preparation Transfer ConnectivityThelephora terrestris F16 Till mounding yes noF39 Till mounding no yesF42 Till mounding no noF54 Till none no noF66 Till none no yesF66 Till none no yesF70 Till none yes yesF90 Till mounding yes yesF94 Till none yes noF96 Till none yes noF122 Tailings tailings removal yes noF124 Tailings tailings removal no noF183 Tailings none yes yesF197 Tailings mounding yes yesWilcoxina rehmii F35 Till mounding yes yesF35 Till mounding yes yesF53 Till mounding yes yesF110 Tailings tailings removal no yesF181 Tailings none yes yesF207 Tailings mounding yes noF215 Tailings tailings removal no yesWilcoxina sp. 1 F1 Till none yes noF2 Till none yes noF44 Till mounding no noF96 Till none yes noF133 Tailings none yes yesF143 Tailings none yes yesF153 Tailings tailings removal yes noWilcoxina sp. 2 F16 Till mounding yes noF45 Till mounding no noF60 Till none yes noF108 Tailings mounding yes noTuber sp. F36 Till mounding yes yesF195 Tailings mounding no noTuber anniae F129 Tailings mounding no yesF131 Tailings mounding no yesF219 Tailings none no yesAmphinema sp. 1 F210 Tailings mounding no yesAmphinema byssoides F89 Till mounding yes yesPseudotomentella sp. F38 Till mounding no yesF141 Tailings none yes yesTomentella sp. F126 Tailings mounding yes yesSebacina sp. F37 Till mounding yes yesInocybe rufoalba F48 Till mounding yes yesClavulina coralloides F206 Tailings mounding yes no211 Table C-7 High-throughput sequencing results for ectomycorrhizal (EM) fungal morphotype vouchers from the roots of harvested spruce seedlings. Samples with poor amplification or no dominant EM taxa (> 10% of reads) were assigned as non-mycorrhizal (grey highlighting). Samples with poor amplification but a dominant EM taxon that aligned with morphology from morphotyping were retained. Blue highlighting denotes secondary EM taxa (not the most abundant) also present in vouchers i.e., mixed vouchers. Sample F148 was assigned to Russula nauseosa despite it being the second most abundant EM taxa based on morphology recorded during morphotyping. Seedling ID ZoneSitepreparation Transfer Connectivity EM fungal taxaProportionof reads (%)Rank AbundancePairwiseidentity (%)TotalreadsF43 Till mounding no no Thelephora terrestris 81.8 1 97.87 10,561 F44 Till mounding no no Thelephora terrestris 72.7 1 0.9848 12,646 Inocybe petiginosa 21.0 2 99.95F53 Till none no no Thelephora terrestris 97.0 1 0.9951 15,664 F70 Till none yes yes Thelephora terrestris 64.3 1 99.51 16,830 Amphinema sp. 2 30.5 2 84.76F84 Till mounding no yes Thelephora terrestris 56.1 1 99.51 11,955 Pustularia sp. 14.2 2 100Tuber anniae 10.1 3 99.97F90 Till mounding yes yes Thelephora terrestris 65.0 1 99.51 13,187 F101 Till mounding no no Thelephora terrestris 64.3 1 98.48 7,160 F148 Tailings tailings removal no yes Thelephora terrestris 43.6 1 99.73 4,599 Russula nauseosa 24.3 2 99.91F194 Tailings mounding no no Thelephora terrestris 66.1 1 0.9951 2,638 F1 Till none yes no Wilcoxina rehmii 47.6 1 100 18,818 Wilcoxina sp. 1 19.3 2 96.37Tuber anniae 14.0 3 99.27F28 Till none no yes Wilcoxina rehmii 31.0 1 100 13,628 F34 Till mounding yes yes Wilcoxina rehmii 46.0 1 100 9,985 Amphinema byssoides 30.0 2 99.98Wilcoxina sp. 1 11.1 3 94.51F81 Till mounding no yes Wilcoxina rehmii 55.1 1 100 11,898 Pustularia sp. 34.6 2 100F191 Tailings tailings removal yes no Wilcoxina rehmii 11.5 2 99.99 5,809 Tuber anniae 11.0 3 99.99Thelephora terrestris 10.1 4 99.51F201 Tailings tailings removal no no Wilcoxina rehmii 24.0 2 99.99 1,711 F173 Tailings mounding yes yes Wilcoxina sp. 1 51.2 1 93.63 6,552 212 Table C-7 cont’d Seedling ID ZoneSitepreparation Transfer Connectivity EM fungal taxaProportionof reads (%)Rank AbundancePairwiseidentity (%)TotalreadsF201 Tailings tailings removal no no Wilcoxina sp. 1 52.8 1 96.37 11,434 Amphinema byssoides 37.6 2 99.35F108 Tailings mounding yes no Wilcoxina sp. 2 50.9 1 94.1 7,741 Thelephora terrestris 21.8 2 99.5Peziza sp. 12.8 3 91.04F38 Till mounding no yes Amphinema sp. 4 98.6 1 99.97 6,552 F40 Till mounding no yes Amphinema sp. 2 48.7 1 84.76 10,519 Amphinema sp. 4 22.5 2 99.97F78 Till none no yes Amphinema sp. 2 46.6 1 84.76 974 Pezoloma ciliifera 14.9 2 98.89F78 Till none no yes Amphinema sp. 2 40.8 1 84.76 11,026 Wilcoxina rehmii 38.9 2 99.99F113 Tailings mounding no no Amphinema byssoides 32.7 1 99.99 3,292 Tuber anniae 31.9 2 100F194 Tailings mounding no no Wilcoxina sp. 1 62.5 1 96.37 8,836 Amphinema sp. 2 18.4 2 84.76F201 Tailings tailings removal no no Amphinema sp. 2 29.0 1 84.52 252 F30 Till mounding no no Tuber anniae 50.9 1 100 8,645 F30 Till mounding no no Tuber anniae 89.5 1 100 18,038 F41 Till mounding no yes Tuber anniae 11.6 2 99.27 4,782 F195 Tailings mounding no no Tuber sp. 74.1 1 82.54 1,800 F40 Till mounding no yes Cenocuccum sp. 84.5 1 90.24 606 F15 Till none yes yes Sebacina epigaea 35.5 1 100 19,555 F51 Till none no no Lactarius sp. 50.7 1 71.51 2,400 F103 Till mounding no no Endogone sp. 48.7 1 83.57 9,329 F30 Till mounding no no Non-mycorrhizal - - - 4,270 F44 Till mounding no no Non-mycorrhizal - - - 5,127 F77 Till none no yes Non-mycorrhizal - - - 864 F107 Tailings mounding yes no Non-mycorrhizal - - - 5,491 F143 Tailings none yes yes Non-mycorrhizal - - - 219 F193 Tailings mounding no no Non-mycorrhizal - - - 7,901 F198 Tailings mounding yes no Non-mycorrhizal - - - 908 213 Table C-8 Post hoc contrasts of site preparation, soil transfer and forest belowground connectivity effects on spruce seedling ectomycorrhizal fungal percent colonization, richness and Shannon diversity. Contrasts were run on binomial generalized linear mixed models (colonization) and linear mixed models (richness and diversity). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Table C-9 Relative abundance and taxa richness of saprotrophic, ectomycorrhizal (EM), and plant pathogenic fungi amplicon sequence variants (ASVs) in spruce seedling rooting zone soil in the tailings and till zones compared to the reference forest. Factor Contrast P P PTill ZoneSite preparation None - mounding 1.04 Inf , 0.296 -1.32 107 , 0.191 -2.12 107 , 0.037Soil transfer No - yes -1.80 Inf , 0.071 -0.85 107 , 0.399 -1.91 107 , 0.059Connectivity No - yes -1.40 Inf , 0.162 0.37 107 , 0.712 1.33 107 , 0.186Tailings zoneSite preparation None - tailings removal -6.62 117 , <0.001 -3.66 117 , 0.001 -1.87 117 , 0.152None - mounding -5.22 117 , <0.001 -3.11 117 , 0.007 -1.34 117 , 0.377Tailings removal - mounding 2.01 117 , 0.116 0.79 117 , 0.708 0.73 117 , 0.749Soil transfer No - yes -1.08 117 , 0.283 -2.31 117 , 0.023 -1.73 117 , 0.087Connectivity No - yes -2.04 117 , 0.045 1.43 117 , 0.155 1.57 117 , 0.118Interactions:Site prep. = none Transfer: no - yes -2.86 117 , 0.005 -1.59 117 , 0.114 -1.13 117 , 0.263Site prep. = removal Transfer: no - yes 0.17 117 , 0.862 -0.84 117 , 0.400 -0.28 117 , 0.781Site prep. = moundingTransfer: no - yes 1.59 117 , 0.115 -1.58 117 , 0.118 -1.65 117 , 0.102Transfer = no Connectivity: no - yes -3.11 117 , 0.003 1.41 117 , 0.162 1.65 117 , 0.101Transfer = yes Connectivity: no - yes 0.29 117 , 0.770 0.62 117 , 0.537 0.57 117 , 0.567Z / T df T df T dfPercent colonization Richness Shannon diversityZoneForest 20.3 ± 7.1 46.5 ± 6.2 7.9 ± 3.6 20.7 ± 2.8 30.8 ± 3.8 8.4 ± 1.3Till 17.9 ± 1.2 44.5 ± 2.5 14.6 ± 1.2 28.6 ± 1.1 23.0 ± 1.1 15.5 ± 0.6Tailings 21.3 ± 1.4 33.6 ± 2.0 7.4 ± 0.8 32.7 ± 1.4 23.2 ± 1.0 11.5 ± 0.6Relative abundance (%) RichnessSaprotrophs EM fungi Plant pathogens Saprotrophs EM fungi Plant pathogens214 Table C-10 PERMANOVA results of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of spruce seedling rooting zone soil fungal communities (Hellinger transformed) with distance from forest edge as a covariate. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Df SS MeanSq F R2PDistance 3 1.28 0.43 1.19 0.036 0.002Mounding 1 0.52 0.52 1.44 0.014 0.001Soil transfer 1 0.69 0.69 1.91 0.019 0.001Connectivity 1 0.37 0.37 1.03 0.010 0.051Mounding x Soil transfer 1 0.30 0.30 0.83 0.008 0.531Mounding x Connectivity 1 0.42 0.42 1.16 0.012 0.01Soil transfer x Connectivity 1 0.33 0.33 0.92 0.009 0.212Mounding x Soil transfer x Connectivity 1 0.30 0.30 0.82 0.008 0.539Residuals 88 31.68 0.36 0.883Total 98 35.88 1.000Tailings zoneDistance 3 1.39 0.46 1.30 0.034 0.001Site preparation 2 1.44 0.72 2.01 0.036 0.001Soil transfer 1 0.67 0.67 1.89 0.017 0.001Connectivity 1 0.43 0.43 1.19 0.011 0.024Site preparation x Soil transfer 2 0.95 0.48 1.33 0.024 0.001Soil transfer x Connectivity 1 0.48 0.48 1.33 0.012 0.003Residuals 98 35.01 0.36 0.867Total 108 40.36 1.000Till zone215 Table C-11 Post hoc contrasts of mounding, soil transfer and forest belowground connectivity effects on richness, diversity and abundance of spruce seedling rooting zone soil fungal amplicon sequencing variants (ASVs) and guilds in the till zone. Contrasts were run on linear mixed models (richness and diversity) and binomial generalized linear mixed models (abundance). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Contrast P P P PTill zoneSite preparation None - mounding 0.77 107 , 0.445 0.47 107 , 0.639 0.70 109 , 0.483 0.80 107 , 0.426Soil transfer No - yes -2.18 107 , 0.031 -1.52 107 , 0.133 -0.78 109 , 0.435 -3.25 107 , 0.002Connectivity No - yes -0.54 107 , 0.589 -1.66 107 , 0.100 0.88 109 , 0.381 0.55 107 , 0.584Interactions:Site prep. = no Connectivity: no - yes -2.35 107 , 0.021 -2.78 107 , 0.006 -0.72 109 , 0.473 -1.32 107 , 0.191Site prep. = mounding Connectivity: no - yes 1.53 107 , 0.129 0.39 107 , 0.698 1.93 109 , 0.057 2.05 107 , 0.043Connectivity = no Site prep.: no - mounding -1.40 107 , 0.165 -1.24 107 , 0.218 -0.84 109 , 0.405 -1.12 107 , 0.264Connectivity = yes Site prep.: no - mounding 2.47 107 , 0.015 1.89 107 , 0.061 1.82 109 , 0.071 2.24 107 , 0.027Factor Contrast P P P PTill zoneSite preparation None - mounding -0.06 107 , 0.956 -0.16 86 , 0.872 -1.49 86 , 0.141 1.08 86 , 0.284Soil transfer No - yes -0.39 107 , 0.696 1.87 86 , 0.065 -0.13 86 , 0.901 -0.99 86 , 0.326Connectivity No - yes -1.08 107 , 0.282 -1.50 86 , 0.137 -0.15 86 , 0.879 1.43 86 , 0.157Interactions:Site prep. = no Connectivity: no - yes -2.19 107 , 0.031 1.25 86 , 0.214 -0.37 86 , 0.716 -0.45 86 , 0.653Site prep. = yes Connectivity: no - yes 0.62 107 , 0.538 -1.51 86 , 0.136 -1.72 86 , 0.089 1.91 86 , 0.060Connectivity = no Site prep.: no - mounding -1.44 107 , 0.154 1.25 86 , 0.214 -0.37 86 , 0.716 -0.45 86 , 0.653Connectivity = yes Site prep.: no - mounding 1.35 107 , 0.181 -1.51 86 , 0.136 -1.72 86 , 0.089 1.91 86 , 0.060Plant pathogens EM fungiRichnessFungal ASVs SaprotrophsRelative abundanceT df T dfT df T dfShannon DiversityT df T dfT df T df216 Table C-12 Post hoc contrasts of distance covariate effects on spruce seedling rooting zone soil fungi, bacteria and fauna in the till and tailings zones. Contrasts were run on linear mixed models (richness and diversity), binomial generalized linear mixed models (relative abundance) and Poisson generalized linear mixed models (faunal abundance). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). ASV = amplicon sequencing variant. Contrast P P P Z P1 m - 3 m -1.39 86 , 0.511 -0.75 82 , 0.876 0.04 82 , 0.043 -0.16 , 0.9991 m - 8 m 1.31 86 , 0.559 -0.92 82 , 0.793 0.01 82 , 0.009 -0.60 , 0.9321 m - 15 m 2.37 86 , 0.091 -2.58 82 , 0.056 0.00 82 , 0.002 3.15 , 0.0093 m - 8 m 2.71 86 , 0.040 -0.19 82 , 0.998 0.92 82 , 0.919 -0.45 , 0.9703 m - 15 m 3.73 86 , 0.002 -1.86 82 , 0.252 0.71 82 , 0.709 3.28 , 0.0068 m - 15 m 1.12 86 , 0.677 -1.64 82 , 0.361 0.98 82 , 0.976 3.62 , 0.002ContrastContrast P P P P P Z P1 m - 3 m 0.07 118 , 1.000 0.51 117 , 0.956 -0.29 119 , 0.992 -1.15 81 , 0.660 0.35 81 , 0.985 -0.54 , 0.9501 m - 8 m 0.81 118 , 0.851 1.55 117 , 0.410 0.53 119 , 0.952 -0.08 81 , 1.000 -1.21 81 , 0.626 -0.83 , 0.8411 m - 15 m 2.69 121 , 0.040 2.37 118 , 0.089 2.05 121 , 0.177 2.31 81 , 0.104 -2.29 81 , 0.109 3.20 , 0.0083 m - 8 m 0.75 117 , 0.879 1.05 117 , 0.720 0.83 119 , 0.842 1.06 81 , 0.715 -1.54 81 , 0.420 -0.29 , 0.9913 m - 15 m 2.66 121 , 0.043 1.94 118 , 0.217 2.32 121 , 0.099 3.25 81 , 0.009 -2.57 81 , 0.057 3.63 , 0.0028 m - 15 m 2.01 121 , 0.189 1.02 118 , 0.738 1.60 121 , 0.380 2.36 81 , 0.093 -1.28 81 , 0.576 3.85 , 0.001T dfT dfChloroflexi relative abundanceTailings zoneFirmicutes relative abundanceTill zoneT dfT df T df T df T dfT dfFaunal abundanceFungal ASV richness EM fungal richnessSaprotroph fungal richnessActinobacteria relative abundanceAcidobacteria relative abundanceFaunal abundanceFungal plant pathogen relative abundance217 Table C-13 Post hoc contrasts of site preparation, soil transfer and forest belowground connectivity effects on richness, diversity and abundance of spruce seedling rooting zone soil amplicon sequencing variants (ASVs) and guilds in the tailings zone. Contrasts were run on linear mixed models (richness and diversity) and binomial generalized linear mixed models (abundance). Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Contrast P P P PTailings zoneSite preparation None - tailings removal -5.06 118 , < 0.001 -4.55 119 , < 0.001 -5.12 119 , < 0.001 -3.82 117 , 0.001None - mounding -4.29 118 , < 0.001 -3.89 119 , 0.001 -3.74 119 , 0.001 -4.45 117 , < 0.001Tailings removal - mounding 1.12 118 , 0.506 0.96 119 , 0.601 1.89 119 , 0.145 -0.70 117 , 0.761Soil transfer No - yes -3.46 117 , 0.001 -3.29 119 , 0.001 -2.60 119 , 0.011 -3.22 117 , 0.002Connectivity No - yes 0.16 118 , 0.875 0.37 119 , 0.714 -1.05 119 , 0.296 -0.49 117 , 0.626Interactions:Site prep. = none Transfer: no - yes -3.97 118 , < 0.001 -4.05 119 , < 0.001 -2.88 119 , 0.005 -3.84 117 , < 0.001Site prep. = removal Transfer: no - yes -0.62 118 , 0.534 -0.73 119 , 0.466 -0.36 119 , 0.720 0.25 117 , 0.802Site prep. = mounding Transfer: no - yes -0.51 118 , 0.610 0.09 119 , 0.925 -0.68 119 , 0.500 -1.13 117 , 0.261Connectivity = no Transfer: no - yes -3.79 118 , < 0.001 -3.35 119 , 0.001 -2.91 119 , 0.004 -3.23 117 , 0.002Connectivity = yes Transfer: no - yes -0.57 117 , 0.570 -0.91 119 , 0.363 -0.34 119 , 0.735 -0.97 117 , 0.336Factor Contrast P P P PTailings zoneSite preparation None - tailings removal -4.10 117 , < 0.001 1.11 96 , 0.510 -2.95 96 , 0.011 -0.34 96 , 0.937None - mounding -3.11 117 , 0.007 0.78 96 , 0.717 -2.15 96 , 0.086 -1.66 96 , 0.227Tailings removal - mounding 1.37 117 , 0.359 -0.43 96 , 0.903 1.14 96 , 0.493 -1.67 96 , 0.222Soil transfer No - yes -3.20 117 , 0.002 2.14 96 , 0.035 -3.01 96 , 0.003 -0.49 96 , 0.623Connectivity No - yes -0.23 117 , 0.822 0.34 96 , 0.735 -0.84 96 , 0.402 -0.43 96 , 0.666Interactions:Site prep. = none Transfer: no - yes -4.53 117 , < 0.001 1.77 96 , 0.080 -2.81 96 , 0.006 -0.65 96 , 0.514Site prep. = removal Transfer: no - yes 0.28 117 , 0.777 0.53 96 , 0.601 -0.35 96 , 0.731 0.34 96 , 0.738Site prep. = mounding Transfer: no - yes -0.04 117 , 0.972 1.27 96 , 0.209 -1.59 96 , 0.116 -0.40 96 , 0.693Connectivity = no Transfer: no - yes -2.88 117 , 0.005 1.73 96 , 0.087 -2.66 96 , 0.009 0.48 96 , 0.632Connectivity = yes Transfer: no - yes -1.43 117 , 0.155 1.20 96 , 0.232 -1.65 96 , 0.102 -1.57 96 , 0.120Fungal ASVs Saprotrophs Plant pathogens EM fungiT df T dfT df T dfRichnessRelative abundanceShannon DiversityT df T dfT df T df218 Table C-14 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity combined), distance from forest edge, soil properties (pH, organic matter, moisture and electrical conductivity) and naturally ingressing plant community on spruce seedling rooting zone soil fungal and bacterial communities (Bray Curtis distances of Hellinger-transformed community data). Marginal significance was tested with db-RDAs for each factor conditioned on trial block. Conditional significance was tested with RDAs of each factor conditioned on the other factors and trial block. Significance was tested with ANOVA run on 999 permutations restricted within levels of block. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor df SumofSqs F P df Variance F P df SumofSqs F P df Variance F PTill zoneTreatment 7 2.92 1.37 0.001 7 0.06 1.16 0.006 7 2.25 1.53 0.001 7 0.05 1.29 0.001Distance 3 1.26 1.36 0.001 1 0.01 1.66 0.001 3 0.99 1.53 0.001 1 0.01 1.70 0.001Soil 4 2.05 1.68 0.001 4 0.05 1.61 0.001 4 2.18 2.68 0.001 4 0.05 2.25 0.001Vegetation 2 0.73 1.16 0.064 2 0.02 1.08 0.209 2 0.54 1.24 0.091 2 0.02 1.57 0.0010.58 (total variance) 0.40 (total variance)Tailings zone 7 4.00 1.70 0.001 5 0.05 1.47 0.001 7 3.97 2.42 0.001 5 0.04 1.43 0.001Treatment 7 4.00 1.70 0.001 5 0.05 1.47 0.001 7 3.97 2.42 0.001 5 0.04 1.43 0.001Distance 3 1.37 1.31 0.001 1 0.01 1.37 0.003 3 1.05 1.36 0.014 1 0.01 1.20 0.069Soil 4 2.31 1.69 0.001 4 0.04 1.27 0.001 4 3.21 3.42 0.001 4 0.04 1.79 0.001Vegetation 2 1.23 1.77 0.001 2 0.02 1.60 0.001 2 1.83 3.74 0.001 2 0.02 1.85 0.0010.66 (total variance) 0.46 (total variance)Soil fungal community Soil bacterial communityMarginal effect Conditional effect Marginal effect Conditional effect219 Table C-15 PERMANOVA results of site preparation (mounding in the till zone, tailings removal and mounding in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of spruce seedling rooting zone soil bacterial communities (Hellinger transformed) with distance from forest edge as a covariate. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Df SS MeanSq F R2PDistance 3 1.03 0.34 1.16 0.036 0.001Mounding 1 0.55 0.55 1.86 0.019 0.001Soil transfer 1 0.44 0.44 1.48 0.016 0.002Connectivity 1 0.21 0.21 0.71 0.007 0.536Mounding x Soil transfer 1 0.28 0.28 0.94 0.010 0.128Mounding x Connectivity 1 0.42 0.42 1.41 0.015 0.003Soil transfer x Connectivity 1 0.19 0.19 0.65 0.007 0.683Mounding x Soil transfer x Connectivity 1 0.21 0.21 0.71 0.007 0.450Residuals 84 24.96 0.30 0.88Total 94 28.30 1.00Tailings zoneDistance 3 1.17 0.39 1.48 0.043 0.004Site preparation 2 2.25 1.13 4.30 0.084 0.001Soil transfer 1 0.36 0.36 1.36 0.013 0.031Connectivity 1 0.22 0.22 0.85 0.008 0.513Site preparation x Soil transfer 2 0.67 0.33 1.28 0.025 0.028Soil transfer x Connectivity 1 0.42 0.42 1.61 0.016 0.015Residuals 83 21.74 0.26 0.810Total 93 26.83 1.000Till zone220 Table C-16 Post hoc contrasts of linear mixed models of site preparation, soil transfer and forest belowground connectivity effects on richness, diversity and abundance of spruce seedling rooting zone soil bacterial amplicon sequencing variant (ASV) richness and Shannon diversity. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Contrast P PTill ZoneSite preparation None - mounding 0.07 106 , 0.941 -0.38 106 , 0.707Soil transfer No - yes -1.06 106 , 0.293 -1.77 106 , 0.080Connectivity No - yes -1.16 105 , 0.248 -0.94 105 , 0.349Interactions:Site prep. = no Connectivity: no - yes -1.38 106 , 0.170 -1.76 105 , 0.081Site prep. = mounding Connectivity: no - yes -0.25 105 , 0.803 0.45 105 , 0.653Tailings zoneSite preparation None - tailings removal -1.59 105 , 0.254 -2.67 104 , 0.024None - mounding -1.25 104 , 0.428 -2.24 103 , 0.069Tailings removal - mounding 0.51 103 , 0.868 0.66 103 , 0.790Soil transfer No - yes -2.65 103 , 0.010 -3.00 102 , 0.003Connectivity No - yes -0.28 103 , 0.783 -0.53 102 , 0.599Interactions:Site prep. = none Transfer: no - yes -3.03 102 , 0.003 -3.60 102 , 0.001Site prep. = removal Transfer: no - yes -1.41 104 , 0.162 -0.94 103 , 0.352Site prep. = mounding Transfer: no - yes 0.58 103 , 0.563 0.23 103 , 0.819Transfer = no None - tailings removal -2.46 103 0.041 -3.78 102 0.001Transfer = no None - mounding -3.04 102 0.009 -3.98 102 0.000Transfer = no Tailings removal - mounding -0.64 102 0.796 -0.13 102 0.991Transfer = yes None - tailings removal 0.13 105 0.991 -0.11 104 0.993Transfer = yes None - mounding 1.14 104 0.494 0.67 103 0.783Transfer = yes Tailings removal - mounding 1.37 104 0.359 1.06 103 0.539Richness Shannon diversityT df T df221 Table C-17 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity combined), distance from forest edge and soil properties (pH, organic matter, moisture and electrical conductivity) and naturally ingressing plant community effects on spruce seedling rooting zone soil faunal communities (Bray Curtis distances). Marginal significance was tested with db-RDAs for each factor conditioned on trial block. Conditional significance was tested with RDAs of each factor conditioned on the other factors and trial block. Soil pH had a significant marginal effect in the tailings zone (P = 0.045) and was used to assess conditional effects. Significance was tested with ANOVA run on 999 permutations restricted within levels of block. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor df SumofSqs F P df Variance F PTill zoneTreatment 7 2.78 0.99 0.502 - - - -Distance 3 1.20 1.00 0.461 - - - -Soil 4 1.66 1.05 0.425 - - - -Vegetation 2 0.49 0.61 0.922 - - - -Tailings zoneTreatment 7 3.32 1.64 0.001 5 0.97 1.46 0.097Distance 3 1.01 1.11 0.280 - - - -Soil 4 1.32 1.09 0.286 1 0.15 1.12 0.250Vegetation 2 1.19 2.00 0.005 2 1.08 4.06 0.00313.06 (total variance)Marginal effect Conditional effect222 Table C-18 PERMANOVA results of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of the log-transformed naturally ingressing plant community with distance from forest edge as a covariate. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Df SS MeanSq F R2PDistance 2 0.31 0.16 0.87 0.025 0.244Mounding 1 0.24 0.24 1.34 0.019 0.037Soil transfer 1 0.19 0.19 1.04 0.015 0.15Connectivity 1 0.12 0.12 0.68 0.010 0.5Mounding x Soil transfer 1 0.06 0.06 0.36 0.005 0.909Mounding x Connectivity 1 0.23 0.23 1.28 0.018 0.044Soil transfer x Connectivity 1 0.07 0.07 0.40 0.006 0.858Mounding x Soil transfer x Connectivity 1 0.19 0.19 1.05 0.015 0.16Residuals 62 11.11 0.18 0.887Total 71 12.53 1.000Distance 2 0.61 0.31 1.72 0.035 0.001Site preparation 2 1.64 0.82 4.61 0.095 0.001Soil transfer 1 0.24 0.24 1.36 0.014 0.078Connectivity 1 0.19 0.19 1.09 0.011 0.17Site preparation x Soil transfer 2 0.24 0.12 0.68 0.014 0.736Soil transfer x Connectivity 1 0.17 0.17 0.94 0.010 0.346Residuals 80 14.22 0.18 0.821Total 89 17.32 1.000Till zoneTailings zone223 Table C-19 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity combined), distance from forest edge and soil properties (pH, organic matter, moisture and electrical conductivity) on log-transformed naturally ingressing plant communities (Bray Curtis distances). Marginal significance was tested with db-RDAs for each factor conditioned on trial block. Conditional significance was tested with RDAs of each factor conditioned on the other factors and trial block. Significance was tested with ANOVA run on 999 permutations restricted within levels of block. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor df SumofSqs F P df Variance F PTill zoneTreatment 7 1.34 1.14 0.136 13 13.23 1.85 0.019Distance 2 0.40 1.19 0.201 - - - -Soil 13 2.82 1.37 0.004 7 4.76 1.23 0.05632.32 (total variance)Tailings zoneTreatment 7 2.74 2.05 0.001 5 4.43 1.06 0.296Distance 2 0.67 1.65 0.002 1 2.35 2.82 0.001Soil 13 4.20 1.73 0.001 13 20.29 1.87 0.00159.80 (total variance)Marginal effect Conditional effect224 Table C-20 PERMANOVA results of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfer and forest belowground connectivity effects on Bray Curtis distances of baseline soil chemical parameters with distance from forest edge as a covariate. Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. Bold = significant P-value (P < 0.05); italics = marginally significant (0.05 < P < 0.1). Factor Df SS MeanSq F R2PDistance 2 0.07 0.04 2.00 0.053 0.031Mounding 1 0.04 0.04 2.12 0.028 0.048Soil transfer 1 0.03 0.03 1.41 0.019 0.128Connectivity 1 0.01 0.01 0.77 0.010 0.329Mounding x Soil transfer 1 0.02 0.02 1.10 0.015 0.216Mounding x Connectivity 1 0.02 0.02 1.30 0.017 0.158Soil transfer x Connectivity 1 0.01 0.01 0.35 0.005 0.576Mounding x Soil transfer x Connectivity 1 0.03 0.03 1.59 0.021 0.1Residuals 62 1.10 0.02 0.831Total 71 1.33 1.000Distance 2 0.21 0.10 2.74 0.057 0.031Site preparation 2 0.32 0.16 4.22 0.088 0.003Soil transfer 1 0.02 0.02 0.49 0.005 0.503Connectivity 1 0.00 0.00 0.04 0.000 0.947Site preparation x Soil transfer 2 0.02 0.01 0.25 0.005 0.869Soil transfer x Connectivity 1 0.04 0.04 0.96 0.010 0.294Residuals 80 3.07 0.04 0.834Total 89 3.68 1.000Till zoneTailings zone225 Figure C-1 Field trial block locations in areas impacted by the Tailings Storage Facility embankment failure at the Mount Polley Mine, British Columbia, Canada. Note that some trial blocks locations are in the forest; these areas were logged after aerial imagery was taken due to forest disturbance to facilitate recontouring of steep, eroded banks (till zone) or tree die-off from anaerobic soil conditions created by saturated tailings deposition (tailings zone). 226 Figure C-2 db-RDA of spruce seedling rooting zone soil fungal community (Hellinger-transformed) Bray Curtis dissimilarities between the reference forest, tailings zone and till zone site. The reference forest soil fungal community was highly dissimilar from the tailings zone (Bray Curtis dissimilarity = 0.92) and till zone (Bray Curtis dissimilarity = 0.92) soil, and the till and tailings zones were dissimilar from each other (Bray Curtis dissimilarity = 0.89). 227 Figure C-3 Relative abundance of dominant bacterial phyla (> 1.5% of total sequencing reads) in spruce seedling rooting zone soil in the till zone, tailings zone and reference forest. 228 Figure C-4 Binomial generalized linear mixed model T-value results of effects of site preparation, soil transfers and forest belowground connectivity on bacterial phyla abundance in spruce seedling rooting zone soil. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). OM = organic matter; EC = electrical conductivity. 229 Figure C-5 Relative abundance of soil faunal broad groups in spruce seedling rooting zone soil in the till zone, tailings zone and reference forest. All broad groups are at the order level except Chilopoda is a class, and Acari and Collembola are subclasses. 230 Figure C-6 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity) and soil properties (pH) on soil faunal community composition Bray Curtis dissimilarities in the tailings zone. 231 Figure C-7 db-RDA of natural ingressing plant community (log-transformed) Bray Curtis dissimilarities between the reference forest, tailings zone and till zone site. Plant community composition was highly dissimilar between till and tailings zones (Bray Curtis dissimilarity = 0.78) and from the reference forest (till zone Bray Curtis dissimilarity = 0.93; tailings zone Bray Curtis dissimilarity = 0.92). 232 Figure C-8 Effects of site preparation (mounding in the till zone, mounding and tailings removal in the tailings zone), soil transfers and forest belowground connectivity on plant percent cover (A, C), non-native plant percent cover (B) and Shannon diversity (D) of naturally ingressing plants in the till zone (A, B) and tailings zone (C, D). Bar plots represent main effects and line plots represent interactions. Bars/points are the back-transformed means ± 1 SE from binomial generalized linear mixed models (cover) and linear mixed models (diversity). Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). OM = organic matter; EC = electrical conductivity. 233 Figure C-9 Variation partitioning between treatment effects (soil transfers, site preparation and forest belowground connectivity), distance from forest edge and soil properties (pH, organic matter, moisture and electrical conductivity) on Bray Curtis distances of log-transformed naturally ingressing plant community in the till zone (A) and tailings zone (B). communities. Only marginally significant factors, as tested by db-RDAs for each factor conditioned on trial block, were included in variation partitioning. 234 Figure C-10 Effects of site preparation (mounding and tailings removal) on bulk density in the till and tailings zones, with comparison to reference forest soil. Box centerlines = mean, hinges = 25th and 75th percentiles, whiskers = 1.58 x interquartile range (≈ 95% confidence interval) and points = outliers. 235 Figure C-11 Correlation matrices of soil baseline chemical parameter Pearson correlation coefficients in the till zone (A) and tailings zone (B). Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. EC = electrical conductivity (μS/cm); CEC = effective cation exchange capacity (cmol+/kg); N_tot = total nitrogen (%); C_tot = total carbon (%); P = PO4-P (mg/kg). All metals = exchangeable ions (cmol+/kg). 236 Figure C-12 Effects of site preparation (mounding in the till zone; mounding and tailings removal in the tailings zone; C–H) and mounding x soil transfer interactions (A, B) on spruce seedling rooting zone soil parameters in the till zone (A–D) and tailings zone (E–H). Bar plots represent main effects and line plots represent interactions. Bars/points are back-transformed means ± 1 SE from linear mixed models. Post hoc testing significance is indicated by *** (P < 0.001), ** (0.001 < P < 0.01), * (0.01 < P < 0.05), . (0.05 < P < 0.1) or NS (not significant). OM = organic matter; EC = electrical conductivity. C.1 Supplemental baseline soil data Mounding tended to reduce bulk density across both zones (P = 0.095; Figure C-10). In the tailings zone, tailings removal also reduced bulk density (P = 0.04) and there was non-significant trend towards lower bulk density with tailings removal than mounding (Figure C-10). Soil bulk density was lower in the reference forest than in the till zone (P < 0.001) and the tailings zone (P = 0.007; Figure C-10). Site preparation, but not forest belowground connectivity or distance from forest edge, significantly affected baseline soil chemical parameters in both zones (till 237 PERMANOVA: R2 = 0.03, P = 0.048; tailings PERMANOVA R2 = 0.09, P = 0.003; Figure C-13; Table C-20). Baseline soil chemistry also varied significantly with distance from forest edge in both zones (till: PERMANOVA R2 = 0.05, P = 0.031; tailings: PERMANOVA R2 = 0.06, P = 0.031), supporting the inclusion of distance as a covariate in statistical models of treatment effects. For tailings, there was a significant positive relationship between distance and pre-site preparation tailings depth (F2 = 6.15, P = 0.003). In the till zone, soil pH was higher with site preparation (T79.3, = 2.99, P = 0.004) and forest belowground connectivity (T79.3, = 2.17, P = 0.033), and was higher at 11–15 m from the forest edge compared to 1–5 m (T72.1, = 4.76, P < 0.001) and 6–10 m (T72.1, = 3.27, P = 0.005). pH was significantly correlated with all parameters (P ≤ 0.001), where it was positively correlated with electrical conductivity, Ca, K, Mg Na and effective CEC, and negatively correlated with Al, Fe, Mn, total N and available P (Figure C-11). Total C was less strongly related to pH (P = 0.031; Figure C-11). Baseline total C was not affected by distance from forest edge, but it was lower with site preparation in treatments with forest belowground connectivity (T79.3, = -3.19, P = 0.002). Similarly, treatments with connectivity tended to have lower total soil C with site preparation (T79.3, = -1.78, P = 0.079), but higher total C in treatments without site preparation (T79.3, = 1.81, P = 0.075). Conversely, in the tailings zone, pH was not affected by distance from forest edge and was lower with site preparation (mounding: T91.2, = -11.56, P < 0.001, removal: T91.2, = -14.25, P < 0.001) with lower pH following removal than mounding (T91.2, = -3.30, P = 0.004). All parameters (Al, Ca, Fe, K, Mg, Mn, NA, effective CEC, total N, total C and available P) were significantly negatively correlated with pH (P < 0.001; Figure C-11) except electrical conductivity, which was not affected by site preparation but was higher at 11–15 m from forest 238 edge compared to 1–5 m (T92.1, = 3.28, P = 0.004). pH was also positively related to pre-site preparation tailings depth (P = 0.002; Figure C-11). Chemistry data (by zone and site preparation method) are presented in Table C-21 and indicates that forest soil used for transfers generally had lower pH, total N and C, available P and effective CEC compared to the treatment zones. 239 Table C-21 Soil chemical properties of forest soil used in transfers for block and mean baseline soil properties of the till and tailings zones by site preparation type. Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. CEC = cation exchange capacity; EC = electrical conductivity. pH EC (μS/cm) Al Ca Fe K Mg Mn Na CEC Total N (%) Total C (%) PO4-P (mg/kg)Transfer soilTill block 1 5.33 169.1 0.071 31.71 0.004 0.70 4.12 0.213 0.060 36.89 0.510 11.81 26.0Till block 2 5.34 98.0 0.785 13.14 0.007 0.45 1.62 0.267 0.064 16.34 0.327 7.38 13.1Till block 3 5.60 137.8 0.089 25.02 0.007 0.66 5.82 0.125 0.060 31.78 0.484 10.01 61.3Tailings block 1 5.64 165.3 0.140 41.41 0.004 0.82 8.30 0.235 0.189 51.09 0.829 22.53 29.6Tailings block 2 5.75 106.5 0.098 34.95 0.003 0.68 7.21 0.051 0.112 43.11 0.559 9.49 17.7Tailings block 3 5.24 86.4 0.565 5.70 0.006 0.23 1.11 0.221 0.033 7.86 0.133 2.87 105.4Till zoneNo site preparation 6.87 121.0 0.200 8.14 0.002 0.15 1.73 0.022 0.051 10.29 0.050 0.93 14.8Mounding 7.16 110.7 0.059 8.81 0.001 0.14 2.09 0.021 0.059 11.18 0.044 0.79 7.9No site preparation 8.03 212.4 0.006 6.62 0.001 0.18 0.40 0.005 0.051 7.25 0.029 0.81 3.2Mounding 6.72 210.5 0.019 15.25 0.001 0.18 1.73 0.058 0.081 17.32 0.124 2.49 10.2Tailings removal 6.41 198.5 0.047 17.01 0.001 0.24 2.27 0.084 0.100 19.75 0.165 3.17 10.2Barium Chloride Exchangeable Cations and Effective CEC (cmol+/kg)BlockTailing zone240 Figure C-13 db-RDA of site preparation effects on Bray Curtis distances of baseline soil chemical properties. Baseline soil samples were collected following site preparation and installation of forest belowground connectivity trenching treatments. Full models included soil transfer, forest belowground connectivity and treatment interactions, and a distance from forest edge covariate (Table C-20). PERMANOVA analysis found significant effects of site preparation in the till zone (P = 0.048) and the tailings zone (P = 0.003). 241 Appendix D Chapter 5 PLFA analyses D.1 Chapter 5 PLFA analyses PLFA analysis was conducted to assess potential changes in the soil microbial community of the control soil stored at 4 °C during the conditioning phase. PLFAs were run on forest soil samples from each reference site (n = 3; Table A-1) following soil collection and after the storage period. The storage period was twelve months due to: two months between initial collection and experiment initiation; having to re-start the conditioning phase after two months due to poor germination and survival; a six month growing period; and two months to process conditioning phase seedlings, including morphotyping of EM fungi on spruce seedling roots. Comparisons of PLFA total or broad group biomass from fresh and stored forest soil were done using linear models with storage treatment (baseline or stored) as the response variable. PLFA analysis of forest soil samples to assess potential control soil inoculum loss from refrigeration during the conditioning phase showed that total PLFA biomass significantly increased (P = 0.016; Figure D-1). Similarly, biomass of all broad microbial groups increased (P < 0.05), yet, there were no significant differences in the proportion of each broad microbial group (Figure D-1). The only exception was protozoa where the proportion significantly increased (P < 0.001); however, protozoa make up < 1% of the overall microbial community. 242 Figure D-1 Phospholipid fatty acid (PLFA) total biomass (A) and microbial broad group biomass (B) results for control forest soil before (= Baseline) and after (= Storage) storage at -4 °C during the conditioning phase of the experiment. 243 Appendix E Chapter 5 supplementary material Table E-1 Next-generation sequencing identification of spruce ectomycorrhizal (EM) fungi morphotype vouchers. Grey cells indicate non-mycorrhizal samples. Voucher ID EM fungal taxa Proportion of reads (%) Abundance rank Pairwise identity (%) Total reads Sx-E2 Tomentella sublilacina 19.5 1 99.5 220 Aureoboletus mirabilis 13.6 2 100.0 220 Amphinema sp. 8.6 5 99.6 220 Hebeloma birrus 5.5 8 96.8 220 Sx-E3 Tomentella sublilacina 4.4 3 97.0 893 Clavulina cinerea 3.7 4 100.0 893 Pustularia sp. 3.5 5 100.0 893 Sx-H4 Non-mycorrhizal 0.0 NA NA 264 Sx-K1 Tomentella sublilacina 59.7 1 97.0 203,006 Wilcoxina sp. 2 32.1 2 87.2 203,006 Sx-K2 Non-mycorrhizal 0.0 NA NA 5,692 Sx-M2 Tomentella sublilacina 0.5 9 96.3 1,927 Sx-P1 Wilcoxina sp. 2 40.0 1 87.2 13,510 Wilcoxina rehmii 37.6 2 100.0 13,510 SxSx-C4 Sistotrema adnatum 0.2 22 94.0 42,822 SxSx-D2 Wilcoxina rehmii 49.6 1 100.0 47,733 Pustularia sp. 31.8 2 100.0 47,733 SxSx-G1 Wilcoxina sp. 1 67.6 1 96.4 30,867 SxSx-H2 Amphinema sp. 24.1 1 100.0 195 SxSx-H3 Wilcoxina rehmii 57.2 1 100.0 21,557 Pustularia sp. 41.9 2 100.0 21,557 SxSx-K1 Tomentella sublilacina 1.6 4 99.5 2,404 SxSx-K2 Non-mycorrhizal 0.0 NA NA 2,513 SxSx-M1 Cadophora finlandica 65.2 1 97.1 9,071 SxSx-M3 Russula sapinea 13.2 2 100.0 1,061 SxSx-O3 Wilcoxina rehmii 60.5 1 100.0 3,263 WSx-A2 Wilcoxina sp. 1 6.3 4 96.4 10,235 WSx-B4 Wilcoxina sp. 3 3.4 5 97.2 6,715 WSx-D2 Tomentella sublilacina 1.1 8 99.5 1,695 WSx-G1 Non-mycorrhizal 0.0 NA NA 10,814 WSx-G2 Tomentella sublilacina 0.2 6 99.5 10,313 WSx-J1 Tomentella sublilacina 0.2 8 98.3 4,372 Lactarius lilacinus 0.2 8 99.5 4,372 WSx-J5 Wilcoxina sp. 3 4.2 5 97.2 3,026 WSx-L4 Non-mycorrhizal 0.0 NA NA 4,986 WSx-M2 Tuber anniae 28.8 3 100.0 11,575 WSx-N1 Tuber sp. 1.2 4 86.9 11,140 WSx-N2 Wilcoxina sp. 2 0.6 6 87.2 9,086 WSx-N4 Non-mycorrhizal 0.0 NA NA 386 244 Table E-2 Conditioning species post hoc comparisons of shoot biomass linear models by feedback species (willow, spruce and redcedar). Bold values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). Shoot biomass feedback post hoc comparisons Feedback species Conditioning species contrast estimate SE df T P Willow Willow - None 1.52 0.39 64 3.884 <0.001 Spruce Willow - None 1.88 0.238 112 7.917 <0.001 Spruce - None 0.35 0.206 112 1.698 0.211 Willow - Spruce 1.53 0.226 112 -6.791 <0.001 Redcedar Willow - None 3.375 0.268 100 12.596 <0.001 Spruce - None 0.602 0.194 100 3.105 0.007 Willow - Spruce 2.773 0.242 100 -11.459 <0.001 Table E-3 Shoot biomass linear model results by feedback phase species (willow, spruce and redcedar) with soil type (forest, overburden, tailings) and forest soil addition (yes, no) treatments combined into the soil treatment model term. Foliar carbon to nitrogen (C:N) ratio, soil depth (settling in pot) and drought damage (willow only) were included as covariates. Bold values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). Shoot biomass ANOVA Willow Spruce Redcedar Model term df F P df F P df F P Soil treatment 6 0.6031 0.727 6 5.0138 <0.001 6 5.0948 <0.001 Conditioning species 1 4.6957 0.034 2 4.8833 0.009 2 14.8475 <0.001 Foliar C:N ratio 1 0.0444 0.834 1 7.4687 0.007 1 1.6851 0.197 Soil depth 1 5.0996 0.027 1 5.0506 0.027 1 0.0002 0.989 Soil type x conditioning species 6 1.7323 0.128 12 1.7466 0.066 12 1.7038 0.077 245 Table E-4 Shoot biomass linear model results by feedback phase species (willow, spruce and redcedar) with conditioning species, soil type (overburden, tailings; forest soil not analyzed here) and inoculation (yes, no) treatments as explanatory variables. Foliar carbon to nitrogen (C:N) ratio, soil depth (settling in pot) were included as covariates. Bold italic values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). Shoot biomass ANOVA Willow Spruce Redcedar Model term df F P df F P df F P Conditioning species 1 5.284 0.025 2 2.8794 0.061 2 19.641 <0.001 Soil type 2 2.0631 0.136 2 1.685 0.191 2 1.3276 0.270 Forest soil addition 1 1.1068 0.297 1 0.099 0.754 1 0.2546 0.615 Foliar C:N ratio 1 0.6756 0.414 1 8.1911 0.005 1 0.8072 0.371 Soil depth 1 6.3309 0.015 1 3.962 0.049 1 1.5007 0.224 Conditioning species x soil type 2 5.5106 0.006 4 0.978 0.423 4 2.4951 0.049 Conditioning species x forest soil addition 1 0.7958 0.376 2 2.3503 0.101 2 1.0927 0.340 Soil type x forest soil addition 2 1.0022 0.373 2 2.7842 0.067 2 0.4595 0.633 Conditioning species x soil type * forest soil addition 2 6.986 0.002 4 1.5374 0.197 4 1.3589 0.255 246 Table E-5 Indicator species analysis of willow and redcedar root fungal communities from next-generation sequencing for a subset of soil treatments (forest soil, overburden and tailings only i.e., no forest soil additions) and spruce ectomycorrhizal fungi communities based on morphotyping (combined across all soil types). Feedbackspecies SoilConditioning species Taxon GuildIndicator species statistic P -valueWillow Forest None Pezicula sp. Pathotroph-Saprotroph 0.926 0.002Tomentella sublilacina Symbiotroph 0.926 0.001Phialocephala sp. Symbiotroph 0.914 0.001Helotiales sp. Unassigned 0.836 0.003Meliniomyces bicolor Symbiotroph 0.655 0.024Neonectria candida Pathotroph 0.655 0.029Cadophora sp. Symbiotroph 0.949 0.001Trichocladium opacum Saprotroph 0.946 0.001Pseudogymnoascus roseus Pathotroph-Saprotroph-Symbiotroph 0.829 0.004Solicoccozyma terricola Unassigned 0.828 0.004Cadophora finlandica Symbiotroph 0.734 0.007Willow Mortierella rishikesha Saprotroph-Symbiotroph 0.919 0.002Tetracladium sp. Saprotroph 0.707 0.005Peziza badia Saprotroph-Symbiotroph 0.707 0.013Moesziomyces aphidis Pathotroph 0.707 0.011Mortierella hyalina Saprotroph-Symbiotroph 0.624 0.025Cadophora sp. Symbiotroph 0.949 0.001Trichocladium opacum Saprotroph 0.946 0.001Pseudogymnoascus roseus Pathotroph-Saprotroph-Symbiotroph 0.829 0.004Solicoccozyma terricola Unassigned 0.828 0.004Cadophora finlandica Symbiotroph 0.734 0.007Podospora multipilosa Saprotroph-Symbiotroph 0.729 0.022Pleosporales sp. 2 Unassigned 0.901 0.027Plectosphaerella cucumerina Pathotroph 0.972 0.005Overburden None Ascomycota sp. Unassigned 0.756 0.003Podospora multipilosa Saprotroph-Symbiotroph 0.729 0.022Pleosporales sp. 1 Unassigned 0.949 0.001Plectosphaerella cucumerina Pathotroph 0.972 0.005Willow Hyphopichia burtonii Saprotroph 0.729 0.007Tetracladium breve Saprotroph 0.632 0.036Pleosporales sp. 1 Unassigned 0.949 0.001Pleosporales sp. 2 Unassigned 0.901 0.027Plectosphaerella cucumerina Pathotroph 0.972 0.005Tailings None Plectosphaerella cucumerina Pathotroph 0.972 0.005Willow Pleosporales sp. 2 Unassigned 0.901 0.027Plectosphaerella cucumerina Pathotroph 0.972 0.005247 Table E-5 cont’d Feedbackspecies SoilConditioning species Taxon GuildIndicator species statistic P -valueSpruce Combined None Wilcoxina sp. Ectomycorrhizal fungi 0.323 0.014Wilcoxina rehmii Ectomycorrhizal fungi 0.595 0.001Willow Trichophaea sp. Ectomycorrhizal fungi 0.446 0.049Spruce Thelephora terrestris Ectomycorrhizal fungi 0.437 0.005Trichophaea sp. Ectomycorrhizal fungi 0.446 0.049Wilcoxina rehmii Ectomycorrhizal fungi 0.595 0.001Redcedar Forest None Cadophora sp. Symbiotroph 0.758 0.004Phialocephala sp. 2 Symbiotroph 0.577 0.042Ascomycota sp. Unassigned 0.655 0.05Thelonectria rubrococca Saprotroph 0.953 0.001Willow Agaricomycetes sp. Unassigned 0.816 0.004Sebacinales sp. Unassigned 0.816 0.008Mortierella rishikesha Saprotroph-Symbiotroph 0.805 0.009Phialocephala sp. 1 Symbiotroph 0.79 0.004Basidiomycota sp. Unassigned 0.756 0.011Cadophora sp. Symbiotroph 0.758 0.004Phialocephala sp. 2 Symbiotroph 0.577 0.042Thelonectria rubrococca Saprotroph 0.953 0.001Spruce Ilyonectria liriodendri Saprotroph 0.707 0.029Thielaviopsis basicola Saprotroph 0.707 0.022Solicoccozyma terricola Unassigned 0.685 0.016Basidiomycota sp. Unassigned 0.756 0.011Cadophora sp. Symbiotroph 0.758 0.004Ascomycota sp. Unassigned 0.655 0.05Thelonectria rubrococca Saprotroph 0.953 0.001Overburden None Pleosporales sp. 2 Unassigned 0.764 0.013Olpidium brassicae Pathotroph 0.899 0.001Thelonectria rubrococca Saprotroph 0.953 0.001Willow Pleosporales sp. 2 Unassigned 0.764 0.013Paranamyces uniporus Unassigned 0.676 0.036Spruce Thelonectria rubrococca Saprotroph 0.953 0.001Tailings None Cadophora luteo-olivacea Symbiotroph 0.739 0.016Olpidium brassicae Pathotroph 0.899 0.001Spruce Pleosporales sp. 1 Unassigned 0.756 0.025Cadophora luteo-olivacea Symbiotroph 0.739 0.016248 Table E-6 Sanger sequencing identification of ectomycorrhizal fungi vouchers from morphotyping of spruce seedling roots. Table E-7 Conditioning species post hoc comparisons of shoot biomass linear models by feedback species (willow, spruce and redcedar) with no foliar nutrient covariate in the model. Bold values are significant (P <0.05) and italics values are marginally significant (0.05 < P <0.1). Shoot biomass feedback post hoc comparisons Feedback species Conditioning species contrast estimate SE df T P Willow Willow - None 1.61 0.352 69 4.592 <0.001 Spruce Willow - None 1.761 0.24 114 7.335 <0.001 Spruce - None 0.214 0.205 114 1.041 0.553 Willow - Spruce -1.547 0.231 114 -6.693 <0.001 Redcedar Willow - None 3.505 0.26 102 13.466 <0.001 Spruce - None 0.601 0.195 102 3.079 0.008 Willow - Spruce -2.904 0.233 102 -12.465 <0.001 Voucher ID Species Pairwise identity (%) SxSx-A2 Trichophaea sp. 1 99.9 SxSx-A4 Thelephora terrestris 100 SxSx-A5 Trichophaea sp. 2 99.7 SxSx-A6 Wilcoxina rehmii 99.7 SxSx-A7 Trichophaea sp. 1 98.8 SxSx-B1 Tuber anniae 99.4 SxSx-C1 Trichophaea sp. 2 99.7 Sx-F1 Trichophaea sp. 2 98.5 WSx-H1 Trichophaea sp. 1 99.8 SxSx-I3 Cadophora finlandica 100 SxSx-I4 Cadophora finlandica 98.7 Sx-I4 Cadophora finlandica 99.6 SxSx-J2 Tuber anniae 99.6 WSx-K1 Trichophaea sp. 2 99.3 SxSx-L1 Hebeloma sp. 1 99.2 SxSx-L2 Hebaloma sp. 2 98.2 WSx-O1 Trichophaea sp. 2 99.3 SxSx-P1 Wilcoxina rehmii 99.8 WSx-P1 Trichophaea sp. 2 98.8 Sx-P4 Wilcoxina rehmii 99.3 SxSx-A1 Trichophaea sp. 2 99.7 SxSx-M2 Pustularia sp. 100 WSx-O2 Peziza sp. 99.7 Sx-P2 Wilcoxina mikolae 98.8 249 Figure E-1 Seedling height loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 250 Figure E-2 Seedling root collar diameter loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 251 Figure E-3 Seedling root biomass loge response ratios of willow (A), spruce (B) and redcedar (C) in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 252 Figure E-4 Regressions of shoot biomass feedbacks against ectomycorrhizal fungi (EMF; A, C), arbuscular mycorrhizal fungi (AMF; E) and root fungal endophyte (B, F) percent colonization feedbacks, and shoot biomass feedbacks against EMF species richness (E) for willow (A, B), spruce (C, D) and redcedar (E, F) seedling roots. Each data point represents loge response ratios of conditioning treatment vs. control for one soil type x forest soil amendment treatment. 253 Figure E-5 Representative willow, spruce and redcedar seedlings (replicate with median shoot biomass in treatment) grown in control, willow-conditioned or spruce-conditioned forest soil for six months in a greenhouse. 254 Figure E-6 NMDS analysis of high-throughput sequencing fungal (ITS2) community data for willow (A) and redcedar (B) in willow- and spruce-conditioned soils (forest soil, overburden and tailings) compared to unconditioned control soils. Ellipses could not be draw for willow and control (none) conditioning treatments in redcedar due to poor survival. 255 Figure E-7 Amplicon sequence variant (ASV) richness (A, C, E) and relative abundance (B, D, E) of pathogenic (A, B), symbiotic (C, D) and saprotrophic (E, F) fungi on roots of willow seedlings grown in unconditioned control and willow-conditioned soils (tailings, overburden and tailings + overburden). “+ forest soil” treatments were mixed with 25% v/v forest soil. Bars represent mean ± 1 SE. 256 Figure E-8 Spruce seedling ectomycorrhizal fungal (EMF) species richness loge response ratios in willow- and spruce-conditioned soils (forest soil, overburden, tailings and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. Figure E-9 Spruce seedling ectomycorrhizal fungal (EMF) species richness loge response ratios of in willow- and spruce-conditioned reclamation soils (tailings, overburden and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 257 Figure E-10 Amplicon sequence variant (ASV) richness (A, C, E) and relative abundance (B, D, E) of pathogenic (A, B), symbiotic (C, D) and saprotrophic (E, F) fungi on roots of redcedar seedlings grown in unconditioned control, willow-conditioned and spruce-conditioned soils (tailings, overburden and tailings + overburden). “+ forest soil” treatments were mixed with 25% v/v forest soil. Bars represent ± 1SE. 258 Figure E-11 Spruce (A) and redcedar (B) specific root length loge response ratios of in willow- and spruce-conditioned reclamation soils (tailings, overburden and tailings + overburden) compared to unconditioned control soils. “+ forest soil” treatments were mixed with 25% v/v forest soil. Feedbacks are significant (P < 0.05) where the 95% confidence interval does not cross zero. 259 Figure E-12 Regressions of shoot biomass (A, C) and mycorrhizal fungal percent colonization (B, D) feedbacks against specific root length feedbacks for spruce (A, B) and redcedar (C, D) seedlings. Each data point represents loge response ratios of conditioning treatment vs. control for one soil type x forest soil addition treatment. EMF = ectomycorrhizal fungi; AMF = arbuscular mycorrhizal fungi.
Thesis/Dissertation
eng
Forestry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Investigation of ecosystem legacy-based mine reclamation techniques : effects of soil transfers, forest connectivity and plant–soil feedbacks on soil food web recovery and plant establishment
Text