Science, Irving K. Barber Faculty of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Yamaoka, Naomi K.
Master of Science - MSc
University of British Columbia
Although ectomycorrhizal associations have been studied in great detail in mature stands, lab settings, and greenhouse studies, very little research has been conducted examining the early stages of the symbiosis in the field. With a changing climate, fire regimes, and harvesting practices, it is imperative we understand how this relationship establishes in a natural setting. By collecting lodgepole and ponderosa pine seedlings during their first growing season in the appropriate biogeoclimatic zones, in areas that were affected by a clearcut or severe wildfire the year before, I was able to identify when signs of colonization on pine roots first began, the rate at which fungi colonized new germinants, and how this related to nutrition and growth. Seedlings collected in this study started to show signs of colonization in July, a month after germination was first observed. There was a mixture of colonized and uncolonized seedlings collected in the summer; lodgepole pine seedlings that were colonized generally had higher nitrogen contents and biomass compared to uncolonized seedlings collected at the same time. This suggests that the fungi were aiding in nutrient acquisition, even within the first weeks after mycorrhiza formation. By the end of the season, all collected seedlings had ectomycorrhizae present on their roots, so fungal inoculum did not appear to be limiting on severely burned or recently clearcut sites. The genera of fungal species present on the plant root tips did not seem to have an effect on the amount of seedling N, or biomass.
Nitrogen and mycorrhization in Pinus ponderosa and Pinus contorta germinants the first growing season after wildfire or clearcutting by Naomi K. Yamaoka B.Sc., University of British Columbia Okanagan, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) November 2022 © Naomi Yamaoka, 2022 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Nitrogen in Pinus ponderosa and Pinus contorta germinants in the first growing season after wildfire or clearcutting: the effect of mycorrhization submitted by Naomi K. Yamaoka in partial fulfillment of the requirements of the degree of Master of Science . Dr. Melanie Jones, Irving K. Barber Faculty of Science, Biology Supervisor Dr. Daniel Durall, Irving K. Barber Faculty of Science, Biology Supervisory Committee Member Dr. Barbara Hawkins, UVic, Science, Biology Supervisory Committee Member Dr. Miranda Hart, Irving K. Barber Faculty of Science, Biology Supervisory Examiner Dr. Sue Grayston, UBC V, Forestry/Forest and Conservation Sciences University Examiner iii Abstract Although ectomycorrhizal associations have been studied in great detail in mature stands, lab settings, and greenhouse studies, very little research has been conducted examining the early stages of the symbiosis in the field. With a changing climate, fire regimes, and harvesting practices, it is imperative we understand how this relationship establishes in a natural setting. By collecting lodgepole and ponderosa pine seedlings during their first growing season in the appropriate biogeoclimatic zones, in areas that were affected by a clearcut or severe wildfire the year before, I was able to identify when signs of colonization on pine roots first began, the rate at which fungi colonized new germinants, and how this related to nutrition and growth. Seedlings collected in this study started to show signs of colonization in July, a month after germination was first observed. There was a mixture of colonized and uncolonized seedlings collected in the summer; lodgepole pine seedlings that were colonized generally had higher nitrogen contents and biomass compared to uncolonized seedlings collected at the same time. This suggests that the fungi were aiding in nutrient acquisition, even within the first weeks after mycorrhiza formation. By the end of the season, all collected seedlings had ectomycorrhizae present on their roots, so fungal inoculum did not appear to be limiting on severely burned or recently clearcut sites. The genera of fungal species present on the plant root tips did not seem to have an effect on the amount of seedling N, or biomass. iv Lay Summary The aim of this study was to observe how and when pine seedlings formed root associations with fungi that are typically considered to be beneficial symbiotic partners, in burned and clearcut sites in the Okanagan Valley. Results from this research indicate that even within the first year, colonization of roots by fungal symbionts likely promoted nitrogen accumulation and seedling growth. Because nitrogen is generally considered to be a limiting nutrient in such forests, swift colonization may increase seedling survival. By the end of the growing season, all collected seedlings had fungal symbioses established on their roots, which indicates that neither severe wildfire nor clearcutting eliminated these root fungal symbionts from the soil. v Preface The study design was created with collaboration between Dr. Melanie Jones and myself, with input from my committee. Field work was conducted by Erica Packard, Sarah Neumann and me, with help from Dan Durall, Nigel Sapriken and Maddi Arnold. Lab work and analysis was conducted by Erica Packard and me, with assistance from Dr. Andy Midwood, Tirhas Mebratu Gebretsadikan, Nora Skuridina, and Kat Chhen. Next generation sequencing preparation and bioinformatics were done by Erica Packard, who also collected and analysed the majority of the naturally regenerating seedlings in this study. I conducted all the statistical analyses, composed all the figures and tables, and wrote the text in the thesis, with editing from Dr. Jones, and input from the rest of my committee. vi Table of Contents Abstract .............................................................................................................................................. iii Lay Summary .................................................................................................................................... iv Preface ..................................................................................................................................................v Table of Contents .............................................................................................................................. vi List of Tables ................................................................................................................................... viii List of Figures .................................................................................................................................... xi Acknowledgements .......................................................................................................................... xv Dedication......................................................................................................................................... xvi Chapter 1: Introduction .....................................................................................................................1 1.1 Background ...........................................................................................................................1 1.1.1 Forest disturbances in western North America ..........................................................3 1.1.1.1 Wildfires ...................................................................................................................3 1.1.1.2 Clearcuts ...................................................................................................................3 1.1.2 Soil N and the effects of disturbance ..........................................................................4 1.1.3 Pinus contorta and Pinus ponderosa ..........................................................................6 1.1.4 Nitrogen content and acquisition in young conifers ..................................................8 1.1.5 Ectomycorrhizae and nitrogen acquisition by plants .................................................9 1.1.6 Ectomycorrhizal fungal communities and colonization following a disturbance . 12 1.2 Thesis overview, objectives, and hypotheses .................................................................. 15 Chapter 2: Seedling establishment and early colonization ....................................................... 17 2.1. Background ........................................................................................................................ 17 2.2. Material and methods ........................................................................................................ 19 2.2.1. Study sites .................................................................................................................. 19 2.2.2. Sowing of seeds ........................................................................................................ 20 2.2.3. Sampling of seedlings ............................................................................................... 22 2.2.4. Soil sampling ............................................................................................................. 23 2.2.5. Processing and analysis of seedlings ....................................................................... 23 2.2.6. Soil analysis ............................................................................................................... 24 2.2.7. Data analysis ............................................................................................................. 25 2.3. Results ................................................................................................................................ 26 2.3.1. Soil chemistry ............................................................................................................ 26 2.3.2. Germination ............................................................................................................... 29 2.3.3. Colonization by ectomycorrhizal fungi ................................................................... 31 2.3.4. Seedling biomass accumulation ............................................................................... 34 2.3.5. Seedling nutrient status ............................................................................................. 37 2.3.6. Effect of mycorrhization on seedlings ..................................................................... 44 2.3.6.1. Lodgepole pine seedlings ................................................................................. 46 vii 2.3.6.2. Ponderosa pine seedlings ................................................................................. 47 2.4. Discussion .......................................................................................................................... 58 2.4.1. Early growth and N accumulation by pine seedlings after disturbance ................ 58 2.4.2. Timing of mycorrhiza formation in young pine germinants .................................. 61 2.4.3. Did mycorrhizal and nonmycorrhizal germinants differ from each other? ........... 62 2.4.3.1. Accumulation of C and biomass ...................................................................... 63 2.4.3.2. N accumulation ................................................................................................. 64 Chapter 3: Ectomycorrhizal fungal species following a disturbance ...................................... 67 3.1. Background ........................................................................................................................ 67 3.2. Materials and methods ...................................................................................................... 68 3.2.1. Site selection and field work .................................................................................... 68 3.2.2. Molecular analysis .................................................................................................... 68 3.2.3. Bioinformatics ........................................................................................................... 70 3.2.4. Data analysis ............................................................................................................. 70 3.3. Results ................................................................................................................................ 71 3.3.1. Fungal species present .............................................................................................. 71 3.3.2. Fungal species and seedling N ................................................................................. 75 3.4. Discussion .......................................................................................................................... 76 3.4.1. Fungi present following the disturbance ................................................................. 77 3.4.2. Effect of ectomycorrhizal fungal identity on seedling nitrogen ............................ 80 Chapter 4: Conclusion..................................................................................................................... 81 Bibliography ..................................................................................................................................... 85 Appendices ...................................................................................................................................... 104 Appendix A- Additional figures ................................................................................................. 104 Appendix B- Methods ................................................................................................................. 117 viii List of Tables Table 2.1. Characteristics of study sites ..................................................................................... 21 Table 2.2: KCl- extractable nitrate-, ammonium-, and amino acid-N; total N (dry combustion elemental analysis) and pH of soils from ponderosa pine wildfire (PPWF), lodgepole pine wildfire (LPWF) and lodgepole pine clearcut (LPCC) sites, and soils from adjacent undisturbed area. n=3 sites. .......................................................................................................................... 28 Table 2.3: Soil nutrient concentrations (mg kg-1 (standard error)) determined by microwave digestion followed by ICP-MS for soils from disturbed ponderosa pine wildfire (PPWF), lodgepole pine wildfire (LPWF) and lodgepole pine clearcut (LPCC) sites, and soils from adjacent undisturbed area. n=3 sites. Disturbed soils were collected in the spring of 2019, undisturbed in the spring 2020. ................................................................................................. 29 Table 2.4 Seedling germination and numbers at each site. Seeds were sown in 2019 between April 26-30 (ponderosa pine wildfire sites), May 2-29 (lodgepole pine wildfire sites), and May 27-30 (lodgepole pine clearcut sites). ........................................................................................ 30 Table 2.5. Percent (mean (standard deviation)) of root tips colonized per seedling for sown mycorrhizal seedlings. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcut (LPCC) sites. .............................................. 33 Table 2.6 Percent (mean (standard deviation)) of root tips colonized per seedling for naturally regenerating mycorrhizal seedlings. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole from wildfire (LPWF) and clearcut (LPCC) sites. ................................ 34 Table 2.7. Results from linear mixed models using type III ANOVA with Satterthwaite’s method of seedling characteristics of seedlings collected throughout the growing season. Significant differences and interactions (p £ 0.05) are indicated in bold. Data shown for ponderosa pine ix seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). .................................................................................................................................... 35 Table 2.8: Weight, carbon and nitrogen content of the ponderosa and lodgepole pine seeds used in this study. Standard error is shown in brackets, n=5. ............................................................. 39 Table 2.9. Concentrations (mg kg-1 (standard error)) of macronutrients in root and shoot tissues of seedlings collected in Fall 2019, as determined by microwave digestion followed by ICP-MS. n=3 sites. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). ............................................................................ 42 Table 2.10. Concentrations (mg kg-1 (standard error)) of micronutrients and Al in root and shoot tissues of seedlings collected in September as determined by microwave digestion followed by ICP-MS. < DL = below detection limit. n=3 sites. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). .................. 43 Table 2.11. Results of PERMANOVA1 comparing nonmycorrhizal and mycorrhizal sown seedlings. P values less than 0.10 are in bold. ............................................................................ 45 Table 2.12. Results of PERMANOVA comparing nonmycorrhizal and mycorrhizal naturally regenerating seedlings sampled in Jun-Aug. P values < 0.10 (ANOVA) are in bold. ................. 46 Table 2.13. Results from linear mixed models using type III ANOVA with Satterthwaite’s method of seedling characteristics of sown mycorrhizal and nonmycorrhizal seedlings sampled in July and August. Significant differences and interactions (p £ 0.05) are indicated in bold. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). .................................................................................... 49 Table 2.14. Results from linear mixed models using type III ANOVA with Satterthwaite’s ANOVA of seedling characteristics of mycorrhizal and nonmycorrhizal naturally regenerating x seedlings for status, date sampled, and interaction term. P values £ 0.10 are in bold. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF). ................................................................................................................................................. 50 Table 2.15. Results from linear mixed models using type III ANOVA with Satterthwaite’s method of d 13C and d 15N isotopic signatures of mycorrhizal and nonmycorrhizal sown seedlings. Significant differences (p £ 0.05) are indicated in bold. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). .................................................................................................................................... 51 Table 3.1. Results of a PERMANOVA comparing fungal community between months where mycorrhizal seedlings were collected (Jul-Oct). ........................................................................ 72 Table 3.2. Results from linear model with fungal species present on the roots as the fixed effect and shoot % N as the variable effect for lodgepole pine seedlings from (LPCC) clearcut sites and (LPWF) wildfire sites, and (PPWF) ponderosa pine from wildfire sites. .................................... 75 xi List of Figures Figure 2.1 Biomass (log(g)) of sown ponderosa pine seedlings from burned sites, and lodgepole pine from burns and clearcuts, the first growing season following a disturbance. Percent (%) of seedlings colonized across all three sites is shown at the top of each month. Red asterisks indicate significant differences (p £ 0.05) from June according to results from the linear mixed effects model. ..................................................................................................................................... 32 Figure 2.2 Root and shoot d13C of sown ponderosa pine seedlings from (a, b) burned sites and lodgepole pine from (c, d) burned and (e, f) clearcut sites the first growing season following a disturbance. The hashed line indicates the seed d13C of the seedlot from which these seedlings germinated (-22.732 ± 0.76 in ponderosa pine, -22.12 ± 0.54 in lodgepole pine). Each open circle represents one seedling. Black points and error bars indicate mean and standard error of each month. Red asterisks indicate significant differences (p £ 0.05) from June according to linear models. ................................................................................................................................................ 36 Figure 2.3 Percent (%) N of sown ponderosa pine shoots from burned sites, lodgepole pine from burned sites, and lodgepole pine from clearcuts over the first growing season following the disturbances. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of seedlings sampled in each month. Letters indicate months that are significantly different from each other, as determined by Tukey’s HSD post-hoc test. The hashed lines indicate the seed N. ................................................................................................................... 39 Figure 2.4 Total N (log(mg)) of sown ponderosa pine seedlings from burned sites, and lodgepole pine from burned sites and clearcuts the first growing season following a disturbance. The hashed line indicates the amount of seed N (1.26 ± 0.09 mg in ponderosa pine, 0.13 ± 0.02 mg in lodgepole pine). Each open circle represents one seedling. Black points and error bars indicate xii mean and standard error of each month. Red asterisks indicate significant differences (p £ 0.05) from June according to linear models. .............................................................................................. 40 Figure 2.5 Root and shoot d15N of sown (a, b) ponderosa pine seedlings from burned sites, (c, d) lodgepole pine seedlings from burned sites and (e, f) clearcuts the first growing season following the disturbance. The hashed line indicates the isotopic signatures in the seeds (-3.08 ± 0.30 in ponderosa pine, -2.56 ± 0.28 in lodgepole pine). Each open circle represents one seedling. Black points and error bars indicate mean and standard error of each month. Red asterisks indicate significant differences (p £ 0.05) from June, red circles indicate p £ 0.1 according to linear models. ................................................................................................................................................ 41 Figure 2.6 Total biomass (roots + shoots) (log(g)) of sown (a) ponderosa pine seedlings from burned sites, and lodgepole pine from (b) burned, and (c) clearcut sites in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. Red asterisks indicate a significant (p £ 0.05) difference between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. ...................................................................... 53 Figure 2.7 Total N (roots + shoots) (log(mg)) of sown (a) ponderosa pine seedlings from burned sites, and lodgepole pine seedlings from (b) burned sites, and (c) clearcuts in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. Red asterisks indicate a significant (p £ 0.05) difference between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. ...................................................................... 54 Figure 2.8 Shoot percent (%) N of sown (a) ponderosa pine shoots from burned sites, and lodgepole pine seedlings from (b) burned sites, and (c) clearcuts in months where both xiii mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. ............... 55 Figure 2.9 Root and shoot δ15N values of sown (a, b) ponderosa pine seedlings from burned sites, and lodgepole pine seedlings from (c, d) burned sites, and (e, f) clearcuts, in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. The hashed line indicates the seed signatures (-3.08 ± 0.30 in ponderosa, -2.56 ± 0.28 in lodgepole). Red asterisks indicate significant (p £ 0.05) differences between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. ...................................................................... 56 Figure 2.10 Root and shoot δ13 C values of sown (a, b) ponderosa pine seedlings from burned sites, and lodgepole pine from (c, d) burned sites and (e, f) clearcuts in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. Red asterisks indicate significant (p £ 0.05) differences between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. ...................................................................... 57 Figure 3.1 The number of seedlings on which each fungal taxon was found over the sampling period following a disturbance: July-October for lodgepole pine seedlings, and July-September for ponderosa pine seedlings. When there was more than one ectomycorrhizal fungal species on a seedling, the dominant species was selected to represent that seedling. If more than one species of each genus was found, the number of species is shown in brackets, with the exception of Wilcoxina spp. Species that only occurred once across all three site types were omitted. Lodgepole pine seedlings from clearcuts (60), and wildfire sites (53), and ponderosa pine seedlings from wildfire sites (38) were included. ............................................................................ 73 xiv Figure 3.2 The shoot % N of seedlings colonized by different ectomycorrhizal genera. Points represent individual seedlings from all months and any of the three replicate sites because % N did not vary over time or by site. When there was more than one species on one seedling, the dominant species was selected to represent that seedling. Fungal genera that occurred on fewer than three seedlings per site type were omitted. The red asterisk indicates a significant difference among seedlings colonized by different ectomycorrhizal fungal species within a site type based on a linear model with analysis of variance (ANOVA). Groups of points that do not share letters represent seedlings with different concentrations of N, according to Tukey’s HSD tests (p £ 0.05). ................................................................................................................................................... 75 xv Acknowledgements I would like to express the deepest gratitude to my supervisor, Dr. Melanie Jones for giving me the opportunity and space to grow both as a researcher and a person. She has truly changed my life and career for the better, and I will be infinitely grateful. I am appreciative of the support and guidance from my committee members, Dr. Daniel Durall, Dr. Barbara Hawkins, and Dr. Miranda Hart. I would also like to thank other members of the Jones lab group, especially Sarah Neumann, Tirhas Mebratu Gebretsadikan, Nora Skuridina, and Kat Chhen for the assistance with field and lab work and Dr. Andy Midwood for the input and guidance with isotopic work. A special thank you to Dan Durall for rescuing our field crew when our vehicle broke down. I am grateful for the support of fellow graduate students, particularly Maddi Arnold and Portiaa McGonigal. I would also like to express immense gratitude to Erica Packard, who assisted me in more ways that I can count, both in research and morale. A very special thank you to my parents, Bea and Brian; siblings, Julie, Jenny, Ashley and Keith; and close friends, especially Morgan, Maddi and Alex, for all the incessant and indispensable support. xvi Dedication This thesis is dedicated to my parents for their endless love and support, without which this would not have been possible. 1 Chapter 1: Introduction 1.1 Background Forested land makes up 35%, or 3.47 million km2 of the total area in Canada, with 68% of that being coniferous (Statistics Canada, 2018). As a result, forests play an essential role in Canada both environmentally and economically. In 2017, forestry was responsible for 32% ($14.1 billion) of British Columbia’s (BC) exports, with an estimated provincial revenue of $992 million from forests in 2018/2019 (BC Ministry of Finance, Budget Estimates 2018). Two of the most prevalent forms of stand-replacing disturbance in Canadian coniferous forests are wildfires and timber harvest using clearcutting. Seedling establishment has typically been successful following these major disturbances; they promote growth of new seedlings by opening up the canopy, thereby allowing increased sunlight to reach the forest floor. However, over the past two decades forest regeneration after wildfire has declined due to the increased temperatures and drought conditions associated with climate change (Gillett and Weaver 2004; Kemp et al. 2019). Primary productivity in forests in western Canada is usually limited by nitrogen (N). There is often considerable total N, but it is unavailable because it is bound in organic matter (Sponseller et al. 2016). Fungi symbiotic with plant roots (mycorrhizal fungi) can help to alleviate N stress in most tree species by providing increased access to this limiting nutrient (Abuzinadah et al. 1986). Ectomycorrhizal fungi (EMF), which are symbionts on most temperate coniferous trees, play a particularly important role in plant N acquisition. They do this in two major ways: (i) exploratory hyphae increase the volume of soil beyond nutrient depletion zones around roots, and (ii) hyphae access N immobilized in organic matter using secreted enzymes (Read and Perez-Moreno 2003; Bödeker et al. 2014). The reliance of most conifer trees in northern forests on EMF for increased 2 access to N is a large reason why they are often regarded as obligate symbionts. (Miller et al. 1998). Conifer seedlings contain high concentrations of N, derived from their seed reserves, and are able to live for a short amount of time without EMF. While colonization within the first two growing seasons is thought to be required for seedling survival (Hatch 1936; Miller et al. 1998; Guignabert et al. 2018), no study has specifically determined the timing of initial colonization of naturally regenerating conifer seedlings in the field, and relationship between this colonization and N accumulation by seedlings. Understanding this relationship is important in light of the decreased forest regeneration under climate change, and because major loss of N has occasionally be observed in ectomycorrhizal (EM) seedlings (Zackrisson 2013; Smith et al. 2020). In my thesis research, I examined first year ponderosa and lodgepole pine seedlings growing in the field the year following a clearcut or severe wildfire. Seedling growth, nutrient content, and mycorrhizal colonization were monitored, taking note of when seedlings started to become mycorrhizal, and how this affected growth and N content. This study helps give insight into the early relationship between prevalent conifer species and their fungal symbionts during the crucial first year following a disturbance. 3 1.1.1 Forest disturbances in western North America 1.1.1.1 Wildfires Although fires are part of natural forest regeneration, they can result in substantial ecosystem N losses through volatilization and soil leaching (Imeson et al. 1992; Huffman et al. 2001). Fires are also highly destructive and can pose a considerable risk to humans by destroying and damaging structures, and decreasing air quality. 2017, 2018 and 2021 were the three worst fire years on record in British Columbia in terms of area burned (Statistics Canada, 2021), and recent studies predict that the frequency and severity of forest fires will only keep increasing as a result of climate change (Abatzoglou and Williams 2016; Coogan et al. 2019). In the dry ponderosa pine forests of southern BC, fires historically occurred in the same stand roughly every 21 years, and were usually low in severity (Heyerdahl et al. 2012). Increasing average fire severity means that tree species, such as lodgepole and ponderosa pine, and their fungal symbionts, especially those at the edge of their biogeoclimatic zone at lower elevations, are high at risk for regeneration failure following a fire (Stevens-Rumann et al. 2018). 1.1.1.2 Clearcuts The stability of forests is of great economic importance in Canada. The forestry industry contributed $24.6 billion dollars to Canada’s GDP in 2017, with a large portion of timber production coming from British Columbia (Natural Resources Canada 2019). The vast majority (85% in 2015) of logging in Canada is done by completely removing all trees in an area, a practice known as clearcutting. Regeneration success following clearcutting can be largely dependent on the type of site preparation applied following logging. For example, there is evidence that sites exposed to chain-drag, or drag scarification, before being left to naturally 4 regenerate, can result in 12 times more surviving seedlings compared to sites with no preparation (Landhäusser 2009). It is required by law that all areas harvested on public land be reforested, either naturally or by re-planting (Natural Resources Canada 2019), which stresses the need for understanding how seedlings re-establish following a disturbance. 1.1.2 Soil N and the effects of disturbance During primary succession, N is almost entirely absent from the substrate, and gradually increases as microbial biomass accumulates and N fixation occurs (Lambers et al. 2008). Eventually, most N in temperate forest soils becomes immobilized in organic matter, and is considered to be mostly unavailable to plants without the help of microbial symbionts (Brockley 2001b; Sponseller et al. 2016). The classic theory of soil N cycling suggested that plants are limited by the mineralization of organic N by microbes, with which they must compete in order to gain access to the newly available N (Schimel and Bennett 2004). This was based on the understanding that roots could take up only inorganic N (i.e., ammonium or nitrate). However, a more recent paradigm incorporates the knowledge that roots can also absorb small soluble organic molecules such as amino acids. It defines the depolymerization of N-containing soil organic matter into usable monomers as the limiting step in soil N cycling (Schimel and Bennett 2004). Although there can be large fluxes of inorganic N, it is still a small fraction of the overall N that remains in forest soils. For these reasons, ecosystem productivity is often limited by the breakdown of organic matter into usable forms of N, which is facilitated by soil microbes, including fungi (Schimel and Bennett 2004). During a severe fire, the heat of combustion converts organic N into soluble inorganic forms 5 (Certini 2005; Peay et al. 2010). The amount of organic N converted to inorganic N as a result of wildfires depends largely on fire severity, which can determine the temperature at the soil surface. Soil moisture can also affect loss of N during wildfire, as heat is conducted better in moist soils, and is able to reach greater depths; however, the latent heat of vaporization prevents the temperature of the moist soil from exceeding 95 °C (Franklin et al. 1997). By contrast, drier soils such as those in the Okanagan Valley reach much higher temperatures at the surface, while lower soil layers remain protected because of reduced conductivity. This high surface temperature results in the combustion of organic matter, increasing both total available N, and inorganic N in spite of potential losses due to volatilization (Weston and Attiwill 1990; Covington and Sackett 1992). An immediate surge in ammonium is observed as a direct product of this combustion. However, it is important to note that these changes in N availability can vary within a site due to the nature of the disturbance, and factors such as ash deposition (Grogan et al. 2000). These disturbances can also indirectly affect the N availability of the soil by creating a temporary hydrophobic layer parallel to the surface, which obstructs infiltration and increases runoff for up to 22 months (Imeson et al. 1992; Huffman et al. 2001) after a severe wildfire. Following clearcutting, there is both a large accumulation of dead organic matter, which provides the raw material required for N mineralization and nitrification, and a reduction in vegetative biomass to take up N released by mineralization (Bradley and Fyles 1996). This results in the pulse of mineral N that is observed in the forest floor following clearcutting, which has been referred to as ‘the assart flush’ (Bradley et al. 2001). The resulting amount of mineral N can be influenced by soil temperature and moisture, and the amount of surface litter present. Unlike fires, the peak of N availability following clearcutting is not immediate, but rather occurs 6 3-5 years after harvest (Bradley et al. 2001). Following both types of disturbance, conditions such as increased amounts of ammonium, increased pH, and favourable soil temperature and moisture result in increased nitrification (Bradley et al. 2001). This nitrification decreases the amount of ammonium in the soil as it is converted into nitrate; however, this surplus of inorganic N is short-lived, as ammonium that has eluded nitrification is largely bound to negatively charged surface particles, and much of the newly-formed nitrate is leached downward in the soil horizon (Piirainen et al. 2004). Thus, the high rates of mineralization may also leave the ecosystem susceptible to large losses of N due to increased mobility and subsequent leaching (Covington and Sackett 1992; Bradley et al. 2001; Certini 2005). Regardless, this ephemeral surge of available inorganic N following both types of disturbance has the potential to create favourable conditions for seedling establishment (Barker et al. 2014). 1.1.3 Pinus contorta and Pinus ponderosa Lodgepole pine is the most widespread of the North American conifer species, and can be found as far north as Alaska and the Mackenzie district in the Yukon, extending down the west coast to northern Baja in California, and throughout the Rocky Mountains into Colorado (Owens 2006). The particular subspecies that occurs in the BC interior is Pinus contorta var latifolia. Lodgepole pine trees have little taper and thin bark, which results in a higher volume of wood per stem than other tree species (Strong 2014). These characteristics, wide range, and abundance make lodgepole pine one of the most economically significant species in the B.C forestry industry. It is planted on 53%, or 3.8 million ha, of British Columbia’s secondary forest (Owens 2006, BC 7 Ministry of Forests, Mines and Lands 2010). Pinus ponderosa (ponderosa pine) has a limited distribution in BC, only present at low elevations in the southern interior of the province, but ranges as far south as Mexico (Norris et al. 2006). It is of particular interest because it can establish on sites with high temperatures and drought conditions (Kolb and Robberecht 1996). Although water is generally considered to be the limiting nutrient for ponderosa pine trees, N has also been found to be a constraining factor (Marshall et al. 2019). Because they are only prevalent at lower elevations in the southern interior of BC, ponderosa pine trees are not often harvested in the province. Ponderosa and lodgepole pine are typically well adapted to low severity wildfires. Ponderosa pine trees have thick bark that sloughs off when exposed to fire, and deep root systems which remain intact following a surface fire (reviewed in Fitzgerald 2005). Lodgepole pine cone are serotinous, meaning that they require intense heat from fire to open and release seeds, which promotes new growth following a fire (Knapp and Anderson 1980). In addition to this, both lodgepole and ponderosa pine are shade-intolerant species, and can generally only establish in open areas where there is enough sunlight reaching the forest floor, such as after a disturbance, which results in even-ages stands (Heyerdahl et al. 2012). Augustine and Reinhardt (2019) investigated seedling physiology of both pine species and found that photosynthesis and carbon (C) accumulation was prioritized even when under drought stress, indicating that seedling survival is largely dependent on C limitations. 8 1.1.4 Nitrogen content and acquisition in young conifers During early seedling growth, developmental controls ensure that there is a relatively large amount of N available in young pine germinants (Canãs et al. 2006). Most of the high N observed in seedlings is likely derived from the seed itself, with Abuzinadah et al. (1986) estimating a mean seed N content of 200 ± 23 µg in lodgepole pine seeds. This N is speculated to move primarily to the roots of the growing germinants, and may be in excess of what is required for development (Canãs et al. 2006). After a brief period (estimated to be 11-12 days), of relying on seed nutrient reserves during development, pine seedlings gradually become dependent on an external supply (Flaig and Mobr 1992). Conifer trees can move N to different parts of the plant to meet nutrient demands and prevent loss. Nitrogen is generally stored in the older foliage of evergreen trees (reviewed in Millard and Grelet 2010), and is remobilized and translocated to areas of new growth (Mead and Preston 1994; Proe et al. 2000). Nitrogen remobilization was traditionally considered to be negligible in younger trees, as litterfall is insignificant and foliar biomass is increasing exponentially (Miller et al. 1998). However, Proe et al. (2000) supplied two-year-old Scots pine (Pinus sylvestris) seedlings with labelled 15N, and determined that up to 83% of the N in new tissues had been supplied from remobilization. Plants that were nutrient limited were also perceived to obtain a larger proportion of their N from mobilization, rather than by direct uptake. This indicates that N redistribution in conifers is highly adaptable and is influenced by sink strength, even in seedlings. The seedlings in this study had only germinated weeks to months before collection, so it likely that most if not all of their N was mobilized from seed reserves before beginning to acquire N from the soil, a process that could be influenced by any colonizing fungi on their roots. 9 1.1.5 Ectomycorrhizae and nitrogen acquisition by plants A mycorrhiza is a symbiotic relationship between roots of a photobiont and a mycobiont. At least 80% of land plants are known to form mycorrhizae, and it is postulated that the movement of plants onto land was made possible by this relationship (Field et al. 2015). Mycorrhizal fungi help to promote plant survival in a variety of ways, such as by reducing the susceptibility of plants to pathogens and toxins, increasing soil stability (Yang et al. 2017), and most importantly, by promoting enhanced nutrient and water acquisition (Reid et al. 1983; Abuzinadah et al. 1986; Näsholm et al. 2009). In parallel, the plant partner provides the fungi with photosynthetically-fixed carbon required for survival. It is through these reciprocal rewards that this relationship has evolved into one of the most stable and ubiquitous symbioses on Earth (Bücking et al. 2012). Mycorrhizal fungi can colonize plants either intracellularly, as seen in ericoid mycorrhizae, orchid mycorrhizae, and arbuscular mycorrhizae (AM), or extracellularly as in ectomycorrhizae (ECM). Although AM fungi have the largest global distribution and colonize a wider range of plant species (Wang and Qiu 2006), ECM fungi are very important in temperate and boreal forests (Taylor and Alexander 2005) and are the focus of this study. Northern forests are particularly N limited due their slow accumulation of biologically-fixed N during ecosystem development (Chapin et al. 1986). Because of this, plant nutrition is largely dependent on the portion of the N that is immobilized in organic matter, which is mostly inaccessible to the plant without the aid of microbial symbionts, including EMF (Lipson and Näsholm 2001). Ectomycorrhizal fungi have evolved numerous times from saprotrophic ancestors (Hibbett et al. 2000; Kohler et al. 2015), and have retained some decomposer abilities 10 (Abuzinadah et al. 1986; Nicolás et al. 2019). This allows them to break down complex organic matter in the soil, giving both them, and their photosymbiont, increased access to organic N present in the soil (Abuzinadah and Read 1989; Bending and Read 1995; Näsholm et al. 2009). The ability of EMF to use organic N is considered to be ubiquitous, with fungi from different lineages demonstrating the capability (Nicolás et al. 2019). Nevertheless, proteolytic ability can vary among EMF species. Lilleskov et al. (2002) found that many EMF common in soils with low inorganic N were able to grow on protein, glutamine, and serine, while taxa from areas with high inorganic N grew poorly when given protein and serine as the N source. This disparity could potentially be attributed to different abilities to utilize organic N (Lilleskov et al. 2002). Kranabetter et al. (2009) observed that the abundance of EMF was either positively and negatively affected by site productivity, depending on the fungal species. Genera with higher enzymatic capability, including Suillus, Cortinarius, and Tricholoma, appear to be replaced by nitrophilic species such as Russula, Tomentalla, and Inocybe as the amount of inorganic N increases (Kranabetter et al. 2009; Lilleskov et al. 2011; Horton et al. 2013; reviewed in Hawkins et al. 2015). In addition, certain forms of inorganic of nitrogen (NH4) are more readily taken up; therefore, preferred by some species of EMF, which was observed by Bödeker et al. (2014) by measuring a decreased expression of lignocellulytic genes in EM when ammonium was present in the field. Considering that soil inorganic N at the sites in this study is expected to be higher than intact forests, it is likely that there will be an abundance of nitrophilic fungal species encountered. 11 Using d15N in arctic forests, it has been estimated that between 61% and 86% of the N in mature ectomycorrhizal plants is derived from the mycorrhizal symbioses (Hobbie and Hobbie 2006), with the quantity of N transferred by the fungal partner to its host depending on a number of factors. Traditionally, it has been thought that the soil nutrients are “exchanged” by the fungus for carbon-rich sugars from the plant, resulting in both partners benefitting from the symbiosis. The biological market model (Noë and Hammerstein 1994) recognizes the exchange of goods or services between species. Using this model to study the ectomycorrhizal symbiosis, Franklin et al. (2014) predicted that increased competition among plant or fungal symbionts would increase the amount of resource made available. This suggests that having more plant partners would increase the C contribution of each plant below ground, and more fungal partners per phytobiont would increase the amount of N transferred to the plant by each fungus. The quantity of N transferred to the plant is also dependent on the fungal species (Abuzinadah and Read 1989) and can be influenced by factors such source and sink strength. Considering young seedlings in this study will likely be colonized by fewer fungal species than mature trees, it is possible that the transfer of N from mycobiont to photobiont will be less than would occurs in mature stands. It is also important to note that the mycorrhizal relationship may not always be a mutualistic one. For example, using varying CO2 levels, Alberton et al. (2007) demonstrated that elevated atmospheric carbon increased photosynthesis and ultimately the quantity of C supplied to the fungal symbiont. The subsequent increased rate of fungal growth resulted in more N being immobilized in the soil, thereby reducing the amount available to the plant. After injecting labelled 15NO3 into soil around Scots pine trees exposed to varying levels of shading and N additions, Hasselquist et al. (2016) observed a similar pattern. They found that greater C 12 allocation to fungal symbionts resulted in reduced N allocation from fungi to the plants, which indicated that the relationship is not necessarily reciprocal. Similarly, extensive extramatrical mycelium growth in lab experiments can result in the reduction of N in host plant shoots, and subsequent reduction in plant growth rate (Abuzinadah and Read 1989; Colpaert et al. 1996). In two extreme cases, EMF seemed to play a role in the loss of N from pine seedlings over their first growing season. Zackrisson et al. (1997) severed hyphal connections between naturally regenerating Pinus sylvestris (Scots pine) seedlings and other plants in the field. By the end of the first growing season, seedlings with intact hyphae had lower amounts of nutrients, including N, compared to levels initially present in the seeds. Seedlings with severed hyphae had no net loss of nutrients relative to the seeds. More recently, Smith et al. (2020) conducted an experiment in microcosms, where the N contents of Pinus contorta seedlings inoculated with Suillus tomentosus were monitored. When the hyphae were primed with N after a period of starvation, colonized seedlings experienced a rapid drop in N content compared to seedlings in which the hyphae had been severed. Both of these studies indicate that there can be a decrease in N content of colonized seedlings during early development, but further research is needed to determine if this is a common phenomenon. 1.1.6 Ectomycorrhizal fungal communities and colonization following a disturbance Although wildfires have long been viewed as having a positive effect on long-term forest health and regeneration, the effects on EMF communities can be detrimental immediately following the event. The heat generated by fires can reduce the total number of fungal propagules up to 10 cm deep from the soil surface, which is where most of the EMF community is located (Bergner et al. 13 2004). The re-establishment of EMF communities after wildfire may be slower than after other types of disturbance. For example, Barker et al. (2013) found temporarily lower colonization rates of Douglas-fir seedlings establishing from seed in burned sites compared to seedlings from clearcut sites. When colonization eventually does occur, EMF species diversity and abundance can also be reduced (Miller et al. 1998; Barker et al. 2013). However, the effects of fires should not be generalized, as differences in fire severity between sites, as well as spatial heterogenicity and microclimates within sites, can result in varying effects on the soil microbial community (Smithwick et al. 2005). This disparity can be seen in studies that report no long term negative effects of fire on EMF communities (Glassman et al. 2016). Similar to wildfires, most results show that there is a shift in EMF community composition following harvest, due to loss of inoculum and changes in soil chemistry (reviewed in Jones et al. 2003). In addition, there is evidence of overall loss of fungal biomass immediately following a clearcut because tree root death following harvesting reduces the abundance of possible inoculum; however, this decrease appears to be less than at wildfire sites (Rodriguez-Ramos et al. 2021). Barker et al. (2013) examined the impacts of disturbance level of both wildfire and clearcutting on first-year sown Douglas-fir ectomycorrhizal fungal communities in the interior of British Columbia. They observed two levels of each disturbance: low and high severity fires, and clearcutting with or without screefing. Overall, they found that the difference in EMF response between both types of disturbance is likely a result of the level of forest floor disturbance. Specifically, the two more severe disturbances, severe burns and screefed clearcuts, decreased 14 community diversity and richness on root tips, particularly in the first growing season. These shifts were largely due to an increased dominance of Wilcoxina spp. on seedlings from disturbed compared to undisturbed sites. This contrasts with a study by Glassman et al. (2016), who found a decrease in most Wilcoxina spp., and an increased prevalence of only Rhizopogon olivaceotinctus in the soil immediately following a severe fire compared to before the fire in a monodominant ponderosa pine forest. Similar results by Rodriguez-Ramos et al. (2021) indicate a decrease in all ECM in disturbed compared to intact sites, resulting in a total shift from EMF to saprotrophic dominance in the soil. These shifts in community composition following heavy disturbance are likely attributable to the loss of live hosts and a reduced fungal spore bank, which is likely the main source of inoculum after severe fires (Glassman et al. 2016). It is also hypothesized that fungal species that are able to withstand harsh conditions can colonize swiftly post-disturbance (Courty et al. 2015). This includes Rhizopogon and Cenococcum species (Miller et al. 1998), which have been shown to be tolerant to water stress. Availability of water may be a limiting factor for other fungal species in early succession (Pigott 1982), especially in drier areas such as those where ponderosa pine seedlings establish. There is evidence indicating that certain pine seedlings germinating following a disturbance are dependent on swift ECM formation for success. This was demonstrated by Guignabert et al. (2018), who found that mycorrhization in living maritime pine (Pinus pinaster) seedlings the first year after clearcutting increased plant nutrient status. Miller et al. (1998), observed the colonization of all surviving lodgepole pine germinants by September of the first growing 15 season, with the number of ectomycorrhizae positively correlated with the number of primary needles and root/shoot ratio. Fast colonization after disturbances may promote a higher resilience of seedlings after frequent disturbances, as those who are able to survive the first growing season generally do so while mycorrhizal. However, the timing of colonization of pine seedlings that have germinated in the field, and how this affects seedlings in terms of N status and overall growth has not been documented. 1.2 Thesis overview, objectives, and hypotheses The main objective of my thesis research was to observe the growth and N content of Pinus ponderosa and Pinus contorta seedlings germinating in post-disturbance environments in the Okanagan Valley, and to determine how these variables related to the timing of their colonization by ectomycorrhizal fungi. Two types of disturbances (wildfires and clearcutting), and two pine species (Pinus ponderosa var. ponderosa Dougl. ex C. Law. (ponderosa pine) and Pinus contora Dougl. var. latifolia Englm. (lodgepole pine)) were studied. Because ponderosa pine trees are rarely harvested in British Columbia, sowing and collecting them at clearcut sites did not seem relevant; therefore, ponderosa pine seedlings were followed at burned sites only. Seedlings were collected monthly from these sites, and their colonization status, fungal species present, nitrogen content, d15N, d13C, and growth were observed. My thesis addresses the following questions. Q1. When do pine germinants become colonized (with ECM fungi) during their first year of growth on disturbed sites in the Okanagan Valley? 16 H1. Seedlings will gradually become colonized during the first growing season, and all will show signs of colonization by the end. Q2. Does N or biomass in newly colonized pine seedlings differ from non-mycorrhizal seedlings? H2. Colonized seedlings will have higher biomass and N content when compared to uncolonized seedlings collected from the same sites at the same time because previous field studies have shown an advantage to early colonization. Q3. Which fungal species are the first to colonize young pine germinants at these disturbed sites? H3. Early fungal colonization will be by nitrophilic species, or species resistant to disturbances, such as Wilcoxina spp., Suillus spp., or Cenococcum spp. H4. The species of fungi found on root tips will change as the growing season progresses. Q4. Does the N content of seedlings relate to the EMF colonizing their roots? H5. Seedling N content will depend on the fungal species colonizing the seedling roots. 17 Chapter 2: Seedling establishment and early colonization 2.1. Background Pine regeneration success in western North America has been suffering in recent decades. This is partially due to a combination of increased drought conditions and rising temperatures (Stevens-Rumann et al. 2018) leading to a rise in the prevalence and severity of forest fires, which are only predicted to worsen (Kemp et al. 2019). Climate change, combined with frequent logging and other anthropogenic activities are increasing the amount of disturbance in northern montane and boreal forests (Abatzoglou and Williams 2016). Because, for many pine species, seedlings establish simultaneously after a stand-replacing disturbance, first-year seedling survival has a major influence on forest regeneration and subsequent succession (Hope et al. 1991a) One of the important stages in conifer seedling development is thought to be the formation of symbioses with ectomycorrhizal fungi (EMF), although the exact timing of initial colonization has not been studied in the field. While a greenhouse study by Ekwebelam and Reid (1983) and a meta-analysis by Karst et al. (2008) indicate that early mycorrhization is advantageous for seedling survival, an in vitro study by Alberton et al. (2007) found that EMF colonization hindered biomass and N uptake of seedlings. In the most extreme case, substantial loss of N from young seedlings was mediated by EMF (Smith et al. 2020). Field studies, which offer a better overall glimpse into this relationship with less manipulation, are scarce compared to lab and greenhouse studies. Nevertheless, results from field studies that examined the effects of colonization are also mixed when looking at the effects of colonization; however, it is likely that this can be explained by difference among species of colonizing fungi, or publication bias or methodological issues (Karst et al. 2008). 18 Mycorrhizal fungi aid in acquisition of limiting nutrients, which in northern montane and boreal forests, tends to be nitrogen (N). In the soils of these forests, most N occurs in organic forms (Brockley 2001b; Sponseller et al. 2016) that are inaccessible to nonmycorrhizal roots (Abuzinadah and Read 1989). During wildfires, the combustion of organic soil layers results in a large shift from bound N in organic matter to soluble, inorganic forms of N (Certini 2005; Peay et al. 2010). Similarly, following a clearcut event, a decrease in dissolved organic N (DON) is observed, this time due to increased nitrification (Bradley et al. 2001). As a result, both of these events can lead to an increase in plant-available N in the soil compared to undisturbed soils. Because N is typically limiting for conifers, and because most N is acquired via EMF (Hobbie and Hobbie 2006), studying the N dynamics as seedlings become colonized can serve as an indicator for the impact of mycorrhization on seedlings. One of the ways the contribution of EMF to N nutrition of plants can be monitored is by measuring the isotopic signature of N in plant and fungal tissues. Because of the fractionation that occurs as N moves through the hyphae, plant d15N values are lower when N is transferred from mycorrhizal fungi than when taken up directly from the soil (Proe et al. 2000; Hobbie and Hobbie 2006), and can be used as a way to gauge the proportion of N received from the fungi. Likewise, d13C values can be used to determine photosynthetic rates and subsequent carbon acquisition in seedlings because discrimination lowers the plant d13C values (Sharma and Williams 2009). In order to better understand the effects of initial EMF colonization on pine germinants in the field, this study monitored the growth and N dynamics in seedlings of two pine species, 19 ponderosa and lodgepole pine, as they became naturally colonized over the first season following a disturbance. Seeds of lodgepole pine were sown in areas affected by wildfires or clearcut logging the year before. Because ponderosa pine trees are rarely harvested in British Columbia, first year germinants were studied only in recent wildfire sites. Once germinated, seedlings from sown seed, as well as naturally regenerating pine seedlings, were collected monthly. Colonization of roots, biomass, N content, and isotopic signatures of N and C were measured in seedlings as they gradually became mycorrhizal. 2.2. Material and methods 2.2.1. Study sites All of the sites used in this study were located in the Okanagan Valley in the southern interior of British Columbia, Canada, which has a warm continental climate with hot, dry summers and cool winters (Hope et al. 1991b). Sites were characterized by disturbance type (wildfire or clearcut logging), and dominant tree species in the pre-disturbance stand (Pinus ponderosa var. ponderosa Dougl. ex C. Law. (ponderosa pine) or Pinus contorta Dougl. var. latifolia Englm. (lodgepole pine)). There were nine study sites: three each in burned ponderosa pine stands (PPWF), burned lodgepole pine stands (LPWF), and clearcut lodgepole pine stands (LPCC). The three ponderosa pine sites were at lower elevations and classified as Ponderosa Pine (PPxh1) (Table 2.1) under British Columbia’s Biogeoclimatic Ecosystem Classification (BEC) system (Pojar et al. 1987). The six lodgepole pine sites were at higher elevations and within the Montane Spruce (MSdm2) or Interior Douglas Fir (IDFdk2) zones (Table 2.1). The wildfire sites had burned in the summer of 2018, and were identified using iMap BC (https://maps.gov.bc.ca/ess/hm/imap4m/) and the BC wildfire dashboard 20 (https://governmentofbc.maps.arcgis.com/apps/opsdashboard/index.html#/f0ac328d88c74d07aa2ee385abe2a41b). Clearcut sites had been harvested in 2018 and prepared for natural germination and establishment of seedlings using chain drag in May 2019 (Stephanie Bulmer, Gorman Brothers Ltd., pers. comm.). Sites of the same disturbance type were at least 1 km apart, but sites from different disturbance types were not interspersed. Hence, the three types of sites were considered separately during statistical analysis. Both naturally regenerating seedlings and seedlings from sown seeds were sampled from each site. 2.2.2. Sowing of seeds Seeds of the appropriate tree species were sown at each site to ensure that there were enough seedlings present for frequent sampling, and to reduce the impact of my sampling on forest regeneration at these sites. For both species, seedlots from similar elevations and BEC zones within the Okanagan Valley were supplied by the British Columbia Ministry of Forests, Lands and Natural Resource Operations, Tree Seed Centre, Surrey, British Columbia, Canada: seedlot #3692 for lodgepole pine and seedlot #41708 for ponderosa pine. Seeds were surface sterilized by soaking in 30% H2O2 for 1.5 min, followed by five rinses in deionized water (Anand et al. 2013). The seeds were then stratified by soaking in tap water for 24 hours, draining, and placing in a loosely tied plastic bag at 4 °C for 28 days. Four (five at Mount Eneas-East) 1.1 x 1.4 m seedbeds were established at each of the nine sites. Seedbeds were at least 10 m apart from each other, and 20 m from the nearest living tree. At wildfire sites, seedbeds were located in relatively flat areas of severely burned forest. An area was classified as severely burned if the forest floor had been completely combusted to expose 21 mineral soil, and all canopy trees had been killed (Johnstone and Chapin 2006). At clearcut sites, seedbeds were established in areas with minimal downed logs or coarse woody debris. Table 2.1 Characteristics of study sites. Site name Pine species Disturbance type BEC zone Elevation (m) Coordinates Size of disturbance (ha) Mount Eneas (West) Ponderosa Wildfire PPxh1 780 49.43° N 119.46° W 1800 Mount Eneas (East) Ponderosa Wildfire PPxh1 792 49.43° N 119.47° W 1800 Chute Lake Ponderosa Wildfire PPhx1 720 49.38° N 119.36° W 5.5 Gottfriedsen Mountain (East) Lodgepole Wildfire MSdm2 1400 49.58° N 119. 56°W 600 Gottfriedsen Mountain (West) Lodgepole Wildfire MSdm2 1409 49.58° N 119. 58°W 600 Law Creek Lodgepole Wildfire IDFdk2 1000 49.49° N 119.41° W 15.4 12N-2 Lodgepole Clearcut MSdm2 1450 49.43° N 120.06° W 8.5 12N-3 Lodgepole Clearcut MSdm2 1500 49.43°N 120.05° W 5.2 12N-4 Lodgepole Clearcut MSdm2 1425 49.44° N 120.03° W 3.7 Ponderosa pine seeds were sown April 26-30, and lodgepole pine seeds were sown May 2-29 (wildfire sites), and May 27-30 (clearcut sites), 2019. Within each seedbed, 160 ponderosa pine or 198 lodgepole pine seeds were sown 1-2 cm deep, approximately 5 cm (ponderosa pine) or 8 22 cm (lodgepole pine) apart within rows that were placed 10 cm apart. Seedbeds were then covered with a wire or plastic mesh (2.54 cm diameter openings) to exclude rodents, birds, and larger predators, overlain with nylon window screening to exclude ants and smaller predators. Both layers were stapled to 2.5 X 5 cm wooden posts on each corner, and buried at the edges. The nylon screening was removed once the seedlings emerged to prevent shading. 2.2.3. Sampling of seedlings Two types of germinating seedlings were sampled: seedlings from the sown seeds (“sown seedlings” hereafter) and seedlings from seeds naturally present on the site (“naturally regenerating seedlings” hereafter). Sampling of sown seedlings began on June 3, 2019 for ponderosa pine, and on June 17 for lodgepole pine, when seedlings from sown seeds were just beginning to emerge from the soil. Sampling then took place every month, finishing in October for the lodgepole pine (five sampling times) and in September for the ponderosa seedlings (four sampling times). To obtain a sample representative of each site, where possible, 1-3 seedlings were collected from each seedbed, totaling 7-10 sown seedlings per site, at each sampling time. In addition, 1-4 naturally regenerating seedlings were collected from within a 5-m radius of each seedbed at ponderosa and lodgepole pine wildfire sites from Jun-Sept; naturally regenerating seedlings were not collected from burned lodgepole pine sites in October or from clearcut sites. In total, 42-60 seedlings from sown seeds and 29-43 naturally regenerating seedlings were collected from each site over the growing season. Harvested seedlings were kept on ice in a cooler for transport to the lab before being stored at 4 °C. 23 2.2.4. Soil sampling Soil was collected from the area just outside each seedbed at the time of first seedling collection in June 2019, the last collection in the fall (September or October 2019), and from adjacent undisturbed forests in July, 2020. A 5-cm diameter soil auger was used to collect 0-20 cm deep soil samples within 30 cm of the perimeter on all four sides of each seedbed. The four samples per seedbed were combined in the field to form one composite sample. No forest floor soil remained at the severely burned sites because it had been combusted; therefore, those soil samples contain only mineral soil. At clearcut sites, the forest floor had been highly disturbed and mixed into the mineral soil, so no attempt was made to separate mineral soil and forest floor. 2.2.5. Processing and analysis of seedlings Within 48 hrs of collection, all seedlings were thoroughly rinsed with tap water and root systems examined in water under a dissecting microscope to quantify mycorrhiza formation. The presence of a mantle on root tips was used to classify seedlings as mycorrhizal. In cases of uncertainty, root tips were examined at 40x or 100x magnification under a compound microscope in order to detect early mantle formation. The number of nonmycorrhizal and mycorrhizal root tips per seedling were counted, as were the abundances of different ectomycorrhizal morphotypes, which were distinguished based on the basis of colour and mantle pattern. Root tips with the same ectomycorrhizal morphotype from each seedling (2-40 per morphotype, depending on the number available) were then frozen together in one microcentrifuge tube at -80 °C. Roots from a subset of seedlings (1-2 per site per sampling time) were set aside to check for the presence of arbuscular mycorrhizae using ink and vinegar staining (Vierheilig et al. 1998). The shoots of those seedlings were processed as described below. 24 After examination under the microscope, roots and shoots were separated, weighed, and oven dried at 60 °C for at least 72 hrs. The dried roots and shoots of sown seedlings were re-weighed, ground to a fine powder in a ball mill, and analysed for % N content, as well as the ratios of 14N/15N, and 13C/14C using dry combustion in a Euro EA (CHNS) Elemental Analyser spectrometer (Milan, Italy) linked to an IsoPrime continuous-flow mass spectrometer (GV Instruments, Manchester, UK). Soil standards with known C and N content were used as quality controls. Biomass and % N were used to calculate total N content per shoot or root. The dry weights of naturally regenerating seedlings sampled from June to August were not recorded; therefore, % N and isotopic signatures are presented for those seedlings, but biomass and total N for those months are not. 2.2.6. Soil analysis Within 24 hrs of being collected, soils were homogenized, and large roots and rocks were removed before 5.5 g from each composite sample was shaken with 25 mL 2 M KCl. The KCl extracts were then frozen at -20 °C for later analysis of ammonium, nitrate (Miranda et al. 2001) and dissolved organic N (Darrouzet-Nardi et al. 2013). The remaining soil in each sample was then sieved through a 2-mm sieve and dried at 60 °C for 48 hrs. A subsample of this dried soil was milled, and then analyzed for total N, 14N, and 15N. A second subsample of dried soil was sent to the Analytical Chemistry Services Laboratory of the British Columbia Ministry of Environment and Climate Change Strategy, Victoria, British Columbia for nutrient (Al, B, Ca, Cu, Fe, Mg, Mo, Na, P, S, Zn) analysis using microwave digestion followed by inductively coupled plasma mass spectrophotometry. 25 2.2.7. Data analysis Nitrogen and carbon isotope values are expressed as d15N and d13C, which were calculated using δ14N or δ13C (‰)=(Rsample/Rstandard - 1) * 1000 ‰, with R=14N/15N or 13C/14C where the standard reference material is atmospheric N2 for N and PeeDee Belemnite for C. Data from each disturbance type-tree species combination (i.e., ponderosa pine wildfire sites, lodgepole pine wildfire sites, and lodgepole pine clearcut sites) were analyzed separately because the three types of sites were not geographically interspersed. In order to examine when seedlings had accumulated N or newly fixed C above that present in the seed, generalized linear mixed models were used for analysis of univariate seedling data (nutrient concentration or content, d15N, d13C, and biomass), using site as a random factor, and month as a fixed factor. Significant differences between months, and between seedlings were evaluated using using type III analysis of variance (ANOVA) with Satterthwaite’s method. Tukey’s Honest Significant Difference (HSD) tests using p £ 0.05 were used to determine which months were different from each other. False discovery rate (FDR) correction was used on results of mixed effect linear models because multiple variables were tested on the same seedlings. Seedling values were compared to seed values using one-tailed t-tests. To determine if there were differences between mycorrhizal and nonmycorrhizal seedlings using the measured characteristics, multivariate analyses were performed using shoot biomass, % N, d15N and d13C for sown seedlings, and shoot % N, d15N and d13C for naturally regenerating seedlings. Principal component analysis (PCA) and permutational multivariate analysis of variance (PERMANOVA) were conducted using combined data from July and August because both mycorrhizal and nonmycorrhizal seedlings occurred in sufficient numbers in only those 26 months. Multivariate analyses were conducted on only shoot data because there were fewer replicates of roots due to loss of samples during analysis and allocation to root staining. To confirm differences between mycorrhizal and nonmycorrhizal seedling for specific variables, ANOVAs were used to evaluate mixed effects models with mycorrhizal status as fixed factor, and site as a random factor. Normality was tested using a Shapiro-Wilk test, and homoskedacity was evaluated by plotting the residuals. Variance was expected to slightly increase throughout the growing season, as seedlings got bigger and started to differentiate more. All statistical analyses were completed in R version 1.2.1335, with a significance level of p £ 0.05. 2.3. Results 2.3.1. Soil chemistry The KCl-extractable N pool was dominated by ammonium at both the burned and clearcut lodgepole pine sites (Table 2.2). By contrast, ammonium was only 50 % higher than nitrate at the burned ponderosa pine sites in June and only 27 % higher in September. By the fall, the concentration of extractable inorganic N (i.e. ammonium and nitrate) had decreased by an average of 73 % across all sites, compared to the spring. The concentrations of extractable amino acids were low, and did not change substantially between spring and fall, meaning that they represented a slightly higher percentage of extractable N in soil by the fall (2.6 % on average across all sites) compared to June (0.4 %). The total soil concentrations of extractable N, and most other nutrients appeared to be more heterogenous in clearcut than burned soils from lodgepole pine sites (Tables 2.2, 2.3), although site types could not be compared statistically because they were not interspersed geographically. Similarly, data from disturbed and 27 undisturbed parts of the sites could not be compared statistically because they were collected in different years. Nevertheless, it appeared that extracted inorganic N was highest in samples collected in June the year after disturbance, at the same time the seedlings were germinating, when compared to samples from the subsequent October, and adjacent undisturbed soils (Table 2.2). The soil pH was acidic in at all sites, although slightly less so in burned compared to undisturbed sites, and more so in clearcut sites compared to adjacent undisturbed areas (Table 2.2). 28 Table 2.2 KCl- extractable nitrate-, ammonium-, and amino acid-N; total N (dry combustion elemental analysis) and pH of soils from ponderosa pine wildfire (PPWF), lodgepole pine wildfire (LPWF) and lodgepole pine clearcut (LPCC) sites, and soils from adjacent undisturbed area. Means (standard error), n=3 sites. Date collected NO3 -N (mg kg-1) NH4-N (mg kg-1) Amino acid-N (mg kg-1) Total N (mg kg-1) d 15N d 13C pH PPWF Jun 2019 141.99 (35.64) 218.96 30.65 1.07 (0.16) 1507.73 (174.06) 0.54 (1.01) -23.74 (0.76) n/a PPWF Sep 2019 43.72 (7.73) 55.67 (7.81) 0.98 (0.24) 1178.89 (181.14) - 1.13 (0.82) -23.74 (0.76) 7.01 (0.10) PPWF Undisturbed Jul 2020 3.34 (1.30) 43.93 (12.05) 1.75 (0.45) 1672.40 (500.88) -1.08 (1.01) -25.46 (0.16) 6.10 (0.06) LPWF Jun 2019 0.66 (0.18) 250.96 (105.18) 1.01 (0.18) 1076.42 (99.75) 0.21 (1.13) -22.56 (0.27) n/a LPWF Oct 2019 5.46 (1.82) 36.96 (4.34) 0.91 (0.13) 798.52 (75.22) 0.77 (0.37) -24.81 (0.70) 5.88 (0.22) LPWF Undisturbed Jul 2020 0.09 (0.05) 18.88 (0.33) 1.25 (0.16) 925.26 (209.76) 0.38 (0.74) -26.32 (0.12) 5.64 (0.07) LPCC Jun 2019 12.48 (5.01) 74.05 (15.54) 1.11 (0.08) 1499.06 (28.42) -1.78 (0.52) -24.60 (0.41) n/a LPCC Oct 2019 8.34 (1.87) 38.89 (5.58) 2.98 (0.31) 1743.74 (148.56) -3.23 (0.67) -26.02 (0.42) 5.02 (0.14) LPCC Undisturbed Jul 2020 10.13 (4.78) 21.69 (3.19) 1.44 (0.28) 1094.82 (123.77) -0.13 (0.39) -26.39 (0.10) 5.52 (0.04) 29 Table 2.3 Soil nutrient concentrations (mg kg-1 (standard error)) determined by microwave digestion followed by ICP-MS for soils from disturbed ponderosa pine wildfire (PPWF), lodgepole pine wildfire (LPWF) and lodgepole pine clearcut (LPCC) sites, and soils from adjacent undisturbed area. n=3 sites. Disturbed soils were collected in the spring of 2019, undisturbed in the spring 2020. Al Ca Cu Fe K Mg Mn Na P Zn PPWF 20333.33 (1855.92) 15166.67 (7917.98) 37.00 (18.25) 16333.33 (1855.92) 1966.67 (166.67) 3966.67 (1271.92) 653.33 (14.53) 1453.33 (246.67) 570.00 (92.92) 68.33 (7.86) PPWF Undisturbed 19666.67 (666.67) 5966.67 (1072.90) 9.70 (1.15) 16000.00 (577.35) 2133.33 (338.30) 3066.67 (317.98) 656.67 (84.13) 1000.00 (400.37) 636.67 (197.68) 78.33 (14.05) LPWF 21333.33 (333.33) 8266.67 (185.59) 18.33 (3.28) 17333.33 (881.92) 1433.33 (240.37) 3833.33 (290.59) 373.33 (108.68) 1233.33 (33.33) 1063.33 (172.47) 53.33 (15.34) LPWF Undisturbed 21333.33 (3527.67) 5766.67 (581.19) 21.67 (3.93) 18333.33 (2185.81) 1500.00 (251.66) 3666.67 (683.94) 460.00 (126.62) 1286.67 (217.97) 593.33 (112.89) 60.00 (13.65) LPCC 18000.00 (3785.94) 6166.67 (2967.79) 15.93 (9.05) 14900.00 (3865.66) 1633.33 (296.27) 2866.67 (1386.04) 656.33 (317.90) 1350.00 (510.72) 796.67 (302.78) 68.33 (20.74) LPCC Undisturbed 15666.67 (1763.83) 2133.33 (185.59) 12.47 (2.49) 11733.33 (1392.04) 1366.67 (66.67) 1376.67 (321.26) 562.67 (329.63) 1766.67 (371.18) 810.00 (313.74) 76.67 (32.42) 2.3.2. Germination Sown seeds started germinating at all sites 5 to 6 weeks after sowing, with maximum germination rates averaging 28.67 ± 4.26 % for ponderosa pine sites, 32.67 ±12.14 % for lodgepole pine at burned sites, and 33.3 ± 0.67 % at clearcut sites (Table 2.4). Lodgepole pine seeds continued to germinate into July, while maximum germination for ponderosa pine seeds was in early June. The lowest germination rate for lodgepole pine seeds was observed at Law Creek, which was located in a drier biogeoclimatic zone (IDF) than the other lodgepole pine sites 30 (MS). Small numbers of naturally regenerating seedlings were first observed when sowing seeds, and they continued to appear into the early summer. Table 2.4 Seedling germination and numbers at each site. Seeds were sown in 2019 between April 26-30 (ponderosa pine wildfire sites), May 2-29 (lodgepole pine wildfire sites), and May 27-30 (lodgepole pine clearcut sites). Site name Site type # of seeds sown Maximum # of seedlings observed Overall % germination Date maximum observed Mount Eneas (West) Ponderosa wildfire 640 227 35 Jun 5 Mount Eneas (East) Ponderosa wildfire 800 187 23 Jun 5 Chute Lake Ponderosa wildfire 640 161 25 Jun 3 Gottfriedsen Mountain (East) Lodgepole wildfire 792 428 54 Jul 15 Gottfriedsen Mountain (West) Lodgepole wildfire 792 267 34 Jul 15 Law Creek Lodgepole wildfire 792 85 11 Jul 18 12N-2 Lodgepole clearcut 792 269 34 Jul 17 12N-3 Lodgepole clearcut 792 273 34 Jul 17 12N-4 Lodgepole clearcut 792 252 32 Jul 17 31 2.3.3. Colonization by ectomycorrhizal fungi In July, a month after germination was first observed, seedlings from sown seeds first showed signs of ectomycorrhiza formation at all sites (numbers at top of Figure 2.1). More seedlings became colonized over the summer; by September all sampled seedlings were colonized. Naturally regenerating ponderosa, but not lodgepole pine seedlings were colonized earlier than sown seedlings, with a few colonized ponderosa pine seedlings observed in June, and the majority colonized by July (Figure A1). As with the sown seedlings, 100 % of the naturally regenerating seedlings were colonized by the fall. Root systems became more colonized as the year went on, although there was very large variation in the percentage of root tips colonized on mycorrhizal seedlings (Tables 2.5, 2.6). This is mainly because root systems varied substantially in size, and those with only a few root tips either had very low or very high percentage of colonization. No formation of arbuscular mycorrhizae was observed in any of the stained roots. 32 −1.5−1.0−0.50.0Jun Jul Aug Sep OctMonthSeedling biomass (log(g))−2−1Jun Jul Aug Sep OctMonthSeedling biomass (log(g))−2−1Jun Jul Aug Sep OctMonthSeedling biomass (log(g))* * * * * 29 67 100 100% 0 42 70 100% 100 0 0 16 82 100% * * * * * * Ponderosa Pine Wildfire Lodgepole Pine Wildfire Lodgepole Pine Clearcut Figure 2.1 Biomass (log(g)) of sown ponderosa pine seedlings from burned sites, and lodgepole pine from burns and clearcuts, the first growing season following a disturbance. Percent (%) of seedlings colonized across all three sites is shown at the top of each month. Red asterisks indicate significant differences (p £ 0.05) from June according to results from the linear mixed effects model. 33 Table 2.5 Percent (mean (standard deviation)) of root tips colonized per seedling for sown mycorrhizal seedlings. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcut (LPCC) sites. The range of number (n) of seedlings colonized per site per month are shown. n Site Type Jul Aug Sept Oct Chute Lake 2-9 PPWF 24.50 (14.85)2 59.60 (4.04) 69.11 (13.77) n/a1 Eneas (East) 2-10 PPWF 27.50 (20.51)2 42.43 (17.21) 42.80 (23.77) n/a1 Eneas (West) 8-9 PPWF n/a1 34.63 (16.45) 62.11 (23.72) n/a1 Law 4-10 LPWF 32.25 (27.94) 52.50 (27.18) 30.50 (21.27) 57.00 (33.41) Gotffriedson (East) 2-10 LPWF 68.00 (16.97)2 65.22 (13.51) 82.90 (15.76) 82.40 (19.78) Gotffriedson (West) 6-10 LPWF n/a1 52.33 (23.00) 70.88 (13.43) 48.30 (27.74) 12-N2 6-10 LPCC 55.17 (25.18) 79.63 (20.47) 69.10 (14.24) 96.60 (6.13) 12-N3 1-10 LPCC 44.002 46.57 (34.58) 64.10 (21.09) 78.00 (22.95) 12-N4 6-10 LPCC 68.50 (29.49) 74.00 (22.66) 60.44 (12.02) 84.90 (13.71) 1. No sampled seedlings were colonized at this site in this month. 2. < 3 colonized seedlings were sampled at this site in this month. 34 Table 2.6 Percent (mean (standard deviation)) of root tips colonized per seedling for naturally regenerating mycorrhizal seedlings. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole from wildfire (LPWF). The range of number (n) of seedlings colonized per site per month are shown. n Site Type Jun Jul Aug Sept Oct Chute Lake 3-4 PPWF n/a1 80 (12.03) 78.33 (15.82) 96 (6.08) n/a1 Eneas (East) 3 PPWF n/a1 37 (29.22) 48 (30.51) 40.67 (32.96) n/a1 Eneas (West) 3 PPWF n/a1 65.67 (15.04) 66.67 (27.10) 83.33 (11.67) n/a1 Law 1-4 LPWF 92 61.52 (44.55) 49 (36.59) 33.67 (29.30) 70.25 (34.64) Gotffriedson (East) 3-4 LPWF 40 (13.11) 47.67 (24.70) 89.33 (8.22) 84 (15.52) 99.67 (0.58) Gotffriedson (West) 3-4 LPWF n/a1 46.25 (24.70) 72.67 (12.10) 78 (9.53) 99 (1.73) 1. No sampled seedlings were colonized at this site in this month. 2. < 3 colonized seedlings were sampled at this site in this month. 2.3.4. Seedling biomass accumulation Once germinated, seedling biomass increased in a log-linear fashion throughout the growing season (Figures 2.1, A2, Table 2.7). Biomass of sown seedlings reached 520.03 ± 36.68 mg for ponderosa pine, 117.85 ± 22.96 mg for lodgepole pine in burned sites, and 93.64 ± 83.77 mg for 35 lodgepole pine seedlings in clearcuts by the final sampling. Both sown seedlings and naturally regenerating seedlings had accumulated significant amounts of new biomass by July, one month following germination. Seedling d13C had already decreased well below seed values by June (Figure 2.2; one-tailed t-test, p = 0.05), indicating that the seedlings had already started to acquire photosynthetic C. d13C continued to decrease over the growing season in shoot and root tissues from both species and both disturbance types, especially in sown seedlings (Figures 2.2, A3, Table 2.15). Table 2.7 Results from linear mixed models using type III ANOVA with Satterthwaite’s method of characteristics of seedlings collected throughout the growing season. Significant differences among seedlings sampled in different months and interactions (p £ 0.05) are indicated in bold. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). Seedling biomass (log (g)) Total N content (log(mg)) Sums of Squares F Df P Sums of Squares F Df P PPWF 16.42 161.6 3 <0.001 10.66 110.7 3 <0.001 LPWF 31.97 167.9 4 <0.001 16.66 81.18 4 <0.001 LPCC 33.49 103.8 4 <0.001 14.47 29.27 4 <0.001 36 −30−25Jun Jul Aug Sep OctMonthShoot −30−25Jun Jul Aug Sep OctMonthShoot −30−25Jun Jul Aug Sep OctMonthShoot δ 13C δ 13C δ 13C * * * * * * * * * * * * * * * * * * * * * * δ 13C δ 13C δ 13C a. b. c. d. e. f. Figure 2.2 Root and shoot d13C of sown ponderosa pine seedlings from (a, b) burned sites and lodgepole pine from (c, d) burned and (e, f) clearcut sites the first growing season following a disturbance. The hashed line indicates the seed d13C of the seedlot from which these seedlings germinated (-22.732 ± 0.76 in ponderosa pine, -22.12 ± 0.54 in lodgepole pine). Each open circle represents one seedling. Black points and error bars indicate mean and standard error. Red asterisks indicate significant differences (p £ 0.05) from June according to linear models. 37 2.3.5. Seedling nutrient status Sown seedlings sampled in June had similar total N and % N as the seeds (Figures 2.3, 2.4, Table 2.8); seedlings initially accumulated N at a slower rate than biomass (Figure 2.4), resulting in a decrease in the concentration of N in shoot tissues by July (Figure 2.3). By August all groups of sown seedlings had accumulated amounts of N significantly above seed N content (Figure 2.4), with a similar trend in naturally regenerating seedlings (Figure A4); this was two months after germination and one month after initial mycorrhizal colonization. Nitrogen concentrations in shoot tissues of sown seedlings were stable from this point onward, as seedlings continued to grow (Figure 2.3). Sampling of naturally regenerating seedlings did not coincide with the period of initial high concentrations of N in shoots but, by the fall, the N concentration in all groups of seedlings was approximately 2.2-2.5 % (Figures 2.3, A1), which is considered adequate (Carter 1992). d15N values of roots and shoots of sown seedlings were very similar to those of the seeds (-3.09 ± 0.305 for ponderosa pine and -2.56 ± 0.28 for lodgepole pine) at the first sampling in June (Figure 2.5). In both sown and naturally regenerating seedlings, there was a lag of a couple months before d15N increased slightly in shoots and roots as the summer progressed, except in the roots of lodgepole pine seedlings from clearcuts (Figures 2.5, A5). d15N was stable in the fall in lodgepole pine tissues. Concentrations of most other macronutrients (Table 2.9) and micronutrients (Table 2.10) in the shoots of both sown and naturally regenerating seedlings sampled in September were within the ‘adequate’ range for foliar nutrients in mature ponderosa and lodgepole pine (Carter 1992; 38 Moore et al. 2004). An exception was that shoots of both groups of lodgepole pine seedlings collected from burned sites, as well as sown seedlings from clearcuts, were below the adequate range for Ca, while sown seedlings of both species from both disturbance types were B deficient. Shoots from all seedling groups had very high concentrations of Cu, Fe, Mn and Zn (Carter 1992; Moore et al. 2004). Table 2.8: Weight, carbon and nitrogen content of the ponderosa and lodgepole pine seeds used in this study. Standard error is shown in brackets, n=5 seeds. Total dry weight (mg) % N d 13C d 15N Total C (mg) Total N (mg) Ponderosa 2.27 (0.17) 5.6 (0.3) -22.32 (0.34) -3.08 (0.30) 12.16 (0.93) 1.26 (0.09) Lodgepole 0.213 (0.025) 6.2 (0.1) -22.12 (0.57) -2.56 (0.28) 1.10 (0.13) 0.13 (0.02) 39 Ponderosa Pine Wildfire Lodgepole Pine Wildfire Lodgepole Pine Clearcut a a a b b b b b b b b c c c Figure 2.3 Percent (%) N of sown ponderosa pine shoots from burned sites, lodgepole pine from burned sites, and lodgepole pine from clearcuts over the first growing season following the disturbances. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of seedlings sampled in each month. Letters indicate months that are significantly different from each other, as determined by Tukey’s HSD post-hoc test. The hashed lines indicate the seed N. 40 0.00.51.0Jun Jul Aug Sep OctMonthTotal seedling N (log (mg))−1.0−0.50.00.51.0Jun Jul Aug Sep OctMonthTotal seedling N (log (mg))−1.0−0.50.00.51.0Jun Jul Aug Sep OctMonthTotal seedling N (log (mg))* * * * * * * * Ponderosa Pine Wildfire Lodgepole Pine Wildfire Lodgepole Pine Clearcut Figure 2.4 Total N (log(mg)) of sown ponderosa pine seedlings from burned sites, and lodgepole pine from burned sites and clearcuts the first growing season following a disturbance. The hashed line indicates the amount of seed N (1.26 ± 0.09 mg in ponderosa pine, 0.13 ± 0.02 mg in lodgepole pine). Each open circle represents one seedling. Black points and error bars indicate mean and standard error of each month. Red asterisks indicate significant differences (p £ 0.05) from June according to linear models. 41 −15−10−50510Jun Jul Aug Sep OctMonthRoot −15−10−50510Jun Jul Aug Sep OctMonthRoot δ 15N δ 15N * * * * * * −10−50510Jun Jul Aug Sep OctMonthShoot −10−50510Jun Jul Aug Sep OctMonthShoot δ 15N * * * * δ 15N * * ◦ * ◦ −10−50510Jun Jul Aug Sep OctMonthShoot * ◦ δ 15N δ 15N −15−10−50510Jun Jul Aug Sep OctMonthRoot a. b. c. d. e. f. Figure 2.5 Root and shoot d15N of sown (a, b) ponderosa pine seedlings from burned sites, (c, d) lodgepole pine from burned sites and (e, f) clearcuts the first growing season following the disturbance. The hashed line indicates the isotopic signatures in the seeds (-3.08 ± 0.30 in ponderosa pine, -2.56 ± 0.28 in lodgepole pine). Each open circle represents one seedling. Black points and error bars indicate mean and standard error. Red asterisks indicate significant differences (p £ 0.05) from June, red circles indicate p £ 0.1 according to linear models. δ 15N 42 Table 2.9 Concentrations (mg kg-1 (standard error)) of macronutrients in root and shoot tissues of seedlings collected in Fall 2019, as determined by microwave digestion followed by ICP-MS. n=3 sites. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). Values were compared to Brockley (2001a) for foliar nutrient concentrations, and all were determined to be adequate. Site Ca K Mg P S Sown PPWF roots 3866.67 (440.96) 7933.33 (66.67) 1400.00 (100.00) 2333.33 (145.30) 1500.00 (57.74) Sown PPWF shoots 2633.33 (333.33 9866.67 (133.33) 1100.00 (57.74) 2933.33 (202.76) 1966.67 (33.33) Naturally regenerating PPWF roots 3266.67 (176.38) 7366.67 (440.96) 1233.33 (66.67) 2200.00 (57.74) 1566.67 (88.19) Naturally PPWF shoots 2466.67 (260.34 9466.67 (338.30) 1166.67 (66.67) 2633.33 (145.30) 1833.33 (66.67) Sown LPWF roots 2043.33 (684.75) 6900.00 (1662.33) 1163.33 (243.61) 1933.33 (417.67) 1650.001 (50.00) Sown LPWF shoots 1766.67 (218.58) 9666.67 (176.38) 1133.33 (88.19) 2833.33 (202.76) 1800.00 (152.75) Naturally regenerating LPWF roots 3700.00 (1100.00) 11166.67 (2445.63) 2100.00 (251.66) 4066.67 (817.18) 2100.00 (602.77) Naturally regenerating LPWF shoots 1733.33 (166.67) 9400.00 (300.00) 1153.33 (100.88) 2833.33 (233.33) 1733.33 (88.19) Sown LPCC roots 2063.33 (647.05) 8100.00 (2281.08) 1036.67 (325.08) 3033.33 (829.32) 1430.00 (331.51 Sown LPCC shoots 1566.67 (66.67) 9833.33 (638.57) 1166.67 (66.67) 3100.00 (100.00) 1700.00 (100.00) 1. n < 3 43 Table 2.10 Concentrations (mg kg-1 (standard error)) of micronutrients and Al in root and shoot tissues of seedlings collected in September as determined by microwave digestion followed by ICP-MS. < DL = below detection limit. n=3 sites. Data shown for ponderosa pine seedlings from wildfire (PPWF), and lodgepole pine from wildfire (LPWF) and clearcuts (LPCC). Values were compared to Brockley (2001a) for foliar nutrient concentrations (of all except Al, Mo and Na), and all that had reached the detection limit were determined to be adequate. Site Al B Cu Fe Mn Mo Na Zn Sown PPWF roots 3366.67 (260.34) 13.001 (0.00) 20.33 (5.46) 3266.67 (218.58) 133.00 (24.27) 1.87 (0.93) 260.00 (36.06) 69.00 (7.00) Sown PPWF shoots 316.67 (24.04) 18.00 (1.15) 7.33 (0.28) 333.33 (24.04) 196.67 (48.07) 0.79 (0.26) 40.00 (4.62) 80.67 (3.71) Naturally PPWF roots 2333.33 (272.85) 12.00 (1.53) 28.33 (1.76) 2366.67 (284.80) 130.00 (11.55) 2.40 (1.01) 153.33 (20.28) 61.00 (5.51) Naturally PPWF shoots 403.33 (46.31) 25.00 (5.51) 10.00 (1.01) 426.67 (71.26) 183.33 (18.56) 1.121 (0.38) 35.33 (3.38) 82.00 (9.07) Sown LPWF roots 3433.33 (633.33) < DL 14.07 (3.54) 3633.33 (520.68) 210.00 (110.15) < DL 283.33 (49.10) 63.00 (22.61) Sown LPWF shoots 370.00 (45.09) 19.00 (2.52 5.70 (0.12) 190.00 (30.55) < DL < DL 41.33 (8.09) 63.67 (9.91) Naturally regenerating LPWF roots 6333.33 (328.30) 32.33 (6.67) 52.67 (10.09) 6700.00 (305.51) 336.67 (197.43) 2.05 (1.24) 456.67 (34.80) 114.67 (42.70) Naturally regenerating LPWF shoots 443.33 (29.06) 22.33 (0.88) 7.73 (0.19) 170.00 (0.00) 200.00 (55.08) < DL 40.67 (1.67) 57.67 (0.67) Sown LPCC roots 907.38 (363.33) < DL 16.60 (5.30) 2333.33 (592.55) 310.00 (87.18) < DL 280.00 (101.16) 121.00 (44.84) Sown LPCC shoots 363.33 (69.60) 14.501 (1.50) 6.17 (0.43) 143.33 (8.82) 330.00 (81.85) < DL 52.33 (7.31) 80.00 (2.08) 1. n < 3 44 2.3.6. Effect of mycorrhization on seedlings During July and August, both mycorrhizal and nonmycorrhizal sown seedlings were present in sufficient numbers to test whether the characteristics of these two groups differed. Using shoot biomass, % N, d15N, and d13C as response variables, PERMANOVAs indicated that sown mycorrhizal and nonmycorrhizal lodgepole pine seedlings differed from each other at both the burned and clearcut sites, but ponderosa pine seedlings did not (Table 2.11, Figure A6). Using the same variables, with the exception of shoot biomass (i.e., % N, d15N, and d13C of shoots), PERMANOVAs also detected differences between mycorrhizal and nonmycorrhizal seedlings regenerating naturally after fire, for both tree species, but only later in the summer (Table 2.12, Figure A7). 45 Table 2.11 Results of PERMANOVA1 comparing nonmycorrhizal and mycorrhizal sown seedlings. P values £ 0.05 are shown in bold. 1. Biomass, Shoot % N, and d 15N and d 13C of shoot tissues were included as response variables. Month Mycorrhizal n Nonmycorrhizal n Sums of Squares F Value R2 P Ponderosa pine wildfire July 3 23 4.661 1.1738 0.04856 0.33 Ponderosa pine wildfire August 20 5 4.932 1.2457 0.05138 0.27 Lodgepole pine wildfire July 10 13 20.392 6.0974 0.19607 0.003 Lodgepole pine wildfire August 19 8 15.014 4.24 0.15014 0.018 Lodgepole pine clearcut July 6 21 11.608 3.191 0.13191 0.012 Lodgepole pine clearcut August 18 7 9.776 2.5822 0.08729 0.063 46 Table 2.12 Results of PERMANOVA1 comparing nonmycorrhizal and mycorrhizal naturally regenerating seedlings sampled in Jun-Aug. P values £ 0.05 (ANOVA) are in bold. 1. % N, d 15N, and d 13C of shoot tissues were included as response variables. 2.3.6.1. Lodgepole pine seedlings Depending on disturbance type, tree species and seedling type, mycorrhizal seedlings differed from nonmycorrhizal seedlings in all of the variables measured. Over July and August, shoots of mycorrhizal lodgepole pine seedlings germinating from sown seed were larger and had accumulated more N than nonmycorrhizal seedlings (Table 2.13, Figure 2.6 b, c, Figure 2.7 b, c). Mycorrhizal lodgepole pine seedlings sampled from burned sites were 63% larger than nonmycorrhizal seedlings and contained 39% more N, while mycorrhizal seedlings from clearcuts were 61% larger and contained 46% more N than nonmycorrhizal seedlings. By contrast, neither shoot nor root % N (data not shown) differed between mycorrhizal and nonmycorrhizal lodgepole pine sown seedlings (Table 2.13, Figure 2.8 b, c). In naturally Month Mycorrhizal n Nonmycorrhizal n Sums of Squares F Value R2 p value Ponderosa pine wildfire July 21 9 0.22 0.068 0.0052 0.97 Ponderosa pine wildfire August 22 6 7.16 2.77 0.22 0.063 Lodgepole pine wildfire June 4 23 5.71 2.06 0.15 0.117 Lodgepole pine wildfire July 16 7 9.90 3.95 0.22 0.01 47 regenerating lodgepole pine seedlings % N in shoot tissues was also similar in mycorrhizal and non-mycorrhizal seedlings in June, but increased in mycorrhizal relative to non-mycorrhizal seedlings in July and August (Table 2.14 Figure A8 b). For sown seedlings, shoots of mycorrhizal lodgepole pine from burned sites had higher δ15N values compared to those of nonmycorrhizal seedlings in both July and August (Table 2.15, Figure 2.9). Root tissues were enriched in δ15N in mycorrhizal lodgepole pine seedlings from clearcuts. Enrichment in 15N relative to the seed N, seemed to occur in roots of sown seedlings with time at the burned sites and with mycorrhization at the clearcut sites (Fig. 2.9 d, f). In naturally regenerating lodgepole pine seedlings at burned sites, δ15N was higher in both shoots and roots of mycorrhizal than nonmycorrhizal seedlings (Table 2.16, Fig. A9 c, d) across June through August, but for shoots especially as the summer progressed. Mycorrhizal lodgepole pine seedlings at burned sites were generally depleted in 13C compared to nonmycorrhizal seedlings, regardless of whether they were naturally regenerating (Figure A10, Table 2.16) or had grown from sown seed (Figure 2.10 c, d, Table 2.15). The differences in δ13C between mycorrhizal and nonmycorrhizal naturally regenerating seedlings was negligible in July (Figure A10). There was no difference in δ13C between mycorrhizal and nonmycorrhizal sown lodgepole pine seedlings from clearcut sites (Table 2.15, Figure 2.10 e, f). 2.3.6.2. Ponderosa pine seedlings Although differences in biomass and total N were not detected between mycorrhizal and nonmycorrhizal ponderosa pine seedlings growing from sown seeds (Table 2.13; Figures 2.6 a, 48 2.7 a), naturally regenerating mycorrhizal ponderosa pine seedlings had accumulated more N than nonmycorrhizal seedlings across July and August but did not have higher biomass (Table 2.14, Figures A11, A12). Differences in % N in roots and shoots were not conclusive for either regeneration type, and were dependent on the month sampled (Tables 2.13, 2.14), but tended to be higher in mycorrhizal than nonmycorrhizal seedlings in August (Figures 2.8 a, A8 a). δ15N values were enriched in the roots of mycorrhizal sown and naturally regenerating seedlings, relative to nonmycorrhizal seedlings, but for sown seedlings, this was observed in August only, (Tables 2.15, 2.16; Figures 2.9 a, b, A9 b). For ponderosa pine seedlings, mycorrhization did not have a discernable or consistent effect on δ13C (Tables 2.15, 2.16, Figures 2.10 a, b, A10 a, b). 49 Table 2.13 Results from linear mixed models using type III ANOVA with Satterthwaite’s method of seedling characteristics of sown mycorrhizal and nonmycorrhizal seedlings sampled in July and August. Significant differences and interactions (p £ 0.05) are indicated in bold. Data are shown for ponderosa pine seedlings from wildfire sites (PPWF), and lodgepole pine from wildfire sites (LPWF) and clearcuts (LPCC). 1. Mycorrhizal status Seedling biomass (log (g)) Total N content (log(mg)) Shoot % N Root % N Sums of Squares F Value P Value Sums of Squares F Value P Value Sums of Squares F Value P Value Sums of Squares F Value P Value PP WF Status1 Month Status*Month 0.069 0.33 0.088 2.73 13.02 3.48 0.11 0.00089 0.070 0.12 0.26 0.021 3.99 8.99 0.72 0.11 0.0049 0.40 0.20 0.32 1.974 0.71 1.15 7.13 0.40 0.29 0.010 0.20 0.32 1.97 0.714 1.12 7.13 0.60 0.29 0.011 LP WF Status1 Month Status*Month 0.22 1.025 0.0029 6.27 29.50 0.084 0.033 0.00003 0.77 0.170 0.34 0.036 4.58 9.047 0.95 0.039 0.0048 0.34 0.76 8.75 2.510 0.57 6.60 1.89 0.45 0.013 0.18 0.28 2.44 0.015 0.43 3.76 0.23 0.52 0.06 0.88 LP CC Status1 Month Status*Month 1.067 1.03 0.035 15.61 15.12 0.51 0.00027 0.00033 0.48 1.14 0.076 0.096 19.25 1.30 1.63 0.00027 0.26 0.21 0.013 1.70 2.016 0.016 2.14 2.52 0.90 0.15 0.12 0.37 9.60 0.028 0.34 8.79 0.026 0.56 0.0073 0.87 50 Table 2.14 Results from linear mixed models using type III ANOVA with Satterthwaite’s method of seedling characteristics of mycorrhizal and nonmycorrhizal naturally regenerating seedlings for status, date sampled, and interaction term. P values £ 0.05 are in bold. Data are shown for ponderosa pine seedlings (PPWF), and lodgepole pine seedlings (LPWF) from wildfire sites. 1. Mycorrhizal status Biomass (log (g)) Total N (log(mg) Shoot % N Root % N Sums of Squares F value p-value Sums of Squares F value p-value Sums of Squares F value p-value Sums of Squares F value p-value PPWF Status1 Month Status* Month 0.20 0.11 0.058 7.30 4.02 2.11 0.133 0.058 0.16 0.26 0.071 0.034 6.45 1.76 0.85 0.019 0.20 0.37 5.38 0.0002 0.032 0.093 0.99 0.86 0.34 0.00010 0.002 0.13 0.091 0.0039 4.72 3.17 0.14 0.068 0.094 0.72 LPWF Status1 Month Status* Month n/a n/a n/a n/a n/a n/a 1.17 1.16 4.06 0.29 0.32 0.026 0.22 0.44 0.52 0.021 1.05 0.25 0.11 2.85 0.69 0.74 0.075 0.51 51 Table 2.15 Results from linear mixed models using type III ANOVA with Satterthwaite’s method of d 13C and d 15N isotopic signatures of mycorrhizal and nonmycorrhizal sown seedlings. Significant differences (p £ 0.05) are indicated in bold. Data are shown for ponderosa pine seedlings from wildfire sites (PPWF), and lodgepole pine from wildfire sites (LPWF) and clearcuts (LPCC). d 15N d 13C Site Type Seedling Sums of Squares F- value P-value Sums of Squares F-value P-value PPWF Shoots Status Month Status*Month 0.45 58.52 0.47 0.14 18.16 0.15 0.71 0.00010 0.70 1.19 7.00 0.94 1.50 8.81 1.19 0.60 0.0048 0.28 PPWF Roots Status Month Status*Month 5.46 42.04 26.96 1.18 9.06 5.81 0.47 0.0047 0.021 0.47 2.0072 0.044 0.53 2.27 0.050 0.47 0.14 0.82 LPWF Shoots Status Month Status*Month 65.80 44.67 0.38 7.24 4.92 0.042 0.015 0.031 0.84 13.00 5.58 0.014 9.45 4.053 0.0098 0.010 0.050 0.92 LPWF Roots Status Month Status*Month 9.029 195.60 12.78 0.90 19.48 1.27 0.35 0.000086 0.2667 10.47 7.81 0.15 9.97 7.48 7.45 0.0062 0.0096 0.71 LPCC Shoots Status Month Status*Month 2.60 12.37 10.56 0.23 1.096 0.94 0.90 0.30 0.34 2.76 6.61 2.44 2.90 6.61 2.57 0.28 0.011 0.12 LPCC Roots Status Month Status*Month 39.45 34.57 0.70 7.95 6.97 0.14 0.019 0.014 0.71 0.400 21.32 0.13 0.30 16.14 0.097 0.59 0.000040 0.76 52 Table 2.16 Results from linear mixed models using type III ANOVA with Satterthwaite’s method comparing characteristics of mycorrhizal and nonmycorrhizal naturally regenerating seedlings. Month indicates the month sampled. Significant differences (p £ 0.05) are indicated in bold. Data are shown for ponderosa pine seedlings (PPWF), and lodgepole pine seedlings (LPWF) from wildfire sites. 1. Mycorrhizal status Shoot d 15N Root d15N Shoot d 13C Root d 13C Sums of Squares F value p-value Sums of Squares F value p-value Sums of Squares F value p-value Sums of Squares F value p- value PPWF Status1 Month Status*Month 5.59 6.84 0.24 1.75 2.14 0.075 0.20 0.16 0.79 37.92 0.11 1.08 16.077 0.047 0.45 0.0031 0.83 0.51 0.66 1.87 4.53 0.91 2.58 6.25 0.35 0.12 0.02 0.038 1.48 2.62 0.82 2.30 4.07 0.38 0.14 0.064 LPWF Status1 Month Status*Month 54.15 8.60 39.32 14.51 1.15 5.27 0.019 0.33 0.010 75.61 13.54 8.01 9.18 0.82 0.49 0.012 0.45 0.62 6.99 7.66 1.56 9.71 10.64 2.16 0.0056 0.00025 0.13 2.62 13.70 5.28 8.03 21.05 8.11 0.012 0.00024 0.0017 53 a. b. c. * * Figure 2.6 Total biomass (roots + shoots) (log(g)) of sown (a) ponderosa pine seedlings from burned sites, and lodgepole pine from (b) burned, and (c) clearcut sites in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. Red asterisks indicate a significant (p £ 0.05) difference between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. 54 * * a. b. c. Figure 2.7 Total N (roots + shoots) (log(mg)) of sown (a) ponderosa pine seedlings from burned sites, and lodgepole pine seedlings from (b) burned sites, and (c) clearcuts in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. Red asterisks indicate a significant (p £ 0.05) difference between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. 55 a. b. c. Figure 2.8 Shoot percent (%) N of sown (a) ponderosa pine shoots from burned sites, and lodgepole pine seedlings from (b) burned sites, and (c) clearcuts in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. 56 Ponderosa Pine WildfirLodgepole Pine WildfirLodgepole Pine Clearcut δ 15N δ 15N δ 15N * * δ 15N δ 15N δ 15N a. b. c. d. e. f. Figure 2.9 Root and shoot δ15N values of sown (a, b) ponderosa pine seedlings from burned sites, and lodgepole pine seedlings from (c, d) burned sites, and (e, f) clearcuts, in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. The hashed line indicates the seed signatures (-3.08 ± 0.30 in ponderosa, -2.56 ± 0.28 in lodgepole). Red asterisks indicate significant (p £ 0.05) differences between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. 57 δ 13C δ 13C δ 13C δ 13C δ 13C a. b. c. d. * * e. f. δ 13C Figure 2.10 Root and shoot δ13 C values of sown (a, b) ponderosa pine seedlings from burned sites, and lodgepole pine from (c, d) burned sites and (e, f) clearcuts in months where both mycorrhizal and nonmycorrhizal seedlings were present. Each open circle represents one seedling. Solid points and error bars indicate mean and standard error of each month. Red asterisks indicate significant (p £ 0.05) differences between mycorrhizal and nonmycorrhizal seedlings across months according to linear models. 58 2.4. Discussion 2.4.1. Early growth and N accumulation by pine seedlings after disturbance This study is one of the few to report on germination, growth, and mycorrhizal colonization of pine seedlings over their first season of growth in the field following stand-destroying disturbance (Miller et al. 1998). The majority of seedlings used in this study were collected from seedbeds of sown seeds, but a smaller number of naturally regenerating seedlings were collected to detect any major effects of sowing. The sown seedlings were slightly younger and smaller than naturally regenerating seedlings, but became mycorrhizal at similar times and, therefore, generally showed similar differences between mycorrhizal and nonmycorrhizal seedlings. Seedlings gradually became mycorrhizal over the first growing season until all were colonized by the fall, which supported my first hypothesis that seedlings would all show signs of colonization by the end of the growing season. They started to accumulate C almost immediately after germination, but had not accumulated more N than was in the seed until August, which was also the month when there were more mycorrhizal than nonmycorrhizal seedlings. There was more N and higher biomass in colonized than uncolonized lodgepole pine seedlings, with similar trends observed in ponderosa pines. Although this finding supports the hypothesis that seedling N and biomass would increase after colonization, the difference in % N between mycorrhizal and nonmycorrhizal seedlings was not as apparent. Germination at each site ranged from 11-54% of sown seeds, which is comparable to studies involving sowing of Pseudotsuga menzieii (Douglas-fir) seeds in burned or clearcut sites in the same region (Barker et al. 2014). After germination, seedling biomass immediately and steadily 59 increased in a log-linear manner over the growing season, whereas N started accumulating later. Steadily decreasing δ 13C values throughout the growing season are consistent with accumulation of newly fixed C by the seedlings, as values shifted from the enriched signatures typically seen in seeds to depleted values usually observed in older trees (Vaganov et al. 2009; Voronin et al. 2017). Under greenhouse conditions, Augustine and Reinhardt (2019) concluded that first year lodgepole and ponderosa pine germinants prioritized carbon acquisition and above ground growth over developing drought tolerance, even when under water stress. This is particularly relevant to the current study, considering the seedlings were collected from biogeoclimatic zones that are typically dry and experience little rain during the growing season (Pojar et al. 1987). Notably, 2019, the year the seedlings in my field study were collected, was wetter than most, and seedings did not experience a reduction in growth rates due to drought conditions. I focussed my examination on N accumulation in pine seedlings because, in northern coniferous forests, N is frequently the limiting nutrient (Smithwick et al. 2009). The shoot N concentration was very high in early seedlings collected in June (6.2 ± 1.9 % in lodgepole pines from wildfires and clearcuts and 5.2 ± 1.5 % in ponderosa pine) when compared to N levels generally found in foliage of mature trees, which ranged from 0.9-1.3 % in lodgepole pine and 1.0-1.5 % in ponderosa pine collected over a 16 year period in late fall across the north west of the United States (Moore et al. 2004). The high early N concentrations were likely a result of residual seed reserves because concentrations of N in the seeds averaged 6.2 % for lodgepole pine and 5.6 % for ponderosa pine. The concentration of N in seedling shoots collected from all sites had decreased by July, and then remained at around 2 % N for the rest of the growing season (Figure 2.3). Nevertheless, considering that lodgepole pine seedlings with foliar N concentrations less 60 than 1.35 % are considered to be somewhat nutrient deficient (Carter 1992), young seedlings in this study appeared to have adequate amounts of N. There are very few published values of N concentrations in naturally regenerating pine seedlings, especially ponderosa pine, but lodgepole pine seedlings taken from a clearcut 16-20 weeks after germination in the same region by Smith et al. (2020) had similar % N (1.5-4 %) as seedlings of the same age in this study. I found no evidence for a loss of N from ectomycorrhizal seedlings, as had been observed in laboratory-grown lodgepole pine seedlings by Smith et al. (2020) and in field-grown Scots pine by Zackrisson et al. (1997). The initial lag in N accumulation (Figure 2.4) when compared to biomass accumulation (Figure 2.1) could be because seed reserves were large enough that the seedlings did not have to take up N immediately following germination. The N concentration remained stable after June, which suggests that N was limiting growth, as one would expect to see luxury consumption of N if a different nutrient was limiting growth. It is also possible that seedling roots had grown enough to be better able to take up N. First signs of N accumulation above that found in the seed were seen in August, the same month that most seedlings had become mycorrhizal. Although the timing of this could be circumstantial, it is also likely that the seedlings started to accumulate more N once colonized. The two types of disturbances investigated in this study, wildfire and clearcut logging, often result in ephemeral increases in available soil N (Piirainen et al. 2004; Smithwick et al. 2005). The increase of N in clearcut soils can be delayed up to four years as mineralization occurs, while effects of combustion during a fire are immediate (Bradley et al. 2001). As a result, the 61 amount of N available to first year germinants was expected to be lower in clearcut soils compared to burned soil. Although I cannot compare the disturbance types statistically because the sites were not geographically interspersed, the extractable N appeared to be greater in the spring following a severe wildfire when compared to adjacent unburned soils, while this increase was less pronounced in clearcut soils. Although differences in soil N between these disturbance types can have an effect on N uptake by first year conifer germinants (Barker 2010), the lodgepole pine seedlings collected from wildfires or clearcuts in this study showed no obvious difference in N content (1.04 ± 0.22 mg in seedlings from wildfires and 0.89 ± 0.13 mg in seedlings from clearcuts) or concentration (3.0 % in seedlings from wildfires and 2.7 % from clearcuts). Furthermore, there were no large differences between lodgepole pine seedlings collected from clearcut or burned areas for most nutrients measured, although seedlings collected from burned sites did have slightly higher aluminum, iron, and sulfur concentrations, while seedlings from clearcuts had higher zinc concentrations (Table 2.9, 2.10). Therefore, I found no evidence that N uptake in first year pine seedlings differed between disturbance from wildfire or clearcutting in this study. 2.4.2. Timing of mycorrhiza formation in young pine germinants The timing of colonization of young seedlings by ectomycorrhizal fungi has rarely been studied in the field. In my study, seedlings started becoming colonized in July, until all were mycorrhizal by September. The mycorrhization of all seedlings by the fall is consistent with previous studies done on lodgepole pine (Miller et al. 1998) or Douglas-fir (Barker et al. 2013) seedlings in similar biogeoclimatic zones; however, those earlier studies did not observe colonization until August. It is possible that colonization was detected earlier in this study because seedlings were 62 observed within the week of collection, rather than stored until the end of the growing season, which could leave mantles susceptible to drying out (Barker et al. 2013), or because of the thorough inspection of every root tip in this study. It is also possible that colonization did indeed occur earlier than previous studies, possibly due to the wetter than usual growing season. It is surprising that colonization happened at a similar rate regardless of tree species or disturbance type, considering the greater loss of forest floor in the burned than the clearcut sites. Rodriguez-Ramos et al. (2021) concluded that loss of forest floor was a major determinant of colonization by EMF, likely because the forest floor is an important source of EMF inoculum. A study looking at the effects of different disturbances on EMF community assembly done in the interior of BC by Barker et al. ( 2013), which included screefed and non-screefed clearcuts, low and high severity wildfires, and undisturbed areas, indicated that the EMF community in the high severity burned sites was the most distinct from communities in low severity burned and clearcut sites, although the greatest similarity in colonization rates were seen between screefed clearcut and high severity burned sites because both had experienced high levels of forest floor disturbance. 2.4.3. Did mycorrhizal and nonmycorrhizal germinants differ from each other? Ectomycorrhizal symbioses have been traditionally described as essential for the early establishment of pine and other ECM tree species (Hatch 1936; Pec et al. 2020). There is considerable evidence that early ectomycorrhizal colonization of conifer seedlings in the greenhouse can result in greater plant nutrient content and growth rate (Ekwebelam and Reid 1983; Hobbie et al. 2008; Karst et al. 2008; Jenkins et al. 2018). Furthermore, in some 63 observational field studies of subalpine fir and lodgepole pine, all surviving seedlings were mycorrhizal by the end of the first growing season or the beginning of the second growing season (Cui and Smith 1991; Miller et al. 1998;); however, as emphasized by Miller et al. (1998), this is only circumstantial evidence that ectomycorrhizae are required for survival over the winter. In addition, at some sites with short or wet growing seasons, nonmycorrhizal fir, spruce or hemlock seedlings were still present and vigorous in the second summer after germination (Kropp and Trappe 1982; Cui and Smith 1991; Hagerman et al. 1999; Jones et al. 2009; Nicholson and Jones 2017), while some controlled inoculation studies show that mycorrhization can lead to lower biomass and N uptake (Alberton et al. 2007). Inoculated seedlings transplanted into the field can grow faster, slower, or at the same rate as nonmycorrhizal seedlings depending on the fungal species present on the roots (Jones et al. 2003 and references therein; Jones et al. 2009). Conifer seedlings have even been shown to lose nutrients relative to levels in the seed over the first growing season, a process that may be exacerbated by their fungal symbionts (Zackrisson et al. 1999; Smith et al. 2020). In our study, we looked for evidence that mycorrhization was beneficial to seedling establishment by comparing C and N accumulation in seedlings that became mycorrhizal sooner than others during the first year after stand-destroying disturbances. 2.4.3.1. Accumulation of C and biomass In general, differences between mycorrhizal and nonmycorrhizal seedlings in this study were more pronounced in lodgepole pine seedlings than ponderosa pine seedlings. The pine seedlings that had become mycorrhizal between June and August had greater biomass than nonmycorrhizal seedlings, significantly so in sown lodgepole pine seedlings. On burned sites, mycorrhizal 64 lodgepole pine seedlings, both sown and naturally regenerating, also showed a depletion in 13C relative to nonmycorrhizal seedlings, indicating that the mycorrhizal seedlings were growing faster (Werth and Kuzyakov 2006). This increased biomass accumulation has been shown to result in a survival advantage in first-year pine seedlings (Augustine and Reinhardt 2019). It is also possible that the enrichment of 13C in the nonmycorrhizal lodgepole pine seedlings from burned sites was an indication that they experienced more water stress than mycorrhizal seedlings. Seedlings experiencing drought conditions typically close their stomata to loss of water via transpiration, which also would result in higher 13C values. Mycorrhizal and nonmycorrhizal ponderosa pine seedlings did not differ in biomass. Ponderosa pine seeds are also much larger, and it is possible that the larger amount of nutrients in seed reserves compared to lodgepole pine seeds resulted in a lower dependence on EMF. There were also fewer replicates of ponderosa pine seedlings, which lead to lower statistical power and a higher chance of type II errors. 2.4.3.2. N accumulation A higher rate of N accumulation was an even more consistent trait of mycorrhizal pine seedlings than higher C accumulation. Increases in either total N content of seedlings or tissue d15N, or both, were present in both tree species from both disturbance types. Although colonization by EMF is known to provide seedlings access to organic N, there is also evidence that colonized seedlings take up more ammonium than uncolonized seedlings (Hobbie et al. 2008), which was the most abundant form of soil extractable N in this study. 65 Natural abundances of 15N were measured in this study as an additional way to detect a contribution of ectomycorrhizae to early seedling N acquisition. d15N values are typically lower in the shoots of mycorrhizal seedlings than nonmycorrhizal seedlings (Hobbie et al. 1999). This is because 15N is discriminated against during assimilation and transport from fungus to plant. In this study, d15N was higher in the roots of mycorrhizal than nonmycorrhizal seedlings in both tree species and disturbance types, which would be expected if 15N had accumulated in fungal tissue in the roots during transfer of N to the plant. However, 15N was not depleted in the shoots of any group of mycorrhizal seedlings; instead, d15N was higher in shoots of mycorrhizal than nonmycorrhizal lodgepole and naturally regenerating ponderosa pine seedlings collected from burned sites. Although it is the opposite of what was expected, it is likely that the higher d15N values in mycorrhizal seedlings were observed because the N in the burned soil was enriched in 15N relative to seed values (Tables 2.2, 2.8), which has also been observed in other studies (Huber et al. 2013). Colonized lodgepole pine seedlings growing from both seed types at burned sites, as well as roots of naturally regenerating ponderosa pine seedlings had higher total N values when compared to uncolonized seedlings, indicating they had acquired more N from the soil, which would lead to enriched levels of d15N. Overall, the N isotope data supports a more active accumulation of N by mycorrhizal pine seedlings. Isotopic signatures vary depending on form and availability of nitrogen, which can make natural abundance ratios difficult to interpret in field studies (Craine et al. 2009). Ammonium, which was elevated in these disturbed soils, has a higher d 15N value compared to nitrate and organic N (Tatsumi et al. 2021). This means that the d 15N values of the seedlings in this study could vary depending on the form of N taken up, regardless of colonization status. Other factors unique to 66 young germinants such as seed nutrient translocation could also be affecting the isotopic values in these seedlings. Lack of a colonization effect on the d15N could be because the proportion of seedling N acquired via the fungus relative to that supplied by the seed is minimal at early stages of the symbiosis, with only a portion of root tips covered in a thin fungal mantle. Seedlings collected for this study had only recently undergone germination, and seed reserves might have been high enough to negate the need for immediate fungal transfer from the soil (Baxter and Dighton 2001). Conversely, the subsequent depletion of the seed N reserves could result in increased competition between the fungus and the plant (Alberton et al. 2007). It is also important to be mindful of the recent onset of colonization, taking into consideration the possibility that mycorrhizal seedlings could have become colonized just before being sampled. The short amount of time between the onset of colonization and sampling could mean that any possible differences between mycorrhizal and nonmycorrhizal seedlings may have been difficult to detect. The observational nature of this study also makes it difficult to determine whether any measured differences between seedlings were due to their mycorrhization status, or if seedlings that were larger or had more N were able to become mycorrhizal more readily. 67 Chapter 3: Ectomycorrhizal fungal species following a disturbance 3.1. Background In the previous chapter, I reported that seedlings first showed signs of colonization in July, and all seedlings were mycorrhizal by the fall. The goal of this portion of the field study was to establish which ectomycorrhizal fungal (EMF) species were the first to naturally establish on the root tips of the pine germinants in disturbed areas, and to determine if community composition changed over time. In addition to this, I wanted to investigate if the fungal species present on the root tips appeared to have any influence on seedling N, which was of particular interest because N is generally limiting for pine trees. Past studies have indicated that there is a change in EMF community composition following disturbance. In burned sites, there have been shifts observed towards a higher abundance of pyrophilous, or fire-loving, fungal taxa such as Pustularia, Hebeloma, Laccaria, and Thelephora (Hughes et al. 2020; Hopkins et al. 2021). This is likely due to the spores being better adapted to survive the fire (Glassman et al. 2016), or because of the change in nitrogen dynamics from high organic matter to more readily available nitrate and ammonium (Kranabetter et al. 2009; Hawkins et al. 2015). A similar shift towards nitrophilic genera such as Russula, Inocybe and Tomentella (Kranabetter et al. 2009; Horton et al. 2013) has been observed following a logging event, likely due to the effects of the forest floor disturbance (Barker et al. 2013). Because of this, I hypothesized that the types of EMF observed on ponderosa or lodgepole pine seedlings collected from recently disturbed sites would be pyrophilous such as Pustularia, nitrophilic such as Tomentella, or known to be resistant to disturbance like Rhizopogon, or 68 Wilcoxina. In addition to this, I hypothesized that there would be a change in the community composition as the growing season progressed due to changing soil conditions (Jones et al. 2003), and the subsequent ecosystem succession (LeDuc et al. 2013). The final hypothesis for this chapter was that the species of fungi colonizing the seedling root tips would influence the amount of seedling N. 3.2. Materials and methods 3.2.1. Site selection and field work Sites were selected, and seedlings were sown and collected as described in Section 2.2.1-2.2.5. 3.2.2. Molecular analysis Root tips were examined under dissecting and compound microscopes to determine ectomycorrhizal status using the presence or absence of a mantle, as described in Section 2.2.5. Ectomycorrhizal root tips were characterized into morphotypes using mantle texture and pigment, as well as hyphal characteristics. Root tips from the same seedling that were determined to have the same morphotype were then grouped together, suspended in water and frozen at -80 °C prior to molecular analysis. To isolate fungal DNA, root tips were subjected to CTAB buffer and chloroform using the method of Kranabetter et al. (2018). The internal transcribed spacer (ITS) region of rDNA was then amplified using polymerase chain reaction (PCR) with Kappa HiFi Taq polymerase (VWR), and fungal specific primers ITS1-F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’) (Martin and Rygiewicz 2005). PCR involved an initial 69 three-minute denaturation at 94 °C, then 40 seconds each of denaturation (94 °C), annealing (54 °C) and elongation (72 °C) for 34 cycles, with a final elongation (72 °C) for 10 minutes. Negative controls, composed of the reaction mix without any fungal DNA, were used during amplification to rule out contamination, and positive controls using DNA extracted from fruiting bodies collected from the field were used to confirm successful PCR. Samples that were not successfully amplified initially were either diluted to under 20 ng µl-1, or subject to a protein purification reagent (Promega) before being reamplified. The amplified DNA then underwent Sanger sequencing using the forward primer ITS1-F at the Fragment Analysis and DNA Sequencing Service at UBCO using an Applied Biosystems 3130xl sequencer. Sequences were checked in Sequencher or FinchTV for alignment and the presence of chimeras. The final sequences were then BLASTed with the UNITE database to determine species based upon a > 97%, or genus if > 90%, similarity criterion (Köljalg et al. 2013; Nilsson et al. 2019). Because seedlings were very young and mycorrhizas were newly formed, it was difficult to characterize and distinguish mycorrhizas formed by different fungal species, especially some ascomycetes. As a result, multiple EMF species were occasionally grouped into the same morphotype sample, resulting in unreadable Sanger sequences. DNA extractions that showed signs of having more than one fungal species present were sent to the Genomics Core Facility at Dalhousie University for Illumina sequencing of the ITS2 region using the primers ITS86 and ITS4. 70 3.2.3. Bioinformatics Illumina MiSeq samples were returned demultiplexed as single-end forward and reverse reads. Adapters and primers were first trimmed from sequences using the `Cutadapt` plugin (v3.1) implemented in R (v 3.6.0; R Core Team 2019). Using the ITS fungal DADA2 pipeline in R, sequences were quality filtered and trimmed with the following constraints: minimum length of 50 base pairs, no maximum length, a maximum of two “expected errors” per read, and reads were truncated following any base pairs with a quality score of two or less (Callahan et al. 2016). Sequences were then dereplicated, paired ends were merged, and chimeras were removed. The sequences were resolved to Amplicon Sequence Variants (ASVs), which were then imported into QIIME2 (https://qiime2.org). Amplicon Sequence Variants were matched to UNITE (http://unite.ut.ee) reference sequences with a 97% clustering threshold using a Ribosomal Database Project (RDP) classifier. I opted for ASVs instead of operational taxonomic units (OTUs) as ASVs can have better taxonomic resolution and are considered more replicable (Callahan et al. 2017; Cline et al. 2017). Abundant EMF ASVs from each sample that were identified, and were found in results from Sanger sequencing, were selected and included in the list of fungal species present on the root tips. Photos of root tips identified using Sanger sequencing were also cross referenced for verification of inferred mantle-forming fungal species or genus. 3.2.4. Data analysis All statistical analyses were completed in R version 1.2.1335, with a significance level of p £ 0.05. A PERMANOVA was conducted to determine if fungal species observed on fungal root tips varied across months using the adonis function in the vegan package. A linear model with 71 fungal species as the fixed factor and shoot % N as the response variable was used to determine if seedling N varied depending on the fungal species present on the root tips. When there was more than one fungal species or genus on a seedling, the dominant one was selected to represent that seedling. Species were included in the analysis if there was only one in the genus, or there were high numbers of them present (e.g., Wilcoxina); otherwise, genera were used. Percent seedling N was used as the response variable because it did not vary across time or site, so samples from different sites and months could be combined. Species that occurred fewer than three times per site type were omitted from the analysis for statistical purposes. Tukey’s Honest Significant Difference (HSD) tests using p £ 0.05 were used to determine seedlings with different concentrations of N in cases where the linear model detected significant fungal effects. 3.3. Results 3.3.1. Fungal species present There were 28 distinct fungal OTUs (Sanger sequencing) or ASVs (Illumina sequencing) found on the mycorrhizas collected. Pustularia sp. and Wilcoxina rehmii were the most abundant species; they were common in all three species/disturbance combinations (Fig 3.1) and were detected throughout the season (data not shown). Wilcoxina rehmii appeared to be more prevalent on lodgepole pine root tips collected from clearcut sites when compared to burned sites. By contrast, Pustularia sp., Coltricia confluens, Hebeloma mesophaeum, Inocybe spp. and Trichophaea spp. ectomycorrhizae were found more commonly in sites affected by wildfire compared to logged areas. Phialocephala sp., Rhizopogon spp., and Wilcoxina mikolae were found only on lodgepole pine roots from both disturbance types, not on ponderosa pine root tips. 72 There did not appear to be any differences in the EMF community across months collected (Table 3.1). Table 3.1 Results of PERMANOVAs comparing fungal communities on roots of mycorrhizal pine seedlings between months (Jul-Oct for lodgepole pine seedlings, and Jul-Sept for ponderosa pine seedlings). Df Sums of Squares F Value R2 P Ponderosa pine wildfire 2 0.494 1.709 0.406 0.199 Lodgepole pine wildfire 3 0.1792 0.286 0.087 0.945 Lodgepole pine clearcut 3 0.354 0.386 0.126 0.997 73 Figure 3.1 The number of seedlings on which each fungal taxon was found over the sampling period following a disturbance: July-October for lodgepole pine seedlings, and July-September for ponderosa pine seedlings. When there was more than one ectomycorrhizal fungal species on a seedling, the dominant species was selected to represent that seedling. If more than one species of each genus was found, the number of species is shown in brackets, with the exception of Wilcoxina spp. Species that only occurred once across all three site types were omitted for simplicity. Lodgepole pine seedlings from clearcuts (60), and wildfire sites (53), and ponderosa pine seedlings from wildfire sites (38) were included. Frequency of occurrence (number of seedlings on which each taxon was found) 74 * Ponderosa Pine Wildfire Lodgepole Pine Wildfire Lodgepole Pine Clearcut a. b. c. a ab ab b 75 Table 3.2 Results from linear model with ectomycorrhizal fungal taxa present on the roots as the fixed effect and shoot % N as the response variable for lodgepole pine seedlings from (LPCC) clearcut sites and (LPWF) wildfire sites, and (PPWF) ponderosa pine from wildfire sites. Site Type Degrees of Freedom Sum of Squares Mean Square F-value P-value PPWF 2 0.514 0.257 1.39 0.270 LPWF 4 0.360 0.0900 0.610 0.659 LPCC 3 4.52 1.51 4.05 0.0135 3.3.2. Fungal species and seedling N Comparing the effects of EMF species on N accumulation by the seedlings was greatly limited by the low number of replicate seedlings colonized by each fungal species at each sampling time from each disturbance type. Because shoot % N did not change across months, I used seedlings Figure 3.2 The shoot % N of seedlings colonized by different ectomycorrhizal genera. Points represent individual seedlings from all months and any of the three replicate sites because % N did not vary over time or by site. When there was more than one species on one seedling, the dominant genus was selected to represent that seedling. Fungal genera that occurred on fewer than three seedlings per site type were omitted. Wilcoxina rehmii was included in the analysis. The red asterisk indicates a significant difference among seedlings colonized by different ectomycorrhizal fungal genera within a site type based on a linear model with analysis of variance (ANOVA). Groups of points that do not share letters represent seedlings with different concentrations of N, according to Tukey’s HSD tests (p £ 0.05). 76 from all sampling dates to ensure sufficient replicates to compare N status of seedlings colonized by different EMF species. On burned sites, there was no difference in shoot % N between seedlings colonized by different fungal species for either ponderosa or lodgepole pine (Figure 3.2 a, b). On the clearcut sites, lodgepole pine seedlings that had Wilcoxina rehmii forming the ectomycorrhizae had higher % N in their shoots compared to seedlings colonized by Cenococcum geophilium, with seedlings colonized by Pustularia sp. and Phialocephala sp. intermediate in N concentration. 3.4. Discussion This study examined the ectomycorrhizal fungi that colonized roots of pine seedlings in the year following two types of standing-destroying disturbances that are common in the interior of British Columbia: clearcutting or severe wildfire. Ectomycorrhizae on first-year pine seedlings were dominated by Ascomycota, including Wilcoxina spp. and a Pustularia sp., which were found most frequently, as well as Cenococcum geophilum, Sphaerosporella brunnea, and Trichophaea spp. Across all seedlings sampled, ascomycetes formed 81 % of the ectomycorrhizae. Most of the ascomycetes found were in the Pyronemataceae, which is generally regarded as a broad group, with few unifying characteristics (Hughes et al. 2020). Both Wilcoxina and Pustularia species have been previously documented to be prevalent after various kinds of disturbances (Barker et al. 2013; Pulido-Chavez et al. 2021). The most frequently encountered basidiomycetous genera were Rhizopogon and Tomentella. Wilcoxina and Rhizopogon are considered to be early-successional, likely due to their persistence in the spore bank after a disturbance (Glassman et al. 2016; Nicholson and Jones 2017). This provided partial support for my third hypothesis, that colonization on roots would be by disturbance resistant or 77 nitrophilic species. There was no evidence that the fungal species found on pine root tips shifted over the first growing season. Determining if seedling N was affected by the species of fungi on root tips was difficult due to a low number of fungal replicates, but there was some evidence that lodgepole pine seedlings colonized by Cenococcum had lower % N compared to those colonized by Wilcoxina spp. 3.4.1. Fungi present following the disturbance Fungi that are abundant following wildfire are referred to as pyrophilous fungi. In my study known pyrophilous fungi included Coltricia confluens, Hebeloma mesophaeum, Inocybe spp. and Trichophaea spp. (Glassman et al. 2016; Hughes et al. 2020), all of which colonized both species of pine seedlings on burned sites, but were not observed on the clearcut sites. A species of Pustularia, which is also recognized as a pyrophilous genus, appeared to be more abundant on seedlings from burned than clearcut sites. Inocybe spp. have been found early in succession and are known to be nitrophilous (Kranabetter 2014), which may explain their occurrence on only burned sites in my study (Certini 2005). Wilcoxina spp. are perhaps the most commonly recorded fungi on ectomycorrhizal roots in disturbed soils (Barker et al. 2013; Pulido-Chavez et al. 2021; Rudawska and Leski 2021), so it was unsurprising that Wilcoxina mikolae and Wilcoxina rehmii were two of the most common species found across the sites. W. rehmii was particularly prevalent in this study, and is thought to dominate young conifers in disturbed soils because of its high nutrient demands and low C requirements (Kazantseva et al. 2009; Barker et al. 2013; Pulido-Chavez et al. 2021). W. rehmii was encountered 1.45 times more frequently on lodgepole pine seedlings in clearcuts than burned 78 sites, whereas W. mikolae was more common on burned sites. It is possible that there was a decrease in W. rehmii propagules following the fire, or that there was a higher abundance of W. rehmii at clearcut sites compared to wildfire sites before the disturbance (Smith et al. 2005). Although Pustularia sp. was one the most commonly encountered EMF across the three site types, it has not been as commonly reported in the past as other genera I found in my study. It has been reported more frequently in recent papers (Marčiulynienė et al. 2021; Cahanovitc et al. 2022), possibly due to the general shift away from morphotyping towards DNA sequencing. In my study, recently formed mantles of members on very young seedlings proved hard to differentiate; molecular analysis was required for identification to genus. As a result, it is possible that Pustularia has been mistaken for more commonly identified species in early stages of ecosystem succession in the past, especially considering their mycorrhizas have a similar mantle structure to that of Geopora, and other members of the Pyronemataceae (Tedersoo and Smith 2013). There is also evidence pointing to their fleeting appearance in recently disturbed soils (Pulido-Chavez et al. 2021), meaning they had disappeared by the time most field studies are conducted. Additionally, Pustularia has been used as a synonym for Tazzetta, (Yao and Spooner 2002) so it is possible that it is a new name for a previously reported fungus. Cenococcum geophilum was also one of the most frequent species found on root tips in this study, while previous has studies on fungal communities after wildfires have found either an increase (Buscardo et al. 2010), or decrease (Glassman et al. 2016) in its occurrence after a wildfire. In general, it is very commonly observed across a range of forests, although there is some evidence that it may be nitrophobic (de Witte et al. 2017). As a result, it is possible that the 79 abundance of C. geophilum following a disturbance is dependent on factors such as the quantity present before the disturbance, and the changes in N availability that occur following the disturbance. Tomentella spp., which are also considered nitrophilic, were found on seedlings in both burned and clearcut sites (Jones et al. 2012; Horton et al. 2013; Kranabetter 2014). This is not surprising, as both types of disturbance can result in increased nitrification in the soil (Bradley et al. 2001). A study by Baar et al. (1999) observed abundant Tomentella spp. after a stand-replacing fire; however, they noted that it was also abundant before the fire. Pec et al. (2020) found Tomentella spp. to be an indicator genus for high tree mortality in pine beetle-killed stands, suggesting these fungi were more abundant in areas with young trees. The most commonly found species hypothesis of Tomentella in this study was SH1502300.08FU, which has been found mostly in similar climates in western North America and Sweden. Commonly known as a dark septate root endophyte, Phialocephela sp. was found at both lodgepole clearcut and wildfire sites, and has been found on other Pinaceae species following a disturbance in past studies (Lazaruk et al. 2008; Olchowik et al. 2021). Sometimes considered to be saprotrophic (Day and Currah 2011) or parasitic (Wang et al. 1985), it has also been shown to enhance nutrient uptake by young seedling (Jumpponen et al. 1998). Interestingly, the seedlings colonized by Phialocephela in this study had average % N in their shoots compared to seedlings colonized by other genera. This could support the theory that the mutualistic or parasitic tendencies of Phialocephela depend on the nutrient limitations of the environment (Johnson et al. 1997). The moderate shoot % N and significant variation could also be due to the large number 80 of distinct cryptic species of Phialocephela fortinii (Grünig et al. 2003), which could each be different in their N acquisition. 3.4.2. Effect of ectomycorrhizal fungal identity on seedling nitrogen Different EMF species are known to have different impacts in seedling N uptake. In my study, comparing the effect of colonizing fungal species on seedling N was limited to comparing shoot % N due to limited sample sizes. This is not the most sensitive indicator of N uptake, particularly in young seedlings that have relatively high concentrations of N due to seed reserves. Total N content would have been a preferable comparison to determine if fungal species had any effect on seedling N, but was not possible because the seedlings were growing at such a rapid rate and total N was increasing too quickly to allow me to combine seedlings across sample dates to generate sufficient replication. In addition to this, recently disturbed soils have high amounts of available N (Bradley et al. 2001; Certini 2005), which could have negated any potential differences in uptake between fungal species. Finally, it is possible that any potential effects of fungal species on seedling N uptake differ even between closely related species, and could have been missed by grouping fungi at the genus level (Plett et al. 2022). Nevertheless, I found lodgepole pine seedlings from clearcut sites that were colonized by Wilcoxina spp. had a higher % N in their shoots when compared to those colonized by Cenococcum geophilum. This result is very similar to that of Jones et al. (2009), who found that Engelmann spruce seedlings colonized naturally by Wilcoxina spp. in the field took up labelled N at faster rates than seedlings colonized by Cenococcum sp. It is possible that the difference in N uptake between the two species was due to different forms of preferred N (Yu et al. 2001; Lilleskov et al. 2002), or expression of transporters (Nehls and Plassard 2018). 81 Chapter 4: Conclusion Although our knowledge and understanding of ectomycorrhizae has been expanding, there is still very little known about the early stages of the symbiosis, especially in a field setting. The goal of this research was to gain a better understanding of how pine germinants establish with their fungal symbionts in the field. This was accomplished by conducting a study in which seedlings were harvested each month during their first growing season in the appropriate biogeoclimatic zone, the year after a clearcut or severe wildfire. This research has contributed to our understanding of the establishment and first year of ectomycorrhizal pine symbiosis in the most applicable setting possible. My first hypothesis that seedlings would gradually become colonized over the season, eventually all showing signs of colonization by the end of the year was accepted. Although the timing and onset of colonization has been shown to vary in previous studies, the seedlings in this study all showed signs of ectomycorrhizae on their root tips by fall, after gradually becoming colonized throughout the summer, which suggests that fungal inoculum was not limiting at these sites. My second hypothesis was that seedling N and biomass would increase after colonization because the fungi would promote N uptake as they formed ectomycorrhizae. Pine seedlings that showed early signs of colonization were generally larger and had accumulated more N when compared to uncolonized seedlings collected at the time from the same site. Although this may suggest that mycorrhizal fungi promoted nutrient acquisition and growth, even within weeks of colonization, the observational nature of this study means that I cannot determine causality. It is also possible that larger seedlings with greater root systems just became colonized more readily. 82 My third, fourth and fifth hypotheses were covered in Chapter Three, while examining the fungal species colonizing the collected seedling root tips. I predicted that fungi forming ectomycorrhizae on pine roots would be species generally thought to be nitrophilic, pyrophilous, or known to be resistant to disturbances. This hypothesis was partially supported, as Wilcoxina, Pustularia, and Cenococcum were observed; these are fungal genera known to be nitrophilous, common after disturbance, or fires. However, genera such as Russula, Thelephora and Hebeloma, which are also considered to be early colonizers in disturbed soils, were scarcely found, if at all. There was no evidence that the community of fungal species colonizing the root tips shifted over the growing season. The number of colonized tips found in the samples may have been too low to detect such changes. My final hypothesis was that the seedling N content would be influenced by the species of fungus colonizing the root tips. This was difficult to test due to the low number of replicates of each fungal species, and there was little evidence supporting this hypothesis. Lodgepole pine seedlings collected from clearcut sites with Cenococcum present on their roots did appear to have lower % N compared to those colonized by Wilcoxina spp., but there was no difference between ponderosa pine seedlings colonized by those two fungal species at wildfire sites. Overall, I think the study design and resulting data were robust and convincing; however, there were both strengths and limitations. The observational nature of this field study was both a strength and a limitation. Due to the complex nature of ectomycorrhizal relationships, most studies in this research area are either conducted in growth chambers or greenhouses. These studies most often include artificial settings, with results that are not always directly applicable 83 to the relationship in the field. Although seeds were sown in this study, they were chosen from seedlots within BC as close in proximity and elevation as possible to the field sites, and surface sterilized before stratifying and sowing. Sites were deliberately chosen in the correct biogeoclimatic zones where each species of pine is normally a dominant canopy tree, and the study commenced as early in the spring as possible. Minimal manipulation prior to and during seedling collection meant that I was able to provide a glimpse into the earliest days of the ectomycorrhizal relationship. However, by not experimentally manipulating the conditions, I was not able to determine whether the mycorrhizal fungi were actually providing more N to the seedlings, resulting in higher biomass. Another potential limitation of this study is that the data were also contingent on the timing and rate of mycorrhization naturally occurring in my study plots. When I set up the experiment, I gambled that there would be a mix of both uncolonized and colonized seedlings in the summer months, which then allowed for direct comparisons of mycorrhizal and nonmycorrhizal seedlings. However, because all seedlings are initially nonmycorrhizal, it was not surprising that there were disproportionally more uncolonized seedlings in early months (Jun/Jul), and more colonized later in the summer (Aug), which led to slightly unbalanced sample sizes of the two groups, especially for ponderosa pine. Finally, sample sizes of different colonizing fungi proved to be too small within site types at a given time, so comparing the effect of different fungal species proved to be difficult. Percent (%) N was the only variable that was able to be compared between fungal species, because it was the only one that did not change over the sampling period. 84 These issues could be addressed by conducting a similar field study, but with a larger number of seedlings collected during the month(s) where there are both mycorrhizal and nonmycorrhizal seedlings present. This would increase the chances of having many seedlings colonized by the same dominant mycorrhizal fungi, and would allow for better comparisons to be drawn between colonizing fungal species. Increasing the sampling frequency during these months would also give better insight into the timing of colonization by increasing the replication, and also increase the chances of a more balanced ratio of colonized and uncolonized seedlings. In future studies, conducting a greenhouse experiment with pine seeds sowed in disturbed field soil, inoculated in test groups with the most common types of fungi found in this study would better answer my question about whether ectomycorrhizal fungal species have an effect on the growth and amount of N in pine seedlings. This research gives new insight into the early relationship between pine seedlings and colonizing mycorrhizal fungi in the field, which is vital information when studying ecosystem succession following a stand-replacing event. Recognizing the importance of seedlings becoming colonized by ectomycorrhizal fungi present in the soil after a disturbance, even within the first few weeks of germination, may have implications for tree planting after logging, or potentially any restoration effort involving conifers. 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Interference mechanisms in conifer-Ericaceae-feathermoss communities. Oikos 78(2): 209–220. 104 Appendices Appendix A- Additional figures 82 b. 69 63 68 27 0 100 % 100 % 91 a. * * Shoot N Concentration (%) Figure A1. Percent (%) N of naturally regenerating (a) ponderosa pine and (b) lodgepole pine shoots the first growing season following wildfire. Percent (%) of seedlings colonized across all three sites is shown at the top of each figure. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of each month. Red asterisks indicate a significant difference (p < 0.05, ANOVA) from June. 105 * * * * b. a. Figure A2. Biomass (log(g)) of (a) naturally regenerating lodgepole pine, and (b) ponderosa pine seedlings, both from burned sites over the first growing season following the wildfires. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of each month. Red asterisks indicate a significant difference (p < 0.05, ANOVA) from the first month sampled. 106 Figure A3. d13C of roots and shoots of naturally regenerating (a, b) ponderosa pine, and (c, d) lodgepole pine seedlings from burned sites the first growing season following the fires. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of seedlings sampled each month. Red asterisks indicate significant differences (p < 0.05, ANOVA) from June. a. d. 107 * * ◦ b. a. Figure A4. Total N (log(mg)) of naturally regenerating (a) ponderosa pine and (b) lodgepole pine shoots the first growing season following wildfire. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of each month. Red asterisks indicate significant differences (p < 0.05, ANOVA) from June; red circles indicate p <0.1. 108 δ 15N δ 15N a. d. c. b. * * * * * * * * * * * * δ 15N δ 15N Figure A5. Root and shoot d 15N of naturally regenerating (a, b) ponderosa pine and (c, d) lodgepole pine seedlings over the first growing season following wildfire. Each open circle represents one seedling. Black points and error bars indicate mean and standard error of seedlings sampled during each month. Red asterisks indicate a significant difference (p < 0.05 ANOVA) from June. 109 Total Total 151513C 13C % N % N Total N 15 N % N 13 C eTotal N % N 15 N 13 C . x Total N % N 15 N 13 C 13 C Total N % N 15 N b. c. d. a. e. f 110 Figure A6. Principal coordinate analysis based on seedling characteristics for sown ponderosa pine seedlings collected from burned sites in (a) July and (b) August, lodgepole pine seedlings collected from burned sites in (c) July and (d) August, and lodgepole pine seedlings collected from clearcuts in (d) July and (e) August. Mycorrhizal seedlings are shown in black and nonmycorrhizal seedlings in grey. 111 13C 15N 13C % N 15N 13C % N % N 15N 13C % N 15N b. c. d. a. Figure A7. Principal coordinate analysis based on seedling characteristics for naturally regenerating ponderosa pine seedlings from collected burned sites in (a) July and (b) August, lodgepole pine seedlings collected from burned sites in (c) June and (d) July. Mycorrhizal seedlings are shown in black and nonmycorrhizal seedlings in grey. 112 b. a. Figure A8. Shoot percent (%) N of naturally regenerating (a) ponderosa pine, and (b) lodgepole pine shoots from burned sites in months where both mycorrhizal and nonmycorrhizal seedlings were present. Points with error bars indicate mean and standard error. There was no main effect of mycorrhizal status, but there was a significant interaction between month and mycorrhizal status for lodgepole pine seedlings (see Table 2.14). 113 Ponderosa Pine Lodgepole Pine * * δ 15 δ 15 δ 15 δ 15 * bc. d. a. * Figure A9. Root and shoot d 15N values of naturally regenerating (a) ponderosa pine and (b) lodgepole pine seedlings from burned sites, in months where both mycorrhizal and nonmycorrhizal seedlings were present. Points attached to error bars indicate mean and standard error of each month. Red asterisks indicate significant (p < 0.05, ANOVA) differences between mycorrhizal and nonmycorrhizal seedlings across months. 114 Ponderosa Pine Lodgepole Pine δ 13C δ 13C δ 13C δ 13C * * b. c. d. a. Figure A10. d 13C values of roots and shoots of naturally regenerating (a, b) ponderosa pine and (c, d) lodgepole pine seedlings from burned sites in months where both mycorrhizal and nonmycorrhizal seedlings were present. Points associated with error bars indicate mean and standard error of each month. Red asterisks indicate significance (p < 0.05, ANOVA) difference between mycorrhizal and nonmycorrhizal seedlings across months. 115 * Figure A11. Total N (log(mg)) of shoots of naturally regenerating ponderosa pine seedlings from burned sites in months where both mycorrhizal and nonmycorrhizal seedlings were present. Points attached to error bars indicate mean and standard error of each month. Red asterisks indicate significance (p < 0.05, ANOVA) difference between mycorrhizal and nonmycorrhizal seedlings across months. 116 Figure A12. Biomass (log(g)) of sown ponderosa pine seedlings (shoot + root) from burned sites in months where both mycorrhizal and nonmycorrhizal seedlings were present. Points attached to error bars indicate mean and standard error of each month. 117 Appendix B- Methods Extraction and determination of soil NH4+ and NO3- (Dr. Tim Cavagnaro, The University of Adelaide. Lab Protocols & Kranabetter et al. 2007): Soil was sieved through a 2 mm sieve within 24 hours of being collected. Roughly 5 g dry weight equivalent (5.75 g wet weight) soil was placed into 50 mL centrifuge tubes, and 25 mL of 2 M KCl was added. Tubes were shaken end-to-end on a platform shaker for 60 min at 100 rpm. They were then centrifuged at 850 g for 15 min to form a pellet. The supernatant was transferred to 15 mL centrifuge tubes, and frozen at -20 °C until analysis. Ammonium: 11 µL of KCl extract was added to a 96-well plate containing 140 µL each of reagent A (100 mL dH2O, 6.5 g Na salicylate, 5 g Na citrate, 5 g Na tartrate dibasic dihydrate, 0.025 g Na nitroprusside), and reagent B (6 g NaOH, 2.4 mL bleach). Samples were then read at 650 nm after 1 hr. Blanks and standards were included using 2 M KCl and NH4 standard solution. Nitrate: 33.22 µL of Reagent A (0.4 g of VCl3 added to 50 mL of 1 M HCl, swirled by hand and let sit for 10 min) was added to 265.77µL of reagent B (0.2 g of sulphanilamide and 0.01 g of N---(1---naphthyl)ethylenediamine dihydrochloride) dissolved in 400 mL milliQj). 299 µL of the reagent mix was added to each well of a 96- well plate, then 6 µL of KCl sample was be added. The plate was be moved to the fumehood and left overnight before being read at 540 nm. Amino Acid extraction and quantification- modified from (Darrouzet-Nardi et al. 2013): 118 Based off a fluorometric amino acid extraction and quantification method using o-phthaldialdehyde and b-mercaptoethanol (OPAME) in 96-well microplates with a 90 min incubation time using leucine as a standard.
Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
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Graduate
Jones, Melanie D.
Nitrogen and mycorrhization in Pinus ponderosa and Pinus contorta germinants the first growing season after wildfire or clearcutting
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