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Relationships between cyanolichen communities and nutrient cycling in sub-boreal spruce forests Campbell, Jocelyn 2010

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Relationships between Cyanolichen Communities and Nutrient Cycling in Sub-boreal Spruce Forests by  Jocelyn Campbell B.Sc.H. Queen’s University, 1995 M.Sc. The University of Northern British Columbia, 1999  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  The Faculty of Graduate Studies (Forestry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2010  © Jocelyn Campbell, 2010  ABSTRACT Cyanolichens (lichens with cyanobacterial symbionts) form a distinct assemblage of epiphytes strongly associated with humid microclimatic conditions in inland British Columbia. Disparate abundance patterns in sub-boreal forests are examined in relation to the influence of overstorey tree species. A comparison of lichens on conifer saplings beneath five overstorey tree species revealed that saplings beneath Populus support a disproportionately abundant and species-rich community of cyanolichens. Cyanolichens also grew more rapidly and had lower rates of mortality beneath Populus than beneath conifer overstorey trees. That cyanolichens were observed beneath Populus in stands that were otherwise climatically unsuitable suggests that Populus facilitates cyanolichen communities by providing a factor that compensates for suboptimal conditions. Chemical analyses of throughfall precipitation from beneath Populus, Picea, Abies, Pseudotsuga and Betula failed to explain the variation in lichen community structure. However, glucose-rich nectar, exuded from extrafloral nectaries on Populus leaves, may instead be supporting cyanolichen communities. The nectar accumulates during dry periods, is washed off during subsequent rain events, and may be intercepted and metabolized by cyanolichens on conifer saplings beneath mature Populus canopies. C-flux measurements and phospholipid fattyacid analyses with experimental applications of 13C6-labelled glucose revealed a strong physiological response to glucose and a rapid incorporation of exogenous-13C into cyanolichen fatty-acid tissues. Field evidence further supports this hypothesis with higher rates of cyanolichen establishment observed on Picea branches under treatment of 2% glucose solution compared to water. The exogenous C may enable cyanolichens to become established in regions that are otherwise too dry to support them by providing a source of C despite drought-induced inactivity of the cyanobacterial partner. The abundant communities of nitrogen-fixing cyanolichens in wet, mature forests and beneath Populus are important to ecosystem function. The contribution of cyanolichens to N-cycling is calculated at sites with varying lichen abundances from measured rates of lichen litter deposition, decomposition and nutrient release. Cyanolichen litter biomass represents up to 11.5% of the total N-input from aboveground litterfall and is estimated to release 2.1 kg N ha-1 yr-1 of newly-fixed N that would otherwise be unavailable in these mature sub-boreal forests.  ii  TABLE OF CONTENTS  ABSTRACT ................................................................................................................................... ii  TABLE OF CONTENTS ............................................................................................................ iii  LIST OF TABLES ........................................................................................................................ v  LIST OF FIGURES .................................................................................................................... vii  LIST OF ABBREVIATIONS AND TERMS ............................................................................ ix  ACKNOWLEDGEMENTS ......................................................................................................... x  DEDICATION............................................................................................................................. xii  CO-AUTHORSHIP STATEMENT ......................................................................................... xiii  CHAPTER 1  1.1  1.2  1.3  1.4  1.5   INTRODUCTION............................................................................................. 1   The Lichen symbiosis ..................................................................................................... 1  Cyanolichen habitat ........................................................................................................ 2  Contribution of cyanolichens to forest nutrient cycling ................................................. 4  Research approach and objectives .................................................................................. 5  References ....................................................................................................................... 7   CHAPTER 2   The Influence of overstorey Populus on epiphytic lichens .......................... 10   2.1  Introduction ................................................................................................................... 10  2.2  Methods and materials .................................................................................................. 12  2.2.1  Study area.................................................................................................................. 12  2.2.2  Sampling method ...................................................................................................... 14  2.2.3  Environmental conditions ......................................................................................... 15  2.2.4  Precipitation, bark and soil chemistry ....................................................................... 16  2.2.5  Data analysis ............................................................................................................. 17  2.3  Results ........................................................................................................................... 19  2.3.1  Environmental variation............................................................................................ 19  2.3.2  Lichen community patterns....................................................................................... 20  Site effects ......................................................................................................................... 20  Overstorey tree effects ...................................................................................................... 20  Conifer saplings versus overstorey Populus ..................................................................... 30  2.3.3  Precipitation, bark and soil chemistry ....................................................................... 31  2.4  Discussion ..................................................................................................................... 35  2.4.1  Lichen abundance patterns ........................................................................................ 35  2.4.2  Relationship to precipitation chemistry .................................................................... 37  2.5  References ..................................................................................................................... 39  CHAPTER 3   Growth and mortality of cyanolichens under Populus ................................ 42   3.1  Introduction ................................................................................................................... 42  3.2  Methods and materials .................................................................................................. 43  3.2.1  Study area.................................................................................................................. 43  3.2.2  Cyanolichen growth .................................................................................................. 44   iii  3.2.3  Data analysis ............................................................................................................. 46  3.3  Results ........................................................................................................................... 46  3.3.1  Large thalli ................................................................................................................ 46  3.3.2  Small thalli ................................................................................................................ 47  3.3.3  Mortality ................................................................................................................... 50  3.4  Discussion ..................................................................................................................... 52  3.4.1  Effect of overstorey tree species ............................................................................... 52  3.4.2  Facilitation ................................................................................................................ 57  3.5  References ..................................................................................................................... 57  CHAPTER 4   The influence of exogenous glucose on epiphytic cyanolichens .................. 60   4.1  Introduction ................................................................................................................... 60  4.2  Methods and materials .................................................................................................. 63  4.2.1  Physiological response to 13C6 -glucose .................................................................... 63  4.2.2  Fatty-acid extraction and analysis............................................................................. 65  4.2.3  Establishment response to glucose............................................................................ 66  4.3  Results ........................................................................................................................... 67  4.3.1  Physiological response to 13C6-glucose .................................................................... 67  4.3.2  Fatty-acid analysis .................................................................................................... 70  4.3.3  Glucose fertilization experiment ............................................................................... 74  4.4  Discussion ..................................................................................................................... 76  4.4.1  Conclusion ................................................................................................................ 79  4.5  References ..................................................................................................................... 80  CHAPTER 5   Decomposition and nutrient release from four lichen litters ...................... 84   5.1  Introduction ................................................................................................................... 84  5.2  Methods and materials .................................................................................................. 86  5.2.1  Study area.................................................................................................................. 86  5.2.2  Litterfall .................................................................................................................... 88  5.2.3  Litter decomposition and nutrient release ................................................................. 89  5.2.4  Soil nitrogen .............................................................................................................. 90  5.2.5  Data analysis ............................................................................................................ 90  5.3  Results ........................................................................................................................... 91  5.3.1  Decomposition rates.................................................................................................. 91  5.3.2  Nutrient release rates................................................................................................. 95  5.3.3  Litterfall rates ............................................................................................................ 98  5.3.4  Soil nitrogen .............................................................................................................. 99  5.4  Discussion ................................................................................................................... 102  5.4.1  Decomposition of lichen litter ................................................................................ 102  5.4.2  Element changes ..................................................................................................... 105  5.4.3  Litterfall .................................................................................................................. 106  5.4.4  Significance of lichen N inputs ............................................................................... 107  5.5  References ................................................................................................................... 108  CHAPTER 6  6.1   CONCLUSIONS ........................................................................................... 112   References ................................................................................................................... 117   iv  LIST OF TABLES  Table 2.1 Total light availability (direct plus diffuse light expressed as percent of maximum ±SD) recorded under conifer saplings beneath Populus tremuloides or P. trichocarpa, Betula papyrifera, Abies lasiocarpa, and Picea glauca x engelmannii at Fraser, Aleza and Herrick sites. ......................................................................................................................... 19  Table 2.2 The mean thallus area (average of total cm2 plot-1, N=9) of epiphytic foliose lichen species observed on saplings (branches up to 3.5 m and trunks up to 2 m) under Populus tremuloides or P. trichocarpa, Betula papyrifera, Abies lasiocarpa, Picea glauca x engelmannii and Pseudotsuga menziesii at Fraser, Aleza and Herrick sites. The lichen communities beneath Populus tremuloides and P. trichocarpa were found to be statistically similar (MRPP, T=-0.61 A=0.01 P=0.19). ........................................................................... 21  Table 2.3 Total number of foliose macrolichen species (and mean species per plot ± SD) observed within four functional groups on conifer saplings beneath five overstorey tree species at Fraser, Aleza and Herrick sites. ............................................................................ 24  Table 2.4 Total numbers of small cyanolichen thalli (< 0.5 cm2) observed within three cyanolichen functional groups on conifer saplings beneath five overstorey tree species at Fraser, Aleza and Herrick sites (N=9). ................................................................................. 27  Table 3.1 Repeated Measures ANOVA statistics at Fraser, Aleza and Herrick sites. Models tested significant differences in the change in transplant mass (large thalli) or transplant area (small thalli) over 3, 12, 15, 24 and 27 months (time as the repeated measure) between lichen species (Lobaria pulmonaria and Lobaria hallii) and tree species (Populus and Picea). Differences associated with Pseudotsuga were not tested due to low sample size. . 49  Table 3.2 Comparative studies on growth rates of cyanolichens in old forest environments. Precipitation is given as mm year-1 unless (except in cases where the number of measurement days is included in parentheses). Approximate annual growth rates are based on calculations from seasonal or multi-year growth rates. Annual growth rates in parentheses indicate those that are extrapolated from shorter-term studies and may therefore overestimate annual growth. See Coxson and Stevenson 2007a for other records of L. pulmonaria growth................................................................................................................ 53  Table 4.1 One-tailed t-test statistics between water- and 13C6-glucose-treated cyanolichens at six levels of photosynthetically active radiation (PAR). ............................................................ 69  Table 4.2 Relative abundance (%) of phospholipid fatty acids (PLFA) and neutral lipid fatty acids (NLFA) in four 13C6-glucose treated cyanolichen species. ND- not detected (N=6). . 72  Table 4.3 Mean quantity (±SD) of 13C assimilated into PLFAs, NLFAs and total lichen fatty acids, the ratio of 13C assimilated into PLFA (μg 13C g sample -1) to NLFA (μg 13C g sample -1 ), and the ratio of total PLFA-C g sample-1 to total NLFA-C g sample-1 in four cyanolichen species (N=6). ....................................................................................................................... 74  v  Table 5.1 The initial C:N and decay rate constants (k) for four lichen species (AS = Alectoria sarmentosa, PG = Platismatia glauca, LP = Lobaria pulmonaria and NH = Nephroma helveticum). Decay constants are calculated according to Olson (1963) for four lichen litters during four time intervals and cumulatively over the two year incubation. ......................... 92  Table 5.2 Chemistry of litter from four lichen species (AS = Alectoria sarmentosa, PG = Platismatia glauca, LP = Lobaria pulmonaria and NH = Nephroma helveticum) at five sequential decay stages. ........................................................................................................ 94  Table 5.3 Lichen, leaf, needle and twig litterfall at each of three sites. Litterfall values are mean kg ha-1 yr-1± SD. Different superscript letters represent significant differences in litterfall mass between site-types (ANOVA, p<0.05)......................................................................... 98  Table 5.4 Estimated annual nitrogen input and release from lichen, leaf, needle and twig litterfall at each of three sites. Nitrogen release (kg N ha-1 yr-1) is calculated from the annual litterfall mass multiplied by the initial N content and the change in N content during the first year of decay. The mass of chlorolichen and cyanolichen (including Lobaria pulmonaria) litterfall was increased by 10% and 20% respectively to account for the expected litterfall mass loss between collection periods (based on median mass loss for each species over the initial 4 months). .............................................................................................................................. 100   vi  LIST OF FIGURES  Figure 2.1 Location of the study sites near Prince George, British Columbia, Canada. .............. 13  Figure 2.2 Total abundance of four functional groups of epiphytic foliose macrolichens observed on sapling branches beneath five tree species at the three site-types. Tree species codes: Pt (Populus tremuloides or P. trichocarpa), Bp (Betula papyrifera), Al (Abies lasiocarpa), Pgxe (Picea glauca x engelmannii), Pm (Pseudotsuga menziesii). Letters above the bars denote significant multivariate differences in lichen diversity and abundance across plot types (overall MRPP; T= -18.11, p<0.0000, A= 0.39). ........................................................ 26  Figure 2.3 NMS ordination of 46 species observed in 117 plots at three site-types. The ordination resulted in a 2-dimensional solution with a final instability criterion <0.0001 after 250 runs with real data. NMS axes 1 and 2 accounted for 47% and 42% of the variation in the distance matrix respectively. Letters (H- chlorolichen, Y – stratified cyanolichen, G – nonstratified cyanolichens) denote individual lichen species and symbols represent plots shown by site-type and overstorey tree species combinations. Dashed lines indicate approximate separation of the three site-types........................................................................................... 29  Figure 2.4 Diversity of foliose chlorolichens, non-stratified cyanolichens, stratified bipartite cyanolichens and tripartite cyanolichens observed on Populus trunks versus on the conifer saplings beneath Populus canopies at Fraser, Aleza and Herrick sites. ................................ 30  Figure 2.5 Concentration (± SD) of Ca, P, Mn, and Mo in precipitation captured in the open and beneath four tree species at three site-types. Elements were extracted from resin capsules installed in the field from November 2007 to May 2008 (Winter) and from May to September 2008 (Summer). Dissimilar letters represent significant differences (p<0.05) between tree species within site-types. ................................................................................. 33  Figure 2.6 Comparisons of (a) throughfall precipitation pH (±SD) and (b) conifer sapling bark pH (±SD) beneath five tree species at three site-types. Tree species codes: Pt (Populus tremuloides or P. trichocarpa), Bp (Betula papyrifera), Al (Abies lasiocarpa), Pgxe (Picea glauca x engelmannii), Pm (Pseudotsuga menziesii). Dashed lines represent the lower pH limit for cyanolichen habitat (Gauslaa 1985). Dissimilar letters represent significant differences (p<0.05) between tree species and site-types. .................................................... 34  Figure 3.1 Cumulative percent growth rate of large thalli (a) and transplant mass (b) over 27 months beneath mature Populus, Picea, and Pseudotsuga trees at Fraser (left panel), Aleza (middle panel), and Herrick (right panel) sites. .................................................................... 48  Figure 3.2 Cumulative percent growth rate of small thalli (a) and transplant mass (b) over 27 months beneath mature Populus, Picea, and Pseudotsuga trees at Fraser (left panel), Aleza (middle panel), and Herrick (right panel) sites. .................................................................... 48   vii  Figure 3.3 Cumulative mortality of small Lobaria hallii and L. pulmonaria thalli beneath Populus, Picea and Pseudotsuga trees at a) Fraser, b) Aleza and c) Herrick sites over 27 months. .................................................................................................................................. 51  Figure 4.1 Net photosynthesis of water- and 2% 13C6-glucose-treated a) Lobaria hallii b) Lobaria pulmonaria c) Leptogium saturninum and d) Nephroma helveticum. Error bars represent standard deviation and stars represent significant differences between treatments at individual light levels for individual species (N=6). ............................................................ 68  Figure 4.2 The % 13C enrichment in extracted (a) phospho-lipid fatty acids (PLFA) and (c) neutral-lipid fatty acids (NLFA) and the % of total 13C enrichment in each (b) PLFA and (d) NLFA from 13C6-glucose treated samples of Lobaria pulmonaria, Lobaria hallii, Nephroma helveticum and Leptogium saturninum. Associations of specific fatty acids with specific lichen bionts are indicated. Error bars represent standard deviation. Natural abundance of 13 C (1.15%) was subtracted from values prior to analysis (N=6). ........................................ 73  Figure 4.3 The number of small (<1mm long) thalli observed on control and 2%-glucose treated Picea branches. Establishment on glucose-treated branches was significantly higher than control (p=0.019). ................................................................................................................. 75  Figure 5.1 Monthly mean air temperature (top) and relative humidity (bottom) at the Fraser (dark symbols), Aleza (open symbols) and Herrick (shaded symbols) sites.................................. 87  Figure 5.2 Mean (±SD) decomposition rates over two years at (a) three site types for (b) four species of lichen litter. Dotted lines represent decomposition of vascular plant litter incubated from 1993-1997 (from Prescott et al. 2004). Dissimilar letters represent significant differences between site types (a) or species (b). ................................................ 93  Figure 5.3 Changes in elemental concentrations (mg g-1 ±SD; panel a) and elemental mass remaining in the litterbags (mg ±SD; panel b) of N, P and K over two years of lichen litter decomposition. ...................................................................................................................... 96  Figure 5.4 The ratio of C:N as a function decreasing C concentration (%) in litter of four epiphytic lichen species. Twelve-, 15-, and 24-month decay periods are denoted by circles, triangles and square symbols respectively. ........................................................................... 97  Figure 5.5 Nitrogen availability (±SD) in mineral soil measured as NO3- and NH4+ supply rates onto PRSTM ion exchange probes incubated in mineral soil within root-exclusion tubes for 10 weeks.............................................................................................................................. 101   viii  LIST OF ABBREVIATIONS AND TERMS ANOVA  Analysis of variance  AS  Alectoria sarmentosa  Bipartite cyanolichen  Lichen with cyanobacterial and fungal symbionts  Chlorolichen  Lichen with green algal and fungal symbionts  DBH  Diameter at breast height  EFN  Extrafloral nectary  k  Decomposition constant  LH  Lobaria hallii  LP  Lobaria pulmonaria  LS  Leptogium saturninum  MRPP  Multi-response permutation procedures  NH  Nephroma helveticum  NLFA  Neutral lipid fatty acid  NMS  Nonmetric multidimensional scaling  PG  Platismatia glauca  PLFA  Phospholipid fatty acid  SBS  Sub-boreal spruce (biogeoclimatic zone)  SBSvk  Very-wet, cool subzone of the sub-boreal spruce zone  SBSwk  Wet, cool subzone of the sub-boreal spruce zone  SD  Standard deviation  Tripartite cyanolichen  Lichen with cyanobacterial, green algal and fungal symbionts  ix  ACKNOWLEDGEMENTS This has been an incredibly enlightening and life-altering experience. It was made possible by the assistance of countless people at both the University of British Columbia and the University of Northern British Columbia. I’d like to specifically thank the following colleagues, friends and family who have helped make this project a success. Thank you: Dr. Cindy Prescott for teaching me to question science more critically, for trusting in my ideas, for patience and encouragement, and for being an advocate for the other parts of graduatestudent life! Dr. Art Fredeen for supporting my research ideas for so many years, for the enthusiastic reception of each new piece of data, and for encouraging me to pursue a Ph.D. in the first place. Dr. Gary Bradfield for the perspective on ecology, for the statistical and editorial advice and for the good-humoured support of this work. Rachel Botting for braving the bugs and the rain and my indecision as we wandered around the landscape looking for cottonwood, for the incredibly long hours in the field, and for the wisdom and experience in helping to design this project. Caitlin French for the good-humoured assistance, for being an organizational-crutch, for the endless laughs over the endless tasks, and for the ‘Wanna see something interesting?’ diversions. Carol Sapergia for being an unstintingly good-natured field and lab assistant. The women of the UBC Belowground Ecosystem Group, especially Beth Brockett, Denise Brooks, Toktam Sajedi, Richa Anand and Julie Deslippe for broadening my scientific world and for being a source of inspiration along the way. The members of the UNBC Soil discussion group, particularly Sue Robertson, Nabla Kennedy, Katherine Stewart, Paul Sanborn, Darwyn Coxson, Susan Stevenson and Hugues Massicotte. Many of the ideas presented here would have remained buried without our enlightening and often diversionary discussions. Trevor Goward for the inspiration, for the lichenological knowledge to do this project in the first place and for lichen identification. x  Suzanne Simard, Roy Turkington and Yngvar Gauslaa for providing thoughtful suggestions that improved the text and for their questions that initiated interesting discussions at my dissertation defense. Melanie Karjala and David Radies for assisting with site selection. Becky Bowler, Kazu Ishimaru and Kate Delbel for instruction and assistance with lab work. Per Bengtson for analysis and interpretation of PLFA data and for thorough and patient reviews of the resulting manuscript. Darwyn Coxson, Susan Stevenson and Sue Grayston for lab and equipment use. Paul Sanborn for planting the carbon idea and Rob Guy for the discussions over its possibility. John Orlowsky for access to growth chambers and forestry equipment (despite my affiliation!) Allen Esler, Clive Dawson and Eric Bayrd for assistance in solving analytical chemistry problems and for conducting chemical analyses. NSERC for funding this work and providing me with the financial resources to do it. The Edward W. Bassett Memorial Scholarship, The Donald S. McPhee Fellowship, The Ralph M. and Elizabeth E. Cochrane Scholarhip, The Association of Professional Biologists of British Columbia, The Natural Resources Canada Science and Technology Internship Program, and The Aleza Lake Research Forest for their financial support. My many good friends for your support, for the places to stay, for not asking ‘when will you be done?’ too often and for running, biking, skiing, skating and drinking wine with me when I needed the mental break. My family, who always supported my desire to do science, for encouraging me to ‘keep at it’! And to my husband Marc for supporting me in my decision to pursue this degree, for helping and supporting me in countless ways at every step along the way, for picking up the slack to make sure our lives ran smoothly despite the long work hours, and for the understanding, patience, encouragement, pep-talks and reality checks required to dive this deep into lichens!  xi  DEDICATION  For my parents, who introduced me to the natural world  and for Marc who’s help made this all possible  xii  CO-AUTHORSHIP STATEMENT Co-authorships of manuscripts reflect editorial advice, financial assistance, intellectual contributions and guidance in project design, analysis and write-up consistent with responsibilities of supervisory committee members (C.E. Prescott, G.E. Bradfield and A.L. Fredeen) and analysis of 13C-enrichment of lichen fatty acids and interpretation of these data (P.Bengtson). The principal work in all aspects of the research, including identifying research questions, designing experiments, collecting and analysing the data, and writing the thesis – was done by Jocelyn Campbell.  xiii  Introduction  CHAPTER 1  INTRODUCTION  1.1 The Lichen symbiosis  Lichens are a symbiotic association between a heterotrophic fungal partner (mycobiont) and an autotrophic photobiont. The photobionts are either cyanobacteria (bipartite cyanolichens), green algae (chlorolichens), or both (tripartite cyanolichens). The fungus provides an environment in which the photobiont cells can survive in an appropriate light and moisture regime. This is particularly crucial with tree-dwelling species where most photobionts would otherwise not survive the desiccating environment of the forest canopy. The hydrophobic fungal layer also ensures adequate gas exchange at the photobiont-fungus interface and facilitates carbon and nutrient transfer between the symbionts (Honegger 1991). The fungal partner is thought to exert some level of control over cell reproduction and growth by regulating the nutrient supply (Ahmadjian 1993, Hill 1989). As the only photosynthetic cells within the lichen symbiosis, the algae and/or cyanobacteria provide fixed carbon for maintenance and growth of all partners and may supply 70-90% of assimilated carbon to the mycobiont (Smith 1980). Nutrient and carbon transfer occurs in chlorolichens via fungal extensions (haustoria) that penetrate the algal cell. There are no specialized tissues connecting the symbionts in bipartite cyanolichens (Honegger 1985) and mobile carbohydrates (principally glucose) are released by the photobiont into the apoplastic space between the two partners. The fixed carbon is then picked up by the hyphae of the mycobiont (Honegger 1991). That this transfer of soluble materials requires a film of moisture may be one of the physiological reasons for higher moisture requirements in cyanolichens compared with chlorolichens.  1  Introduction 1.2 Cyanolichen habitat  Cyanolichens form a distinct assemblage of epiphytes that are strongly associated with humid microclimatic conditions (Goward and Spribille 2005) and mature forest ecosystems (Campbell and Fredeen 2004). Lichens are physiologically active only when wet, and being poikilohydric, are unable to control the degree and duration of hydration (Kappen 1988). The prevailing climate thus determines the period of physiological activity and the ability to utilize light or nutrient resources toward growth and development. Although all lichens may become completely desiccated and regain physiological activity upon rehydration, photosynthesis in cyanolichens only occurs following contact with liquid water (Lange et al. 1986). Furthermore, while chlorolichens can reach maximal activity at 50-70% thallus moisture, this only occurs in cyanolichens when thallus-water content exceeds 150% (Lange et al. 2004). Consequently, the composition of epiphytic lichen communities is often determined by climatic conditions, with drier zones being largely occupied by chlorolichens and wetter areas by cyanolichens (Sillett and Neitlich 1996; Lehmkulh 2004). In the interior, mixed-conifer forests of British Columbia, cyanolichens are generally restricted to the lower canopy of forests more than 140 years old (Goward 1994; Campbell and Fredeen 2004) where the probability of stand-level disturbance is low (Sanborn et al. 2006). Such forests provide an irregular stand structure that allows sun-flecks and indirect light to penetrate to the lower-canopy branches that are critical as cyanolichen substrate. By contrast, younger forests are often even-aged with a single canopy layer and few gaps to allow light penetration (Benson and Coxson 2002). Under such conditions light levels in the lower canopy may be below the threshold requirements for cyanolichen growth (Gaio-Oliveira et al. 2004). Light availability has been repeatedly demonstrated to limit lichen growth. Gauslaa et al. (2006) observed that biomass gain in Lobaria pulmonaria was directly proportional to the 2  Introduction amount of light intercepted by the lichen thallus. However, given that cyanolichens must be highly hydrated for growth, the positive influence of light will depend on moisture availability. Indeed, Dahlman and Palmqvist (2003) demonstrated that up to 66% of the variation in cyanolichen mass gain was attributable to the combination of light and water availability. Consequently, cyanolichens are often most abundant and diverse in old forests where conditions provide sufficient light for growth while preventing thallus damage due to desiccation and moisture stress (Gauslaa et al. 2006). Lichen community composition is also strongly influenced by biotic factors, particularly relating to host or neighboring tree species. Hauck and Spribille (2005) reported higher overall epiphyte abundance and greater cyanolichen diversity on subalpine fir relative to Engelmann spruce in NW Montana. A significant interaction between tree species and soil type affecting epiphyte abundance in a sub-boreal forest makes interpreting the phorophyte effect difficult, but the results of Campbell and Fredeen (2007) also suggest preferential bipartite cyanolichen colonization on subalpine fir compared to spruce. Phorophyte preferences may be particularly evident when cyanolichen communities are compared between coniferous and deciduous trees. The structural differences between coniferous and deciduous tree canopies create disparate patterns of rain and light interception. Epiphytes beneath a broadleaf canopy experience greater seasonal fluctuation in rainfall and light and may be exposed to higher irradiance than under dense coniferous canopies. In addition, needles and leaves determine, through the amount and composition of litter, the nutrient dynamics of the stand (Prescott 2002). Broadleaf trees may result in localized regions of nutrient enrichment and increased base-cation saturation compared to conifers within the same ecosystem (Fujinuma et al. 2005). Whether due to climatic or chemical factors, deciduous trees, particularly in the  3  Introduction Populus and Salix genera, support a unique, species-rich community of epiphytic lichens. In Scandinavia (Kuusinen 1994; Gauslaa 1995; Hedenås and Ericson 2000), Estonia (Jüriado et al. 2003) and the United Kingdom (Ellis and Coppins 2007) Populus trunks provide habitat for cyanobacterial lichens that are otherwise absent in the predominantly conifer forest. In British Columbia, cyanolichens are often asymmetrically distributed on conifer branches with the heaviest loading in the direction of a neighbouring Populus balsamifera (Goward and Arsenault 2000). Such differences in lichen community structure between phorophytes have been attributed to inhibition by high manganese concentration (Hauck 2003), and promotion by increased calcium concentration, decreased acidity (Gauslaa 1995; Goward and Arsenault 2000), and increased phosphorus concentrations (Benner and Vitousek 2007). However, neither the identity, nor the concentrations of chemicals that are influencing lichen communities are well understood. Thus the composition of epiphyte communities beneath different tree species is examined here with reference to climatic and chemical site factors to provide a deeper understanding of the habitat requirements of cyanolichens in sub-boreal forests of British Columbia. 1.3 Contribution of cyanolichens to forest nutrient cycling  Decay of, and nutrient release from, nitrogen-fixing lichens may be important to ecosystem N-cycling. In late-seral forest ecosystems, where atmospheric inputs are as low as 0.8 kg N ha-1 yr-1 (Hope 2001), N is often considered a growth-limiting element. Symbiotic N-fixing organisms are less abundant in older forests and so N is tightly cycled (Davidson et al. 1992) and largely supplied by decomposing forest litter (Sollins et al. 1980). Nitrogen-fixing cyanolichen biomass, on the other hand, increases with forest age. McCune (1994) reported epiphytic lichen loadings of 2250 kg ha-1 in wet-coastal forests and Campbell and Fredeen (Campbell and Fredeen 2004) recorded 1400 kg ha-1 in wet-interior forests of British Columbia. While these  4  Introduction biomass loadings are small relative to vascular plants which can exceed 1500 Mg ha-1 in interior forests (Fredeen et al. 2005), N contribution from cyanolichens may represent approximately 50% of the total N input (Denison 1979). The quantity of in situ cyanolichen-N has been calculated for wet interior forests of British Columbia (Campbell and Fredeen 2007), but these data provide only limited insight into the role of lichens in N-cycling. Thus the rates of cyanolichen litter deposition, decay, and nutrient release are examined here to provide an accurate estimate of cyanolichen-N contributions to nutrient cycling. 1.4 Research approach and objectives  This research can be divided into two components; an investigation of the ecology of cyanolichens with reference to climatic and chemical site factors, and an evaluation of the contribution of cyanolichens to nutrient cycling. The first component begins with a description of the lichen communities in sub-boreal spruce forests and an evaluation of lichen community structure across site-types and between overstorey tree species (Chapter 2). A strong positive association between cyanolichens and overstorey Populus led to an examination of the climatic conditions (temperature, relative humidity and light availability) and the chemistry of throughfall precipitation as potential factors influencing lichen community composition. Chapter 3 documents differences in rates of growth and mortality for two cyanolichen species across site-types and between tree species. Finding no climatic or chemical differences between tree species that could account for the abundant and species-rich cyanolichen communities observed beneath Populus, Chapter 4 evaluates whether the proliferation of cyanolichens beneath Populus is due to facilitation by an exogenous source of labile carbon.  5  Introduction The second component evaluates the role of nitrogen-fixing cyanolichens in N-cycling in sub-boreal spruce forests. Rates of lichen litterfall, decomposition and nutrient release are analysed in Chapter 5 to predict the contribution of cyanolichen litter to ecosystem N. The objectives and working hypotheses for each chapter are as follows:  CHAPTER 2. To evaluate the extent to which variation in lichen communities on understorey conifer saplings is related to neighbouring, overstorey tree species and to identify patterns in microclimate and precipitation chemistry to which this variation might be related. Hypotheses: 1. There is a strong positive spatial-association between the epiphytic cyanolichens observed on conifer saplings and overstorey Populus that occur intermittently in the mixed-conifer forest. 2. Overstorey Populus trees sustain patches of high cyanolichen diversity on understrorey conifer saplings in stands that are less favourable to cyanolichen establishment or growth. 3. Patterns in cyanolichen community species-richness and abundance on understorey conifer saplings are related to the chemistry of throughfall precipitation beneath different overstorey tree species. CHAPTER 3. To evaluate the influence of overstorey tree species, relative to regional climatic conditions, on the rate of cyanolichen growth. Hypotheses: 1. Cyanolichen thalli transplanted beneath Populus canopies will have faster growth rates than thalli transplanted beneath the canopy of overstorey Picea or Pseudotsuga regardless of site-type. 2. Tripartite cyanolichen species will have a faster growth rates than bipartite cyanolichen species regardless of overstory tree-species.  6  Introduction CHAPTER 4. To examine the possibility that cyanolichen communities proliferate beneath overstorey Populus under sub-optimal climatic conditions due to facilitation by exogenousglucose from Populus extrafloral nectaries. Hypotheses: 1. Cyanolichen net photosynthesis will decrease and nitrogen fixation will markedly increase in response to exogenous glucose. 2. Exogenous glucose will be readily taken up and assimilated into lichen fatty-acid tissues. 3. The rate of cyanolichen establishment will significantly increase with the provision of exogenous glucose. CHAPTER 5. To examine rates of epiphytic lichen litter deposition, decay and nutrient release and to evaluate the contribution of cyanolichens to forest N-cycling. Hypotheses: 1. Cyanolichen litter will decompose faster and release N and P faster than chlorolichens, resulting in a convergence of N and P concentrations in lichen litter as decomposition proceeds. 2. Decomposing cyanolichen litter will rapidly release N and thereby provide a source of N for forest ecosystem functioning. 1.5 References  Ahmadjian, V. 1993. The Lichen symbiosis. John Wiley and Sons. New York. Benner, J.W., and Vitousek, P.M. 2007. Development of a diverse epiphyte community in response to phosphorus fertilization. Ecology Letters, 10: 628-636. Benson, S., and Coxson, D.S. 2002. Lichen colonization and gap structure in wet-temperate rainforests of northern interior British Columbia. Bryologist, 105: 673-692. Campbell, J., and Fredeen, A.L. 2004. Lobaria pulmonaria abundance as an indicator of macrolichen diversity in Interior Cedar-Hemlock forests of east-central British Columbia. Canadian Journal of Botany, 82: 970-982. Campbell, J., and Fredeen, A.L. 2007. Contrasting the abundance, nitrogen and carbon of epiphytic macrolichen species between host trees and soil types in a sub-boreal forest. Canadian Journal of Botany, 85: 31-42.  7  Introduction Dahlman, L., and Palmqvist, K. 2003. Growth in two foliose tripartite lichens, Nephroma arcticum and Peltigera aphthosa: empirical modelling of external vs internal factors. Functional Ecology, 17: 821-831. Davidson, E.A., Hart, S.C., and Firestone, M.K. 1992. Internal cycling of nitrate in soils of a mature coniferous forest. Ecology, 73: 1148-1156. Denison, W.C. 1979. Lobaria oregana, a nitrogen-fixing lichen in old-growth Douglas-fir forests. In Symbiotic nitrogen fixation in the management of temperate forests. Edited by Gordon, J.C., Wheeler, C.T., and Perry, D.A. Forest Research Laboratory, Oregon State University, Corvallis, OR. pp. 266-275. Ellis, C.J., and Coppins, B.J. 2007. Reproductive strategy and the compositional dynamics of crustose lichen communities on aspen (Populus tremula L.) in Scotland. Lichenologist, 39: 377-391. Fredeen, A.L., Bois, C.H., Janzen, D.T., and Sanborn, P.T. 2005. Comparison of coniferous forest carbon stocks between old-growth and young second-growth forests on two soil types in central British Columbia, Canada. Canadian Journal of Forest Research, 35: 14111421. Fujinuma, R., Bockheim, J., and Balster , N. 2005. Base-cation cycling by individual tree species in old-growth forests of Upper Michigan, USA. Biogeochemistry, 74: 357-376. Gaio-Oliveira, G., Dahlman, L., Palmqvist, K., and Maguas, C. 2004. Growth in relation to microclimatic conditions and physiological characteristics of four Lobaria pulmonaria populations in two contrasting habitats. Ecography, 27: 13-28. Gauslaa, Y. 1995. The Lobarion, an epiphytic community of ancient forests threatened by acid rain. Lichenologist, 27: 59-76. Gauslaa, Y., Lie, M., Solhaug, K.A., and Ohlson, M. 2006. Growth and ecophysiological acclimation of the foliose lichen Lobaria pulmonaria in forests with contrasting light climates. Oecologia, 147: 406-416. Goward, T. 1994. Notes on oldgrowth-dependent epiphytic macrolichens in inland British Columbia, Canada. Acta Botanica Fennica, 150: 31-38. Goward, T., and Arsenault, A. 2000. Cyanolichen distribution in young unmanaged forests: A dripzone effect? Bryologist, 103: 28-37. Goward, T., and Spribille, T. 2005. Lichenological evidence for the recognition of inland rain forests in Western North America. Journal of Biogeography, 32: 1209-1219. Hauck, M. 2003. Epiphytic lichen diversity and forest dieback: the role of chemical site factors. Bryologist, 106: 257-269. Hauck, M., and Spribille, T. 2005. The significance of precipitation and substrate chemistry for epiphytic lichen diversity in spruce-fir forests of the Salish Mountains, Northwestern Montana. Flora, 200: 547-562. Hedenås, H., and Ericson, L. 2000. Epiphytic macrolichens as conservation indicators: successional sequence in Populus tremula stands. Biological Conservation, 93: 43-53. Hill, D.J. 1989. The control of the cell cycle in microbial symbionts. New Phytol. 112: 175-184. 8  Introduction  Honegger, R. 1985. Fine structure of different types of symbiotic relationships in lichens. In Lichen Physiology and Cell Biology. Edited by Brown, D.H. Plenum Publishing, New York. pp. 287-302. Honegger, R. 1991. Functional aspects of the lichen symbiosis. Annual Review of Plant Physiology and Plant Molecular Biology, 42: 553-578. Hope, G. 2001. The soil ecosystem of an ESSF forest and its response to a range of harvesting disturbances. British Columbia Ministry of Forests. Extension note 53. http://www.for.gov.bc.ca/hfd/pubs/Docs/En/En53.htm. Jüriado, I., Paal, J., and Liira, J. 2003. Epiphytic and epixylic lichen species diversity in Estonian natural forests. Biodiversity and Conservation, 12: 1587-1607. Kappen, L. 1988. Ecophysiological relationships in different climatic regions. In CRC Handbook of Lichenology. Edited by Galun, M. CRC press, Boca Raton, Florida. pp. 37-100. Kuusinen, M. 1994. Epiphytic lichen flora and diversity on Populus tremula in old-growth and managed forests of southern and middle boreal Finland. Annales Botanici Fennici, 31: 245260. Lange, O.L., Kilian, E., and Ziegler, H. 1986. Water vapor uptake and photosynthesis of lichens: performance differences in species with green and blue-green algae as phycobionts. Oecologia, 71: 104-110. Lange, O.L., Burkhard, B., Meyer, A., Zellner, H., and Zotz, G. 2004. Lichen carbon gain under tropical conditions: water relations and CO2 exchange of Lobariaceae species of a lower montane rainforest in Panama. Lichenologist, 36: 329-342. Lehmkulh, J.F. 2004. Epiphytic lichen diversity and biomass in low-elevation forests of the eastern Washington Cascade Range, USA. Forest Ecology and Management, 187: 381-392. McCune, B. 1994. Using epiphyte litter to estimate epiphyte biomass. Bryologist, 97: 396-401. Prescott, C.E. 2002. The influence of the forest canopy on nutrient cycling. Tree Physiology, 22: 1193-1200. Sanborn, P., Geertsema, M., Jull, A.J.T., and Hawkes, B. 2006. Soil and sedimentary charcoal evidence for Holocene forest fires in an inland temperate rainforest, east-central British Columbia, Canada. The Holocene, 16: 415-427. Sillett, S.C., and Neitlich, C. 1996. Emerging themes in epiphyte research in westside forests with special reference to cyanolichens. Northwest Science, 70: 54-60. Smith, D.C. 1980. Mechanisms of nutrient movement between lichen symbionts. In Cellular interactions in symbiosis and parasitism. Edited by Cook, C.B., Pappas, P.W., and Rudolph, E.D. Columbus Ohio State University Press, Columbus, Ohio. Sollins, P., Grier, C.C., McCorison, F.M., Cromack, K.J., and Fogel, R. 1980. The internal element cycles of an old-growth Douglas-fir ecyosystem in Western Oregon. Ecological Monographs, 50: 261-285.  9  The influence of overstorey Populus on epiphytic lichens  CHAPTER 2  The Influence of overstorey Populus on epiphytic lichens1  2.1 Introduction  Cyanolichens (lichens with cyanobacterial symbionts) form a distinct assemblage of epiphytes strongly associated with humid microclimatic conditions (Goward 1994; Arsenault and Goward 2000) and mature forest ecosystems (McCune 1993; Campbell and Fredeen 2004). Unlike chlorolichens (lichens with green-algal symbionts), cyanolichens require contact with liquid water to become physiologically active (Lange et al. 1993). Consequently, the composition of epiphytic lichen communities is often determined by climatic conditions, with drier zones being largely occupied by chlorolichens and wetter areas by cyanolichens (Sillett and Neitlich 1996; Lehmkulh 2004). Cyanolichens are also abundant and diverse in later-seral forests (McCune 1993; Campbell and Fredeen 2004) where the discontinuous canopy structure allows greater penetration of indirect light through to the more humid lower-canopy (Benson and Coxson 2002). Cyanolichen community composition is also strongly influenced by biotic factors, particularly relating to host or neighbouring canopy tree species. For example, higher overall epiphyte abundance and greater cyanolichen diversity has been observed on mature Abies lasiocarpa trees compared to mature Picea trees in east-central British Columbia (Campbell and Fredeen 2007) and in Montana (Hauck and Spribille 2005). Hauck and Spribille (2005) attributed these differences to the inhibitory effect of higher manganese concentrations in Picea bark. Enriched cyanolichen communities have also been noted on the trunks of Populus and Salix compared to those of surrounding conifer trees (Kuusinen 1994; Gauslaa 1995; Hedenås and Ericson 2000; Jüriado et al. 2003; Ellis and Coppins 2007). The higher pH of Populus bark has 1  A version of this chapter has been published by the Canadian Journal of Forest Research as Campbell, J. Bradfield, G.E., Prescott, C.E. and Fredeen, A.L. 2010. The influence of overstorey Populus on epiphytic lichens in sub-boreal spruce forests of British Columbia Can. J. For. Res. 40:143-152.  10  The influence of overstorey Populus on epiphytic lichens been cited as an important factor promoting cyanolichens in acid-affected European and Scandinavian forests (Gauslaa 1995). Most comparisons of epiphytic lichen communities between particular tree species relate to observations on the trunks and branches of the host trees themselves and make no note of the potential influence of surrounding trees. Goward and Arsenault (2000) provide an exception, reporting an asymmetrical distribution of epiphytic cyanolichens on temperate conifers, with the heaviest loading in the direction of a neighbouring Populus balsamifera ssp. trichocarpa. These observations, coupled with accounts of unique cyanolichen flora on Populus trunks as mentioned above, suggest that cyanolichen communities may benefit from the chemical environment created by Populus trees. More specifically, as suggested by Goward and Arsenault (2000) and Gauslaa (1995), cyanolichens may be responding to an increase in pH resulting from higher base cation concentrations. However, neither the identity, nor the concentration of chemicals that are required for the development of cyanolichen communities are well understood. The objectives of this study were to evaluate the extent to which variation in lichen communities on understorey conifer saplings is related to neighbouring, overstorey tree species and to identify patterns in microclimate and precipitation chemistry to which this variation might be related. Specifically, the study was designed to test the following hypotheses: (1) there is a strong, positive spatial association between the epiphytic cyanolichens observed on conifer saplings and overstorey Populus trees that intermittently occur in the mixed-conifer forests, (2) overstorey Populus trees sustain patches of high cyanolichen diversity on understorey conifer saplings in stands that are less favourable to cyanolichen growth, (3) patterns in cyanolichen community species-richness and abundance on understorey conifer saplings are related to the chemistry of throughfall precipitation beneath different overstorey tree species.  11  The influence of overstorey Populus on epiphytic lichens 2.2 Methods and materials  2.2.1 Study area The study area was located north-east of Prince George, British Columbia, in old-growth (mean tree age >240 years) forests of the Sub-Boreal Spruce (SBS) biogeoclimatic zone (Meidinger and Pojar 1991). These forests are characterized by cool, moist summers and cold, snowy winters. Three mature, conifer-leading forest site-types representing different levels of moisture and light deficiencies for lichen growth were selected for study within the wet Subboreal spruce zone (Fig. 2.1). For each site-type, three replicate stands (nine in total, hereafter referred to as study sites) were randomly chosen for sampling from a pool of candidate sites meeting the criteria of showing no evidence of recent disturbance, and having intermittent occurrence of deciduous trees in an otherwise conifer-dominated forest canopy. The three ‘Aleza’ sites were located in the wet, cool subzone (SBS wk) in the Aleza Lake Research Forest at 680 m in elevation. The Aleza sites receive approximately 897 mm in annual precipitation (Murphy 1996) and so were considered the most moisture-deficient of the three site-types. The three ‘Herrick’ sites were located along the Herrick Forest Service Road approximately 40 km north-east of the Aleza sites in the very-wet, cool SBS subzone (SBS vk) at an elevation of 850 m. Selective logging approximately 100 years ago at the Herrick sites resulted in a relatively closed overstorey canopy. Thus, Herrick sites were considered the most light-deficient of the three site-types. Finally, the three ‘Fraser’ sites (680 m elevation) were located in an ecotonal region between the two aforementioned site-types and neither moisture nor light were considered deficient. Annual precipitation at both the Fraser and Herrick sites was approximately 964 mm (Murphy 1996).  12  The influence of overstorey Populus on epiphytic lichens  Herrick sites (Herrick Forest Service Road) UTM: 10U 0586226, 5997613  SBSvk Fraser sites (North Fraser and Bowron Forest Service Roads) UTM: 10U 0576928, 5995740  SBSwk  Aleza sites (Aleza Lake Research Forest) British Columbia  UTM: 10U 0558760, 5994066  Prince George  1:300,000 kilometers US  0  10  20  Figure 2.1 Location of the study sites near Prince George, British Columbia, Canada. Picea glauca (Moench) Voss x engelmannii Parry ex Engelmann (interior hybrid spruce) and Abies lasiocarpa (Hook.)Nutt. (subalpine fir) were the dominant conifer species at all three site-types, and Betula papyrifera Marsh. (paper birch) made up most of the deciduous canopy. Populus balsamifera L. ssp. trichocarpa Brayshaw (black cottonwood) was a minor component at the Fraser and Herrick sites while Populus tremuloides Michx. (trembling aspen) was present at the Aleza sites. Pinus contorta Dougl. Ex Loud. var. latifolia Engelm. (lodgepole pine), Pseudotsuga menziesii (Mirbel.) Franco var. glauca (Beissn.) Franco. (Douglas-fir) and Tsuga heterophylla (Raf.) Sarg. (western hemlock) were minor components at the Fraser, Aleza and  13  The influence of overstorey Populus on epiphytic lichens Herrick sites respectively. For simplicity, tree species are hereafter referred to by genus name only, with P. tremuloides and P. trichocarpa collectively referred to as Populus. 2.2.2 Sampling method At each study site, three individual trees of each overstorey species were randomly selected from a pool of candidate trees satisfying the following criteria: 1) >20 cm diameter at breast height (DBH), 2) at least 5 m from another canopy tree of a different species and at least 10 m from a mature Populus, and 3) surrounded (within 3 m from the bole) by three conifer saplings (<10 cm DBH). Overstorey tree species included Populus, Abies, Picea and Betula at all sites, and Pseudotsuga at the Aleza sites. At each study tree, the three closest live conifer saplings within a 3-m-radius plot (centered on the study tree) were selected for epiphyte community sampling. Live candidate saplings that had more than 10 branches below 3.5 m were preferentially selected to provide a range of potential habitat for epiphytic lichens. The predominant conifer saplings (Abies and Picea) were used as a standardized sampling unit to assess the influence of overstorey tree species, rather than the influence of the phorophyte, on lichen community structure. In plots where Populus was the central tree, lichens were also sampled on the Populus trunk and lower branches (if present) to compare canopy influence versus direct phorophyte contact on the lichen communities. As the abundance and composition of epiphytic lichen communities are known to vary between the trunks and branches of trees (McCune 1993), lichens were assessed within two sampling zones on the conifer saplings: on trunks up to 2 m and on branches up to 3.5 m in height. Similar sampling zones were also assessed on Populus where it was the central study tree. Following Campbell and Fredeen (2007), abundance was recorded for each species as the total area (cm2) of thallus cover for each species within each sampling zone. Species richness was recorded as the number of epiphytic lichen species observed per plot. Hair lichens (e.g. 14  The influence of overstorey Populus on epiphytic lichens Alectoria sarmentosa) and Cladonia spp. were excluded due to the ubiquitous nature of the genera, and Parmelia spp. were only identified to genus. To assess recruitment success, an estimate (nearest 10) of the number of small (<0.5 cm2) thalli within each sampling zone was also recorded for each foliose lichen species (or genera in cases where small thallus size prevented in situ identification to species). 2.2.3 Environmental conditions Air temperature and relative humidity data were recorded at the nine study sites using HOBO Pro RH and Temperature Data-loggers (Onset Computer Corporation, Bourne, MA). At each site, data loggers were installed 1.8 m above ground-level on the north side of the trunk of one Populus, Abies and Picea overstorey tree; additional units were installed in a similar fashion on Pseudotsuga trunks at the three Aleza sites. Data were collected continuously from July 2006 to June 2008. Light availability was assessed using digital hemispherical photographs taken beneath the canopy of the overstorey tree and under each of the three saplings in each plot. Photographs were taken on the north side of each tree both before (summer) and after (winter) leaf abscission to quantify phenological differences in light availability. Photos were taken using a Nikon Coolpix 8700 camera with a Nikon Fisheye converter (UR-E12) and lens (FC-E9). Images were analysed for light availability using Gap Light Analyzer Version 2.0 (Simon Fraser University, Burnaby, Canada). Differences in the light environment between tree species and site-types were evaluated using total light availability (direct plus diffuse light) as both constituents have been demonstrated to affect cyanolichen growth (Gauslaa et al. 2006). Total tree (trunks >20cm DBH) and sapling (trunks <10cm DBH) density, species composition, and overstorey tree ages (using an increment borer) were also determined at each study site.  15  The influence of overstorey Populus on epiphytic lichens 2.2.4 Precipitation, bark and soil chemistry Throughfall precipitation collectors were established beneath 5 overstory Populus, Betula, Abies and Picea at each site. An additional 3 collectors were placed in the open to collect precipitation unaffected by the foliage of any overstorey tree species, for a total of 23 collectors per site. Collectors consisted of a high-density polypropylene funnel (with a 15-cm diameter opening) secured to a 125-ml high-density polyethylene NalgeneTM bottle (Fisher Scientific, Ottawa, Canada). Glass wool was placed at the funnel mouth to filter debris and a 2-cm diameter polyester capsule containing 10 ml of mixed bed ionic resins (PST-2; Unibest, Bozeman, Montana, USA) was placed in each bottle. Resin capsules were surrounded in a bed of glass wool to maximize water-holding capacity as a 3-mm hole was drilled in the bottom of each bottle to allow water to pass freely throughout the collection period. Supports constructed from PVC piping were installed on each tree trunk at approximately 3 m in height to minimize disturbance by wildlife. Collectors were held in supports approximately 1 m from the trunk and placed to intercept throughfall precipitation from a single tree species and to avoid branches with abundant epiphytes. Winter precipitation collectors were placed in the field following the first snowfall in November 2007 and removed prior to leaf flush in May 2008. Summer collectors were placed at this time and were removed in September 2008 prior to leaf abscission. Similar precipitation collectors were used in July 2007 to collect water for pH analysis. The pH of duplicate water samples was recorded using an Oakton pHTestr 3 (Oakton Instruments, Vernon Hills Il. USA) calibrated with pH 4 and 7 buffers. Nutrients were extracted from the resin capsules using 2M HCl according the protocol described by Skogley and Dobberman (1996). Element concentrations were analysed at the University of Northern British Columbia Central Equipment Laboratory using inductively  16  The influence of overstorey Populus on epiphytic lichens coupled plasma-mass spectrometry (ICP-MS, 7500 Series, Agilent Technologies, Santa Clara, CA) as described by Dolan and Capar (2002). Samples of conifer sapling branches were collected from the field in July 2007. Branches (approximately 1 cm in diameter) were stripped of foliage and cut to four 15-cm lengths. The branch ends and other bark wounds were coated with paraffin wax to ensure that only the outer bark would contact the water. Samples were immersed in deionized water in HDPE NalgeneTM bottles and stored at 8oC. After 1 week, samples were removed and the pH was recorded for duplicate water samples using an Oakton pHTestr 3. Organic matter and mineral soil were collected within the drip-zone of three individuals of each tree species at each site in July 2007. Duplicate 25 ml samples were air dried, sifted through a 5 mm sieve, diluted to 10% and 50% (organic matter and mineral soil respectively) and agitated periodically for 1 hour. The pH was recorded using an Oakton pHTestr 3. 2.2.5 Data analysis Differences in stand age across site-types were tested with a non-parametric KruskalWallis ANOVA and variation in monthly temperature and relative humidity between site-types and tree species were evaluated with a repeated-measures ANOVA and a Bonferonni post-hoc comparison. Differences in light availability among site-types and tree species were assessed with a factorial ANOVA and a Bonferonni post-hoc test. Lichen species were divided into four functional groups (chlorolichens, non-stratified cyanolichens, bipartite stratified cyanolichens, and tripartite stratified cyanolichens sensu McCune 1993) according to the type and distribution of the primary photosynthetic symbiont (Brodo et al. 2001). The total abundance and total number of species observed on Abies and Picea saplings were compared with one-way ANOVAs following log transformation to achieve a normal distribution (Statistica v. 6.1, StatSoft Inc. Tulsa, OK, USA). 17  The influence of overstorey Populus on epiphytic lichens Two multivariate methods, non-metric multidimensional scaling (NMS) and multiresponse permutation procedures (MRPP), were used to evaluate overall patterns of variation among the epiphytic lichen communities (PC-ORD v.5.0, McCune and Grace 2002). NMS generates a reduced number of ordination axes (“dimensions”) that are well suited for visual display of relationships among plots according to similarities in lichen assemblages. The NMS used a random starting configuration and the Sørensen distance measure calculated on a matrix of 117 plots and 46 species. For consistency, only those lichen species that were readily identifiable in the field were included in the analysis. Rare species (<2 thalli in the entire study area) were either removed for ordination analysis or grouped and analyzed as genera (e.g. Peltigera spp.). MRPP, a non-parametric method for testing group differences, was used to test for quantitative differences in lichen community composition among plots grouped by site-type and overstorey tree species (e.g. Fraser - Populus) yielding a total of 13 groups of 9 plots each. Differences in precipitation chemistry, soil and bark pH between site-types and tree species were evaluated following log normal transformation (where necessary) with a fullfactorial ANOVA and a Bonferonni post hoc test. The relationship between Ca, P, Mn, and Mo concentrations and the total abundance of bipartite stratified cyanolichens, non-stratified cyanolichens, tripartite cyanolichens and chlorolichens were evaluated using Spearman rank order correlations. Relationships between soil pH, precipitation pH and sapling bark pH were tested using Pearson’s correlation (Statistica, StatSoft Inc. Tulsa, OK, USA). Values reported in text and tables are given as the mean ± standard deviation unless otherwise noted.  18  The influence of overstorey Populus on epiphytic lichens  2.3 Results  2.3.1 Environmental variation The three site-types had similar mean overstorey tree ages (134±72 y, N=27; H(2,44)=2.0, p=0.34) and had 461±32 mature trees (DBH >20cm) and 1843±147 saplings (DBH <10cm) ha-1. The Herrick sites had the lowest average summer light availability (F(2, 72)= 5.5, p=0.005; Table 2.1). Winter light availability did not differ among sites. Average daily temperatures were higher at the Aleza sites than at the Fraser sites (F(46,552)=50.8, p=0.000). Monthly relative humidity was consistently and significantly lower at the Aleza sites compared to the Herrick and Fraser sites (F(46,552)=7.1, p=0.000; data not shown). There were no climatic differences in temperature or relative humidity among tree species at any site-type.  Table 2.1 Total light availability (direct plus diffuse light expressed as percent of maximum ±SD) recorded under conifer saplings beneath Populus tremuloides or P. trichocarpa, Betula papyrifera, Abies lasiocarpa, and Picea glauca x engelmannii at Fraser, Aleza and Herrick sites.  % winter lightavailability  % summer lightavailability  Site-type  Populus  Betula  Abies  Picea  Site mean  Fraser  19.1±3.3  20.5±2.8  18.9±3.2  18.1±1.2  19.2±2.8  Aleza  19.3±4.4  17.2±1.5  18.9±1.7  17.1±1.7  18.5±3.8  Herrick  18.7±4.4  20.2±4.1  18.7±2.9  20.3±3.8  19.4±2.7  a  b  ab  a  Fraser  20.7±3.0  17.5±2.6  19.8±3.6  20.7±2.7  20.3±3.5a  Aleza  21.6±5.1a  21.4±5.7a  21.6±2.2a  21.2±3.3a  21.6±3.4a  Herrick  16.9±3.5b  17.7±2.2b  19.2±3.5b  19.6±3.9b  18.4±3.6b  Note: Winter light availability refers to photographs taken after leaf abscission and summer light availability refers to those taken while deciduous leaves were still on the trees.  19  The influence of overstorey Populus on epiphytic lichens 2.3.2 Lichen community patterns Sixty-one taxa of epiphytic foliose macrolichens were recorded across the study area (Table 2.2). These included 26 chlorolichens, 19 bipartite stratified cyanolichens, 1 tripartite cyanolichen and 15 bipartite non-stratified cyanolichens, including 4 species that are not yet described (T. Goward, personal communication 2009). As there were no differences in lichen abundance or species richness observed on Abies and Picea saplings (F(1, 66) =0.5, p=0.49) the two sapling species were grouped for all subsequent analyses. Site effects More epiphytic foliose lichen species were observed at the Fraser sites (total 50, mean 41±5) than at the Aleza (total 41, mean 32±4) and Herrick (total 47, mean 39±3) sites. Community composition differed between site-types with cyanolichens comprising two-thirds (32 species) of the total macrolichen flora at Fraser sites and approximately half of the species at Aleza and Herrick sites (19 and 24 species, respectively; Table 2.3). Moreover, the species richness of non-stratified cyanolichens at Fraser sites (15 species) was double that at the Aleza and Herrick sites (8 and 7 species, respectively). Overstorey tree effects Lichen communities beneath overstorey Populus trees were the most species-rich with 90% (55 species) of the total observed foliose lichen flora occurring on conifer saplings under Populus. Saplings beneath Abies, Betula and Picea trees supported 77% (47 species), 74% (45 species) and 69% (42 species) respectively, of all lichen species observed. Saplings beneath Pseudotsuga had only 24% (15 species) of the total foliose lichen flora. This low diversity was partly related to the occurrence of Pseudotsuga at only one site-type; however, saplings beneath Pseudotsuga also supported lower lichen richness compared to other tree species at the Aleza sites (Table 2.3). 20  The influence of overstorey Populus on epiphytic lichens Table 2.2 The mean thallus area (average of total cm2 plot-1, N=9) of epiphytic foliose lichen species observed on saplings (branches up to 3.5 m and trunks up to 2 m) under Populus tremuloides or P. trichocarpa, Betula papyrifera, Abies lasiocarpa, Picea glauca x engelmannii and Pseudotsuga menziesii at Fraser, Aleza and Herrick sites. The lichen communities beneath Populus tremuloides and P. trichocarpa were found to be statistically similar (MRPP, T=-0.61 A=0.01 P=0.19). Site-type  Fraser sites  Tree species  Populus  Betula  Abies  Picea  Populus  Foliose chlorolichens Cavernularia hultenii Degel. Hypogymnia austerodes (Nyl.)Rae.  H. canadensis Goward H. metaphysodes (Asah.) Rass. H. occidentalis L. Pike H. physodes (L.) Nyl. H. rugosa† (G. Merr.) L. Pike H. tubulosa (Shaerer) Hav. H. vitatta (Ach.) Parrique Melanelia elegantula (Zahlbr.)  1  2  1  0.2 1 3  2 21 4  0.2 24 10  0.1 43 36  0.1  2 23  0.3 2  2 19  Essl.  M. exasperatula (Nyl.) Essl. M. fuliginosa (Fr. ex Duby) Essl. M. subaurifera (Nyl.) Essl. M. subelegantula (Essl.) Essl. M. trabeculata (Ahti) Essl. Parmelia spp. Parmeliopsis ambigua (Wulfen) Ach.  P. hyperopta (Ach.) Arnold Phycsia aipolia† (Ehrn.) Hampe. Platismatia glauca (L.)Culb.&C.F.Culb  Ramalina dilacerata (Hoffm.) Hoffm.†  Herrick sites  Aleza sites  0.2 1 0.1 0.4  2  0.2  3 0.3 1 45 42 9 1 0.4 0.4  Betula 0.1 2 0.1 1 76 45 1 16 4 1  Abies  Picea  Pseudotsuga  Betula  Abies  1 77 34  1 3 1 4 26 36  6 1  10 2  42 16  1 2  5 0.1 4 5 1 433  2 1 278  346  0.4 0.2 2 112 38  0.4  14 3  6 1  1 0.1  0.1  0.1  4 1 1 2 107 40 1 13 4  Picea  0.3 0.4 0.1 0.3 7 27  4 0.8 2 104 55  0.1 0.3  0.1  Populus  3 1 10  3 3 2 3 118 50 12 4 4 4 0.1  41  179  0.2 132  0.4 0.3  7 26  6 2  35 9  18 9  39 39  80 44  68 54  138 64  3 1 0.1  10 23  72 24  47 12  6  176  181  347  101  1402  1367  848  1383  17  278  287  542  0.1  205  154  384  365  168  223  123  21  The influence of overstorey Populus on epiphytic lichens Site-type  Fraser sites  Tree species  Populus  Betula  Abies  Picea  Ramalina intermedia (Delise ex (Willd.) Hale T. ciliaris (Ach.) Gyelnik T. orbata (Nyl.) M. J. Lai Vulpicida pinastri (Scop.) Gray  Stratified bipartite cyanolichens Fuscopannaria spp.† P.M. Jorg. Lempholemma spp.† Körb. Lobaria hallii (Tuck.) Zahlbr. L. scrobiculata (Scop.) DC. Nephroma bellum (Sprengel) Tuck.  N. helveticum Ach. N. isidiosum (Nyl.) Gyeln. N. parile (Ach.) Ach. N. resupinatum (L.) Ach. Peltigera canina‡ (L.) Willd P. collina (Ach.) Schrader P. membranacea‡ (Ach.) Nyl. P. neckeri Hepp‡ ex Mull. Arg. P. polydactylon‡ (Necker) Hoffm. P. praetextata‡ (Sommerf.) Zopf Pseudocyphellaria anomala Brodo& Ahti.  Sticta fuliginosa (Hoffm.) Ach. S. limbata (Sm.) Ach. S. oroborealis Goward & Tønsberg  0.4  0.3  10  8  20  0.1  Populus  Betula  Abies  Picea  Pseudotsuga  Populus  Betula  Abies  Picea  16  0.2 18  66  38  33  59  6  84  108  60  2 4  1 2  0.2 0.4  0.1 5  0.3  1  1 1  1 1 6  0.4 0.3  0.1 3 1  2  45 5  18 3  1 5  0.1 7  254 8 18 278 3  44 52 23 121  2 19 4 24  2 3 8 34  16 11  16 13  Nyl.) Nyl.†  Tuckermanopsis chlorophylla  Herrick sites  Aleza sites  0.1 0.1 445 39 81 202 12 265 109 130 11  114 2  105 3  118 25  11 1  2  53 708 8 529 9 5 17  7 212 0.3 207 15  27 487 6 210 9  4 41 9 105 9  1 14 1 0.1  17  1 0.3 8 1 3  0.1 3 1 2  20 12  27  39 25 1 0.2  3 0.3 1  0.4 12 8  3 5 12 1 0.1  2  32 10 0.1 0.2  82 31 0.3 1  6 4  1 0.2  1  1  17 7  24 8 1  22  The influence of overstorey Populus on epiphytic lichens Site-type  Fraser sites  Tree species  Populus  Betula  Bipartite non-stratified cyanolichens Collema auriforme (With.) 2  Coppins & J.R. Laundon C. coniophilum Goward C. curtisporum Degel. C. flaccidum† (Ach.) Ach. C. furfuraceum (Arnold) Du Rietz C. spp. nova 1 Goward ined.† ¶ C. subflaccidum Degel. C. spp. nova 2 Goward ined. ¶ Leptogium burnetii C.W. Dodge L. intermedium† (Arnold) Arnold L. spp nova 1Bjork & Goward ined.† ¶ L. occultatum† Bagl. L. saturninum† (Dickson) Nyl. L. teretiusculum† (Wallr.) Arnold L. spp. nova 2 Bjork & Goward ined.† ¶  Tripartite cyanolichen L. pulmonaria (L.). Hoffm  16 0.4 0.2 11 4 10 0.4 40 0.2 0.1 0.2 116 0.1  Abies  Picea  Populus  Betula  Abies  Picea  Pseudotsuga  Populus  Betula  Abies  Picea  0.1 5 0.1  4 1  4  1 2  2  2 0.1 1 0.4 6  1 2 2 0.1 2  1 1 1.2 0.1 3  0.4  1  0.1 0.1 7  0.3  45  27 0.1  13 0.1  68 0.1  0.1 4118  Herrick sites  Aleza sites  13  0.1  49 0.1  0.1 4518  3434  1  0.1 5678  † - not included in the NMS or MRPP analysis due to rarity in the data set ‡- included in the NMS and MRPP analysis as Peltigera spp. ¶ - not yet described  2578  1705  952  493  78  880  216  114  144  23  The influence of overstorey Populus on epiphytic lichens Table 2.3 Total number of foliose macrolichen species (and mean species per plot ± SD) observed within four functional groups on conifer saplings beneath five overstorey tree species at Fraser, Aleza and Herrick sites.  Site-type  Bipartite stratified cyanolichens  Bipartite non-stratified cyanolichens  Tripartite stratified cyanolichens  Chlorolichens  Populus  14 (8±1)  15 (7±3)  1  12 (3±3)  42 (18±4)  Betula  14 (8±2)  8 (3±2)  1  16 (6±4)  39 (18±5)  Abies  12 (6±3)  11 (3±3)  1  13 (6±3)  37 (16±4)  Picea  13 (8±3)  8 (2±3)  1  14 (7±4)  36 (18±3)  Populus  10 (5±2)  8 (3±2)  1  17 (9±3)  36 (16±7)  Betula  7 (2±1)  0  1  19 (11±2)  27 (13±2)  Abies  6 (2±2)  1 (0.1±0.3)  1  16 (11±2)  24 (13±2)  Picea  6 (2±2)  0  1  15 (10±1)  22 (12±3)  1 (0.4±0.5)  0  1  13 (9±1)  15 (10±2)  Populus  13 (7±2)  7 (3±2)  1  18 (8±4)  39 (19±5)  Betula  12 (6±2)  1 (0.4±0.5)  1  21 (13±3)  35 (20±4)  Abies  8 (5±3)  0  1  21 (12±2)  30 (18±3)  Picea  8 (4±2)  0  1  19 (13±2)  28 (17±4)  Tree species  Total  Fraser  Aleza  Pseudotsuga  Herrick  Differences in cyanolichen communities on conifer saplings beneath the different overstorey tree species varied with site-type (Table 2.3). At the Fraser sites, cyanolichen communities were similar regardless of the overstorey tree species. By contrast, the cyanolichen communities on saplings beneath overstorey Populus at the Aleza and Herrick sites were  24  The influence of overstorey Populus on epiphytic lichens significantly more abundant and species-rich compared to those on saplings beneath other tree species (MRPP, T=-20.04, A=0.46, p=0.0001). Furthermore, 24% and 27% of all cyanolichen species observed at the Aleza and Herrick sites, respectively, were observed exclusively on saplings beneath Populus. Epiphyte communities on saplings beneath Betula, Abies and Picea at the Aleza and Herrick sites were mainly composed of chlorolichens and were largely devoid of non-stratified cyanolichen species; only two small Leptogium saturninum thalli (<1 cm2) were recorded beneath these trees at the Aleza and Herrick sites. Cyanolichen abundance, like species-richness, was greater on saplings beneath Populus at the Aleza and Herrick sites, and at Fraser sites regardless of tree species (Fig. 2.2). The degree to which lichen abundance on conifer saplings differed by overstorey tree species varied with site-types; cyanolichens were 44%, 96% and 89% more abundant on saplings beneath Populus than beneath conifers or Betula at the Fraser, Aleza and Herrick sites respectively. Chlorolichens, in contrast, were most abundant on saplings beneath Betula or conifer canopies at the Aleza and Herrick sites (Fig. 2.2). Small thalli of all cyanolichen functional groups were highly abundant on saplings beneath all overstorey tree species at the Fraser sites (Table 2.4). In contrast, small cyanolichen thalli (especially the non-stratified cyanolichens) were limited to saplings beneath Populus trees at the Aleza and Herrick sites.  25  The influence of overstorey Populus on epiphytic lichens  8000 b  Chlorolichen Gel cyanolichen Bipartite stratified Tripartite stratified  -1  Mean total lichen abundance (cm plot )  ab  2  6000  ab  a 4000  c a  c d d  2000  e df  f f  0 Pt  Bp  Al  Pgxe  Fraser sites  Pt  Bp  Al  Aleza sites  Pgxe  Pm  Pt  Bp  Al  Pgxe  Herrick sites  Figure 2.2 Total abundance of four functional groups of epiphytic foliose macrolichens observed on sapling branches beneath five tree species at the three sitetypes. Tree species codes: Pt (Populus tremuloides or P. trichocarpa), Bp (Betula papyrifera), Al (Abies lasiocarpa), Pgxe (Picea glauca x engelmannii), Pm (Pseudotsuga menziesii). Letters above the bars denote significant multivariate differences in lichen diversity and abundance across plot types (overall MRPP; T= 18.11, p<0.0000, A= 0.39).  26  The influence of overstorey Populus on epiphytic lichens Table 2.4 Total numbers of small cyanolichen thalli (< 0.5 cm2) observed within three cyanolichen functional groups on conifer saplings beneath five overstorey tree species at Fraser, Aleza and Herrick sites (N=9).  Site-type  Bipartite stratified cyanolichens  Bipartite non-stratified cyanolichens  Tripartite stratified cyanolichens  Populus  5270  640  4720  Betula  2500  70  2780  Abies  2770  220  5170  Picea  7970  260  6870  Populus  220  620  2940  Betula  0  0  70  Abies  0  0  50  Picea  40  0  10  Pseudotsuga  0  0  5  Populus  1080  540  190  Betula  340  0  170  Abies  140  0  240  Picea  80  0  30  Tree species  Fraser  Aleza  Herrick  27  The influence of overstorey Populus on epiphytic lichens The NMS ordination provided a graphical summary of the overall variation in lichen community composition and abundance (Fig. 2.3). Fraser plots largely grouped with the cyanolichens while Aleza and Herrick sites were more widely spread out, covering a range of species groups. Plots representing saplings beneath overstorey Populus from Aleza and Herrick sites clustered with all plots at the Fraser sites due to their association with cyanolichens. In contrast, plots representing saplings beneath overstorey conifer and Betula trees at the Aleza and Herrick sites grouped with the chlorolichens and were clearly separated from those representing saplings beneath Populus and all the Fraser-site plots. The MRPP comparisons confirmed the quantitative differences in lichen communities across site-types and between overstorey tree species (Fig. 2.2). Whereas differences between the plots representing saplings beneath Populus and those representing saplings beneath other overstorey tree species were non-significant at the Fraser sites, this was not the case at Aleza and Herrick sites, where Populus plots differed significantly from the other overstorey trees.  28  The influence of overstorey Populus on epiphytic lichens  Axis 2  H G Y  Fraser, Populus Aleza, Populus Herrick, Populus Fraser, Betula Aleza, Betula Herrick, Betula Fraser, Conifer Aleza, Conifer Herrick, Conifer Chlorolichen Gel cyanolichen Stratified cyanolichen  Aleza sites  H H H  H HH H  H H  H H  H HH  H H  H H  H  H  Y Y  YY Y  Y GG G  H  Y Y  G  Y  Y G Y G YG  Y  G YG  Herrick sites  Fraser sites  Axis 1 Figure 2.3 NMS ordination of 46 species observed in 117 plots at three site-types. The ordination resulted in a 2-dimensional solution with a final instability criterion <0.0001 after 250 runs with real data. NMS axes 1 and 2 accounted for 47% and 42% of the variation in the distance matrix respectively. Letters (H- chlorolichen, Y – stratified cyanolichen, G – non-stratified cyanolichens) denote individual lichen species and symbols represent plots shown by site-type and overstorey tree species combinations. Dashed lines indicate approximate separation of the three site-types.  29  The influence of overstorey Populus on epiphytic lichens Conifer saplings versus overstorey Populus Cyanolichen abundance and species-richness were higher on conifer saplings beneath the overstorey Populus canopy than on the trunks of the Populus trees themselves. Populus trunks (and branches where present) supported 11, 11 and 14 cyanolichen species at Fraser, Aleza and Herrick site-types, respectively. In contrast, conifer saplings beneath Populus canopies supported 31, 19 and 21 cyanolichen species for the same site-types (Fig.2.4).  Chlorolichen Gel cyanolichen Bipartite stratified Tripartite stratified  Number of macrolichen species  50  40  30  20  10  0 Trunk  Sapling  Fraser sites  Trunk  Sapling  Aleza sites  Trunk  Sapling  Herrick sites  Figure 2.4 Diversity of foliose chlorolichens, non-stratified cyanolichens, stratified bipartite cyanolichens and tripartite cyanolichens observed on Populus trunks versus on the conifer saplings beneath Populus canopies at Fraser, Aleza and Herrick sites.  30  The influence of overstorey Populus on epiphytic lichens 2.3.3 Precipitation, bark and soil chemistry In general, no clear relationships were evident between throughfall precipitation chemistry and foliose macrolichen community patterns. Calcium concentrations beneath Populus were similar to all other overstorey tree species at the Fraser sites, Picea at the Aleza sites, and Abies and Picea at the Herrick sites (F(8,122)=3.36, p=0.002; Fig. 2.5a & b). Phosphorus and manganese concentrations were both significantly lower in precipitation throughfall from beneath Populus and Betula compared to beneath Abies and Picea (P: F(4,122)=94.71, p=0.000; Fig. 2.5c; Mn: F(4,122)=58.12, p=0.000; Fig. 2.5e). The highest overall phosphorus and manganese concentrations were observed in throughfall precipitation at the Fraser sites (F(2,122)=6.51, p=0.002; Fig. 2.5c) and at the Herrick sites (F(2,122)=9.1, p=0.000; Fig. 2.5e), respectively. No differences in throughfall concentrations of molybdenum were detected across site-types or tree species (Fig. 2.5g). In contrast to the other elements studied, throughfall molybdenum levels were significantly lower than in the non-throughfall (open) precipitation across all three site-types (F(4,122)=11.14, p=0.000), indicating some degree of uptake by trees from the ambient precipitation. Also unlike the other four elements, molybdenum concentrations were higher in the winter precipitation (Fig. 2.5h). Precipitation chemistry was poorly correlated with lichen abundance; manganese concentrations were negatively correlated with non-stratified cyanolichen abundance (r= -0.29, p=0.003), and phosphorus was negatively correlated with bipartite stratified cyanolichen abundance (r= -0.27; p=0.004). All other correlations were non-significant. The pH of ambient precipitation was 6.5±0.2 and did not differ between site-types. Throughfall precipitation beneath Abies and Picea was more acidic than that beneath Populus and Betula at all site-types (F(4,48)=6.73, p=0.0001; Fig. 2.6a). Conifer sapling bark pH was higher at the Fraser sites regardless of over overstorey tree species. At the Aleza and Herrick 31  The influence of overstorey Populus on epiphytic lichens sites, bark pH of saplings beneath Populus was similar to that at the Fraser sites, and was significantly higher than that beneath all other tree species (F(6,271)=7.84, p<0.0001; Fig. 2.6b). Sapling bark pH was positively correlated with cyanolichen abundance (r=0.62, p=0.0001) but only weakly correlated with precipitation pH (r= 0.29; p=0.04), and not significantly correlated with soil pH. Soil organic matter was more acidic at the Fraser sites (pH 4.8±0.1) than at the Aleza (5.0±0.1) or Herrick (5.2±0.1) sites (F(2, 114)=7.52, p=0.0009), with no difference between tree species. Conversely, mineral soil pH was significantly higher beneath Populus at Fraser sites (5.2±0.1) than beneath Pseudotsuga at Aleza sites (4.6±0.1) or Picea at Herrick sites (4.6±0.1; F(3,104)=2.91, p=0.04).  32  The influence of overstorey Populus on epiphytic lichens Summer 90x103  a  Winter b  a  Populus Betula Abies Picea Open  Ca (μg/L)  75x103 60x103  b  ac  b 45x103  bc bd  ab  ab  d  a  30x10  a  bc  15x103 0 35x103  a  ab  a  30x103  c  c b  c  ac bc  a a a  b  b  a b  b  ab ab ab  d  3  b  P (μg/L)  25x103 20x103  b  b b  15x103  b  10x103  b  a a  5x103  Mn (μg/L)  0 8000  a  a  a  c  b a  b  e  b a ac bc a  a  f  b  4000  b  b a  2000  aa  b  a  a c  0  c  aa  b  b  6000  ab c  aa  c  c  a  b aa  c  c  g  b  c  aa  bb c  aa  b c  h  8 7  Mo (μg/L)  6 5 4  b  3 2  aaa a  b  b a  a a  a  a  a a a  1 0  Fraser  Aleza  Site type  Herrick  Fraser  Aleza  Herrick  Site type  Figure 2.5 Concentration (± SD) of Ca, P, Mn, and Mo in precipitation captured in the open and beneath four tree species at three site-types. Elements were extracted from resin capsules installed in the field from November 2007 to May 2008 (Winter) and from May to September 2008 (Summer). Dissimilar letters represent significant differences (p<0.05) between tree species within site-types.  33  The influence of overstorey Populus on epiphytic lichens  8.0 A  ab  a  B  Fraser ALRF Herrick  7.5  pH  7.0  ab a  a  a  ab  a  6.5 bc bc bc  6.0  a  c  c  a  c  a a  5.5  b  5.0  a  a bc c  c  c  c c  c  4.5 Pt  Bp  Al  Tree species  Pgxe  Open  Pt  Bp  Al  Pgxe  Pm  Tree species  Figure 2.6 Comparisons of (a) throughfall precipitation pH (±SD) and (b) conifer sapling bark pH (±SD) beneath five tree species at three site-types. Tree species codes: Pt (Populus tremuloides or P. trichocarpa), Bp (Betula papyrifera), Al (Abies lasiocarpa), Pgxe (Picea glauca x engelmannii), Pm (Pseudotsuga menziesii). Dashed lines represent the lower pH limit for cyanolichen habitat (Gauslaa 1985). Dissimilar letters represent significant differences (p<0.05) between tree species and site-types.  34  The influence of overstorey Populus on epiphytic lichens  2.4 Discussion  2.4.1 Lichen abundance patterns Abundant and species-rich cyanolichen communities were observed on conifer saplings beneath overstorey Populus trees across the study area. This spatial association intensified in areas where sub-optimal environmental conditions (i.e. moisture or light deficiencies) appeared to preclude cyanolichens in the surrounding conifer forests. This suggests that Populus may facilitate the development of cyanolichen thalli beneath its canopy. This may be particularly the case with the non-stratified (homoiomerous) lichen species, more than 85% of which were observed exclusively under Populus in these sub-boreal forests. The distribution of non-stratified lichens is known to be limited in inland temperate forests (for example, see Spribille et al. 2009 for ecology and distribution of Collema coniophilum), however, the nearly exclusive existence beneath Populus at sites with moisture or light deficiencies merits further investigation. The importance of moisture availability in determining the composition of lichen communities is borne out in our results. Lichens are less physiologically active when thallus moisture falls below a certain threshold (Kappen 1988). This threshold varies considerably with species and is generally higher for cyanolichens (>150% dry weight, Lange et al. 2004) than for chlorolichens with green-algal partners (50-70% dry weight, Lange et al. 1986). Accordingly, the epiphyte communities beneath a coniferous overstorey at the Fraser sites were composed largely of cyanolichens while those at the drier, Aleza sites were primarily chlorolichens. However, that overstorey conifers at the Herrick sites, with similar rainfall and relative humidity as the Fraser sites, also supported predominantly chlorolichen communities indicates that other environmental factors are involved. The removal of individual trees from the Herrick sites in the early 1900’s effected stand structure such that light was 9% lower than at the other sites. For cyanolichens  35  The influence of overstorey Populus on epiphytic lichens that almost invariably occupy the lower canopy where direct sunlight is infrequent, this reduced light availability could result in reduced establishment, growth (Gauslaa et al. 2006) and photosynthesis (Gaio-Oliveira et al. 2004). While climate is a key determinant of regional distribution patterns, moisture and light availability cannot account for the enriched cyanolichen communities observed beneath Populus as there were no differences in these factors between overstorey tree species. Differences in lichen distribution patterns have previously been related to variation in the physical characteristics of the phorophyte itself (Gauslaa 1995; Hauck and Spribille 2005). However, the rich cyanolichen community observed beneath Populus cannot be solely attributed to substrate characteristics as all host saplings here were either Abies or Picea, and no significant floristic differences were noted between them. Furthermore, the greater diversity of cyanolichens observed on saplings beneath the Populus canopy, rather than on the Populus trunks, indicates that the facilitative role involves more than the physical attributes of the host tree species. The abundant and species-rich cyanolichen communities observed on conifer saplings beneath overstorey Populus trees suggest that Populus facilitates one or more of lichen dispersal, establishment or growth. The close proximity between the cyanolichen-rich communities beneath Populus and the cyanolichen-poor communities beneath conifers indicates that dispersal may not be a limiting factor. However, the probability of dispersed diaspores becoming established may be low (Scheidegger 1995) and successful recruitment of cyanolichens on conifer branches in a moisture- or light-deficient conifer forest may depend on the possible amelioration of habitat conditions by overstorey Populus. This possibility is supported by the greater number of small (and presumably newly established) cyanolichen thalli observed beneath Populus compared to all other overstorey trees.  36  The influence of overstorey Populus on epiphytic lichens 2.4.2 Relationship to precipitation chemistry These results support the concept of a drip-zone of nutrient-enriched precipitation surrounding Populus (Goward and Arsenault 2000). Several chemical factors have been highlighted in the literature as potentially important to cyanolichens. Cyanolichens are sensitive to acidic conditions below pH 5.0 (Gauslaa 1985; Farmer et al. 1991). Goward and Arsenault (2000) attributed substrate pH differences to an increase in available calcium in throughfall precipitation. Our data also show a strong correlation between cyanolichen abundance and sapling bark pH. However, a similar relationship with the pH or calcium concentration of throughfall precipitation was not observed. Overall, the weak correlation between precipitation pH, soil pH, and sapling bark pH suggests that factors other than precipitation or soil chemistry may be involved. It is plausible that the lichens themselves are altering the pH of sapling bark, rather than responding to it. Gauslaa and Holien (1998) reported that cyanolichens can raise the pH of their bark substrate by releasing more cations than protons at ion-exchange sites, whereas chlorolichens have the opposite effect. Thus, the abundant cyanolichens at Fraser sites and beneath Populus at Aleza and Herrick sites might have increased the sapling bark pH, while the abundant chlorolichens beneath conifers and Betula at the Aleza and Herrick sites might have decreased the substrate pH. Cyanolichens are capable of N2-fixation and so may be limited by the availability of phosphorus or molybdenum (Kurina and Vitousek 1999). Indeed, cyanolichen diversity and abundance increased in direct response to long-term phosphorus fertilization in late-seral Hawaiian forests (Benner and Vitousek 2007). In this study, however, the weak correlation between phosphorus concentrations and cyanolichen abundance suggests that phosphorus was not a limiting factor (see also Arocena and Sanborn 1999). Molybdenum additions have likewise been shown to increase the nitrogen-fixing activity of asymbiotic N2-fixing bacteria (Silvester 37  The influence of overstorey Populus on epiphytic lichens 1989) and lichens (Horstmann et al. 1982). However, the similar molybdenum concentrations across tree species and study sites here suggest that it is unlikely to be a limiting factor for cyanolichen growth. Finally, the weak inverse relationship between manganese concentrations and non-stratified cyanolichen abundance provides tentative agreement with previous records of lichen-thallus inhibition in response to high manganese concentration (reviewed in Hauck 2003). Nonetheless, significantly different lichen communities were noted beneath Betula and Populus canopies despite similar manganese concentrations. Cyanolichen communities often occur only on the trunks of Populus trees and are otherwise absent on the surrounding coniferous tree species. Cyanolichens are not similarly restricted to Populus trunks in sub-boreal British Columbia and, indeed, are more species-rich on the branches of conifer saplings beneath Populus than on the Populus trunks themselves. There are several plausible explanations. Sapling conifer branches provide a larger, horizontal surface area which may allow for higher cyanolichen colonization compared to Populus trunks. In a similar vein, the varied orientation and exposure of conifer branches beneath Populus trees may provide a more heterogeneous habitat which, as ecological theory predicts, can result in greater diversity (Ricklefs 1977). Alternatively, the unexpectedly low diversity of lichens observed on Populus trunks may be in response to the ameliorated environment created by frequent interception of stemflow and throughfall precipitation through the Populus canopy. In such environments, stress is likely to be minimal and as predicted by Grime (1973), inter-specific competition will strongly define community structure. One or a few strongly competitive species would thus be expected to become dominant to the exclusion of other, less competitive, species (Paine 1966).  38  The influence of overstorey Populus on epiphytic lichens While competition may be a dominant factor determining lichen species-richness on Populus trunks, the lichens observed on conifer saplings beneath Populus suggest that facilitation is equally important in defining lichen community structure. The near-exclusive occurrence of cyanolichens beneath overstorey Populus at sites with moisture (Aleza) or light (Herrick) deficiencies suggests that Populus supplies some as yet unknown, resource that is critical to cyanolichen establishment or growth. As such, overstorey Populus trees appear to be crucial for providing cyanolichen habitat in regions where environmental conditions otherwise limits their establishment in the surrounding conifer forest. 2.5 References  Arocena, J.M. and Sanborn, P. 1999. Mineralogy and genesis of selected soils and their implications for forest management in central and northeastern British Columbia. Canadian Journal of Soil Science 79:571-592. Arsenault, A. and Goward, T. 2000. Ecological Characteristics of Inland Rain Forests. In Biology and management of species and habitats at risk, Kamloops, B.C., 15-19 February,1999. Edited by L.M. Darling. B.C. Ministry of Environment, Lands and Parks, Victoria, BC and the University college of the Cariboo, Kamloops, BC. pp. 437-439. Benner, J.W. and Vitousek, P.M. 2007. Development of a diverse epiphyte community in response to phosphorus fertilization. Ecology Letters 10:628-636. Benson, S. and Coxson, D.S. 2002. Lichen colonization and gap structure in wet-temperate rainforests of northern interior British Columbia. Bryologist 105:673-692. Brodo, I.M., Sharnoff, S.D. and Sharnoff, S. 2001. Lichens of North America. Yale University Press, New Haven, Conneticut. Campbell, J. and Fredeen, A.L. 2004. Lobaria pulmonaria abundance as an indicator of macrolichen diversity in Interior Cedar-Hemlock forests of east-central British Columbia. Canadian Journal of Botany 82:970-982. Campbell, J. and Fredeen, A.L. 2007. Contrasting the abundance, nitrogen and carbon of epiphytic macrolichen species between host trees and soil types in a sub-boreal forest. Canadian Journal of Botany 85:31-42. Dolan, S., P. and Capar, S., G. 2002. Multi-element analysis of food by microwave digestion and inductively coupled plasma-atomic emmision spectrometry. Journal of Food Composition and analysis 15:593-615.  39  The influence of overstorey Populus on epiphytic lichens Ellis, C.J. and Coppins, B.J. 2007. Reproductive strategy and the compositional dynamics of crustose lichen communities on aspen (Populus tremula L.) in Scotland. Lichenologist 39:377-391. Farmer, A.M., Bates, J.W. and Bell, J.N.B. 1991. Seasonal variations in acidic pollutant inputs and their effects on the chemistry of stemflow, bark and epiphyte tissues in three oak woodlands in N.W. Britain. New Phytologist 188:441-451. Gaio-Oliveira, G., Dahlman, L., Palmqvist, K. and Maguas, C. 2004. Growth in relation to microclimatic conditions and physiological characteristics of four Lobaria pulmonaria populations in two contrasting habitats. Ecography 27:13-28. Gauslaa, Y. 1985. The ecology of Lobarion pulmonariae and Parmelion caperatae in Quercus dominated forests in South-West Norway. Lichenologist 17:117-140. Gauslaa, Y. 1995. The Lobarion, an epiphytic community of ancient forests threatened by acid rain. Lichenologist 27:59-76. Gauslaa, Y. and Holien, H. 1998. Acidity of boreal Picea abies-canopy lichens and their substratum, modified by local soil and airborne acidic depositions. Flora 193:249-257. Gauslaa, Y., Lie, M., Solhaug, K.A. and Ohlson, M. 2006. Growth and ecophysiological acclimation of the foliose lichen Lobaria pulmonaria in forests with contrasting light climates. Oecologia 147:406-416. Goward, T. 1994. Notes on oldgrowth-dependent epiphytic macrolichens in inland British Columbia, Canada. Acta Botanica Fennica 150:31-38. Goward, T. and Arsenault, A. 2000. Cyanolichen distribution in young unmanaged forests: A dripzone effect? Bryologist 103:28-37. Grime, J.P. 1973. Competitive exclusion in herbaceous vegetation. Nature 242:344-347. Hauck, M. 2003. Epiphytic lichen diversity and forest dieback: the role of chemical site factors. Bryologist 106:257-269. Hauck, M. and Spribille, T. 2005. The significance of precipitation and substrate chemistry for epiphytic lichen diversity in spruce-fir forests of the Salish Mountains, Northwestern Montana. Flora 200:547-562. Hedenås, H. and Ericson, L. 2000. Epiphytic macrolichens as conservation indicators: successional sequence in Populus tremula stands. Biological Conservation 93:43-53. Horstmann, J.L., Denison, W.C. and Silvester, W.B. 1982. 15N2 Fixation and molybdenum enhancement of acetylene reduction by Lobaria spp. New Phytologist 92:235-241. Jüriado, I., Paal, J. and Liira, J. 2003. Epiphytic and epixylic lichen species diversity in Estonian natural forests. Biodiversity and Conservation 12:1587-1607. Kappen, L. 1988. Ecophysiological relationships in different climatic regions. In CRC Handbook of Lichenology. Edited by M. Galun. CRC press, Boca Raton, Florida. pp. 37-100. Kurina, L.M. and Vitousek, P.M. 1999. Controls over the accumulation and decline of a nitrogen-fixing lichen, Stereocaulon vulcani, on young Hawaiian lava flows. Journal of Ecology 87:784-799.  40  The influence of overstorey Populus on epiphytic lichens Kuusinen, M. 1994. Epiphytic lichen flora and diversity on Populus tremula in old-growth and managed forests of southern and middle boreal Finland. Annales Botanici Fennici 31:245260. Lange, O.L., Kilian, E. and Ziegler, H. 1986. Water vapor uptake and photosynthesis of lichens: performance differences in species with green and blue-green algae as phycobionts. Oecologia 71:104-110. Lange, O.L., Büdel, B., Meyer, A. and Kilian, E. 1993. Further evidence that activation of net photosynthesis by dry cyanobacterial lichens requires liquid water. Lichenologist 25:175189. Lange, O.L., Burkhard, B., Meyer, A., Zellner, H. and Zotz, G. 2004. Lichen carbon gain under tropical conditions: water relations and CO2 exchange of Lobariaceae species of a lower montane rainforest in Panama. Lichenologist 36:329-342. Lehmkulh, J.F. 2004. Epiphytic lichen diversity and biomass in low-elevation forests of the eastern Washington Cascade Range, USA. Forest Ecology and Management 187:381-392. McCune, B. 1993. Gradients in epiphyte biomass in three Pseudotsuga-Tsuga forests of different ages in Western Oregon and Washington. Bryologist 96:405-411. McCune, B. and Grace, J.B. 2002. Analysis of Ecological Communities. MjM Software Design, Gleneden Beach, Oregon. Meidinger, D. and Pojar, J. 1991. Ecosystems of British Columbia. Ministry of Forests, Research Branch, Victoria, B.C. Murphy, B. 1996. Prince George Forest District: Climate normals 1951-1980. Report for McGregor Model Forest. Prince George, B.C. Paine, R.T. 1966. Food web complexity and species diversity. American Naturalist 100:65-75. Ricklefs, R.E. 1977. Environmental heterogeneity and plant species diversity: A hypothesis. American Naturalist 111:376. Scheidegger, C. 1995. Early development of transplanted isidioid soredia of Lobaria pulmonaria in an endangered population. Lichenologist 27:361-374. Sillett, S.C. and Neitlich, C. 1996. Emerging themes in epiphyte research in westside forests with special reference to cyanolichens. Northwest Science 70:54-60. Silvester, W.B. 1989. Molybdenum limitations of asymbiotic nitrogen fixation in forests of Pacific Northwest America. Soil Biology & Biochemistry 21:283-289. Skogley, E.O. and Dobermann, A. 1996. Synthetic ion-exchange resins: Soil and environmental studies. Journal of Environmental Quality 25:13-24. Spribille, T., Björk, C.R., Ekman, S., Elix, J.A., Goward, T., Printzen, C., Tonsberg, T. and Wheeler, T. 2009. Contributions to an epiphytic lichen flora of northwest North America: I. Eight new species from British Columbia inland rain forests. Bryologist 112:109-137.  41  Growth and mortality of cyanolichens under Populus  CHAPTER 3  Growth and mortality of cyanolichens under Populus2  3.1 Introduction  Populus has been shown to support unique and species-rich lichen communities. In many parts of the world, Populus trunks provide habitat for cyanobacterial lichens that are otherwise absent in the predominantly conifer forests (Kuusinen 1996; Hedenås and Ericson 2000). Although many cyanolichen species are also frequently observed on conifer branches in wet, interior forest ecosystems of British Columbia, cyanolichen communities are disproportionately species-rich and abundant on conifers growing beneath an overstorey Populus tree compared to those beneath a conifer canopy (Goward and Arsenault 2000; Campbell et al. 2010). The factors underlying the strong association between cyanolichens and Populus are not understood, but it is probable that Populus supports the cyanolichen symbiosis at one or more developmental stage. Sillett et al. (2000) describe the development of epiphytic cyanolichens in three stages; propagule dispersal, establishment, and thallus growth. Inadequate propagule dispersal has been shown to contribute to differences in epiphyte community structure (Sillett et al. 2000). However, dispersal limitations may not be involved in defining the observed differences between overstorey tree species; Populus trees, heavily loaded with L. pulmonaria, are often located within 0.1 ha of other tree species that support cyanolichen-poor epiphyte communities (e.g. Kuusinen 1996; Goward and Arsenault 2000; Campbell et al. 2010). The proximity of these disparate communities is well within the dispersal range of many cyanolichens (Ockinger et al. 2005). Any potential positive influence of Populus on cyanolichen communities is thus more likely to occur at a later phase of thallus development. Indeed, Schiedegger (1995) demonstrated that very few dispersed propagules successfully develop into a 2  A version of this chapter will be submitted for publication as Campbell, J. Growth and mortality of cyanolichens under Populus in sub-boreal spruce forests.  42  Growth and mortality of cyanolichens under Populus mature lichen thallus, and that this success depends on substrate and microclimatic conditions. The strong spatial association between abundant cyanolichen communities and Populus suggest that colonization success is greater near Populus than near other trees. However it is unknown whether this success is due to greater rates of establishment or growth. Previous studies have suggested that lichen growth rates are dependent on the conditions of the study area. Transplant experiments have demonstrated the relative influence of forest age (Sillett et al. 2000), light availability (Coxson and Stevenson 2007a), moisture availability (Gauslaa et al. 2007), seasonality (Muir et al. 1997) and nutrient availability (McCune and Caldwell 2009) on lichen growth. However, despite repeated accounts of highly disparate lichen communities on broadleaf versus conifer trees, few studies have controlled for the influence of different host or overstorey tree species on lichen growth. This study examines the effect of overstorey Populus by comparing growth rates of cyanolichens transplanted beneath the canopies of mature Populus tremuloides or P. balsamifera ssp. trichocarpa, Picea glauca x engelmannii and Pseudotsuga menziesii var. glauca. The influence of overstorey tree species is evaluated, relative to regional climate, by assessing growth rates in three sub-boreal spruce forests with varying climatic conditions. Furthermore, the influence of overstorey tree species on growth rates of tripartite (both a cyanobacterial and green-algal partner) and bipartite (only a cyanbacterial partner) cyanolichen species are compared. 3.2 Methods and materials  3.2.1 Study area The study area was located north-east of Prince George British Columbia in old growth (mean tree age >240 years) forests of the Sub-Boreal Spruce (SBS) biogeoclimatic zone (Meidinger and Pojar 1991). Three replicate stands were selected in each of three site-types. The  43  Growth and mortality of cyanolichens under Populus ‘Aleza’ sites were located in the Aleza Lake Research Forest at 680 m in elevation. Relatively humidity was consistently lower at these three sites and so they were considered to be the most moisture-deficient of the three site-types (Campbell et al. 2010). ‘Herrick’ sites were located along the Herrick Forest Service Road approximately 28 km north-east of the Aleza sites at an elevation of 850 m. Lower light levels were recorded at these sites compared to the other site types and so they were considered to be light-deficient (Campbell et al. 2010). Finally, three sites where neither moisture nor light were limiting (the Fraser sites) were located in an ecotonal region between the two aforementioned subzones at 680 m in elevation (see Campbell et al. 2010 for detailed site information). 3.2.2 Cyanolichen growth Healthy, intact thalli of Lobaria pulmonaria (L.) Hoffm. and Lobaria hallii (Tuck.) Zahlbr. were collected from a single spruce-subalpine fir dominated forest stand in early June 2006. Lobaria pulmonaria is a tripartite cyanolichen species and is among the more highly studied of all lichen species and is abundant beneath all tree species in many sub-boreal spruce forests (Campbell et al. 2010). Lobaria hallii is a bipartite cyanolichen with more limited distribution; it is highly abundant beneath Populus but is otherwise infrequent in sub-boreal British Columbia (Campbell et al. 2010). Forty large (initial mass: L. pulmonaria 0.42±0.07 g, L. hallii 0.23±0.07 g) and 120 small (initial mass: L. pulmonaria 0.003±0.001 g, L. hallii 0.004±0.001 g) thalli of each lichen species were collected to represent mature and establishing lichens, respectively. Samples were cleaned, carefully removed from the substrate, and placed in a climate-controlled environment (25oC and 40% relative humidity) where thallus-water content was allowed to stabilize for at least 72 h prior to weighing as per Coxson and Stevenson (2007b). Selected thalli were secured to strips of silicone-coated (TSNet-Tech Spray, Amarillo TX) aluminium mesh using clear silicone sealant 44  Growth and mortality of cyanolichens under Populus (GE Sealants and Adhesives, Huntesville, NC). Strips were attached to a 28 cm x 18 cm plastic tray (1 cm x1 cm grid) using binder clips and enclosed by UV-resistant, translucent mesh. Light transmission in these growth enclosures was >95% of ambient light (Coxson and Stevenson 2007b). Growth enclosures contained one large and three small thalli of either L. pulmonaria or L. hallii (see Coxson and Stevenson 2007b for photograph of growth enclosure). Two enclosures (one of each lichen species) were hung from wooden supports attached at approximately 3 m in height on the north side of each of two mature trees of each tree species at each site (N=6 large thalli, 18 small thalli per lichen species x tree species x site-type). Tree species included Picea glauca (Moench) Voss x engelmannii Parry ex Engelmann (interior hybrid spruce) and Populus spp. at each site (Populus tremuloides Michx. (trembling aspen) at Aleza sites and P. balsamifera L. ssp. trichocarpa Brayshaw (black cottonwood) at Fraser and Herrick sites). Enclosures were also hung beneath Pseudotsuga menziesii (Mirbel.) Franco var. glauca (Beissn.) Franco. (Douglas-fir) at the Aleza sites only. For simplicity, supporting tree species are hereafter referred to by genus name only, with P. tremuloides and P. trichocarpa collectively referred to as Populus. Transplants were initially placed in the field in late June 2006 and brought back into the lab for remeasurement in early October 2006, late June 2007, early October 2007 and late June 2008. Each time they were cleaned of debris and pollen and placed in the climate-controlled environment for 72 hours prior to remeasurement. Transplants were replaced in the field after no more than 96 hours in the lab. Large lichen thalli were weighed at each time interval to calculate growth rate. Growth of small thalli was represented by changes in surface area over time as recorded in digital photographs. Areas rather than masses were thus used for small thalli to  45  Growth and mortality of cyanolichens under Populus prevent growth being obscured by small variations in measuring conditions. Briefly, thallus size was standardized by resizing a digital photograph (CorelDRAW™ 12; Corel Corporation Inc., Mountain View, CA) so that 50 mm on a plastic ruler included in the photograph equaled a 50 mm digital standard. Lichen thalli were then digitally traced and exported to ArcView (3.2; ESRI, Redlands, CA) to quantify the surface area of each thallus at each time period. The initial surface areas of small L. pulmonaria and L. hallii were 31.8±10.2 mm2 and 27.1±13.0 mm2, respectively. Lichen thalli were removed from the experiment if they were disturbed by wildlife or falling trees, visibly lost large fragments, or were completely discolored (either entirely bleached or entirely black) and visibly unresponsive to rewetting. 3.2.3 Data analysis Lichen transplant growth was analyzed at each site-type using a repeated-measures ANOVA with a Fishers LSD post hoc test. The main effects and interactions between lichen species (Lobaria pulmonaria and L. hallii) and tree species (Populus and Picea) were tested across the 27 month transplant period. A Chi-square was used to test group differences in mortality frequency between tree species and site types. Values are given as mean± standard deviation unless otherwise stated. 3.3 Results  3.3.1 Large thalli Growth rates of both species varied considerably, ranging from 29 to 238% and 5 to 225% in Lobaria pulmonaria and L. hallii, respectively, over 27 months. However, faster growth rates were observed with L. pulmonaria transplants than L. hallii at all three sites (Fig. 3.1a). Accordingly, the mass of L. pulmonaria transplants was significantly larger than that of L. hallii transplants throughout the entire transplant period at all three site types (Fig. 3.1b)  46  Growth and mortality of cyanolichens under Populus The growth rate of large lichens was also consistently faster beneath Populus than beneath Picea for both lichen species (Fig. 3.1a; see Table 3.1 for ANOVA statistics). Transplant mass of L. pulmonaria increased by 117±40% and 80±38% beneath Populus and Picea, respectively, over 27 months, while L. hallii mass increased by 112±46% and 45±36% for the same comparison. The slowest growth for both lichen species was observed beneath Pseudotsuga with cumulative growth rates of 36±2% and 3±2% for L. pulmonaria and L. hallii, respectively. The final transplant mass of both lichen species was greater beneath Populus than Picea at all three site types (Fig. 3.1b). There were no significant differences in cyanolichen growth between site types. 3.3.2 Small thalli Growth of small Lobaria pulmonaria and L. hallii thalli was significantly faster beneath Populus than beneath Picea at all site types (Fig. 3.2a; see Table 3.1 for ANOVA statistics). While most small thalli beneath Populus had modest area gains over 27 months, the majority of small thalli beneath conifers exhibited slow or negative-growth resulting in final thallus areas that were as small, or smaller, than the initial thallus size (Fig. 3.2b). After 27 months, 2% of small L. pulmonaria and 27% of small L. hallii showed a net decrease in total area. Under the Pseudotsuga overstorey, small L. pulmonaria thalli had modest area-gains (16±17%) over the first 12 months and lost area thereafter. Lobaria hallii thallus-areas consistently decreased over 15 months after which point all small L. hallii were removed from the experiment due to visible necrosis or death.  47  Growth and mortality of cyanolichens under Populus  Fraser  Cummulative growth rate (%)  a  Aleza  L. hallii - Populus L. hallii - Picea L. hallii - Pseudotsuga L. pulmonaria - Populus L. pulmonaria - Picea L. pulmonaria - Pseudotusga  150  100  Herrick  50  0  1.2  b  0.8  0.6  0.4  0.2  0.0 0  5  10  15  20  25  0  5  10  Transplant period (mo.)  15  20  25  0  5  10  Transplant period (mo.)  15  20  25  Transplant period (mo.)  Figure 3.1 Cumulative percent growth rate of large thalli (a) and transplant mass (b) over 27 months beneath mature Populus, Picea, and Pseudotsuga trees at Fraser (left panel), Aleza (middle panel), and Herrick (right panel) sites.  Cummulative growth rate (%)  a  L. hallii - Populus L. hallii - Picea L. hallii - Pseudotsuga L. pulmonaria - Populus L. pulmonaria - Picea L. pulmonaria - Pseudotusga  Fraser  150  100  Aleza  Herrick  50  0  -50 80  b  70  Transplant area (cm2)  Transplant mass (g)  1.0  60 50 40 30 20 10 0 0  5  10  15  Transplant period (mo.)  20  25  0  5  10  15  20  25  0  Transplant period (mo.)  5  10  15  20  25  Transplant period (mo.)  Figure 3.2 Cumulative percent growth rate of small thalli (a) and transplant mass (b) over 27 months beneath mature Populus, Picea, and Pseudotsuga trees at Fraser (left panel), Aleza (middle panel), and Herrick (right panel) sites. 48  Growth and mortality of cyanolichens under Populus  Table 3.1 Repeated Measures ANOVA statistics at Fraser, Aleza and Herrick sites. Models tested significant differences in the change in transplant mass (large thalli) or transplant area (small thalli) over 3, 12, 15, 24 and 27 months (time as the repeated measure) between lichen species (Lobaria pulmonaria and Lobaria hallii) and tree species (Populus and Picea). Differences associated with Pseudotsuga were not tested due to low sample size.  Fraser sites time Time*lichen time*tree time*lichen* tree Error Aleza sites time Time*lichen time*tree time*lichen* tree Error Herrick sites time Time*lichen time*tree time*lichen* tree Error  Large thalli SS DF  MS  F  p  Small thalli SS DF  MS  F  p  1.534 0.393 0.181  5 5 5  0.307 0.079 0.036  44.750 11.450 5.279  0.0000 0.0000 0.0002  3093.7 77.3 1522.9  4 4 4  773.4 19.3 380.7  12.398 0.310 6.103  0.0000 0.8710 0.0001  0.008  5  0.002  0.226  0.9502  381.4  4  95.3  1.528  0.1965  0.651  95  0.007  9731.9  156  62.4  0.626 0.102 0.022  5 5 5  0.125 0.020 0.004  70.417 11.515 2.483  0.0000 0.0000 0.0414  1563.9 1398.4 898.5  4 4 4  391.0 349.6 224.6  6.720 6.008 3.861  0.0001 0.0002 0.0056  0.035  5  0.007  3.920  0.0038  150.3  4  37.6  0.646  0.6311  0.107  60  0.002  6516.7  112  58.2  1.170 0.254 0.186  5 5 5  0.234 0.051 0.037  39.110 8.499 6.223  0.0000 0.0000 0.0001  1183.0 924.8 2105.3  4 4 4  295.8 231.2 526.3  5.679 4.439 10.106  0.0003 0.0020 0.0000  0.011  5  0.002  0.369  0.8683  88.3  4  22.1  0.424  0.7913  0.419  70  0.006  8541.0  164  52.1  49  Growth and mortality of cyanolichens under Populus 3.3.3 Mortality Cumulative small-thallus mortality of both lichen species tended to be higher beneath Picea than Populus at all site-types and was significantly greater beneath conifers than Populus at the Aleza sites (χ2 (6) = 26.35, p=0.0002). All small lichen thalli transplanted to beneath Pseudotsuga were either dead or highly necrotic following the second summer (Fig. 3.3). Indeed, with few exceptions, the highest mortality rates were observed between June and October 2007 (12 – 15months; Fig. 3.3). The highest mortality rates were consistently observed beneath conifers at the Aleza sites, where 90-100% of both species were dead after 27 months. Although several large lichen thalli appeared at least partially necrotic after 27 months, there was no largethallus mortality in either lichen species. Unfortunately all large L. hallii thalli transplanted to beneath Pseudotsuga were disturbed by wildlife over the second winter and were excluded from the experiment.  50  Growth and mortality of cyanolichens under Populus  Cumulative thallus mortality (%)  100  a  L.hallii - Populus L.hallii - Picea L.hallii - Pseudotsuga L.pulmonaria - Populus L.pulmonaria - Picea L.pulmonaria - Pseudotsuga  80  60  40  20  0  b  Cumulative thallus mortality (%)  100  80  60  40  20  0  Cumulative thallus mortality (%)  100  c  80  60  40  20  0 0  4  8  12  16  20  24  28  Transplant period (mo.)  Figure 3.3 Cumulative mortality of small Lobaria hallii and L. pulmonaria thalli beneath Populus, Picea and Pseudotsuga trees at a) Fraser, b) Aleza and c) Herrick sites over 27 months.  51  Growth and mortality of cyanolichens under Populus  3.4 Discussion  3.4.1 Effect of overstorey tree species Growth of both Lobaria pulmonaria and Lobaria hallii was faster beneath Populus than beneath Picea at all three site types (Fig. 3.1a and Fig. 3.2a). In addition, growth rates of large thalli for both lichen species beneath Populus were faster than most other published rates (Table 3.2). The sole exception was 68% growth of Lobaria pulmonaria over 15 months in Western Portugal (Gaio-Oliveira et al. 2004) where calcareous soils and high pH may have promoted cyanolichen growth (Gauslaa 1995). The mean annual growth of L. hallii under Populus was almost three times the mean of published annual bipartite cyanolichen growth rates (14%; Table 3.2). Lobaria pulmonaria is among the more studied of all forest lichens and growth rates have been documented across a wide range of forest types under different moisture regimes (Table 3.2). The most comparable are from other wet interior forests, where mean growth rates range from 16 to 19% over 12 months in even-aged, and old-growth Thuja-Tsuga forests, respectively (Coxson and Stevenson 2007b). The mean first-year L. pulmonaria growth recorded here under Picea was faster than that of Coxson and Stevenson, but is similar to other studies under similar moisture regimes (Table 3.2). Annual growth of Lobaria hallii beneath Picea varied with site-type but, like L. pulmonaria, remained within the range of reported growth rates for other bipartite cyanolichen species (Table 3.2). Specifically, growth rates under Picea were similar to those recorded for two closely related species, Lobaria retigera and L. scrobiculata, which had annual growth rates of 4.5-10.5% (Coxson and Stevenson 2007a) and 19% (Hilmo 2002) respectively.  52  Growth and mortality of cyanolichens under Populus The slowest growth rates for both cyanolichen species were recorded beneath overstorey Pseudotsuga. The response of L. hallii was particularly striking, with growth rates that were as low as the lowest published rate of annual growth for a bipartite cyanolichen species (Table 3.2). These observations, in combination with high juvenile thallus mortality, suggest that cyanolichens are inhibited by Pseudotsuga at the establishment and growth life stages (Sillett et al. 2000), but the causal factor remains unknown. Table 3.2 Comparative studies on growth rates of cyanolichens in old forest environments. Precipitation is given as mm year-1 unless (except in cases where the number of measurement days is included in parentheses). Approximate annual growth rates are based on calculations from seasonal or multi-year growth rates. Annual growth rates in parentheses indicate those that are extrapolated from shorter-term studies and may therefore overestimate annual growth. See Coxson and Stevenson 2007a for other records of L. pulmonaria growth. Citation Lobaria pulmonaria Antoine & McCune (2004) Asplund & Gauslaa (2008) Coxson & Stevenson (2007a) Denison (1988) Gaio-Oliviero et al. (2004) Gauslaa (2006) Gauslaa et al. (2006) Gauslaa et al. (2007)  Location  Pseudotsuga-tsuga forest, northern Oregon Broadleaf forest, south & south-east Norway Thuja-Tsuga forest, British Columbia western Oregon Quercus faginea forest, western Portugal Picea abies forest, northern Sweden Picea abies forest, south-east Norway Old Picea abies forest, south-east Norway Picea abies forests, northern Sweden Picea abies forests, western Norway  Precipitation (mm)  Growth (%)  Time period (mo.)  Annual growth  2500  4 -13  12  4-13  346 (104d.)  5-8.2  3.5  (23)  840  16.1-19  24  9  1300 927  8 68  12 15  8 54  1204 487 (100d.)  1.9-9.5 11.6-18.5  10.5 3.5  6 (52)  487 (100d.)  16  3.5  (55)  276 (110d.)  16.5  3.5  (57)  440 (110d.)  34.6  3.5  (119)  53  Growth and mortality of cyanolichens under Populus Citation  Location  Gauslaa et al. (2009)  Picea abies forests, northern Sweden & western & southern Norway Fraxinus latifolia forest, western Oregon Pseudotsuga-Tsuga forest, western Oregon Fraxinus latifolia, western Oregon Picea abies forest, northern & southern Sweden Picea abies forest, northern Sweden  McCune & Caldwell (2009) McCune et al. (1996) Muir et al. (1997) Palmqvist & Sundberg (2000) Renhorn et al. (1997) Shirazi et al. (1996) Sillett et al. (2000) Sundberg et al. (1997) This study  Lobaria hallii This study  Precipitation (mm) 276-440 (110d.)  Growth (%) 21.2  Time period (mo.) 3.5  Annual growth (73)  1080  14.8  12  14.8  1800  13-41  9-12  13-41  1000 650  28 -1.2 & -0.4  12 12  28 -1.2 &-0.4  600  28  16  21  Fraxinus latifolia, western Oregon Pseudotsuga-Tsuga forest, western Oregon Picea abies forest, northern Sweden Populus, British Columbia Picea, British Columbia Pseudotsuga, British Columbia  1080 1000  24.5 15.2  4 12  (74) 15.2  600 897-964 897-964 897-964  2.9 42.5 32.3 21.9  16 12 12 12  2 42.5 32.3 21.9  Populus, British Columbia Picea, British Columbia Pseudotsuga, British Columbia  897-964 897-964 897-964  39.4 17.3 4.3  12 12 12  39.4 17.3 4.3  L. retigera Stevenson & Coxson (2008)  Thuja-Tsuga forest, British Columbia  840  4.5-10.5  12  4.5-10.5  L. scrobiculata Hilmo (2002)  Picea abies forest, western Norway  788  19  12  19  Nothofagus forest, Pategonia, Argentina  3000  12.6  12  12.6  Picea abies forest, western Norway  440 (110d.)  35.7  3.5  (122)  Pseudotsuga-Tsuga forest, western Oregon Pseudotsuga-Tsuga forest, western Oregon Pseudotsuga-Tsuga forest, western Oregon  1800  4-8  12  4-8  1000  14  12  14  2000  5.7  12  5.7  Pseudocyphellaria berberina Caldiz (2004) P. crocata Gauslaa et al. (2007) P. rainierensis McCune et al. (1996) Sillett & McCune (1998) Sillett (1994)  54  Growth and mortality of cyanolichens under Populus  Lobaria pulmonaria generally grew faster than L. hallii beneath Picea. Stevenson and Coxson (2008) similarly reported slower growth rates in the bipartite cyanolichen species L. retigera compared to L. pulmonaria in the same environment. The faster growth of the tripartite cyanolichen may be attributed to the supplemental photosynthetic activity of the green-algal biont. That the growth rate of both L. pulmonaria and L. hallii beneath Populus was considerably higher than most published accounts may account for the disproportionately high cyanolichen abundance observed under Populus at each of the three site-types (Campbell et al. 2010). The decrease in size, and high mortality rates, of small cyanolichen thalli beneath conifers may also provide insight into cyanolichen abundance patterns in these sub-boreal forests. Denison (1988) attributed a decrease in the mass of lichen transplants to early stages of thallus necrosis and death and indeed, a large proportion of small thalli under Picea and Pseudotsuga died during the 27-month experiment. The mortality rates beneath conifers here substantially exceed mortality in other transplant experiments involving large thalli; Sillett et al. (2000) recorded 26% and 42% mortality of Lobaria oregana and Psuedocyphellaria rainierensis, respectively. This suggests that small thalli are disproportionately sensitive. Indeed, Scheidegger (1995) observed that 90% of diaspores died within the first 20 months and concluded that L. pulmonaria distribution was limited by establishment and early thallus growth. The small thalli in this experiment were initially >5 mm in diameter and therefore likely to be at least 12 months old at the time of transplant (Scheidegger 1995). Nevertheless, the high mortality rate of these thalli supports Scheidegger’s conclusions, and may explain the near absence of small cyanolichen thalli beneath conifers at the Aleza sites (Campbell et al. 2010). More generally,  55  Growth and mortality of cyanolichens under Populus high mortality of lichen thalli during the establishment and juvenile growth phases may be a key factor limiting cyanolichen populations under conifers in sub-boreal forests. Sillett et al. (2000) showed that cyanolichens will survive and grow in unsuitable climates but it is important to note that the L. pulmonaria transplant samples in that study were ‘adult’ thalli, having mean dry masses of 0.123g. As shown here, large thalli will survive and grow when transplanted, even to unfavourable environmental conditions. In contrast, as demonstrated here, and by Scheidegger (1995), thallus mortality is frequent in small thalli. The greater surface area decreases the water-holding capacity of small thalli which may shorten the duration of physiological activity following each wetting event in young thalli compared to older thalli (Gauslaa and Solhaug 1998). Such shorter hydration events may prevent photosynthetic carbon gains from overcoming respiratory carbon losses. Young thalli may therefore be more susceptible to sub-optimal moisture (Gauslaa and Solhaug 1998) and light (Coxson and Stevenson 2007b) conditions. In this study, this is most evident at the drier Aleza sites where large thalli beneath conifers survived the experiment while 80-100% of small thalli died. Moisture-related thallus mortality may be an important reason why L. pulmonaria and bipartite cyanolichens were 12 and 171 times less abundant, respectively, beneath Picea at Aleza than at Fraser sites (Campbell et al. 2010). Bipartite cyanolichens may be particularly susceptible to moisture-related mortality as net photosynthesis is only achieved in these species at high thallus hydration. In contrast, tripartite species such as Lobaria pulmonaria can depend on photosynthetic output from the green-algal biont which can rehydrate from water vapour alone and remain active at lower thallus moisture (Lange et al. 1986). The higher rates of thallus mortality in L. hallii compared to L. pulmonaria, and the more limited distribution of bipartite cyanolichens across the sub-boreal spruce landscape (Radies et al. 2009; Campbell et al. 2010)  56  Growth and mortality of cyanolichens under Populus suggests that these species are less able to survive the phases of establishment and juvenile growth. 3.4.2 Facilitation The rapid cyanolichen growth rates beneath Populus suggest that Populus may have a facilitative influence on cyanolichen communities. Indeed, Populus may act as a nurse-species, promoting cyanolichen establishment and growth by either directly or indirectly controlling the availability of some unknown resource. Disparate cyanolichen growth rates have been attributed to the availability of exogenous nutrients. McCune and Caldwell (2009) demonstrated faster cyanolichen growth rates with a treatment of phosphorus and Gauslaa et al. (2006) saw increased growth rates with nitrogen additions. However, Campbell et al. (2010) examined the chemical composition of precipitation throughfall between Populus and Picea at these sites and concluded that mineral nutrient availability explained little of the variation in lichen community structure. Although the specific resource remains unknown, some insight may be gleaned from a qualitative comparison between treatments here. Small cyanolichen transplants beneath conifers died or decreased in size by month 12 at the moisture-deficient (Aleza) and light-deficient (Herrick) sites. Both moisture- and light-limitations have previously been demonstrated to limit photosynthetic activity in cyanolichens (Lange et al. 1986; Palmqvist and Sundberg 2000). That transplants beneath Populus at these sites continued to increase in size throughout the experiment, despite such limitations, suggests that some factor within the drip-zone of a Populus tree compensates for the more limited photosynthesis in these transplanted thalli. 3.5 References  Antoine, M.E., and McCune, B. 2004. Contrasting fundamental and realized ecological niches with epiphytic lichen transplants in an old-growth Pseudotsuga forest. Bryologist 107: 163-173. Asplund, J., and Gauslaa, Y. 2008. Mollusc grazing limits growth and early development of the old forest lichen Lobaria pulmonaria in broadleaved deciduous forests. Oecologia 155: 93-99.  57  Growth and mortality of cyanolichens under Populus Caldiz, M.S. 2004. Seasonal growth pattern in the lichen Pseudocyphellaria berberina in northwestern Patagonia. Lichenologist 36: 435-444. Campbell, J., Bradfield, G.E., Prescott, C.E., and Fredeen, A.L. 2010. The influence of overstorey Populus on epiphytic lichens in sub-boreal spruce forests of British Columbia. Canadian Journal of Forest Research 40:143-152. Coxson, D.S., and Stevenson, S.K. 2007a. Influence of high-contrast and low-contrast forest edges on growth rates of Lobaria pulmonaria in the inland rainforest, British Columbia. Forest Ecology and Management 253: 103-111. Coxson, D.S., and Stevenson, S.K. 2007b. Growth rate responses of Lobaria pulmonaria to canopy structure in even-aged and old-growth cedar-hemlock forests of central-interior British Columbia, Canada. Forest Ecology and Management 242: 5-16. Denison, W.C. 1988. Culturing the lichens Lobaria oregana and L. pulmonaria on nylon monofilament. Mycologia 80: 811-814. Gaio-Oliveira, G., Dahlman, L., Palmqvist, K., and Maguas, C. 2004. Growth in relation to microclimatic conditions and physiological characteristics of four Lobaria pulmonaria populations in two contrasting habitats. Ecography 27: 13-28. Gauslaa, Y. 1995. The Lobarion, an epiphytic community of ancient forests threatened by acid rain. Lichenologist 27: 59-76. Gauslaa, Y., and Solhaug, K.A. 1998. The significance of thallus size for the water economy of the cyanobacterial old-forest lichen Degalia plumbea. Oecologia 116: 76-84. Gauslaa, Y. 2006. Trade-off between reproduction and growth in the foliose old forest lichen Lobaria pulmonaria. Basic and Applied Ecology 7: 455-460. Gauslaa, Y., Lie, M., Solhaug, K.A., and Ohlson, M. 2006. Growth and ecophysiological acclimation of the foliose lichen Lobaria pulmonaria in forests with contrasting light climates. Oecologia 147: 406-416. Gauslaa, Y., Palmqvist, K., Solhaug, K.A., Holien, H., Hilmo, O., Nybakken, L., Myhre, L.C., and Ohlson, M. 2007. Growth of epiphytic old forest lichens across climatic and successional gradients. Canadian Journal of Forest Research 37: 1832-1845. Gauslaa, Y., Palmqvist, K., Solhaug, K.A., Hilmo, O., Holien, H., Nybakken, L. and Ohlson, M. 2009. Size-dependent growth of two old-growth associated macrolichen species. New Phytologist 181:683-692. Goward, T., and Arsenault, A. 2000. Cyanolichen distribution in young unmanaged forests: A dripzone effect? Bryologist 103: 28-37. Hedenås, H., and Ericson, L. 2000. Epiphytic macrolichens as conservation indicators: successional sequence in Populus tremula stands. Biological Conservation 93: 43-53. Hilmo, O. 2002. Growth and morphological response of old-forest lichens transplanted into a young and an old Picea abies forest. Ecography 25: 329-335. Kuusinen, M. 1996. Epiphyte flora and diversity on basal trunks of six old-growth forest tree species in southern and middle boreal Finland. Lichenologist 28: 443-463.  58  Growth and mortality of cyanolichens under Populus Lange, O.L., Kilian, E., and Ziegler, H. 1986. Water vapor uptake and photosynthesis of lichens: performance differences in species with green and blue-green algae as phycobionts. Oecologia 71: 104-110.  Meidinger, D. and Pojar, J. 1991. Ecosystems of British Columbia. Ministry of Forests, Research Branch, Victoria, B.C. McCune, B., Derr, C.C., Muir, P.S., Shirazi, A.M., Sillett, S., and Daly, W.J. 1996. Lichen pendants for transplant and growth experiments. Lichenologist 28: 161-169. McCune, B., and Caldwell, B. 2009. A single phosphorus treatment doubles growth of cyanobacterial lichen transplants. Ecology 90: 567-570. Muir, P.S., Shirazi, A.M., and Patrie, J. 1997. Seasonal growth dynamics in the lichen Lobaria pulmonaria. Bryologist 100: 458-464. Ockinger, E., Niklasson, M., and Nlisson, S.G. 2005. Is local distribution of the epiphytic lichen Lobaria pulmonaria limited by dispersal capacity or habitat quality? Biodiversity and Conservation 14: 759-773. Palmqvist, K., and Sundberg, B. 2000. Light use efficiency of dry matter gain in five macrolichens: relative impact of microclimate conditions and species-specific traits. Plant, Cell and Environment 23: 1-14. Radies, D.N., Coxson, D.S., Johnson, C., and Konwicki, K. 2009. Predicting canopy macrolichen diversity and abundance within old-growth inland temperate rainforests. Forest Ecology and Management 259:86-97. Renhorn, K.E., and Esseen, P.-A. 1997. Growth and vitality of epiphytic lichens. I. Responses to microclimate along a forest edge-interior gradient. Oecologia 109: 1-9. Scheidegger, C. 1995. Early development of transplanted isidioid soredia of Lobaria pulmonaria in an endangered population. Lichenologist 27: 361-374. Shirazi, A.M., Muir, P.S., and McCune, B. 1996. Environmental factors influencing the distribution of the lichens Lobaria oregana and L. pulmonaria. Bryologist 99: 12-18. Sillett, S.C. 1994. Growth rates of two epiphytic cyanolichen species at the edge and in the interior of a 700- year- old Douglas-Fir forest in the Western Cascades of Oregon. Bryologist 97: 321324. Sillett, S.C., and McCune, B. 1998. Survival and growth of cyanolichen transplants in Douglas-fir forest canopies. Bryologist 101: 20-31. Sillett, S.C., McCune, B., Peck, J.E., Rambo, T.R., and Ruchty, A. 2000. Dispersal limitations of epiphytic lichens result in species dependent on old-growth forests. Ecological Applications 10: 789-799. Stevenson, S.K., and Coxson, D.S. 2008. Growth responses of Lobaria retigera to forest edge and canopy structure in the inland temperate rainforest, British Columbia. Forest Ecology and Management 256: 618-623. Sundberg, B., Palmqvist, K., Esseen, P.-A., and Renhorn, K.-E. 1997. Growth and vitality of epiphytic lichens II. Modelling of carbon gain using field and laboratory data. Oecologia 109:10-18.  59  The influence of exogenous glucose on epiphytic cyanolichens  CHAPTER 4  The influence of exogenous glucose on epiphytic cyanolichens3  4.1 Introduction  Lichens are formed by a relationship between a fungus and either a cyanobacterium (cyanolichens), green alga (chlorolichens), or both (tripartite cyanolichens). The prevailing moisture conditions under which certain lichen species are found is determined, in part, by the photosynthetic partner. Chlorolichens require only 50-70% thallus hydration (Lange et al. 1986; Lange et al. 2001) while cyanolichens require up to 150% thallus hydration and contact with liquid water for physiological activity (Lange et al. 1986). Drier forest ecosystems in sub-boreal British Columbia are characterized by chlorolichen communities (Lehmkulh 2004) while cyanolichens are most abundant in wet forest ecosystems (Sillett and Neitlich 1996; Goward and Spribille 2005). A comparison of epiphytic lichens on conifer saplings beneath five mature tree species in sub-boreal spruce forests revealed that cyanolichens are uniformly abundant throughout some wet mixed-conifer forests but are restricted to saplings beneath the drip-zone of Populus trees in drier forest types (Campbell et al. 2010). Such patterns suggest that Populus trees provide some factor that compensates for inadequate moisture. Abundant cyanolichen communities have previously been attributed to available calcium (Goward and Arsenault 2000), manganese (Hauck 2003), molybdenum (Horstmann et al. 1982) and phosphorus (Benner and Vitousek 2007). However, analysis of throughfall precipitation beneath different tree species suggests that mineral nutrients are not responsible for the patterns observed in the sub-boreal forests (Campbell et al. 2010). A plausible alternative is found by examination of the chemical secretions of Populus trees themselves. Leaves of many Populus species have large extrafloral nectaries (EFN) at the 3  A version of this chapter will be submitted for publication as Campbell, J., Bengtson, P., Fredeen, A.L. and Prescott, C.E. Unfaithful fungi: the influence of exogenous-glucose in supporting cyanolichen communities beneath overstorey Populus.  60  The influence of exogenous glucose on epiphytic cyanolichens junction of the petiole and leaf blade (Trelease 1881; Curtis and Lersten 1974). Nectar is secreted from Populus EFNs regardless of tree size, including from leaves in the crowns of mature trees (Curtis and Lersten 1978). Of the 93 families that develop foliar EFNs (Pemberton 1998), only Salicaceae, containing the genera Salix and Populus, is present in sub-boreal forests of British Columbia (Elias 1983). Both Salix and Populus are observed to support abundant cyanolichen communities despite the rarity or absence of cyanolichens from surrounding conifer forests (Kuusinen 1994; Hedenås and Ericson 2000). Although there are no published accounts of the carbohydrate concentrations of Populus extrafloral nectar, extrafloral nectar from a phylogenetically related species, Prockia crucis, contains up to 49.6% sugar, 16.2% of which is glucose (Thadeo et al. 2008). This concentrated nectar is secreted onto the leaf surface where it dries to an extremely viscous film during dry atmospheric conditions. EFNs and the associated glucose-rich secretions are theorized to be a reward for insects and thus provide a protective function to the plant (Thadeo et al. 2008). However, the accumulation of nectar on the leaf surfaces will also wash off during subsequent rain events and thus may drip onto epiphytic cyanolichens on lower branches. Whether one ascribes to a mutualistic or a more parasitic paradigm (see Richardson 1999), lichens are a symbiotic relationship. Under appropriate climatic conditions, this relationship provides a somewhat reliable source of fixed C for fungal metabolism in return for a conduit for mineral nutrients to, and a protective shell of hyphae for, the photosynthetic partner (Honegger 1985). The widespread success of this arrangement implies that these are reasonable trade-offs for the fungus, particularly when no other photosynthate source is readily available. However, lichenized fungi may retain the opportunistic approach to C acquisition characteristic of their free-living counterparts. Indeed lichen-forming fungi have been shown to preferentially  61  The influence of exogenous glucose on epiphytic cyanolichens metabolize exogenous glucose over glucose from the photobiont under laboratory conditions (Drew and Smith 1967). Exogenous glucose has also been shown to promote nitrogen-fixation by epiphyllic cyanobacteria (Bentley 1987). Cyanolichens on the Populus trunks and on conifer saplings beneath mature Populus canopies may intercept and metabolize the glucose-rich extrafloral nectar, thereby acquiring an exogenous source of reduced C despite long periods of drought-induced inactivity of photobiont photosynthesis. To our knowledge, the only record of C concentration in throughfall precipitation beneath a Populus canopy showed saccharide concentrations of 9-24 mg L-1 beneath mature Populus balsamifera (Sanborn and Pawluk 1983). However, these values represent an average concentration across multiple rain events over two growing seasons and are not necessarily representative of the glucose concentrations on which cyanolichen communities might depend. Several authors have demonstrated that C concentration in throughfall precipitation beneath broadleaf trees fluctuates dramatically throughout the growing season (Carlisle et al. 1966; DeBoois and Jansen 1976). Furthermore, rainfall following a dry period of some duration will both wash the existing accumulation of nectar from the leaf surface, as well as stimulate further release from EFN (Trelease 1881). Both factors may result in glucose concentrations that are orders of magnitude higher than reported averages. Relatively dry conditions will both reduce photosynthetic activity of cyanobacteria (Lange et al. 1986) and apparently increase the availability of glucose from EFN (Trelease 1881). Taken together, these factors may favour alternative nutritional strategies to meet the metabolic requirements of cyanolichens under less suitable environmental conditions. In this paper, we explore whether the proliferation of cyanolichens beneath Populus is a consequence of facilitation by the exogenous source of labile C provided by poplar EFNs. We experimentally  62  The influence of exogenous glucose on epiphytic cyanolichens test three hypotheses which, if true, provide supporting evidence for the hypothesized maintenance of cyanolichen communities by exogenous-glucose. These include: a) net photosynthesis will decrease and nitrogen fixation will increase in cyanolichen thalli with the addition of exogenous glucose, b) exogenous glucose will be readily taken up and assimilated into lichen fatty-acids, and c) the rate of cyanolichen establishment will be significantly enhanced with the provision of exogenous glucose. 4.2 Methods and materials  4.2.1 Physiological response to 13C6 -glucose Healthy and intact specimens of four cyanolichens species were collected from Picea glauca (Moench) Voss x engelmannii Parry ex Engelmann and Abies lasiocarpa (Hook.) Nutt. branches in a single old (mean tree age >240 years) mixed-conifer forest in September 2008. Lichens growing on branches within 5 m of a Populus tree were avoided. Samples included; two stratified bipartite cyanolichen species (Nephroma helveticum Ach. and Lobaria hallii (Tuck.) Zahlbr.), where the cyanobacteria are located in a discrete layer beneath the thallus surface; one stratified tripartite cyanolichen (Lobaria pulmonaria (L.) Hoffm.) where the thallus consists of a fungal and an algal photobiont (photosynthetic partner) and the cyanobacteria are in discrete pockets embedded at the surface of the fungal matrix; and one homoeomerous (gel) cyanolichen species (Leptogium saturninum Dickson) Nyl.) where the cyanobacteria are located throughout the lichen thallus. Nostoc spp. was the cyanobacterial partner in all lichens species (Brodo et al. 2001). Samples were maintained in an open container on a 12-hour light: 12-hour dark day: night cycle with day temperatures of 12oC (6am to 6pm) at 200 µmol m-2 s-1 of photosynthetically active radiation (PAR) and night temperatures of 8oC. Relative humidity remained at approximately 60-70% throughout and thalli were periodically re-wetted (approximately every 3  63  The influence of exogenous glucose on epiphytic cyanolichens days) with a de-ionized water spray to prevent desiccation. Lichen thalli were stored under these conditions for a 10-day acclimatization period. Thalli were moved to a sealed container 24 hours prior to the experiment and were pre-treated with either 10 ml of deionized water (control) or 10 ml of 2% glucose (13C6-glucose for C fixation). Thalli were pre-incubated at 19 oC and 200 µmol m-2 s-1 of light for the 3 hours immediately preceding experiments. Net photosynthesis and respiration Cyanolichen thalli were treated with either water or 13C6-glucose to determine the photosynthetic response to exogenous glucose. Glucose was used because it is the principal form in which fixed C moves between symbionts (Drew and Smith 1967) and because it is absorbed preferentially over other saccharides (Harley and Smith 1956). A glucose concentration of 2% was used to ensure carbohydrate-saturation of the lichen thallus (Harley and Smith 1956) and to ensure a measurable physiological response over the measurement period. A second 10-ml application of either water or 2% 13C6-glucose was applied less than a minute prior to CO2-flux measurements. Thalli were spot-dried and sealed inside a 2 x 3-cm LiCOR 6400 – 02B LED gas-exchange chamber in concert with the Li-6400 gas-exchange system (LiCOR Inc., Lincoln, NE.). Photosynthetically active radiation (PAR) was manipulated through sequential 5 - 10-min equilibration intervals at 400, 200, 100, 50, 25, and 10 µmol PAR m-2 s-1. Net photosynthesis was measured at 20 oC and an external CO2 concentration of 400 µmol mol-1. Differences in net photosynthesis between control and glucose-treated thalli were evaluated using a one-way t-test at each of the six levels of photosynthetically active radiation (PAR). Net photosynthesis differences between lichen species were evaluated with a one way ANOVA (Statistica v 6.1, StatSoft Inc. Tulsa, OK).  64  The influence of exogenous glucose on epiphytic cyanolichens Nitrogen-fixation Nitrogen fixation of lichen thalli was measured using the acetylene reduction assay (ARA) method according to Stewart et al. (1967). Following pre-treatment as above, five additional transplants of each lichen species were treated with water or 2% glucose (n=6 per species per treatment) and placed in 250-ml canning jars modified by inserting rubber septa into the lid and sealing with silicone sealant (GE Sealants and Adhesives, Huntersville, NC). Air in the jar was replaced with acetylene gas (10% v/v). Gas samples (0.7 cc) were taken after a 3hour incubation at 20oC and 200 µmol PAR m-2s-1 and immediately analyzed for ethylene with a gas chromatograph (SRI 8610A Wennick Scientific Corporation, Ottawa, ON.) fitted with a Porapak Column (Alltech Canada, Guelph, ON.) and a flame ionization detector. Hydrogen was used as the carrier gas (pressure 179 kPa) and the column was maintained at 68oC. Mass was recorded for each transplant after drying for 48 hr at 105oC. Differences in ARA rates between lichen species and between control and glucose-treated samples were detected with a factorial ANOVA with a Fishers LSD post hoc test. 4.2.2 Fatty-acid extraction and analysis Assimilation of 13C into lichen tissue was assessed by extracting fatty acids from the same lichen samples for which the photosynthetic responses to exogenous glucose were recorded. Samples were removed from the Li-6400 chamber, weighed, flash-frozen in liquid nitrogen to prevent further respiration, and freeze dried for 24 hours. Freeze-dried lichen samples were ground to a fine powder with a grinding mill (Retsch MM200 stainless steel mixer mill, Sigma Aldrich, St. Louis, MO). Phospho- and neutral-lipids were extracted from the 13Cglucose-treated lichens using techniques proposed by Bligh and Dyer (1959) and described by Olsson et al. (1995). Briefly, 100-200-mg samples were vortex mixed in a 0.8:1:2 (v/v/v) solution of citrate buffer, chloroform and methanol. Extracted acids were eluted with chloroform  65  The influence of exogenous glucose on epiphytic cyanolichens and methanol through pre-packed Accubond II Solid Phase Extraction silica columns (Agilent Technologies Inc., Santa Clara, CA) to fractionate into neutral-lipid fatty acids (NLFA) and phospholipid fatty acids (PLFA), respectively. Following alkaline methanolysis, fatty-acid residues were flash-evaporated under N2-gas and stored in 200 µl hexane at -20oC until analysis. The PLFAs and NLFAs were separated with an Agilent 6890 N gas chromatograph and an Agilent 5975 Inert XL Mass Selective Detector (Agilent Technologies Inc., Santa Clara, CA) with a 30-m J&W HP-5 column as described by Bengston et al. (2009). Specific fatty acids were identified using a combination of mass spectra and retention times relative to an internal standard (19:0) and authentic standards. The 13C concentration in the individual fatty acids was determined on an IsoprimeTM stable-isotope ratio mass spectrometer (GV instruments) connected to an Agilent 6890A gas chromatograph (Agilent Technologies Inc.) under conditions described by Bengston et al. (2009). The µg 13C g-1 of lichen sample and the %13C enrichment were determined for each 13C-labelled fatty acid. PLFA and NLFA nomenclature identifies the number of C atoms in the fatty acid chain (e.g. 18 in 18:1ω7), the number of double bonds in the chain (e.g. 1) and the position of the first double-bonded C from the methyl end of the fatty-acid molecule (e.g. ω7). Fatty-acid common names, where available, follow Robinson (1982). 4.2.3 Establishment response to glucose To evaluate whether an exogenous source of labile C would enhance establishment of cyanolichen thalli, Picea branches were inoculated with propagules of four cyanolichen species; Lobaria pulmonaria, L. hallii, Nephroma helveticum and Leptogium saturninum. Following Benner and Vitousek (2007), propagules were obtained by grinding air-dried samples of mature thalli to a fine powder with a grinding mill (as above). Picea branches were scrubbed with a wire brush and rinsed with 0.05% HCl and deionized water to eliminate any existing lichen propagules. Five sets of two branches were installed in a 100 x 100-m area approximately 2 km 66  The influence of exogenous glucose on epiphytic cyanolichens west of the University of Northern British Columbia (UTM: 10U 510330, E5971738) in May 2008. The site neither contained cyanolichens nor was it generally permissive to their occurrence. Mean annual precipitation for the experimental area is 600 mm based on monthly averages for 1961-2000 from Meteorological Services of Canada, Environment Canada. Each set included one control branch and one glucose-treated branch that were sufficiently spaced to prevent glucose-contamination of controls. Branches were hung between mature, live Pinus contorta trees in the mixed conifer forest. Deionized water was sprayed along a 100-cm segment of each branch to increase propagule retention and approximately 1.5 g of each lichen species was evenly sprinkled over the upper surface. Branches were sprayed once per week with either 30 ml of deionized water (control) or 30 ml of 2% glucose solution (in de-ionized water) from May 5 to Aug 30, 2008 and removed from the field on May 4, 2009. The total number of small (<1 mm long) cyanolichen thalli were counted and identified to species (when possible) by observing each branch through a dissecting microscope. Differences in the number of established thalli between treatment and control were evaluated with a two-sample t-test assuming unequal variances. 4.3 Results  4.3.1 Physiological response to 13C6-glucose Net photosynthesis and respiration Lobaria pulmonaria, Leptogium saturninum and Nephroma helveticum thalli treated with 13  C-glucose had significantly lower rates of net photosynthesis than control thalli (Fig. 4.1; Table  4.1). Furthermore, there was a greater difference in net photosynthesis between control and glucose-treated thalli at 400 µmol PAR m-2 s-1 than at 10 µmol PAR m-2 s-1 (Fig. 4.1). Net photosynthesis in the 13C6-glucose treated thalli was below the light compensation point at all  67  The influence of exogenous glucose on epiphytic cyanolichens light levels in the bipartite stratified cyanolichens (Nephroma helveticum and Lobaria hallii) and at light levels below 200 µmol PAR m-2 s-1 in Leptogium saturninum and Lobaria pulmonaria. Net photosynthetic responses of control thalli were only below the light compensation point when light levels dropped below 50 µmol PAR m-2 s-1. There were no statistical differences in photosynthetic light responses between cyanolichen species (F(3, 40) = 0.84, p = 0.48).  Net photosynthesis -2 -1 μmol CO2m s  4  A. Lobaria hallii  B. Lobaria pulmonaria  C. Leptogium saturninum  D. Nephroma helveticum  2  0  -2  -4  -6  4  *  Net photosynthesis -2 -1 μmol CO2m s  * 2  *  *  *  * *  0  *  -2  -4  Control 2% 13C6-glucose solution  -6  0  100  200  300  400  0  100  200  300  400  Photosynthetically active radiation (μmol m-2 s-1)  Figure 4.1 Net photosynthesis of water- and 2% 13C6-glucose-treated a) Lobaria hallii b) Lobaria pulmonaria c) Leptogium saturninum and d) Nephroma helveticum. Error bars represent standard deviation and stars represent significant differences between treatments at individual light levels for individual species (N=6).  68  The influence of exogenous glucose on epiphytic cyanolichens Table 4.1 One-tailed t-test statistics between water- and 13C6-glucose-treated cyanolichens at six levels of photosynthetically active radiation (PAR). Species  PAR  Lobaria hallii  400  1.87  -0.32  0.09  200  1.47  -0.77  100  0.90  50  Lobaria pulmonaria  Leptogium saturninum  Nephroma helveticum  Mean Control Glucose  Variance Control Glucose  df  t  p  8.79  4  1.65  0.09  0.10  7.96  4  1.76  0.08  -1.40  0.14  7.44  4  1.86  0.07  -0.05  -2.15  0.22  6.98  4  1.75  0.08  25  -0.96  -2.62  0.39  6.67  4  1.40  0.12  10  -2.10  -3.11  0.45  6.17  4  0.88  0.21  400  0.42  0.06  0.26  0.14  5  1.42  0.09  200  0.50  -0.01  0.24  0.10  5  2.10  0.03  100  0.34  -0.21  0.18  0.10  5  2.55  0.02  50  -0.23  -0.66  0.10  0.19  5  2.01  0.04  25  -0.73  -1.15  0.03  0.31  5  1.73  0.07  10  -1.28  -1.62  0.02  0.44  5  1.23  0.14  400  0.90  0.29  0.28  0.11  5  2.40  0.02  200  0.88  0.11  0.32  0.11  5  2.85  0.01  100  0.57  -0.08  0.25  0.14  5  2.58  0.01  50  0.13  -0.35  0.31  0.13  5  1.76  0.06  25  -0.39  -0.67  0.22  0.13  5  1.13  0.14  10  -1.17  -1.06  0.25  0.12  5  -0.43  0.34  400  2.85  -0.34  1.30  1.11  5  4.61  <0.0001  200  2.16  -1.06  0.46  1.89  5  4.69  0.002  100  0.87  -2.00  0.14  2.65  5  3.84  0.01  50  -0.33  -2.79  0.07  3.44  5  2.94  0.02  25  -1.18  -3.14  0.09  3.59  5  2.29  0.04  10  -1.77  -3.54  0.11  4.03  5  1.94  0.06  69  The influence of exogenous glucose on epiphytic cyanolichens Nitrogen-fixation Nitrogen-fixation differed between lichen species. The mean (±SD) ARA rate in Nephroma helveticum (2.10±0.90 μmol C2H4 g-1 hr-1) was significantly higher than in Lobaria hallii (1.57±0.44 μmol C2H4 g-1 hr-1) and Leptogium saturninum (1.14±0.66 μmol C2H4 g-1 hr-1; F(3, 32 = 3.61, p = 0.02). Acetylene reduction by the tripartite species Lobaria pulmonaria (1.67±0.44 μmol C2H4 g-1 hr-1) did not differ from any other cyanolichen species and there was no difference in ARA between the control and glucose-treated thalli for any cyanolichen species (F(1,32) = 1.85, p=0.7; data not shown). 4.3.2 Fatty-acid analysis The composition of phospholipids (PLFA) and neutral lipids (NLFA) varied among cyanolichen species, but the dominant fatty acids in all cyanolichen species were 16:0, 18:1ω9 and 18:2ω6,9 (Table 4.2). Each of the fatty acids detected showed 13C-enrichment with the exception of 18:3ω3 which was only 13C-enriched in Nephroma helveticum and Leptogium saturninum PLFAs (Fig. 4.2a). This fatty acid was also a minor constituent of the NLFAs in Lobaria hallii, L. pulmonaria and Nephroma helveticum (0.03%, 0.1% and 1.58%, respectively; Table 4.2) but was only 13C-enriched in the latter species (Fig. 4.2c). Of the analyzed PLFAs, 20:0 and 20:3 were the most enriched with 13C in all lichen species (Fig. 4.2a). The highly 13Cenriched NLFAs included 26:0 in Lobaria pulmonaria and Lobaria hallii¸ 16:1ω9, 18:1ω9, 18:0 and 20:0 in Leptogium saturninum and 18:0 and 20:0 in Nephroma helveticum (Fig. 4.2c). The largest proportion of total 13C-uptake was assimilated into the PLFAs and NLFAs 18:1ω9 and 18:2ω6,9 in all four cyanolichen species (Fig. 4.2b and d). The amount of 13C that was applied to fatty-acid synthesis by Lobaria pulmonaria and Nephroma helveticum was nearly double that of L. hallii and Leptogium saturninum (Table 4.3). The three bipartite cyanolichens invested more of the assimilated 13C into structural  70  The influence of exogenous glucose on epiphytic cyanolichens (phospholipids) as opposed to storage (neutral) lipids with 68%, 74% and 56% of total assimilated-13C detected in PLFAs in Lobaria hallii, Leptogium saturninum and Nephroma helveticum, respectively. By contrast, the assimilation of 13C in Lobaria pulmonaria was similar between the two lipid types. The ratios of 13C assimilated into PLFA and NLFA and of total PLFA-C to NLFA-C reflect these disparate investments (Table 4.3).  71  The influence of exogenous glucose on epiphytic cyanolichens Table 4.2 Relative abundance (%) of phospholipid fatty acids (PLFA) and neutral lipid fatty acids (NLFA) in four 13C6-glucose treated cyanolichen species. ND- not detected (N=6). Lobaria hallii PLFA  NLFA  Lobaria pulmonaria PLFA NLFA  14:0  0.05  0.69  0.42  0.44  0.16  0.08  0.15  0.04  16:0  21.51  13.48  18.75  12.73  19.61  10.99  20.26  8.89  16:1ω5  0.18  0  0.12  0  0.75  0  0.30  0  16:1ω7  3.46  0  0.01  0  0  0  0  0  16:1ω9  4.81  0.07  0.82  0.66  8.77  0.47  7.00  1.00  17:0  0.72  0.15  0.60  0.16  0.60  0.08  0.48  0.05  18:0  3.88  5.34  3.41  3.29  2.17  6.90  4.99  3.48  18:1ω7  6.64  0  3.03  0  6.77  0  6.24  0  18:1ω9  20.73  36.05  18.61  33.02  18.61  22.19  11.36  9.21  18:2ω6,9 34.38  41.20  51.17  47.82  31.81  53.44  42.57  70.87  18:3ω3  0.01  0.10  0.02  0.03  0.03  0.18  1.78  1.58  20:0  0.08  0.10  0.18  0.12  0.21  0.13  0.29  0.11  20:2  0.26  0  0.19  0  0.79  0  0.24  0  20:3  0.50  0  0.24  0  0.52  0  0.15  0  21:0  0.52  0.47  0.43  0.09  2.02  2.57  0.30  0.08  22:0  1.19  1.14  0.95  0.37  2.40  1.31  1.55  0.83  23:0  0.58  0.17  0.54  0.10  2.42  0.12  0.98  0.09  24:0  0.50  0.43  0.47  0.14  2.37  0.77  1.36  0.78  26:0  0.59  1.05  Leptogium saturninum PLFA NLFA  Nephroma helveticum PLFA NLFA  0.77  2.99  72  %13C enrichment  The influence of exogenous glucose on epiphytic cyanolichens  50  a  c  b  d Lobaria pulmonaria Lobaria hallii Nephroma helveticum Leptogium saturninum  40 30 20 10  13  % of total C enrichment  0 50 40 30 20 10  Bacterial  Fungal  14 16 0 -1w 16 5 -1w 7 16 -:1 w9 16 -0 17 -0 18 18 0 -1w 18 7 -1 18 w9 -2w 6, 18 9 -3w 3 20 -0 20 -2 20 -3 21 -0 22 -0 23 -0 24 -0 26 -0  23 -0 24 -0  21 -0 22 -0  20 -2 20 -3  17 -0 18 -0 18 -1w 18 7 -1 18 w9 -2w 6, 18 9 -3w 3 20 -0  14 -0 16 -1w 16 5 -1w 7 16 -:1 w9 16 -0  0  Nostoc spp.  PLFA  NLFA  Figure 4.2 The % 13C enrichment in extracted (a) phospho-lipid fatty acids (PLFA) and (c) neutral-lipid fatty acids (NLFA) and the % of total 13C enrichment in each (b) PLFA and (d) NLFA from 13C6-glucose treated samples of Lobaria pulmonaria, Lobaria hallii, Nephroma helveticum and Leptogium saturninum. Associations of specific fatty acids with specific lichen bionts are indicated. Error bars represent standard deviation. Natural abundance of 13C (1.15%) was subtracted from values prior to analysis (N=6).  73  The influence of exogenous glucose on epiphytic cyanolichens  Table 4.3 Mean quantity (±SD) of 13C assimilated into PLFAs, NLFAs and total lichen fatty acids, the ratio of 13C assimilated into PLFA (μg 13C g sample -1) to NLFA -1  (μg 13C g sample -1), and the ratio of total PLFA-C g sample to total NLFA-C g  sample-1 in four cyanolichen species (N=6).  μg 13C in PLFA g sample -1  μg 13C in NLFA g sample -1  μg 13C total g sample -1  13  Lobaria hallii  102.5±34.2  48.6±22.4  151.1±56.0  2.2  6.0  Lobaria pulmonaria  108.6±59.4  103.2±51.9  211.9±94.8  1.1  1.4  Leptogium saturninum  86.3±.31.3  30.9±21.0  117.2±44.3  4.0  8.1  Nephroma helveticum  133.7±49.0  104.0±76.9  237.7±118.5  1.6  5.3  C in PLFA: C in NLFA  13  TOTAL PLFA:NLFA  74  The influence of exogenous glucose on epiphytic cyanolichens 4.3.3 Glucose fertilization experiment Significantly more cyanolichen thalli became established on the glucose-treated branches than on the control branches (t(4)=-2.5, p=0.019). A total of 242 cyanolichen thalli were observed on the glucose-treated branches, while only 35 cyanolichen thalli became established on the control branches (Fig. 4.3). Nephroma helveticum was the most abundant species, making up 66% and 69% of all cyanolichens on control and glucose-treated branches, respectively. Leptogium saturninum thalli were observed only on the glucose-treated branches.  Number of established small thalli  60  50  Leptogium saturninum Lobaria hallii Lobaria pulmonaria Nephroma helveticum  40  30  20  10  0 control  2% glucose  Treatment  Figure 4.3 The number of small (<1mm long) thalli observed on control and 2%glucose treated Picea branches. Establishment on glucose-treated branches was significantly higher than control (p=0.019).  75  The influence of exogenous glucose on epiphytic cyanolichens  4.4 Discussion  Success of cyanolichen establishment was substantially enhanced by the provision of exogenous glucose. Although sugars may improve the adherence of lichen diaspores to their substrate (Y. Gauslaa, Pers.comm. March 2010), the uptake and physiological response to exogenous glucose observed here suggest that the benefit of glucose is related to the ecophysiological limitations of cyanolichens. Lichens can completely desiccate during dry periods and reactivate once thallus moisture increases, but the thallus is subject to respiratory Closs during rewetting (Brown et al. 1983). The degree to which photosynthetic C-gains compensate for rewetting respiration depends on the duration of hydration; long hydration events may allow for significant gain of C while shorter events truncate gains and may even result in a net loss of C. Although the proportion of fixed C that is lost during respiration varies with lichen species and physiology (Palmqvist 2000), cyanolichens may be more susceptible to C loss because significant fungal respiration can occur under humid conditions (Lange et al. 1986) but upregulation of the photosynthetic pathways in Nostoc spp. requires contact with liquid water (Lange et al. 2004). In contrast, chlorolichens may rehydrate with water vapour alone and require only 50-70% thallus moisture for photosynthesis (Lange et al. 1986). Photosynthesis in these green-algal species may therefore quickly compensate for respiratory C loss while cyanolichens require longer wetting periods to achieve positive C gain. Establishing thalli may be particularly vulnerable to rewetting C-loss; the large surface area to volume ratio of small thalli limits the duration of thallus hydration following wetting events (Gauslaa and Solhaug 1998). The exogenous glucose provided in this experiment may provide a source of reduced C that begins to compensate for rewetting respiration in establishing cyanolichen thalli before up-regulation of photosynthesis occurs and before subsequent thallus  76  The influence of exogenous glucose on epiphytic cyanolichens dehydration. Establishing cyanolichens on glucose-treated branches may thus overcome C loss more rapidly than control thalli, thereby achieving a positive C balance. Thalli on control branches, by contrast, would experience a consistent loss of C. Cyanolichen thalli treated with 2% 13C6-glucose had lower or more negative net photosynthesis than those treated with deionized water. While the decreased net photosynthesis in glucose-treated thalli may be interpreted as an increase in respiration at low levels (no photosynthesis occurred in either treatment at 10 umol PAR m-2s-1), a proportion of the response to exogenous glucose must also be attributed to a reduction in photosynthesis. A larger difference between control and glucose-treated responses was observed at high compared to low light intensities, indicating that glucose also decreased photosynthesis in cyanolichens. This is consistent with Nátr et al. (1974) who showed a considerable reduction in photosynthesis rate in barley leaves following glucose uptake, however the specific mechanism remains unknown. The ability of the studied cyanolichen species to use exogenous glucose as a C source was confirmed by the 13C experiment. Although the total 13C incorporated into lipids varied between samples and species, almost all lichen fatty-acids were highly 13C enriched. The three bipartite cyanolichens invested more 13C into structural (PLFA) lipids compared to storage (NLFA) lipids despite the fact that neutral lipids make up the highest percentage of total lipids in lichens (Dembitsky 1992). The ratio of PLFA to the corresponding NLFA has been reported as an indicator of physiological condition of the fungus (Olsson et al. 1998) because it indicates an investment into structure rather than storage. The PLFA:NLFA ratio was relatively high for each of the bipartite cyanolichen species signifying a substantial investment into structural lipids. By contrast, Lobaria pulmonaria appeared to invest equally between storage and structural compounds. Taken together, the lower PLFA:NLFA ratio and the weaker respiratory response to  77  The influence of exogenous glucose on epiphytic cyanolichens exogenous glucose in Lobaria pulmonaria compared to other stratified species, might reflect a lesser dependence of the former on exogenous C at sub-optimal moisture conditions. Indeed, this may explain why Lobaria pulmonaria is commonly observed throughout sub-boreal spruce forests while bipartite cyanolichen species are largely restricted to regions beneath a Populus drip-zone in moisture-deficient forests (Campbell et al. 2010). The composition of fatty acids varied across the four cyanolichen species, but the primary fatty acids in each case were 16:0, 18:1ω9 and 18:2ω6,9. These results are consistent with Dembitsky (1992) who observed that 16:0 was the predominant fatty acid in many lichen species and reflects the presence of 16:0 in both fungal (Riley et al. 2000) and cyanobacterial (Gugger et al. 2002) fatty-acid extractions. That the fungal fatty acids 18:1ω9 and 18:2ω6,9 made up a large proportion of the fatty acid composition in this study is also consistent with previous work on cyanolichens (Rezanka and Dembitsky 1999) and likely reflects the greater fungal biomass in the lichen thallus (Honegger 1985). The largest proportion of 13C taken up by the cyanolichen thalli was assimilated into 18:1ω9 (oleic acid) and 18:2ω6,9 (linoleic acid). These lipids are commonly used to identify fungi in soil microbial communities (Frostegård and Bååth 1996) and have been shown to be relatively abundant in stratified lichens (Bowker et al. 2008) and in the homiomerous lichen Leptogium saturninum (Rezanka and Dembitsky 1999). The disproportionate 13C enrichment of these fatty acids suggests that the mycobiont absorbed and assimilated the majority of exogenous 13  C6-glucose. The neutral lipid fatty acids 20:0 (icosanoic acid) and 20:3 (homo-γ-linolenic acid) were  also highly enriched in most lichen species. These results are consistent with the fatty-acid composition of Peltigera spp. which had abundant long-chain neutral lipids in comparison with  78  The influence of exogenous glucose on epiphytic cyanolichens the chlorolichens investigated, many of which had no fatty acids longer than 18-carbons (Dembitsky 1992). Some enrichment in the fatty-acid 18:3ω3 (linolenic acid) was observed. Though absent from fungi, 18:3ω3 is the major fatty acid in thylakoid membranes and present in green algae and many filamentous cyanobacteria, including Nostoc spp. (Potts et al. 1987). The absence of 13  C enrichment NLFA 18:3ω3 is expected as lipids are not a primary C-storage compound in  cyanobacteria (Neidhardt et al. 1990). Enrichment of PLFA 18:3ω3 may indicate some degree of glucose uptake by the cyanobacteria. Alternatively, 13C enrichment of 18:3ω3 may have resulted from photosynthetic fixation of the 13CO2 that was respired by the mycobiont following uptake of 13C6-glucose. Exogenous glucose did not affect the rate of nitrogen-fixation in any of the four lichen species investigated. Kershaw et al. (1977) similarly showed that 2%-glucose failed to elevate N2-fixation in a bipartite cyanolichen following a drop in nitrogenase activity in the dark. The authors concluded that exogenous glucose could not replace the C contribution from light energy. These results are inconsistent with those of Bentley (1987) who demonstrated that exogenous glucose supports cyanobacterial nitrogen-fixation despite a darkness-induced reduction in photosynthate production. These disparate results may be explained by the fact that the cyanobacteria in Bentley’s study were epiphyllic and not surrounded by absorptive fungalhyphae. As demonstrated by the fatty-acid extractions here, most of the exogenous-glucose is taken up by the fungus in lichen studies, leaving comparatively little to potentially affect nitrogen-fixation in the cyanobacterial partner. 4.4.1 Conclusion Cyanolichens are strongly associated with moist environmental conditions in interior British Columbia (Goward and Spribille 2005) where drying events are infrequent and the 79  The influence of exogenous glucose on epiphytic cyanolichens duration of thallus hydration may be adequate to maintain positive C balance. Cyanolichens are comparatively rare under less suitable climatic conditions where cyanobacterial photosynthesis may be insufficient for metabolism in both symbionts. Other nutritional strategies may be necessary to maintain the symbiosis in such cases. The observation of abundant and species-rich cyanolichen communities beneath Populus in otherwise unsuitable moisture conditions (Campbell et al. 2010) suggests that Populus compensates for a drought-induced reduction in photosynthetic activity. Furthermore, experimental evidence presented here supports the hypothesis that glucose-rich nectar produced by Populus EFNs facilitates cyanolichen establishment and survival beneath Populus canopies. Our results demonstrate that fungal respiration and fatty-acid metabolism are enhanced by exogenous glucose, and suggest that lichenized fungal partners may not always be ‘faithful’ to the symbiosis. Under suboptimal moisture conditions, nutritional philandering may be necessary to maintain the relationship. 4.5 References  Bengtson, P., Basiliko, N., Dumont, M.G., Hills, M., Murrell, C.J., Roy, R. and Grayston, S.J. 2009. Links between methanotroph community composition and CH4 oxidation in a pine forest soil. FEMS Microbiology ecology 70:24-34. 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Annals of Botany 38:589-593. Neidhardt, F.C., Ingraham, J.L. and Schaechter, M. 1990. Physiology of the bacterial cell. A molecular approach. Sinauer Associates, Sunderland, Massachusetts, USA. Olsson, P.A., Bååth, E., Jakobsen, I. and Söderström, B. 1995. The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil. Mycological Research 99:623-629. Olsson, P.A., Francis, R., Read, D.J. and Söderström, B. 1998. Growth of arbuscular mycorrhizal mycelium in calcareous dune sand and its interaction with other soil microorganisms as estimated by measurement of specific fatty acids. Plant and Soil 201:9-16. Palmqvist, K. 2000. Carbon economy in lichens. New Phytologist 148:11-36. Pemberton, R.W. 1998. The occurrence and abundance of plants with extrafloral nectaries, the basis for antiherbivore defensive mutualisms along a latitudinal gradient in east Asia. Journal of Biogeography 25:661-668. Potts, M., Olie, J.J., Nickels, J.S., Parsons, J. and White, D.C. 1987. Variation in phospholipid ester-Linked fatty acids and carotenoids of desiccated Nostoc commune (cyanobacteria) from different geographic locations. Applied and Environmental Microbiology 53:4-9.  82  The influence of exogenous glucose on epiphytic cyanolichens Rezanka, T. and Dembitsky, V.M. 1999. Fatty acids of lichen species from Tian Shan Mountains. Folia Microbiologica 44:643-646. Richardson, D.H.S. 1999. War in the world of lichens : parasitism and symbiosis as exemplified by lichens and lichenicolous fungi. Mycology Research 103:641-650. Riley, M.B., Collins, I.J., Richardson, Y.T. and Stutzenberger, F.J. 2000. Extraction of fungal fatty acids for gas chromatography analysis. Mycologia 92:301-304. Robinson, P.G. 1982. Common names and abbreviated formulae for fatty acids. Journal of Lipid Research 23:1251-1253. Sanborn, P. and Pawluk, S. 1983. Process studies of a chernozemic pedon, Alberta (Canada). Geoderma 31:205-237. Sillett, S.C. and Neitlich, C. 1996. Emerging themes in epiphyte research in westside forests with special reference to cyanolichens. Northwest Science 70:54-60. Stewart, W.P.D., Fitzgerald, G.P. and Burris, R.H. 1967. In situ studies on nitrogen fixation using acetylene-reduction technique. Proceedings of the National Academy of Science U.S.A. 58:2071-2078. Thadeo, M., Cassino, M.F., Vitarelli, N.C., Azevedo, A., A., Araújo, J., M., Valente, V.M.M. and Meira, R.M.S.A. 2008. Anatomical and histochemical characterization of extrafloral nectaries of Prockia crucis (Salicaceae). American Journal of Botany 95:1515-1522. Trelease, W. 1881. The foliar nectar glands of Populus. Botanical Gazette 6:284-290.  83  Decomposition and nutrient release from four lichen litters  CHAPTER 5  Decomposition and nutrient release from four lichen litters4  5.1 Introduction  Litter decomposition provides an important source of nutrients for plant growth in forest ecosystems, e.g. 69-87% of the annual nutritional requirements of forest plants come from nutrients mineralized from decomposing litter (Waring and Schlesinger 1985). The rate at which litter is broken down and nutrients are released by microbial communities is regulated by climatic factors (i.e. Aerts 1997; Berg et al. 2000) and by the initial chemical composition of the litter (i.e. Swift et al. 1979; Taylor et al. 1989). In general, litter decomposition rates are positively correlated with increasing moisture and increasing temperature (Waksman and Gerretsen 1931; Zhang et al. 2008). In a meta-analysis of litter decomposition from 70 studies, Zhang et al. (2008) confirmed that mass loss at specific sites was best explained by a combination of climatic factors (latitude, mean annual temperature) and chemical factors. The initial chemical make-up of the litter is often well correlated with the rate of initial decomposition; litter types with high initial N concentrations tend to have higher rates of initial decomposition than those with lower initial N (Taylor et al. 1989). The rate at which nutrients are released from decomposing litter also varies among nutrients and litter types. Generally, relatively mobile nutrients such as Mg, Ca and K are rapidly released during early decay, often at a rate faster than mass loss, while other elements (notably N and P) are retained or even immobilized in litter with high initial C:N or C:P ratios (Manzoni et al. 2008). If, however, the ratio of C to N or P is low, these elements may be released from litters during early decay (Prescott 2005). The release or retention of mineral nutrients during early 4  A version of this chapter has been accepted for publication by The Canadian Journal of Forest Research as Campbell, J., Prescott, C.E. and Fredeen, A.L. Decomposition and nutrient release from four lichen litters in subboreal spruce forests.  84  Decomposition and nutrient release from four lichen litters decay results in a convergence of litter chemistry on common C:P and C:N ratios (Vesterdal 1999). Convergence values vary substantially in forest litter and tend to increase with the proportion of recalcitrant material in the litter. For example, broadleaf litters tend to converge at C:N and C:P ratios of 20-25 and 400, respectively, while needle litters converge at C:N and C:P of 28-35 and 500, respectively (Prescott 2005). Epiphytic lichens generally decay rapidly (McCune and Daly 1994; Esseen and Renhorn 1998; Holub and Lajtha 2003; Caldiz et al. 2007). This may be related to the chemical make-up of the lichen thallus. Cyanolichens (species with cyanobacterial symbionts), in particular, are able to fix atmospheric nitrogen and consequently have a very high N content (up to 3.5%, Campbell and Fredeen 2007). More than 80% of this lichen-N is in highly labile forms such as amino acids and proteins (Dahlman et al. 2003). Given that initial N-concentration and C:N ratios are a strong predictor of initial decay rates, particularly in litters with low lignin content (Taylor et al. 1989), cyanolichen litters are expected to decompose and release nutrients faster than other lichen and vascular plant litters. In many late-seral temperate forest ecosystems, N is thought generally to be a growthlimiting element. While reported rates of symbiotic-N2-fixation in young forest ecosystems range from 0.8-2 kg N ha-1 yr-1 in Lupinus arcticus and Sheperdia canadensis (Hendrickson and Burgess 1989) to 10-15 kg N ha-1 yr-1 in Alnus viridis ssp. sinuate (Sanborn et al. 2002), these species are not abundant following crown closure (Sanborn et al. 2002). In late-seral forests, N is tightly cycled (Davidson et al. 1992) and largely supplied by decomposing forest litter (Sollins et al. 1980). There are therefore limited new N inputs into older sub-boreal forest ecosystems of central British Columbia where atmospheric N inputs are as low as 0.9 kg N ha-1 yr-1 (Hope 2001). The abundance of N2-fixing cyanolichens increases with forest age (Sillett and Neitlich  85  Decomposition and nutrient release from four lichen litters 1996; Campbell and Fredeen 2004) and may therefore be a substantial source of new N to these forests. For example, Denison (1979) estimated the annual N-inputs from epiphytic cyanolichens in mature conifer forests of the Pacific Northwest to be 3-4 kg N ha-1 yr-1. The quantity of in situ cyanolichen-N has been calculated from biomass estimates in wet-temperate interior forests of B.C. (Campbell and Fredeen 2007), but this provides only limited insight into the role of these lichens in N-cycling. There are no estimates of lichen-litter contributions to forest nutrient cycling in sub-boreal spruce ecosystems. In this study we compare mass loss rates and nutrient release dynamics of four lichen species (two cyanolichens and two chlorolichens) with different N concentrations in three subboreal spruce forests. We hypothesize that cyanolichens will decompose faster and lose N and P faster than chlorolichens, resulting in a convergence of N and P concentrations in the four litters as decomposition proceeds. 5.2 Methods and materials  5.2.1 Study area The study was located north-east of Prince George British Columbia in old-growth forests (mean tree age >240 years) of the Sub-Boreal Spruce (SBS) biogeoclimatic zone (Meidinger and Pojar 1991). The sub-boreal forests are characterized by cool, moist summers and cold, snowy winters. Annual precipitation in the study area ranges from 897 mm in the western, wet-cool subzone (SBS wk), to 964 mm in the eastern, very wet-cool subzone (SBS vk, Murphy 1996). Three site-types were established in the SBS to evaluate the relative rates of litterfall, decomposition and nutrient release across varying climatic conditions. “Herrick” sites were located at the north-eastern end of the study area in the SBS vk at an elevation of 850 m. Mean  86  Decomposition and nutrient release from four lichen litters summer temperatures and relative humidity levels were 10.8±5.3oC and 77.6%, respectively (Fig. 5.1). “Aleza” sites were located approximately 40km south-west in the Aleza Lake Research Forest (SBS wk) at 680 m elevation. Mean summer conditions were slightly warmer (12.4±5.5oC) and drier (relative humidity 69.1%) than at the Herrick sites (Fig. 5.1). The “Fraser” sites were located at 680 m elevation in the ecotonal region between the SBS wk and the SBS vk. Summer temperatures were 11.8±5.3oC and relative humidity was 77.6% (Fig. 5.1). Relative humidity was consistently lower at the Aleza Lake sites than at the Fraser or Herrick sites. Records of light availability also show that total (direct and indirect) light levels were 9% less beneath the forest canopy at the Herrick sites compared to the Fraser and Aleza sites (Campbell et al. 2010).  Temperature (oC)  30 20 10 0 -10 -20 -30  Maximum Mean Minimum  Relative Humidity (%)  100 80 60 40 20 0  July  October 2006  January  April  July 2007  October  January  April 2008  Date  Figure 5.1 Monthly mean air temperature (top) and relative humidity (bottom) at the Fraser (dark symbols), Aleza (open symbols) and Herrick (shaded symbols) sites.  87  Decomposition and nutrient release from four lichen litters  Picea glauca Parry x engelmannii (Moench) Voss. (interior hybrid spruce) and Abies lasiocarpa Hook.(Nutt.; subalpine fir) are the dominant conifer species at all sites. Betula papyrifera Marsh (paper birch) made up most of the deciduous canopy and Populus balsamifera L. ssp. trichocarpa Brayshaw (black cottonwood) was a minor component at the Fraser and Herrick sites while Populus tremuloides Michx. (trembling aspen) was infrequently present at the Aleza sites. Three stands (hereafter referred to as study sites) were chosen at each site-type, yielding a total of nine study sites. Fraser site soils varied from fine-loamy glaciolacustrine materials at two sites to an orthic humo-ferric podzol formed from sandy-skeletal glaciofluvial materials at the third. Herrick site soils were orthic humo-ferric podzols formed from sandycolluvial materials and Aleza site soils were a fine-textured orthic luvic gleysols formed from glaciolacustrine parent materials. For more detailed site information and locations see Campbell et al. (2010). 5.2.2 Litterfall To capture canopy (tree and lichen) litter, 1-m x 1-m litterfall traps were constructed from medium-duty landscape fabric stapled to PVC piping frames. Three parallel 40-m transects were established 10 m apart at each of the nine study sites. Five litterfall traps were randomly placed along each of the three transects for a total of 15 traps per site. Placements were cleared of vegetation, plant litter and coarse woody debris prior to trap placement. Traps were installed in June 2006 and all lichen and vascular plant litter falling into the trap was collected in October 2006 and June 2007. Litter was separated into seven categories: leaves, conifer needles, twigs and other small woody debris, ‘other materials’ (consisting mainly of cones and buds), bipartite cyanolichens (with cyanobacterial symbionts), tripartite cyanolichens (with both cyanobacterial  88  Decomposition and nutrient release from four lichen litters and green-algal symbionts) and chlorolichens (with green-algal symbionts). Terrestrial litter (shrubs and herbs) was discarded. Sorted samples were dried at 65ºC for 72 hours and weighed. 5.2.3 Litter decomposition and nutrient release Samples of four lichen species were collected from Abies lasiocarpa branches from a single site in the SBS vk. A hair chlorolichen (Alectoria sarmentosa (Ach.) Ach.), a foliose chlorolichen (Platismatia glauca (L.) Culb.&C.F.Culb), a tripartite cyanolichen (Lobaria pulmonaria (L.) Hoffm.), and a bipartite cyanolichen (Nephroma helveticum Ach.) were chosen to represent four epiphytic macrolichen functional groups. A 1.4-1.6 g sample of each lichen was placed into a 10-cm x 10-cm woven fiberglass-mesh bag with 160±40 μm opening size. Lichens were dried at 65ºC for 72 hours, weighed, and placed in litterbags which were sewn closed with polyester thread. Litterbags were tied to a length of polyester thread (spaced approximately 30 cm apart) to increase the success of litterbag retrieval. The thread was staked to the forest floor adjacent to 14 of the 15 litterfall traps at each site. Each position initially contained one bag each of A. sarmentosa, L. pulmonaria and P. glauca and a litterbag containing N. helveticum was also sewn onto the polyester thread at 6 of the 14 positions. Fewer bags of N. helveticum were prepared because of the lower abundance of the species and the conservation concern over removing substantial quantities from the forest canopy. Litterbags were retrieved from the field in October 2006, June 2007, October 2007 and June 2008. Bags were re-dried and weighed to determine mass loss during each interval. One litterbag of each lichen species from each of the nine sites was destructively sampled at each time interval for nutrient analysis. These litter samples were oven-dried at 65oC for 72 hours and ground to a fine powder with a grinding mill (Retsch MM200 agate mixer mill, Sigma Aldrich, St. Louis, MO). Nitrogen and C content were determined by a flash combustion of duplicate 6-8 mg subsamples (NA 1500 NC elemental analyzer, Fisons Instruments, Italy). Samples were 89  Decomposition and nutrient release from four lichen litters prepared by microwave digestion and analysed for Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn content using inductively coupled plasma-mass spectrometry (ICP-MS, 7500 Series, Agilent Technologies, Santa Clara, CA) as described by Dolan and Capar (2002). Samples were analyzed at the University of Northern British Columbia Central Equipment Laboratory. 5.2.4 Soil nitrogen The availability of soil nitrogen was measured as the quantity of NO3- and NH4+ adsorbed to PRSTM ion exchange probes (Western Ag Innovations Inc., Saskatoon, Canada) over a 10week period from June 16 - August 25, 2006. Probes were inserted into the mineral soil inside root-exclusion tubes (constructed from 10-cm diameter and 20-cm long PVC piping) to a depth of approximately 10 cm. Three sets of probes were buried near each of three randomly selected trees of each dominant species (Picea glauca x engelmannii, Abies lasiocarpa, and Populus tremuloides or trichocarpa). Three root-exclusion tubes and probe sets were also buried outside of the influence of all canopy trees. Probes were extracted, washed with deionized water, and shipped to Western Ag. Innovations for elemental analysis. Compounds are represented as the µg of N adsorbed to the resin probes over the surface area of resin (10cm2) over the burial period (10 weeks). 5.2.5 Data analysis Differences in decay rates between litter species and across site-types were analysed using two, one-way repeated measures ANOVAs with four repeated-measure decay periods and a Bonferroni post-hoc test in each analysis. One-way ANOVAs with Bonferroni post-hoc tests were used to evaluate differences in total litter, vascular litter and lichen litter biomass (analysed as pooled totals by litter category at each site; n = 3 per site-type) and soil nitrogen (log transformed for normality; n = 12 per site-type) across site-types. The relationship between mean cyanolichen litter biomass and soil N was evaluated for each site using a Product-Moment 90  Decomposition and nutrient release from four lichen litters correlation. Cyanolichen litter biomass, soil NO3- and NH4+ were averaged for each site and log transformed for normality prior to correlation analysis. All results are presented as means ± standard deviations. Decomposition rate constants (k) were calculated according to Olson (1963) from the difference between initial and measured litter mass at each stage. The quantity of N-input was calculated by multiplying litter-N content by annual litterfall biomass. Nitrogen release over two years was calculated by subtracting the final mass of N in cyanolichen litter [mg-N ha-1 yr-1] (calculated from cyanolichen litter-mass [kg-cyanolichen litter ha-1 yr-1] x % mass remaining x final N concentration [mg-N kg-cyanolichen litter-1]) from the initial mass of N in the cyanolichen litter (initial N concentration x initial lichen litterfall mass). N-input and potential Nrelease was corrected by increasing the mass of cyanolichen litterfall by 20% to account for the expected litterfall mass-loss between collection periods (based on median mass-loss over the initial four months). 5.3 Results  5.3.1 Decomposition rates More lichen litter mass remained after four months of decay at the Aleza sites than at the other two sites. Mass loss at the three sites was similar after 1 year, but after 2 years less mass remained at the Fraser sites than at the other sites (Fig. 5.2a). Decomposition at all three sites followed a two-stage pattern with an initial rapid mass loss that slowed such that only an additional 11-18% (of the original mass) was lost in year 2 (Fig. 5.2a). During the first four months, decomposition of the two cyanolichens, N. helveticum and L. pulmonaria was faster than that of the two chlorolichens, A. sarmentosa and P. glauca. This is reflected in the higher decay constants for the cyanolichen species (Table 5.1). Decay of A.  91  Decomposition and nutrient release from four lichen litters sarmentosa was more rapid over the first winter (months 4-12) than for the other three species. Consequently, after 24 months only P. glauca differed significantly with more mass remaining (F(3,326)=57.52, p<0.0001; Fig. 5.2b), and a consistently lower cumulative decay constant (Table 5.1) than the other three litter types. After two years in the field, mass remaining ranged from 42.0% (P. glauca) to 26.6% (N. helveticum; Fig. 5.2b). Initial rates of decay (during the first 4 months; Fig. 5.2b) and decay constants (Table 5.1) were highly correlated with initial %N (R2=0.75; see Table 5.2). Low correlations thereafter are attributed to the relatively fast decay of A. sarmentosa during the first winter (Fig. 5.2b). Mass loss of lichens was faster than those measured in past studies of lodgepole-pine needle litters at Aleza Lake (Prescott et al. 2004). Decomposition rates of A. sarmentosa and the two cyanolichens were also more rapid than those observed for aspen leaf litter, but mass loss of P. glauca was similar to that of aspen leaf litter (Fig. 5.2b). Table 5.1 The initial C:N and decay rate constants (k) for four lichen species (AS = Alectoria sarmentosa, PG = Platismatia glauca, LP = Lobaria pulmonaria and NH = Nephroma helveticum). Decay constants are calculated according to Olson (1963) for four lichen litters during four time intervals and cumulatively over the two year incubation. k for time intervals  Cumulative k  Initial C:N  0-4mo  4-12mo  12-16mo  16-24mo  12mo  16mo  24mo  AS  129.4  0.59  0.48  0.17  0.17  0.84  0.79  0.63  PG  88.0  0.79  0.22  0.13  0.12  0.56  0.55  0.44  LP  17.3  1.49  0.25  0.17  0.15  0.83  0.77  0.61  NH  12.4  1.77  0.27  0.19  0.15  0.96  0.93  0.69  92  Decomposition and nutrient release from four lichen litters  A  100  % mass remaining  80  Fraser (lichen litter) Aleza (lichen litter) Herrick (lichen litter) Aleza (vascular litter) a b b  60 c c c  40  cd df  fg g  e  h 20  Repeated-measures whole-model ANOVA F(6,978)=12.68, p<0.0001 0  B  Alectoria sarmentosa Platismatia glauca Lobaria pulmonaria Nephroma helveticum Populus tremuloides Pinus contorta  % mass remaining  100  80  a a  60  b c  bce  e  de d df  40  d  f f fg  g g g  20  Repeated-measures whole-model ANOVA F(9,975)=82.06, p<0.0001 0 0  5  10  15  20  25  Decomposition time (months)  Figure 5.2 Mean (±SD) decomposition rates over two years at (a) three site types for (b) four species of lichen litter. Dotted lines represent decomposition of vascular plant litter incubated from 1993-1997 (from Prescott et al. 2004). Dissimilar letters represent significant differences between site types (a) or species (b).  93  Decomposition and nutrient release from four lichen litters Table 5.2 Chemistry of litter from four lichen species (AS = Alectoria sarmentosa, PG = Platismatia glauca, LP = Lobaria pulmonaria and NH = Nephroma helveticum) at five sequential decay stages. Decay Lichen period species (mo) 0 AS 4 12 16 24 PG 0 4 12 16 24 LP 0 4 12 16 24 NH 0 4 12 16 24  Element Concentration (mg g-1) N P Ca Mg 3.3 0.8 3.4 0.4 0.4 5.2 0.6 5.9 0.7 10.1 1.7 9.5 0.8 9.9 1.9 7.9 0.9 8.0 1.5 4.9 1.0 2.4 0.5 0.6 3.4 0.7 6.0 0.6 6.5 1.3 7.7 0.7 6.5 1.4 7.1 0.8 7.3 1.6 25.9 1.5 1.0 0.4 0.9 2.0 0.6 21.3 0.9 3.3 0.8 23.7 0.8 3.3 0.9 22.1 0.9 3.0 0.8 35.2 2.1 2.0 1.1 1.2 3.3 1.2 25.2 0.9 4.4 1.5 29.3 1.2 6.0 1.9 27.3 1.2 4.8 1.7  K 2.0 0.9 1.1 1.3 0.8 1.9 1.1 1.1 1.4 0.8 4.5 1.5 1.2 1.0 0.7 7.0 2.1 1.6 2.1 1.4  Al 0.04 0.09 1.0 1.0 1.5 0.2 0.4 1.4 1.4 1.2 0.1 0.2 0.7 0.9 0.9 0.6 1.3 3.1 3.7 3.5  Fe 0.03 0.7 0.3 0.4 0.7 0.2 0.5 0.9 1.1 1.2 0.1 0.2 0.5 0.6 1.1 1.2 2.3 3.1 4.3 6.1  (mg kg-1) B Cu 7.7 3.2 8.4 2.4 9.5 9.6 4.6 10.5 18.7 22.0 10.0 5.3 11.5 4.7 8.9 10.4 4.6 10.0 20.4 19.0 10.1 15.9 13.8 20.4 9.4 25.8 5.1 24.4 17.5 42.5 29.9 5.2 16.1 5.4 15.4 9.2 11.7 12.1 28.0 27.5  Mn 204.4 344.1 583.0 654.1 545.8 271.7 405.8 517.1 643.5 613.6 93.1 223.3 441.1 411.5 450.4 296.2 310.7 425.3 633.9 560.8  Na 68.1 54.1 60.8 244.2 54.0 94.5 79.4 120.3 266.7 73.4 98.7 90.5 100.8 176.5 69.3 133.8 132.1 240.1 326.8 135.3  Zn 28.1 28.7 72.8 85.8 89.3 27.3 41.0 64.9 67.8 121.6 21.4 26.6 44.9 45.9 69.7 49.8 56.5 79.7 113.1 111.3  94  Decomposition and nutrient release from four lichen litters 5.3.2 Nutrient release rates Initial N concentrations of lichen litter varied by an order of magnitude from 3.3 mg g-1 in the chlorolichen A. sarmentosa to 35.2 mg g-1 in the cyanolichen N. helveticum (Table 5.2). Lichens with high initial N rapidly lost N during the first year while species with a lower initial N retained N (Fig. 5.3a). After the first year of decay, N concentrations slightly increased indicating that C was lost more rapidly than N during later decay in all lichen litter types (Fig. 5.3b). Nitrogen concentrations after 2 years of decomposition ranged from 7.1 mg g-1 in P. glauca to 27.3 mg g-1 in N. helveticum. Cyanolichen litters had initial C:N ratios of 17 and 12 (for L. pulmonaria and N. helveticum, respectively), which did not change significantly as decomposition proceeded. In contrast, the C:N ratio of chlorolichen litter decreased significantly as a function of decreasing C content (R2=0.4, F(1,28)=20.47, p=0.0001). Neither the N content nor the C:N ratios of chlorolichens and cyanolichens had converged by the end of the second year (Fig. 5.4). Phosphorus and potassium were also lost from all lichen litters during the first year and the rate of loss was positively related to the initial content. Contents (and concentrations) by the end of the first year had converged at approximately 500mg P (1 mg g-1) and 1000 mg K (1 mg g-1; Fig. 5.3c-f). The concentrations of other elements including Al, Ca, Cu, Mg, Mn, Zn increased during decay (Table 5.2). However, there was no evidence of net element gain as an associated increase in the elemental mass was not detected. Initial Fe concentration was higher in N. helveticum than in other lichen litters, an observation likely related to the role of Fe in N2-fixation. While the low Fe concentration remained largely stable in most lichen litters with no change relative to C, there was a consistent increase in concentration with decay time observed in N. helveticum.  95  Decomposition and nutrient release from four lichen litters  60  a  N remaining (mg)  20  10  Alectoria sarmentosa Platismatia glauca Lobaria pulmonaria Nephroma helveticum  40 30 20 10  0  0  2.0  3 P remaining (mg)  -1  P concentration (mg g )  30  1.5 1.0 0.5  2  1  0  0.0 7  10  -1  K concentration (mg g )  b  50  -1  N concentration (mg g )  40  K remaining (mg)  6 5 4 3  8 6 4  2 2  1  0  0 0  5  10  15  Decomposition time (months)  20  25  0  5  10  15  20  25  Decomposition time (months)  Figure 5.3 Changes in elemental concentrations (mg g-1 ±SD; panel a) and elemental mass remaining in the litterbags (mg ±SD; panel b) of N, P and K over two years of lichen litter decomposition.  96  Decomposition and nutrient release from four lichen litters  100 Alectoria sarmentosa Lobaria pulmonaria Nephroma helveticum  80  Platismatia glauca  C:N  60  40  20  0 70  60  50  40  30  20  10  0  C concentration (as % of original)  Figure 5.4 The ratio of C:N as a function decreasing C concentration (%) in litter of four epiphytic lichen species. Twelve-, 15-, and 24-month decay periods are denoted by circles, triangles and square symbols respectively.  97  Decomposition and nutrient release from four lichen litters 5.3.3 Litterfall rates The total mass of aboveground litterfall collected did not differ between site-types. Conifer needle litter made up the majority (58-61%) of litter at all three site types (Table 5.3). However, there was significantly more deciduous leaf litter at the Fraser and Herrick sites than at the Aleza sites (F(2, 132)=9.86, p=0.0001). Lichen litter made up 2.8% and 2.2% of total aboveground litterfall at Fraser and Aleza sites, respectively. Significantly less lichen litter was collected at Herrick sites where it accounted for only 0.4% of total aboveground litter (F(2, 132)=19.12, p=0.0001).  Table 5.3 Lichen, leaf, needle and twig litterfall at each of three sites. Litterfall values are mean kg ha-1 yr-1± SD. Different superscript letters represent significant differences in litterfall mass between site-types (ANOVA, p<0.05). Fraser sites  Litter type  Litterfall biomass (kg ha-1 yr-1)  Aleza sites % of total litter  Litterfall biomass (kg ha-1 yr-1)  Herrick sites % of total litter  Litterfall biomass (kg ha-1 yr-1)  % of total litter  Chlorolichens  21±6a  0.6  59±12b  1.7  12±3a  0.3  Bipartite cyanolichens  6±3a  0.2  0.5±0.1b  0  1±1b  0  L. pulmonaria  77±41a  2.1  19±12b  0.5  2±3b  0.1  Total lichen  104±38a  2.8  78±13a  2.2  15±2b  0.4  Leaves  548±156a  15.0  161±84b  4.6  653±475a  16.3  Conifer needles  2165±142  59.2  2164±235  61.4  2301±231  57.6  Twig  739±211  20.2  932±206  26.4  663±277  16.6  Other  103±50  2.8  192±63  5.4  361±352  9.0  Total  3659±275  100.0  3526±441  100.0  3993±856  100.0  98  Decomposition and nutrient release from four lichen litters The composition of lichen litter also differed significantly between site-types. Chlorolichens comprised most of the lichen litter at the Aleza sites (75%), whereas cyanolichen litter (particularly L. pulmonaria) made up 74% of the lichen litter at the Fraser sites. Although this cyanolichen biomass made up only 2.3% of the total annual aboveground litter mass at the Fraser sites, it represented 11.5% of the total estimated N-input from aboveground litterfall (Table 5.4). The estimated N-input from N2-fixing lichens (comprised of various bipartite cyanolichen species plus the tripartite lichen, L. pulmonaria) was 2.6, 0.6, and 0.1 kg N ha-1 at Fraser, Aleza and Herrick sites, respectively (Table 5.4). Cyanolichen litters after two years of mass- and nutrient-loss would be expected to contain 0.5, 0.2 and 0.02 kg N ha-1 at Fraser, Aleza and Herrick sites, respectively. The difference between initial and final N-contents suggests that cyanolichen litter releases approximately 2.1, 0.4 and 0.08 kg N ha-1 over two years at Fraser, Aleza and Herrick sites, respectively 5.3.4 Soil nitrogen Nitrate (NO3-) was significantly more available in mineral soils at the Fraser sites than at the Aleza and Herrick sites (F(2, 40) = 5.24, p = 0.01; Fig. 5.5). Ammonium (NH4+) was most available at the Fraser and Herrick sites (F(2, 35) = 11.312, p = 0.0001). Soil NO3- was positively correlated (r=0.7, p=0.03) with total cyanolichen litter biomass.  99  Decomposition and nutrient release from four lichen litters Table 5.4 Estimated annual nitrogen input and release from lichen, leaf, needle and twig litterfall at each of three sites. Nitrogen release (kg N ha-1 yr-1) is calculated from the annual litterfall mass multiplied by the initial N content and the change in N content during the first year of decay. The mass of chlorolichen and cyanolichen (including Lobaria pulmonaria) litterfall was increased by 10% and 20% respectively to account for the expected litterfall mass loss between collection periods (based on median mass loss for each species over the initial 4 months).  Litter type  N conc. (mg g-1)  Fraser sites N input % of total -1 ( kg N ha N input yr-1) from litter  N release ( kg N ha-1 yr-1)  Aleza sites N input ( kg N ha-1 yr-1)  % of total N input from litter  N release ( kg N ha-1 yr-1)  Herrick sites N input % of total -1 ( kg N ha N input yr-1) from litter  N release ( kg N ha-1 yr-1)  Chlorolichens  4.9  0.11  0.48  0.05  0.32  1.64  0.13  0.06  0.28  0.03  Bipartite cyanolichens  35.2  0.25  1.08  0.15  0.02  0.10  0.01  0.04  0.19  0.03  L. pulmonaria  25.9  2.39  10.37  1.43  0.59  3.024  0.3  0.06  0.28  0.03  Total lichen  --  2.75  11.93  1.63  0.93  4.76  0.44  0.16  0.74  0.09  Leaves  7.81  4.3  18.66  --  1.3  6.66  --  5.1  23.77  --  Conifer needles  5.12  11.1  48.16  --  11.1  56.84  --  11.8  54.99  --  Twig  6.63  4.9  21.26  --  6.2  31.75  --  4.4  20.50  --  Total  --  23.05  100  --  19.53  100  --  21.46  100  --  1 From Prescott et al. 2004 Can. J. For. Res. 34:1714-1729 – mean of values for Trembling Aspen and Black Cottonwood 2 From Prescott et al. 2004 Can. J. For. Res. 34:1714-1729 – mean of values for Subalpine fir and Engelmann spruce 3 From Laiho and Prescott 1999. Can.J.For.Res. 29:1592-1603 – N concentration in small woody debris from the ‘fir’ site with an overstorey composed of Subalpine fir and Engelmann spruce.  100  Decomposition and nutrient release from four lichen litters  110  a  Fraser sites Aleza sites Herrick sites  Available N -2 -1 (μg 10 cm 10 weeks )  40  b  30 ab 20  10 a 0  b  b  NO3-  NH4+  Figure 5.5 Nitrogen availability (±SD) in mineral soil measured as NO3- and NH4+ supply rates onto PRSTM ion exchange probes incubated in mineral soil within rootexclusion tubes for 10 weeks.  101  Decomposition and nutrient release from four lichen litters  5.4 Discussion  5.4.1 Decomposition of lichen litter The relative rates of initial lichen litter decay were positively correlated with the initial N content of the litter which varied from very low (0.5% in P. glauca) to very high (3.5% in N. helveticum). As such, the two lichens with a cyanobacterial component, L. pulmonaria and N. helveticum had a much faster rate of initial decay, than did the green-algal lichen P. glauca. Mass remaining after 12 months of decay ranged from 40-45% of cyanolichen litter to 57% of P. glauca litter. These results concurred with those of Esseen and Renhorn (1998) and McCune and Daly (1994) who showed faster rates of cyanolichen decomposition (39% and 60% mass remaining after one year for L. pulmonaria and L. oregana respectively) than for P. glauca (58% and 80% mass remaining for the two studies respectively). Guzman et al. (1990) also reported rapid decay rates for cyanolichen species with more than 70% mass loss over one year in a beech forest in southern Chile. That similar mass of N. helveticum, L. pulmonaria and A. sarmentosa remained after two years is consistent with the results of Lang et al. (2009) who observed no differences in mass loss over two years between chlorolichens and tripartite cyanolichens. A meta-analysis of decomposition studies indicate that physical characteristics of plants and bryophytes associated with nutrient-use efficiency, leaf mass per area and phenology strongly influenced decay rates (Cornwell et al. 2008). Lichen litters were not considered in that study but several characteristics of lichens, notably the high N, the relatively labile chemistry of many species and the lack of woody or otherwise recalcitrant tissues suggest that decay of lichen litter would be rapid relative to others evaluated by Cornwell et al. (2008). The rapid decay of A. sarmentosa in this study in particular may be attributed to the physical features of this finelybranched hair-chlorolichen. Following 12 months of decay, the percent mass remaining of A. 102  Decomposition and nutrient release from four lichen litters sarmentosa was not different from that of the cyanolichens, despite an N-content that was 87% lower. Similar results observed by Esseen and Renhorn (1998) and McCune and Daly (1994) were also explained by the morphology of the hair lichen thallus. The highly branched and friable thallus structure of hair lichens makes them more susceptible to mechanical breakdown which may speed up decay by softening the litter (Cortez 1988) or increasing the surface area for microbial action (Fyles and McGill 1987). Other lichens, notably L. pulmonaria, possess physical traits such as a thickened thallus structure or chemical composition that may inhibit decay. Initial decomposition of L. pulmonaria was slower than that of N. helveticum despite both species being N-rich compared to most vascular litters (see Harmon et al. 2009). Lobaria pulmonaria litter decay may have been retarded by the relatively thick outer cortex and chemical resistance of the species. A closely related species, L. oregana, contains stictic acid, norstictic acid and constictic acid (Culberson 1969) which have all been shown to slow decomposition (Hättenschwiler and Vitousek 2000) and to reduce the rate of N mobilization from chitin (Greenfield 1993). The existence of toxic or unpalatable phenols would also explain why such an N-rich lichen is not commonly foraged upon, while A. sarmentosa is readily consumed by caribou (Edwards et al. 1960; Rominger and Oldemeyer 1990), and other mammals (Stevenson and Rochelle 1984). Chlorolichens and tripartite cyanolichens have been previously shown to decompose at similar rates to vascular litter (Lang et al. 2009). Although no side-by-side comparisons are available here, the lichen litter in this study appears to decompose more rapidly than vascular litters. The mean first-year decay constant of lichen litters in this study (k= 0.79 year-1) are larger than those computed for vascular plant litters (k=0.51 year-1) in a comprehensive meta-analysis (Harmon et al. 2009). In addition, there was less mass remaining of three lichen litters than of  103  Decomposition and nutrient release from four lichen litters vascular plant litter after an earlier two-year incubation at the same site (data from Prescott et al. 2004). While inter-annual climatic differences likely influenced the disparate decay rates recorded in this study and that of Prescott et al. (2004), the faster decay of cyanolichen litter may be related to its much lower C:N ratio (12 to 17) compared to that of Pinus contorta (104) or Populus tremuloides (87, Prescott et al. 2004). Fast decay of lichens may also be related to the relatively labile thallus structure of lichens. The hyphal matrix that makes up 95% of the lichen thallus is composed of N-rich chitin and the algal or cyanobacterial cells enclosed in this fungal matrix are largely comprised of labile cellulose and protein (König and Peveling 1984). Decomposition of vascular litter is generally described in two phases. Mass loss during the first phase is adequately described by negative exponential decay models, while mass loss during the second phase (late-stage) is imperceptibly slow (Aber et al. 1990). Decay of lichen litter here did not appear to enter late-stage decay as no asymptote was detected in the percent mass remaining. In evaluating rates of decay in 234 cases, Harmon et al. (2009) demonstrated that overall decay rate constants for litters in late-stage decay were approximately 70% of the short-term (first-year) decomposition rates constants. Overall decay constants for the lichen litters ranged from 0.44 year-1 for P. glauca to 0.69 year-1 for N. helveticum. These constants are 79% to 71% of the first-year constants for the two species, respectively. This suggests that the two-year incubation was insufficient for lichen litter to enter late-stage decay. Alternatively, it may also be that some high-N lichen litters decompose completely and so do not enter a latestage of slow decay, as demonstrated by Holub and Lajtha (2003). Indeed, Greenfield (1993) noted that most of cyanolichen mass was lost by the end of 135 days under laboratory conditions. This suggests that some lichen tissues may rapidly and, potentially, completely decay.  104  Decomposition and nutrient release from four lichen litters The slower lichen decomposition rates observed at the driest sites (Aleza) indicate that moisture is also an important rate-determining factor for lichen decay in these forests. The slowing of decay rates at the Herrick sites may be related to the deeper and more persistent snow pack at these higher-elevation sites. 5.4.2 Element changes Unlike vascular litters which often have net immobilization of nitrogen and phosphorus during early decay (Lousier and Parkinson 1978), the cyanolichens in this study showed immediate and significant loss of N (up to 28% of total N lost during the first 12 months). This was consistent with what was expected given that approximately 80-90% of the N in the lichen thallus is in readily leached compounds such as labile proteins, chitin and nucleic acids (Dahlman et al. 2003) which can be solubilized and rapidly released during early decay (Greenfield 1989; Rai 1998). There was no evidence that N was immobilized in the chlorolichen litter over two years. The small increase in N concentration in these species, however, indicates that N was retained during mass loss (relative to C) resulting in a narrowing of the C:N ratios. Both A. sarmentosa and P. glauca had a high initial C:N (129 and 88 respectively), which declined to 40-50 through the gradual loss of C. The C:N ratios of both chlorolichen litters were above the critical C:N ratio (23-35, Prescott 2005) and so, as expected, there was no net N-release from these litters during 24 months of decay. Phosphorus was released from all lichen litters during the first four months of decay. Although the mass of P inside litter bags consistently decreased during decomposition thereafter, neither the P concentration nor the C:P ratio changed significantly between 4 and 24 months suggesting that P and C were lost at similar rates.  105  Decomposition and nutrient release from four lichen litters There are few previous studies of N and P dynamics in decomposing lichens with which to compare these results. Mat-forming lichens in dry spruce forests are extremely nutrient-poor and were found to retain P and accumulate N (up to 200% of initial concentrations) during decomposition (Moore 1984). Net N-uptake was also recorded during the first 100 days of decay of L. oregana, despite an initial N content of 2.28% (Holub and Lajtha 2003). Although this N was subsequently released, at a rate 19% faster than mass loss, the initial rapid release of N from high-N lichens in our study is more consistent with elemental changes commonly observed during decomposition of high-N vascular plant litters (Prescott 2005). The rate and quantity of the accumulation or release of other nutrients during decay has been recorded for vascular plant litter (i.e. Lousier and Parkinson 1978; Berg and Laskowski 1997; Titus and Malcolm 1999). Generally, K, Ca, Mn, and Mg are readily released during decomposition while Fe, Zn, Cu and Na are retained in the litter. In the lichen litters studied here, only K was rapidly released during early decay and all other nutrients were retained in the litter. Caldiz et al. (2007) also reported rapid K loss from all lichen litters during initial decay in wet, Nothofagus forests in Patagonia. However, while Ca and Mg were consistently retained in our lichen litters during decomposition, these elements were immobilized in chlorolichen litter and retained in cyanolichen litter in the Pategonian forest study. 5.4.3 Litterfall Annual biomass of vascular plant litterfall in these wet, sub-boreal forests (3448-3978 kg ha-1 yr-1) is within the range of litterfall estimates from other coniferous forests in North America (Prescott et al. 1989; Ferrari 1999). Lichen litterfall is likewise comparable to estimates from coastal, wet temperate forests (McCune 1994). However, the total lichen litterfall (78 kg ha-1yr-1) observed at the Aleza sites here represents 13-17% of the 472-603 kg ha-1 of in situ lichen recorded by Campbell and Fredeen (2007) at the same sites (biomass estimates are unavailable 106  Decomposition and nutrient release from four lichen litters for the other two site types). This is over an order of magnitude higher than the 1% of total standing epiphytic lichen biomass observed to enter litter pools in other wet, temperate forests (McCune 1994). The biomass of lichen litter made up 0.4 - 2.8% of the total litter collected at the three sites. This is low compared to nearby high-elevation, spruce-fir forests where lichens made up 2.4 - 6.1% of vascular plant litter (Stevenson and Coxson 2003). 5.4.4 Significance of lichen N inputs The relative contribution of lichen litter to ecosystem N-cycling is perhaps higher than litterfall biomass would suggest. Lichens accounted for up to 12% of litter-N (2.75 kg N ha-1yr-1) despite contributing less than 3% of total litterfall (Table 5.4). As discussed earlier, the N in lichens is mostly in labile and leachable forms and so may be readily available. This cyanolichen-N may therefore contribute to the observed positive correlation between available NO3--N and total cyanolichen litter biomass. Decomposition of cyanolichen litter at the Fraser sites is estimated to release approximately 2.1 kg N ha-1 yr-1 to the ecosystem. This is less than the estimated input (3-4 kg N ha-1 yr-1) from cyanolichens in coastal forests of the Pacific Northwest (Denison 1979) but is within the range of most symbiotic and free-living N2-fixation reported for late-seral interior B.C. forests (Fisher and Binkley 2000). Although Sanborn et al. (2002) reported rates of annual N2-fixation in Sitka alder as high as 10-15 kg N ha-1 yr-1 in a 9yr-old Pinus contorta plantation, symbiotic N2-fixation by vascular plants is not likely to be a substantial source of N in late-seral, closed-canopy forests. Therefore, with N release rates as high as 2.1 kg N ha-1 yr-1, the deposition of cyanolichen litter onto the forest floor presents a significant source of newly fixed-N that would not otherwise be available to these mature, wet sub-boreal forests.  107  Decomposition and nutrient release from four lichen litters  5.5 References  Aber, J.D., Melillo, J. and McClaugherty, C.A. 1990. Predicting long-term patterns of mass loss, nitrogen dynamics, and soil organic matter formation from inital fine litter chemistry in temperate forest ecosystems. Canadian Journal of Botany 68:2201-2208. Aerts, R. 1997. Climate, leaf litter chemistry and leaf litter decomposition in terrestrial ecosystems: a triangular relationship. Oikos 79:439-449. 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Springer-Verlag, Berlin, Heidelberg. pp. 15-41. Rai, A.N. 1998. Nitrogen Metabolism. In CRC Handbook of Lichenology Vol II. Edited by M. Galun. CRC Press, Boca Ratan, Florida. pp. 201-237. Rominger, E.M. and Oldemeyer, J.L. 1990. Early-winter diet of woodland caribou in relation to snow accumulation, Selkirk Mountains, British Columbia, Canada. Canadian Journal of Zoology 68:2691-2694. Sanborn, P., Preston, C. and Brockley, R. 2002. N2-fixation by Sitka alder in a young lodgepole pine stand in central interior British Columbia, Canada. Forest Ecology and Management 167:223-231.  110  Decomposition and nutrient release from four lichen litters Sillett, S.C. and Neitlich, C. 1996. Emerging themes in epiphyte research in westside forests with special reference to cyanolichens. Northwest Science 70:54-60. Sollins, P., Grier, C.C., McCorison, F.M., Cromack, K.J. and Fogel, R. 1980. The internal element cycles of an old-growth Douglas-fir ecyosystem in Western Oregon. 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Influence of soil type on mass loss and nutrient release from decomposing foliar litter of beech and Norway spruce. Canadian Journal of Forest Research 29:95-105. Waksman, S.A. and Gerretsen, F.C. 1931. Influence of temperature and moisuture upon the nature and extent of decompositioin of plant residues by microorganisms. Ecology 12:3360. Waring, R.H. and Schlesinger, W.H. 1985. Forest ecosystems: concepts and management. Academic press, New York, New York, USA. Zhang, D., Dafeng, H., Luo, Y. and Zhou, G. 2008. Rates of litter decomposition in terrestrial ecosystems: global patterns and controlling factors. Journal of Plant Ecology 1:85-93.  111  Conclusions  CHAPTER 6  CONCLUSIONS  This dissertation evaluates cyanolichen ecology in sub-boreal spruce forests with reference to the role of these nitrogen-fixing communities in nutrient and carbon cycling. This work provides evidence that improves our understanding of two important questions, namely: what is the contribution of cyanolichens to nutrient cycling and what are the environmental factors that promote cyanolichen communities? Cyanolichens form a distinct assemblage of epiphytes most frequently observed in mature forest ecosystems under humid microclimatic conditions (Goward and Spribille 2005). With biomass loadings as high as 1400 kg ha-1 in wet-interior forests of British Columbia (Campbell and Fredeen 2004) these nitrogen-fixing species have the potential to contribute to ecosystem N-cycling. Indeed, the cyanolichens studied here rapidly decomposed and showed immediate and significant N-loss. Analysis of litterfall rates, decomposition rates and N-release rates revealed that up to 2.1 kg N ha-1 yr-1 may be made available through lichen litter deposition and decay. As demonstrated by Holub and Lajtha (2004) a large proportion of the N released from cyanolichens remains in the litter and organic layers and thus may be a significant source of newly-fixed N that would not otherwise be available. Cyanolichen communities are most abundant in wet, old forests and as shown here, cyanolichen communities were more abundant at the Fraser sites where relative humidity was higher than at the Aleza sites, and where the more mature stand structure allowed greater penetration of light to the lower canopy than at the Herrick sites. Although such site characteristics have been previously shown to promote cyanolichens (Goward 1994, Campbell and Fredeen 2004), other site factors may also have been involved in defining regional differences in cyanolichen communities. For example, the higher pH of mineral soil at the Fraser 112  Conclusions sites may have elevated the pH of bark substrate as has been observed in Norway (Gauslaa 1985). Regardless of the microclimatic or chemical factors influencing regional cyanolichen distribution patterns, it is clear that cyanolichens are strongly spatially associated with Populus. Cyanolichens are restricted to the trunks of Populus species in areas where acid deposition is thought to preclude their wide-spread occurrence (Gauslaa 1995). In British Columbia however, cyanolichens are also observed on conifer branches, but are more abundant within a Populus drip-zone (Goward and Arsenault 2000). Indeed, a comprehensive survey of cyanolichen communities on conifer saplings beneath overstorey Populus compared to overstorey Betula, Picea, Abies and Pseudotsuga provides evidence for a strong spatial association between cyanolichens and Populus. Cyanolichen communities beneath overstorey Populus are disproportionately species-richness and abundant. Furthermore, the association between cyanolichens and Populus varies with climatic regime. At sites with adequate moisture and light, cyanolichen communities are equally abundant and species-rich beneath all overstorey tree species. By contrast, at sites where either moisture or light were deficient, chlorolichens comprised most of the epiphyte communities and most cyanolichens were abundant only within the Populus drip-zone. This suggests that Populus provides some factor not otherwise available under unfavourable climatic conditions. The factors underlying the strong association between cyanolichens and Populus are not well understood, but it is probable that Populus supports cyanolichens during one or more developmental stages. Sillett et al. (2000) describe the development of epiphytic cyanolichens as going through the stages of dispersal, establishment and thallus growth. Dispersal rates were not evaluated here, but cyanolichens grew faster beneath Populus than beneath conifers (Picea and  113  Conclusions Pseudotsuga) in these sub-boreal forests. More revealing, cyanolichen growth rates beneath Populus were considerably higher than most previously published accounts (Table 3.2). In addition, while most small cyanolichen thalli either decreased in size or died when transplanted beneath conifer trees, many small thalli beneath Populus survived the experiment and even experienced modest area gains. This indicates that conditions during the most vulnerable lifestages of establishment and early growth (Scheidegger 1995) may be ameliorated by Populus. Taken together, the rapid growth rates and lower rates of mortality suggest that both establishment and thallus growth are important factors contributing to the abundance of cyanolichens observed beneath Populus. Differences in lichen distribution patterns have previously been related to the climate (Goward and Spribille 2005; Gauslaa et al. 2006) and chemical characteristics of the host tree (Gauslaa 1995; Hauck and Spribille 2002). However, these results, showing greater cyanolichen abundance beneath, compared to on, the Populus, suggest that cyanolichens are not responding exclusively to substrate characteristics. Rather, these results suggest that some factor present in the Populus canopy facilitates cyanolichen communities. A higher density of diaspores released from a diverse community of mature lichens beneath the Populus canopy may promote cyanolichen species-richness on the saplings below. However, the results presented here provide evidence in support of the drip-zone effect (Goward and Arsenault 2000) in which precipitation that is chemically altered by falling through the Populus canopy, is intercepted by the cyanolichen communities. The concentration of calcium (Gauslaa 1995; Goward and Arsenault 2000), molybdenum (Horstmann et al. 1982), manganese (Hauck and Spribille 2002) and phosphorus (Benner and Vitousek 2007) have all been shown to strongly influence cyanolichen community structure. However, differences in mineral nutrient availability in throughfall  114  Conclusions precipitation beneath Populus, Betula, Picea, Abies and Pseudotsuga were poorly correlated with lichen community structure. The extensive sampling regime for each climatic (relative humidity, temperature, light availability) and chemical (soil nutrients, branch and soil pH, throughfall precipitation chemistry) factor suggests that these results accurately reflect the ecological patterns and provide confidence in the conclusion that these factors do not drive the Populuscyanolichen association. As an alternative, this work presents data in support of a novel hypothesis regarding the factors influencing the association. Populus trees belong to the Salicaceae family, many members of which possess extrafloral nectaries (EFN) at the junction of the leaf-blade and petiole (Trelease 1881). Glucose-rich nectar secreted from EFNs accumulates on the leaf surface (Curtis and Lersten 1978) and may be subsequently washed off and intercepted by epiphytic cyanolichens on the conifer branches below. Experimental evidence presented here demonstrates that fungal respiration is enhanced by exogenous glucose and that glucose is rapidly assimilated into lichen fatty-acids. Furthermore, the establishment success of cyanolichens was substantially enhanced by the provision of exogenous glucose. A mechanistic basis for this difference may be found in the significant C-loss that occurs during rehydration (Brown et al. 1983). Although all lichens can withstand desiccation, cyanolichens may be more susceptible to respiratory losses during rewetting than chlorolichens. In the former, contact with liquid water is required to become photosynthetically active, but significant fungal respiration may occur with a lower thallus-water content reached under high atmospheric humidity (Lange et al. 1986). In contrast, chlorolichens may rehydrate with water vapour alone and require only 50-70% thallus-moisture for respiratory and photosynthetic activity (Lange et al. 1986). Thus, cyanolichens are rarely observed in drier ecosystems where  115  Conclusions the duration of hydration is insufficient to allow photosynthetic C-gains to compensate for respiratory C-losses. Exogenous glucose may compensate for respiration C-losses, allowing the lichen thallus to achieve a positive C-balance despite the moisture-related inactivity of the cyanobacterial partner. The experimental and ecological results support the hypothesis that cyanolichen communities are sustained by glucose from Populus EFNs. However, no data showing the concentration of carbohydrates in throughfall precipitation beneath the trees studied here are available. Although resin capsules were used to quantify carbohydrates in precipitation, analysis of extracts revealed no carbohydrates above detectable limits. This may actually reflect the chemical composition of the throughfall precipitation, but many other reports of saccharide concentrations in precipitation beneath broadleaf canopies makes this unlikely (Carlisle et al. 1966; DeBoois and Jansen 1976; Sanborn and Pawluk 1983). Rather, this omission is likely related to the timing of collection as the concentration of organic carbon in throughfall precipitation fluctuates seasonally (Carlisle et al. 1966; DeBoois and Jansen 1976). Alternatively, this may have been due to an issue with the materials used. The resin capsules were specifically designed to capture hydrocarbons from solution, but to date, have only been tested on more complex molecules and so may not be effective at adsorbing the simpler sugar molecules. Regardless of causal factors, the lack of detection of glucose-data means that this work must remain hypothetical. Discovering the concentration of glucose in throughfall precipitation and tracing nectar from Populus EFNs to understorey cyanolichen communities thus provides an avenue for future work. Nevertheless, this work constitutes a significant contribution to our understanding of cyanolichens and cyanolichen ecology. The highly abundant and species-rich cyanolichen  116  Conclusions communities beneath Populus, and the faster cyanolichen growth rates and lower mortality rates beneath Populus, strongly suggest that Populus facilitate cyanolichen communities. Furthermore, the experimental use and uptake of exogenous glucose, and the increased establishment success of cyanolichens with exogenous glucose, provide support for the hypothesis that it is the glucoserich nectar produced by Populus EFNs that facilitates cyanolichen establishment and survival beneath Populus canopies. This may challenge our view of what a lichen is and may substantiate Goward’s (2009) evaluation of the lichen symbiosis as shifting according to environmental conditions. The predominant view of the lichen symbiosis is one of mutual and somewhat exclusive benefit. That is, apart from water and exogenous mineral nutrients, all of the requirements of the mycobiont may be obtained from within the symbiosis. However, if under unfavourable environmental conditions, cyanolichen mycobionts are instead reliant on an external source of photosynthate, then the accepted paradigm of the nutritional fidelity in the lichen symbiosis may not always be correct. 6.1 References  Benner, J.W. and Vitousek, P.M. 2007. Development of a diverse epiphyte community in response to phosphorus fertilization. Ecology Letters 10:628-636. Brown, D.H., MacFarlane, J.D. and Kershaw, K.A. 1983. Physiological-environmental interactions in lichens. XVI. A re-examination of resaturation respiration phenomena. New Phytologist 93:237-246. Campbell, J. and Fredeen, A.L. 2004. Lobaria pulmonaria abundance as an indicator of macrolichen diversity in Interior Cedar-Hemlock forests of east-central British Columbia. Canadian Journal of Botany 82:970-982. Carlisle, A., Brown, H.H. and White, E.J. 1966. The organic matter and nutrient elements in the precipitation beneath a sessile oak (Quercus petraea) canopy. Journal of Ecology 54:87-98. Curtis, J.D. and Lersten, N.R. 1978. Heterophylly in Populus grandidentata (Salicaceae) with emphasis on resin glands and extrafloral nectaries. American Journal of Botany 65:10031010. DeBoois, H.M. and Jansen, E. 1976. Effect of nutrients in throughfall rainwater and of leaf fall upon fungal growth in a forest soil layer. Pedobiologia 16:161-166. 117  Conclusions Gauslaa, Y. 1985. The ecology of Lobarion pulmonariae and Parmelion caperatae in Quercus dominated forests in South-West Norway. Lichenologist 17:117-140. Gauslaa, Y. 1995. The Lobarion, an epiphytic community of ancient forests threatened by acid rain. Lichenologist 27:59-76. Gauslaa, Y., Lie, M., Solhaug, K.A. and Ohlson, M. 2006. Growth and ecophysiological acclimation of the foliose lichen Lobaria pulmonaria in forests with contrasting light climates. Oecologia 147:406-416. Goward, T. 1994. Notes on oldgrowth-dependent epiphytic macrolichens in inland British Columbia, Canada. Acta Botanica Fennica 150:31-38. Goward, T. and Arsenault, A. 2000. Cyanolichen distribution in young unmanaged forests: A dripzone effect? Bryologist 103:28-37. Goward, T. and Spribille, T. 2005. Lichenological evidence for the recognition of inland rain forests in Western North America. Journal of Biogeography 32:1209-1219. Goward, T. 2009. Twelve readings on the lichen thallus. IV. Re-emergence. Evansia 25:1-6. Hauck, M. and Spribille, T. 2002. The Mn/Ca and Mn/Mg ratios in bark as possible causes for the occurrence of Lobarion lichens on conifers in the dripzone of Populus in Western North America. Lichenologist 34:527-532. Holub, S.M. and Lajtha, K. 2004. The fate and retention of organic and inorganic 15N-nitrogen in an old-growth forest soil in Western Oregon. Ecosystems 7:368-380. Horstmann, J.L., Denison, W.C. and Silvester, W.B. 1982. 15N2 Fixation and molybdenum enhancement of acetylene reduction by Lobaria spp. New Phytologist 92:235-241. Lange, O.L., Kilian, E. and Ziegler, H. 1986. Water vapor uptake and photosynthesis of lichens: performance differences in species with green and blue-green algae as phycobionts. Oecologia 71:104-110. Sanborn, P. and Pawluk, S. 1983. Process studies of a chernozemic pedon, Alberta (Canada). Geoderma 31:205-237. Scheidegger, C. 1995. Early development of transplanted isidioid soredia of Lobaria pulmonaria in an endangered population. Lichenologist 27:361-374. Sillett, S.C., McCune, B., Peck, J.E., Rambo, T.R. and Ruchty, A. 2000. Dispersal limitations of epiphytic lichens result in species dependent on old-growth forests. Ecological Applications 10:789-799. Trelease, W. 1881. The foliar nectar glands of Populus. Botanical Gazette 6:284-290.  118  

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