Mycorrhizal Fungi: Unlocking Their Ecology and Role in the Establishment and Growth Performance of Different Conifer Species in Nutrient-Poor Coastal Forests

MYCORRHIZAL FUNGI: UNLOCKING THEIR ECOLOGY AND ROLE IN THE ESTABLISHMENT AND GROWTH PERFORMANCE OF DIFFERENT CONIFER SPECIES IN NUTRIENT-POOR COASTAL FORESTS

Shannon Heather Ann Guichon

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

The Faculty of Graduate and Postdoctoral Studies

(Forestry)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

December 2015

This thesis explored the fungal communities of arbuscular mycorrhizal-dominated Cedar- Hemlock (CH) and ectomycorrhizal-dominated Hemlock-Amabilis fir (HA) forests on northern Vancouver Island, British Columbia, Canada and examined the role of mycorrhizal inoculum potential for conifer seedling productivity. Objectives of this research project were to: (1) examine the mycorrhizal fungal communities and infer the inoculum potential of CH and HA forests, (2) determine whether understory plants in CH and HA forest clearcuts share compatible mycorrhizal fungi with either western redcedar (Thuja plicata) or western hemlock (Tsuga heterophylla), (3) test whether differences in mycorrhizal inoculum potential between forest types influence attributes of seedling performance during reforestation and (4) test effectiveness of providing appropriate mycorrhizal inoculum at the time of planting on conifer seedling performance. Molecular and phylogenetic techniques were utilized to compare mycorrhizal fungal diversity between forest types and to identify mycorrhizal fungal associates of the plant species occurring in clearcuts. In a field trial utilizing seedling bioassays, the role of mycorrhization of western redcedar and western hemlock on seedling growth was evaluated; reciprocal forest floor transfers from uncut forests were incorporated into the project design as inoculation treatments. Though diversity was similar, ectomycorrhizal and saprophytic fungal community composition significantly differed between CH and HA forests; arbuscular mycorrhizae were widespread in CH forests, but rare in HA forests. There was high similarity of arbuscular mycorrhizal fungi to those found in western redcedar among the dominant plant species colonizing CH clearcuts, including the ericoid plant Gaultheria shallon and in Blechnum spicant growing sparsely in HA clearcuts. No alternative ectomycorrhizal host species were detected. Mycorrhization greatly influenced productivity of western redcedar seedlings; without mycorrhizal inoculum, redcedar did not achieve its full growth potential in HA clearcuts. Mycorrhization of western hemlock seedlings did not differ between forest clearcut type or treatment group; however, an inhibitory effect of forest floor collected under mature western redcedar trees on the growth of western hemlock seedlings was unexpectedly detected. These results have implications for sustainable forest management practices, including retention of legacy trees and plants with timber harvesting and inoculation of seedlings with mycorrhizal fungi at the time of planting.

ii Preface

The research presented in this dissertation was developed and carried out by Shannon Guichon, the author of thesis, under the supervision of Suzanne Simard and the advisement of committee members. Shannon Guichon was responsible for all fieldwork, establishment of research trials, laboratory work, data analyses and manuscript preparation. Writing of this dissertation was accomplished with the help of Suzanne Simard.

The research presented in chapter 2, was initiated by Shannon Guichon. Richard Hamlin contributed to the targeted metagenomics sampling design. Suzanne Simard contributed to the project development and editing of the manuscript. Gary Bradfield assisted with the data analyses.

The research presented in chapter 3, was initiated by Shannon Berch. Suzanne Simard contributed to the project design and editing of the manuscript.

The research presented in chapter 4, was initiated jointly by Suzanne Simard and Shannon Guichon. Suzanne Simard contributed to the trial design and editing of the manuscript. Valerie LeMay assisted with the data analyses.

iii Table of contents

Abstract ...... ii

Preface ...... iii

Table of contents ...... iv

List of tables ...... vi

List of figures ...... ix

List of acronyms ...... xvi

Acknowledgements ...... xvii

Dedication ...... xix

Chapter 1: Introduction ...... 1

1.1 Preamble...... 1

1.2 Research rationale ...... 2

1.3 Literature review ...... 4

1.3.1 Review of studies from the Salal Cedar Hemlock Integrated Research Project ..... 4

1.3.2 Importance of mycorrhization ...... 8

1.3.3 Antagonistic interactions between mycorrhizal guilds ...... 9

1.3.4 Mycorrhizal guilds and their role in nutrient cycling ...... 10

1.3.5 Mycorrhizal studies in the Salal Cedar Hemlock Integrated Research Project..... 12

1.4 Research objectives ...... 16

Chapter 2: Forest floor fungal community composition and mycorrhizal inoculum potential of CH and HA forests ...... 18

2.1 Introduction ...... 18

2.2 Methods ...... 21

iv 2.2.1 Site description ...... 21

2.2.2 Sampling design ...... 23

2.2.3 Targeted metagenomic characterization ...... 24

2.2.4 DNA sequence analyses of clone libraries ...... 25

2.2.5 Amplification of arbuscular mycorrhizal DNA...... 26

2.2.6 Statistical analyses ...... 28

2.3 Results ...... 30

2.3.1 Tree species and the understory plant community ...... 30

2.3.2 Sequence analyses among clone libraries ...... 31

2.3.3 Diversity of fungal ecological roles ...... 31

2.3.4 Multivariate analyses of fungal communities in CH and HA forests ...... 33

2.3.5 PCR amplification DNA extracts using arbuscular mycorrhizal fungi specific primers ...... 34

2.4 Discussion ...... 34

2.4.1 Fungal communities differ between CH and HA forests ...... 35

2.4.2 Saprophytic and ectomycorrhizal fungi important for decomposition ...... 37

2.4.3 Very low abundance of ericoid mycorrhizal fungi ...... 40

2.4.4 Arbuscular mycorrhizal fungi present primarily in CH forests ...... 41

2.4.5 Conclusion ...... 42

Chapter 3: Understory plant species as reservoirs of compatible mycorrhizal fungi for conifer seedling establishment following forest disturbance ...... 57

3.1 Introduction ...... 57

3.2 Methods ...... 60

3.2.1 Site description ...... 60

ii 3.2.2 Sampling design ...... 60

3.2.3 Molecular techniques ...... 62

3.2.4 DNA sequence analyses of clone libraries ...... 64

3.2.5 Phylogenetic analyses of arbuscular mycorrhizal fungi ...... 65

3.2.6 Analyses of fungal diversity ...... 66

3.3 Results ...... 66

3.4 Discussion ...... 68

3.4.1 Lack of alternative ectomycorrhizal hosts to maintain fungal inoculum following clearcutting ...... 69

3.4.2 High species richness of arbuscular mycorrhizae with differing levels of host specificity ...... 70

3.4.3 Compatible arbuscular mycorrhizal fungi present in deer fern...... 72

3.4.4 Could salal and huckleberry truly have arbuscular mycorrhizal mycobionts? ..... 73

3.4.5 Conclusion ...... 76

Chapter 4: Importance of mycorrhizal inoculum for conifer seedling establishment in CH and HA clearcuts...... 83

4.1 Introduction ...... 83

4.2 Methods ...... 87

4.2.1 Study sites ...... 87

4.2.2 Experimental design and treatments...... 87

4.2.3 Seedling measurements ...... 89

4.2.4 Examination of root tips...... 89

4.2.5 Statistical analyses ...... 90

4.3 Results ...... 91

4.4 Discussion ...... 93

iii 4.4.1 Arbuscular mycorrhizal colonization of western redcedar ...... 94

4.4.2 Soil transfer from mature western redcedar trees ...... 96

4.4.3 Poor ectomycorrhizal colonization of western hemlock seedlings ...... 98

4.4.4 Conclusion ...... 101

Chapter 5: Conclusion ...... 114

5.1 Synopsis of chapter 2 ...... 114

5.2 Synopsis of chapter 3 ...... 116

5.3 Synopsis of chapter 4 ...... 117

5.4 Implications and recommendations ...... 118

5.4.1 Legacy of prior forest community on site regeneration ...... 118

5.4.2 Mycorrhizal inoculation of planted western redcedar ...... 119

5.4.3 Arbuscular mycorrhizal fungi affect decomposition ...... 120

5.4.4 Ectomycorrhizal fungi: do they aggravate or alleviate nitrogen deficiency? ..... 121

5.4.5 Absence of ectomycorrhizal hosts following disturbance ...... 122

5.5 Research strengths and limitations ...... 123

5.5.1 Forest types occur across a range of site properties ...... 123

5.5.2 Molecular characterization of fungal communities ...... 124

5.5.3 Community composition versus functional roles ...... 126

5.5.4 Temporal variation of mycorrhizal communities...... 127

5.5.5 Seedling bioassays and soil transfers as inoculation assessment ...... 128

5.6 The bigger picture: mycorrhizal fungi are an important biological factor ...... 130

Bibliography ...... 133

iv Appendices ...... 200

Appendix A: Primer sequences for PCR amplification of fungi ...... 200

Appendix B: DNA binning program ...... 203

Appendix C: Fungal operational taxonomic units from CH and HA forests ...... 206

v List of tables

Table 2.1 Summary of mean values and standard error (mean + SE) for selected tree characteristics of western redcedar (Cw), amabilis fir (Ba) and western hemlock (Hw). The understory plant species composition of the CH and HA forests are also included. Significant differences between forest types (p < 0.1) are indicated with bold text...... 51

Table 2.2 Mean diversity and richness indices of fungal operational taxonomic units found in Cedar Hemlock (CH) and Hemlock-Amabilis fir (HA) forest sites. Significant differences between forest types (p < 0.1) are indicated with bold text. Results are provided for both the total fungal community present within the LFH layers of the forest floor, as well as for general fungal groups based on ecological role, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the internal transcribed spacer regions...... 52

Table 2.3 Summary of operational taxonomic units (OTU) and taxonomic groups, found in either Cedar Hemlock (CH) or Hemlock-Amabilis fir (HA) forests, with statistically significant (p < 0.1) Indicator Species Analyses indices. Operational taxonomic units with sequence homology matches of the internal transcribed spacer with known fungal species in either GenBank or Unite have the homology match information provided. Clones of indicator species at the OTU level of identification comprised 30.4% of all fungal clones sequenced; clones of indicator taxon increased the total number by 6.1%...... 54

Table 3.1 Abundance of vascular plant species found within three-year post-harvest clearcuts of Hemlock-Amabilis fir forests (HA) and of Cedar-Hemlock forests (CH)...... 81

Table 3.2 Richness estimates and diversity indices of arbuscular mycorrhizal fungal operational taxonomic units detected in clone libraries of the small rRNA subunit gene amplified from DNA extracted from roots of western redcedar seedlings grown in greenhouse bioassays (Cw- greenhouse), from western redcedar seedlings harvested from old growth forests (Cw-natural)

vi and from roots of the dominant plant species in the three-year post-harvest clearcut sites of Hemlock-Amabilis fir (HA) and Cedar-Hemlock (CH) forests...... 82

Table 4.1 Effects of ecosystem type prior to forest harvest and source of mycorrhizal inoculum on seedling growth response and mycorrhizal colonization of western hemlock seedling roots after the first growing season (2009) in three-year post-harvest clearcuts on northern Vancouver Island. Results shown are mean values with standard errors in parentheses for seedling growth response (dry above-ground biomass, stem diameter and height) and percent mycorrhizal colonization of roots. Summary of restricted maximum likelihood (REML) results contrast the mean values for main fixed effects (ecosystem and inoculum) and their interaction (ecosystem*inoculum)...... 110

Table 4.2 Effects of ecosystem type prior to forest harvest and source of mycorrhizal inoculum on seedling growth response and mycorrhizal colonization of western hemlock seedling roots after the second growing season (2010) in three-year post-harvest clearcuts on northern Vancouver Island. Results shown are mean values with standard errors in parentheses for seedling growth response (dry above-ground biomass, stem diameter and height). Summary of restricted maximum likelihood (REML) results contrast the mean values for main fixed effects (ecosystem and inoculum) and their interaction (ecosystem*inoculum)...... 111

Table 4.3 Effects of ecosystem type prior to forest harvest and source of mycorrhizal inoculum on seedling growth response and mycorrhizal colonization of western redcedar seedling roots after the first growing season (2009) in three-year post-harvest clearcuts on northern Vancouver Island. Results shown are mean values with standard errors in parentheses for seedling growth response (dry above-ground biomass, stem diameter and height), mycorrhizal colonization of roots (the amount of mycorrhizal colonization within roots and the percent of seedlings colonized with arbuscular mycorrhizae) and degree of root colonization with dark septate endophytes. Summary of restricted maximum likelihood (REML) results contrast the mean values for main fixed effects (ecosystem and inoculum) and their interaction (ecosystem*inoculum)...... 112

vii Table 4.4 Effects of ecosystem type prior to forest harvest and source of mycorrhizal inoculum on seedling growth response and mycorrhizal colonization of western redcedar seedling roots after the second growing season (2010) in three-year post-harvest clearcuts on northern Vancouver Island. Results shown are mean values with standard errors in parentheses for seedling growth response (dry above-ground biomass, stem diameter and height), mycorrhizal colonization of roots (the amount of mycorrhizal colonization within roots and the percent of seedlings colonized with arbuscular mycorrhizae) and degree of root colonization with dark septate endophytes. Summary of restricted maximum likelihood (REML) results contrast the mean values for main fixed effects (ecosystem and inoculum) and their interaction (ecosystem*inoculum)...... 113

Table A.1. Primer sequences for PCR amplification of fungal DNA, used in chapters 2 and 3. . 200

viii List of figures

Figure 2.1 Illustration of plot layout used for collecting forest floor samples. Plot size was 20 m by 20 m. Within each plot, there were sixteen 5 m by 5 m subplots. Each subplot was assigned a number (1-16), and then 12 were chosen randomly. In each of the chosen subplots, a forest floor sample was collected from the center of one randomly selected quadrant (a-d) within the subplot (shaded gray)...... 43

Figure 2.2 Coleman collector curves for fungal operational taxonomic units detected in clone libraries from plots sampled from Cedar Hemlock forests (CH, indicated by red solid shapes) and Hemlock-Amabilis fir forests (HA, indicated by green open shapes). Collector curves from different sites are indicated by shape of marker (triangles for site RMnD, circles for site B405 and squares for site W80)...... 44

Figure 2.3 Proportion of fungal types, found in Cedar Hemlock (CH) and Hemlock-Amabilis fir (HA) forests, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the internal transcribed spacer regions of whole fungal communities present within the LFH layers of the forest floor. Percentages shown are the mean values from the three replicate sites of each forest type. Relative ratios are shown for (a) general fungal group based on ecological role in CH and HA forests, as well the relative percentage of fungi belonging to different taxon within each of these fungal groups for both (b) CH and (c) HA forests. Fungi classified as “unknown”, are from DNA sequences that lacked sequence homology matches to known fungi in sequence data bases and were classified using phylogenetic analyses of the large subunit region; the species identity and mode of nutrition of these fungi are unknown. Endophytic fungi and ericoid mycorrhiza (portions not visible on the graphs) made up less than 1% combined in both forest types...... 46

Figure 2.4 Proportion of ectomycorrhizal fungi found in (a) Cedar Hemlock (CH) and (b) Hemlock-Amabilis fir (HA) forests, as determined by sequence homology matches of cloned

ix DNA fragments from PCR amplification of the ITS regions of whole fungal communities present within the LFH layers of the forest floor. Percentages shown are the mean values from the three replicate sites of each forest type. Sequences of known ectomycorrhizal fungal made up 45% and 56% of total fungal sequences from CH and HA forest floor clone libraries, respectively. .. 47

Figure 2.5 Proportion of saprophytic fungi found in (a) Cedar Hemlock (CH) and (b) Hemlock- Amabilis fir (HA) forests, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the ITS regions of whole fungal communities present within the LFH layers of the forest floor. Percentages shown are the mean values from the three replicate sites of each forest type. Sequences of known saprophytic fungi sequences made up 34% and 25% of total fungal sequences from CH and HA forest floor clone libraries, respectively...... 48

Figure 2.6 Non-metric Multidimensional Scaling ordination of fungal communities found within individual clone libraries from Cedar Hemlock forests (CH, indicated by red solid shapes) and Hemlock-Amabilis fir forests (HA, indicated by green open shapes). Clone libraries from different sites are indicated by shape of marker (triangles for site RMnD, circles for site B405 and squares for site W80). Ordinations are shown for pair wise axis combinations (a-c)...... 49

Figure 2.7 Proportion of indicator to non-indicator fungal species, as defined by operational taxonomic units, found either exclusively in Cedar Hemlock (CH) or Hemlock-Amabilis fir (HA) forests (ecosystem type) or found in both ecosystems. Indicator species are operational taxonomic units that had Indicator Species Analyses (ISA) indices that were statistically significant with p-values < 0.1. Higher taxonomic groups with statistically significant ISA index values (not shown), comprised 12.6% of all clones and overlapped all fungal groups as defined in this figure legend. Of the indicator species (which comprise 30.4% of all fungal clones), 45.2% are ectomycorrhizal (13.7% of all fungal clones) and the remaining 54.8% are saprophytic (16.6% of all fungal clones)...... 50

x Figure 3.1 Maximum likelihood phylogenetic tree of Glomeromycota fungi. Phylogeny was constructed using a 485bp fragment of the small rRNA subunit gene, which is a region amplified using the primers AM1 and WANDA. One representative sequence of each operational taxonomic unit (OTU) sharing 99% sequence similarity detected in this study is indicated by OTU ID. Colored boxes indicate whether the OTUs were detected in roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), in roots of western redcedar seedlings harvested from old growth forests (Cw-natural) and/or in roots of the dominant vegetation species collected from three-year post-harvest clearcut sites. Family clades to which the OTUs detected in this study belong to are indicated. Support values >60% based on 1000 bootstrap values are shown. The choanozoan Corallochytrium limacisporum, a close relative to fungi (Cavalier-Smith and Allsopp, 1996), was used as an outgroup...... 78

Figure 3.2 Proportion of clone sequences of operational taxonomic units (OTUs) of Glomeromycota fungi found in roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), in roots of western redcedar seedlings harvested from old growth forests (Cw-natural) and in roots of the dominant vegetation species collected from clearcut sites. Colored sectors indicate specific OTUs shared between western redcedar and alternative host species. Gray sectors indicate OTUs that were not found in western redcedar; gray sectors with OTU labels next to them indicate OTUs shared between different host species, but not with western redcedar...... 79

Figure 3.3 Coleman collector curves for arbuscular mycorrhizal fungal operational taxonomic units detected in clone libraries of the small rRNA subunit gene amplified from DNA extracted from roots of different plant species. Collector curves for clone libraries generated from roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), from roots of western redcedar seedlings harvested from old growth forests (Cw-natural) and from roots of the dominant vegetation species from three-year post-harvest clearcut sites are indicated by curve color...... 80

xi Figure 4.1 Average above-ground dry biomass of western hemlock seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. Different letters above standard error bars indicate significant difference between treatments and forest types (p < .05)...... 103

Figure 4.2 Average height of western hemlock seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar- dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. Different letters above standard error bars indicate significant difference between treatments and forest types (p < .05)...... 104

xii of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. No statistically significant differences between treatments were detected (p < .05)...... 105

Figure 4.4 Average height of western redcedar seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar- dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. No statistically significant differences between treatments were detected (p < .05)...... 106

Figure 4.5 Average level of symbiotic infection with arbuscular mycorrhizae within roots of western redcedar seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled;

xiii seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. Different letters above standard error bars indicate significant difference between treatments and forest types (p < .05)...... 107

Figure 4.6 Average percent of western redcedar seedlings colonized with arbuscular mycorrhizae when planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. Different letters above standard error bars indicate significant difference between treatments and forest types (p < .05)...... 108

Figure 4.7 Correlation between root colonization with arbuscular mycorrhizae and dry above- ground biomass of western redcedar seedlings in a field trial carried out in three-year post- harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Results are shown for correlation between percent arbuscular mycorrhizal root colonization and seedling biomass after (a) the first growing season in CH clearcuts (F = 1.61, p = .208) (b) the first season in HA clearcuts (F = 17.95, p < .001), (c) the second growing season in CH clearcuts (F = 10.99, p = .002) and (d) the second growing season in HA clearcuts (F = 71.39, p < .001)...... 109

Figure A.1 Map of the 18S small subunit (SSU) ribosomal RNA gene with primer positions. Map includes primers used in this project and two other primers (NS1 and NS4) that are commonly used to amplify regions of this gene. Arrows indicate relative position of primers...... 201

xiv Figure A.2 Map of the internal transcribed spacer (ITS) regions with primer positions. Map includes primers included in this project and other primers that are commonly used to amply regions in this gene (ITS1, ITS2, ITS3 and ITS4). Arrows indicate relative position of primers to the ITS regions and the ribosomal RNA subunits 18S, 5.8S and 28S. Lengths of ITS regions are highly variable between different fungi; typically an amplification of both regions, including the 5.8S (approximately 158 bp (Korabecna, 2007)), spans 900-1200 bp...... 202

Figure C.1 Rank abundance chart of operational taxonomic units (OTUs) detected in forest floor samples collected from Cedar Hemlock forests (CH, indicated by yellow bars) and Hemlock-Amabilis fir forests (HA, indicated by green bars). Percent clone abundance is shown on a logarithmic axis. Identity of each OTU based on sequence homology matches of the internal transcribed spacer region and Bayesian analyses of the large subunit of ribosomal RNA gene are listed on the vertical axis. The data label for each bar includes the GenBank accession number for each OTU in brackets and their most probable ecological. Fungi classified as “unknown”, are from DNA sequences that lacked sequence homology matches to known fungi in sequence data bases and were classified using phylogenetic analyses of the large subunit region; the species identity and mode of nutrition of these fungi are unknown...... 219

xv List of acronyms

AM arbuscular mycorrhizae

ANOVA analyses of variance

BEC Biogeoclimatic Ecosystem Classification system

C:N carbon to nitrogen ratio

CH Cedar-Hemlock forest

CWD coarse woody debris

DNA deoxyribonucleic acid

EM ectomycorrhizae

HA Hemlock-Amabilis fir forest

ITS internal transcribed spacer

LSU large subunit of ribosomal RNA gene

MRPP Multi-response Permutation Procedures

NMR Non-metric Multidimensional Scaling

OTU operational taxonomic unit

PCR polymerase chain reaction

SCHIRP Salal Cedar Hemlock Integrated Research Project

SSU small subunit of ribosomal RNA gene

RNA ribonucleic acid rRNA ribosomal RNA gene

xvi Acknowledgements

Direction and hypotheses examined in this research project were originally initiated with the consultation and assistance of Suzanne Simard, Shannon Berch, Darlene Southworth and Thom O’Dell. Hamish Kimmins, Gordon Weetman and Cindy Prescott provided background information and documents of previous research completed on the forests studied in my research.

I developed the research projects under the advisement of committee members. For chapter 2, Richard Hamlin provided guidance with molecular techniques and sequence analyses. Chapter 3 was based on a hypothesis regarding potential compatible arbuscular mycorrhizal hosts, suggested by Shannon Berch. Though not a committee member, Maarja Öpik provided input for the selection of arbuscular specific primers. The method of inoculation and field trial design in chapter 4 was recommended by Suzanne Simard.

Suzanne Simard supervised the research, providing assistance and recommendations with sampling designs, data analyses and project direction. Suzanne Simard substantially contributed to the interpretation of results and manuscript preparation. Gary Bradfield and Valarie LeMay provided guidance with statistical analyses.

During the fieldwork on northern Vancouver Island, Jonathan Flintoft provided field training, company assistance and supervision from Western Forest Products Inc. Both Jonathon Flintoft and Nick Russell assisted with the selection of field sites. Annette van Niejenhuis, Erik Gagne and Dave Mogensen provided internal reports, company documents and datasets from Western Forest Products Inc. Seedlings used for bioassays in chapter 4 were provided by Annette van Niejenhuis from the Western Forest Products Ltd. Saanich tree farm.

Several research assistants provided help with fieldwork and lab work. Dru Yates, Carol Wright, Michael Wright and Patrick Guichon assisted with the planting and harvest of tree seedlings and collection of forest floor samples. Dru Yates assisted with all aspects of the lab work. Maddie Crowell and Allison Chen assisted with the processing of dry foliar samples

xvii discussed in chapter 4. Melissa Dergousoff and Carol Wright assisted with the staining and microscopy of root samples discussed in chapter 4. Annick St-Denis assisted with DNA extractions and PCR amplifications using arbuscular mycorrhizal specific primers discussed in chapter 3.

Both James Wright and Patrick Guichon designed and wrote computer programs used to merge and separate DNA sequence files. Patrick Guichon wrote a computer program to automate the binning of similar DNA sequences within the datasets generated in this project.

Funding and resources for this research was provided by several sources. Funding for the targeted metagenomic analyses in chapter 2 was provided by a Genome BC Science Opportunities Fund grant. The IMAJO Cedar Fund provided funding for the targeted molecular characterization of western redcedar mycorrhizae, as discussed in chapter 3. Three years of funding and support for research stipends, lab supplies, and fieldwork expenses was covered by the Forest Science Program. Advance Systems Integrators Ltd. assisted with fieldwork travel expenses, as well as computer and software expenses. Midvalley Rebar Ltd. donated the rebar that was used in the field bioassay experiment discussed in chapter 4. Western Forest Products Ltd. sponsored a NSERC Industrial Post Graduate Scholarship II. Scholarship support was also provided by the Edward W Bassett Memorial Scholarship in Reforestation, the Pacific Century Graduate Scholarship, a Donald S McPhee Fellowship and a University of BC Graduate Fellowship. Supplementary support was provided by the NSERC Discovery Grant of Suzanne Simard.

xviii Dedication

To God

To my family

Be inspired to pursue your interests and talents.

Regardless of challenges faced,

strive for excellence, persevere and never lose hope.

xix Chapter 1: Introduction

1.1 Preamble

So often, when people think about forests and the associated forces driving ecological processes, most do not realize the complex interactions occurring below ground. Focus is often on primarily the trees present (giants that rule the land), a landscape with an associated climate and the bounty of plants intermixed among the forest floor. But deep at work, in the unseen world below the forest floor, is a complex story of a diverse number of microscopic players busy at work, driving the forest dynamics above through processes of resource transformations, cooperative treaties and intense rivalry. A major group of these microbial organisms is the fungi, a group of filamentous organisms, existing as branching threads called hyphae. Many people may readily recognize fungi as the “plant-like” organisms that grow into mushrooms and rot decaying matter. Most people, however, will not be aware of the intimate association that some fungal species form with plants that can be key to the plant’s growth, competitiveness and place within the forest community. This symbiotic association, called mycorrhiza, occurs within the plant’s roots, whereby the fungi will take residence, draw upon part of the plant’s energy resources and in exchange, pay its lease by supplying crucial resources from the surrounding soil that the plant is unable to acquire on its own. The provision supplied by the fungi can often tip the balance as to whether the plant will succeed in a harsh environment, where resources are scarce and competition is substantial. The diversity and form of these associations between fungal and plant species is immense, with some partners being promiscuous and others being mutually exclusive. Dynamics that take place between these players below ground greatly contribute to the composition and structure of the above ground plant community, the development of an ecosystem, as well as the associated soil properties. Furthermore, following catastrophic disturbances, these fungi can play an important role for the future recovery of forest communities.

1 1.2 Research rationale

Understanding the ecology and role of mycorrhizal fungi for forest regeneration is important to successful re-establishment of a diversity of conifer species following forest harvesting in nutrient limited ecosystems. My research provides insight into mycorrhizal fungal community composition and diversity in coastal ecosystems on Vancouver Island and their role in the establishment and growth of western redcedar (Thuja plicata Donn ex D.Don) and western hemlock (Tsuga heterophylla (Raf.) Sarg.) during forest regeneration.

Mycorrhizal associations between symbiotic fungi and tree roots are crucial for seedling nutrient uptake, survival and growth, and tree productivity in nutrient-limited environments (van der Heijden et al., 1998b; Jonsson et al., 2001; Read and Perez-Moreno, 2003; Smith and Read, 2008). Many mycorrhizal fungal species allow trees to access nutrients from organic substrates, thus improving seedling competitiveness with other plants that increase dramatically following clear-cutting. There are several guilds of mycorrhizal fungi, which differ by taxa, physiological characteristics of structures formed with plant roots and biochemical traits important for nutrient cycling. Three guilds that play a significant role in my study include arbuscular mycorrhizae (AM), which colonize western redcedar, ectomycorrhizae (EM), which associate with western hemlock, and ericoid mycorrhizae. The AM fungi belong to a phylogenetically divergent division of aseptate fungi called Glomeromycota (Schüβler et al., 2001) that reproduce through very large asexual spores containing hundreds to thousands of nuclei (Bécard and Pfeffer, 1993). The symbiosis is characterized by fungal hyphal structures that penetrate the cell wall of roots, but not the cell membrane, forming complex, highly branched hyphal structures called arbuscules within the root cells (Smith and Read, 2008). Arbuscular mycorrhizal associations are the most common, forming with 74% of land plants (Brundrett, 2009), especially herbaceous species (Francis and Read, 1995; Wang and Qiu, 2006; Smith and Read, 2008). Ectomycorrhizae and ericoid mycorrhizae form associations among various fungi in the divisions Basidiomycota and Ascomycota (Smith and Read, 2008), however, there is little phylogenetic overlap between fungi of these two mycorrhizal guilds (Berch et al., 2002; Cairney and Meharg, 2003; Tedersoo et al., 2010). Ericoid mycorrhizae are associations

2 found among plants in the family Ericaceae; they are similar to AM, in that they form complex intracellular structures (Perotto et al., 2002). Ectomycorrhizae, on the other hand, form a visible mantle of tissue surrounding the exterior of actively growing plant root tips; hyphae grow between the cortical cells in roots, forming a Hartig net that does not penetrate root cells (Smith and Read, 2008). Many plant species are guild specific, forming an association with a particular mycorrhizal guild and often with select fungal species within that guild (Molina and Trappe, 1982; Gianinazzi-Pearson, 1984; McGonigle and Fitter, 1990; Molina et al., 1992; Bidartondo and Bruns, 2002; Taylor et al., 2003; Wang and Qiu, 2006). Western redcedar and western hemlock are considered to have strict fidelity to their specific mycorrhizal guild (AM vs EM) of fungi (Molina et al., 1992), but potentially with considerable fungal species variability within each mycorrhizal guild.

My research contributed to the forestry regeneration project known as the Salal Cedar Hemlock Integrated Research Project (SCHIRP), which is addressing a well-known problem with growth of conifers planted in clear-cuts on northern Vancouver Island, British Columbia, Canada. The two particular forest types of interest, which were first described and characterized by Lewis (1982), are the Cedar-Hemlock (CH) forests (which are western redcedar-dominated) and the Hemlock-Amabilis fir (HA) forests (which are western hemlock- dominated). Seedlings of Sitka spruce (Picea sitchenisis), western hemlock and amabilis fir (Abies amabilis Douglas ex J.Forbes) planted on CH cutovers are in poor condition within approximately 5-8 years after clear-cutting, indicated by chlorosis, reduced growth rates, and severe foliar nitrogen and phosphorus deficiencies. This nutrient deficiency coincides with the dominance of salal (Gaultheria shallon Pursh) on CH sites. When planted on adjacent HA cutovers, these same tree species do not show these growth problems (Prescott, 1996a; Mallik and Prescott, 2001; Blevins and Prescott, 2002). Western redcedar regenerates without problem on CH clearcuts; however, there is high mortality of natural germinants of western redcedar in HA forests (Weber et al., 2003).

Initially there were two major research objectives in SCHIRP. One objective was to identify factors and the underlying ecological mechanisms that differentiate CH and HA forests that influence the growth and nutrient acquisition of different regenerating conifer species

3 (Prescott, 1996a; Mallik and Prescott, 2001; Blevins and Prescott, 2002). The second objective was to establish research trials to study the growth response of selected conifers to various fertilizer and scarification treatments for determining best management strategies (Prescott, 1996a; Mallik and Prescott, 2001; Blevins and Prescott, 2002). The latest work, which was initiated by Western Forest Product Inc., was to identify potential linkages between site quality and tree response to refine associated ecosystem-based interpretations for improved forest management (McWilliams and Klinka, 2005b; McWilliams et al., 2007). Management recommendations were proposed in a new Biogeoclimatic Ecosystem Classification key, however, past researchers have not been able to fully distinguish mechanisms underlying regeneration success between these forests based on abiotic variables alone. Based on forest floor characteristics (including pH and humus forms), Blackwell & Associates proposed that nutrient cycling is driven primarily by fungi (McWilliams et al., 2007). If this is true, then understanding mycorrhizal fungal biology is essential for uncovering reasons behind soil nutrient limitations, poor plantation performance and designing successful forest regeneration practices.

1.3 Literature review

1.3.1 Review of studies from the Salal Cedar Hemlock Integrated Research Project

Much research invested in identifying factors that might be different between HA and CH forests has revealed distinct differences in nutrient availability and tree growth between these forest types, yet has shown that there is considerable overlap in physical site properties. It is well established that tree seedlings in CH sites are nitrogen and phosphorus limited, leading to significantly poorer conifer regeneration in CH sites compared to HA sites (Blevins et al., 2006). Originally it was hypothesized that the cultivation effect of repeated wind-throw caused higher nutrient availability in windstorm-derived, natural second-growth stands of HA, compared with adjacent old-growth CH stands (Lewis, 1982). However, in a study by Messier et al. (1995), mixing of the surface layers had little effect on nutrient availability. Though mixing has been shown to slightly improve growth of western hemlock and reduce salal biomass up to

4 5 years after treatment, it also decreased the soil nutrient status (assessed by soil nutrient availability indices) of CH sites, whereas it did not have a long term impact on HA sites. Availability of nitrogen and phosphorus decreased in both forest types two years after treatment, but HA forests recovered whereas CH forests did not (Messier et al., 1995).

Until recently, it was hypothesized that the CH and HA phenomenon was forests representing two distinct successional stages, whereby in the absence of disturbance, HA forests will develop into CH forests as western redcedar invades small gaps that open during forest succession (Lewis, 1982). More recent evidence based on chronosequence data indicates that without disturbance, the forests are self-replacing stand types and disturbance plays a role in allowing western redcedar establishment into HA stands (Weber et al., 2014). Lately, a predominant hypothesis is that different abiotic site conditions, especially moisture, favor the development of either CH or HA forests (Klinka, 2006; Sajedi et al., 2012; Prescott et al., 2013). Originally, Prescott and Weetman (1994) observed many CH forests growing on lower slopes, indicating that CH forests are wetter and poorly drained compared to HA forests (Battigelli et al., 1994). Based on a survey of 128 regenerating research plots at the SCHIRP Installation near Port McNeill, Vancouver Island, British Columbia to characterize CH and HA forests using the Biogeoclimatic Ecosystem Classification (BEC) system (McWilliams and Klinka, 2005b), a range of soil moisture regimes and soil nutrient regimes overlapped both forest types (McWilliams and Klinka, 2005b; Klinka, 2006). Without statistical analyses of the data collected, Klinka (2006) concluded that HA sites are predominantly drier and nutrient-medium corresponding to a BEC site series of 01 (Blueberry) whereas CH sites are predominantly wetter and nutrient-poor corresponding to a BEC site series of 06 (Deer fern). However, statistical analyses of the data collected did not support this claim. There was no correlation between moisture regime, nutrient regime or site series with either CH and HA sites; within each forest type, soil nutrient regime was correlated with tree productivity, but not correlated with soil moisture regime or forest type (Guichon et al., unpublished manuscript). Sajedi et al. (2012) detected significant differences in mean soil moisture, depth of aeration and redox potential between forest stand types. However, Weber et al. (2014) argued that despite mean differences detected in the study by Sajedi et al. (2012), there was high overlap between samples from both stand types;

5 aeration of CH stands sampled ranged from 6 to 80 cm, whereas HA stands ranged from 31 to 73 cm. Furthermore, other studies have failed to find differences in parent material and topography (Keenan et al., 1993; Albani and Lavery, 2000; Weber et al., 2003; Weber et al., 2005). In a comprehensive survey of forests throughout northern Vancouver Island, old-growth stands of both CH and HA forests have been located on sites with similar moisture and soil quality (McWilliams et al., 2007). Though environmental conditions may influence the development of some stands, the variability of moisture and aeration found between sites is not consistent enough to be solely responsible for patterns of stand differentiation (Weber et al., 2014).

Soil moisture is not always reflective of abiotic site properties alone, but is also influenced by evapotranspiration rates. Stand density greatly impacts soil moisture availability and aeration depth due to associated evapotranspiration rates (Douglas, 1965). Various trials have shown that reducing stand density via thinning results in a decrease in aeration depth; the difference of soil aeration can be a matter of several feet depth (Douglas, 1965). In humid regions, a 20% reduction in stand density is sufficient to reduce evapotranspiration and increase streamflow from excess moisture (Douglas, 1965). Furthermore, as forests age, reduction in evapotranspiration rates are associated with the decreases in stand density (Vertessy et al., 2001), basal area (Moore et al., 2004), stomatal conductance and hydraulic conductivity from increased tree height (Gower et al., 1996; Murty et al., 1996). Age is also associated with an increase in stand structural complexity, which in turn increases soil moisture availability, especially in the top forest floor (Unsworth et al., 2004). In a Pacific Northwest forest, Moore et al. (2004) estimated that young forests use 3.27 times more water than old- growth forests, thereby altering site water balance via elevated evapotranspiration in vigorous young stands. With regards to the CH and HA forests with overlapping site conditions, differences in forest floor properties such as moisture, decomposition rates and nutrient availability could be a result of the forest community, age and structure, rather than the cause of the forest community, even though western redcedar could be the species best adapted to some of the physical site differences (e.g., moisture levels found in depressions versus crests). The younger HA forests, which characterize the end of the stem exclusion stage of stand

6 development, have a dense canopy with minimal shrub cover due to lack of light availability, whereas old-growth CH forests have 30-50% crown closure and a dense understory shrub layer dominated by salal (McWilliams et al., 2007). Such drastic differences in tree density and canopy cover play important roles in soil hydrology of uncut forests, leading to drier soil conditions in mature HA forests. Supporting this hypothesis, moisture content and redox potential were not different between CH and HA sites following clearcutting, even though moisture difference were detected in uncut forests; moisture content increased in HA sites following clearcutting (Sajedi et al., 2012).

A good deal of variation has been found and often physical site properties were not significantly different between forest types. In both forest types, there are high woody debris and detritus accumulations. Keenan et al. (1993) found that CH sites on average had significantly higher forest floor mass in the LFH layers, indicating higher inputs or slower turnover rates. However, even within their study, they found high variability in the amount of woody debris within CH and HA sites (Keenan et al., 1993). They considered that a large number of tree boles was deposited on the CH forest floor under cool, wet climatic conditions, when the logs never dried out, leading to low rates of decay (Keenan et al., 1993). However, decomposition rates between CH and HA material are similar (Prescott et al., 1995; Keenan et al., 1996) as is the carbon concentration found in both HA and CH soils (Prescott et al., 1995). In the old growth forests studied by Brunner and Kimmins (2003), coarse woody debris (CWD) biomass on CH sites was not necessarily greater than that on HA sites. In one HA forest, CWD biomass exceeded that of the CH sites. Nitrogenase activity rates, which are responsible for biological nitrogen fixation, have also found to be similar between forest types (Brunner and Kimmins, 2003). Additionally, Leckie et al. (2004) found that depth of forest floor was similar between CH and HA sites, but that CH had higher moisture content and there was greater fungal biomass. Though Leckie et al. (2004) compared the total microbial community composition and found no significant differences between CH and HA sites, the molecular techniques employed did not provide insight into differences between AM, EM, ericoid mycorrhizal and saprophytic fungal communities. Differences between mycorrhizal

7 communities could be important to ecosystem processes governing above-ground plant community composition of the CH and HA forests.

1.3.2 Importance of mycorrhization

Forming mycorrhizal associations is considered an adaptation of plants to nutrient- limiting conditions (Wallenda and Kottke, 1998). Nutrient rich sites have their own suite of adapted mycorrhizal fungal species (Kranabetter et al., 2009a; Kranabetter et al., 2009b; Avis, 2012), however, under conditions of sufficient nutrient availability, the benefit of mycorrhization can be less pronounced and, in the case of EM, some mycorrhizal fungal species can shift from a mutualistic relationship to a parasitic one (Jones and Smith, 2004; Smith and Read, 2008). In contrast, under nutrient-poor conditions, the benefits of mycorrhization are more prominent and the competitive interactions between roots of different mycorrhizal plants can have a significant impact on plant performance and establishment (Janos, 1980). It has been proposed that the spatial distribution, structure and function of ericoid, EM and AM mycorrhizal fungi influence the colonization and performance of their respective hosts (Janos, 1980; Read, 1991; Brundrett and Abbott, 1995; Lovelock et al., 2003; Wardle et al., 2004), especially since the establishment of seedlings may depend on the availability of the appropriate mycorrhizal inoculum (Haskins and Gehring, 2005).

The relative amount of infective propagules available for potential mycorrhizal fungal inoculation is correlated with aboveground plant species composition (Moyersoen et al., 1998; Korb et al., 2003). Mycorrhizal inoculum sources include below-ground mycorrhizal networks (Brundrett and Abbott, 1994; Kytöviita et al., 2003; Giovannetti et al., 2004; Simard and Durall, 2004), residual mycorrhizal associations on root systems from harvested trees (Biermann and Linderman, 1983; Friese and Allen, 1991), fungal spores (Daniels et al., 1981; Fox, 1983; Friese and Allen, 1991), and resistant propagules such as sclerotia (Trappe, 1969). In a study by Teste et al. (2009a), wind- and soil-borne EM inoculum already present in soil was the most important inoculum source for newly colonized seedlings in interior Douglas-fir (Pseudotsuga menziesii var. glauca) forests. Unfortunately, mycorrhizal inoculum sources can be limited during

8 reforestation, even though they are often considered widely abundant. Actively growing hyphae and mycelia are greatly reduced by severe disturbances such as clear-cutting (Byrd et al., 2000; Jones et al., 2003), burning (Stendell et al., 1999; de Román and de Miguel, 2005), and application of fertilizer (Gill and Lavender, 1983; Kernaghan et al., 1995; Frey et al., 2004). Spore dispersal, though considered a plentiful source of inoculum, is also limited in its effectiveness at colonizing seedlings. Depending on fungal species, very few spores may germinate successfully, with viability dependent on suitability of nutrient and environmental conditions (Fox, 1983; Giovannetti, 2000; Edman et al., 2004), interaction with other soil organisms (Fitter and Garbaye, 1994; Garbaye, 1994), and proximity to roots of specific host plant species (Theodorou and Bowen, 1987). Furthermore, inoculum availability is not the only mycorrhizal factor important for plant establishment; both diversity and community composition of mycorrhizal fungi influence plant establishment and productivity (van der Heijden et al., 1998b; Baxter and Dighton, 2001; Lovelock and Miller, 2002; van der Heijden et al., 2003; Johnson et al., 2005a; Scheublin et al., 2007).

1.3.3 Antagonistic interactions between mycorrhizal guilds

Interactions between the different guilds of mycorrhizae, such as competitive exclusion of the fungi themselves, or prevention of the establishment of plants that are host to alternate mycorrhizal guilds, are poorly understood. Though this area of research has received little attention, there are a few studies examining the effects of AM and EM interactions on below- ground fungal diversity and the establishment or productivity of various plant species.

Competitive interactions between plants can be greatly influenced by both AM (Allen and Allen, 1984; Newman et al., 1992; Hartnett et al., 1993; van der Heijden et al., 1998b; Hartnett and Wilson 1999; O'Connor et al., 2002; Bray et al., 2003; van der Heijden et al., 2003; Scheublin et al., 2007) and EM colonization (Perry et al., 1989; Pedersen et al., 1999). There is also evidence that mycorrhizal guilds can have antagonistic interactions with each other. Although EM and AM can occupy similar niches, Moyersoen et al. (1998) found that a soil volume occupied by roots of an EM species was unlikely to also be occupied by roots of an AM

9 species. Under lab conditions, isolates of ericoid fungi have been shown to inhibit the growth of EM fungi on the same culture plates (Xiao, 1994). In the temperate forest of northwestern Connecticut, EM networks had strong, negative effects on the survival of AM red maple (Acer rubrum) (normally a superior competitor compared to the other tree species tested), whereas EM networks had neutral or positive effects on the survival of three EM tree species, Betula allegheniensis, Pinus strobus, and Tsuga canadensis (Booth, 2004). Booth (2004) proposed that interspecific differences in fungal composition of mycorrhizal networks during seedling establishment influenced forest community development by promoting coexistence of EM tree species and limiting persistence of AM species. Haskins & Gehring (2005) discovered that pinyon pine (Pinus edulis) seedlings were six times less likely to be colonized by EM fungi and had reduced levels of mycorrhizal root tips when grown in soil from AM juniper (Juniperus monosperma)-dominated forest zones. In the field, trenching reduced below-ground competition with juniper roots and resulted in a two-fold increase in abundance of EM roots on pinyon pine (Haskins and Gehring, 2004). When ryegrass (Lolium sp.) was planted in restoration sites of establishing sugar pine (Pinus lambertiana), it resulted in reduced growth rates, high mortality rates, and reduced root tip and mycorrhizal formation of pine seedlings (Amaranthus et al., 1993). Conversely, Pedersen et al. (1999) found that providing additional EM inoculum using Pisolithus arhizus improved the competitiveness and uptake of phosphorus of slash pine (Pinus elliottii) when grown with the AM grass, Panicum chamaelonche.

1.3.4 Mycorrhizal guilds and their role in nutrient cycling

Ecosystem nutrient cycling is affected by the dominant type of mycorrhizal association in the plant community (Read, 1991; Cornelissen et al., 2004; Averill et al., 2014). External hyphal growth of both EM (Finlay et al., 1992; Bending and Read, 1995; Perez-Moreno and Read, 2000) and AM fungi (St. John et al., 1983; Warner, 1984; Joner and Jakobsen, 1995; Hodge et al., 2001) can be stimulated by organic matter, but they differ in their ability to decompose organic substrates.

10 Although AM hyphae preferentially associate with organic particles (St. John et al., 1983), there is little evidence that AM fungi are efficient at decomposing organic substrate (Ames et al., 1983; Francis and Read, 1994; Cliquet et al., 1997; Smith and Read, 2008). Arbuscular mycorrhizal fungi can improve nutrient uptake of their host plant in the presence of organic material (Näsholm et al., 1998; Hodge et al., 2001), but this is quite possibly because of the release of nutrients by stimulation of root exudates from the host plant and/or decomposition by other microbes (Joner and Jakobsen, 1995).

Ectomycorrhizal fungi, on the other hand, are better able to decompose organic substrates (Singer and Aguiar, 1986; Bending and Read, 1995; Näsholm et al., 1998; Tibbett et al., 2000; Tibbett and Sanders, 2002; van Hees et al., 2003). Both the decomposition rates of various organic substrates and plant growth can be significantly enhanced by EM colonization (Dighton et al., 1987). For example, when colonized with EM fungi, Betula pendula (Abuzinadah and Read, 1989a, 1989b), Pinus contorta (Abuzinadah et al., 1986), Picea mariana and P. sitchensis (Abuzinadah and Read, 1986a, 1986b) were all able to use protein nitrogen readily. In tropical rainforests, such as the Amazon (Singer and Aguiar, 1986; Newbery et al., 1988) and secondary Australian forests (Hopkins et al., 1996), EM fungi have been suggested as the primary organisms responsible for cycling nutrients for tree productivity (Singer and Aguiar, 1986; Newbery et al., 1988). In boreal forests, mineral weathering and biodegradation of organic ligands by EM fungi are central for plant acquisition of limited nutrients and for podzolization (Heinonsalo et al., 2004).

The capacity of EM fungi to break down organic material is relatively small compared to saprophytic fungi (Colpaert and van Tichelen, 1996). Based on analyses of fungal genomes, compared to saprophytic fungi, EM fungi have a reduced complement of genes encoding for plant cell wall-degrading enzymes; nonetheless, a unique array of these enzymes has been retained allowing varying abilities to degrade lignin and cellulose among different EM fungal species (Plett and Martin, 2011; Kohler et al., 2015). Consequently, litter decomposition in nutrient-poor soil has been suppressed where EM are the dominant fungi (Singer and Aguiar, 1986; Newbery et al., 1988; Clemmensen et al., 2015; Fernandez and Kennedy, 2015). A

11 phenomenon known as the ‘Gadgil’ effect (Gadgil and Gadgil, 1971, 1975), attributes the accumulation of slowly degraded humus horizons in forests dominated by EM tree species to the inhibition of saprotrophic organisms by EM fungi (Bending, 2003). Ectomycorrhizal fungi have been shown to outcompete saprophytic fungi (Lindahl et al., 1999; Lindahl et al., 2001; Lindahl et al., 2002; Finlay, 2005) and they can inhibit soil bacterial activity (Rasanayagam and Jeffries, 1992; Olsson et al., 1996). Colonization of litter by EM fungi further reduces the quality of the litter by increasing the carbon to nitrogen ratio and depleting nitrogen contents (Bending and Read, 1995). Arbuscular mycorrhizal fungi have also been shown to inhibit saprophytic fungal growth (Fracchia et al., 1998; Olsson et al., 1998; Green et al., 1999), however, little is known about the degree to which this occurs or its impact on nutrient cycling in the forest floor. Discrepancies in the ability of EM versus AM to decompose organic substrates while suppressing saprophytic fungal communities could potentially lead to distinct differences in nutrient cycling and availability between AM- and EM-dominated ecosystems with similar litter inputs.

1.3.5 Mycorrhizal studies in the Salal Cedar Hemlock Integrated Research Project

In several past studies, researchers have investigated the role of mycorrhizae in HA and CH forests. Previous mycorrhizal studies at SCHIRP have provided insight into the diversity of EM and ericoid mycorrhizal fungi, as well as the importance of AM fungi to seedling survival. However, further research is required to test the potential ecological roles of these fungi in plant competition and tree establishment during forest regeneration.

The earliest SCHIRP studies focused on ericoid mycorrhizae associated with salal (Xiao and Berch, 1992; Xiao and Berch, 1995; Xiao and Berch, 1996; Xiao and Berch, 1999). Ericoid mycorrhizae allow their hosts to dominate in nutrient-limited environments where nutrients are bound in complex organic substrates. For example, through the production of a range of extracellular enzymes, the ericoid fungus, Pezoloma ericae (D.J. Read) Baral (formerly known as Hymenoscyphus ericae), can degrade complex organic substrates and provide the nutrients to their host plants from otherwise inaccessible sources (Leake and Read, 1989, 1990; Bending

12 and Read, 1996; Kerley and Read, 1997; Cairney and Burke, 1998; Kerley and Read, 1998; Cairney and Ashford, 2002; Sokolovski et al., 2002; Cairney and Meharg, 2003). The CH sites, which have large accumulations of organic material with low decomposition rates, provide an ideal environment for salal to grow vigorously with help from its mycorrhizal fungi. Furthermore, using in vitro tests, Xiao (1994) found evidence of growth inhibition of EM fungi by isolates of ericoid fungi. Similarly, in a field study by Walker et al. (1999), suppression of EM roots and reduced seedling growth rates of northern red oak (Quercus rubra) and eastern hemlock (Tsuga canadensis) occurred when planted near the ericoid plant, Rhododendron maximum.

In a molecular survey of ITS regions from pooled ectomycorrhizal root tips sampled from 18-year-old western hemlock collected at SCHIRP regeneration sites, Wright et al. (2009) not only found high EM richness, but also detected several fungi matching ericoid mycorrhizal sequences (including Pezoloma ericae) identified previously on salal sampled near the same area on Vancouver Island (Monreal et al., 1999; Allen et al., 2003). Because of the methods used, it could not be determined whether the ericoid fungi detected in the western hemlock roots were from EM morphotypes or were root tip contaminants (Wright et al., 2009). Attempts to isolate EM fungi from western hemlock root tips resulted in cultures of the EM fungus, Cenococcum geophilum, the ericoid fungi Pezoloma ericae, Phialophora spp. and Oidiodendrum spp., as well as many contaminating molds (Wright, unpublished results). Interestingly, ericoid fungal isolates have also been identified as potential EM fungi (Bergero et al., 2000; Cairney and Meharg, 2003). Currently, we do not know whether the fungal strains colonizing ericoid plants are same ones that can form EM associations with western hemlock.

There is supporting evidence for low fidelity to a mycorrhizal guild among ericoid isolates. Through phylogenetic analyses of the ITS region of ribosomal DNA, the EM fungus Piceirhiza bicolorata isolated from EM hosts Picea abies, Pinus sylvestris, Betula pubescens, Populus tremula, Quercus robur and Salix phylicifolia formed subclades within the Pezoloma ericae aggregate (Vrålstad et al., 2000). When Vrålstad et al. (2002) tested eight isolates of the Pezoloma ericae aggregate for their ability to form mycorrhizal associations with sterile EM and

13 ericoid host plants, they formed either EM or ericoid associations, but not both. Piercey et al. (2002) found that isolates of Pezoloma ericae and a Variable White Taxon from ericoid roots developed an intracellular association (which is not characteristic of EM symbiosis) with roots of Pinus mariana. Similarly, Bergero et al. (2000), found one sterile ericoid fungal isolate that formed atypical colonization patterns on the EM plant, Quercus ilex. In contrast, Villarreal-Ruiz et al. (2004) isolated a Piceirhiza bicolorata-like ectomycorrhiza from Pinus sylvestris which, through phylogenetic analyses of the ITS region of ribosomal DNA, grouped with Cadophora finlandia in the Pezoloma ericae aggregate. In aseptic synthesis, the isolate formed both an EM association with Pinus sylvestris and an ericoid association with Vaccinium myrtillus (Villarreal- Ruiz et al., 2004). Mycobionts from the Pezoloma ericae aggregate could potentially impart similar benefits to ectomycorrhizal hosts as has been found in ericoid associations.

When studying factors that influenced the establishment of western redcedar in old- growth HA and CH sites, Weber et al. (2005) found a strong correlation between AM colonization and survivorship of western redcedar seedlings. Greenhouse bioassays that compared the mycorrhizal colonization of western redcedar seedlings in CH versus HA substrates indicated low colonization by AM in the HA substrate (Weber et al., 2005). However, the failure to form a mycorrhizal association does not necessarily reflect the lack of inoculum, but rather might be inhibition of AM (Daniels and Trappe, 1980; Tommerup, 1985; Hetrick and Wilson, 1989; Koide and Schreiner, 1992) in the HA substrate. Furthermore, though HA forests are EM-dominated, both AM and ericoid plant species co-exist within these forests and could potentially provide sources of mycorrhizal inoculum. For example, HA sites have pteridophyte taxa, including deer fern (Blechnum spicant) (McWilliams et al., 2007; Sajedi et al., 2012) which is an AM host (Harley and Harley, 1987a, 1987b); whether the AM taxa are compatible between western redcedar and ferns is unknown. Although many AM fungal species are non-host specific, recent bioassay, inoculation trials and molecular work has revealed greater host specificity among AM fungal strains than previously believed (McGonigle and Fitter, 1990; Eom et al., 2000; Klironomos, 2000; Vandenkoornhuyse et al., 2003; Sanders, 2003a; Sanders, 2003b; Helgason et al., 2007; Öpik et al., 2009). Though plausible, it is unknown whether western redcedar and deer fern share mycobionts.

14 Western hemlock, which was used as a non-AM control by Weber et al. (2005), grew equally well in CH and HA substrates, but EM colonization was not examined. They concluded that western hemlock was not limited by EM inoculum potential; however, under greenhouse conditions, it is common for greenhouse EM fungal species to become established while displacing the natural fungal species (Trappe and Strand, 1969; Chu-Chou and Grace, 1985; Hung and Trappe, 1987). Also, though it is believed that western redcedar and western hemlock are mutually exclusive with respect to their mycorrhizal fungal symbionts (Weber et al., 2005; Smith and Read, 2008), there is evidence indicating the potential of western hemlock germinants to form AM associations during early establishment. For example, seedlings grown in pots with mixtures of salal and Rhododendron macrophylum resulted in AM colonization of 25% of the western hemlock seedlings; cross-sections of roots revealed both vesicles and dark septate endophytes growing under EM mantles (Cázares and Smith, 1996). Contrary to Weber et al. (2005), Prescott et al. (1993) showed that both western redcedar and western hemlock seedlings grew more slowly with reduced nitrogen and phosphorus uptake when grown in forest floor material collected from CH sites than from HA sites. Prescott et al. (1993) did not examine mycorrhizal colonization, making it difficult to compare results with Weber et al. (2005).

These past studies have provided a glimpse into the possible roles mycorrhizae play in ecosystem processes at CH and HA sites and the potential significance of mycorrhizal inoculum to forest regeneration. Further study is warranted to better understand these relationships. Furthermore, there is no information regarding the distribution of AM and EM fungi in CH and HA forests. It is possible that both ecosystems harbour both AM and EM, but as with other complex ecosystems, the inoculum is likely patchy. Practices that result in insufficient inoculum potential of the host-specific guild remaining on-site could contribute to failure of the host species to regenerate.

15 1.4 Research objectives

The objective of this research project was to understand the mycorrhizal community composition of western redcedar-dominated CH forests and western hemlock-dominated HA forests and to examine its role in conifer seedling establishment following clearcutting. The close proximity of CH and HA forests provided a unique opportunity to study and compare AM- dominated versus EM-dominated ecosystems. The specific questions that were addressed were as follows:

1) Are there differences in AM and EM fungal communities or inoculum potential between CH and HA sites? I hypothesized that the guild of mycorrhizal fungi and inoculum present in the forest floor is governed by the dominant host tree species, such that CH sites, which are dominated by western redcedar and have dense understory vegetation comprised primarily of the ericoid plant salal, contain predominantly AM and ericoid mycorrhizal fungi, whereas HA sites dominated by western hemlock contain predominantly EM fungi. My null hypothesis was that there are no differences between mycorrhizal communities or inoculum potential of EM and AM between CH and HA forests. This objective was accomplished by sampling forest floors from replicate blocks of these forests and using targeted metagenomic characterization of DNA extracted (chapter 2). By integrating molecular tools, I had the opportunity to characterize mycorrhizal communities, extrapolate their potential functional role for nutrient cycling in the forest floor and infer mycorrhizal inoculum potential between forest types.

2) Are there alternative AM or EM plant species that harbour mycorrhizal inoculum compatible with western redcedar or western hemlock? I hypothesized that understory plants in CH and HA forest clearcuts have compatible mycorrhizal fungi for either western redcedar or western hemlock, thus providing a potential source of mycorrhizal inoculum for tree seedlings. My null hypothesis was that mycorrhizal fungi of the understory plant community are host specific and therefore do not contribute to the pool of inoculum sources for tree seedlings. This objective was accomplished by

16 molecular characterization of mycorrhizal fungal DNA extracted from plant roots collected from clearcuts of CH and HA forests (chapter 3). By identifying mycorrhizal species using sequencing homology matches and phylogenetic analyses, I was able to examine the specificity of mycorrhizal associations found among the dominant plant species and extrapolate the likelihood that mycorrhizal networks may influence seedling mycorrhization in a given site.

3) How does mycorrhizal colonization in different ecosystems influence seedling establishment, survival, and growth? I hypothesized that mycorrhizal colonization is critical for conifer seedling establishment in nutrient-poor conditions, and that differences in mycorrhizal inoculum potential between ecosystems greatly influences colonization and attributes of seedling performance. My null hypothesis was that mycorrhizal colonization does not limit or influence seedling establishment and growth. This objective was accomplished using field bioassays of planted conifer seedlings in both CH and HA clearcuts (chapter 4). By utilizing seedling bioassays, I was able to examine the role of mycorrhizal inoculum potential for the establishment and growth of both western redcedar and western hemlock during forest regeneration.

4) Can modifying mycorrhizal inoculum at the time of planting improve seedling establishment and productivity? I hypothesized that providing appropriate host-specific mycorrhizal inoculum at the time of planting improves seedling establishment and productivity. My null hypothesis was that modifying the mycorrhizal inoculum has neutral or even negative effects on seedling establishment. This objective was accomplished using field bioassays of planted conifer seedlings in both CH and HA clearcuts, applying different treatments of reciprocal forest floor transfers from mature AM- and EM- dominated forests at the time of planting (chapter 4). Western redcedar and western hemlock seedlings were planted in CH and HA clearcuts with (a) forest floor transferred from nearby HA forests, (b) forest floor transferred from nearby CH forests and (c) no forest floor transfer.

17 Chapter 2: Forest floor fungal community composition and mycorrhizal inoculum potential of CH and HA forests

2.1 Introduction

Fungi are heterotrophic microorganisms that perform essential roles in decomposition, nutrient cycling and ecosystem processes (Foster, 1949; Moore-Landecker, 1972). Two important groups of fungi, which differ by mode of nutrition, are saprophytes and symbionts called mycorrhizae (Högberg et al., 1999). Saprophytic fungi are free-living organisms that obtain their nutritional requirements, especially nitrogen and carbohydrates, from degradation of organic matter (Foster, 1949; Swift, 1982). Mycorrhizal fungi are symbionts that form associations within the roots of plants and are dependent upon host plants for photosythetically fixed carbohydrates (Smith and Read, 2008).

Mycorrhizal fungi influence soil properties and decomposition processes. Mycorrhizal fungi can immobilize nutrients into their mycelium (Näsholm et al., 2013) and produce novel recalcitrant compounds, thereby effecting soil nutrient availability and altering humification and soil aggregate stability (Wright and Upadhyaya, 1996; Wright and Upadhyaya, 1998; Wright et al., 1999; Miller and Jastrow, 2000). Through the production of extracellular enzymes, EM and ericoid mycorrhizal fungi have varying abilities to degrade organic litter (Bending and Read, 1996; Colpaert and van Tichelen, 1996; Bending and Read, 1997), albeit, they are typically less efficient at decomposition compared to saprophytic fungi (Colpaert and van Tichelen, 1996). Arbuscular mycorrhizal fungi, are predominantly believed to have no saprophytic ability (Smith and Read, 2008), yet there is evidence that they can capture nitrogen from organic matter (Johansen et al., 1994; Hawkins et al., 2000; Hodge et al., 2001; Tanaka and Yano, 2005; Leigh et al., 2009; Hodge and Fitter, 2010; Whiteside et al., 2012) and alter nearby fungal and bacterial saprophytic community structure (Fracchia et al., 1998; Olsson et al., 1998; Green et al., 1999; Hodge, 2000). There is often niche overlap between saprophytic and mycorrhizal fungi (Dighton et al., 1987; Shaw et al., 1995). Though in situ dynamics between various fungal groups are

18 poorly understood, it is currently believed that competition and inhibition from mycorrhizal fungi can result in shifts in decomposition processes by saprophytes (Gadgil and Gadgil, 1971, 1975; Cairney and Meharg, 2002; Bending, 2003; Koide and Wu, 2003; Leake et al., 2003b; Lindahl et al., 2010).

Recent analyses of global datasets indicate that dominance of a mycorrhizal guild in a given ecosystem substantially influences litter decomposition (Averill et al., 2014). In temperate and boreal forests dominated by EM trees, EM fungi are believed to drive a higher carbon to nitrogen (C:N) ratio in decomposing organic matter compared with saprophytic fungi (Bending and Read, 1995; Orwin et al., 2011), and this is due to their higher nitrogen and lower carbon demands from soil organic material (Hobbie et al., 1999; Högberg et al., 1999). Furthermore, through aggregate formation and stabilization, it has been suggested that EM fungi protect soil organic carbon from decomposition (Fernandez and Kennedy, 2015). As a result, large biomass of nitrogen poor humus commonly accumulates in the forest floor. Conversely, although AM fungi can supress saprophytic fungal decomposition (Leifheit et al., 2015; Verbruggen et al., 2015), organic matter in AM dominated ecosystems typically has faster rates of decay and lower C:N ratios (Averill et al., 2014). However, the trend of faster rates of decomposition in AM dominated ecosystems does not always hold true. Trees in the conifer family Cupressaceae have AM associations (Wang and Qiu, 2006), yet humus accumulations and C:N ratio in the forest floor in single species stands of these conifers can be similar or greater to those found in stands dominated by EM conifer species in the Pinaceae family (Alban, 1969; Norris et al., 2001; Prescott, 2002; Smith and Heath, 2002).

In addition to their role in decomposition, mycorrhizal fungi can play a crucial role in seedling establishment, survivorship and growth (Trappe and Strand, 1969; Hayman, 1974; Fenner, 1987; Perry et al., 1987; Amaranthus and Perry, 1989b; Thiet and Boerner, 2007). Propagules of mycorrhizal fungi in soil serve as an important inoculum source for the establishment of plant communities. Typically, the abundance and distribution of mycorrhizal fungal propagules is reflective of the aboveground plant community (Moyersoen et al., 1998; Korb et al., 2003; Thiet and Boerner, 2007; Dickie et al., 2013; Nave et al., 2013). Following

19 severe disturbance, such as a deforestation event, there can be a legacy effect in which remnant mycorrhizal propagules in the soil facilitate the re-establishment of compatible host plants (Janos, 1980; Allen and Allen, 1984; Wilson and Hartnett, 1998; van der Heijden et al., 1998a; Stampe and Daehler, 2003; Wardle et al., 2004).

In this study, I compared forest floor fungal communities between an AM dominated and an EM dominated forest type. I then predicted the mycorrhizal inoculum potential in the forest floor following a deforestation event. The EM forest type is dominated by EM western hemlock and amabilis fir (referred to as Hemlock-Amabilis fir or HA forests). The AM forest is dominated by AM western redcedar with a minor component of subordinate western hemlock and amabilis fir (referred to as Cedar-Hemlock or CH forests) (Lewis, 1982; McWilliams et al., 2007). These forest types occur adjacent to each other, often with similar physical site characteristics (Keenan et al., 1993; Albani and Lavery, 2000; Weber et al., 2003; Weber et al., 2005; McWilliams and Klinka, 2005b; Weber et al., 2014), yet there is compelling evidence that decomposition rates and/or processes differ, leading to greater humus accumulation in the forest floor of CH forests (Leckie et al., 2004; Sajedi et al., 2012).

Previous work on the fungal communities in these forests has indicated significant differences between CH and HA forests. Using ribosomal intergenic spacer analysis, Leckie et al. (2004) detected profile differences in fungal communities; unfortunately, the technique is limited in that it does not elucidate the species identity or functional groups of fungi present. Using DNA cloning and sequence analyses of EM root tips, Lim and Berbee (2013) identified EM fungal species and detected subtle differences in species composition colonizing root tips of mature western hemlock and amabilis fir growing in these forests. However, their study did not provide insight into the overall abundance and distribution of EM in the forest floor. Using seedling bioassays, Weber et al. (2003) and Weber et al. (2005) showed that western redcedar seedlings became colonized by AM fungi in the forest floor under CH forests but not HA forests, suggesting absence of AM in HA forests. Furthermore, the ericaceous plant salal forms a dense understory layer in CH forests, but is uncommon in HA forests (McWilliams et al., 2007). It has been suggested that the ericoid mycorrhizal fungi potentially have an inhibitory effect on EM

20 fungi (Xiao, 1994) and promote immobilization of nitrogen where salal predominates (Prescott et al., 1996). From these studies, there is evidence that CH and HA forests differ in fungal community structure, which could have substantial implications for ecosystem processes. However, there is still much that is unknown about the overall fungal soil communities in these forests.

The hypothesis I tested for this project is that fungal communities, mycorrhizal guilds and functional groups of fungi in the forest floor differ between CH and HA forests; this is based on the premise that abundance of mycorrhizal mycelium in the forest floor is reflective of the dominance of the above-ground mycorrhizal plant community. Therefore, I predicted that AM and ericoid mycorrhizae are the predominant mycorrhizal guilds in CH forests, but that these guilds are substantially lower and rare in HA forests. Furthermore, I predict greater dominance of EM fungi in HA forests than in CH forests. Associated with these differences in dominance of mycorrhizal guilds, I predicted that the ratio and species composition of the saprophytic fungal communities would differ between forest types. Using a targeted metagenomic approach (Lindahl and Kuske, 2013), I carried out a large DNA sequencing effort to characterize fungal communities dominating the forest floor of CH and HA forests. By identifying forest floor fungal species composition and frequency detected among seventy two forest floor samples, I was able to extrapolate whether significant differences in fungal ecological groups, mycorrhizal guilds and mycorrhizal inoculum potential occurred between forest types.

2.2 Methods

2.2.1 Site description

The field research took place in forests located in the Nahwitti Lowland throughout areas around Port Hardy and Port McNeill on northern Vancouver Island in British Columbia, Canada. Forests in this region occur in the Very Wet Maritime Coastal Western Hemlock (CWHvm) biogeoclimatic subzone. Soils are moderately to imperfectly drained Duric Humo- ferric Podzols with a reddish brown mineral Bf horizon (Lewis, 1982). The area receives approximately 1900 mm of precipitation annually (Negrave, 2004), with a monthly low of 50

21 mm from May to August and a high of 150-250 mm during October to February (Lewis, 1982). Most of the annual precipitation is in the form of rainfall. The average monthly temperature maximum of just over 17C occurs during July and August, and the minimum of just below 0C occurs during October to February (Lewis, 1982).

Three replicate pairs of adjacent naturally regenerated CH and HA forests with matching site characteristics were selected. The HA forest type is 100-year-old second growth forest resulting from a wind-throw event in 1908 (Lewis, 1982) and is comprised almost exclusively of EM western hemlock and amabilis fir. These forests were reaching the end of the stem exclusion stage of stand development (Oliver and Larson, 1996) and thus have a dense canopy that permits very little light penetration to the understory, resulting in the absence of understory vegetation. The CH forest is old-growth with western redcedar trees over 1000 years-old and is dominated by AM western redcedar with a minor component of subordinate western hemlock and amabilis fir (Lewis, 1982; McWilliams et al., 2007). Cedar-Hemlock forests are structurally complex with 30-50% canopy closure and a dense understory of predominantly salal, some bunchberry and few ferns (Lewis, 1982; McWilliams et al., 2007; Weber et al., 2014). Assessment of tree age in CH forests revealed that western hemlock trees present are up to 400 years of age (Keenan, 1993).

Study sites were selected in collaboration with forest managers at Western Forest Products Inc. The sites were similar to those characterized by B.A. Blackwell and Associates Ltd. for developing the Biogeoclimatic Ecosystem Classification (BEC) for the Nahwitti Lowland (McWilliams et al., 2007). The pairs of sites were located on a mid-slope position at similar elevation and were classified as zonal (01) site series with a poor soil nutrient regime and mesic soil moisture regime (McWilliams et al., 2007). In addition to using site classification data provided by Western Forest Products Inc., one soil pit per site was also excavated to a depth of 100 cm where possible and soil properties and forest floor were examined following protocols outlined in the Canadian System of Soil Classification (1998) and by Green and Klinka (1994). A water table present within less than 60 cm prevented deeper excavation of some soil pits. At all sites, the forest floor was >40 cm depth and classified as Hemimor. The forest floor directly overlaid an upper Bf soil horizon of the Humo-Ferric Podzolic soil order.

22 2.2.2 Sampling design

This is considered a natural experiment with two treatments (CH and HA) replicated three times in a randomized block design. Forest floor samples were collected from the three replicate sites with adjacent old-growth CH and HA forests. At each site, two 20 m by 20 m plots were established; one in the CH ecosystem and one in the HA ecosystem. Each plot was at least 50 m from the ecosystem boundary as determined by site series (Pojar et al., 1991; McWilliams and Klinka, 2005a) and the Lewis ecosystem classification (Lewis, 1982). The GPS coordinates were recorded for the four corners of each plot. A random sampling design (Zar, 1999; EPA, 2002) was used to select twelve sample locations for collecting cores of the forest floor, which included the L, F and upper H (LFH) layers. To accomplish this, each plot was divided into sixteen 5 m by 5 m subplots. Twelve of the sixteen subplots were chosen randomly, and then one sample of forest floor was collected (20 cm x 20 cm to a 15 cm depth) from one randomly selected quadrant in the subplot (Figure 2.1).

Tree and vegetation characteristics were recorded at each site. Percent cover of understory plant species, the abundance of each tree species present and their associated Diameter at Breast Height (DBH) in cm were recorded. The tree data was used to calculate the quadratic mean diameter in cm (Curtis and Marshall, 2000), basal area (BA = π x (DBH)2 / 144) in m2/hectare (Franklin et al., 1965) and density (trees per hectare) per plot .

Plot establishment, forest floor sample collection and measurements of tree species composition, DBH and understory plant cover were accomplished in December 2008. Plots were revisited in September 2009 to ensure that annual and/or deciduous plant species were not overlooked during the winter survey. Forest floor samples were stored at 4°C during the first week after collection (until transported back to the lab). Each sample was used for targeted metagenomic characterization of mycorrhizal communities. Aliquots of 200 ml of each sample were stored at -20°C until used for DNA extraction.

23 2.2.3 Targeted metagenomic characterization

Forest samples contained the LFH layers combined. Woody debris and larger root fragments were carefully removed and forest floor cores were homogenized by hand in sterile beakers. Fine roots were included in the final mixture used for DNA extraction, especially since a large proportion of AM fungal biomass (Frey et al., 1994) and ericoid fungal biomass (Smith and Read, 2008) is closely associated with the fine roots. Total fungal DNA was extracted from 7g of each sample using the MO BIO Power Max Soil DNA isolation kit (MO BIO Laboratories, Inc., California). Successful extraction of DNA from each sample was confirmed by visual inspection of photographs from electrophoresis gels. DNA extracts were loaded into a 1% agarose electrophoresis gel with EZ-Vision3 loading buffer with dye (Mandel Scientific Inc.), following the manufacturer’s protocol. Electrophoresis gels were run at 70 V for 1 hour and then photographed under ultraviolet light.

The ribosomal internal transcriber spacer (ITS) regions ITS1 and ITS2, the 5.8S ribosomal unit and the 5’ end of the 18S large ribosomal subunit (LSU) were PCR amplified with primers TW13 (Taylor and Bruns, 1999) and fungal-specific ITS1-F (White et al., 1990) and PuReTaq Ready-To-Go PCR Beads (Amersham Biosciences Ltd.), following the manufacturer’s protocol. The total volume of each PCR mixture was 25µl; this contained 12.5µl of DNA extract that had been diluted 1:10 and 0.5 µM of each primer. The reaction was started using an initial denaturation step of DNA at 95°C for 2 minutes, followed by 30 cycles with the following parameters: (i) denaturation for 30 seconds, (ii) annealing of primers at 55°C for 30 seconds, (iii) elongation at 72°C for 30 seconds plus 4 additional seconds per cycle. A 7 minute elongation at 72°C was carried out at the end of the 30 cycles. Successful amplification was confirmed using the same electrophoresis technique described above. The PCR products of the DNA extractions were purified using EZNA Cycle-Pure Kit (Omega Bio-tek, Inc, Doraville USA).

Ligation mixtures were prepared for each sample using pCR®2.1 vector from TOPO TA Cloning kit (Invitrogen), following the manufacturer’s protocol. Once completed, the ligation mixtures were placed on ice and immediately submitted to Canada's Michael Smith Genome Sciences Centre, in Vancouver Canada, for cell transformation (cloning), automated clone

24 picking and subsequent bidirectional sequencing. For each clone library, 96 clones (i.e. enough to fill one 96 well microplate) were randomly selected and sequenced using the universal vector sequencing primers M13F and M13R (Messing and Vieira, 1982).

2.2.4 DNA sequence analyses of clone libraries

Automated assembly and trimming of bidirectional sequence pairs into contigs was accomplished using the DNA Baser Sequence Assembler (Heracle BioSoft SRL, Romania). Unassembled bidirectional sequences were trimmed to include ends with 100% good bases in a 20 base window. Assembled contig ends were trimmed to exclude fungal primer sequences, thus removing the ends of vector DNA. Using the web-based workbench PlutoF (Abarenkov et al., 2010), the ITS1, 5.8S and ITS2 sub regions of each sequence were extracted using the ITS Extractor and then chimeric sequences were detected using the chimera checker utility. Chimeric sequences were removed from further analyses.

Sequences were binned based on a 99% sequence identity match of the ITS1, 5.8S and ITS2 sub regions using a dynamic programming algorithm for the longest common subsequence (Hirschberg, 1977; Smith and Waterman, 1981) written in the computer programming language Java (Appendix B). One representative sequence from each bin was used for sequence identification by sequence homology matches of known sequenced voucher specimens in DNA databases using BLAST searches (Altschul et al., 1990) within GenBank (Sayers et al., 2009; Benson et al., 2012) and UNITe (Kõjalg et al., 2005; Abarenkov et al., 2010; Kõljalg et al., 2013). Representative clone sequences were submitted to GenBank (accession numbers KP889328 - KP889980).

Clone sequences were given a species identification and assigned to an operational taxonomic unit (OTU) (Blaxter et al., 2005) based on their sequence homology match of >90% query sequence length of the ITS1, 5.8S and ITS2 regions. If a clone sequence had a homology match to the genus Cortinarius, a > 99% sequence homology match was used as the species identification (Wright, 2006; Harrower et al., 2011; Lim and Berbee, 2013); for all other fungal taxa, a > 97% sequence homology match was used for species identification (Nilsson et al.,

25 2008). Sequences that could not be assigned to species level, but had a 95–97% identity match where assigned to genera. Sequences of Cortinarius species remained binned at 99% sequence similarity and were assigned a corresponding OTU designation; sequences of all other fungal taxa were subsequently re-binned and given an OTU designation based on 97% sequence similarity.

Sequences that lacked > 95% homology matches to known voucher specimens in the databases were assigned to a taxonomic group based on a naïve Bayesian analysis of the sequence fragment of their large subunit (LSU) region, using the LSU Fungal Classifier utility on the online RDP Classifier Version 2.6 (Wang et al., 2007). Sequences were hierarchically classified to the highest taxonomic resolution based on at least 80% bootstrap support in 100 iterations.

Once taxonomically identified, fungal OTUs were categorized by their ecological role, as determined by previous publications of known fungal species (for example, Rinaldi et al. (2008) and (Tedersoo et al., 2010)). The primary categories identified were ectomycorrhizal, saprophytic, conifer root associate, ericoid mycorrhizae, pathogen and unknown. Conifer root associates were fungi previously identified to species that have been isolated into culture or sequenced from DNA extracts from roots of conifers, but their role in the association is unknown and their role in the environment is unknown. The OTUs that could not be identified to species and sometimes genus level, or have no known information on their ecological role, were categorized as unknown.

2.2.5 Amplification of arbuscular mycorrhizal DNA

Upon the completion of the identification of OTUs within the targeted metagenomic profiles, it was discovered that the arbuscular mycorrhizal fungal communities had not been amplified with the PCR primers selected. Therefore, original DNA extracts were re-amplified using arbuscular specific PCR primers to detect the possible presence of arbuscular mycorrhizal fungi within the samples. Partial small subunit ribosomal (SSU rRNA) gene fragments were amplified using nested PCR (Kemp et al., 1989). Touchdown PCR (Don et al., 1991) parameters

26 were used to increase yield of amplified target DNA and decrease the amplification of non- target DNA. Because amplified DNA was neither cloned nor sequenced, arbuscular mycorrhizal fungal species present could not be identified or quantified.

All PCR reactions were performed in 0.5 ml thin wall PCR tubes, using Fermentas PCR Master Mix (2X) (Thermo Scientific), in a final volume of 25 µl. The final composition of each PCR reaction contained 0.5 µM of each primer, 0.625 units Taq DNA polymerase in reaction buffer, 2 mM MgCl2, and 2 mM each dNTP. In the first amplification, 1 µl of 1:10 dilution of DNA extract was used as the template. In the nested amplification, 1 µl of 1:1000 diluted PCR product from the first amplification was used. To check for contamination in PCR mixtures, negative controls consisting of PCR grade nuclease-free water were used. In the nested PCR, a second negative control consisted of a re-amplified negative control from the first round of PCR.

The first PCR amplification was accomplished using the arbuscular mycorrhizal specific primer AML2 (Lee et al., 2008) and a modification of the universal eukaryotic primer NS31 (Simon et al., 1992). The modification of NS31 was an omission of first three base pairs on the 5’ end (new primer sequence named SG1: 5’- GAGGGCAAGTCTGGTGCC -3’) for the purpose of decreasing the primer melting temperature to make it compatible with AML2. The touchdown PCR parameters used for the first round of DNA amplification were as follows: (i) the reaction was started using an initial denaturation step of DNA at 95°C for 2 minutes, then followed by 10 cycles with the following touchdown PCR parameters: (ii) denaturation at 95°C for 30 seconds, (iii) annealing of primers at 66°C for 30 seconds minus 1°C per cycle until reaching 56°C, and (iv) elongation at 72°C for 60 seconds. This was followed with 20 cycles with the following parameters: (v) denaturation at 95°C for 30 seconds, (vi) annealing of primers at 56°C for 30 seconds, and (vii) elongation at 72°C for 60 seconds. This amounted to 30 cycles total. A 7 minute elongation at 72°C was carried out at the end of the final cycle.

27 using an initial denaturation step of DNA at 95°C for 2 minutes, then followed by 10 cycles with the following touch-down PCR parameters: (ii) denaturation at 95°C for 30 seconds, (iii) annealing of primers at 69°C for 30 seconds minus 1°C per cycle until reaching 59°C, and (iv) elongation at 72°C for 60 seconds. This was followed with 10 cycles with the following parameters: (v) denaturation at 95°C for 30 seconds, (vi) annealing of primers at 56°C for 30 seconds, and (vii) elongation at 72°C for 60 seconds. This amounted to 20 cycles total. A 7 minute elongation at 72°C was carried out at the end of the final cycle. Successful amplification was confirmed using the same electrophoresis technique described above.

2.2.6 Statistical analyses

For each forest site, the following richness estimates and diversity indices were calculated for the total fungal communities present and also for different categories of fungal ecological roles. Total richness in each plot was estimated using non-parametric species estimators: Chao1 (Chao, 1984), first-order Jackknife (Jack1) and second order Jackknife (Jack2) (Burnham and Overton, 1978; Heltshe and Forrester, 1983). Diversity and evenness were calculated using Shannon’s H’ (H’ = - ∑ pi ln pi) and Simpson’s D (D = 1 - ∑ pi2) diversity indices (where pi is the proportion of individual species in a random clone library). Using the Data Analyses Toolpack for descriptive statistics in Microsoft Excel 2013, one-way analysis of variance (ANOVA) was used to compare forest types for OTU richness detected, total richness estimates and species diversity and evenness indices. Similarly, one-way ANOVA was used to

28 compare stand characteristics between forest types. To ensure that ANOVA requirements were met, data were tested for normality using the Shapiro-Wilk test, and for homogeneity of variance using Bartlett’s test.

Multivariate analyses were utilized to study fungal species composition differences between forest types. All multivariate analyses were accomplished using PC-ORD statistical software (version 4.34, MjM Software Design, Oregon, USA). Multi-response Permutation Procedures (MRPP), a nonparametric statistical method for testing the null hypothesis of no difference between groups (McCune et al., 2002), was used with Sorenson’s distance measure to determine whether species composition between forest types differed. Non-metric Multidimensional Scaling (NMS) was used as an ordination technique to determine and display the overall structure of species abundance among samples. It was also used as both a method for determining the dimensionality of the data set and to detect whether the structure in the response data was stronger than expected by chance. Sorenson’s distance measure was used with the automatic settings of PC-ORD (McCune and Mefford, 1999). This included a maximum number of iterations of 400, the starting number from a random seed, a starting number of axes of 6, and with 40 real runs and 50 randomized runs. The final instability criterion was 0.00001. Overlays of forest types for each replicate were performed on the NMS ordinations to detect patterns between forest types and sites. Indicator Species Analyses (Dufrêne and Legendre, 1997; McCune et al., 2002) were performed to determine whether particular species were significantly correlated with forest type. Monte Carlo test were run to determine whether indicator values were likely to occur by chance alone.

29 2.3 Results

2.3.1 Tree species and the understory plant community

Tree species composition, density, basal area and percent cover of understory plant species were reflective of associated differences between old growth (i.e. CH) forest and second growth (i.e. HA) forests (Table 2.1). Compared to HA forests, CH forests had more than double the total basal area, but almost half the tree density. Most of the total basal area in CH forests was attributed to the large quadratic mean diameter of western redcedar; western redcedar trees accounted for 23.7% of the total trees per hectare, but their large quadratic mean diameter (150 cm) was associated with their dominance of relative basal area (84.9%) in CH forests. Although amabilis fir and western hemlock are up to 400 years old in CH forests (Keenan, 1993), trees did not attain a significantly greater size in the CH forest compared to HA forests, as reflected by quadratic mean diameters. Amabilis fir was a minor component of CH forests studied. Of the sites studied, two of the three replicate CH sites lacked amabilis fir within the plots, however, amabilis fir was observed in the forest area surrounding the plots at all replicate sites. Amabilis fir had lower quadratic mean diameter and basal area in CH forests compared to HA forests, however, differences between CH and HA forest were not significant. Compared to other tree species present, western hemlock accounted for the greatest number of trees in both CH and HA forests (69.2% and 91.1%, respectively). Although quadratic mean diameter of western hemlock did not differ between CH and HA forests, western hemlock occurred more frequently in HA forests than CH forests and therefore also had greater basal area in HA forests.

Species richness of understory plants was low; CH sites had four understory plant species, whereas HA sites had three (Table 2.1). The CH forests had significantly greater percent cover of salal (59.2% in CH compared to 14.8% in HA forests). Forest gaps in CH forest had >90% salal cover; gaps were absent from HA forests. Huckleberries and deer fern were present in very low abundance in both forest types. Bunchberry was present in CH forests, but not in HA forests.

30 2.3.2 Sequence analyses among clone libraries

Clone libraries for all 72 forest floor samples were successfully generated in this study. The automated clone picking and sequencing resulted in 81.7% valid sequences (5647 out of 6912 clone sequences). The remaining 18.3% sequences were false positives (sequenced cloning vector), primer dimers and chimeras; such sequences were excluded from further analyses. Binning of sequences, based on ITS similarity, resulted in a total of 652 operational taxonomic units (see appendix C). Of the OTUs binned, 342 (51.2%) were singletons and 68 (10.2%) were doubletons that occurred only within single clone libraries. Sequence homology matches to known voucher specimens in either UNITe or GenBank resulted in identification to species level for 22.0% of the OTUs, which accounted for 38.5% of the clones sequenced. The remaining 78.0% of OTUs, which accounted for 61.5% of clones sequenced, had to be identified using the LSU Fungal Classifier.

Coleman collector curves generated for most clone libraries did not plateau, indicating insufficient number of clones sequenced within each clone library to adequately capture all of the fungal species amplified from each forest floor sample. Mean OTU richness detected in each clone library was 28 + 7 in CH sites and 27 + 5 in HA sites. Similarly, Coleman collector curves generated for each study site continued to increase almost linearly (Figure 2.2), thus also failing to reach saturation. Mean total OTU richness detected in each plot was 203 + 15 in CH forests and 179 + 9 in HA forests (Table 2.2); richness estimates indicated that the total species richness may be more than double the detected richness in each site (Table 2.2). Increased sampling effort of cores collected within each plot and increased number of clones sequenced within each clone library would be necessary to determine the full richness of species present at each site.

2.3.3 Diversity of fungal ecological roles

The overall proportion of different fungal groups found in clone libraries were compared between HA and CH forests, based on the fungal ecological roles (Figure 2.3). Ectomycorrhizal fungi were the most common clone sequences found in libraries from both CH and HA forests,

31 accounting for 45% and 56% of clones sequenced, respectively (Figure 2.3a). In both forest ecosystems, basidiomycetes in the order Agaricomycetes were the dominant EM fungal clones (40% in CH and 49% in HA) and ascomycetes in the order Pezizomycotina accounted for the remaining EM fungal clones (5% in CH and 7% in HA) (Figure 2.3b and 2.3c). Both CH and HA forests had a large variety of basidiomycete EM fungal genera (Figure 2.4). Species belonging to the family Atheliaceae, which included Piloderma and Tylospora, were the most abundant in both forest types (50% of CH EM clones and 41% of HA EM clones). In HA forests, fungi belonging to the genus Russula were the second most abundant EM fungi (25% of HA EM clones). Both CH and HA forests had a similar proportion of species belonging to the genus Cortinarius (16% and 13% of EM clones, respectively) and of Craterellus tubaeformis (5% of EM clones from CH and 6% of EM clones from HA). Of the ascomcycete EM fungal genera, CH and HA also had similar ratios of Cenococcum geophilum (8% and 9% of EM clones, respectively) and Melinomyces spp. (3% and 2% of EM clones, respectively) (Figure 2.4).

The next most common fungal group was of clone sequences of fungi with unknown ecological roles, accounting for 37% of clone sequences from CH libraries and 27% of clones from HA libraries. Many of these fungi could only be identified to fungal division using the LSU Fungal Classifier. In both CH and HA libraries, saprophytic fungi accounted for 12% of clones sequenced (Figure 2.3). Unlike the EM community, the saprophytic community was predominantly ascomycete fungi (Figure 2.5). The majority of the saprophytic fungi in both CH and HA libraries (54% and 49% of saprophytic sequences, respectively) were primarily in the family Helotiales; next most common were fungi of the subclass Cheatothyriomycetidea (11% and 14% of saprophytic sequences, respectively). In both CH and HA libraries, less than 20% of the saprophytic sequences were basidiomycete fungi. The most common saprophytic basdiomycetes were in the genus Mycena (13% and 10% of saprophytic sequences in CH and HA, respectively). The remaining 5-6% of clone sequences in libraries from both CH and HA forests were primarily conifer root associates and a small portion were pathogenic fungi (<1%).

Using ANOVA, diversity indices of various fungal groups differed between CH and HA forests (Table 2.2). CH forests had higher diversity than HA forests not only of all fungi

32 combined, but also of fungi with unknown ecological roles, and those that are saprophytic, conifer root associates, and ericoid mycorrhizae. Diversity of EM fungi, however, did not differ between CH and HA forests.

2.3.4 Multivariate analyses of fungal communities in CH and HA forests

Useful ordinations using NMS were achieved. Up to six ordination axes were significant with a p-value of < 0.02; however, a 3-dimensional solution was recommended by PC-ORD to best represent the data. The final stress for a 3-dimensional solution was 21.8 and the final instability was 0.00001; these indicate that although useable ordination was achieved, according to Clarke’s rule of thumb, caution is warranted for how much reliance should be placed using NMS alone (McCune et al., 2002). Examination of the ordinations revealed that along the second axis, CH and HA forests had some distinct differences in fungal community composition (Figure 2.6a and 2.6c); however, there was much overlap in some fungal species as shown by the first and third axes (Figure 2.6b). Statistically significant differences (p < .001) in fungal communities between CH and HA forests were detected using MRPP. Extreme heterogeneous differences between groups was indicated by the chance-corrected within- group agreement A-value = 0.012. The significant differences in community composition were driven primarily by the presence and absence of indicator species.

Indicator Species analyses detected 38 indicator species and 12 indicator taxa (Table 2.3). The total number of indicator species accounted for 30.4% of all fungal clones sequenced (Figure 2.7); clones of indicator taxa increased the total number by 6.1%. Of the clones that comprised indicator species, 45.2% were EM fungi (13.7% of all fungal clones) and the remaining 54.8% were saprophytic fungi (16.6% of all fungal clones).

Each indicator species separately accounted for a very small amount of the total number of clones sequenced (Table 2.3). Many fungal species occurred rarely and therefore did not have indicator species values that were significantly greater than random. However, when species were grouped together at higher taxonomic levels, a few of the taxonomic groupings did have significant indicator values. For example, fungal clones belonging to the families

33 Elaphomyceteae and Inocybaceae were found exclusively in HA forests, whereas those belonging to the family Nectriaceae and the genera Ramariopsis and Tomentella were found exclusively in CH forests (Table 2.3).

Fungal OTUs belonging to the EM order Russulales were the most common indicator species and taxa (accounting for 8.3% of clone sequences) and were found primarily in HA forests. The next most common indicator species (accounting for 6.1% of clone sequences) was an OTU found in HA forests that was best identified to the division Ascomycota (clone bin ID SG017_A11). All other indicator species and taxonomic groupings each accounted for roughly 2% or less of the clones sequenced; most indicator species each accounted for less than 0.5% of the clones sequenced.

2.3.5 PCR amplification DNA extracts using arbuscular mycorrhizal fungi specific primers

Amplification of fungal DNA using the primers ITS1F and TW13 failed to capture the arbuscular mycorrhizal communities in the targeted metagenomic libraries. Subsequent PCR amplification of the original DNA extracts for each forest floor sample using the arbuscular mycorrhizal specific primers resulted in the potential detection of arbuscular mycorrhizal fungi; cloning and sequence analyses would be required to confirm that amplifications were in fact DNA of the target organism. Amplifications were positive for 94% of samples collected from CH forests, but only 3% of amplifications were positive for samples collected from HA forests. The positive PCR amplification using of arbuscular mycorrhizal specific primers had a significant indicator value (p < .001) of 97.1 in CH forests.

2.4 Discussion

By using targeted metagenomic characterization of fungal community composition, my study provided insight into the mycorrhizal inoculum potential of CH and HA forests and detected significant differences between forest types in species composition of major fungal groups that are important in soil processes, especially decomposition. Approximately one third of clone sequences of both EM and saprophytic fungi were indicator species or taxa primarily

34 associated with either CH or HA forests. Excluding AM fungi, CH forests had slightly higher diversity of fungi present, particularly among unidentified ascomycete fungi. Although the diversity and richness of EM fungi were similar between CH and HA forests, there was lower abundance of EM fungal clone sequences and higher abundance of saprophytic clone sequences from CH forests than from HA forests. Arbuscular mycorrhizal fungi occurred primarily in CH forests. Unfortunately, species identification of AM fungi and estimated ratio to non-AM fungi was not possible in this study. If it were possible to calculate the contribution of AM fungi into the diversity indices and abundance measures, then CH forests would likely have even higher fungal diversity indices. Given that relative clone abundance of DNA sequences of different fungal groups of saprophytic and EM fungi should be fairly reflective of their ratio of mycelial abundance found in the soil (Anderson et al., 2003), we can make inferences about which are the dominant fungi in CH and HA forests.

2.4.1 Fungal communities differ between CH and HA forests

My prediction that CH forests would have substantially lower EM fungal abundance and inoculum was not well supported by my study when considered in concert with prior studies that estimated total fungal biomass. The CH forests had approximately 10% lower clone sequence abundance of EM fungi. Given that Leckie et al. (2004) estimated that total biomass of EM and saprobic fungi was greater in CH than HA forests, taken together, our studies suggest total EM biomass might be similar between CH and HA forests. With regards to EM diversity, the old-growth CH forests and 100-year-old second growth HA forests had similar measures of EM diversity, a trend seen in other comparisons of old-growth and mature forests (Luoma et al., 1991; Goodman and Trofymow, 1998). However, in my study, certain fungal species and taxa were associated with specific forest types. Differences in fungal communities may be influenced not only by forest floor properties that differ between CH and HA forests (Leckie et al., 2004), but also dominance by western redcedar, nitrogen availability or carbohydrate allocation associated with western hemlock productivity. For example, Russula species were among the most abundant EM fungi and indicator taxa in HA forests, but were rare in CH forests. In an earlier study of EM fungi colonizing western hemlock root tips in CH and HA forests, Lim and

35 Berbee (2013) also found a greater clone abundance of Russula species on root tips associated with mature HA forests as opposed to young regenerating CH sites.

There is considerable evidence that a succession of EM fungi occurs during forest development (Blasius and Oberwinkler, 1989) , however, stand age alone is not enough to explain the rare incidence of Russula species in CH forests. Russula species are considered “late- stage” EM fungi (Visser, 1995; Bergemann and Miller, 2002), with minimal occurrence during stand regeneration, and common in older forest age classes (Twieg et al., 2007). Among various studies, numerous Russula species have been detected in old-growth forests in western North America (Smith et al., 2002; Izzo et al., 2006). The presence of Russula species, however, may be affected by the presence of western redcedar. Studies that characterized the EM diversity in both old-growth and second growth mixed forests with a major component of western redcedar typically had minimal representation of Russula species (Goodman and Trofymow, 1998; Trudell and Edmonds, 2004), whereas forests with minor amounts of western redcedar had higher representation of Russula species (Coates et al., 1997; Durall et al., 1999; O'Dell et al., 1999). Russula species are not the only taxon potentially influenced by abundance of western redcedar. Kranabetter and Kroeger (2001) detected lower ectomycorrhizal diversity in forest blocks with higher dominance of western redcedar; they proposed that western redcedar has a negative effect on many ectomycorrhizal taxa by reducing hyphal contact between roots of neighboring EM trees of EM fungal species better adapted to spreading via networks than by spores. In my study, other EM and saprophytic fungal taxa associated with forest type included fungi in the families Clavariaceae, Elaphomyceteae and Inocybaceae which were found exclusively in HA forests, as well as fungi belonging to the family Nectriaceae and the genera Ramariopsis and Tomentella, which were found exclusively in CH forests.

The lower abundance of Russula in CH than HA forests may be related to lower nitrogen availability. Not only do the CH forests have lower soil nitrogen availability (Prescott et al., 1993; Prescott et al., 1995; Leckie et al., 2004), but the western hemlock trees in CH forests have lower foliar nitrogen concentration (Lim and Berbee, 2013) and slower growth rates compared to those in HA forests (Keenan, 1993), and this potentially contributes to the disparity in taxa between CH and HA forests. Many Russula species are sensitive to soil

36 nitrogen availability. Certain Russula species are more prevalent in soils with high nitrogen availability (Cox et al., 2010; Suz et al., 2014), especially following nitrogen fertilization (Avis et al., 2003; Avis and Charvat, 2005; Avis et al., 2008). Other Russula species appear to be nitrophobic, as observed by the reduction in root tip abundance (Suz et al., 2014) or sporocarp production with increased soil nitrogen (Wallenda and Kottke, 1998; Lilleskov and Bruns, 2001; Lilleskov et al., 2001; Peter et al., 2001; Lilleskov et al., 2002a). However, caution is needed with interpreting results of sporocarp surveys given that below-ground fungal biomass might not be reflected by sporocarp abundance (Gardes and Bruns, 1996). Lilleskov and Bruns (2001) suggested that shifts in EM fungal communities may be more related to changes in carbon supply associated with increased host productivity rather than the direct effect of nitrogen availability on the fungi.

The percent of EM to non-EM fungal clone sequences in my study is similar to the ratio of EM to saprophytic fungal biomass that have been quantified in other coniferous forests, thereby providing further support that clone abundance was likely reflective of mycelial abundance. In my study, 45% of fungal sequences were ectomycorrhizal in CH forests and 56% were ectomycorrhizal in HA forests. In a study by Nilsson et al. (2012), EM fungal biomass constituted 57% of total fungal biomass in nutrient poor EM dominated forests. Likewise, in the uppermost mineral soil of 30- and 40-year-old single species EM forest plantations in Norway, EM fungal biomass constituted approximately half of the total fungal biomass measured (Nilsson, unpublished results, personal communication 2015). Given that Leckie et al. (2004) measured higher total fungal biomass in CH forests, based on clone sequence abundance detected in this study, EM fungi do not appear to be limited in CH forests even though growth of EM western hemlock and amabilis fir hosts is poor and these EM trees represent less than half the basal area and density compared to HA forests.

2.4.2 Saprophytic and ectomycorrhizal fungi important for decomposition

In both forest types, fungi important for decomposition were detected both among the saprophytic ascomycete fungi, which are known to have an important role in initial litter degradation, and certain EM basidiomycete fungi known to degrade organic material similar to

37 white rot fungi. Approximately half of the clone sequences in my study were non-EM fungi, identified as either saprophytic fungi or of unknown species or genera. These two groups of fungi had slightly higher diversity in CH forests and there were a number of indicator species associated with a given forest type, thus supporting my hypothesis that non-mycorrhizal fungal communities differ between forest type. Though functional differences can exist at the species and strain level for fungi, functional similarity is also possible in the fungal communities found in different forest types (Nannipieri et al., 2003); this is a conundrum that cannot be addressed with targeted metagenomic data alone. However, there was similarity in ratios of these two fungal groups at the genus and family level in both forest types.

The majority of sequences of unidentified species were phylogenetically grouped in the division Ascomycota. Although the ecological role of the unidentified ascomycete fungi cannot be determined with certainty at this time, most of them were potentially saprophytes involved with degradation of foliar litter in the forest floor. Litter decomposition is complex and though the process is not fully understood, typically saprophytic ascomycete fungi break down the least recalcitrant components at the beginning of decomposition, whereas basidiomycete fungi, with characteristics most closely associated with white rot fungi, degrade the lignin (Hammel, 1997). It is less likely that the unidentified sequences were EM fungi; though some ascomycete fungi are ectomycorrhizal, ectomycorrhizal associations more typically form with basidiomycete fungi (Smith and Read, 2008). Furthermore, if these unidentified ascomycete fungi were ectomycorrhizal, then they would also have likely been detected in the studies by Wright et al. (2009) and Lim and Berbee (2013), who sequenced root tips of western hemlock from the CH and HA forests.

Of the saprophytic fungi identified, fungi in the order Helotiales were the most common in both CH and HA forests. Helotiales is one of the predominant orders that occurs in litter at early stages of decomposition (Lindahl et al., 2007); nevertheless, knowledge of the functional capacity of these fungi is very limited (Boberg, 2009). Some of these fungi appear to have some ability to degrade cellulose but not lignin (Boberg, 2009). Many ericoid mycorrhizae also belong to the order Helotiales, such as the Pezoloma ericae complex and the Phialocephala–Acephala complex (Walker et al., 2011). In my study, I attempted to account for the detection of known

38 ericoid mycorrhiza fungal genera and, by sequence homology matches, to identify ericoid mycorrhizal fungi in salal roots from previous studies (Monreal et al., 1999; Berch et al., 2002; Allen et al., 2003) available in GenBank. Given that Walker et al. (2011) detected more genera of ericoid mycorrhizal fungi in the order Helotiales outside of the more well-known ericoid mycorrhizal fungal taxa, it is possible that some of the Helotiales fungal sequences that I grouped as saprophytes may have been ericoid mycorrhizal fungi. However, the most abundant ericoid mycorrhizal species would have likely been detected in the previous studies in the same ecosystems as my study.

Based on DNA clone sequence abundance, there was extremely low abundance of saprophytic basidiomycete fungi compared to EM basidiomycete fungi. Of the few saprophytic basidiomycete fungi present, the predominant genus detected in both forest types was Mycena, which contains well-known white rot fungi important for degrading foliar litter (Hintikka, 1970; Kirk, 1971; Ghosh et al., 2003; Lindahl and Boberg, 2008). Given that basidiomycete fungi are extremely important for the final degradation of lignin in both wood and foliar litter (Hammel, 1997), EM fungi may play a greater role in the final breakdown of lignin than saprophytic fungi in CH and HA forests. However, given that woody debris was removed from the forest floor samples prior to DNA extractions, the low detection of decay basidiomycete fungi could also potentially be an artifact created by removing substrate for these fungi from the samples being analyzed.

A particular group of EM fungi detected in high abundance in my study that likely contribute significantly to lignin degradation were fungi in the family Atheliaceae, which comprised approximately one quarter of DNA sequences in clone libraries from both CH and HA forests. Fungi in the family Atheliaceae produce laccase enzymes that greatly contribute to lignin degradation, similar to white rot fungi (Chen et al., 2003; Luis et al., 2005). In addition, among Atheliaceae fungi, nitrogen availability has been shown to upregulate the transcription of genes for production of laccase enzymes; higher nitrogen availability increases the fungal breakdown of lignin by these fungi (Chen et al., 2003). Given that CH and HA forests differ in nitrogen availability (Leckie et al., 2004), further research is warranted to determine whether metabolic activities differ, which could contribute to rates of decomposition observed.

39 2.4.3 Very low abundance of ericoid mycorrhizal fungi

There were very few clone sequences of ericoid mycorrhizae in either CH or HA forests; DNA sequences of known ericoid mycorrhizal species comprised less than 1% of sequences in clone libraries. Previous in vitro studies that characterized the enzymes produced, substrate utilized and competitive effects of ericoid mycorrhizae from salal suggested that ericoid mycorrhizal fungi are likely the dominant fungi of CH forests. In vitro, ericoid fungi utilized complex organic substrate (Xiao and Berch, 1999) and inhibited the growth of a few select cultures of EM fungi (Xiao, 1994). As such, inferences were made that where salal forms dense understory cover in CH forests, their mycorrhizal fungi inhibit EM fungal growth in the forest floor (Prescott et al., 1996). However, results of my study do not support this hypothesis, especially with regards to differences between CH and HA forests. Based on ratios of fungal groups detected in clone libraries, especially in CH forests, ericoid fungi were neither dominant, nor were EM fungi suppressed. With such low abundance of ericoid mycorrhizal sequences, there is little support for the hypothesis that ericoid mycorrhizal fungi are driving major differences in decomposition between CH and HA forests. These results provide further support for a theory, proposed by Prescott and Sajedi (2008), that the presence of salal is not the cause of low nutrient availability.

Caution, however, is warranted for interpreting the ratio of EM to ericoid mycorrhizal fungi detected. Generally, quantification of ericoid mycorrhizal biomass to EM biomass is challenging (Wallander, 2006). Although it has been shown that ericoid extra-radical mycelia extend only a few millimetres to centimeters from the root surface (Read and Stribley, 1975), the fact that fine roots were included in the DNA extraction suggests the ericoid mycorrhizal fungal DNA would not likely have been missed due to sampling technique. Conversely, given that sheaths of EM root tips often have substantially higher fungal biomass (Fogel and Hunt, 1979), the inclusion of EM fine roots could also have resulted in higher EM biomass that could have potentially overpowered the ericoid mycorrhizae during DNA extractions and amplification. However, based on lab incubation trials designed to quantify mycorrhizal fungi in soil (Wallander, 2006), data from Nilsson et al. (2005) suggest that ericoid mycorrhiza can be

40 substantially higher than EM fungi in nutrient poor coniferous forests when using soil samples processed in a similar manner as carried out in this research project.

2.4.4 Arbuscular mycorrhizal fungi present primarily in CH forests

My results support the hypothesis that HA forests have minimal AM inoculum potential. Though Glomeromycota DNA failed to be amplified with the primers utilized for generating clone libraries of the ITS region of ribosomal DNA, the subsequent PCR amplification using AM fungal specific primers for a region of the SSU ribosomal DNA indicated that AM fungi are present throughout CH forests, but are rare in HA forests. Without characterizing the AM fungal species in CH forests, it cannot be determined how much of the AM fungi present is associated with western redcedar versus understory AM plants. Furthermore, results from my study did not give insight into the potential ratio of AM fungi to other fungal groups in the forest floor samples. It has been suggested that AM mycelial biomass increases in less fertile soil (Lovelock et al., 2004), which may imply that AM biomass in CH forest could be high.

In AM coniferous dominated forest ecosystems, AM fungal biomass has not been quantified, especially relative to other fungal groups. Some studies attempted to quantify fungal abundance in soil under western redcedar and western hemlock using culture based techniques such as plate counts (Turner and Franz, 1985; Leckie et al., 2004); given that AM fungi cannot be cultured (Brundett et al., 1996; Walker, 1999) nor can various EM fungal species (Palmer, 1969), these studies would have primarily characterized the saprobic fungal communities. Other studies quantified forest floor fungal biomass in CH and HA forests (Leckie et al., 2004) and in forest floor under single species plantations of western redcedar (Leckie et al., 2004; Grayston and Prescott, 2005) using a technique that estimates EM and saprobic fungal biomass, but excludes the AM fungal biomass. Fungal biomass estimates in these studies were based on phospholipid fatty acid analysis, a technique that measures microbial biomass using the abundance of fatty acids that are unique to different groups of microbes (Frostegård et al., 2011). These studies quantified the fatty acid PFLA 18:2ω6,9, which is useful for estimating fungi in the divisions Ascomycota and Basidiomycota, but it is not found in the AM fungal division Glomeromycota (Larsen et al., 1998; Olsson, 1999; Nilsson et al., 2005;

41 Frostegård et al., 2011). In other ecosystems, studies on the relative ratio of AM fungal biomass to total fungal biomass have given a wide range of biomass estimates, from very low soil AM fungal biomass (<20% of total fungal biomass) in deciduous AM dominated forests (Nilsson, unpublished results, personal communication 2015) to very high soil AM fungal biomass (nearly double the amount of AM fungal biomass to EM biomass) in mixed forests with high cover of AM herbaceous understory (Nilsson et al., 2005). In some non-forest soils, AM fungi can comprise over 50% of fungal mycelia and up to 30% of the total microbial biomass (Olsson and Wilhelmsson, 2000); in a study by Olsson et al. (1999), extraradial AM fungal mycelia constituted the largest fraction of soil microbial biomass.

2.4.5 Conclusion

By characterizing the fungal communities based on DNA sequences, distinct differences in fungal species composition were detected between CH and HA forests, which were likely driven by differences in the dominant tree species with particular mycorrhizal guilds and host productivity. The CH forests had a higher diversity of saprophytic fungi and a slightly lower proportion of EM fungi than HA forests. There was no evidence that CH forests were deficient in EM inoculum, however, there were differences in species composition. Arbuscular mycorrhizal fungi, and thus inoculum, were widely distributed in CH forests, whereas they were rare in HA forests, and these differences were related to the abundance of western redcedar. The proportion of ericoid mycorrhizal fungi was extremely low in both forest types, and therefore ericoid mycorrhizal fungi are not likely inhibiting EM fungi nor driving major soil processes in CH forests. Given the low abundance of saprophytic basidiomycete fungi detected in the forest floor of these forests, mycorrhizal fungi may be greatly contributing to differences in decomposition processes and nutrient availability.

20 m

a b a b a b a b 1 2 3 4 d c d c d c d c

a b a b a b a b 8 7 6 5 d c d c d c d c 20 m a b a b a b a b 9 10 11 12 d c d c d c d c

a b a b a b a b 5 m 16 15 14 13 d c d c d c d c

5 m

Figure 2.1 Illustration of plot layout used for collecting forest floor samples. Plot size was 20 m by 20 m. Within each plot, there were sixteen 5 m by 5 m subplots. Each subplot was assigned a number (1- 16), and then 12 were chosen randomly. In each of the chosen subplots, a forest floor sample was collected from the center of one randomly selected quadrant (a-d) within the subplot (shaded gray).

250

200

150

100

50 Numberfungal of operationaltaxonic units detected 0 0 2 4 6 8 10 12 Number of clone libraries per plot

Figure 2.2 Coleman collector curves for fungal operational taxonomic units detected in clone libraries from plots sampled from Cedar Hemlock forests (CH, indicated by red solid shapes) and Hemlock- Amabilis fir forests (HA, indicated by green open shapes). Collector curves from different sites are indicated by shape of marker (triangles for site RMnD, circles for site B405 and squares for site W80).

44 a.

b. CH c. HA

Figure 2.3

45 Figure 2.3 Proportion of fungal types, found in Cedar Hemlock (CH) and Hemlock-Amabilis fir (HA) forests, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the internal transcribed spacer regions of whole fungal communities present within the LFH layers of the forest floor. Percentages shown are the mean values from the three replicate sites of each forest type. Relative ratios are shown for (a) general fungal group based on ecological role in CH and HA forests, as well the relative percentage of fungi belonging to different taxon within each of these fungal groups for both (b) CH and (c) HA forests. Fungi classified as “unknown”, are from DNA sequences that lacked sequence homology matches to known fungi in sequence data bases and were classified using phylogenetic analyses of the large subunit region; the species identity and mode of nutrition of these fungi are unknown. Endophytic fungi and ericoid mycorrhiza (portions not visible on the graphs) made up less than 1% combined in both forest types.

a. CH b. HA

CH 41% HA

Figure 2.4 Proportion of ectomycorrhizal fungi found in (a) Cedar Hemlock (CH) and (b) Hemlock-Amabilis fir (HA) forests, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the ITS regions of whole fungal communities present within the LFH layers of the forest floor. Percentages shown are the mean values from the three replicate sites of each forest type. Sequences of known ectomycorrhizal fungal made up 45% and 56% of total fungal sequences from CH and HA forest floor clone libraries, respectively.

a. CH b. HA

CH HA

Figure 2.5 Proportion of saprophytic fungi found in (a) Cedar Hemlock (CH) and (b) Hemlock-Amabilis fir (HA) forests, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the ITS regions of whole fungal communities present within the LFH layers of the forest floor. Percentages shown are the mean values from the three replicate sites of each forest type. Sequences of known saprophytic fungi sequences made up 34% and 25% of total fungal sequences from CH and HA forest floor clone libraries, respectively. 48

a. b.

Figure 2.6 Non-metric Multidimensional Scaling ordination of fungal communities found within individual clone libraries from Cedar Hemlock forests (CH, indicated by red solid shapes) and Hemlock- Amabilis fir forests (HA, indicated by green open shapes). Clone libraries from different sites are indicated by shape of marker (triangles for site RMnD, circles for site B405 and squares for site W80). Ordinations are shown for pair wise axis combinations (a-c).

17% Fungi found in both ecosystems CH 45% 37% 12% 5% Indicator species, exclusive to ecosystem type 14% HA 56% 27% 12% 3% Non-indicator species, exclusive to ecosystem type Indicator species, found in both ecosystems 0% 20% 40% 60% 80%13% 100% Fungi found in a single research site

12%

50 Table 2.1 Summary of mean values and standard error (mean + SE) for selected tree characteristics of western redcedar (Cw), amabilis fir (Ba) and western hemlock (Hw). The understory plant species composition of the CH and HA forests are also included. Significant differences between forest types (p < 0.1) are indicated with bold text.

CH HA p

Quadratic Mean Diameter (cm) Cw 150 + 29 Ba 13 + 13 44 + 8 0.75 Hw 33 + 2 37 + 1 0.12

Basal Area (m2/hectare) 214.4 + 25.2 98.8 + 3.3 0.01

Species Basal Area (m2/hectare) Cw 175.5 + 34.9 Ba 5.6 + 5.6 10.7 + 2.2 0.30 Hw 33.3 + 1.8 88.1 + 4.3 0.00

Relative Basal Area (%) Cw 84.9 + 1.2 Ba 1.7 + 1.7 10.9 + 2.4 0.04 Hw 13.4 + 1.5 89.1 + 2.4 0.00

Density (trees per hectare) 583 + 82 900 + 66 0.04

Species Density (trees per hectare) Cw 142 + 36 Ba 50 + 50 83 + 33 0.61 Hw 392 + 22 817 + 36 0.00

Relative Density (trees per hectare %) Cw 23.7 + 4.3 Ba 7.1 + 7.1 8.9 + 2.9 0.83 Hw 69.2 + 7.3 91.1 + 2.9 0.05

Percent cover of understory vegetation (%)

Salal (Gaultheria shallon) 59.2 + 8.1 14.8 + 9.8 0.03 Vaccinium spp. 1.3 + 1.0 5.2 + 4.0 0.40 Bunchberry (Cornus canadensis) 0.3 + 0.2 Deer fern (Blechnum spicant) 1.5 + 0.7 0.6 + 0.6 0.39

51 Table 2.2 Mean diversity and richness indices of fungal operational taxonomic units found in Cedar Hemlock (CH) and Hemlock-Amabilis fir (HA) forest sites. Significant differences between forest types (p < 0.1) are indicated with bold text. Results are provided for both the total fungal community present within the LFH layers of the forest floor, as well as for general fungal groups based on ecological role, as determined by sequence homology matches of cloned DNA fragments from PCR amplification of the internal transcribed spacer regions.

All Fungi Ectomycorrhiza Unknown Fungi Saprobe

CH HA p CH HA p CH HA p CH HA p

Shannon Index H' 4.43 4.00 0.07 3.20 3.07 0.46 3.42 2.75 0.04 3.67 3.53 0.29 Shannon Evenness (H'/Hmax) E 0.83 0.78 0.03 0.78 0.77 0.64 0.79 0.65 0.03 0.93 0.91 0.05

Simpson's Diversity Index D (1-D) 0.98 0.96 0.04 0.93 0.93 0.60 0.94 0.78 0.03 0.97 0.97 0.50 Simpson's Reciprical 1/D 43.5 25.0 0.06 15.4 13.6 0.93 15.8 5.0 0.01 29.9 23.9 0.12 Simpson's Evenness E 0.20 0.10 0.04 0.26 0.25 0.92 0.21 0.07 0.23 0.56 0.48 0.07

OTU Richness 203 179 0.25 59 54 0.27 75 67 0.90 53 50 0.66

Jack 1 Richness Estimate 323 289 0.34 90 82 0.34 123 114 0.62 85 81 0.66 Jack 2 Richness Estimate 387 354 0.46 103 97 0.62 152 144 0.75 103 97 0.69 Chao 1 Richness Estimate 443 488 0.65 101 141 0.51 250 252 0.98 141 114 0.54

Relative Clone Abundance (%) 100 100 0.73 45.1 56.5 0.05 36.9 26.9 0.40 12.3 12.0 0.86

Table 2.2 Continued.

Conifer Pathogen Ericoid Root Associate Mycorrhiza CH HA p CH HA p CH HA p

Shannon Index H' 1.02 0.00 0.00 0.66 1.32 0.19 1.34 0.10 0.00 Shannon Evenness (H'/Hmax) E 0.71 0.00 0.00 0.95 0.79 0.03 0.98 0.14 0.00

Simpson's Diversity Index D (1-D) 0.58 0.00 0.00 0.76 0.70 0.58 0.89 0.06 0.00 Simpson's Reciprical 1/D 2.3 1.0 0.00 2.2 2.8 0.51 3.8 0.7 0.01 Simpson's Evenness E 0.54 1.00 0.00 0.95 0.53 0.00 0.95 0.53 0.22

OTU Richness 4 1 0.00 2 5 0.05 4 1 0.22

Jack 1 Richness Estimate 6 1 0.00 4 9 0.02 7 2 0.06 Jack 2 Richness Estimate 8 2 0.01 4 12 0.01 8 3 0.10 Chao 1 Richness Estimate ------

Relative Clone Abundance (%) 4.5 3.3 0.56 0.4 1.1 0.02 0.6 0.5 0.75

ITS Sequence Homology Match

Taxonomic OTU Indicator Ecosystem % Clone % Sequence Level Taxon Accession # Value Found Abundance Accession # Match Database

Division Blastocladiomycota - 88.9 HA 0.16% - - -

Subclass Eurotiomycetidae - 90.9 HA 0.32% - - -

Order Dothideomycetes incertae sedis - 84.2 HA 0.46% - - - Order Russulales 63.2 HA 8.32% - - -

Family Clavariaceae - 95.2 CH 0.58% - - - Family Elaphomycetaceae - 100.0 HA 0.44% - - - Family Inocybaceae - 100.0 HA 0.16% - - - Family Nectriaceae - 100.0 CH 0.06% - - -

Genus Melinomyces - 68.3 HA 1.27% - - - Genus Ramariopsis - 100.0 CH 0.55% - - - Genus Tomentella - 100.0 CH 0.18% - - -

OTU Archaeorhizomyces sp. (SG017_A03) KP889943 100.0 CH 0.25% GQ159998 99% GenBank OTU Archaeorhizomyces sp. (SG018_B05) KP889358 100.0 CH 0.81% GQ160111 99% GenBank OTU Archaeorhizomyces sp. (SG043_A05) KP889558 100.0 HA 1.56% FJ152543 99% GenBank OTU Chaunopycnis alba (SG026_D08) KP889637 100.0 HA 0.07% HQ608141 98% GenBank

OTU Cortinarius iliopodius (SG030_C09) KP889969 100.0 HA 0.39% FJ039555 99% GenBank OTU Cortinarius angelesianus (SG025_B08) KP889610 100.0 HA 0.13% JF907945 100% GenBank

OTU Cortinarius flexipes (SG029_C02) KP889956 100.0 HA 0.18% FJ717557 99% GenBank

OTU Cortinarius junghuhnii (SG019_H06) KP889632 100.0 CH 0.17% HQ604725 99% GenBank OTU Elaphomyces muricatus (SG025_F11) KP889625 100.0 HA 0.37% UDB000092 99% UNITe

Table 2.3 Continued.

ITS Sequence Homology Match

Taxonomic OTU Indicator Ecosystem % Clone % Sequence Level Taxon Accession # Value Found Abundance Accession # Match Database

OTU Inocybe lanuginosa (SG027_E12) KP889930 100.0 HA 0.13% HQ604314 99% GenBank

OTU Mycena leptocephala (SG017_E08) KP889533 100.0 CH 0.24% HQ604773 99% GenBank

OTU Piloderma olivaceum (SG035_C05) KP889628 97.9 CH 0.81% DQ469291 98% GenBank

OTU Russula fragilis (SG017_B12) KP889933 88.9 HA 2.42% KC581327 99% GenBank OTU Russula paludosa (SG025_A02) KP889680 97.6 HA 3.46% UDB011179 98% UNITe OTU cf. Fungi (SG031_D09) KP889845 100.0 HA 0.15% - - - OTU cf. Fungi (SG025_F11) KP889626 100.0 HA 0.39% - - - OTU cf. Ascomycota (SG017_A01) KP889866 100.0 CH 1.34% - - - OTU cf. Ascomycota (SG017_A07) KP889440 100.0 CH 2.12% - - - OTU cf. Ascomycota (SG017_A12) KP889796 100.0 CH 1.18% - - - OTU cf. Ascomycota (SG017_E01) KP889887 100.0 CH 0.61% - - - OTU cf. Ascomycota (SG022_E04) KP889542 100.0 CH 0.24% - - - OTU cf. Ascomycota (SG035_C02) KP889361 100.0 CH 0.08% - - - OTU cf. Ascomycota (SG036_B12) KP889495 100.0 CH 0.37% - - - OTU cf. Ascomycota (SG017_A11) KP889959 87.9 HA 6.14% - - - OTU cf. Ascomycota (SG031_E06) KP889888 100.0 HA 0.10% - - - OTU cf. Ascomycota (SG032_E09) KP889469 100.0 HA 0.05% - - - OTU cf. Dothideomycetes (SG017_B10) KP889461 100.0 CH 0.20% - - - OTU cf. Dothideomycetes (SG023_C01) KP889937 100.0 CH 0.12% - - - OTU cf. Dothideomycetes (SG032_C09) KP889360 100.0 HA 0.10% - - - OTU cf. Leotiomycetes (SG024_E01) KP889837 100.0 CH 0.17% - - - OTU cf. Chaetothyriales (SG022_D07) KP889664 100.0 CH 0.10% - - - OTU cf. Helotiales (SG018_D11) KP889570 100.0 CH 0.24% - - - OTU cf. Helotiales (SG020_B05) KP889436 100.0 CH 0.15% - - - OTU cf. Helotiales (SG022_A03) KP889569 100.0 CH 0.37% - - - OTU cf. Helotiales (SG026_B07) KP889395 100.0 HA 0.07% - - - OTU cf. Helotiales (SG029_D10) KP889932 100.0 HA 0.07% - - - OTU cf. Herpotrichiellaceae (SG021_A08) KP889759 100.0 CH 0.54% - - - OTU cf. Herpotrichiellaceae (SG017_A05) KP889582 100.0 CH 0.20% - - -

Table 2.3 Continued.

ITS Sequence Homology Match

Taxonomic OTU Indicator Ecosystem % Clone Accession % Sequence Level Taxon Accession # Value Found Abundance # Match Database

OTU cf. Agaricomycetes (SG017_G04) KP889514 100.0 CH 1.16% - - - OTU cf. Agaricomycetes (SG032_A10) KP889738 100.0 HA 0.08% - - - OTU cf. Agaricales (SG023_C09) KP889635 100.0 CH 0.13% - - - OTU cf. Auriculariales (SG019_C07) KP889747 100.0 CH 0.15% - - - OTU cf. Atheliaceae (SG017_H08) KP889604 100.0 CH 0.12% - - - OTU cf. Atheliaceae (SG025_F06) KP889517 95.7 HA 0.39% - - - OTU cf. Atheliaceae (SG027_A04) KP889683 100.0 HA 0.62% - - - OTU cf. Cortinariaceae (SG037_C12) KP889847 100.0 CH 0.07% - - - OTU cf. Hygrophoraceae (SG035_A02) KP889772 100.0 CH 0.76% - - - OTU cf. Hyaloriaceae (SG026_E01) KP889428 100.0 HA 0.13% - - - OTU cf. Ramariopsis (SG020_G08) KP889612 100.0 CH 0.12% - - -

Chapter 3: Understory plant species as reservoirs of compatible mycorrhizal fungi for conifer seedling establishment following forest disturbance

3.1 Introduction

Mycorrhizal associations are essential for seedling establishment and survivorship of plant species that are dependent upon their mycobionts for acquisition of limiting nutrients for growth (Hayman, 1974; Fenner, 1987; Perry et al., 1987; Amaranthus and Perry, 1989b). Establishing plants are most vulnerable to resource limitations between the ontological stages of seed germination and juvenile establishment (Kitajima and Fenner, 2000). Following disturbance events such as forest clearcutting, remnant propagules of soil mycelium and spores serve as mycorrhizal inoculum that can facilitate the re-establishment of mycorrhizal plant communities (Biermann and Linderman, 1983; Friese and Allen, 1991; Teste et al., 2009b). In the absence of host plants, these propagules rapidly diminish (Harvey et al., 1980; Perry et al., 1987; Amaranthus, 1991b; Hashimoto and Hyakumachi, 1998; Hagerman et al., 1999; Dahlberg, 2002; Jones et al., 2003). Where mycorrhizal plants do exist, both the diversity of mycorrhizal fungi and the presence of mycorrhizal networks formed by underground fungal mycelium shared between compatible plants have been shown to increase seedling colonization and establishment as well as alter plant competition and diversity (van der Heijden et al., 1998b; Baxter and Dighton, 2001; Lovelock and Miller, 2002; van der Heijden et al., 2003; van der Heijden, 2004; Johnson et al., 2005a; Scheublin et al., 2007). In the case of regeneration of forests following disturbance, these networks, where they are supported by successional plant species or remaining legacy trees of a compatible mycorrhizal guild, may serve as an important inoculum source to facilitate seedling establishment (Simard and Durall, 2004; Teste and Simard, 2008). Conversely, there is evidence that if mycorrhizal networks are of a mycorrhizal guild that is incompatible with a given plant species, the mycorrhizal fungi of the network may inhibit root proliferation and mycorrhizal colonization of the incompatible host. This can

57 reduce nutrient uptake, productivity and survivorship of the host plant species belonging to the incompatible mycorrhizal guild (Francis and Read, 1994; Francis and Read, 1995; Haskins and Gehring, 2005; McHugh and Gehring, 2006). Furthermore, fungal-host specificity have been detected even within a given mycorrhizal guild (Sanders, 2003a; Sanders, 2003b). For example, although two different plant species may both be AM, the fungal species or genotype they form a symbiosis with might not be compatible between plant species. This means that if a common AM network exists, it might not be compatible with a given host AM plant species.

My research compares regenerating mycorrhizal plant communities between two forest types on northern Vancouver Island, British Columbia, which differ in the pre-harvest forest age and composition. One forest type is a 100 year-old second growth forest resulting from a wind- throw event in 1908 (Lewis, 1982) and is dominated by EM western hemlock and amabilis fir (referred to as Hemlock-Amabilis fir or HA forests). These forests were reaching the end of the stem exclusion stage of stand development (Oliver and Larson, 1996) and thus have a dense canopy that permits very little light penetration to the understory, resulting in the absence of understory vegetation. The other forest type is old-growth and is dominated by AM western redcedar with a minor component of subordinate western hemlock and amabilis fir (referred to as Cedar-Hemlock or CH forests) (Lewis, 1982; Keenan et al., 1994; McWilliams et al., 2007). Cedar-Hemlock forests are structurally complex with 30-50% canopy closure and a dense understory of predominantly salal, some bunchberry and few ferns (Lewis, 1982; McWilliams et al., 2007; Weber et al., 2014). These forest occur adjacent to each other, often with similar physical site characteristics (Keenan et al., 1993; Albani and Lavery, 2000; Weber et al., 2003; Weber et al., 2005; McWilliams and Klinka, 2005b; Weber et al., 2014). Following forest harvest, however, conifer regeneration is problematic in clearcuts of CH forests but not HA forests. In particular, western hemlock seedling growth is poor and nutrient limited in CH forest clearcuts, but not in HA forest clearcuts. On the other hand, growth of planted western redcedar seedlings does not substantially differ between forest types (Prescott et al., 1996; Blevins and Prescott, 2002). Following forest harvest, distinct plant communities develop in HA and CH sites (Sajedi et al., 2012), indicating that mycorrhizal guilds may differ. Such differences

58 in mycorrhizal communities could potentially influence the conifer seedling dynamics observed between clearcuts of forest types.

Western redcedar seeds naturally disperse into disturbed sites previously occupied by both CH or HA forests, but the seedlings generally do not become established in HA sites (Weber et al., 2014). The western redcedar have been shown to remain uncolonized by AM fungi when grown in forest floor of HA forests. Under shaded conditions, as occurs in mature HA forests or in dense juvenile stands, absence of AM root colonization is associated with reduced western redcedar seedling survivorship (Weber et al., 2003; Weber et al., 2005). Based on their research, it was concluded that HA forests lack AM fungi (Weber et al., 2003; Weber et al., 2005; Weber et al., 2014). However, deer fern, which has been reported to be a facultative AM host (Harley and Harley, 1987a, 1987b), occurs in HA forests and clearcuts (Weber et al., 2003; Sajedi et al., 2012) and may serve as a source of AM inoculum. However, it is not known if deer fern in HA forests actually harbour AM fungi, and if so, whether the AM fungi are compatible with western redcedar.

The objective of this research was to characterize mycorrhizal fungal communities associated with the dominant plant species in clearcuts of HA and CH forests. Specifically, I sought to determine whether the different plant species harboured mycorrhizal networks that could serve as sources of mycorrhizal inoculum for regenerating western hemlock and western redcedar seedlings. It was an exploratory research study using DNA sequencing for fungal species identification in roots of the different plant species. These techniques were used to not only identify the mycorrhizal guild, but also to determine whether the mycorrhizal fungi detected are host specific or generalists capable of colonizing western redcedar and western hemlock seedlings.

59 3.2 Methods

3.2.1 Site description

Three replicate pairs of clearcuts of adjacent CH and HA forests with matching site characteristics were selected; for description of study sites and forests prior to harvest, refer to chapter 2 study sites. The pairs of sites were located on a mid-slope position at similar elevation and were classified as zonal (01) site series with a poor soil nutrient regime and mesic soil moisture regime. These sites had been harvested three years prior in 2006 and had an immediate adjacent forested area in which soil properties were characterized. A soil pit was excavated to a depth of 100 cm where possible and soil properties and forest floor were examined following protocols outlined in the Canadian System of Soil Classification (1998) and by Green and Klinka (1994). A water table present within less than 60 cm prevented deeper excavation of some soil pits. At all sites, the forest floor was >40 cm depth and classified as Hemimor. The forest floor directly overlaid an upper Bf soil horizon of the Humo-Ferric Podzolic soil order. The clearcut sites largely lacked the upper forest floor, resulting in a patchily exposed Bf soil horizon, probably due to the clearcut harvesting methods used.

Most of the vegetation sampled in this study were collected from these sites on northern Vancouver Island. However, salal root samples were collected from a 100-year-old second growth stand dominated by western redcedar, western hemlock and Douglas-fir (Pseudotsuga menziesii) in Pacific Spirit Park, Vancouver, British Columbia, Canada. The subzone of this site is the Dry Maritime Coastal Western Hemlock (CWHdm) subzone, soil order is Humo-ferric Podzol and the humus forms is Hemimor (Green and Klinka, 1994).

3.2.2 Sampling design

Plant root samples were collected in August 2009 from all replicate study sites. Roots samples from the dominant native plant species were collected at each site. At each site, 50 m x 50 m plots were established; one each in the CH and HA replicate clearcut sites. Three samples of each plant species were randomly selected from each plot. The plant species sampled were bunchberry (Cornus canadensis L.), deer fern (Blechnum spicant (L.) Smith), spiny wood fern

60 (Dryopteris expansa (C.Presl) Fraser-Jenk. & Jermy), red huckleberry (Vaccinium parvifolium Sm.), fireweed (Epilobium angustifolium L.), pearly everlasting (Anaphalis margaritacea (L.) Benth. & Hook.f.), elderberry (Sambucus racemosa L.), trailing black current (Ribes laxiflorum Pursh) and salmonberry (Rubus spectabilis Pursh). Entire root systems were excavated and samples were stored at 4°C until transported to the University of British Columbia within one week.

Initially, sampling of salal (Gaultheria shallon Pursh) roots was not part of the project design, given that molecular characterization of ericoid mycorrhizal associates had already been extensively examined in other research using a set of general fungal primers for the internal transcribed spacer (ITS) region (Berch et al., 2002; Allen et al., 2003). However, in 2010, root samples of salal were collected from Pacific Spirit Park to test for the amplification of Glomeromycota fungi from DNA extracted from roots using arbuscular specific primers for the 18S small subunit ribosomal (SSU rRNA) gene, an approach that had not been done in previous studies. When positive PCR amplifications occurred, these samples were fully screened as per other vegetation collected in this study.

Similarly, using the same harvest approach, naturally growing western redcedar roots were collected from saplings growing in uncut old growth CH forests adjacent to the clearcut sites; sampling of western redcedar was not possible in HA forests due to its absence. Saplings from uncut forests were chosen, as opposed to those growing in clearcuts, given that close proximity to mature conspecific individuals may potentially increase the diversity of AM fungi associated through AM networks (Kytöviita et al., 2003; van Der Heijden and Horton, 2009; Hausmann and Hawkes, 2010), thereby potentially resulting in a more extensive database of AM associates of western redcedar. Three additional samples of western redcedar roots from a greenhouse seedling bioassay were also included in the study to assist with identifying arbuscular mycorrhizae from forest floor inoculum propagules (Guichon, unpublished data).

Western hemlock seedlings were not collected for EM fungal identification because the EM fungal associates in these ecosystems have already been extensively characterized for this species (Wright et al., 2009; Lim and Berbee, 2013). However, samples of western hemlock

61 roots of seedlings greater than 15 cm tall and of the orchid Listera cordata, a plant species that does not form mycorrhizal associations with AM fungi (Harley and Harley, 1987a, 1987b), were collected from mature HA and CH forests, respectively, to be used as negative controls for PCR amplification of AM fungal DNA. Root systems were rinsed with tap water to remove soil particles and debris; roots were examined under a dissecting microscope to detect EM mantles on root tips and then fine roots were excised. For each plant species, roots collected from a particular site were pooled together and then ten fragments of fine roots, each 2 cm long, were randomly selected and placed into a 1.5 ml microtube. Roots were then stored at -20°C until used for DNA extraction.

3.2.3 Molecular techniques

Mycorrhizal fungal species within root samples were determined using DNA extraction, cloning, DNA sequencing, sequence homology matches and phylogenetic analyses. The total fungal DNA was extracted from root samples using the MO BIO PowerSoil DNA isolation kit (MO BIO Laboratories, Inc., California), following manufacturers protocol. DNA extracts were loaded into a 1% agarose electrophoresis gel with EZ-Vision3 loading buffer with dye (Mandel Scientific Inc.), following the manufacture’s protocol. Electrophoresis gels were run at 70 V for 1 hour and then photographed under ultraviolet light. Successful extraction of DNA for each sample was accomplished by visual inspection of photographs of electrophoresis gels.

PCR amplifications of fungal DNA were carried out separately for the detection of EM and AM fungi. For the EM fungi, the internal transcribed spacer (ITS) region was amplified using fungal specific primers ITS1F and TW13 (see chapter 2 for PCR amplification parameters). For the AM fungi, partial small subunit ribosomal (SSU rRNA) gene fragments, approximately 485bp in length, were amplified using nested PCR with AM specific primers. In addition to the nested PCR approach, touchdown PCR (Don et al., 1991) parameters were used to increase yield of target DNA and decrease the amplification of non-target DNA. All PCR reactions were performed in 0.5 ml thin wall PCR tubes, using Fermentas PCR Master Mix (2X) (Thermo Scientific), in a final volume of 25 µl. The final composition of each PCR reaction contained 0.5 µM of each primer, 0.625 units Taq DNA polymerase in reaction buffer, 2 mM MgCl2, and 2 mM

62 each dNTP. In the first amplification, 1 µl of 1:10 dilution of DNA extract was used as the template. In the nested amplification, 1 µl of 1:1000 diluted PCR product from the first amplification was used. To check for contamination in PCR mixtures, negative controls consisting of PCR grade nuclease-free water were used. In the nested PCR, a second negative control consisted of a re-amplified negative control from the first round of PCR.

The nested PCR amplification was carried out using the arbuscular mycorrhizal specific primers WANDA (Dumbrell et al., 2011) and AM1 (Helgason et al., 1998). The touchdown PCR parameters used for the nested DNA amplification were as follows: (i) the reaction was started using an initial denaturation step of DNA at 95°C for 2 minutes, then followed by 10 cycles with the following touch-down PCR parameters: (ii) denaturation at 95°C for 30 seconds, (iii) annealing of primers at 69°C for 30 seconds minus 1°C per cycle until reaching 59°C, and (iv) elongation at 72°C for 60 seconds. This was followed with 10 cycles with the following parameters: (v) denaturation at 95°C for 30 seconds, (vi) annealing of primers at 56°C for 30 seconds, and (vii) elongation at 72°C for 60 seconds. This amounted to 20 cycles total. A 7 minute elongation at 72°C was carried out at the end of the final cycle. Successful amplification was confirmed using the same electrophoresis technique described above.

63 Following visual inspection of electrophoresis gels to confirm successful PCR amplification, PCR products from strong amplifications were selected for cloning. Three PCR products of different samples were selected from naturally growing western redcedar seedlings and pooled together; three PCR products of samples collected from the greenhouse bioassay were also selected and pooled together. Similarly, for each plant species, three PCR products of amplification types (SSU versus ITS) were selected and pooled prior to ligation for cloning (i.e. for each plant species, one cloning reaction was carried out for AM fungi and one cloning reaction was out for EM fungi; each cloning reaction contained PCR products from 3 different samples). The PCR products were sent to Macrogen, Seoul Korea, for ligation, cloning and automated sequencing of random clone libraries. For each plant species examined, 96 clones (i.e. enough to fill a 96 well microplate) were randomly selected from clone libraries for bidirectional sequencing.

3.2.4 DNA sequence analyses of clone libraries

Bidirectional sequences generated for each clone were assembled into contigs and then duplicate contig sequences were sorted into bins. Automated assembly and trimming of bidirectional sequence pairs into contigs were accomplished using the DNA Baser Sequence Assembler (Heracle BioSoft SRL, Romania). Unassembled bidirectional sequences were trimmed to include ends with 100% good bases in a 20 base window. Assembled contig ends were trimmed to exclude fungal primer sequences, thus also removing the ends of vector DNA. Sequences were binned based on a 99% sequence identity match using a dynamic programming algorithm for the longest common subsequence (Hirschberg, 1977; Smith and Waterman, 1981) written in the computer programming language Java (Appendix B).

One representative sequence from each bin was used for sequence homology matches to known sequenced voucher specimens in DNA databases using Basic Local Alignment Search Tool (BLAST) searches (Altschul et al., 1990) within GenBank (Sayers et al., 2009; Benson et al., 2012) at the National Center for Biotechnology Information and within UNITe (Kõjalg et al., 2005; Abarenkov et al., 2010; Kõljalg et al., 2013). Clone sequences of nonspecific amplifications, as detected by homology matches to non-fungal organisms, were removed from

64 further analyses. A representative clone sequence of each mycorrhizal fungal operational taxonomic units (OTUs) detected in this study was submitted to GenBank (http://www.ncbi.nlm.nih.gov) under the accession numbers KP889278 - KP889327.

To identify fungal clone sequences from the clone libraries containing the ITS region, sequence homology matches of known sequenced voucher specimens in DNA databases using BLAST searches (Altschul et al., 1990) within GenBank (Sayers et al., 2009; Benson et al., 2012) and UNITe (Kõjalg et al., 2005; Abarenkov et al., 2010; Kõljalg et al., 2013) were used (see chapter 2 for further detail). Clone sequences that lacked > 95% homology matches to known ITS regions of voucher specimens in the databases were assigned to a taxonomic group based on a naïve Bayesian analysis of the sequence fragment of their large subunit (LSU) region (see chapter 2). In contrast, phylogenetic analyses were used to identify AM fungal clone sequences of the SSU.

3.2.5 Phylogenetic analyses of arbuscular mycorrhizal fungi

Operational taxonomic units (OTUs) of AM fungi were identified to family and when possible to genera using phylogenetic analyses of the sequences generated in this study with representative SSU sequences of Glomeromycota available in GenBank. The majority of the sequences from GenBank that were utilized for the phylogeny were the SSU sequences from voucher specimens identified to species that were utilized by Kruger et al. (2012) for the establishment of a reference data set for molecular systematics and environmental community analyses of arbuscular mycorrhizae. Additional sequences from GenBank that were not associated with voucher specimens were also included if they had a sequence homology match of at least 98% based on BLAST searches.

Sequence alignments and phylogenetic analyses were carried out in Geneious version 8.0 (Kearse et al., 2012) using appropriate software plugins. A multiple sequence alignment was accomplished using the Multiple Sequence Comparison by Log-Expectation (MUSCLE) alignment plugin (Edgar, 2004a; Edgar, 2004b). The alignment was refined manually upon visual inspection. Phylogenetic relationships between the OTUs detected in this study and the

65 sequences of known arbuscular mycorrhizal fungi in GenBank were determined using maximum likelihood analyses with 1000 bootstraps using the PhyML plugin (Guindon et al., 2010). Similar to Vandenkoornhuyse et al. (2002), the choanozoan Corallochytrium limacisporum was used as the outgroup, based on the widely accepted hypothesis that choanozoans are closely related to fungi, having a contemporary evolutionary radiation (Cavalier-Smith and Allsopp, 1996). Following phylogenetic analyses, the community composition of arbuscular mycorrhizal fungal OTUs detected in roots were compared among host plant species and western redcedar.

3.2.6 Analyses of fungal diversity

To assess whether sequencing effort was sufficient to detect all fungal OTUs present within each clone library, Coleman collector curves (Coleman et al., 1982) were generated for each clone library using EstimateS, Biodiversity Estimation Software Version 9.1 (Colwell, 2013). To analyze community composition, diversity and richness, the abundance of clone sequences for each OTU within a library was treated as the abundance of each fungal species. For each plant host species, richness estimates and diversity indices were calculated for the total fungal communities present. Total richness in each clone library was estimated using non-parametric species estimators Chao1 estimator (Chao, 1984), first-order Jackknife (Jack1) and second order Jackknife (Jack2) (Burnham and Overton, 1978; Heltshe and Forrester, 1983). Diversity and evenness were calculated using Shannon’s H’ (H’ = - ∑ pi ln pi) and Simpson’s D (D = 1 - ∑ pi2) diversity indices (where pi is the proportion of individual species in a random clone library).

3.3 Results

Cover of many of the target plant species in the three-year-old CH and HA clearcuts was sparse. The CH sites had substantial slash and extensive plant cover (>90%) comprised of salal and bunchberry, but only sporadic occurrences of deer fern, pearly everlasting, red huckleberry, spiny wood fern and elderberry (Table 3.1). HA sites, on the other hand, had minimal slash, exposed mineral soil and relatively low plant cover (<50%) comprised of

66 fireweed, deer fern, red huckleberry and salal along with the occasional salmonberry, trailing black currant and spiny wood fern (Table 3.1).

None of the understory plant species were found to be ectomycorrhizal based on microscopic examination of root tips and molecular identification of fungal species. The PCR amplifications and subsequent cloning of DNA extracted from roots using the ITS primers ITS1F and TW13 yielded neither ectomycorrhizal fungal species nor arbuscular species. Most sequences had homology matches to mostly ascomycete fungi, including dark septate endophytes, as well as some saprophytic basidiomycete fungi (results not shown).

DNA extracts from the majority of the plant roots sampled had positive PCR amplifications of SSU rRNA belonging to arbuscular mycorrhizae; the negative controls, western hemlock and Listera cordata, yielded negative PCR amplifications. Positive PCR amplifications occurred among all samples of all species collected, except for DNA extracts from red huckleberry; only one out of six samples of red huckleberry had a positive PCR amplification. Of the 1152 clones sequenced, 1073 (93%) were DNA sequences of Glomeromycota. The remaining sequences (7%) were non-target amplifications with sequence homology matches to the unicellular eukaryotes Rhizaria; sequences of these non-target amplifications were discarded. Clone sequences of arbuscular mycorrhizae lacked sequence homology match above 97% sequence similarity to sequences of known species in GenBank or UNITe. Therefore, the fifty operational taxonomic units (OTUs) detected in this study were identified using phylogenetic analyses with sequences of known Glomeromycota species. Based on a maximum likelihood phylogenetic tree, forty nine of the OTUs detected belonged to three family clades (Figure 3.1). Highest species richness was found among sequences of Glomeraceae, which was comprised of forty OTUs. Of the remaining OTUs, five belonged to Acaulosporaeceae, four belonged to Claroideoglomeraceae and one OTU was not part of any known clade (Figure 3.1). All OTUs detected in western redcedar roots belonged to the Glomeraceae clade.

Many of the OTUs detected were found among multiple hosts (Figure 3.1 and 3.2). Sixteen OTUs were detected in more than one host, ten of which were found in western redcedar (Figure 3.2). Many of the plant species that grew in low abundance in clearcuts (Table

67 3.1) had the lowest number of shared mycorrhizal OTUs within their roots (Figure 3.2). Spiny wood fern, fireweed, pearly everlasting, elderberry, trailing black currant and salmon berry all had up to four shared arbuscular mycorrhizae OTUs with western redcedar. The plant species growing in highest abundance, which included salal, bunchberry and deer fern, had the highest number of shared arbuscular mycorrhizal OTUs with western redcedar. Red huckleberry was an exception, which had low abundance but shared six OTUs with western redcedar. However, the fungal species composition associated with red huckleberry roots were based on PCR amplification results of only one positive amplification out of six samples amplified.

Based on richness estimates (Table 3.2) and Coleman collector curves (Figure 3.3), there was sufficient sampling effort of sequencing clones from clone libraries to detect the majority of arbuscular mycorrhizal fungal species present within all clone libraries. Exceptions were for the clone libraries created from DNA extracted from roots of deer fern and, to a lesser degree, bunchberry. Random clone libraries from deer fern and bunchberry had the highest species richness (23 and 12 OTUs, respectively) and diversity; richness estimates indicate that the total species richness in each library may be double that detected (Table 3.2).

3.4 Discussion

Different plant species that grow in CH and HA clearcuts host AM fungi that are compatible with western redcedar, therefore providing a source of inoculum for regenerating western redcedar seedlings. The CH clearcuts had greater richness of AM plant species than the HA clearcuts. The cover of AM plants in CH clearcuts exceeded 90%, whereas AM hosts in HA clearcuts occurred sporadically. Plants that dominated in CH clearcuts, especially bunchberry, salal and deer fern, had high AM fungal species similarity to western redcedar. The sequencing results in this study also indicate there may be different levels of AM host specificity; although there were many AM fungal OTUs detected in one host species, the majority of the abundant OTUs were non-specific.

68 3.4.1 Lack of alternative ectomycorrhizal hosts to maintain fungal inoculum following clearcutting

Of the plant species sampled, none hosted EM fungi, and hence none could serve as alternative EM hosts for western hemlock regeneration. Though members in the genera Ribes, Rubus, Sambucus and Dryopteris have been reported to host both AM and EM fungi (Harley and Harley, 1987a, 1987b), EM fungi were not detected among the species of these plant genera found in the CH or HA clearcuts using either microscopic examination of the roots or DNA sequencing.

The absence of EM fungi among the target plant species suggests that wind dispersed spores, remnant mycorrhizal propagules and advance regeneration or mature legacy trees of western hemlock and amabilis fir are the primary source of EM inoculum for regenerating conspecific seedlings on CH and HA clearcuts. Though patches of advance regeneration can be common in HA clearcuts, they are often uncommon in CH clearcuts (Mogensen, personal communication 2007; personal observation). Partial forest harvest where mature legacy western hemlock and amabilis fir trees are retained could help conserve the pre-harvest EM fungal community inoculum (Kranabetter and Friesen, 2002; Teste and Simard, 2008; Simard, 2009a; Teste et al., 2009b). Following severe disturbance such as clearcutting, EM inoculum rapidly diminishes without presence of suitable hosts (Harvey et al., 1980; Perry et al., 1987; Amaranthus, 1991b; Hashimoto and Hyakumachi, 1998; Hagerman et al., 1999; Dahlberg, 2002; Jones et al., 2003), and dies within two years or less (Perry et al., 1987; Kabir et al., 1997). Cenococcum geophilum is one exception because it forms resistant sclerotia that remain viable many years without a host (LoBuglio, 1999). As shown in chapters 2 and 4, C. geophilum is widely distributed in both HA and CH forests and clearcuts. Even so, this species may impart little benefit to the seedlings (Marx et al., 1978; Dickie et al., 2002; Jones et al., 2009; Dalong et al., 2011). Given that EM colonization of roots of newly establishing western hemlock seedlings can greatly improve seedling survivorship and growth (Christy et al., 1982; Kropp, 1982; Smith and Read, 2008), immediate replanting of western hemlock following clearcutting is advisable. By temporarily capturing part of the original EM fungal community from the previous forest

69 found in remnant fine roots within the soil (Harvey et al., 1980; Hagerman et al., 1999), rapid replanting into existing mycorrhizal networks ought to facilitate seedling establishment until pioneer EM species establish (Jones et al., 2003).

The difference in abundance of AM plants on HA and CH clearcuts suggest that AM plants were of little competitive threat to western hemlock seedlings in HA clearcuts, but were important competitors in CH clearcuts. Messier (1993) showed that complete vegetation removal in both young and old CH clearcuts resulted in increased western hemlock growth as well as nitrogen and phosphorus availability. How domination of plant communities with AM fungal hosts influence EM colonization on planted seedlings, especially the colonization of pioneer EM fungal species, is unknown. However, there is evidence that prevalence of AM hosts can delay root colonization of conifer seedlings by native EM fungal species (Richter and Bruhn, 1993). Furthermore, direct interaction between mycorrhizal roots of coexisting AM and EM hosts can have an inhibitory effect on the proliferation of EM fungi among the roots and an associated reduced growth response of the EM host (Trappe and Perry, 1993; Haskins and Gehring, 2004, 2005; McHugh and Gehring, 2006). If similar dynamics are occurring between AM and EM fungi in CH sites, the dominance of AM hosts in regenerating forests may intensify competition between western hemlock seedlings and AM plants. This higher order interaction could partially account for the poor growth, nutrient uptake and survivorship of western hemlock seedlings extensively reported in earlier studies (Messier, 1993; Prescott et al., 1993; Prescott et al., 1996).

3.4.2 High species richness of arbuscular mycorrhizae with differing levels of host specificity

The fifty arbuscular mycorrhizal OTUs detected in our study is considered to be a relatively high, yet common richness estimate compared to AM species richness detected in other studies using similar methodology (Husband et al., 2002; Öpik et al., 2008; West et al., 2009; Castillo et al., 2010; Patreze et al., 2012; Chaiyasen et al., 2014). Unidentified Glomus species were the most abundant group detected, while Claroidioglomus and Acaulospora species were the next most common; this pattern of community composition is similar to other

70 studies in AM dominated ecosystems (Vandenkoornhuyse et al., 2002; Öpik et al., 2008; Öpik et al., 2009; West et al., 2009; Chaiyasen et al., 2014). Nevertheless, an entire suite of AM fungi may have been missed with the AM specific primers selected. Two specific AM phyla, Archaeosporaceae and Paraglomaceae, have been shown to have poor amplification results due to high sequence divergence affecting the primer binding site for the PCR primer AM1 (Redecker et al., 2000). Regardless of potentially overlooked AM fungal species, such high diversity of AM fungi detected should facilitate the dominance of AM plant species that were found in CH clearcuts. This is because AM diversity has been shown to influence plant productivity and competition; moreover, more diverse AM fungal communities have been shown to promote higher plant diversity (van der Heijden et al., 1998b).

Several of the plant species in my study are common to disturbed sites and have been previously classified as both AM hosts and non-mycorrhizal; these included spiny wood fern (Harley and Harley, 1987a, 1987b; Turnau et al., 1999), fireweed (Titus and Moral, 1998), pearly everlasting (Harley and Harley, 1987a, 1987b; Titus and Moral, 1998), elderberry, deer fern and species closely related to bunchberry of the genus Cornus (Harley and Harley, 1987a, 1987b). Succession stage may influence the AM status of these hosts. The factors that determine whether these host plant species become mycorrhizal is not fully understood, however, it has been suggested that being AM facultative aids the plant species in invading new sites devoid of mycorrhizal inoculum. Once colonized by AM fungi, the competitive status of the establishing plants is expected to be altered (Miller, 1987; Molina et al., 1992). In my study, all samples of these host species were colonized by AM fungi; in the case of CH clearcuts, this likely occurred because there are compatible remnant AM propagules and networks remaining from residual mature western redcedar trees and refuge plant species from the previous harvest.

Differing levels of AM fungal specificity were observed among the plant species surveyed. Sixteen of the fifty OTUs were shared between host plants of different species; ten of these OTUs were found in western redcedar roots. Plants species that were the most abundant in clearcuts (salal, bunchberry and deer fern) had the highest number of shared AM fungal OTUs. As seen in chapter 2, the salal, bunchberry and deer fern were present in uncut forests

71 and increased in abundance following clearcutting (Table 2.1 and 3.1); these species likely served as refuge species present before and after clearcutting. On the other hand, plant species that were the least abundant (fireweed, pearly everlasting, elderberry, salmonberry and trailing black current) where typically post-disturbance species colonizing the clearcuts and they had the fewest number of shared OTUs. By hosting nonspecific AM fungi, extensive networks of shared AM fungi can facilitate the recruitment of new seedlings through supply of limiting nutrients (van der Heijden, 2004).

3.4.3 Compatible arbuscular mycorrhizal fungi present in deer fern

In HA sites, most sampled plants were colonized by AM fungi, although AM diversity and shared compatibility with western redcedar was much lower than among plants in CH clearcuts. As mentioned earlier, all of the plants sampled in HA clearcuts are facultative AM hosts, suggesting they are able to colonize sites lacking mycorrhizal inoculum. Of all the plant species sampled, deer fern had the most diverse AM fungal assemblage and the OTUs were very similar to those found in western redcedar. Prior to this study, Weber et al. (2005) questioned the possibility that western redcedar shared compatible AM fungi with deer fern. Though there was previous microscopic evidence of AM structures within roots of deer fern (Harley and Harley, 1987a, 1987b; Wang and Qiu, 2006), there was also speculation that the AM fungal species had a high degree of specificity that could prevent co-colonization with different host species (Weber et al., 2005). The deer fern samples I collected from HA clearcuts provide evidence that the same AM fungal OTUs occur across multiple sites, but these fungi are likely restricted to small isolated patches surrounding compatible hosts, especially deer fern. Though previous studies did not detect AM fungal colonization of western redcedar seedlings planted in uncut forests (Weber et al., 2003) or in greenhouse pots using HA forest substrate (Weber et al., 2005), this may be attributed to sampling from locations lacking deer fern. Moreover, deer fern has previously been reported as facultative, where the plants have been found growing without mycobionts (Harley and Harley, 1987a, 1987b). Facultative mycorrhizal ability would allow deer fern to colonize new locations were AM propagules have not yet dispersed and suggests that not all deer fern present in HA forests are colonized by AM fungi.

72 In mixed forests of western redcedar and western hemlock, there is typically less plant cover and diversity in the understory of western hemlock trees than under western redcedar, even with similar light conditions (Turner and Franz, 1986). Suggested reasons include physical soil properties that are modified by the tree species, allelopathy or mycorrhizal competition (Turner and Franz, 1986). Given that most understory plant species in these forests are AM hosts, low availability of AM fungal inoculum in ectomycorrhizal HA forests may explain the exclusion of understory AM plant species, especially under low light conditions (Weber et al., 2003).

3.4.4 Could salal and huckleberry truly have arbuscular mycorrhizal mycobionts?

Of the plant species that were screened for AM fungi, salal had the most surprising results. Not only was the AM fungal species composition similar to western redcedar, but the sequence similarity was notable given the geographical separation of salal sample points compared to the other sample collections. Salal was collected from the south-western mainland of British Columbia, whereas the rest of the plant species were collected from northern Vancouver Island in British Columbia. With the distant geographical isolation of these populations, greater divergence of the communities was expected (Molina et al., 1992; Lekberg et al., 2007), though Hazard et al. (2013) demonstrated that geographical distance can have little impact.

Ericoid mycorrhizae of salal collected from CH and HA forests has been extensively studied, yet AM fungal species had not been previously identified. Culturing and axenic re- synthesis experiments in the lab previously detected ascomycete ericoid mycobionts (Xiao and Berch, 1992; Xiao and Berch, 1995). The occasional electron microscopy images that were taken confirmed that the fungal structures observed did not penetrate the cellular membrane (a defining characteristic of mycorrhizal associations) and the fungal hyphae involved had ascomycete cellular characteristics (Xiao and Berch, 1992). Sequencing results from past studies used the fungal specific primers ITS1F and TW13; these studies also only detected ascomycete associates and some uncultureable basidiomycete associates (Monreal et al., 1999; Berch et al.,

73 2002; Allen et al., 2003). However, similar to my study, there was no microscopic confirmation of basidiomycete cellular structures within salal roots (Allen et al., 2003). As seen in my results and other studies, the ITS primers did not amplify AM fungal DNA during PCR (Redecker et al., 2000; Anderson et al., 2003); therefore, AM fungi would not have been detected in previous molecular studies on salal mycorrhizal associates. My study is not the first to report AM fungi in the genus Gaultheria; using microscopic examination of roots and identification of associated Glomus spores, dual colonization by both ericoid mycorrhizal fungi and AM fungi in roots of Gaultheria fragrantissima (Das and Kayang, 2012) and Gaultheria poeppiggi (Urcelay, 2002) have been recorded. Though AM fungi were detected in my study using AM specific primers, confirmed imaging of Glomeromycota fungal structures within salal roots is needed to determine whether the AM sequences detected were from symbiotic associations. Synthesis experiments (Mosse, 1962) with AM fungi and salal and the application of fluorescent in-situ hybridization (Vagi et al., 2014) using both AM fungal specific probes and salal plant DNA probes on roots collected from environmental samples would provide supporting evidence to confirm the AM status of salal.

There is evidence that many ericoid plant species have the potential to form different types of mycorrhizal associations, including AM. Ericoid shrubs been reported to have AM associations include Styphelia tameiameiae (Koske et al., 1990; Koske et al., 1992), Astroloma spp (McGee, 1986), Leucopogon juniperinus, Epacris microphylla and Brachyloma daphnoides (Bellgard, 1991). Various species in the genus Vaccinium have also been reported to be colonized by both ericoid mycorrhizal and AM fungi (Koske et al., 1990; Koske et al., 1992). In my study, one out of six huckleberry plants surveyed had AM fungal OTUs detected, indicating a facultative AM status. Certain plants form facultative root associations with mycorrhizal fungi (Zhi-wei, 2000; Brundrett, 2004; Smith et al., 2009), especially when growing in close proximity to a different plant species that is the primary host of the mycorrhizal fungus (Hartnett et al., 1993). Furthermore, AM root colonization depends on environmental factors (Muthukumar et al., 2004); for example, mycorrhizal colonization can decrease and in some cases be absent when hosts are growing epiphytically versus terrestrially (Kessler et al., 2009; Kessler et al., 2010). Given that most huckleberry in HA sites were found growing on raised tree stumps, AM

74 colonization may be depend on where the plants are growing, availability of AM inoculum and proximity to other AM hosts. Vaccinium species have also been found to form ectomycorrhiza- like symbioses, characterized by sheath-like mantles, with basidiomycetes closely related to the order Trechisporales; this symbiosis proved more effective at degrading recalcitrant substrate and increasing plant growth than common ericoid ascomycete mycorrhizae (Vohník et al., 2012). Ectomycorrhizal associations with huckleberry roots, however, were not detected in my study.

Some mycologists question whether the reported AM structures in ericoid roots are true symbionts or merely the result of root penetration by AM fungi when in close proximity to the “true” host (Francis and Read, 1995; Read, 1996). Read (1996) emphasized not only the importance of meeting the requirements of Koch’s postulates (Koch, 1912), but that a healthy relationship is produced and a measure of host response is needed to confirm the AM status of ericoid hosts (Read, 1996). However, it is now more widely accepted that mycorrhizal symbioses occur along a spectrum, from beneficial to parasitic (Francis and Read, 1995; Jones and Smith, 2004) and that AM fungi affect plant species differently (Lee et al., 2013). With this in mind, it would be worthwhile to further examine the mycorrhizal status and role of the AM fungi found in salal roots, especially with regards to overall plant community structure and environmental conditions that may influence the association.

The implications of AM fungi colonizing salal are substantial, potentially explaining the coexistence between salal and western redcedar. Previous research has indicated that in CH sites, there is strong competition between western hemlock and salal, but not between western redcedar and salal (Fraser et al., 1995; Keenan et al., 1995). Shared AM fungal compatibility between salal and western redcedar could allow for development of shared AM networks, potentially reducing competition between these two host plant species and facilitating their coexistence in these nutrient poor ecosystems. Although there is little evidence for carbon transfer between hosts in a common AM network, there is evidence for N transfer (Hodge and Storer, 2015), the nutrient that is most limiting in clearcuts of these forests (Prescott et al., 1996; Blevins and Prescott, 2002). Furthermore, given that salal has greater

75 capacity than western redcedar to invade HA forests and disturbed sites (Blevins and Prescott, 2002; McWilliams and Klinka, 2005a), and given that it might be a facultative AM host, its establishment could provide a pathway for introducing AM inoculum that is necessary for establishment of other obligate AM host species.

3.4.5 Conclusion

I found high AM fungal species richness and high abundance of AM fungi shared among the plant species that commonly occur in CH clearcuts, yet the AM fungal diversity might be even higher. The shared compatibility among hosts indicated varying degrees of host specificity. Further research is warranted to determine if certain AM fungal species greatly impact ecosystem processes, plant competition and productivity essential to forest regeneration and succession. Furthermore, in many ecosystems, delayed re-establishment of tree seedlings following clearcutting has been shown to rapidly reduce available mycorrhizal inoculum, therefore reducing survivorship and growth of tree seedlings. With the lack of alternative EM host plant species in CH and HA clearcuts, EM propagules will rapidly diminish following clearcutting if western hemlock is not immediately replanted or re-established naturally. On the other hand, the high abundance of AM fungal propagules associated with western redcedar in CH forests would facilitate rapid colonization with compatible AM plants and western redcedar following disturbance such as clearcutting. During forest regeneration, the predominance of an AM plant community in CH clearcuts with shared mycorrhizal symbionts suggests that AM networks can form and influence competition among different plant species and western redcedar. The presence of compatible AM networks likely facilitates the re-establishment of western redcedar in these sites where nutrients are low and plant competition is high (Messier, 1993; Fraser et al., 1995).

76 Key: Glomeromycota DNA extracted from Figures and Tables (Thuja plicata) (Thuja plicata)

(Cornus canadensis) (Blechnum spicant) (Dryopteris expansa) (Gaultheria shallon)

(Vaccinium parvifolium) (Epilobium angustifolium)

(Anaphalis margaritacea) (Sambucus racemosa) (Ribes laxiflorum)

(Rubus spectabilis) Glomeraceae

Acaulosporaceae

Claroideoglomeraceae

Unknown family

Figure 3.1 77

Figure 3.1 Maximum likelihood phylogenetic tree of Glomeromycota fungi. Phylogeny was constructed using a 485bp fragment of the small rRNA subunit gene, which is a region amplified using the primers AM1 and WANDA. One representative sequence of each operational taxonomic unit (OTU) sharing 99% sequence similarity detected in this study is indicated by OTU ID. Colored boxes indicate whether the OTUs were detected in roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), in roots of western redcedar seedlings harvested from old growth forests (Cw-natural) and/or in roots of the dominant vegetation species collected from three-year post-harvest clearcut sites. Family clades to which the OTUs detected in this study belong to are indicated. Support values >60% based on 1000 bootstrap values are shown. The choanozoan Corallochytrium limacisporum, a close relative to fungi (Cavalier-Smith and Allsopp, 1996), was used as an outgroup.

Cw - greenhouse Cw - natural Bunchberry Deer fern OTU 01 Thuja plicata Thuja plicata Cornus canadensis Blechnum spicant OTU 06

OTU 31 OTU 02 OTU 10 OTU 14 OTU 17 OTU 18

OTU 12 OTU 26 OTU 12 OTU 32 OTU 37 Spiny wood fern Salal Red huckleberry Fireweed OTU 39 Dryopteris expansa Gaultheria shallon Vaccinium parvifolium Epilobium angustifolium OTU 50

OTU 02 OTU 26

Pearly everlasting Elderberry Trailing black currant Salmonberry Anaphalis margaritacea Sambucus racemosa Ribes laxiflorum Rubus spectabilis OTU 50

OTU 31 OTU 41

OTU 41

OTU 26

Figure 3.2 Proportion of clone sequences of operational taxonomic units (OTUs) of Glomeromycota fungi found in roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), in roots of western redcedar seedlings harvested from old growth forests (Cw- natural) and in roots of the dominant vegetation species collected from clearcut sites. Colored sectors indicate specific OTUs shared between western redcedar and alternative host species. Gray sectors indicate OTUs that were not found in western redcedar; gray sectors with OTU labels next to them indicate OTUs shared between different host species, but not with western redcedar.

25 Key: Collector curves of clone libraries from

(Thuja plicata) 20 (Thuja plicata) (Cornus canadensis)

(Blechnum spicant) 15 (Dryopteris expansa)

(Gaultheria shallon) (Vaccinium parvifolium) 10 (Epilobium angustifolium)

(Anaphalis margaritacea) 5 (Sambucus racemosa)

(Ribes laxiflorum) operational taxonomic units detected units taxonomic operational

Number of arbuscular mycorrhizal fungal fungal mycorrhizal arbuscular of Number (Rubus spectabilis) 0 0 10 20 30 40 50 60 70 80 90 100 Number of clones sequenced per random clone library

Figure 3.3 Coleman collector curves for arbuscular mycorrhizal fungal operational taxonomic units detected in clone libraries of the small rRNA subunit gene amplified from DNA extracted from roots of different plant species. Collector curves for clone libraries generated from roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), from roots of western redcedar seedlings harvested from old growth forests (Cw-natural) and from roots of the dominant vegetation species from three- year post-harvest clearcut sites are indicated by curve color.

Table 3.1 Abundance of vascular plant species found within three-year post-harvest clearcuts of Hemlock-Amabilis fir forests (HA) and of Cedar-Hemlock forests (CH).

Cover (%) Common name Scientific name HA sites CH sites

Salal Gaultheria shallon 1-5 50-70 Bunchberry Cornus canadensis 0 30-50 Deer fern Blechnum spicant 10-15 5-10 Fireweed Epilobium angustifolium 5-15 0 Red huckleberry Vaccinium parvifolium 5-10 1-5 Salmonberry Rubus spectabilis 1 0 Pearly everlasting Anaphalis margaritacea 0 1-5 Spiny wood fern Dryopteris expansa 1 5 Trailing black currant Ribes laxiflorum 1 0 Elderberry Sambucus racemosa 0 <1

Table 3.2 Richness estimates and diversity indices of arbuscular mycorrhizal fungal operational taxonomic units detected in clone libraries of the small rRNA subunit gene amplified from DNA extracted from roots of western redcedar seedlings grown in greenhouse bioassays (Cw-greenhouse), from western redcedar seedlings harvested from old growth forests (Cw-natural) and from roots of the dominant plant species in the three-year post-harvest clearcut sites of Hemlock-Amabilis fir (HA) and Cedar-Hemlock (CH) forests.

)

) )

greenhouse natural

- -

Blechnum spicant Rubus spectabilis Ribes laxiflorum Vaccinium parvifolium Epilobium angustifolium canadensisCornus Dryopteris expansa Sambucus racemosa Anaphalis margaritacea Thuja plicata Thuja plicata Gaultheria shallon

Deer fernDeer Salmonberry blackTrailing currant huckleberryRed Fireweed Bunchberry Spiny wood fern Elderberry Pearly everlasting Cw Cw Salal

( ( ( ( ( ( ( ( ( ( ( (

OTU Richness 23 8 7 6 6 12 7 6 2 4 10 10

Jack 1 Richness Estimate 36 8 7 6 7 17 7 6 2 4 10 10 Jack 2 Richness Estimate 47 8 7 6 6 22 7 6 2 4 10 10 Chao 1 Richness Estimate 49 8 7 6 6 22 7 6 2 4 10 10

Shannon Index H' 2.69 1.58 1.25 1.60 1.15 1.99 1.49 1.51 0.50 1.21 1.99 1.78 Shannon Evenness (H'/Hmax) E 0.86 0.76 0.64 0.89 0.64 0.80 0.77 0.84 0.73 0.88 0.86 0.77

Simpson's Diversity Index D (1-D) 0.91 0.72 0.61 0.78 0.61 0.84 0.73 0.75 0.33 0.70 0.84 0.78 Simpson's Reciprical 1/D 9.99 3.44 2.51 4.30 2.49 5.98 3.56 3.81 1.48 2.91 5.80 4.39 Simpson's Evenness E 0.43 0.43 0.36 0.72 0.41 0.50 0.51 0.63 0.74 0.73 0.58 0.44

Chapter 4: Importance of mycorrhizal inoculum for conifer seedling establishment in CH and HA clearcuts

4.1 Introduction

When plant seedlings are establishing, they are most vulnerable to mortality during the period between germination and development as a juvenile plant (Kitajima and Fenner, 2000). During this period, mycorrhizal colonization is often critical for successful seedling establishment (Trappe and Strand, 1969; Hayman, 1974; Fenner, 1987; Perry et al., 1987; Amaranthus and Perry, 1989b; Thiet and Boerner, 2007). Following forest disturbance, such as fire, wind throw or clearcutting, the re-establishment of conifer seedlings can be problematic if appropriate mycorrhizal inoculum is not available (Janos, 1980; Amaranthus and Trappe, 1993); in some cases, complete failure of seedling survivorship has been observed (Amaranthus and Perry, 1987).

Mycorrhizal fungal inoculum can exist in the soil in many forms; these include fungal spores (Daniels et al., 1981; Fox, 1983; Friese and Allen, 1991), fungal mycelia in mycorrhizal networks (Brundrett and Abbott, 1994; Kytöviita et al., 2003; Giovannetti et al., 2004; Simard and Durall, 2004) or associated with residual root systems of harvested trees (Biermann and Linderman, 1983; Friese and Allen, 1991), and resistant propagules produced by fungi such as sclerotia (Trappe, 1969). Spore viability can be short lived in the soil; some spores, such as the large ones produced by mycorrhizal fungi in the division Glomeromycota, are often readily grazed by invertebrates (Edman et al., 2004). Fungal mycelia require carbohydrate resources from their host plant to grow and persist (Jumpponen and Egerton-Warburton, 2005). Often, this mycelium connects different host plants; in many cases, where different plant species share the same mycorrhizal fungal species, an interspecific mycorrhizal network forms (Perry et al., 1992; Simard et al., 1997a; Simard et al., 1997b; Kennedy et al., 2003; Simard, 2012). Disturbances that kill host plants cause rapid decline of the fungal mycelium and networks within the soil (Harvey et al., 1980; Perry et al., 1987; Amaranthus, 1991b; Hashimoto and

Hyakumachi, 1998; Hagerman et al., 1999; Bruns et al., 2002; Dahlberg, 2002; Jones et al., 2003). Dispersal of spores from forests also play a role in mycorrhizal colonization of plants (Cazares, 1992; Bruns et al., 2002; Jumpponen et al., 2002; Kjoller and Bruns, 2003; Teste et al., 2009b; Barker et al., 2013). Most EM fungal spores produced by mushrooms rapidly disperse by wind (Lilleskov and Bruns, 2005; Geml et al., 2008; Geml et al., 2012), yet many are dependent on animal vectors to travel long distances (Terwilliger and Pastor, 1999; Lilleskov and Bruns, 2005; Galante et al., 2011). Though spores of Glomus are wind-dispersed to some extent (Kivlin et al., 2014), many of the extraordinarily large AM fungal spores typically disperse over much shorter distances, requiring soil translocation or transport by animal vectors (Janos et al., 1995). However, very low success of spore germination for numerous mycorrhizal fungal species is common, as many have narrow ecological niches and require carbohydrate supply from an already established host plant (Deacon et al., 1983; Fox, 1983; Mason et al., 1983; Jumpponen and Egerton-Warburton, 2005). Furthermore, Peay et al. (2012) demonstrated that establishment of air-borne EM spores on regenerating seedlings diminish with increased distance from the forest edge.

Spatial distribution of mycorrhizal fungi and inoculum availability are greatly influenced by the plant community (Moyersoen et al., 1998; Korb et al., 2003; Thiet and Boerner, 2007; Dickie et al., 2013; Nave et al., 2013). Ecosystems with predominantly AM plant species have ample AM inoculum availability, but restricted EM inoculum (Thiet and Boerner, 2007). For example, in mixed temperate deciduous forests, the below-ground biomass of 232hyphae of a particular mycorrhizal guild was reflective of the dominance of AM and EM host trees; as the ratio of AM host trees increased, so did the total biomass of AM hyphae, whereas the EM hyphal biomass decreased (Nave et al., 2013). Likewise, an EM dominated forest that excluded understory AM plants by forming a dense canopy and occluding light was devoid of AM inoculum (Weber et al., 2003; Weber et al., 2005). Following a disturbance, the residual biota remaining from the previous plant community can have a legacy effect on the future development of the regenerating ecosystem, as remnant fungal propagules in the soil serve as inoculum that can favor the establishment of plant species of a compatible mycorrhizal guild, partially by bestowing a competitive edge over non-compatible plant species (Janos, 1980;

Allen and Allen, 1984; Wilson and Hartnett, 1998; van der Heijden et al., 1998a; Stampe and Daehler, 2003; Wardle et al., 2004). Following severe disturbance, loss of mycorrhizal inoculum or delayed re-establishment of host plants of a specific mycorrhizal guild can result in a shift in the ecosystem that favors dominance of plants of the opposite mycorrhizal guild (Perry et al., 1987; Amaranthus, 1991a; Simard, 2009b). This phenomenon has been observed in forest communities where delayed establishment of Douglas fir (Pseudotsuga menziesii) resulted in the loss of compatible EM inoculum and the simultaneous rapid colonization with AM shrubs leading to AM dominated regeneration. Efforts to re-establish Douglas fir seedlings in these conditions has resulted in poor seedling survivorship and in some cases , complete regeneration failure (Amaranthus and Perry, 1987). Studies that examined the role of mycorrhizal inoculum in these problematic regenerating sites have shown that introducing the necessary EM inoculum to seedlings using soil transfers from EM dominated forests at the time of planting can significantly improve seedling survivorship, nutrient uptake and growth (Amaranthus and Perry, 1987, 1989b; Amaranthus et al., 1999).

In this study, I examined the effect that mycorrhizal inoculum at the time of planting had on mycorrhizal root colonization, seedling survivorship and growth performance of western redcedar and western hemlock in clearcuts of both AM and EM dominated forests on northern Vancouver Island, British Columbia. The EM dominated forests were comprised almost exclusively of EM western hemlock and amabilis fir (Abies amabilis Douglas ex J.Forbes) (referred to as Hemlock-Amabilis fir or HA forests). The other forest type was dominated by AM western redcedar with minor amounts of subordinate western hemlock and amabilis fir (referred to as Cedar-Hemlock or CH forests) (Lewis, 1982; Prescott et al., 1996). Following forest harvest, planted western hemlock seedlings grow slowly and are nutrient limited in CH forest clearcuts, but not so in HA forest clearcuts (Prescott and Weetman, 1994). By contrast, growth of planted western redcedar does not differ as substantially between forest types (Prescott et al., 1996; Blevins and Prescott, 2002).

Following disturbance, western redcedar seeds are dispersed and germinate on sites previously occupied by either CH or HA forests, but seedling establishment is not successful on HA sites (Weber et al., 2014). Studies show that AM fungal colonization of western redcedar 85 roots does not happen when seedlings are grown in forest floor of HA forests. Under shaded conditions, as occurs in mature HA forests or under competition from faster growing western hemlock seedlings, absence of AM root colonization greatly reduces western redcedar seedling survivorship (Weber et al., 2003; Weber et al., 2005). From the previous research by Weber et al. (2003, 2005), it was concluded that HA forests lack AM fungal inoculum compatible with western redcedar. However, results from my research indicate that deer fern (Blechnum spicant), which grows sparsely in the understory of HA forests, can host compatible AM fungi for western redcedar (chapter 3).

The objective of this study was to compare mycorrhizal colonization of seedlings during early establishment in clearcuts of HA and CH forests. This was achieved by examining the mycorrhizal colonization of western hemlock and western redcedar grown as seedling bioassays in clearcuts. Furthermore, I tested whether providing mycorrhizal inoculum, in the form of soil transfers from nearby uncut CH and HA forests, improved mycorrhizal colonization of roots and seedling performance, as measured by growth and survivorship, in either clearcut type during early forest regeneration. Using this approach, I tested two hypotheses. The first hypothesis was that there are differences in availability of EM and AM fungal inoculum between clearcuts of CH and HA forests, reflective of the mycorrhizal guild that dominated the previous forest. I predicted that CH forests would have ample AM fungal inoculum for colonizing western redcedar seedlings, but limited EM fungal inoculum for colonizing western hemlock seedlings. Conversely, I predicted that HA forests would have ample EM fungal inoculum for colonizing western hemlock seedlings, but would be devoid of AM fungal inoculum for western redcedar. The second hypothesis was that, where mycorrhizal inoculum of a particular guild was lacking from the clearcut of a given forest type, soil transfers, comprised of LFH layers, at the time of planting from a nearby forest of the opposite forest type would improve mycorrhizal colonization, thereby increasing seedling performance and survivorship. Thus, I predicted that in CH clearcuts, soil transfers from HA forests would improve EM fungal colonization of western hemlock seedlings, and likewise, in HA clearcuts, soil transfers from CH forests would improve AM fungal colonization of western redcedar seedlings. Furthermore, I predicted seedling survivorship and growth would increase with mycorrhizal colonization.

4.2 Methods

4.2.1 Study sites

Three replicate pairs of clearcuts of adjacent CH and HA forests with matching site characteristics were selected on northern Vancouver Island; for description of study sites and forests prior to harvest, refer to chapter 2 and 3 study sites.

4.2.2 Experimental design and treatments

The field experiment was established in the three replicate HA and CH clearcuts in October 2008. The treatments were organized in a split-plot structure in a randomized block design, where replicate site was the random blocking factor. In this structure, the whole plot factor was forest type (CH versus HA), and the split-plot factor was an inoculum treatment in the form of a soil transfer of the LFH layers of the forest floor from uncut, old-growth or mature forests. The same design was utilized for both western redcedar seedlings and western hemlock seedlings. Twelve circular plots, each at least 5 m apart, were distributed throughout each clearcut. Plots were randomly assigned an inoculum treatment. Inoculum treatments were comprised of 150ml of (1) forest floor transferred from a mature HA forest, (2) forest floor transferred from around western hemlock trees in an old-growth CH forest, and (3) forest transferred from around western redcedar trees in an old-growth CH forest. Hereon, the forest floor utilized is referred to as a “soil transfer”. As a control, seedlings were also planted without a soil transfer. At each replicate clearcut site, this amounted to three plots for each of the four inoculum treatments (three soil transfer treatments plus one control treatment). To collect the forest floor for the transfer treatments, one 15 L composite, homogenized forest floor sample was collected from the nearby uncut mature HA forest and two 15 L composite, homogenized forest floor samples were collected from the nearby uncut old-growth CH forest (one composite sample from around western hemlock trees, and one composite sample from around western redcedar trees). To create each composite forest floor sample, three trees were randomly selected, 5 L of forest floor was excavated to a depth of 15 cm from under the

87 drip line of each tree, and then the 5 L forest floor samples were mixed together to a create a composite 15 L sample. Within 12 hours of being collected, these forest floor samples were utilized as soil transfers during the planting of conifer seedlings. Six-month-old greenhouse grown conifer seedlings were provided by Western Forest Products from their nursery located in Saanich on Vancouver Island. Prior to seedling germination, both soil and styroblocks were steam sterilized. Seedlings were grown with optimal fertilization to maximize growth rates and minimize the potential for mycorrhizal colonization. Prior to planting, roots of twenty seedlings of both western redcedar and western hemlock were examined microscopically for mycorrhizal colonization. In addition to examination for EM morphotypes on root tips of western hemlock, roots of both western redcedar and western hemlock examined for AM colonization after clearing and staining (Koske and Gemma, 1989) as described below, given that AM fungal infection has been reported among young seedlings of western hemlock (Cázares and Smith, 1996) and also to confirm that roots of western hemlock did not have Hartig nets in the absence of visible mantles. All roots were found to be devoid of any mycorrhizal colonization and fungal root endophytes. In the center of each plot, a 1.5 m long piece of rebar was driven 30 cm into the ground and the location was recorded using a recreational handheld Global Positioning System (GPS) (Garmin, US). Within a 4 m radius around the central rebar, five seedlings of a given tree species were planted at least 2 m apart from each other in a random pattern (5 seedlings X 3 planting locations X 2 ecosystems X 4 soil transfer treatments X 3 replicate sites = total of 360 seedlings of each species). At the time of planting, each seedling received 150 ml of the assigned soil transfer treatment placed around its root ball in the planting hole. Seedlings were tagged with an identifying number using sequentially numbered, laser-printed, plastic security seals (OneSeal, Canada) and their corresponding plot number and treatment were recorded. To prevent animal browsing, each planted seedling was surrounded by a rigid plastic mesh protection tube (Northwest Plastic Extrusion, US).

4.2.3 Seedling measurements

Two partial seedling harvests were carried out to examine periodic seedling growth and mycorrhizal colonization; the first harvest was after one growing season and the second harvest was after two growing seasons. The first partial harvest was conducted in late October 2009, in which two seedlings of western hemlock and two seedlings of western redcedar were randomly selected per plot. At the end of two growing seasons, the remaining bioassay seedlings were harvested in late October 2010. The first harvest entailed excavation of entire root systems with a shovel. For the second harvest, only partial root systems could be excavated due to their large size and root growth entanglement with old dead roots from residual trees. Seedling height and stem diameter were measured and then the aboveground portion (including foliage and stems) was severed at the root collar and oven dried at 70˚C to weigh for dry biomass. The roots of seedlings were rinsed to remove soil and debris and then stored at -20°C until examined microscopically for mycorrhizal colonization.

4.2.4 Examination of root tips

Western redcedar and western hemlock root tips were examined using different techniques. Ectomycorrhizae of western hemlock were identified using morphotyping (Agerer, 1996). First, the overall root system was visually inspected to estimate the percent colonization of root tips. Using a dissecting microscope, EM root tips were randomly selected. For each sample, root fragments were distributed evenly on a 10 by 10 grid, with 1 cm X 1 cm squares, in a Petri plate filled with water. Using a random number table, up to 25 EM root tips per sample were excised from root fragments overlying the grid locations corresponding to the random numbers. Given low EM colonization of western hemlock roots in this study, slightly less than 25 EM root tips per seedling was common. The numbers of EM root tips per morphotype were tallied. Arbuscular mycorrhizal colonization of western redcedar was examined microscopically following trypan blue staining of roots (Koske and Gemma, 1989). Twenty five seedlings per treatment were randomly selected per year (100 seedlings per year). Initially, 50 seedlings from the first growing season were randomly selected and 10 segments of fine roots, each 5 cm long, 89 were stained and examined microscopically for AM infection to determine the number of fragments necessary for the detection of AM infection. Following, due to the high number of seedlings to process, microscopic examination was carried out for only 5 root fragments per sample. Microscopic examination of stained root segments, each 1 cm long, was carried out to detect the presence/absence of AM structures. Following, for each 1 cm stained root fragment, 4 sub-segments, each 0.2 mm long and approximately 2 mm apart from each other, were randomly selected examined using a 40X objective lens on a compound microscope to estimate percent root infection. The estimated percentage of cells colonized with arbuscules and vesicles encountered within each segment were averaged for each plant examined. Percent root colonization with dark septate endophytes observed during the microscopic examination for AM fungal colonization were also recorded.

4.2.5 Statistical analyses

This experiment utilized a split-plot model with a randomized block design (Quinn and Keough, 2002), in which replicate sites (total of 3) were the blocks. Within sites, the whole-plot factor was the ecosystem prior to harvest (CH or HA), and the split-plot factor was the inoculum treatment (four total) randomly applied to subplots within each ecosystem (4 treatments X 3 subplots per treatment X 2 ecosystems x 3 replicate sites = 72 subplots total). A linear mixed model analysis with restricted maximum likelihood (REML) estimation was utilized to test the main effects (whole-plot and split-plot) and the two-way interaction between the main effects on the response variables. In this mixed model, the main effects were fixed effects, which were ecosystem type prior to harvest and inoculum source (Bennington and Thayne, 1994). The response variables were seedling growth measurements (height, stem diameter and dry aboveground biomass) and percent mycorrhizal root colonization. Site location was a random effect incorporated into the model as the experimental error; subsampling of seedlings harvested each year was a random effect incorporated into the model as the sampling error. REML estimation was used rather than the traditional, more widely known, analysis of variance (ANOVA), given that not only does REML perform equally as well as the ANOVA in balanced models with balanced data, REML better matches the split-plot model utilized to give optimal

90 results (Corbeil and Searle, 1976; Littell, 2002) since it automatically corrects for the right F tests. It is also better suited to unbalanced data (Corbeil and Searle, 1976; Wulff, 2008), as was the case with this study, given that (1) the number of observations per treatment varied upon harvest of seedlings due to mortality, and (2) because only a randomly selected subset of root samples from total number harvested were examined for mycorrhizal colonization. All statistical analyses were performed using JMP® for Windows, Version 12 statistical software (SAS Institute Inc., Cary, N.C.). Data for western redcedar seedlings and western hemlock seedlings were analyzed separately. To ensure that response variables met the required REML assumptions of normality and homogeneity of variance, the Shapiro-Wilk test for normality and Levene’s test for equal variance were utilized in the JMP Distribution platform. Response data that did not meet normality -- all seedling growth measurements and mycorrhizal colonization data -- were transformed by the natural log. Treatment effects were tested on JMP Fit Model platform using model described above. Where REML estimate showed significant effects of ecosystem type and/or inoculum treatment, post hoc analyses using multiple pairwise comparisons were made using the Tukey’s honest significant difference test (α=0.05) to determine where differences between groups occurred. Furthermore, correlation analysis was performed to examine the relationship between seedling performance variables and mycorrhizal colonization.

4.3 Results

Seedling survivorship was high (>95%) among all treatment groups for both western redcedar and western hemlock (results not shown). Western hemlock was taller in HA than CH clearcuts (P=0.011), and its larger size was reflected in non-significant trends in dry above- ground biomass and stem diameter after the first and second growing seasons (Table 4.1 and 4.2). Western redcedar likewise tended to perform better in HA than CH clearcuts, but differences were not significant at α=0.05 (Table 4.3 and 4.4). Soil transfers significantly affected growth of western hemlock and mycorrhizal root colonization of western redcedar (P<0.05) (Table 4.1, 4.2, 4.3 and 4.4). Based on REML analyses, there were no significant

91 interactions between ecosystem type and soil transfer on growth parameters of either western hemlock or western redcedar seedlings.

For western hemlock, soil transfers affected seedling growth parameters in both the first and second growing seasons (Table 4.1 and 4.2; Figure 4.1 and 4.2). In both CH and HA clearcut sites, western hemlock planted with forest floor collected from the base of a western redcedar tree in a mature CH forest had significantly reduced growth compared to all other treatments (Table 4.1 and 4.2). Western hemlock seedlings that received this soil transfer treatment in HA clearcut sites had a mean growth rate comparable to western hemlock seedlings in CH clearcut sites, where they generally grew slowly; western hemlock that received this same soil transfer treatment in a CH clearcut sites grew the least compared with all treatments studied (Figure 4.1 and 4.2). Mycorrhizal colonization of western hemlock roots was detected after the first growing season, but was not affected by either ecosystem type or soil transfer treatment. Ectomycorrhizal colonization of western hemlock seedlings was <10% across all treatments and was almost exclusively with Cenococcum geophilum (Table 4.1). During the harvest after the second growth season, larger seedlings were observed to have high levels of mycorrhizal colonization with morphotypes similar to Lactarius/Russula and Craterellus species, whereas smaller seedlings had lower colonization of C. geophilum and Lactarius morphotypes, but these were not quantified due to cold storage failure and subsequent deterioration of the samples.

For western redcedar, mycorrhizal colonization differed significantly among soil transfer treatments (Table 4.3 and 4.4; Figure 4.5 and 4.6). In HA clearcut sites, western redcedar seedlings grown either with soil transfer from mature HA forests or without soil transfer had minimal mycorrhizal colonization after both the first and second growing seasons. By contrast, soil transfers from mature CH forests significantly increased mycorrhizal colonization (P < .05) (Table 4.3 and 4.4; Figure 4.5 and 4.6). In both CH and HA clearcut sites, forest floor collected from under a western redcedar tree in a mature CH forest significantly increased both the percent of seedlings colonized with AM and percent root colonization after the first growing season. Mycorrhizal colonization of western redcedar seedlings also increased in HA but not CH clearcuts with forest floor transferred from under a western hemlock tree in a mature CH 92 forest. After the second growing season, there were no differences in mycorrhizal colonization within CH clearcut sites; however, western redcedar seedlings in HA clearcuts continued to have significantly higher mycorrhizal colonization where they received inoculum from mature CH forests. In HA clearcuts, growth and biomass of western redcedar tended to be greater after the second growing season with a soil transfer from mature CH forests, however, the difference was not statistically significant (Table 4.4; Figure 4.3 and 4.4).

Arbuscular mycorrhizal colonization of western redcedar was linearly correlated with its growth response (Figure 4.7); the significance and R2 values increased from the first to the second growing season and these were most prominent in HA clearcut sites. Western redcedar seedlings without mycorrhizal colonization in HA clearcut sites had similar growth as those observed among the mycorrhizal seedlings grown in CH clearcut sites. During the microscopic examination of western redcedar roots for arbuscular mycorrhizae, dark septate endophytes were also observed and recorded; these did not vary by ecosystem type or soil transfer treatment.

4.4 Discussion

Small reciprocal soil transfers, comprised of the LFH layers of the forest floor from old- growth CH forests and mature HA forests, at the time of planting greenhouse-grown seedlings into three-year post-harvest clearcut sites significantly influenced mycorrhizal colonization of western redcedar and growth of western hemlock seedlings. Seedlings of both tree species grew larger in HA clearcuts than in CH clearcuts, most likely due to differences in nutrient availability characterized in previous studies (Prescott et al., 1993). This study shows that mycorrhizal fungi as well as other unidentified soil related factors are important factors determining success or failure of conifer regeneration in the coastal temperate rainforests of British Columbia.

4.4.1 Arbuscular mycorrhizal colonization of western redcedar

Forest floor transferred from old-growth CH forests into planting holes of western redcedar seedlings in HA clearcuts greatly improved mycorrhizal root colonization compared to the control group or forest floor transferred from mature HA forests. Given that low AM colonization of western redcedar roots occurred among the control group, it is clear that AM fungal inoculum is present in HA clearcuts, but is limited in availability. This AM inoculum could have been sourced from deer fern refuge plants (see chapter 3). Weber et al. (2003) suggested that if AM propagules were present, AM colonization of redcedar could be inhibited in HA. However, I found high AM root colonization in HA clearcuts when provided with inoculum from CH forests, suggesting that AM fungal inoculum was lacking rather than being present but inhibited in HA substrate. However, my study was carried out in clearcuts three years post- harvest whereas Weber et al. (2003 and 2005) tested substrate from uncut forests, and it is possible that potential factors inhibiting AM growth diminish following forest harvest.

Arbuscular mycorrhizal colonization was positively correlated with growth of western redcedar seedlings, agreeing with Weber et al. (2003) and Kough et al. (1985). This correlation was more pronounced in HA than in CH clearcuts and redcedar planted in HA sites were more responsive to soil transfers. In HA clearcuts, the weaker correlation and smaller growth response with soil transfers in the first than second growing season may be attributed to residual fertilizer seedlings received in the greenhouse prior to planting. Often, AM fungi bestow less benefit to their host when nutrients are high (Whiteside et al., 2012).

Without AM fungal colonization, I found that western redcedar was not achieving its full growth potential in HA clearcuts during early establishment. The positive correlation between AM fungal colonization and seedling biomass indicates that western redcedar has greater growth potential in HA sites if it is provided with the necessary fungal inoculum at the time of planting. The correlation between AM fungal colonization and seedling biomass substantially increased after the second growing season compared to the first, further indicating that an early growth advantage from AM fungi might impart an even greater legacy effect (Cuddington, 2011) on growth and competitiveness in following years. Because AM fungi function primarily

94 in nutrient uptake, my findings suggest growth of western redcedar is nutrient-limited to a degree on CH and HA clearcuts. This is a novel insight because, on balance, growth of western redcedar is less responsive to nutrient deficiencies than western hemlock in CH and HA clearcuts (Weetman et al., 1989; Messier, 1993; Leckie et al., 2004; Blevins et al., 2006; Negrave et al., 2007), potentially due to a higher tolerance of nutrient limited environments (Krajina, 1969; Minore, 1990; Messier and Kimmins, 1992; Klinka and Brisco, 2009).

In contrast to HA clearcuts, AM fungal inoculum was not limited in CH clearcuts; regardless of the inoculum treatment, western redcedar seedlings showed similar growth response and AM root colonization after two growing seasons. Although AM fungal inoculum can diminish following disturbance (Reeves et al., 1979; Schnoor et al., 2011) or removal of its host (Sieverding, 1991; Kabir et al., 1997), CH clearcut sites develop high cover of AM plants that are host to a rich diversity of compatible mycorrhizal fungi with western redcedar (see in chapter 3). The delay in root colonization during the first growing season in CH clearcuts may be explained by soil disturbance that occurred during the seedling planting. Soil disturbance disrupts arbuscular mycorrhizal networks, which in turn reduces infectivity for root colonization (Miller, 1979; Evans and Miller, 1988; Jasper et al., 1989a, 1989b; Evans and Miller, 1990; Jasper et al., 1991; Kabir et al., 1997), especially for newly growing plants (Evans and Miller, 1990).

Arbuscular mycorrhizal fungi in CH sites are likely an important adaptation for redcedar to acquire scarce nitrogen, the nutrient most limiting for plant growth in these sites. Although most research has focused on AM fungal importance for plant phosphorus uptake (Leake et al., 2003a), AM fungi can acquire recalcitrant and labile forms of organic nitrogen (Whiteside et al., 2012), enhance nitrogen mineralization, relieve plant nitrogen deficiency and improve plant growth rates (Atul-Nayyar et al., 2009). Though it is predominantly believed that AM fungi lack any significant saprophytic capability (Smith and Read, 2008), AM fungi can colonize live and dead plant tissues (Parke and Linderman, 1980) and capture nitrogen (Hodge et al., 2001; Leigh et al., 2009; Hodge and Fitter, 2010; Nuccio et al., 2013), but not carbon (Leigh et al., 2009) from complex organic sources. The AM fungal absorption of nitrogen from organic and inorganic sources can account for 7-75% of plant nitrogen uptake (Johansen et al., 1994; Hawkins et al., 2000; Tanaka and Yano, 2005). Isolates of AM fungi from nutrient poor 95 environments have been shown to be more efficient at nitrogen uptake compared to isolates from environments with higher nutrient availability (Hawkins et al., 2000). Where nitrogen limitation was presumably caused by slow mineralization of soil organic matter, Näsholm et al. (1998) showed that mycorrhizal acquisition of organic nitrogen, in the form of the amino acid glycine, was higher in the AM host grass Deschampsia flexuosa than in the EM trees Pinus sylvestris and Picea abies. How AM coniferous trees have adapted to acquiring nitrogen in nitrogen limited conditions is not fully characterized; however, compared to western hemlock, the higher rate of translocation of nitrogen from senescing foliage in western redcedar (Keenan et al., 1995; Prescott et al., 1996) and the benefit bestowed by its AM fungi in nitrogen poor environments may greatly contribute to the success of this tree species.

4.4.2 Soil transfer from mature western redcedar trees

While soil transfer from mature western redcedar trees in old-growth CH forests improved mycorrhizal colonization of western redcedar seedlings, it inhibited growth of western hemlock in both CH and HA clearcuts after the first growing season. Interestingly, irrespective of EM root colonization, western hemlock seedlings that received this soil transfer treatment displayed growth inhibition in both CH and HA clearcut sites relative to all other treatment groups. The mechanism of growth inhibition of western hemlock is unknown. Mycorrhizal antagonism, growth promoting rhizobacteria and allelopathy are among the possible mechanisms involved.

The arbuscular mycorrhizal community associated with western redcedar may have played a role in inhibiting growth of western hemlock. Antagonism by AM fungi has been observed to reduce the growth of non-AM plants (Francis and Read, 1994; Brundrett, 2004). Originally, lack of competitive ability was hypothesized to explain the poor survivorship of non- AM hosts in AM fungal dominated environments (Grime, 1979). However, microcosm experiments showed that non-AM plants grew less vigorously in the presence of AM fungi (Grime et al., 1987). When Allen et al. (1989) characterized the reaction of the non-AM host, Salsola kali, to AM fungal infection they detected localized lesions where the AM fungi penetrated the root and temporarily formed arbuscules, which the host rejected soon after. 96

Additional microcosm experiments by Grime et al. (1988) showed that such AM fungal antagonism was detrimental to non-AM plants; non-AM plants grew slowly, were weak in appearance and had extremely poor survivorship when AM fungi were growing proximal to their roots. When non-AM hosts had AM mycelium in contact with their roots, there was no evidence of fungal penetration or lesions on roots; instead, root extension, root branching and root hair development were inhibited in the presence of AM mycelium (Grime et al., 1988). O'Connor et al. (2002) found that, by inhibiting AM fungi, growth of non-AM host improved, indicating antagonism between AM fungal mycelium and non-AM hosts. All of these studies mentioned so far, examined herbaceous plants of both AM hosts and non-AM hosts. However, similar phenomenon between AM plants and coniferous EM plants have detected. For example, the above-ground and below-ground growth of the EM host pinyon pine (Pinus edulis) is negatively affected by abundance of the AM host one-seed juniper (Juniperus monosperma) (McHugh and Gehring, 2006). Haskins and Gehring (2004) showed that below ground interactions between roots of AM one-seed juniper and the roots of the EM pinyon pine led to reductions in root production and EM colonization of pinyon pine. In a bioassay experiment, pinyon pine seedlings grown in soil from juniper-dominated zones also had reduced EM colonization (Haskins and Gehring, 2005). In my study, while AM antagonism is possible, the probability that the AM fungi and their inhibitory effect would have persisted into the second growing season is low given the low AM host density in HA clearcuts (see chapter 3).

Other biological factors associated with the rhizosphere have also been implicated in the inhibitory or beneficial effects of mycorrhizal fungi on seedlings (Amaranthus and Perry, 1989a). In particular, rhizobacterial associates of roots can promote or inhibit plant growth through (1) the production of phytohormones and phytotoxins (Bolton and Elliott, 1989; Nehl et al., 1997; Carvalho et al., 2011), (2) inhibition or facilitation of mycorrhizal fungal growth and root colonization (Bowen, 1973; Bianciotto et al., 1996; Andrade et al., 1997; Duponnois and Plenchette, 2003; Artursson et al., 2006), or (3) direct competition for nutrients (Nehl et al., 1997). It has been suggested that coevolution between a plant species and strains of rhizobacteria can occur, possibly influencing rhizobacterial dynamics through root exudates produced by a given host species (Nehl et al., 1997). Just as mycorrhizal fungi can influence

97 plant community diversity and structure, rhizobacteria may play an important role in the competition between different plant species.

Thirdly, the production of allelochemicals by western redcedar is a possible cause of western hemlock growth inhibition. Conifer species in the Cupressaceae family, including western redcedar, have been suggested to produce allelopathic compounds that suppress the growth of other plant species. For example, extracts from eastern redcedar (Juniperus silicicola) have inhibited seed germination and seedling growth of lettuce (Lactuca sativa) (Rathinasabapathi et al., 2005); and terpenes extracted from white cedar (Thuja occidentalis) have reduced the growth rate of the weed Silybum marianum (Saad and Abdelgaleil, 2014) and inhibited germination of the annual herbaceous plants Amaranthus caudatus and Lepidium sativum (Oster et al., 1990). Oil extracted from western redcedar in particular contains approximately 70% thujone (French, 2011), a terpene that has been implicated in inhibition of seed germination and subsequent growth of various herbaceous plant species (Seigler, 1995; Dudai et al., 2004; Gilani et al., 2010). By using extracts, many of these studies may have applied higher concentrations of allelopathic compounds than would naturally occur in the environment, amplifying their negative effects. However, in a greenhouse experiment, seeds planted in organic soil collected from under a mature Clanwilliam cypress (Widdringtonia cedarbergensis) had significantly reduced germination rates compared to seeds germinated in soil where the organic layer was burned off (Manders, 1987). Manders (1987) attributed the inhibition to allelopathy from the cypress litter.

4.4.3 Poor ectomycorrhizal colonization of western hemlock seedlings

Soil transfer was ineffective as a source of EM inoculum for the greenhouse grown western hemlock seedlings planted in CH and HA clearcut sites. Western hemlock seedlings that received different soil transfer treatments did not differ in EM species composition or percent colonization of roots after the first growing season. After the first growing season in CH and HA clearcuts, western hemlock roots were primarily colonized by Cenococcum geophilum and average percent colonization was relatively low (< 10%). No correlation was detected between percent root colonization with C. geophilum and seedling growth. Though Amaranthus and 98

Perry (1989b) demonstrated that soil transfer from mature EM forests stimulated ectomycorrhizal formation and root growth of planted seedlings, failure of many EM fungal species in soil transfers to colonize seedlings may be due to the EM fungi’s dependency on living roots to supply carbohydrate from the host (Fleming, 1983; Fleming et al., 1984; Jones et al., 2003). Greater EM fungal diversity and similar fungal species composition to mature forests have been shown on seedlings grown in close proximity to legacy trees (Kranabetter and Friesen, 2002); these legacy trees support mycorrhizal networks that improve conifer seedling survivorship (Teste and Simard, 2008; Simard, 2009a; Teste et al., 2009b). Without a connection to roots of mature trees in my study, the fungal propagules in the soil transferred from mature forests might not have had sufficient energy for infection of regenerating seedlings. Furthermore, there is considerable evidence that a succession of EM fungi occurs during forest development (Mason et al., 1983; Dighton et al., 1986; Blasius and Oberwinkler, 1989; Jones et al., 2003) and it is not uncommon for late-stage fungi to fail as an inoculum source following disturbance (Deacon et al., 1983; Fleming, 1983; Fox, 1983). The inability of EM fungi from mature forests to persist in disturbed sites was demonstrated by Kranabetter and Friesen (2002). When seedlings of naturally regenerated western hemlock in mature forests were transplanted into forest opening, the diversity of EM fungi on the seedlings greatly decreased and the pioneer fungus, Thelephora terresteris, established on the seedlings (Kranabetter and Friesen, 2002). Jones et al. (2003) proposed that shifts in fungal species composition following clearcutting result in ectomycorrhizal fungal communities that are better adapted to the new disturbed conditions as opposed to the original forest fungal community. Previous studies, especially on western hemlock (Kranabetter and Friesen, 2002), have indicated that most pioneer EM fungal species stimulate greater growth of regenerating seedlings in disturbed sites (Kranabetter, 2004, 2005). These studies suggest the western hemlock seedlings in my study were unable to support the full suite of late successional fungi that would have been present in the soil transferred from mature conspecifics.

The C. geophilum that dominated the western hemlock seedlings in my study is a common pioneer EM fungus (Dahlberg and Stenström, 1991; Kranabetter and Wylie, 1998; LoBuglio, 1999) and is a major root symbiont of western hemlock seedlings (Molina, 1980;

Schoenberger and Perry, 1982). It is one of the few EM fungi that often imparts little nutritional or growth benefit to host seedlings compared to other EM fungal species (Marx et al., 1978; Dickie et al., 2002; Jones et al., 2009; Dalong et al., 2011). Under certain growing conditions, dominance by C. geophilum can even be deleterious to plant productivity (Kummel, 2004; Kummel and Lostroh, 2011). However, there have been some instances where C. geophilum has been reported as beneficial for seedling growth, especially for western hemlock (Kropp et al., 1985). It is possible that differences in nutrient availability of a site may play a role in the degree of benefit bestowed by C. geophilum; efficiency at phosphorus uptake has been one of the mechanisms by which C. geophilum has improved seedling growth when phosphorus was limited (Bowen, 1973; Mejstrik and Krause, 1973). In addition, C. geophilum is an asexually reproducing species complex composed of a number of phylotypes (LoBuglio, 1999; Jany et al., 2002; LoBuglio and Taylor, 2002; Douhan and Rizzo, 2004) and ecotypes (Cline et al., 1987; Coleman et al., 1989; LoBuglio, 1999) with similar root tip morphology, but dissimilar effects on seedling performance (LoBuglio, 1999). Different phylotypes have been previously detected between CH and HA forests (Wright et al., 2009; Lim and Berbee, 2013). Therefore, identification of C. geophilum by root tip morphology alone restricted my ability to distinguish the presence of different phylotypes between treatment groups or clearcut sites and, further, whether certain phylotypes were more beneficial than others.

The EM fungal diversity and percent root colonization was exceptionally low in my study after the first growing season. Based on the sequencing results in chapter 2, the forest floor from mature and old-growth forests used in this study had high diversity of EM fungi present. The low EM colonization of western hemlock seedling roots by either pioneer EM species or EM fungi from soil transfer may have been due to fertilization received in the greenhouse prior to transplant. Increased nitrogen reduces ectomycorrhizal diversity, extraradial mycelia and colonization of roots (Marx and Barnett, 1974; Ruehle and Marx, 1977; Gagnon et al., 1988; Wallenda and Kottke, 1998; Peter et al., 2001; Lilleskov et al., 2002a; Avis et al., 2003; Nilsson and Wallander, 2003; Frey et al., 2004). Explanations for a decrease in EM fungal colonization and diversity following nitrogen fertilization include sensitivity of the fungi to high concentrations of nitrogen within the soil (Ek, 1997; Bidartondo et al., 2001; Lilleskov et al.,

100

2002b) and reduced allocation of carbon from the host plant to the mycobionts (Wallenda and Kottke, 1998; Nilsson and Wallander, 2003). Prior to planting, western hemlock seedlings were devoid of EM, even though greenhouse grown western hemlock seedlings are known to be frequently colonized by Thelephora terrestris (Castellano and Molina, 1989; Roth and Berch, 1992). Not only could fertilization have inhibited EM in the greenhouse, the residual effects of the greenhouse fertilizer potentially inhibited EM fungal colonization of western hemlock roots from both naturally occurring pioneer species found within the sites as well as infectious EM propagules from the forest floor transferred during the first growing season. Cenococcum geophilum is an exception in that it is one of the few species that grows abundantly under fertilization (Fransson et al., 2000). It is unfortunate that in this study, the EM colonization of western hemlock could not be determined following the second growing season. Future research is needed to determine whether eventual colonization and diversity of pioneer EM fungal species differs between ecosystems and, if so, how it relates to western hemlock seedling performance.

4.4.4 Conclusion

The CH clearcuts were not lacking in AM inoculum necessary for western redcedar. Nor were HA clearcuts devoid of AM inoculum compatible with western redcedar, but AM inoculum availability a minimal and sporadic. Adding AM fungal inoculum via soil transfer from old- growth CH forests at the time of planting increased AM mycorrhizal colonization of western redcedar seedlings in HA clearcuts. Here, AM fungal colonization was positively correlated with western redcedar biomass. Thus, soil transfer enabled western redcedar seedlings to achieve greater growth potential during the first two years following planting in HA clearcuts. Long term studies are needed to determine whether early AM colonization of western redcedar seedlings imparts a significant legacy effect on growth and nutrient uptake.

Both CH and HA clearcuts were replete in C. geophilum inoculum; however, EM fungal colonization and diversity of western hemlock roots was poor following the first growing season regardless of forest type or soil transfer treatment. Failure of transferred forest floor to act as an inoculum source was likely due to the late stage successional status of the EM fungi present 101 and/or inhibition of EM growth caused by the greenhouse fertilizer. Further research efforts would be best invested in studying the importance of pioneer EM fungi on western hemlock seedling growth, nutrient uptake and competition in both CH and HA clearcuts. In CH clearcuts in particular, research is needed to determine whether AM antagonism influences the colonization of pioneer EM fungi and the growth of western hemlock.

The inhibition of western hemlock growth and facilitation of western redcedar AM colonization in both HA and CH clearcuts following soil transfer from mature western redcedar trees was unexpected. Research is needed to determine the mechanism driving these effects. Whether this phenomenon significantly favors the re-establishment of western redcedar over western hemlock has important implications for harvesting and regeneration methods in old- growth CH forest cutblocks.

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a) Hemlock biomass after first growing season . 80 Figure legend: Soil transfer as an

60 inoculum treatment abc ab CH - Cw a 40 CH - Hw

bcd bcd HA cd 20 abcd Control d

0 Seedling Seedling above groundbiomass dry (g) CH clearcut HA clearcut

b) Hemlock biomass after second growing season .

80 ab a a 60

40 c

20 bc

0 Seedling Seedling above groundbiomass dry (g) CH clearcut HA clearcut

Figure 4.1 Average above-ground dry biomass of western hemlock seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. Different letters above standard error bars indicate significant difference between treatments and forest types (p < .05). 103

a) Hemlock height after first growing season . 80 Figure legend: Soil transfer as an a 60 ab a inoculum treatment bc CH - Cw bc bc 40 CH - Hw c HA 20 Control

(cm) height Seedling

0 CH clearcut HA clearcut

b) Hemlock height after second growing season .

80 a a ab

60 bcd cd abc

40 d

(cm) height Seedling

0 CH clearcut HA clearcut

Figure 4.2 Average height of western hemlock seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. Different letters above standard error bars indicate significant difference between treatments and forest types (p < .05). 104

a) Redcedar biomass after first growing season . 35 Figure legend: 30 Soil transfer as an 25 inoculum treatment

20 CH - Cw 15 CH - Hw HA 10 Control 5

0 Seedling Seedling above groundbiomass dry (g) CH clearcut HA clearcut

b) Redcedar biomass after second growing season . 35

25 20

0 Seedling Seedling above groundbiomass dry (g) CH clearcut HA clearcut

Figure 4.3 Average above-ground dry biomass of western redcedar seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. No statistically significant differences between treatments were detected (p < .05). 105

a) Redcedar height after first growing season . 80 Figure legend: Soil transfer as an 60 inoculum treatment CH - Cw 40 CH - Hw

Seedling Seedling height(cm) 20 Control

0 CH clearcut HA clearcut

b) Recedar height after second growing season .

Seedling Seedling height(cm) 20

0 CH clearcut HA clearcut

Figure 4.4 Average height of western redcedar seedlings planted with different inoculum treatments in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA) and (4) no soil transfer at time of planting as a control. Results are shown for biomass after (a) the first growing season and (b) the second growing season. Seedlings measured in different years were destructively sampled; seedlings measured after the second growing season are not the same samples for which measurements were made after the first growing season. No statistically significant differences between treatments were detected (p < .05). 106

a) AMRedcedar root infection AM root after infection first growing after first season growing . season 60 ab Figure legend: Soil transfer as an a inoculum treatment 40 CH - Cw CH - Hw abc HA 20 bcd cd Control cd d d 0

arbuscular mycorrhizal structures (%) Totalamount root of colonizationwith CH clearcut HA clearcut

b) AMRedcedar root infection AM root after infection second after growing first growing season season

60 a

a a ab ab 40

bc c

arbuscular mycorrhizal structures (%) 0 Totalamount root of colonizationwith CH clearcut HA clearcut

a) Percent of seedlings with AM after first growing season

100 a Figure legend: a ab Soil transfer as an 80 inoculum treatment

60 bc CH - Cw c CH - Hw c 40 HA

c c Control

20 arbuscular mycorrhizalfungi (%) Percent seedlings of colonized with 0 CH clearcut HA clearcut

b) Percent of seedlings with AM after second growing season

100 a a

60 b 40 b

arbuscular mycorrhizalfungi (%) Percent Percent seedlings of colonized with 0 CH clearcut HA clearcut

a) Redcedar in CH clearcut b) Recedar in HA clearcut after first growing season after first growing season

8 8 y = 0.0045x + 1.7688 y = 0.0262x + 2.4 R² = 0.0182 R² = 0.2164 6 6

4 4

2 2

Seedling Seedling biomass (g) Seedling biomass (g) 0 0 0 20 40 60 80 100 0 20 40 60 80 100 Percent root colonization with arbuscular Percent root cells colonized with arbuscular mycorrhizal fungi (%) mycorrhizal fungi (%)

c) Redcedar in CH clearcut d) Redcedar in HA clearcut after second growing season after second growing season

80 80 y = 0.1137x + 5.9048 y = 0.4395x + 12.168 R² = 0.1929 R² = 0.5234 60 60

40 40

20 20

Figure 4.7 Correlation between root colonization with arbuscular mycorrhizae and dry above-ground biomass of western redcedar seedlings in a field trial carried out in three-year post-harvest clearcuts of western redcedar-dominated (CH) and western hemlock dominated (HA) forests on northern Vancouver Island. Results are shown for correlation between percent arbuscular mycorrhizal root colonization and seedling biomass after (a) the first growing season in CH clearcuts (F = 1.61, p = .208) (b) the first season in HA clearcuts (F = 17.95, p < .001), (c) the second growing season in CH clearcuts (F = 10.99, p = .002) and (d) the second growing season in HA clearcuts (F = 71.39, p < .001).

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Dry biomass of stem Stem diameter Seedling height Mycorrhizal root and foliage (mg) (mm) (cm) colonization (%)

Ecosystem

CH clearcut 14.9 (1.5) 6.2 (0.2) 34.7 (1.4) b 6.3 (0.9) HA clearcut 31.8 (2.8) 8.3 (0.3) 47.2 (2.1) a 6.4 (0.9)

F-statistic, df=1 7.09, p = .056 6.23, p = .067 19.61, p = .011 0.37, p = .576

Inoculum

CH-Cw 12.0 (1.7) b 5.8 (0.3) b 31.7 (1.7) b 6.5 (1.4) CH-Hw 26.7 (3.8) a 7.5 (0.5) a 42.4 (2.7) a 6.4 (1.3) HA-Hw 29.0 (3.8) a 8.0 (0.5) a 43.6 (2.8) a 5.5 (1.0) Control 25.8 (3.2) a 7.7 (0.4) a 45.9 (2.7) a 6.8 (1.4)

F-statistic, df=3 6.81, p < .001 5.90, p < .001 6.84, p < .001 0.03, p = .994

Ecosystem*Inoculum

F-statistic, df=3 1.94, p = .122 1.75, p = .156 0.74, p = .530 1.56, p = .198

Note: Forest ecosystem type prior to harvest were western redcedar-dominated (CH) or western hemlock dominated (HA). Inoculum treatments made at the time of planting were 150 ml soil transfers of forest floor collected under (1) western redcedar trees in mature CH forests (CH-Cw), (2) western hemlock trees in mature CH forests (CH-Hw), (3) western hemlock trees in mature HA forests (HA-Hw) and (4) no soil transfer at time of planting as a control. Means and standard errors followed by different letters indicate significant difference between inoculum treatments and/or forest ecosystem clearcut type based on Tukey’s honest significance test (α = 0.05).

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Dry biomass of stem Stem diameter Seedling height and foliage (mg) (mm) (cm)

Ecosystem

CH clearcut 24.5 (2.3) 8.0 (0.3) 45.8 (1.9) HA clearcut 51.9 (3.9) 11.1 (0.4) 64.1 (2.5)

F-statistic, df=1 3.82, p = .123 2.93, p = .162 7.27, p = .055

Inoculum

CH-Cw 19.3 (2.7) b 7.2 (0.4) b 41.6 (2.4) b CH-Hw 45.0 (5.8) a 10.1 (0.6) a 58.5 (3.6) a HA-Hw 46.2 (5.5) a 10.6 (0.7) a 57.5 (3.8) a Control 42.3 (4.5) a 10.4 (0.5) a 62.2 (3.1) a

F-statistic, df=3 11.71, p < .001 11.59, p < .001 10.05, p < .001

Ecosystem*Inoculum

F-statistic, df=3 2.38, p = .071 2.42, p = .068 0.83, p = .480

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Amount of Dark septate Dry biomass of stem Stem diameter Seedling height mycorrhizal root Percent of seedlings endophyte root and foliage (mg) (mm) (cm) colonization (%) with AM (%) colonization (%)

Ecosystem

CH clearcut 1.8 (0.1) 2.9 (0.1) 24.3 (0.5) 14.6 (2.6) 48.8 (0.5) 4.6 (0.5) HA clearcut 2.6 (0.1) 3.3 (0.1) 28.7 (0.6) 13.8 (3.0) 45.3 (0.6) 5.4 (0.6)

F-statistic, df=1 4.07, p = .114 3.86, p = .124 3.86, p = .121 0.01, p = .921 0.44, p = .515 1.50, p = .336

Inoculum

CH-Cw 2.1 (0.2) 3.0 (0.1) 26.0 (1.1) 36.8 (6.0) a 81.0 (5.7) a 4.6 (0.6) CH-Hw 2.2 (0.2) 3.1 (0.1) 26.6 (0.7) 14.9 (3.4) b 57.7 (9.6) b 5.0 (0.8) HA-Hw 1.9 (0.1) 2.9 (0.1) 25.4 (0.6) 3.1 (1.0) c 23.4 (6.0) c 4.3 (0.5) Control 2.4 (0.2) 3.2 (0.1) 26.9 (0.9) 6.2 (2.1) c 26.2 (9.3) c 5.8 (1.1)

F-statistic, df=3 1.11, p = .348 0.95, p = .124 0.68, p = .564 19.54, p < .001 26.59, p < .001 0.18, p = .913

Ecosystem*Inoculum

F-statistic, df=3 0.28, p = .840 0.23, p = .873 1.85, p = .140 2.15, p = .096 9.19, p < .001 1.06, p = .396

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Table 4.4 Effects of ecosystem type prior to forest harvest and source of mycorrhizal inoculum on seedling growth response and mycorrhizal colonization of western redcedar seedling roots after the second growing season (2010) in three-year post-harvest clearcuts on northern Vancouver Island. Results shown are mean values with standard errors in parentheses for seedling growth response (dry above-ground biomass, stem diameter and height), mycorrhizal colonization of roots (the amount of mycorrhizal colonization within roots and the percent of seedlings colonized with arbuscular mycorrhizae) and degree of root colonization with dark septate endophytes. Summary of restricted maximum likelihood (REML) results contrast the mean values for main fixed effects (ecosystem and inoculum) and their interaction (ecosystem*inoculum).

Amount of Dark septate Dry biomass of stem Stem diameter Seedling height mycorrhizal root Percent of seedlings endophyte root and foliage (mg) (mm) (cm) colonization (%) with AM (%) colonization (%)

Ecosystem

CH clearcut 9.2 (1.0) 4.9 (0.2) 43.0 (1.9) 34.8 (4.7) 78.3 (6.3) 6.9 (1.3) HA clearcut 21.6 (1.8) 7.2 (0.3) 60.7 (2.2) 20.9 (3.5) 51.0 (11.0) 6.0 (0.9)

F-statistic, df=1 3.89, p = .118 3.88, p = .117 4.26, p = .106 4.58, p = .098 8.13, p = .012 0.39, p = .567

Inoculum

CH-Cw 15.6 (2.5) 6.0 (0.4) 52.7 (3.5) 32.6 (5.6) a 84.9 (5.5) a 7.3 (1.5) CH-Hw 21.8 (2.9) 7.1 (0.5) 58.0 (3.7) 41.5 (6.0) a 83.1 (6.3) a 5.4 (1.7) HA-Hw 14.6 (2.0) 6.0 (0.4) 52.2 (3.0) 13.7 (4.8) b 40.3 (13.7) ab 5.7 (1.0) Control 13.2 (2.0) 5.8 (0.4) 50.2 (2.8) 17.2 (4.9) b 50.3 (16.8) b 7.1 (1.7)

F-statistic, df=3 2.22, p = .088 1.62, p = .187 0.74, p = .528 7.48, p < .001 5.64, p = .008 0.62, p = .601

Ecosystem*Inoculum

F-statistic, df=3 0.96, p = .412 1.05, p = .373 1.44, p = .234 3.46, p = .019 1.79, p = .189 0.87, p = .460

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Chapter 5: Conclusion

Forest regeneration, productivity and processes of decomposition and nutrient cycling are influenced by more than abiotic site properties; mycorrhizal fungi associated with the above ground plant community structure play an important role in these processes and can have a legacy effect following forest disturbance. Through research presented in this thesis, I provided insight into the significant differences of the mycorrhizal and saprophytic fungal community structure of the arbuscular mycorrhizal (AM)-dominated Cedar Hemlock (CH) forests and ectomycorrhizal (EM)-dominated Hemlock-Amabilis fir (HA) forests on northern Vancouver Island. From this data, I extrapolated inoculum potential in the forest floor for western redcedar and western hemlock seedlings. In a field trial, I examined the role of mycorrhizal inoculum availability on early establishment of western redcedar and western hemlock seedlings in clearcuts of CH and HA forests; I also tested effectiveness of soil transfers of the LFH layer of the forest floor from both uncut CH and HA forests as an inoculum treatment to improve growth of planted western redcedar and western hemlock seedlings in clearcuts of these forests. As an unexpected outcome, I detected an inhibitory effect of forest floor collected under mature western redcedar trees on the growth of western hemlock seedlings. Furthermore, I identified mycorrhizal fungi found in alternative host plants that dominate clearcuts of these forests; by doing so, I discovered high similarity of AM fungi to those found in western redcedar, especially in deer fern growing sparsely in HA clearcuts and among the dominant plant species colonizing CH clearcuts, including the ericoid plant, salal. The shared mycorrhizal fungi between western redcedar and surrounding vegetation potentially alters competition between species and contributes to the success of western redcedar in nutrient poor clearcuts.

5.1 Synopsis of chapter 2

Dominance of mycorrhizal guild has a substantial impact on the microbial community (Paulitz and Linderman, 1991; Fracchia et al., 1998; Olsson et al., 1998; Green et al., 1999; Hodge, 2000; Huang et al., 2009), soil structure, humification, nutrient immobilization and rates of decomposition (Bending and Read, 1995; Wright and Upadhyaya, 1996; Wright and Upadhyaya, 1998; Wright et al., 1999; Miller

114 and Jastrow, 2000; Rillig and Mummey, 2006; Orwin et al., 2011; Averill et al., 2014). In chapter 2, I carried out a high sampling effort and utilized a targeted metagenomic approach to characterize the forest floor fungal communities of AM-dominated CH forests and EM-dominated HA forests. Community composition of mycorrhizal and saprophytic fungi differed between CH and HA forests. One third of clone sequences from DNA extracted from forest floor mycelia were indicator taxa of EM and saprophytic fungi associated with a specific forest type. Differences in fungal species composition may be attributed to productivity of host trees, nutrient availability, incidence of western redcedar and mycorrhizal antagonism between mycorrhizal guilds. By contrast, similar patterns of major fungal taxa of EM and saprophytic fungi were observed in both forest types. The saprophytic fungal communities detected were composed primarily of ascomycete fungi in the family Helotiales, a group of fungi involved in early stages of litter decomposition (Lindahl et al., 2007). There were very few saprophytic basidiomycete fungi, indicating that EM basidiomycete fungi play a greater role in the final breakdown of lignin (Gadgil and Gadgil, 1971, 1975; Bending, 2003). Matching this proposition, wood rot fungi in the family Atheliaceae (Chen et al., 2003; Luis et al., 2005) comprised the largest group of EM fungi detected in both forest types.

Though the below-ground community of mycorrhizal guilds is typically proportionate to the above-ground dominance of mycorrhizal plants (Moyersoen et al., 1998; Korb et al., 2003; Thiet and Boerner, 2007; Dickie et al., 2013; Nave et al., 2013), this trend was not fully supported in the forests in my study. Diversity and richness of EM fungi were similar between AM-dominated CH forests and EM-dominated HA forests. In CH forests, the ratio of EM fungi to saprophytic fungi was somewhat lower than in HA forests; however, when complemented by measures of fungal biomass in these forests (Leckie et al., 2004), there was insufficient support that EM fungal biomass was lower in the AM-dominated CH forest. Furthermore, though salal forms a dense understory vegetation layer in CH forests, ericoid mycorrhizal fungi were not a dominant fungal group inhabiting the forest floor of CH forests. Based on past in vitro culture studies (Xiao, 1994), it has been proposed that ericoid mycorrhizae associated with salal inhibit EM fungi and thus are likely the dominant mycorrhizal guild in CH forests (Prescott et al., 1996). Contrary to this hypothesis, based on the results of my study, ericoid mycorrhizal fungi had extremely low abundance detected in both CH and HA forests. Presence of AM fungi, however, was reflective of the above-ground species composition; arbuscular mycorrhizal fungi

115 primarily occurred in CH forests; incidence of AM fungi in HA forests was rare. Given that AM fungi influence the surrounding microbial community structure and produce novel compounds that alter soil structure and humification (Wright and Upadhyaya, 1996; Amora-Lazcano et al., 1998; Wright and Upadhyaya, 1998; Wright et al., 1999; Miller and Jastrow, 2000), their abundance in CH forests may be a contributing factor to differences in soil processes and nutrient availability; further research to examine this hypothesis is warranted. Additionally, my results support the hypothesis that an AM deficiency exists in HA forests, which may limit successful natural establishment of western redcedar germinants under intense competition with western hemlock (Weber et al., 2003; Weber et al., 2005).

5.2 Synopsis of chapter 3

Mycorrhizal networks shared among plant communities are an important source of inoculum for facilitating the establishment of conifer seedlings (Lovelock and Miller, 2002; Simard and Durall, 2004; van der Heijden, 2004; Nara, 2006; Selosse et al., 2006; Teste and Simard, 2008). In chapter 3, I identified the mycorrhizal fungi found in the dominant plant species on clearcuts of CH and HA forests using phylogenetic analyses of fungal DNA sequences extracted from plant roots. All the plant species examined in clearcuts of both forest types had some overlap in arbuscular mycorrhizal species associated with western redcedar. Clearcuts of CH forests were densely colonized by plants with AM fungi compatible with those found in western redcedar, indicating that shared AM among the plant community dominating clearcuts of CH forests are an important source of AM inoculum for western redcedar. A high number of AM fungi were detected in roots of the two dominant plant species present in CH clearcuts, bunchberry and salal, the latter of which is an ericoid species. In HA clearcuts, facultative mycorrhizal host plant species are capable of facilitating the eventual introduction of AM fungi as inoculum for western redcedar seedlings, though there was low occurrence of AM plants three years post-harvest and mycorrhizal diversity was typically lower than in plants found in CH clearcuts. An exception to this trend was seen in deer fern and salal; my results showed that deer fern and potentially salal are reservoirs of a diverse suite of AM fungi compatible with western redcedar, thus providing small patches of AM inoculum in clearcuts of ectomycorrhizal HA forests.

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5.3 Synopsis of chapter 4

Mycorrhization is crucial for conifer seedling establishment in nutrient poor sites (Trappe and Strand, 1969; Hayman, 1974; Fenner, 1987; Perry et al., 1987; Amaranthus and Perry, 1989b; Thiet and Boerner, 2007). In chapter 4, I conducted a field trial to examine the role of mycorrhizal inoculum in seedling growth and survivorship of western redcedar and western hemlock in clearcuts of CH and HA forests. Small reciprocal soil transfers of the LFH layer of the forest floor from mature forests were used to test whether modification of inoculum at the time of planting could improve mycorrhization and seedling performance. Seedlings of western redcedar planted in HA clearcut sites without CH forest inoculum had low arbuscular mycorrhizal colonization rates. Without mycorrhization, western redcedar did not achieve its full growth potential in nutrient poor clearcuts; western redcedar seedling growth rates were positively correlated with arbuscular mycorrhizal colonization. Small soil transfers from mature western redcedar dominated forests at time of planting improved mycorrhization of western redcedar seedlings in HA clearcut sites which are deficient in AM fungi. On the other hand, for greenhouse grown, planted western hemlock seedlings, the majority of the late stage EM fungi present in soil transfers from mature forests were ineffective at colonizing roots. Western hemlock seedlings did not show differences in ectomycorrhizal root colonization or ectomycorrhizal fungal species composition after the first growing season. Regardless of forest type prior to harvest or soil transfer given at the time of planting, western hemlock seedlings were poorly colonized with the single EM fungus Cenococcum geophilum. Unexpectedly, forest floor from under mature western redcedar trees had an inhibitory effect on western hemlock seedlings. This phenomenon was not observed when western hemlock seedlings were planted in CH or HA clearcuts with forest floor collected from under a mature western hemlock tree in a CH forest. Possible mechanisms to explain the growth inhibition include mycorrhizal antagonism (Francis and Read, 1995; Haskins and Gehring, 2004), growth promoting rhizobacteria (Bowen, 1973; Bolton and Elliott, 1989; Bianciotto et al., 1996; Andrade et al., 1997; Nehl et al., 1997; Duponnois and Plenchette, 2003; Artursson et al., 2006; Carvalho et al., 2011) and allelopathy (Fujii and Hiradate, 2007) from western redcedar.

117

5.4 Implications and recommendations

5.4.1 Legacy of prior forest community on site regeneration

Forest type prior to harvest can have a legacy effect on the establishment of seedlings following disturbance. Following disturbances such as clearcutting, mycorrhizal propagules and remnant mycorrhizal networks may facilitate the establishment of compatible host plants (Biermann and Linderman, 1983; Friese and Allen, 1991), especially where nutrients are limited. By altering competition between plant species (van der Heijden et al., 1998b; van der Heijden et al., 2003) and potentially providing a competitive edge over plants of different mycorrhizal guilds (Janos, 1980; Allen and Allen, 1984; Wilson and Hartnett, 1998; van der Heijden et al., 1998a; Stampe and Daehler, 2003; Wardle et al., 2004), mycorrhizal fungi can influence the plant community structure and tree productivity during reforestation. In CH clearcuts, as seen in chapter 3, a high diversity of compatible AM fungi for western redcedar is maintained by the plant community, potentially explaining why western redcedar is well adapted to growing in these sites. Conversely, in HA sites, deficiency of AM fungi likely puts natural germinating seeds of western redcedar at a disadvantage, where competition from rapidly recolonizing western hemlock seedlings can be high (Weber et al., 2003; Weber et al., 2005).

Furthermore, as seen in chapter 4, interspecific inhibition of seedling growth can occur, potentially from mycorrhizal antagonism, rhizobacteria or allelopathic compounds still present in the soil after the disturbance event. In my study, a very small amount of forest floor transferred from under a mature western redcedar tree inhibited the growth of western hemlock seedlings in clearcuts. Research is needed to identify the cause of the inhibition and to understand its role in spatial patterns of seedling establishment in both undisturbed and clearcut forests. Furthermore, research is needed to determine how long after clearcutting the inhibitory factor will reside and examine how it impacts the survivorship and natural distribution of western hemlock seedlings during forest regeneration.

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5.4.2 Mycorrhizal inoculation of planted western redcedar

Arbuscular mycorrhizal fungi are an important adaptation for western redcedar growing in nutrient limited ecosystems. Mycorrhization of western redcedar improves seedling survivorship (Weber et al., 2003; Weber et al., 2005) and can increase seedling biomass production up to 2000% (Kough et al., 1985). Results from chapter 3 indicate that plants colonizing forest clearcuts can serve as alternative hosts for compatible AM inoculum for establishing western redcedar seedlings. However, in nutrient limited conditions, without mycorrhization, as can happen in HA sites relatively lacking AM networks, western redcedar might not achieve its full growth potential. Results from chapter 4 indicate that inoculation of western redcedar seedlings with AM fungi may be beneficial for improved seedling vigor in nutrient poor clearcuts lacking necessary inoculum. Whether a commercial AM inoculum would prove beneficial for improved reforestation warrants further investigation, and if it does, whether the benefits exceed the treatment costs (Castellano, 1996; Berch et al., 1999). Initial bioassays would be required to determine whether a currently available commercial AM inoculum would result in optimal growth response, or if a specific mixture of AM fungi native to western redcedar in these sites would need to be developed (Miyasaka et al., 2003; Caravaca et al., 2003a; Bhatt et al., 2006; Siddiqui and Kataoka, 2011). Research should also compare the effectiveness and costs of utilizing AM inoculum versus operational fertilization practices; field trials should extend a minimum of five years to fully capture short-term versus long-term effects. Fertilization can provide immediate and substantial growth response (McDonald et al., 1994; Fraser et al., 1995; Prescott, 1996b; Blevins and Prescott, 2002; Blevins et al., 2006), however, increased soil nutrient availability often diminishes rapidly and requires reapplication (Binkley and Reid, 1985; Cole et al., 1990; Valinger et al., 2000; Bennett et al., 2003; Nigh, 2013). Mycorrhizal fungi, on the other hand, once established do not require further treatments for ongoing seedling growth benefit (Plenchette et al., 1981; Stenström and Ek, 1990; Haselwandter and Bowen, 1996; Bhatia et al., 1998; Caravaca et al., 2003b; Ortas, 2012). Additionally, an advantage that mycorrhizal fungi provide over fertilizer treatment is improved resilience to environmental stress (Smith and Read, 2008), including drought resistance (Davies et al., 1993; Ruiz- Lozano et al., 1995; Augé, 2001; Al-Karaki et al., 2004; Augé, 2004) and a level of protection from soil borne pathogens (Fitter and Garbaye, 1994; Azcón-Aguilar and Barea, 1997; Azcón-Aguilar et al., 2002;

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Harrier and Watson, 2004). As such, AM inoculation may improve survivorship of seedlings planted, thus reducing the need and cost for replanting due to seedling mortality.

Nevertheless, as shown in chapter 3, following clearcutting, facultative AM plants that eventually colonize sites originally devoid of AM fungi, are capable of facilitating the migration of compatible AM fungi into the locality. This implies that planted western redcedar seedlings should eventually become colonized by AM fungi in these sites without intervention, likely within a matter of years. Timing for seedling establishment, however, may be a crucial factor. Diminished nutrient availability and tree seedling growth check can occur as soon as five years following clearcutting in the nutrient poor sites on northern Vancouver Island (Prescott et al., 1996). Moreover, forest companies are encouraged to replant within one or two years following harvest in order to avoid high costs of achieving free-growing status (Simard, personal communication 2015). Therefore, it may be more beneficial to inoculate seedlings with AM fungi sooner than would occur by natural dispersal of AM propagules, so that seedlings can better establish and utilize immediately available nutrients.

5.4.3 Arbuscular mycorrhizal fungi affect decomposition

Differences in mycorrhizal fungal guilds between CH and HA forests may have greater implications beyond just inoculum availability for seedling establishment. Given that CH and HA ecosystems differ by the presence of AM fungi, further study is warranted to not only quantify AM fungal abundance in AM conifer dominated ecosystems, but also to determine their influence on other fungal dynamics and decomposition processes.

There is evidence that in forests of similar age class, decomposition processes differ in soil under western redcedar versus soil under western hemlock. Comparing forest floor properties in single species forest plantations of western redcedar, western hemlock, Douglas fir (Pseudotsuga menziesii) and Sitka spruce (Piceae sitchensis), the forest floor under western redcedar was found to be the most distinct, with a higher percent moisture content (Grayston and Prescott, 2005), the greatest mass and the highest rate of nitrogen mineralization; yet, it had the slowest rate of carbon mineralization and litter decay (Prescott et al., 2000). Analyses of the microbial communities revealed that soil under western redcedar had the lowest fungal biomass, a measurement that excluded AM fungal biomass

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(Grayston and Prescott, 2005). By comparing old-growth forests of either western redcedar or western hemlock, Alban (1969) also detected significantly greater organic accumulation under western redcedar than western hemlock. Originally, differences were attributed to decomposition rates associated with litter quality of western redcedar (Keenan et al., 1995; Prescott et al., 1996). However, long term studies indicate that after a decade, regardless of source of litter, different types of litter had comparable decomposition (Prescott, 2010), indicating that the microbial communities in soil under western redcedar drive the slower rate of decomposition.

Arbuscular mycorrhizal fungi have been shown to slow rates of decomposition by supressing saprophytic fungal decomposition (Leifheit et al., 2015; Verbruggen et al., 2015). The AM fungi in CH forests also potentially have a modest contribution to higher C:N ratio in soil. By producing copious amounts of glomalin (Wright and Upadhyaya, 1996; Wright and Upadhyaya, 1998; Rillig, 2004), a glycoprotein that can take decades to degrade (Rillig et al., 2001; Harner et al., 2004), AM fungi reduce soil nitrogen availability by immobilizing 4-5% of soil nitrogen (Rillig et al., 2001). Not only does glomalin increase soil aggregate stability (Wright and Upadhyaya, 1998; Rillig, 2004), the accumulation of glomalin in soil has been correlated with increased soil C:N ratios (Lovelock et al., 2004), partially though the stabilization of litter-derived aggregates (Verbruggen et al., 2015). It has also been suggested that AM fungal hyphae contribute approximately 15% to the soil organic carbon (Miller and Kling, 2000; Rillig et al., 2001). By influencing soil microbial communities and by directly altering soil properties, such as forming aggregates that take decades longer to degrade than tree litter alone, AM fungi may be contributing to differences in decomposition and forest floor characteristics of CH and HA forests.

5.4.4 Ectomycorrhizal fungi: do they aggravate or alleviate nitrogen deficiency?

When ectomycorrhizal host tree productivity is nutrient limited, it often allocates greater amount of carbohydrate below-ground to its mycobiont (Hobbie, 2006). Unfortunately, this in turn can further drive nitrogen deficiencies, given that the fungi immobilize greater amount of nitrogen into the rapidly growing mycelium (Hobbie and Wallander, 2006; Näsholm et al., 2013). When combined with previous fungal biomass estimates (Leckie et al., 2004), the ratio of EM fungi to saprophytic fungi detected in chapter 2 indicate that biomass of EM fungi in CH forests may be comparable to that in HA 121 forests. Given that the density and growth rates of the EM hosts western hemlock and amabilis fir are substantially lower in CH than HA forests, fewer EM host trees in CH forest may be allocating greater amounts of photosynthetic carbon to support the relatively large biomass of EM fungal mycelium. As such, it is possible that high abundance of EM fungi in CH forests could contribute to nitrogen immobilization. Therefore, EM fungi in CH forests may exacerbate nitrogen deficiency. To test this hypothesis, a tree girdling or defoliation study to reduce the below-ground allocation of photosynthates to the EM fungi (Högberg et al., 2001; Markkola et al., 2004; Dannenmann et al., 2009) in CH forests could prove useful to examine how much nitrogen might be immobilized in the EM mycelium.

5.4.5 Absence of ectomycorrhizal hosts following disturbance

No alternative EM host species were detected in clearcuts of CH or HA forests, indicating that where advance regeneration of western hemlock and amabilis fir is lacking, spores of pioneer EM species likely play a greater role for the establishment of western hemlock, especially when reforestation is delayed. Without legacy trees or advance regeneration to maintain EM fungal networks, residual soil EM fungal inoculum will rapidly diminish (Harvey et al., 1980; Perry et al., 1987; Amaranthus, 1991b; Kabir et al., 1997; Hashimoto and Hyakumachi, 1998; Hagerman et al., 1999; Dahlberg, 2002; Jones et al., 2003). In my study, seedlings of western hemlock planted into three-year- old clearcuts became poorly colonized with only the pioneer EM fungus, C. geophilum. Past research has indicated that this fungus often imparts little benefit to its host (Marx et al., 1978; Dickie et al., 2002; Jones et al., 2009; Dalong et al., 2011). Therefore, even though western hemlock becomes colonized with C. geophilum, western hemlock might initially be at a disadvantage growing without other EM species in CH clearcuts where competition with AM plants is substantial. Research is needed to determine if any benefit is provided by the early colonization with C. geophilum and to gain an understanding of the process of colonization by other pioneer EM fungi in these sites. Since the majority of the plants colonizing clearcuts in the sites studied on northern Vancouver Island are AM hosts, a study design should also include examining whether mycorrhizal antagonism from AM fungi reduces the growth and root colonization of pioneer EM fungi.

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Given that EM colonization of roots of newly establishing western hemlock seedlings can greatly improve seedling survivorship and growth (Christy et al., 1982; Kropp, 1982; Smith and Read, 2008), immediate replanting of western hemlock following clearcutting is advisable. By temporarily capturing part of the original EM fungal community from the previous forest found in remnant fine roots within the soil (Harvey et al., 1980; Hagerman et al., 1999), rapid planting into existing mycorrhizal networks ought to facilitate seedling establishment until a diversity of pioneer EM species establish (Jones et al., 2003).

Variable retention harvest, where mature legacy western hemlock and amabilis fir trees are retained, could also help conserve pre-harvest EM fungal community inoculum (Kranabetter and Wylie, 1998; Kranabetter and Friesen, 2002; Teste and Simard, 2008; Simard, 2009a; Teste et al., 2009b). However, a study in these ecosystems is necessary to determine whether the mycorrhizal networks maintained by legacy trees would provide enough benefit to seeding performance to justify the implementation of this management practice. In a five year study by Kranabetter (2005), western hemlock seedlings in an interior forest of British Columbia, Canada had slightly lower foliar nitrogen content when grown in direct root contact with legacy trees compared to seedlings of treatment groups that had restricted root and mycorrhizal contact; also, no significant differences in seedling growth occurred between treatments (Kranabetter, 2005). However, Teste and Simard (2008) showed that distance from legacy trees results in trade-offs between competition from mature tree roots and benefits bestowed by mycorrhizal networks. Furthermore, seedling responsiveness to mycorrhizal networks can be related to environmental stress, whereby seedling benefit from established networks is observed under suboptimal conditions (Bingham and Simard, 2011).

5.5 Research strengths and limitations

5.5.1 Forest types occur across a range of site properties

Distribution of CH and HA forests occur across a range of landscape, with differing slope position, levels of moisture availability, humus development and nutrient availability. There is a great degree of site overlap in CH and HA forests (McWilliams and Klinka, 2005a; McWilliams and Klinka, 2005b; McWilliams et al., 2007; Weber et al., 2014), however, HA forests more frequently occur on

123 higher slope positions and likewise more CH forests are located on lower slope positions (Albani and Lavery, 2000); Weber et al. (2014) proposed that this may potentially be due to western redcedar having higher tolerance to wetter soil conditions. A strength in my study is that site properties were held constant as much as possible during CH and HA block selection, thus ensuring that fungal communities studied were reflective of the forest community, not the physical site differences. Adjacent CH and HA sites with similar slope position were selected with collaboration of Western Forest Products Inc. forest managers, who were familiar with the region and had site characterization data collected from trained company professionals. Furthermore, soil pits were characterized using the updated Biogeoclimatic Ecosystem Classification key for the Nahwitti Lowland of North Vancouver Island in British Columbia, Canada (McWilliams and Klinka, 2005a) to confirm similar soil moisture and nutrient regimes. Differences in fungal communities may be even greater between CH and HA forests differing in slope position.

5.5.2 Molecular characterization of fungal communities

Molecular identification of mycorrhizal fungi used in my research allowed for rapid identification of fungal species present in plant roots and of forest floor mycelia. Given that this technique does not rely upon detecting morphological or reproductive structures, it can detect a greater number of species that are otherwise difficult to identify based on visual characteristics alone, especially given that many mycorrhizal fungi can be cryptic species or can lack necessary traits or reproductive lifecycles necessary for identification (Egger, 1995; Mehmann et al., 1995; Horton and Bruns, 2001; Horton, 2002; Anderson and Cairney, 2004). Until my research, differences in fungal communities had been detected, but the techniques utilized could not elucidate the identity of organisms present (Leckie et al., 2004). By using a targeted metagenomic approach, I was able to provide insight into how fungal community composition differed between forest types and infer whether a difference in ecological roles of identified fungi existed. Furthermore, by characterizing the mycorrhizal fungi present prior to forest harvest, it created a baseline to compare results of mycorrhizal composition detected in the seedling bioassays, especially among the treatment groups that received a soil transfer from mature forests. In addition, until my study, it was unknown whether AM fungal species colonizing plant communities of these forests were compatible with western

124 redcedar (Weber et al., 2003; Weber et al., 2005); by using sequence identification of the fungi in the roots, I was able to reveal the overlap in AM fungal diversity shared within the plant communities of CH and HA clearcuts.

There are several drawbacks to using molecular characterization of fungal communities. Species identification is largely dependent upon DNA sequence homology matches to taxa represented in DNA sequence databases (Anderson and Cairney, 2004; Abarenkov et al., 2010; Begerow et al., 2010; Kõljalg et al., 2013). As seen in my study, a large number of fungal species do not yet have published DNA sequences from voucher specimens. Though phylogenetic analyses can provide insight into varying degrees of systematic relatedness for sequences lacking homology matches to known species (Bruns et al., 1998; Wang et al., 2007), in my study it frequently did not give high enough resolution to species or genus level to extrapolate the potential role of the organism. Also, amplification of DNA using the polymerase chain reaction (PCR) is not free of biases and thus can lead to misrepresentation of actual fungal dominance (Anderson et al., 2003; Anderson and Cairney, 2004). Polymerase chain reaction amplification bias is a phenomenon where DNA from particular species are preferentially amplified over other species, especially due to DNA sequence divergence in primer binding sites (Acinas et al., 2005), GC (guanine-cytosine) content and sequence length (Dabney and Meyer, 2012). Utilizing highly conserved primer binding regions is believed to reduce amplification bias (Acinas et al., 2005). The regions I utilized tend to be reasonably conserved among the higher fungi such as ascomycetes and basidiomycetes (Gardes and Bruns, 1996) and PCR amplifications with the primers utilized are known to have good representation of these fungi (Anderson et al., 2003). Unfortunately, like other studies (Redecker et al., 2000; Anderson et al., 2003), the primers utilized failed to capture the AM fungi. As such, the ratio of clone sequences in my study should be a fair reflection of the actual ratio of major groups of ascomycete and basidiomycete fungi, but excluded the AM fungi and may have misrepresented some species.

Although ratios of fungi can be roughly estimated from DNA clone libraries generated for molecular identification (Anderson et al., 2003), they do not provide actual biomass estimates. For future fungal studies on these forests, complementary techniques such as phospholipid fatty acid (PFLA) analyses (Frostegård and Bååth, 1996; Frostegård et al., 2011) in combination with ingrowth bags could provide supporting measures of AM, EM and ericoid mycorrhizal biomass estimates in soil 125

(Wallander et al., 2001; Wallander et al., 2003). Real-time PCR using taxon-specific primers could also provide valuable biomass estimates of individual selected taxa (Fraga et al., 2008) in both soil and in individual roots, however, this technique is limited to quantifying a few taxa at a time.

Furthermore, when using molecular identification of mycorrhizal fungi in roots, the technique does not differentiate among true mycorrhizal root symbionts, fungal hyphae growing along the surface of the roots, root endophytes or pathogenic hyphal root penetration. Mycorrhizal synthesis experiments, in which plants are grown under axenic conditions and are inoculated with pure cultures of fungal isolates, can elucidate the nature of the association for mycorrhizal fungal species that can be cultured (Pearson and Read, 1973; Fortin et al., 1983; Pons et al., 1983; Wilcox and Lo-Buglio, 1983; Wong and Fortin, 1989; Xiao and Berch, 1995; Brundett et al., 1996; Guerin-Laguette et al., 2000; Vrålstad et al., 2002). However, even this approach can result in atypical associations under laboratory conditions which do not occur under natural conditions (Burggraaf and Beringer, 1989). In situ fluorescent hybridization, on the other hand, holds much promise, as species-specific probes can be used to microscopically visualize fungal structures formed within the plant roots of the target fungal species, thereby confirming whether the association is mycorrhizal. This technique does require a knowledge of specific fungal organisms to target ahead of time. From the results of my work in chapter 3, we now have DNA sequence data for target AM taxa worth investigating to confirm mycorrhizal structures formed within host plant roots as detected in random clone libraries.

5.5.3 Community composition versus functional roles

Determining the fungal species identity does not fully elucidate functional role of the organism. Moreover, functional roles of organisms can change under different environmental conditions and in different microbial community structures (Smith and Smith, 1996; Johnson et al., 1997; Jones and Smith, 2004). Extrapolations based on known roles of identified fungi can be postulated, but to fully understand how the community composition translates into ecosystem processes requires further study. Capturing the full fungal enzymatic activity and processes in soil for decomposition and nutrient cycling, however, is no trivial undertaking. Given the dependency of mycorrhizal fungi on host plants for photosynthates (Smith and Read, 2008), most mycorrhizal fungi rapidly diminish with the utilization of lab incubation trials, and thus lab incubations primarily capture the activity of the saprophytic 126 community present and the simultaneous die off of the symbiotic community (Bååth et al., 2004; Nilsson et al., 2005). Culture based techniques developed for assessing enzymatic activities are appropriate for individual EM root tips (Courty et al., 2005; Pritsch et al., 2011), but when used to assess soil mycelia, the enzyme assays will likely capture the activity of saprophytic fungi better able to grow in culture conditions. Therefore, in order to better understand mycorrhizal processes leading to differences in decomposition, nutrient cycling and thus above-ground productivity, there is a need to utilize techniques such as isoenzyme analyses, proteomics and transcriptomics, which can provide insight into active metabolic activities of soil communities occurring under natural conditions (Lindahl et al., 2005; Urich et al., 2008). Even then, correlations detected might not just be reflective of the community present, but rather a complex interplay of abiotic and biotic factors and of forest history that changes site properties through time.

5.5.4 Temporal variation of mycorrhizal communities

Mycorrhizal community structure, biomass production and activity can have seasonal variation (Griffiths et al., 1990; Abbott and Robson, 1991; Wallander et al., 1997; Genet et al., 2000; Buée et al., 2005; Kaiser et al., 2010). My research provided a snap shot of community composition of saprophytic and mycorrhizal fungal communities occurring in winter, a time when the mycorrhizal biomass and ectomycorrhizal root colonization can be moderate (Fogel and Hunt, 1979; de Román and de Miguel, 2005). Whether dissimilarities in temporal species dominance and biomass in functional groups occur between CH and HA forests is unknown. Swaty et al. (1998) detected significant differences in patterns of seasonal variation in ectomycorrhizal communities in soils that differed by moisture and nutrient stress. Given the differences in age related canopy structure of CH and HA forests, evapotranspiration rates will likely differ (Douglas, 1965; Vertessy et al., 2001; Moore et al., 2004; Unsworth et al., 2004), potentially leading to wetter conditions in CH forests, as detected by Sajedi et al. (2012) and Prescott et al. (2013), during months with higher rainfall. Both the hydrological differences and associated impact of seasonal variation in these forests on the fungal communities and their biological activity needs to be studied to better understand the full fungal community dynamics that occur.

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5.5.5 Seedling bioassays and soil transfers as inoculation assessment

Seedling bioassays have long been used as a means of capturing the diversity of EM inoculum present within soil (Perry et al., 1982; Schoenberger and Perry, 1982; Parke et al., 1984; Pilz and Perry, 1984; Amaranthus and Perry, 1989a, 1989b; Borchers and Perry, 1990; Amaranthus et al., 1993; Baar et al., 1999; Massicotte et al., 1999; Taylor and Bruns, 1999; Weber et al., 2005; Johnson et al., 2005b). Similar patterns of EM colonization of seedlings have been observed between field and greenhouse bioassays (Jones et al., 1997), however, in some cases, when seedling bioassays are carried out under greenhouse conditions, the results are not always reflective of fungi present within soil collected (Taylor and Bruns, 1999; Hagerman and Durall, 2004). Some EM fungal species fail to colonize young seedlings (Deacon et al., 1983; Fleming, 1983; Fox, 1983), especially under greenhouse conditions (Marx, 1979; Danielson et al., 1984), and greenhouse contaminants can establish as a dominant symbiont (Castellano and Molina, 1989; Roth and Berch, 1992; Stottlemyer et al., 2008). By contrast, greenhouse bioassays have the advantage of isolating soil effects from climate or site effects on mycorrhizal communities. A strength in my study, was that by carrying out a seedling bioassay in the field, I was able to study the role of mycorrhizal inoculum under natural environmental conditions, thereby avoiding misrepresentation that can occur in laboratory and greenhouse experiments. Although not reported in this dissertation, I also conducted a complementary greenhouse bioassay, which I will use to disentangle the mycorrhizal effects from site effects that may be present in the field data (Guichon, unpublished data).

The use of soil transfers from mature forests with compatible mycorrhizal fungi at the time of planting a seedling bioassay trial has been used as a simple technique of evaluating whether a deficiency in mycorrhizal fungi and associated stunted tree growth can be alleviated at a given site (Amaranthus and Perry, 1987, 1989a, 1989b; Amaranthus and Trappe, 1993). However, as seen in the seedling bioassay in chapter 4, a drawback to using soil transfers as an inoculum treatment is that more factors than mycorrhizal fungal inoculum alone can exist in the soil transfer. A wide range of other microorganisms can influence mycorrhization, including growth promoting rhizobacteria (Bowen, 1973; Bianciotto et al., 1996; Andrade et al., 1997; Duponnois and Plenchette, 2003; Artursson et al., 2006), mycorrhizal helper bacteria (Garbaye, 1994; Frey‐Klett et al., 2007) and mycorrhizas of other guilds (Haskins and Gehring, 2004, 2005; McHugh and Gehring, 2006). Furthermore, although the 128 amount of soil being transferred is typically insignificant for changing the overall nutrient status, allelopathic compounds can be present that inhibit seedling growth (Oster et al., 1990; Seigler, 1995; Ferguson et al., 2003; Dudai et al., 2004; Rathinasabapathi et al., 2005; Gilani et al., 2010; Saad and Abdelgaleil, 2014). As such, interpretation of results is not restricted to examining the effect of a few mycorrhizal species detected alone, but rather can be the result of a complex community of organisms working in synergy.

In my study, the western hemlock seedling bioassay utilized in chapter 4 did not capture the majority of EM fungi present in soil transfers from mature forests. After the first growing season, only C. geophilum colonized the seedling roots. However, based on the targeted metagenomic results of chapter 2, a highly diverse suite of EM fungal propagules was present in the soil transfers, which did not colonize the planted seedling roots. Failure of mycorrhizal infectivity from residual propagules of the suite of fungi present may have been due to inhibition from fertilization seedlings received prior to planting (Marx and Barnett, 1974; Ruehle and Marx, 1977; Gagnon et al., 1988), EM fungal sensitivity to soil disturbance (Read and Birch, 1988; Boerner et al., 1996), seral stage of the EM fungi present (Deacon et al., 1983; Fleming, 1983; Fox, 1983; Mason et al., 1983) and fungal dependency on mature hosts (Fleming, 1983; Fleming et al., 1984; Jones et al., 2003). Though the seedling bioassay technique was an ineffective approach to capture the diversity of EM fungi present, a strength of this study was that the results were reflective of what happens when fertilized, greenhouse grown western hemlock seedlings are planted in clearcuts as part of forestry management practices. Results from my study indicate that fertilized, greenhouse grown western hemlock seedlings planted in the clearcuts of northern Vancouver Island are poorly colonized by EM fungi from residual EM propagules during early establishment and this may contribute to poor seedling performance and survivorship in the first year or two following planting. Therefore, colonization by pioneer EM fungi will play a greater role for mycorrhizal root colonization and seedling establishment in these clearcuts. Research is needed to examine factors affecting the establishment and impact of pioneer EM species in these nutrient poor sites. Additional research is needed for developing reforestation practices that may enhance EM diversity of pioneer and later-stage fungi on establishing seedlings. Furthermore, given that clearcuts of the AM dominated CH forests become rapidly colonized by AM plants, as seen in chapter 3, studies are needed to elucidate the influence of AM antagonism on the colonization of pioneer EM fungi.

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5.6 The bigger picture: mycorrhizal fungi are an important biological factor

Returning to the original question of what factors are driving differences in the development and nutrient regimes of CH and HA forests, my research on the mycorrhizal communities adds an important piece to a much larger puzzle. My research showed that fungal communities differ between forest types, and these may alter soil properties, decomposition processes and nutrient availability, potentially through different functional roles of the organisms, alteration of other soil microorganisms and production of recalcitrant compounds. Furthermore, the results of my study indicate that networks of compatible AM fungi supported by the plant community may serve as an important inoculum source that can facilitate the establishment of western redcedar seedlings during reforestation, whereas lack of compatible alternative EM hosts may result in a rapid loss of inoculum for western hemlock. Where compatible mycorrhizal inoculum is lacking, addition of infective propagules from mature forests was effective for planted western redcedar, but was ineffective for planted western hemlock.

However, more biological factors not yet studied may very well be contributing to the CH and HA nutrient and regeneration phenomena. Previous research has examined factors contributing to nutrient cycling of nitrogen through tree litter production, decomposition and immobilization (Prescott et al., 1996; Sajedi et al., 2012). However, microbial nutrient additions versus losses have barely been touched upon. In temperate forests, mycorrhizae and nitrogen-fixing microbes combined have been estimated to be responsible for 80% of fixed nitrogen that is acquired by plants annually (van der Heijden et al., 2008). Although Brunner and Kimmins (2003) found no difference in nitrogenase (an enzyme important for microbial nitrogen fixation) activity of soil between forest types, this measure would have only estimated the contribution of free-living nitrogen fixing bacteria. Nitrogen inputs into the system via symbiotic cyanobacteria of feather moss, is an area of research deserving future attention. In particular, forest floors of HA sites are often dominated with feather mosses (up to 70- 100% ground cover), especially Hylocomium splendens (Weber et al., 2003). Although varying measures of moss abundance have been reported (deMontigny, 1992; Keenan, 1993; McWilliams et al., 2007; Sajedi et al., 2012), examination of data of over twenty forests sites, presented by McWilliams et al. (2007) revealed that feather moss abundance is often much lower in CH forests

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(between 0 and 45%). Symbiotic cyanobacteria that grow within feather moss have been shown to be substantial source of fixed nitrogen in boreal forests (Zackrisson et al., 2009). Nitrogen fixation rates of symbiotic cyanobacteria in H. splendens have been estimated around 1.6 to 1.8 kg fixed nitrogen/hectare/year (Lagerström et al., 2007; Zackrisson et al., 2009). Although a small contribution, it can account for up to 30% of the net primary productivity of nutrient poor boreal forests (Wardle et al., 2012) and some researchers argue that the input from the nitrogen-fixing ability facilitates a larger impact on key processes that control nitrogen and carbon cycles (Lindo et al., 2013). Likewise, nitrogen fixation by symbiotic cyanobacteria of feather moss may be an important nitrogen input differing between CH and HA forests.

Yet another area that deserves attention is the role of pathogens in directing forest composition. Though no substantial fungal pathogens were detected in my study, salal is an important vector for several fungal-like oomycete pathogens such as Phytophthora spp., which cause high mortality among germinants of various plant and tree species (Linderman and Zeitoun, 1977; Schlenzig, 2005; Linderman et al., 2006; Chastagner et al., 2011; Osterbauer et al., 2014; Reeser et al., 2015). Western hemlock seedlings are highly susceptible to this pathogen, whereas western redcedar is relatively resistant (Hamm and Hansen, 1982; Denman et al., 2005; Manter et al., 2007; Manter et al., 2008). This may be a contributing factor to the co-occurrence of salal and western redcedar. In areas dominated by salal, incidence of the pathogen may be higher, resulting in selective pressure against western hemlock following seed germination. Western hemlock seedlings growing in CH forests are frequently found on raised nurse logs and stumps, but not on the forest floor, even when there is little competition from other vegetation (personal observation; Prescott, personal communication 2008). These nurse logs act as major seedbeds (Harmon and Franklin, 1989), potentially providing a refuge from soil borne disease (O'Hanlon-Manners and Kotanen, 2004).

Studying the CH and HA phenomena is a difficult challenge, given these forest types differ by centuries in tree age and forest development (Lewis, 1982). Canopy structure and forest productivity will greatly influence site and soil properties, including moisture availability through differences in evapotranspiration (Douglas, 1965; Vertessy et al., 2001; Moore et al., 2004; Unsworth et al., 2004) and microbial activity through differences in photosynthate allocation (Högberg et al., 2001; Hobbie, 2006; Hobbie and Wallander, 2006; Näsholm et al., 2013). As such, when correlations are detected, 131 caution is needed for interpreting whether the factor studied is a driving variable for differences in ecosystem processes, or rather the result of overall processes associated primarily with forest age. Given that forest harvest and regeneration have been underway for decades, in coming years we may have the opportunity to revisit some of the leading hypotheses and test whether they hold true when comparing the different forest communities at a similar age in development.

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Appendices

Appendix A: Primer sequences for PCR amplification of fungi

Table A.1. Primer sequences for PCR amplification of fungal DNA, used in chapters 2 and 3.

Melting Primer name Sequence References Temperature (ºC)

ITS1-F 5’- CTT GGT CAT TTA GAG GAA GTA A -3’ 50 White et al. (1990) TW13 5’- GGT CCG TGT TTC AAG ACG -3’ 53 Taylor and Bruns (1999) M13F 5’- TGT AAA ACG ACG GCC AGT -3’ 54 Messing and Vieira (1982) M13R 5’- AGG AAA CAG CTA TGA CCA T -3’ 50 Messing and Vieira (1982) SG1 (modified NS31) 5’- GAG GGC AAG TCT GGT GCC -3’ 57 AML1 5’- ATC AAC TTT CGA TGG TAG GAT AGA -3’ 53 Lee et al. (2008) WANDA 5’- CAG CCG CGG TAA TTC CAG CT -3’ 59 Dumbrell et al. (2011) NS31 5’- TTG GAG GGC AAG TCT GGT GCC -3’ 61 Simon et al. (1992) AML2 5’- GAA CCC AAA CAC TTT GGT TTC C -3’ 55 Lee et al. (2008) AM1 5’- GTT TCC CGT AAG GCG CCG AA -3’ 60 Helgason et al. (1998)

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NS1 NS31 AM1 NS4 ITS1-F

18S SSU rRNA

AML1 WANDA AML2

0 500 1000 1500 2000

Note: this figure is a simplification and modification of figure 1 found in Lee et al. (2008)

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ITS1-F ITS3 TW13

ITS1 ITS2 18S SSU rRNA 5.8S rRNA 28S LSU rRNA

ITS1 ITS2 ITS4

158 bp

900-1200 bp

Figure A.2 Map of the internal transcribed spacer (ITS) regions with primer positions. Map includes primers included in this project and other primers that are commonly used to amply regions in this gene (ITS1, ITS2, ITS3 and ITS4). Arrows indicate relative position of primers to the ITS regions and the ribosomal RNA subunits 18S, 5.8S and 28S. Lengths of ITS regions are highly variable between different fungi; typically an amplification of both regions, including the 5.8S (approximately 158 bp (Korabecna, 2007)), spans 900-1200 bp.

Note: this figure is a modification of the ITS map found on http://nature.berkley.edu/brunslab/

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Appendix B: DNA binning program

The DNA sequence binning program was written in Java by Patrick Guichon. Coding was compiled and run in Java Standard Ed. 6 Development Kit (Oracle Corporation, CA). The binning program was based on the longest common substring algorithm. It compared DNA sequences, saved in *.txt format, to each other and binned unique sequences into individual folders based on a set threshold for sequence similarity.

Code file

import java.io.*; import java.util.*; public class LCSTest3 { public static void main(String[] args) {

String x = null; String y = null;

String fileName1 = "";

File dir = new File(".");

FilenameFilter filter = new FilenameFilter() { public boolean accept(File dir, String name) { return name.endsWith(".txt"); } };

HashMap processedFileDirectories = new HashMap();

String[] children = dir.list(filter);

if (children == null) { } else { for (int f=0; f

} catch (Exception e) { System.out.println("Error reading file: "+fileName1); }

for(String dnaString : processedFileDirectories.keySet()) { x = dnaString; String directoryName = processedFileDirectories.get(dnaString);

int M = x.length(); int N = y.length();

float correctnessPercent = 0.99f; float smallestSourceLength = Math.min(x.length(),y.length());

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float largestSourceLength = Math.max(x.length(),y.length()); float sizeRatio = smallestSourceLength / largestSourceLength; int maxErrorsBeforeFail = (int)((smallestSourceLength * (1- correctnessPercent)) + (largestSourceLength - smallestSourceLength)); int[][] opt = new int[M+1][N+1];

boolean confirmedWrong = false;

float longestConseqPercent = 0; float percentSequenceMatch = 0;

int lcsStringLength = 0;

int currentConseq = 0; int longestConseq = 0;

if (sizeRatio >= .97) {

// calculate LCS (dynamic programming) stopComparing: { for (int i = 1; i < M+1; i++) { for (int j = 1; j < N+1; j++) { if (x.charAt(i-1) == y.charAt(j-1)) { opt[i][j] = opt[i-1][j-1] + 1; } else { opt[i][j] = Math.max(opt[i-1][j], opt[i][j-1]); } if (Math.min(i,j) - opt[i][j] > maxErrorsBeforeFail) { confirmedWrong = true; break stopComparing; } } } }

lcsStringLength = 0;

currentConseq = 0; longestConseq = 0;

if (!confirmedWrong) { int i = M, j = N; while(i > 0 && j > 0) { if (x.charAt(i-1) == y.charAt(j-1)) { lcsStringLength++; currentConseq++; if (currentConseq>longestConseq) { longestConseq = currentConseq; } i--; j--; } else if (opt[i-1][j] >= opt[i][j-1]) { i--; currentConseq = 0; } else { j--; currentConseq = 0; } } } longestConseqPercent = longestConseq / smallestSourceLength; percentSequenceMatch = lcsStringLength / smallestSourceLength; }

if (sizeRatio >= 0.97 && !confirmedWrong && longestConseqPercent > 0.10 && percentSequenceMatch > 0.99) {

try { File directory = new File(directoryName);

boolean success = directory.exists() || directory.mkdir(); if (!success) { // Directory creation failed System.out.println("Error creating new folder"); }

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BufferedWriter out = new BufferedWriter(new FileWriter(directoryName+"\\"+fileName1+".txt")); out.write(y); out.flush(); out.close(); } catch (IOException e) { System.out.println("Error creating a new file "+fileName1); e.printStackTrace(); }

break nextFile; } }//for each already classified dna strings

//if compared to all other files processed so far and not found, // it's something new! :O processedFileDirectories.put(y, fileName1);

try { File directory = new File(fileName1);

boolean success = directory.exists() || directory.mkdir(); if (!success) { System.out.println("Error creating new folder"); }

BufferedWriter out = new BufferedWriter(new FileWriter(fileName1+"\\"+fileName1+".txt")); out.write(y); out.flush(); out.close(); } catch (IOException e) { System.out.println("Error creating a new file "+fileName1); e.printStackTrace(); }

}//nextFile

} //end of processing all files }//no files in the directory } }

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Appendix C: Fungal operational taxonomic units from CH and HA forests

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0.01 0.1 1 10 100 Percent clone abundance (%) Figure C.1

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