TEMPORAL DETERMINANTS OF FUNGAL COMMUNITY ASSEMBLY
A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL SCIENCES AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
Devin R. Leopold May 2017
© 2017 by Devin Robert Leopold. All Rights Reserved. Re-distributed by Stanford University under license with the author.
This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/kj190rg6492
ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Tadashi Fukami, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Erin Mordecai
I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Kabir Peay
I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy.
Peter Vitousek
Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost for Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives.
iii Abstract
Fungi play key roles in terrestrial ecosystems, driving many biogeochemical processes and influencing the structure of plant and animal communities as mutualists and pathogens. The fungal kingdom is also taxonomically and functionally diverse, and efforts to understand what determines fungal species composition in ecological communities are motivated by the functional consequences of fungal community assembly. Recent advances in DNA sequencing-based microbial community profiling have facilitated rapid advances in the study of fungal community ecology. However, the factors that determine the outcome of community assembly are not static through time, and factors related to the temporal scale of fungal community assembly remain poorly defined. In this dissertation I explore two temporal determinants of fungal community assembly, ecosystem age and species arrival order. First, I explore the effect of ecosystem age using an observational study of the fungi associated with the roots of an ericaceous plant, Vaccinium calycinum, across a 4.1 myr soil chronosequence in Hawaii. I show that soil development promotes greater diversity in ericaceous root-associated fungal communities and that soil-age related nutrient limitation facilitates colonization of ericaceous roots by a greater diversity of non- mycorrhizal fungi in both young and old soils. Second, I use a laboratory microcosm study of V. calycinum root-associated fungi to show that fungal species pools from older ecosystems include species that are more likely to coexist within the roots of a single seedling. Finally, I use a community of wood-decomposing fungi in a microcosm experiment to show that the interactive effects of top-down (grazing) and bottom-up (nutrient availability) forces determine the importance of immigration history (i.e., priority effects) for community composition and function. Taken together these results demonstrate that temporal processes occurring at both short and long time scales can be important determinants of fungal community assembly.
iv Acknowledgments
I would like to thank Tadashi Fukami for encouraging me to pursue a PhD at Stanford and for being an outstanding advisor and friend throughout the process. My committee members, Erin Mordecai, Kabir Peay, and Peter Vitousek, have each provided important feedback and assistance at various points along the way. Kabir, in particular, has contributed a significant amount of his time and resources, for which I am grateful.
The entire Fukami Lab and Stanford community has always made me feel welcomed, despite my infrequent trips to campus. Specifically, Holly Moeller, Matt Knope, Marie-Pierre Gauthier, and Rachel Vannette all helped me grow as a scientist and became good friends along the way.
This work was supported by research grants from the National Science Foundation, The American Society of Naturalists, and the Mycological Society of America. Laboratory space and other resources were generously provided Michael Shintaku and Anne Veillet, at the University of Hawaii, Hilo, Scott Gibb, at the USDA-ARS, Pacific Basin Agricultural Research Center, and Christian Giardina, at the USFS, Institute of Pacific Islands Forestry.
Finally, and most importantly, I have to thank my family for their support and encouragement. Christina Leopold has supported me in countless ways and her confidence in my success has often been greater than my own. Her contribution to this dissertation is immeasurable. My son, Nico Leopold, who was born during the second year of my PhD, has provided much needed distraction from my work, as well as renewed motivation for success. Lastly, I am fortunate to have parents, Karen and Robert Leopold, who have always encouraged me to pursue my interests freely and worked hard to set me up for success in life.
v Table of contents
Chapter 1: General introduction and author contributions...... 1
General Introduction...... 2 Author contributions...... 6 References...... 7
Chapter 2: Ericoid fungal diversity: challenges and opportunities for mycorrhizal research...... 9
Abstract...... 10 Introduction...... 11 Diversity of ericoid mycorrhizal fungi...... 14 Biogeography of ericoid mycorrhizal fungi...... 19 Research opportunities for ericoid mycorrhizal systems...... 24 Conclusions...... 28 Acknowledgments...... 29 References cited...... 30 Figures...... 42
Chapter 3: Diversity of Vaccinium calycinum (Ericaceae) root-associated fungi increases throughout long-term ecosystem development...... 44
Abstract...... 45 Introduction...... 46 Materials and methods...... 49 Results...... 55 Discussion...... 57 Acknowledgments...... 61 References cited...... 62 Tables...... 69 Figures...... 70 Supplemental materials...... 76
Chapter 4: Local richness increases more with regional richness under older species pools: evidence from root-associated fungi...... 77
Abstract...... 78 Introduction...... 79 Material and methods...... 81 Results...... 91 Discussion...... 92 References cited...... 95 Tables...... 102 Figures...... 103
vi Supplemental materials...... 108 Appendix A: Mock community sample preparation...... 114
Chapter 5: Priority effects are interactively regulated by top-down and bottom-up forces: evidence from wood decomposer communities...... 115
Abstract...... 116 Introduction...... 117 Materials and methods...... 119 Results...... 124 Discussion...... 126 Acknowledgments...... 130 References cited...... 131 Figures...... 137 Supplemental materials...... 141
vii List of tables
Chapter 3: Diversity of Vaccinium calycinum (Ericaceae) root-associated fungi increases throughout long-term ecosystem development
1. PerMANOVA results for long-term fertilization plots...... 69
Chapter 4: Local richness increases more with regional richness under older species pools: evidence from root-associated fungi
1. Michaelis-Menten model results using observed richness...... 102 2. Michaelis-Menten model results using inverse Simpson’s diversity...... 108
viii List of figures
Chapter 2: Ericoid fungal diversity: challenges and opportunities for mycorrhizal research
1. Mycorrhizal taxa across the Fungal Tree of Life...... 43
Chapter 3: Diversity of Vaccinium calycinum (Ericaceae) root-associated fungi increases throughout long-term ecosystem development
1. Map of Hawaiian Long Substrate Age Gradient (LSAG) sites...... 70 2. Bipartite graph showing relative OTU abundance across the LSAG ...... 72 3. Relationship between substrate age and fungal richness and diversity...... 73 4. NMDS plot showing fungal OTU composition across the LSAG...... 74 5. Fertilizer effects on fungal richness and diversity...... 75 6. NMDS plot showing putative ErMF OTU composition across the LSAG...76
Chapter 4: Local richness increases more with regional richness under older species pools: evidence from root-associated fungi
1. Estimated phylogenetic relationship among fungal isolates...... 104 2. Effect of species pool age on phylogenetic and functional diversity...... 105 3. Relationship between pool richness and observed richness...... 106 4. Standardized effect size of mean pairwise phylogenetic distance...... 107 5. Graphical summary of experimental pool design...... 109 6. Seedling biomass and leaf tissue chemistry...... 110 7. Results of the mock community analysis...... 111 8. Relationship between pool richness and inverse Simpson’s diversity...... 112 9. Effect of pool age variance on phylogenetic and functional diversity...... 113
Chapter 5: Priority effects are interactively regulated by top-down and bottom-up forces: evidence from wood decomposer communities
1. Effects of treatments on fungal species composition at 12 months...... 137 2. Effects of treatments on the prevalence individual species at 12 months...138 3. Outcomes in single species microcosms at 12 months...... 139 4. Effects of treatments on decomposition at 12 months...... 140 5. Inoculation and sampling positions on wood disks...... 141 6. Effects of treatments on the prevalence individual species at 6 months.....142 7. Outcomes in single species microcosms at 6 months...... 143 8. Effects of treatments on decomposition at 6 months...... 144
ix Chapter 1 General introduction and author contributions
1 General introduction
What determines the composition of ecological communities? This fundamental ecological question is motivated in part by innate human curiosity, but also by the practical need to understand the distribution of natural resources and, increasingly, to forecast and mitigate human impacts on the natural world. Defining the factors that determine the composition of ecological communities is challenging because the primary drivers of community assembly can be contingent on the types of organisms, as well as the spatial and temporal scales being considered (Lawton 1999). For example, differences in size, dispersal mechanisms, and generation times between microbes and larger taxa, such as plants and animals, can result in different patterns of species distributions across space and time (Martiny et al. 2006). Nonetheless, some basic ecological patterns appear to be common across communities of microbial and macrobial taxa, such as power-law species-area relationships and exponential decay in species rank-abundance distributions (Nemergut et al. 2013). One group of organisms, the filamentous fungi can be viewed as a hybrid of the microbial and macrobial lifestyles, interacting with the environment at the micrometer scale, yet existing as sessile, multicellular organisms that can cover much larger areas (Peay et al. 2008). Fungal community assembly has direct ecological relevance because fungi are key regulators of many biogeochemical processes (Gadd 2006; Taylor et al. 2012) and serve as important mutualists and pathogens of plants and animals (Read & Perez- Moreno 2003; Currie 2003). Recent advances in high-throughput molecular methods of microbial community characterization have dramatically increased the pace of research on fungal community ecology (Lindahl et al. 2013). However, while these advances have facilitated significant progress in defining the factors responsible for spatial patterns in microbial community composition (e.g., Talbot et al. 2014; Tedersoo et al. 2014), factors related to the temporal scale of fungal assembly remain poorly defined.
In this dissertation I explore two factors related to the temporal scale of assembly, ecosystem age and species arrival order, through the lens of two distinct fungal
2 communities. First, I explore the influence of ecosystem age on the community of fungi associated with the roots of plants in the family Ericaceae. I begin by reviewing the literature on ericoid mycorrhizal fungi, which form a specialized mycorrhizal symbiosis with the roots of ericaceous plants, but are relatively poorly characterized given their global distribution and ecological significance (Chapter 2). Drawing from a growing body of literature on the diversity of fungi associated with the roots of ericaceous plants I show that the true extent of ericoid mycorrhizal diversity is likely to be much greater than the limited number of species that have been studied thus far. I also highlight emerging patterns in the global distributions of some ericoid fungal taxa and identify regions of the globe where more data is needed. Finally, I identify some aspects of the ericoid mycorrhizal symbiosis that make it well suited as an experimental system for the study of mycorrhizal fungal ecology generally.
In Chapters 3 and 4, I focus on the fungal community associated with a single ericaceous host plant, Vaccinium calycinum, across a 4.1 myr chronosequence of soil development in Hawaii. Despite the fact that root-associated fungi are involved in mediating plant response to changing soil conditions and nutrient limitations (van der Heijden et al. 2008), surprisingly little is known about how the composition and structure of these fungal communities change in response to pedogenesis and long- term ecosystem development (Dickie et al. 2013). This is knowledge gap is particularly acute for plants and fungi that are commonly associated with the later stages of ecosystem development, such as ericaceous plants and their specialized fungal symbiont community. In Chapter 3, I use high-throughput DNA sequencing to characterize fungal communities in field collected roots to test the hypothesis that the diversity of ericaceous root-associated fungi increases with substrate age due to the unique ecology of ericoid mycorrhizal fungi. I found a clear pattern of fungal species turnover and increasing diversity across the chronosequence that was largely driven by putative ericoid mycorrhizal fungi. By using long-term fertilization experiments at the youngest and oldest chronosequence sites, I also demonstrate that the shift from nitrogen to phosphorus limitation with increasing substrate age results in a
3 corresponding shift in nutrient dynamics responsible for structuring these communities.
In Chapter 4, I bring the fungal community associated with V. calycinum into the laboratory by isolating fungi from field collected roots. Using experimental fungal species pools in plant-fungal microcosms, I test the hypothesis that older ecosystems facilitate increased local diversity due to fine-scale coexistence of species comprising the regional species pool. Consistent with this hypothesis, I found that, as species pool richness increased, more species coexisted with an individual seedling when they originated from older sites. However, species pools from older ecosystems also had lower phylogenetic and functional diversity, indicating trait convergence in older species pools. Trait convergence is consistent with an increasing role of equalizing fitness differences, rather than niche differentiation, in the coexistence of ericaceous root-associated fungi in older ecosystems. These results suggest that ecosystem age increases local diversity not just through increased regional diversity, as has been demonstrated before, but also by strengthening coexistence mechanisms among species in the regional species pool.
In Chapter 5, I explore a different temporal aspect of assembly, species arrival order, using a different fungal community, wood-decomposing saprotrophs. This study builds on earlier experimental work linking the effect of species arrival order on community assembly (i.e., priority effects) to functional properties of wood- decomposing fungal communities (i.e., decomposition rate and carbon and nutrient dynamics; Fukami et al. 2010; Dickie et al. 2012). In this study, I experimentally test for the joint effects of top-down (grazing) and bottom-up (resource availability) forces on the strength of priority effects for both community assembly and function. Using a factorial manipulation of immigration history, fungivore presence, and nitrogen availability, I found interactive effects of all three factors on fungal species composition and wood decomposition. The strength of priority effects on community structure was affected primarily by nitrogen availability, whereas the strength of priority effects on decomposition rate was interactively regulated by nitrogen and
4 fungivores. These results demonstrate that top-down and bottom-up forces jointly determine how strongly assembly history affects community structure and function.
Taken together, my experimental results demonstrate that fungal community assembly is affected by processes occurring over multiple temporal scales. One theme that emerges from this work is that factors affecting assembly over short and long temporal scales cannot be viewed as independent of each other. For example, at ecological time- scales (weeks to months), the strength of priority effects for fungal community assembly is affected by nutrient resource availability (Chapter 5). However, shifting nutrient limitations throughout long-term soil development (Chapter 3) may modulate the strength of priority effects in fungal communities over geologic time-scales. Moreover, changes in abiotic conditions throughout long-term ecosystem development may dictate mechanisms of species coexistence in local communities (Chapter 4). At the same time, the outcomes of species interactions at ecological time scales may affect assembly processes over longer temporal scales by influencing the composition of the regional species pool (Chapter 4). By exploring these interconnected processes, this dissertation advances understanding of the temporal determinants of fungal community assembly, while also demonstrating the utility of fungal communities as tractable model systems for addressing difficult questions in community ecology.
5 Author contributions
I am the sole author of Chapter 2, which is a slightly reformatted version of a manuscript published in Fungal Ecology (Leopold 2016). I was invited to prepare this mini-review for a special issue of Fungal Ecology, on the “Diversity and Biogeography of Mycorrhizal Fungi,” after participating in an eponymous symposium at the 8th International Conference on Mycorrhizas in Flagstaff, Arizona in 2015.
Chapter 3 was coauthored with Kabir G. Peay, Peter M. Vitousek, and Tadashi Fukami. PMV provided contextual information about the field system and assisted with field site access. KGP provided guidance and material support for the Illumina MiSeq library preparation. TF provided input and material support for all aspects of the project. I initially planned the project, collected samples, performed laboratory work, analyzed data and prepared the manuscript. All authors provided feedback on early revisions of the manuscript.
Chapter 4 was coauthored by Tadashi Fukami, who assisted with planning the microcosm experiment, provided material support for preliminary phases of the project, provided feedback on data analysis and helped to revise early drafts of the manuscript. I led all aspects of the project, from planning through preparation of the manuscript.
Chapter 5 was coauthored by J. Paula Wilkie (Independent Researcher, New Zealand), Ian A. Dickie (Lincoln University, New. Zealand), Robert B. Allen (Independent Researcher, New Zealand), Peter K Buchanna (Landcare Research, New Zealand), and Tadashi Fukami. TF designed the study with input from RBA, IAD, JPW, and PKB. JPW led implementation of the experiment. IAD developed molecular identification. I performed data analysis with input from IAD and TF and I wrote the manuscript with input from all other authors.
6 References cited
Cornell, H.V. & Harrison, S.P. (2014). What are species pools and when are they important? Annu. Rev. Ecol. Evol. Syst., 45, 45–67. Currie, C.R. (2003). Ancient tripartite coevolution in the attine ant-nicrobe symbiosis. Science, 299, 386–388. Dickie, I.A., Fukami, T., Wilkie, J.P., Allen, R.B. & Buchanan, P.K. (2012). Do assembly history effects attenuate from species to ecosystem properties? a field test with wood-inhabiting fungi. Ecol. Lett., 15, 133–141. Dickie, I.A, Martinez-Garcia, L.B., Koele, N., Grelet, G.A, Tylianakis, J.M., Peltzer, D.A, et al. (2013). Mycorrhizas and mycorrhizal fungal communities throughout ecosystem development. Plant Soil, 367, 11–39. Fukami, T., Dickie, I.A., Wilkie, J.P., Paulus, B.C., Park, D., Roberts, A., et al. (2010). Assembly history dictates ecosystem functioning: evidence from wood decomposer communities. Ecol. Lett., 13, 675–84. Gadd, G.M. (Ed.). (2006). Fungi in biogeochemical cycles (Vol. 24). Cambridge University Press. van der Heijden, M.G.A., Bardgett, R.D. & van Straalen, N.M. (2008). The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol. Lett., 11, 296–310. Lawton, J.H. (1999). Are there general laws in ecology? Oikos, 84, 177–192. Leopold, D.R. (2016). Ericoid fungal diversity: challenges and opportunities for mycorrhizal research. Fungal Ecol., 1–10. Lindahl, B.D., Nilsson, R.H., Tedersoo, L., Abarenkov, K., Carlsen, T., Kjøller, R., et al. (2013). Fungal community analysis by high-throughput sequencing of amplified markers–a user’s guide. New Phytol., 199, 288–299. Martiny, J.B.H., Bohannan, B.J.M., Brown, J.H., Colwell, R.K., Fuhrman, J.A, Green, J.L., et al. (2006). Microbial biogeography: putting microorganisms on the map. Nat. Rev. Microbiol., 4, 102–112.
7 Nemergut, D.R., Schmidt, S.K., Fukami, T., O’Neill, S.P., Bilinski, T.M., Stanish, L.F., et al. (2013). Patterns and processes of microbial community assembly. Microbiol. Mol. Biol. Rev., 77, 342–356. Pärtel, M., Bennett, J.A. & Zobel, M. (2016). Macroecology of biodiversity: disentangling local and regional effects. New Phytol., 211, 404–410. Peay, K.G., Kennedy, P.G. & Bruns, T.D. (2008). Fungal community ecology: a hybrid beast with a molecular master. Bioscience, 58, 799. Read, D. & Perez-Moreno, J. (2003). Mycorrhizas and nutrient cycling in ecosystems– a journey towards relevance? New Phytol., 157, 475–492. Talbot, J.M., Bruns, T.D., Taylor, J.W., Smith, D.P., Branco, S., Glassman, S.I., et al. (2014). Endemism and functional convergence across the North American soil mycobiome. Proc. Natl. Acad. Sci., 1–6. Taylor, L.L., Banwart, S.A., Valdes, P.J., Leake, J.R. & Beerling, D.J. (2012). Evaluating the effects of terrestrial ecosystems, climate and carbon dioxide on weathering over geological time: a global-scale process-based approach. Philos. Trans. R. Soc. Lond. B. Biol. Sci., 367, 565–82. Tedersoo, L., Bahram, M., Polme, S., Koljalg, U., Yorou, N.S., Wijesundera, R., et al. (2014). Global diversity and geography of soil fungi. Science, 346, 1256688. Zobel, M. (2016). The species pool concept as a framework for studying patterns of plant diversity. J. Veg. Sci., 27, 8–18.
8 Chapter 2 Ericoid fungal diversity: challenges and opportunities for mycorrhizal research
9 Abstract
Ericoid mycorrhiza occur only within the plant family Ericaceae, yet are globally widespread and contribute to carbon and nutrient cycling in many habitats where harsh conditions limit decomposition and plant nutrient uptake. An increasingly diverse range of fungi are recognized as ericoid symbionts and patterns in the distribution of ericoid taxa are beginning to emerge across scales. However, the true diversity of ericoid mycorrhizal fungi remains unresolved due to limited sampling from some regions and challenges associated with delineating mycorrhizal taxa from the broader fungal community associated with ericoid plants. Interpreting patterns in the diversity and distributions of ericoid mycorrhizal fungi will ultimately require improved understanding of their functional ecology and functional diversity, which is currently limited to a few well studied species. Fortunately, many ericoid taxa are amenable to experimental manipulation and continued ericoid mycorrhizal research promises to improve general understanding of the ecology and evolution of mycorrhizal symbioses.
Keywords: biogeography; community assembly; diversity; Ericaceae; ericoid mycorrhiza; mycorrhizal fungi; mycorrhizal research; plant-fungal symbioses
10 Introduction
Mycorrhizal fungi colonize the roots of most terrestrial plant species, enhancing nutrient uptake in exchange for photosynthetically derived sugars and making them key drivers of carbon and nutrient cycling in many ecosystems (van der Heijden et al. 2008). In the plant family Ericaceae, the extra-fine terminal roots of most species, which lack root hairs and are known as hair-roots, are colonized by fungi that form ericoid mycorrhiza (ErM; Smith & Read 2008). An ErM, which includes both the plant and fungal components of the symbiotic complex, is a morphologically distinct mycorrhiza characterized by the formation of compact intracellular hyphal coils in enlarged epidermal hair-root cells which function as the sites of nutrient exchange (for detailed ErM morphology see, Bonfante-Fasolo & Gianinazzi-Pearson 1979; Read 1983). Although ErM plants account for just 1% of angiosperm species (Brundrett 2009), they have a nearly global distribution and are often abundant in habitats with harsh edaphic conditions, primarily where acidic soils, low temperatures or low soil moisture limit the uptake of soil nutrients by plants and slow the degradation of organic matter (Cairney & Meharg 2003; Mitchell & Gibson 2006; Read 1991). ErM are particularly abundant in heathlands and the Boreal Forest understory, habitats which account for approximately 70% of the terrestrial surface of the Northern Hemisphere (Read et al. 2004). The proliferation of ericaceous plants in these environments has been attributed to symbiosis with ErM fungi (ErMF), which help to detoxify acidic soil conditions and provide access to recalcitrant organic nutrient pools (Näsholm et al. 1998; Read et al. 2004). However, ErMF remain understudied relative to the more common mycorrhizal types, arbuscular and ecto- mycorrhizae (AM and ECM, respectively), and definitive data on the mycorrhizal status and functional roles of many taxa associated with ErM roots are lacking.
Based primarily on studies of Northern Hemisphere heathlands, ErM were historically viewed as a highly specialized symbiosis, including only a narrow range of plants and fungi (Harley & Smith 1983; Straker 1996). The proliferation of culture-independent molecular methods (Allen et al. 2003; Perotto et al. 1996) and the increasing
11 availability of globally-distributed data (Bruzone et al. 2015) have challenged these early views. While ErM plants remain phylogenetically constrained to the family Ericaceae, an increasingly diverse range of fungi are recognized as forming ErM (but see Lehnert et al. 2009 and Okuda et al. 2011 for descriptions of symbioses resembling ErM in the Diapensiaceae and neotropical ferns, respectively). Furthermore, the identification of ErM associated fungi from contrasting environments and across environmental gradients has revealed patterns in fungal community composition (e.g., Bougoure et al. 2007; Gorzelak et al. 2012), though the factors driving these patterns are not yet well understood. Molecular methods have also revealed a diverse assemblage of other fungal symbionts within ErM roots, including dark-septate endophytes (DSE), ECM fungi and saprotrophs (e.g., Bougoure et al. 2007; Walker et al. 2011). New evidence that some DSE and ECM fungi can form hyphal coils resembling ErM in ericaceous plants (Lukešová et al. 2015; Villarreal- Ruiz et al. 2012), and reports of novel taxa forming ErM in resynthesis trials (Vohník et al. 2012), have begun to blur the distinctions between these classifications, raising new questions about the functional relationships amongst a broad range of fungi found in ErM roots. Greater focus on the functional diversity among putative ErMF, along with the abiotic and biotic factors that influence the structure and function of ErM associated fungal communities as a whole, is needed to advance understanding of the ErM symbiosis.
While uncertainty surrounding the phylogenetic and functional diversity of ErMF presents a clear challenge to ErM research, several features of the symbiosis are well- suited for manipulative experimentation. Most notably, many fungi associated with ErM roots are highly saprophytic and, as a result, many potentially mycorrhizal taxa can be readily isolated and maintained in pure culture (Leake & Read 1991). Although culture collections such as this are known to bias against some taxa (Allen et al. 2003; Bougoure & Cairney 2005a), the ability to consistently and relatively quickly obtain pure-culture collections of potentially mycorrhizal fungi lies in stark contrast to AM fungi, which can only be cultured in planta, or ECM fungi, which are often difficult to
12 isolate in pure culture (Hobbie et al. 2001). One factor that may have slowed progress in ErM research is the fact that many ericaceous plants grow slowly or are difficult to propagate. Fortunately, some species such as Calluna vulgaris and many Vaccinium spp., have proven to function as broadly compatible ErM host plants, amenable to axenic culture for experimentation (e.g., Villarreal-Ruiz et al. 2012) and some Rhododendron and Gaultheria species have also been used to establish in vitro plant- fungal co-cultures (Grunewaldt-Stöcker et al. 2013; Xiao & Berch 1999). The potential to obtain culture collections of ErMF from contrasting environments or plant hosts and experimentally manipulate ErM under controlled conditions represents a largely untapped resource for mycorrhizal research and has the potential to advance both our understanding of the ErM symbiosis and mycorrhizal symbioses more broadly. However, to realize this potential for ErM research, a more complete understanding of the species and functional diversity of ErMF is needed.
Conclusive determination of the mycorrhizal status of a fungus is complicated by the fact that mycorrhizal symbioses exist along a continuum of mutualistic to parasitic interactions and outcomes for either partner can vary with the abiotic and biotic environment (Johnson et al. 1997). Brundrett (2004) recognized this functional variability and suggested an inclusive definition of mycorrhizae that requires experimental evidence of both the formation of a specialized symbiotic interface resulting from synchronized plant-fungal development (i.e., ericoid hyphal coils) and direct plant-fungal resource exchange, without stipulating a net benefit to either partner (Brundrett 2004); however, evidence for the latter is lacking for many ErM associated fungi (Leake & Read 1991). Given the uncertainty surrounding the range of taxa capable of forming ErM, this review will distinguish between those fungi that have been experimentally determined to form functional ErM (sensu Brundrett 2004) and those for which only limited or circumstantial evidence is currently available. In addition, the broader community of fungi associated with ErM roots, including ErMF and taxa with uncertain mycorrhizal status, will be referred to as ErM associated fungi. To facilitate continuing advances in ErM research, this review has three primary goals:
13 (1) Assess the current state of knowledge on ErMF diversity and the unique challenges associated with delineating ErMF from the broader community of ErM associated fungi. (2) Identify emerging biogeographic patterns for ErMF across global, regional and local scales. (3) Identify focal areas for future ErM research and research opportunities where continued study of ErM may enhance general understanding of the ecology and evolution of mycorrhizal symbioses.
Diversity of ericoid mycorrhizal fungi
A broad range of potentially mycorrhizal ascomycetous and basidiomycetous fungi are often identified in ErM roots using both culture based and culture-independent molecular methods. However, mycorrhizal status has only been experimentally confirmed for a few species, making the definitive identification of ErMF from among the broader community of ErM associated fungi challenging. Unlike AM fungi (Glomeromycota), ErMF are not monophyletic (Smith & Read 2008), precluding a simple phylogenetic prescription. In addition, many putative ErMF occur within lineages that encompass functionally diverse groups of plant and soil associated fungi, limiting the ability to infer mycorrhizal status from phylogenetic information alone. Furthermore, unlike ECM, in which individual root tips are typically colonized by a single mycorrhizal species (Smith & Read 2008), ErM roots are characterized by multiple occupancy, with multiple putative mycorrhizal taxa occurring in close proximity (Perotto et al. 2012; Setaro et al. 2006). Even the proper identification of ericoid hyphal coils within roots requires careful attention to detail due to the common presence of non-mycorrhizal endophytes which can form intracellular structures, such as loose hyphal coils or sclerotia, that could be mistaken for ErM (Lukešová et al. 2015; Usuki & Narisawa 2005). Following the basic principals of Koch’s postulates, mycorrhizal resynthesis experiments, in which individual fungi are isolated and reinoculated onto axenic host plants, are the primary method used for determining the mycorrhizal status of fungi associated with ErM roots (Leake & Read 1991). However, the recalcitrance of some putative ErMF to pure culture techniques requires alternative approaches (Selosse et al. 2007; Setaro et al. 2006).
14 Among ascomycetous ErMF, Rhizoscyphus ericae (formerly Hymenoscyphus ericae and Pezizella ericae; Read 1974; Zhang & Zhuang 2004) was the earliest to be identified and experimentally confirmed to be mycorrhizal (Pearson & Read 1973; Stribley & Read 1974). With the advancement of molecular and phylogenetic methods, many additional sterile isolates from ErM roots that could not be classified morphologically were recognized as being closely related to R. ericae, forming a species complex known as the R. ericae aggregate (REA; Vrålstad et al. 2000; Vrålstad et al. 2002). Hambleton & Sigler (2005) further refined the REA which now includes the ErMF species Meliniomyces variabilis, which has also been experimentally show to exchange C and N with host plants (Grelet, Johnson, et al. 2009), along with the ECM species Meliniomyces bicolor and Cadophora finlandica, and numerous other related mycorrhizal species and non-mycorrhizal endophytes. Experimentation with ErM associated REA species other than R. ericae and M. variabilis have largely focused on the formation of ericoid hyphal coils in vitro and a greater effort to understand the functional variability among these species is needed to fully understand the extent of functional ErM formation in this species aggregate.
Although early work focused on R. ericae as the sole ErMF, a second group of ascomycetous species isolated from ErM roots and identified as members of the genus Oidiodendron, were eventually shown to form ericoid hyphal coils in resynthesis experiments (Couture et al. 1983; Dalpé 1986). Although later molecular approaches identified some phylogenetic variability amongst ErM associated Oidiodendron isolates, most appear to be very closely related to O. maius and may or may not represent distinct species (Lacourt et al. 2002). Research on the functional aspects of ErM formation by O. maius has largely focused on the role of the mycobiont in improving heavy metal tolerance in the host (Daghino et al. 2015), though some investigations into enhanced host nutrient uptake have helped confirm the mycorrhizal status of this species (Vohník et al. 2005; Xiao & Berch 1999). O. maius conspecifics and related species have also been isolated from soils and ECM roots, suggesting that
15 this species is not restricted to association with ErM plants (Bergero et al. 2000; Rice & Currah 2006).
There are many reports of other ascomycetous fungi that can form hyphal coils with ErM plants in resynthesis trials. Three Helotialean isolates that could not be resolved between the families Dermataceae or Hyaloscyphaceae were shown by Grelet, Meharg, et al. (2009) to form ericoid hyphal coils and exchange C and N with Vaccinium spp. in vitro, demonstrating that at least some non-REA Helotiales can form functional ErM. In the genus Leohumicola, several isolates were shown to by Grunewaldt-Stöcker et al. (2013) and Bizabani (2015) form ericoid hyphal coils and repress the growth of oomycete root-rot pathogens, though evidence for nutrient exchange was not reported. Isolates with affinity to the genus Capronia have also been reported to form ericoid hyphal coils in resynthesis experiments (Allen et al. 2003). Although no functional data is currently available for Capronia-like species, the observation that similar taxa are often observed as significant components of sequencing based surveys of ErM associated fungal communities (Lukešová et al. 2015; Walker et al. 2011; Wurzburger et al. 2012) has led many ErM researchers to consider these taxa putative ErMF. Other ascomycetous fungi reported to form hyphal coils resembling ErM in resynthesis experiments with ericaceous plants include Acremonium strictum (Monreal et al. 1999; Xiao & Berch 1996), Geomyces pannorum (Vohník et al. 2007), members of the Phialocephala–Acephala complex (Lukešová et al. 2015; Walker et al. 2011) and isolates with affinities to the genera Cadophora (Bizabani & Dames 2015; Monreal et al. 1999), Cryptosporiopsis (Bizabani 2015; Chambers et al. 2008; Walker et al. 2011) and Lachnum (Walker et al. 2011). The mycorrhizal status of these associations remain questionable and many reports of in vitro associations resembling ErM are likely to be the result of opportunistic colonization by non-mycorrhizal species that primarily function as endophytes, necrotrophs or root-associated saprotrophs. However, it is also possible that some of these associations represent nascent mycorrhizal symbioses and further study of their potential functional significance and the conditions under which they
16 occur is warranted. In addition, both culture-based and culture-independent survey methods regularly yield poorly resolved taxa that match closely with other unidentified ascomycetous fungi associated with ErM roots (e.g., Bougoure et al. 2007; Zhang et al. 2009). This suggests that an even broader range of ascomycetous fungi, some of which may be difficult or impossible to culture, may have potential to form ErM.
The presence of basidiomycetous fungi in ErM roots was recognized in some of the earliest ErM research using morphological characteristics of intracellular hyphae (Bonfante-Fasolo 1980; Peterson et al. 1980). However, the inability to successfully culture any basidiomycetes from ErM roots initially prevented their identification. Based on observations of basidiocarps associated with ErM plants and subsequent immunofluorescent staining of colonized roots, a Clavulina sp. was the first basidiomycete suspected of forming ErM (Seviour et al. 1973). Englander & Hull (1980) later provided evidence for bi-directional transfer of isotopically labeled C and P between naturally occurring ericaceous plants and Clavulina sp. fruiting bodies, but they were unable to conclusively determine if the symbiosis was mycorrhizal or represented some other saprotrophic or necrotrophic association. Molecular methods eventually revealed that species in the order Sebacinales are common inhabitants of ErM roots, sometimes as the dominant taxa in sequencing based community profiles (Allen et al. 2003; Berch et al. 2002). Ultrastructural observations further support the formation of ErM by Sebacinales, which can be distinguished by their electron-opaque cell walls and dolipore septa with imperforate parenthesomes, while ascomycetes have electron-translucent cell walls and simple septal pores with Woronin bodies (Setaro et al. 2006). Selosse et al. (2007) combined targeted molecular methods and ultrastructural observations to show that Sebacinales in the family Serendipitaceae nom. prov. (formerly Sebacinales Clade B; Weiß et al. 2016) form ericoid hyphal coils in many ericoid plants and these taxa are now widely regarded as putative ErMF. However, these enigmatic species have not yet been successfully isolated in pure
17 culture, complicating experimental confirmation of their mycorrhizal status and limiting understanding of their functional significance.
Recently, a basidiomycetous species with affinity to the Trechisporales was cultured and identified by Vohník et al. (2012) as a co-dominant symbiont of some Vaccinium spp. in Norway. In resynthesis experiments these isolates were shown to form intracellular structures with a unique morphology that the authors described as a “sheathed-ericoid” mycorrhiza. Although conclusive evidence of resource exchange was not provided, the isolates did enhance plant growth in vitro and, combined with strong circumstantial evidence for ErMF in the Sebacinales, this finding has dramatically broadened the phylogenetic range of putative ErMF.
There is accumulating evidence that some ErMF colonize non-ericaceous hosts and some fungi typically classified as ECM or DSE may also be capable of forming ErM, blurring the distinction between these functional classifications (Grelet et al. 2010; Vohník & Albrechtová 2011; Vohník et al. 2013; Vrålstad 2004; Zijlstra et al. 2005). Villarreal-Ruiz et al. (2004) were the first to conclusively show that a REA isolate collected from an ECM host, Pinus sylvestris, could simultaneously form ECM in an ectotrophic host and ericoid hyphal coils in an ericaceous host. Grelet, Johnson, et al. (2009) later demonstrated that the ECM species Meliniomyces bicolor could form functional ErM with Vaccinium vitis-idaea roots, exchanging C and N with ErM plants. Similarly, Villarreal-Ruiz et al. (2012) found that Laccaria bicolor, a basidiomycetous ECM species, extensively colonized multiple ErM plants in vitro, forming hyphal coils that resembled typical ErM. Among species commonly identified as DSE, numerous species belonging to the Phialocephala–Acephala species complex (Lukešová et al. 2015) along with Heteroconium chaetospira (Usuki & Narisawa 2005) were also experimentally shown to colonize ErM plants, forming intracellular structures resembling ericoid hyphal coils, though the functional significance of these structures are unclear.
18 It is clear from the diversity of confirmed and possible ErMF, and the recent discovery of novel putative ErMF lineages, that the true diversity of ErMF has not yet been circumscribed (Fig. 1). Renewed focus on resynthesis experiments that include tests for bi-directional nutrient transfer is needed to clarify the mycorrhizal status of many fungi capable of forming hyphal coils in ericaceous plants. For unculturable taxa, this may ultimately require methods such as laser microdisection (Gomez & Harrison 2009), stable isotope probing (DNA-SIP; Dumont & Murrell 2005), or taxon specific fluorescent in situ hybridization coupled with nano-scale secondary ion mass spectrometry (FISH-NanoSIMS; Behrens et al. 2008). It is likely that the formation of hyphal coils in ErM roots by some fungi simply represents opportunistic colonization without the formation of true mycorrhizal symbioses, however, it is also possible that the functional classifications of these associations vary with environmental conditions or host ontogeny (Brundrett 2004), forming ecologically significant mycorrhizal symbioses only under particular conditions (Moeller & Neubert 2016). Ultimately, the mycorrhizal status of some ErM associated fungi may remain ambiguous, underscoring both the lack of a sharp distinction between functional classifications and the general difficulties associated with any functional definition of a mycorrhiza (Jones & Smith 2004). However, as a more comprehensive understanding of ErMF ecology and diversity begins to emerge, it may become easier to confidently assign mycorrhizal status to some ErM associated species without direct experimental evidence, an accepted practice for some ECM fungi (Tedersoo & Smith 2013).
Biogeography of ericoid mycorrhizal fungi
Limited sampling of ErM from many regions relative to the geographic distribution of ericaceous plants currently precludes conclusive identification of global biogeographical patterns of ErM mycobiont community composition. The lack of data from much of the Southern Hemisphere is particularly striking considering that diversity hot spots for the Ericaceae include the South African fynbos, montane regions in the Neotropics and Papua New Guinea (Bruzone et al. 2015; Luteyn 2002; Oliver 2000). Some early studies of ErMF from South Africa suggested that ErM
19 associated fungi in the fynbos are predominantly ascomycetes, including Oidiodendron spp. and Helotiales, but not R. ericae conspecifics (Straker 1996). Bizabani (2015) recently confirmed these findings, using both molecular and culture based approaches which revealed Meliniomyces, Phialocephala, Cadophora, Cryptosporiopsis, O. maius, and Chaetothyriales, but not R. ericae. In South America, Bruzone et al. (2015) recently reported for the first time on the identities of fungi associated with two species of Gaultheria from NW Patagonia, expanding the geographic range of data available for the Southern Hemisphere. Using culture independent methods they found that Sebacinales were the dominant members of ErM associated fungal communities, though culturable isolates included primarily non- REA Helotiales, Hypocreales and O. maius. One notable exception to the dearth of data from the Southern Hemisphere is Australia, where fungi associated with a variety of endemic ericaceous genera have been investigated, revealing diverse assemblages of ascomycetous fungi, primarily groups of Helotialean species outside of the REA and some O. maius related species (Bougoure & Cairney 2005a,b; Cairney & Ashford 2002; Chambers et al. 2000; Curlevski et al. 2009; McLean et al. 1999; Midgley et al. 2004).
There is considerably more data available for the Northern Hemisphere and global geographic patterns are beginning to emerge for some ErMF. For example, Sebacinales have been increasingly identified as dominant components of ErM associated fungal communities in North America and the Neotropics (Allen et al. 2003; Berch et al. 2002; Bruzone et al. 2015; Kottke et al. 2008). Setaro & Kron (2011) reported close phylogenetic relationships among ErM associated Sebacinales across this range, leading them to suggest a history of concerted migration. In contrast, members of the REA, including R. ericae and M. variabilis, are dominant ErMF across many Boreal and subarctic habitats and much of temperate Eurasia (Gorzelak et al. 2012; Grelet et al. 2010; Ishida & Nordin 2010; Kjøller et al. 2010). O. maius, on the other hand, appears to be a cosmopolitan species, having been detected in studies that span the globe (e.g., Australia, Bougoure & Cairney 2005b; South Africa,
20 Bizabani 2015; China, Zhang et al. 2009; USA, Wurzburger et al. 2012), though rarely as the dominant mycobiont at any given site. There are also many other groups of poorly understood, possible ErMF which can be locally dominant in sequencing based surveys, such as undescribed members of the Chaetothyriales or Helotiales (Lukešová et al. 2015), and much more data is needed before a comprehensive picture of the global biogeography of ErMF can emerge.
Despite the fact that dispersal mechanisms remain largely unknown for most ErMF due to the cryptic nature of their reproductive strategies, there is some evidence for broad dispersal limitation in ErMF communities (Hutton et al. 1997). However, other factors, such as abiotic filtering or historical contingency (i.e., priority effects), are also likely to have influenced global distribution patterns. The global distribution of R. ericeae, for example, appears to exclude Southern Hemisphere Ericaceae, yet R. ericeae isolates capable for forming ErM in laboratory trials have been isolated from a leafy liverwort present on number of sub-Antarctic islands (Upson et al. 2007). This suggests that factors other than dispersal limitation have shaped the global biogeography of this species.
It is possible that differential global distribution patterns among ErMF have arisen as a result of differential traits among fungal taxa. For example, the cosmopolitan species O. maius can occur as a free-living saprotroph in soil (Rice & Currah 2006), potentially allowing it to disperse widely and independently of ErM plants. Some ErMF in the REA are known to colonize the roots of ECM trees (Grelet et al. 2010; Vohník et al. 2013), which are dominant plants in the Boreal forest, giving REA species multiple niches and potentially contributing to their abundance in this habitat. In contrast, putative ErMF in the Sebacinales have not been observed to colonize co- occurring non-ericaceous plants, even when closely related fungi occur as endophytes or other mycorrhizal types (Garnica et al. 2016; Kottke et al. 2008). In addition, all attempts to culture ErM associated Sebacinales have failed thus far, further suggesting that at least some of these species may be obligate ErM symbionts, restricted to migration with suitable ericaceous plants (Setaro & Kron 2011). However, related
21 orchid mycorrhizal Sebacinales, also in the family Serendipitaceae, which have been successfully cultured, appear to be phylogenetically interspersed with ErM forming lineages (Weiß et al. 2016), and much more study is need to properly characterize the ecological distinctions among these taxa.
At the regional scale, environmental gradients have been used to explore the factors influencing the community composition of ErM associated fungi. Gorzelak et al. (2012) found that fungal communities associated with Vaccinium membranaceum changed across an elevation gradient; dominant culturable fungi shifted from Phialocephala fortinii to R. ericae with increasing elevation. Bougoure et al. (2007) examined the fungal community associated with Vaccinium myrtillus across a vegetation gradient and found distinct communities of putative ErMF in heathland, forest understory and the joining ecotone. These transitions may reflect both a response to abiotic factors and interactions with co-occurring non-ErM vegetation, which may host some ErMF as endophytes or ECM (Grelet et al. 2010; Vohník et al. 2013; Zijlstra et al. 2005). Hazard et al. (2014) identified soil nitrogen as explaining more than 50% of the variation in ErM associated fungal communities sampled across sites with varying land use history in Ireland. However, experimental nitrogen additions in boreal forest plots by Ishida & Nordin (2010) did not alter the community composition of ErM associated fungi. This latter result is surprising given the role of ErMF in facilitating nitrogen uptake in ericaceous plants (Read 1996) and the fact that nitrogen additions are known to impact the community composition of other types of mycorrhizal fungi (Lilleskov et al. 2001; Treseder 2004).
At the local scale, there is also evidence that both abiotic and biotic factors influence the fine scale structure of ErM associated fungal communities. Within individual plants, Wurzburger et al. (2012) found that many putative ErMF were differentially abundant between organic and mineral soil horizons, and that roots from the organic horizon had significantly greater fungal diversity. Gorzelak et al. (2012) used co- occurrence analysis to show that fungi associated with V. membranaceum tended to co-occur more often than expected by chance, suggesting that facilitation may
22 influence ErMF community composition. Evidence for plant-fungal partner specificity in ErMF community composition is currently mixed. Kjøller et al. (2010) found that fungal communities associated with four ErM plants in a subarctic mire were spatially structured at scales of just a few meters, yet were indistinguishable among plants co- occurring in small hummocks. Similarly, Walker et al. (2011) found no difference in fungal communities associating with three ErM plants in the Arctic tundra. However, Bougoure et al. (2007) detected differences in fungal communities associated with two co-occurring ErM plants in a Scott's Pine understory, but was unable to determine if this was a result of host micro-habitat preference. There is also some evidence that intraspecific partner specificity can influence the composition of ErM associated fungal communities. Sun et al. (2012) reported a correlation between the community composition of putative ErMF and intraspecific genetic variation in Rhododendron decorum. Midgley et al. (2004) found that different genotypes of a single putative ErMF species were segregated among two co-occurring ericaceous plants in an Australian dry sclerophyll forest. These contrasting results suggest that host specificity may be relatively weak and context dependent in ericaceous plant-fungal networks. It is likely that fine scale variation in ErMF community composition is linked to both the compositional and functional diversity of the local fungal community and the degree to which host distributions are correlated with fine scale variation in soil conditions, such as organic matter content.
The available data are only beginning to elucidate the factors driving ErMF community composition across scales. Recent analyses of large molecular barcoding databases for AM and ECM fungi have revealed patterns of high and low endemism, respectively, at a global scale (Davison et al. 2015; Tedersoo et al. 2012). The emerging patterns for some ErMF discussed above suggest that the global biogeography of ErMF may be similar to ECM fungi, with patterns of endemism revealed at large spatial scales. This is not surprising considering the phylogenetic overlap between ErMF and ECM fungi. Because ErM are common in Boreal and heathland ecosystems, playing a role in regulating some of the largest terrestrial
23 carbon stocks, a better understanding of how ErMF community composition is linked to environmental conditions could be important for predicting how these ecosystems will respond to anthropogenic global change factors and influence climate change predictions (Clemmensen et al. 2013; Clemmensen et al. 2015; Olsrud et al. 2004). However, detailed understanding of the functional ecology of ErMF is almost entirely limited to studies of R. ericae and O. maius. Unraveling the drivers of ErMF community composition across scales will ultimately require a greater focus on manipulative experiments and diverse mycobiont communities, which have been largely lacking from ErM research and are needed to provide causal explanations for observed patterns.
Research opportunities for ericoid mycorrhizal systems
In a recent review, Perotto et al. (2012) identified O. maius as having significant potential as a model organism for studying the molecular basis of mycorrhizal symbioses; O. maius readily grows and reproduces asexually in culture, producing uninucleated conidia that germinate to produce haploid mycelium in which all nuclei will carry the same mutation. Abbà et al. (2009) took advantage of these features and used Agrobacterium-mediated transformation to disrupt the superoxide dismutase gene in O. maius, resulting in the first targeted knock-down of a fungal gene involved in the establishment of mycorrhizal symbiosis. Advances in understanding the molecular bases of heavy metal tolerance in O. maius (Di Vietro et al. 2014; Khouja et al. 2015; Khouja et al. 2013; Chiapello et al. 2015), a trait linked to the success of ErM host plants in otherwise inhospitable environments (Cairney & Meharg 2003), has also focused attention on this species as a model organism (Daghino et al. 2015). Additionally, the complete O. maius genome was recently published (Kohler et al. 2015), setting the stage for continuing advances using O. maius as a model mycorrhizal fungus. While the study of plant-fungal interactions in mycorrhizal symbioses occurs across scales, from genes and molecular interactions to communities and ecosystems, the potential for ErM symbioses and the broader community of ErM associated fungi to serve as model study systems in a variety of contexts has been
24 largely ignored. The remainder of this review will explore research areas in which ErMF and ErM associated fungal communities could provide particularly well-suited study systems to advance understanding of the ecology and evolution of mycorrhizal symbioses.
Community assembly
Research on mycorrhizal fungi has generally focused on the function of individual fungi and the composition and function of mycobiont communities, with less attention given to processes that influence the local assembly of the mycobiont community itself. In a recent analysis of AM fungal communities at scales ranging from individual plants to global, Davison et al. (2016) found evidence of phylogenetic clustering that increased with spatial extent and habitat heterogeneity, suggesting that processes related to dispersal, host preference and environmental filtering were general drivers of assembly. However, experimental work is needed to understand the conditions under which individual factors are more or less important for assembly processes. For example, Johnson (2015) recently identified the effect of species arrival timing, or priority effects, as an area needing further research. Some experimental work on priority effects in AM and ECM fungi indicates that these historical effects can be important factors influencing the community assembly of mycorrhizal fungi (Kennedy et al. 2009; Werner & Kiers 2015), but the conditions under which priority effects are important and the functional consequences for interacting plant and fungal species are largely unknown. There is also increasing interest in understanding how the composition of regional species pools can influence local community assembly processes (Cornell & Harrison 2014; Pärtel et al. 2016), a topic that is virtually unexplored in microbial symbiont communities. Local factors that influence the assembly of mycorrhizal fungi, such as competitive or facilitative interactions between symbionts, also require further study, particularly in the case of ErM (Thonar et al. 2014).
25 Many unique features of the biology and morphology of the ErM symbiosis are well- suited for the study of symbiont community assembly processes. First, ErMF tend to colonize only the outermost epidermal root cells of ericaceous hair roots by entering from the root surface, with little cell-to-cell hyphal penetration (Massicotte et al. 2005). This suggests that colonized cells can be considered individual units, each challenged and potentially colonized by a variety of fungi present in the rhizosphere (Perotto et al. 2012). As a result, multiple species, or even genotypes of the same species can occupy an individual ericoid root in close proximity (Midgley et al. 2004). Second, the broader community of ErM associated fungi interacting with an individual plant can be extremely diverse (e.g., Bougoure et al. 2007) and many of these symbionts can be isolated in pure culture. Finally, numerous methods have been described for establishing ErMF in microcosms using axenic seedlings and individual fungal isolates (Vohník et al. 2012). By extending these methods to include pairs or entire communities of ErM associated fungi, and tracking community assembly outcomes using molecular methods (e.g., Sikes et al. 2015), many aspects of community assembly in mycorrhizal symbiont communities could be experimentally tested using ErM systems.
Local adaptation and co-evolution
Mycorrhizae have been implicated in the local adaptation of plants to their soil environment (Doubková et al. 2012; Johnson et al. 2010; Schultz & Miller 2001), however many questions remain regarding the conditions under which different patterns of adaptation and co-adaptation occur in mycorrhizal symbioses. Because ErM plants are highly mycotrophic, relying on their fungal symbionts for nutrient uptake (Smith & Read 2008), it can be expected that edaphic conditions are particularly important for the evolutionary trajectory of both plant and fungal traits related to the symbiotic exchange. There is also evidence that intraspecific variation exists in the production of extracellular enzymes and in the uptake of different forms of nutrients by ErM fungi. For example, (Grelet, Meharg, et al. 2009) reported differences in nitrogen use efficiency by three non-REA Helotialean ErMF, including
26 two with identical internal transcribed spacer regions, and further demonstrated that these differences affected Vaccinium host plants in vitro. While data on the functional diversity among the broader community of potential ErMF is sparse, the ability to easily isolate and culture distinct species and genotypes of putative ErMF from sites with contrasting edaphic conditions offers the opportunity to explore the functional diversity of ErMF in the context of the local adaptation of plant-fungal symbioses.
It is also possible that host benefits other than nutrient uptake play a role in the local adaptation of mycorrhizal symbioses. For example, colonization by mycorrhizal fungi is known to increase host resistance to pathogens (Newsham et al. 1995). Grunewaldt- Stöcker et al. (2013) recently demonstrated that some putative ErMF can protect plants from oomycete root rot pathogens in vitro and that the strength of protective benefits varied amongst isolates. Theory suggests that pathogen pressure should increase with nutrient availability while the benefit of nutritional symbionts should decrease (Thrall et al. 2007), suggesting that adaptation to higher nutrient availability could involve increased pathogen protection. Another benefit that can be conveyed to ericaceous plants by ErMF is protection against toxic compounds in soils, particularly heavy metals (Bradley et al. 1981; Sharples et al. 2000). By comparing ErMF from sites contaminated by industrial activity and neighboring uncontaminated sites, strains of both R. ericae and O. maius have been shown to develop adaptive resistance to a variety of heavy metals (Martino et al. 2000; Sharples et al. 2001; Vallino et al. 2011). However, while the mechanisms of adaptive heavy metal resistance are well documented in some ErMF, the potential for co-evolutionary response between symbiont and host requires further study (Vallino et al. 2011).
Evolution of plant-fungal symbioses
The ErM symbiosis is believed to be the youngest of the major mycorrhizal types, originating in the most recent common ancestor of the Ericaceae ~117 mya (Cullings 1996; Schwery et al. 2015). While most plants in the family Ericaceae form ErM, the basal genus in the family, Enkianthus, associates with AM fungi (Glomeromycota), as
27 do related families within the order Ericales (Obase et al. 2013). The mycorrhizal types arbutoid and monotropoid, are also recognized within the Ericaceae in the subfamilies Arbutoideae and Monotropoideae, respectively (Smith & Read 2008). Arbutoideae and Monotropoideae symbionts include typical ECM fungi and root colonization shares some morphological characteristics with both ErM and ECM. The basal position of Enkianthus within the Ericaceae suggests that association with AM fungi is the ancestral mycorrhizal state from which the other mycorrhizal associations evolved (Obase et al. 2013). Furthermore, Enkianthus also harbors typical ErM taxa as endophytes without forming ErM (Obase & Matsuda 2014), presenting an opportunity to study the role of host evolution in the establishment of mycorrhizal symbioses during diversification with a single plant lineage. Through the use of comparative genomics and transcriptomics, it should be possible to identify genes associated with distinct mycorrhizal types and track the evolution of those genes through ancestral state reconstruction across the Ericaceae. In addition, there are some reports of other ericaceous species concurrently forming both ErM and AM (Koske et al. 1990; Urcelay 2002). These species could hold clues to the evolutionary process of transition between mycorrhizal types, however, the functional significance of these dual associations, which may simply represent opportunistic colonization by AM fungi predominantly associated with neighboring hosts, and the extent of the phenomenon within the Ericaceae remain unexplored.
Conclusions
Given their global distribution and prominent role in carbon and nutrient cycling in some of the largest terrestrial biomes, ErM symbioses remain understudied relative to other mycorrhizal types. New evidence has dramatically expanded the phylogenetic range of fungi known to form ErM and has blurred the distinction between ErMF and other co-existing fungal symbionts. Continued efforts to elucidate the factors influencing ErMF community composition using molecular methods will require a more comprehensive documentation of ErM fungal diversity, which can only be achieved through continued sampling of understudied regions and resynthesis
28 experiments that include tests for nutrient exchange. While the lack of functional data for most putative ErMF presents significant challenges to current ErM research, many ErM symbionts are amenable to isolation and laboratory culture, presenting opportunities for experimentation that could be exploited to advance understanding of the ecology and evolution of both ErM and mycorrhizal symbioses more broadly.
Acknowledgments
The author is supported by funding from Stanford University, Department of Biology. Thanks is due to Tadashi Fukami, Gwen Grelet, Christina R. Leopold, Maarja Öpik, Kabir G. Peay and two anonymous reviewers who all provided critical feedback and constructive comments that substantially helped improve this manuscript.
29 References cited
Abbà, S., Khouja, H.R., Martino, E., Archer, D.B. & Perotto, S., 2009. SOD1-targeted gene disruption in the ericoid mycorrhizal fungus Oidiodendron maius reduces conidiation and the capacity for mycorrhization. Molecular Plant-Microbe Interactions, 22, 1412–1421. Allen, T.R., Millar, T., Berch, S.M. & Berbee, M.L., 2003. Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytologist, 160, 255–272. Behrens, S., Lösekann, T., Pett-Ridge, J., Weber, P.K., Ng, W.O., Stevenson, B.S., Hutcheon, I.D., Relman, D.A. & Spormann, A.M., 2008. Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS. Applied and Environmental Microbiology, 74, 3143–3150. Berch, S.M., Allen, T.R. & Berbee, M.L., 2002. Molecular detection, community structure and phylogeny of ericoid mycorrhizal fungi. Plant and Soil, 244, 55– 66. Bergero, R., Perotto, S., Girlanda, M., Vidano, G. & Luppi, A.M., 2000. Ericoid mycorrhizal fungi are common root associates of a Mediterranean ectomycorrhizal plant (Quercus ilex). Molecular Ecology, 9, 1639–1649. Bizabani, C., 2015. The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism (Doctoral dissertation). Rhodes University. Accessed online, https://hdi.handle.net/10962/d1018575. Bizabani, C. & Dames, J., 2015. Effects of inoculating Lachnum and Cadophora isolates on the growth of Vaccinium corymbosum. Microbiological Research, 181, 68–74. Bonfante-Fasolo, P., 1980. Occurrence of a basidiomycete in living cells of mycorrhizal hair roots of Calluna vulgaris. Transactions of the British Mycological Society, 75, 320–325.
30 Bonfante-Fasolo, P. & Gianinazzi-Pearson, V., 1979. Ultrastructural aspects of endomycorrhiza in the Ericaceae. I. Naturally infected hair roots of Calluna vulgaris L. Hull. New Phytologist, 83, 739–744. Bougoure, D.S. & Cairney, J.W.G., 2005a. Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots. Environmental microbiology, 7, 819–827. Bougoure, D.S. & Cairney, J.W.G., 2005b. Fungi associated with hair roots of Rhododendron lochiae (Ericaceae) in an Australian tropical cloud forest revealed by culturing and culture-independent molecular methods. Environmental Microbiology, 7, 1743–1754. Bougoure, D.S., Parkin, P.I., Cairney, J.W.G., Alexander, I.J. & Anderson, I.C., 2007. Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient. Molecular Ecology, 16, 4624–4636. Bradley, R., Burt, A.J. & Read, D.J., 1981. Mycorrhizal infection and resistance to heavy metal toxicity in Calluna vulgaris. Nature, 292, 335–337. Brundrett, M., 2004. Diversity and classification of mycorrhizal associations. Biological Reviews, 79, 473–495. Brundrett, M.C., 2009. Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant and Soil, 320, 37–77. Bruzone, M.C., Fontenla, S.B. & Vohník, M., 2015. Is the prominent ericoid mycorrhizal fungus Rhizoscyphus ericae absent in the Southern Hemisphere’s Ericaceae? A case study on the diversity of root mycobionts in Gaultheria spp. from northwest Patagonia, Argentina. Mycorrhiza, 25, 25–40. Cairney, J.W.G. & Ashford, A.E., 2002. Biology of mycorrhizal associations of epacrids (Ericaceae). New Phytologist, 154, 305–326.
31 Cairney, J.W.G. & Meharg, A.A., 2003. Ericoid mycorrhiza: a partnership that exploits harsh edaphic conditions. European Journal of Soil Science, 54, 735– 740. Chambers, S.M., Curlevski, N.J.A. & Cairney, J.W.G., 2008. Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest. FEMS Microbiology Ecology, 65, 263–270. Chambers, S.M., Liu, G. & Cairney, J.W.G., 2000. Its rDNA sequence comparison of ericoid mycorrhizal endophytes from Woollsia pungens. Mycological Research, 104, 168–174. Clemmensen, K.E., Bahr, A., Ovaskainen, O., Dahlberg, A., Ekblad, A., Wallander, H., Stenlid, J., Finlay, R.D., Wardle, D.A. & Lindahl, B.D., 2013. Roots and associated fungi drive long-term carbon sequestration in boreal forest. Science, 339, 1615–1619. Clemmensen, K.E., Finlay, R.D., Dahlberg, A., Stenlid, J., Wardle, D.A. & Lindahl, B.D., 2015. Carbon sequestration is related to mycorrhizal fungal community shifts during long-term succession in boreal forests. New Phytologist, 205, 1525–1536. Cornell, H. V. & Harrison, S.P., 2014. What are species pools and when are they important? Annual Review of Ecology, Evolution, and Systematics, 45, 45–67. Couture, M., Fortin, J.A. & Dalpe, Y., 1983. Oidiodendron Griseum Robak: an endophyte of ericoid mycorrhiza in Vaccinium spp. New Phytologist, 95, 375– 380. Cullings, K.W., 1996. Single phylogenetic origin of ericoid mycorrhizae within the Ericaceae. Canadian Journal of Botany, 74, 1896–1909. Curlevski, N.J. a, Chambers, S.M., Anderson, I.C. & Cairney, J.W.G., 2009. Identical genotypes of an ericoid mycorrhiza-forming fungus occur in roots of Epacris pulchella (Ericaceae) and Leptospermum polygalifolium (Myrtaceae) in an Australian sclerophyll forest. FEMS Microbiology Ecology, 67, 411–20.
32 Daghino, S., Martino, E. & Perotto, S., 2015. Model systems to unravel the molecular mechanisms of heavy metal tolerance in the ericoid mycorrhizal symbiosis. Mycorrhiza, 26, 263–274. Dalpé, Y., 1986. Axenic synthesis of ericoid mycorrhiza in Vaccinium angustifolium Ait. by Oidiodendron species. New Phytologist, 103, 391–396. Davison, J., Moora, M., Jairus, T., Vasar, M., Öpik, M. & Zobel, M., 2016. Hierarchical assembly rules in arbuscular mycorrhizal (AM) fungal communities. Soil Biology and Biochemistry, 97, 63–70. Dumont, M.G. & Murrell, J.C., 2005. Stable isotope probing–linking microbial identity to function. Nature Reviews Microbiology, 3, 499–504. Englander, L. & Hull, R.J., 1980. Reciprocal transfer of nutrients between ericaceous plants and a Clavaria sp. New Phytologist, 84, 661–667. Garnica, S., Riess, K., Schön, M.E., Oberwinkler, F. & Setaro, S.D., 2016. Divergence times and phylogenetic patterns of Sebacinales, a highly diverse and widespread fungal lineage. PLoS ONE, 11, 1–16. Gomez, S.K. & Harrison, M.J., 2009. Laser microdissection and its application to analyze gene expression in arbuscular mycorrhizal symbiosis. Pest Management Science, 65, 504–511. Gorzelak, M.A., Hambleton, S. & Massicotte, H.B., 2012. Community structure of ericoid mycorrhizas and root-associated fungi of Vaccinium membranaceum across an elevation gradient in the Canadian Rocky Mountains. Fungal Ecology, 5, 36–45. Grelet, G.-A., Johnson, D., Paterson, E., Anderson, I.C. & Alexander, I.J., 2009. Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas. New Phytologist, 182, 359–366. Grelet, G.-A., Meharg, A.A., Duff, E.I., Anderson, I.C. & Alexander, I.J., 2009. Small genetic differences between ericoid mycorrhizal fungi affect nitrogen uptake by Vaccinium. New Phytologist, 181, 708–718.
33 Grelet, G.-A., Johnson, D., Vrålstad, T., Alexander, I.J. & Anderson, I.C., 2010. New insights into the mycorrhizal Rhizoscyphus ericae aggregate: spatial structure and co-colonization of ectomycorrhizal and ericoid roots. New Phytologist, 188, 210–222. Grunewaldt-Stöcker, G., von den Berg, C., Knopp, J. & von Alten, H., 2013. Interactions of ericoid mycorrhizal fungi and root pathogens in Rhododendron: in vitro tests with plantlets in sterile liquid culture. Plant Root, 7, 33–48. Hambleton, S. & Sigler, L., 2005. Meliniomyces, a new anamorph genus for root- associated fungi with phylogenetic affinities to Rhizoscyphus ericae (≡ Hymenoscyphus ericae), Leotiomycetes. Studies in Mycology, 53, 1–27. Harley, J.L. & Smith, S.E., 1983. Mycorrhizal Symbiosis, London: Academic Press. Hazard, C., Gosling, P., Mitchell, D.T., Doohan, F.M. & Bending, G.D., 2014. Diversity of fungi associated with hair roots of ericaceous plants is affected by land use. FEMS Microbiology Ecology, 87, 586–600. van der Heijden, M.G.A., Bardgett, R.D. & van Straalen, N.M., 2008. The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecology Letters, 11, 296–310. Hobbie, E.A., Weber, N.S. & Trappe, J.M., 2001. Mycorrhizal vs saprotrophic status of fungi: the isotopic evidence. New Phytologist, 150, 601–610. Hutton, B.J., Dixon, K.W., Sivasithamparam, K. & Pate, J.S., 1997. Effect of habitat disturbance on inoculum potential of ericoid endophytes of Western Australian heaths (Epacridaceae). New Phytologist, 135, 739–744. Ishida, T.A. & Nordin, A., 2010. No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types. Soil Biology and Biochemistry, 42, 234–243. Johnson, D., 2015. Priorities for research on priority effects. New Phytologist, 205, 1375–1377. Johnson, N.C., Graham, J.H. & Smith, F.A., 1997. Functioning of mycorrhizal associations along the mutualism–parasitism continuum. New Phytologist, 135, 575–585.
34 Jones, M.D. & Smith, S.E., 2004. Exploring functional definitions of mycorrhizas: are mycorrhizas always mutualisms? Canadian Journal of Botany, 82, 1089–1109. Kjøller, R., Olsrud, M. & Michelsen, A., 2010. Co-existing ericaceous plant species in a subarctic mire community share fungal root endophytes. Fungal Ecology, 3, 205–214. Kohler, A., Kuo, A., Nagy, L.G., Morin, E., Barry, K.W., Martin, F., et al., 2015. Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nature Genetics, 47, 410–415. Koske, R.E., Gemma, J.N. & Englander, L., 1990. Vesicular-arbuscular mycorrhizae in Hawaiian Ericales. American journal of botany, 77, 64–68. Kottke, I., Haug, I., Setaro, S., Suárez, J.P., Weiß, M., Preußing, M., Nebel, M. & Oberwinkler, F., 2008. Guilds of mycorrhizal fungi and their relation to trees, ericads, orchids and liverworts in a neotropical mountain rain forest. Basic and Applied Ecology, 9, 13–23. Lacourt, I., Girlanda, M., Perotto, S., Del Pero, M., Zuccon, D. & Luppi, A.M., 2002. Nuclear ribosomal sequence analysis of Oidiodendron: towards a redefinition of ecologically relevant species. New Phytologist, 149, 565–576. Leake, J.R. & Read, D.J., 1991. Experiments with ericoid mycorrhiza. Methods in Microbiology, 23, 435–457. Lehnert, M., Kottke, I., Setaro, S., Pazmiño, L.F., Suárez, J.P. & Kessler, M., 2009. Mycorrhizal associations in ferns from Southern Ecuador. American Fern Journal, 99, 292–306. Lilleskov, E.A., Fahey, T.J. & Lovett, G.M., 2001. Ectomycorrhizal fungal aboveground community change over an atmospheric nitrogen deposition gradient. Ecological Applications, 11, 397–410. Lukešová, T., Kohout, P., Větrovský, T. & Vohník, M., 2015. The potential of dark septate endophytes to form root symbioses with ectomycorrhizal and ericoid mycorrhizal Middle European forest plants. PLoS ONE, 10, e0124752. Luteyn, J.L., 2002. Diversity, adaptation, and endemism in Neotropical Ericaceae: biogeographical patterns in the Vaccinieae. The Botanical Review, 68, 55–87.
35 Martino, E., Turnau, K., Girlanda, M., Bonfante, P. & Perotto, S., 2000. Ericoid mycorrhizal fungi from heavy metal polluted soils: their identification and growth in the presence of zinc ions. Mycological Research, 104, 338–344. Massicotte, H.B., Melville, L.H. & Peterson, R.L., 2005. Structural characteristics of root-fungal interactions for five ericaceous species in eastern Canada. Canadian Journal of Botany, 83, 1057–1064. McLaughlin, D.J., Hibbett, D.S., Lutzoni, F., Spatafora, J.W. & Vilgalys, R., 2009. The search for the fungal tree of life. Trends in Microbiology, 17, 488–497. McLean, C.B., Cunnington, J.H. & Lawrie, A.C., 1999. Molecular diversity within and between ericoid endophytes from the Ericaceae and Epacridaceae. New Phytologist, 144, 351–358. Midgley, D.J., Chambers, S.M. & Cairney, J.W.G., 2004. Distribution of ericoid mycorrhizal endophytes and root-associated fungi in neighboring Ericaceae plants in the field. Plant and Soil, 259, 137–151. Mitchell, D.T. & Gibson, B.R., 2006. Ericoid mycorrhizal association: ability to adapt to a broad range of habitats. Mycologist, 20, 2–9. Moeller, H. V. & Neubert, M.G., 2016. Multiple friends with benefits: an optimal mutualist management strategy? The American Naturalist, 187, E1–E12. Monreal, M., Berch, S.M. & Berbee, M., 1999. Molecular diversity of ericoid mycorrhizal fungi. Canadian Journal of Botany, 77, 1580–1594. Näsholm, T., Ekblad, a, Nordin, a, Giesler, R., Högberg, M. & Högberg, P., 1998. Boreal forest plants take up organic nitrogen. Nature, 392, 914–916. Obase, K. & Matsuda, Y., 2014. Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae). Mycorrhiza, 24, 635–644. Obase, K., Matsuda, Y. & Ito, S., 2013. Enkianthus campanulatus (Ericaceae) is commonly associated with arbuscular mycorrhizal fungi. Mycorrhiza, 23, 199– 208. Okuda, A., Yamato, M. & Iwase, K., 2011. The mycorrhiza of Schizocodon soldanelloides var. magnus (Diapensiaceae) is regarded as ericoid mycorrhiza from its structure and fungal identities. Mycoscience, 52, 425–430.
36 Oliver, E.G.H., 2000. Ericaceae. In O. A. Leistner, ed. Seed plants of southern Africa. National Botanical Institute, pp. 246–248. Olsrud, M., Melillo, J.M., Christensen, T.R., Michelsen, A., Wallander, H. & Olsson, P.A., 2004. Response of ericoid mycorrhizal colonization and functioning to global change factors. New Phytologist, 162, 459–469. Pärtel, M., Bennett, J.A. & Zobel, M., 2016. Macroecology of biodiversity: disentangling local and regional effects. New Phytologist, 211, 404–410. Pearson, V. & Read, D.J., 1973. The biology of mycorrhiza in the Ericaceae. II. The transport of carbon and phosphorus by the endophyte and the mycorrhiza. New Phytologist, 72, 1325–1331. Perotto, S., Actis-Perino, E., Peru, J. & Bonfante, P., 1996. Molecular diversity of fungi from ericoid mycorrhizal roots. Molecular Ecology, 5, 123–131. Perotto, S., Martino, E., Abbà, S. & Vallino, M., 2012. Genetic diversity and functional aspects of ericoid mycorrhizal fungi. In B. Hock, ed. Fungal Associations. Springer Berlin Heidelberg, pp. 255–285. Peterson, T.A., Mueller, W.C. & Englander, L., 1980. Anatomy and ultrastructure of a Rhododendron root-fungus association. Canadian Journal of Botany, 58, 2421–2433. Read, D.J., 1991. Mycorrhizas in ecosystems. Experientia, 47, 376–391. Read, D.J., 1974. Pezizella ericae sp.nov., the perfect state of a typical mycorrhizal endophyte of Ericaceae. Transactions of the British Mycological Society, 63, 381–419. Read, D.J., 1983. The biology of mycorrhiza in the Ericales. Canadian Journal of Botany, 61, 985–1004. Read, D.J., Leake, J.R. & Perez-Moreno, J., 2004. Mycorrhizal fungi as drivers of ecosystem processes in heathland and boreal forest biomes. Canadian Journal of Botany, 82, 1243–1263. Rice, A. V & Currah, R.S., 2006. Oidiodendron maius: saprobe in sphagnum peat, mutualist in ericaceous roots? In B. J. E. Schulz, C. J. C. Boyle, & T. N.
37 Sieber, eds. Microbial Root Endophytes. Berlin, Heidelberg: Springer, pp. 227–246. Schüβler, A., Schwarzott, D. & Walker, C., 2001. A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycological Research, 105, 1413– 1421. Schwery, O., Onstein, R.E., Bouchenak-Khelladi, Y., Xing, Y., Carter, R.J. & Linder, H.P., 2015. As old as the mountains: the radiations of the Ericaceae. New Phytologist, 207, 355–367. Selosse, M.-A., Setaro, S., Glatard, F., Richard, F., Urcelay, C. & Weiß, M., 2007. Sebacinales are common mycorrhizal associates of Ericaceae. New Phytologist, 174, 864–878. Setaro, S., Kottke, I. & Oberwinkler, F., 2006. Anatomy and ultrastructure of mycorrhizal associations of neotropical Ericaceae. Mycological Progress, 5, 243–254. Setaro, S.D. & Kron, K.A., 2011. Neotropical and North American Vaccinioideae (Ericaceae) share their mycorrhizal Sebacinales–an indication for concerted migration? PLoS Currents, 3, RRN1227. Seviour, R.J., Willing, R.R. & Chilvers, G. a., 1973. Basidiocarps associated with ericoid mycorrhizas. New Phytologist, 72, 381–385. Sharples, J.M., Meharg, A.A., Chambers, S.M. & Cairney, J.W.G., 2001. Arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae. New Phytologist, 151, 265–270. Sharples, J.M., Meharg, A.A., Chambers, S.M. & Cairney, J.W.G., 2000. Mechanism of arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericeae. Plant Physiology, 124, 1327–1334. Sikes, B.A., Hawkes, C. V & Fukami, T., 2015. Plant and root-endophyte assembly history: interactive effects on native and exotic plants. Ecology. Smith, S.E. & Read, D.J., 2008. Mycorrhizal Symbiosis 3rd ed., Academic Press. Straker, C.J., 1996. Ericoid mycorrhiza: ecological and host specificity. Mycorrhiza, 6, 215–225.
38 Stribley, D.P. & Read, D.J., 1974. The biology of mycorrhiza in the Ericaceae. III. Movement of carbon-14 from host to fungus. New Phytologist, 73, 731–741. Sun, L., Pei, K., Wang, F., Ding, Q., Bing, Y., Gao, B., Zheng, Y., Liang, Y. & Ma, K., 2012. Different distribution patterns between putative ericoid mycorrhizal and other fungal assemblages in roots of Rhododendron decorum in the southwest of China. PLoS ONE, 7, e49867. Tedersoo, L. & Smith, M.E., 2013. Lineages of ectomycorrhizal fungi revisited: Foraging strategies and novel lineages revealed by sequences from belowground. Fungal Biology Reviews, 27, 83–99. Thonar, C., Frossard, E., Šmilauer, P. & Jansa, J., 2014. Competition and facilitation in synthetic communities of arbuscular mycorrhizal fungi. Molecular Ecology, 23, 733–746. Treseder, K.K., 2004. A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and atmospheric CO2 in field studies. New Phytologist, 164, 347– 355. Upson, R., Read, D.J. & Newsham, K.K., 2007. Widespread association between the ericoid mycorrhizal fungus Rhizoscyphus ericae and a leafy liverwort in the maritime and sub-Antarctic. New Phytologist, 176, 460–71. Urcelay, C., 2002. Co-occurrence of three fungal root symbionts in Gaultheria poeppiggi DC in Central Argentina. Mycorrhiza, 12, 89–92. Usuki, F. & Narisawa, K., 2005. Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi. Mycorrhiza, 15, 61–64. Vallino, M., Zampieri, E., Murat, C., Girlanda, M., Picarella, S., Pitet, M., Portis, E., Martino, E. & Perotto, S., 2011. Specific regions in the Sod1 locus of the ericoid mycorrhizal fungus Oidiodendron maius from metal-enriched soils show a different sequence polymorphism. FEMS Microbiology Ecology, 75, 321–331.
39 Villarreal-Ruiz, L., Anderson, I.C. & Alexander, I.J., 2004. Interaction between an isolate from the Hymenoscyphus ericae aggregate and roots of Pinus and Vaccinium. New Phytologist, 164, 183–192. Villarreal-Ruiz, L., Neri-Luna, C., Anderson, I.C. & Alexander, I.J., 2012. In vitro interactions between ectomycorrhizal fungi and ericaceous plants. Symbiosis, 56, 67–75. Vohník, M. & Albrechtová, J., 2011. The co-occurrence and morphological continuum between ericoid mycorrhiza and dark septate endophytes in roots of six european Rhododendron species. Folia Geobotanica, 46, 373–386. Vohník, M., Albrechtová, J. & Vosátka, M., 2005. The inoculation with Oidiodendron maius and Phialocephala fortinii alters phosphorus and nitrogen uptake, foliar C: N ratio and root biomass distribution in Rhododendron cv. Azurro. Symbiosis, 40, 87–96. Vohník, M., Fendrych, M., Albrechtová, J. & Vosátka, M., 2007. Intracellular colonization of Rhododendron and Vaccinium roots by Cenococcum geophilum, Geomyces pannorum and Meliniomyces variabilis. Folia Microbiologica, 52, 407–414. Vohník, M., Mrnka, L., Lukešová, T., Bruzone, M.C., Kohout, P. & Fehrer, J., 2013. The cultivable endophytic community of Norway spruce ectomycorrhizas from microhabitats lacking ericaceous hosts is dominated by ericoid mycorrhizal Meliniomyces variabilis. Fungal Ecology, 6, 281–292. Vohník, M., Sadowsky, J.J., Kohout, P., Lhotáková, Z., Nestby, R. & Kolařík, M., 2012. Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed basidiomycete with affinities to Trechisporales. PLoS ONE, 7, e39524. Vrålstad, T., 2004. Are ericoid and ectomycorrhizal fungi part of a common guild? New Phytologist, 164, 7–10. Vrålstad, T., Fossheim, T. & Schumacher, T., 2000. Piceirhiza bicolorata–the ectomycorrhizal expression of the Hymenoscyphus ericae aggregate? New Phytologist, 145, 549–563.
40 Vrålstad, T., Myhre, E. & Schumacher, T., 2002. Molecular diversity and phylogenetic affinities of symbiotic root-associated Ascomycetes of the Helotiales in burnt and metal polluted habitats. New Phytologist, 155, 131– 148. Walker, J.F., Aldrich-Wolfe, L., Riffel, A., Barbare, H., Simpson, N.B., Trowbridge, J. & Jumpponen, A., 2011. Diverse Helotiales associated with the roots of three species of Arctic Ericaceae provide no evidence for host specificity. New Phytologist, 191, 515–527. Weiß, M., Waller, F., Zuccaro, A. & Selosse, M.-A., 2016. Sebacinales–one thousand and one interactions with land plants. New Phytologist, 211, 20–40. Wurzburger, N., Higgins, B.P. & Hendrick, R.L., 2012. Ericoid mycorrhizal root fungi and their multicopper oxidases from a temperate forest shrub. Ecology and Evolution, 2, 65–79. Xiao, G. & Berch, S.M., 1996. Diversity and abundance of ericoid mycorrhizal fungi of Gaultheria shallon on forest clearcuts. Canadian Journal of Botany, 74, 337–346. Xiao, G. & Berch, S.M., 1999. Organic nitrogen use by Salal ericoid mycorrhizal fungi from northern Vancouver Island and impacts on growth in vitro of Gaultheria shallon. Mycorrhiza, 9, 145–149. Zhang, C., Yin, L. & Dai, S., 2009. Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China. Mycorrhiza, 19, 417– 423. Zhang, Y.-H. & Zhuang, W.-Y., 2004. Phylogenetic relationships of some members in the genus Hymenoscyphus (Ascomycetes, Helotiales). Nova Hedwigia, 78, 475–484. Zijlstra, J.D., Van’t Hof, P., Baar, J., Verkley, G.J.M., Summerbell, R.C., Paradi, I., Braakhekke, W.G. & Berendse, F., 2005. Diversity of symbiotic root endophytes of the Helotiales in ericaceous plants and the grass, Deschampsia flexuosa. Studies in Mycology, 53, 147–162.
41 Figure 1: Distribution of mycorrhizal taxa across a subset of the Fungal Tree of Life (McLaughlin et al. 2009) including phyla Glomeromycota, Ascomycota and Basidiomycota at the order level. Branches with no known or suspected mycorrhizal taxa are collapsed to class (suffix -mycota) or sub-phyla (suffix -mycotina). Filled circles aligned with fungal orders indicate at least one species is known to form arbuscular mycorrhiza (AM; Schüβler et al. 2001) or ectomycorrhiza (ECM; Tedersoo & Smith 2013). For ericoid mycorrhiza (ErM), fungal orders are identified based on the highest level of confidence in mycorrhizal status available for lower level taxa. Confirmed mycorrhizal taxa have direct experimental evidence of in vitro resynthesis of ericoid hyphal coils and plant-fungal nutrient exchange. Putative mycorrhizal taxa have multiple lines of circumstantial evidence indicating mycorrhizal status, but lack conclusive experimental evidence of resource exchange. Possible mycorrhizal taxa have only been shown to form hyphal coils in ericaceous plants in vitro, which may be the result of opportunistic colonization by non-mycorrhizal species. *The genera Oidiodendron and Leohumicola are incerti ordini and are included here because they contain known and putative ErM fungi, respectively.
42 Figure 1 (continued)
43 Chapter 3 Diversity of Vaccinium calycinum (Ericaceae) root-associated fungi increases throughout long-term ecosystem development
44 Abstract
We use a soil chronosequence to investigate how the composition of fungal communities associated with ericaceous plants changes with substrate age.
We identified fungi associated with the roots of an ericaceous species, Vaccinium calycinum, across a 4.1-myr chronosequence and quantified changes in diversity and species composition. We also used a fertilization experiment to test whether the shift from nitrogen to phosphorus limitation of primary productivity with increasing substrate age affected fungal community composition and structure.
We found a significant increase in diversity and a clear pattern of species turnover across the chronosequence. These patterns were largely driven by putative ericoid mycorrhizal fungi. We also found that fertilization with nitrogen at the youngest site and phosphorus at the oldest site reduced total fungal diversity, but primarily for non-mycorrhizal fungi.
Our results suggest that soil development throughout long-term ecosystem development promotes greater diversity in ericaceous root-associated fungal communities, possibly due to the specialization of ericoid mycorrhizal fungi on recalcitrant organic nutrient pools. Our results also suggest that soil-age related nutrient limitation facilitates colonization of ericaceous roots by a greater diversity of non-mycorrhizal fungi in both young and old soils.
Keywords: chronosequence, Ericaceae, ericoid mycorrhizal fungi, long-term ecosystem development, pedogenesis, root-associated fungi, Vaccinium calycinum
45 Introduction
Over hundreds of thousands to millions of years, many ecosystems progress through predictable stages of development driven by pedogenesis (Walker & Syers 1976; Chadwick et al. 1999) and changes in the composition and function of plant communities (Richardson et al. 2004; Lambers et al. 2008; Zemunik et al. 2015). These stages include an early progressive phase, characterized by nitrogen (N) limitation, increasing productivity and biomass accumulation; a mature phase, characterized by relatively fertile soils and maximal biomass; and a retrogressive phase, resulting from the loss and occlusion of rock-derived nutrients, especially phosphorus (P) (Vitousek & Farrington 1997; Wardle 2004; Peltzer et al. 2010). Throughout this process, root-associated fungal symbionts, including mycorrhizal fungi, mediate plant responses to changing soil conditions by facilitating host nutrient uptake (van der Heijden et al. 2008). In exchange, plant carbon transfer to root- associated fungi (RAF) contributes to below-ground carbon cycling and soil development (Högberg & Read 2006). As a result, RAF facilitate the plant-soil interactions that drive long-term ecosystem development. However, relative to changes in plant communities, changes in RAF communities throughout long-term ecosystem development have received less attention, particularly in mature to retrogressive systems (Dickie et al. 2013).
Plant taxa can host functionally distinct RAF communities that are likely to vary in their response to the changing soil environment throughout long-term ecosystem development. In the plant family Ericaceae, most species associate with a taxonomically diverse RAF assemblage, including a polyphyletic group of fungi that form a unique mycorrhizal symbiosis, known as an ericoid mycorrhiza (ErM; Perotto et al. 2012; Leopold 2016). ErM have been studied primarily in the context of mature to retrogressive ecosystems, where a few well-studied species of ErM fungi (ErMF) have been shown to degrade complex organic matter in soil, accessing recalcitrant nutrient pools that are not otherwise available to plants (Nasholm et al. 1998; Adamczyk et al. 2016). As a result of the nutritional benefits of these fungal
46 associations, ericaceous plants often become dominant in environments where low temperature or excessive soil moisture limit decomposition, leading to the accumulation of organic matter in soil and limited mineral nutrient availability (Cairney & Meharg 2003; Read et al. 2004). However, these habitat associations do not appear to limit the global distribution of Ericaceae, which are found in a wide range of ecosystems and at all stages of ecosystem development (Dickie et al. 2013).
Throughout long-term ecosystem development, changes in edaphic conditions could determine the composition and structure of ericaceous RAF communities through two mechanisms. First, is the accumulation of organic matter in soil, which occurs rapidly at the early stages of soil development (Torn et al. 1997). There is some evidence for vertical niche partitioning of putative ErMF taxa across soil horizons, with greater diversity in the upper organic layers (Wurzburger et al. 2012). ErMF taxa also vary in their affinity for different sources of nutrients, particularly N (Cairney et al. 2000; Grelet et al. 2009), which could facilitate diversity though resource partitioning in complex organic substrates. However, it is not known how the non-mycorrhizal members of the ericaceous RAF interact with the soil environment, although there is some evidence that one common group, the dark-septate endophytes, facilitates host uptake of both N and P from organic and inorganic sources (Newsham 2011).
Second, the relative availability of N and P could influence ericaceous RAF communities. Nutrient availability could limit fungi directly, or indirectly, as a result of host plant nutrient requirements. Currently, most data on ericaceous RAF communities originates from temperate and boreal regions, where cooler temperatures and repeated glaciation throughout the Pleistocene has maintained N limitation, even in older ecosystems (Tamm 1991; Vitousek & Howarth 1991). However, despite fact that ErMF are often associated with these N-limited habitats, where they are directly involved in host N uptake (Read & Perez-Moreno 2003), studies on effect of soil-N availability on the composition of ericaceous RAF communities have reported mixed results (Ishida & Nordin 2010; Hazard et al. 2014). There is also some experimental evidence that ErMF can facilitate P uptake for host plants (Pearson & Read 1973;
47 Myers & Leake 1996). Few studies have characterized ericaceous RAF communities from older tropical regions, or other P-limited systems, and the potential effects of soil P availability on ericaceous RAF communities are unknown. In the context of long- term ecosystem development, studies of arbuscular mycorrhizal fungi (AMF) have suggested direct N and P limitation in young and old ecosystems, respectively (Treseder & Allen 2002), and a peak in AMF richness at fertile, middle-aged sites (Krüger et al. 2015). However, AMF are primarily involved in mineral nutrient uptake (Smith & Read 2006) and appear to lack the extracellular enzymes needed access to the organic nutrient pools available to ErMF (Tisserant et al. 2013). This suggests that ErMF could respond differently to changing mineral nutrient availability with soil aging, but the relative importance of N and P limitation is currently unknown.
Because the direct observation of long-term ecosystem development in a single site is not possible, soil chronosequences, or gradients of soil age in which other putative controlling factors (i.e., parent material, climate, vegetation type, etc.) are held constant, offer a unique opportunity to compare ecosystems at various stages of development (Walker et al. 2010). To explore the effect of long-term ecosystem development on an ericaceous RAF community, we sampled roots of a single species, Vaccinium calycinum, across a 4.1 myr soil chronosequence in the Hawaiian Islands, known as the Long Substrate Age Gradient (LSAG; Vitousek 2004). The LSAG is a useful system to study ericaceous RAF for three reasons. First, a common ericaceous species, V. calycinum, is present at all LSAG sites. This controls for changes in host identity, which can influence the composition of RAF communities (Martínez-García et al. 2015), though intraspecific variation in host traits affecting plant-fungal interactions are also possible (Johnson et al. 2010). Second, vegetation at LSAG sites is dominated by a single canopy tree species and a common suite of understory plants (Kitayama & Mueller-Dombois 1995) which all form mycorrhizal associations with AMF (Koske et al. 1992). The only other ericaceous species present at any of the LSAG sites, Vaccinium dentatum and Leptecophylla tameiameiae, occur sparsely. This further limits the potential impact of changes in host vegetation along the
48 chronosequence. Third, changes in nutrient limitation across the LSAG have been experimentally demonstrated through N and P fertilizer addition experiments (Vitousek 2004). Because these fertilization experiments are ongoing at the oldest and youngest LSAG sites, this system presents a unique opportunity to experimentally test whether ecosystem-level nutrient limitation plays a role in structuring ericaceous RAF communities.
In this paper, we test the hypothesis that the diversity of ericaceous RAF increases throughout long-term ecosystem development, primarily as the result of increasing ErMF diversity in response to the accumulation of complex organic nutrient pools during pedogenesis. We predicted that diversity would increase most rapidly during the early stages of pedogenesis due to the formation and development of the organic soil horizon, but that an increase would continue into the retrogressive stages as soil weathering and occlusion of mineral nutrients promotes niche partitioning in the RAF community. We also use long-term fertilization experiments to test the hypothesis that ecosystem-level nutrient limitation of primary productivity, N at the youngest site and P and the oldest site, dictates the structure of ericaceous RAF communities.
Materials and Methods
Study system
Samples were collected from the LSAG chronosequence at five sites ranging in substrate age from 300 yr to 4.1 myr (Fig. 1). In this system, substrate age is an estimate of how long ago the parent substrate was deposited by volcanic activity (Vitousek 2004). The locations of the LSAG sites were chosen to minimize variation in state factors other than the age of the soil substrate; all sites share similar initial parent substrate, occur on the slope of a constructional shield volcano, are ca. 1200 m asl, receive ca. 2500 mm rain annually and have a mean annual temperature of 16 ºC (for detailed site descriptions see, Crews et al. 1995 & Vitousek 2004). Biotic variation is also highly constrained across the LSAG. Vegetation consists of native forest dominated by the canopy tree species, Meterosideros polymorpha, with an
49 understory that includes Cheirodendron trigynum, Cibotium spp. (tree ferns), Coprosma spp., Ilex anomala, Myrsine spp., and V. calycinum (Kitayama & Mueller- Dombois 1995).
Fertilization plots were established at the 300 yr site in 1985 (Vitousek et al. 1993) and at the 4.1 myr site in 1991 (Herbert & Fownes 1995). At each site, 16 plots (15 x 15 m) were randomly assigned to either an unfertilized control or treatments that received fertilizer in the form of N (half ammonium nitrate and half urea), P (triple superphosphate), or both N and P. Fertilizer was applied twice a year until 2002 and biannually thereafter at a consistent annual rate of 50 kg ha-1 yr-1.
Sample collection
Sampling occurred in August 2014, with the exception of the nutrient addition plots at the 300 yr site, which were sampled in August 2013 as part of a preliminary feasibility study. To account for possible interannual variation, an additional 12 plants from non- fertilized plots at the 300 yr site were collected in 2014. Initial analyses suggested that samples from unfertilized plots collected at the 300 yr site in different years had similar fungal communities (not shown). Nonetheless, comparisons across the chronosequence reported here utilize only the 2014 samples and analyses of samples from fertilizer plots involved comparisons within each site, using the 2013 samples for the 300 yr site and the 2014 samples for the 4.1 myr site.
At each LSAG site, 12 mature V. calycinum plants were randomly selected within a 200 x 200 m area. In the fertilizer plots, two plants were sampled in each 15 x 15 m plot. For each plant, a portion of the root system and the adhering soil (ca. 25 x 25 x 10 cm) was removed with a hand trowel and bagged for transport. Samples were refrigerated (4 °C) until they could be processed, always within 48 hrs of collection. Fine terminal roots were manually separated from the soil and rinsed in tap water to remove all visible soil particles and 12 segments, ca. 2 cm each, were haphazardly selected and pooled for each plant. Pooled root samples were surface sterilized by
50 sequential vortexing for 1 minute in sterile water, 70% EtOH, 50% household bleach, and by 3 rinses in sterile water. Surface sterilized roots were stored at -80 °C.
Fungal community metabarcoding
Frozen roots were homogenized by bead beating in CTAB lysis buffer and total DNA was extracted using the Nucleospin Plant II kit (Macherey-Nagel). In order to provide negative controls for Illumina sequencing, 4 sterile bead beating tubes were processed in parallel with the tubes containing root samples. Following DNA extraction, the first half of the internal transcribed spacer region (ITS1) of fungal nrDNA was amplified using Illumina fusion PCR primers. Primers included the gene primers ITS1F or ITS2, modified to include Illumina adapters and a sample specific 12 bp, error-correcting Golay barcode (Smith & Peay 2014). PCR was carried out in 25 ul reactions using 1 ul of template DNA (diluted 1:20), 0.5 ul of each 10um primer, 12.5 ul OneTaq Hot-Start 2X Master Mix (New England Biolabs) and a cycling program consisting of: initial denaturing at 94 °C (1 min), 30 cycles of 95 °C (30 sec), 52 °C (30 sec) and 68 °C (30 sec) and a final elongation stage of 68 °C (5 min). PCR reactions were carried out in triplicate for each sample and were individually checked for successful amplification using gel electrophoresis. The individual PCR reactions for each sample were cleaned and normalized to 2.5 ng ul-1 using a Just-a-Plate, 96 well normalization and purification plate (Charm Biotech). Samples were then pooled for 300 bp paired-end sequencing on an Illumina MiSeq at the Stanford Functional Genomics Facility. Raw sequence data were deposited in the NCBI short read archive under accession numbers [accession numbers to be determined].
Bioinformatics
Illumina sequencing data were processed using USEARCH v9.2.64, following the UPARSE pipeline (Edgar 2013). Briefly, paired-end sequences were merged after trimming low-quality trailing bases. Any pairs that could not be successfully merged or were < 100 bp after merging were discarded. Merged pairs were then filtered using a maximum expected error of 0.5 bp, dereplicated and clustered into operational
51 taxonomic units (OTUs) at 97% similarity, discarding potential chimeric sequences. The merged, unfiltered reads were then mapped onto OTUs, with an identity threshold of 97%, resulting in a species by sample matrix of read counts (OTU table). The OTU table was filtered of likely contaminants by removing any OTU with an average abundance across all samples that was less than the maximum abundance in any of the negative controls. In addition, observations of OTUs that accounted for < 0.1% of the reads for a given sample were excluded to minimize the influence of low abundance contaminants or sequencing / PCR error and sample “cross-talk,” in which high abundance OTUs appear in low abundance in samples where they are not actually present (Nguyen et al. 2015; Edgar 2016b).
Taxonomic predictions for OTUs were made using the SINTAX algorithm (Edgar 2016a) and the UNITE v7.1 database (Kõljalg et al. 2013) as a reference, disregarding any predictions with less than 50% bootstrap support. As a secondary approach, OTUs were matched against the UNITE v7.1 dynamic species hypothesis database using USEARCH, with modified search heuristics to ensure that the database sequence with the best semi-global alignment with each OTU was found. Identification of potential ErMF in the OTU database was complicated by two factors. First, the short read length and high variability of the ITS region, combined with limited representation of both Hawaiian root / soil associated fungi and fungi associated with ericaceous plants in current reference databases resulted in low confidence for fine scale taxonomic assignments. Second, there is significant uncertainty about the taxonomic range of fungal species capable of forming ErM. We chose an inclusive approach, defining the subset of putative ErMF to include all OTUs assigned to fungal orders reported to include ErM forming fungi (Leopold 2016). This approach is likely to include many non-mycorrhizal endophytes, particularly in the order Helotiales, however some of these species likely have overlapping functional attributes with ErMF (Newsham 2011) and it is not currently possible to distinguish ErM taxa with sequence data alone. An alternative approach, manually curating putative ErMF OTUs by looking at the sources of the individual sequences comprising the closest matching UNITE
52 species hypotheses yielded qualitatively similar results. We focus here on the approach using fungal orders as it is more easily replicated.
Statistical analysis of chronosequence data
To assess if fungal diversity changed across the LSAG, we estimated alpha diversity at the sample level for both the complete data set and the subset of putative ErMF. To account for unequal sequencing depth among samples we estimated alpha diversity using Hill numbers, or the “effective number of species,” at a sampling depth of 5000 sequences per sample using the R-package iNEXT (Chao et al. 2014; Hsieh et al. 2016). We calculated Hill numbers for each sample using a scaling of 0 (equivalent to species richness) and a scaling of 1 (equivalent to the exponent of Shannon entropy, or Shannon diversity) and analyzed the results in parallel. Relationships between alpha diversity and log10 transformed site age were tested using mixed-models in the R- package lme4 (Bates et al. 2015) with site included as a random effect to account for non-independence among samples. The significance of linear and polynomial relationships was tested using likelihood-ratio tests with an intercept-only null model (including the site random effect) and p-values were generated using parametric bootstrapping in the R-package pbkrtest (Halekoh & Højsgaard 2014). Likelihood- ratio based pseudo-r2 values were also calculated for each final model as a measure of variance explained by the fixed-effects using the R-package MuMIn (Bartoń 2016).
To determine how the composition of fungal communities changed across the LSAG, we first calculated Bray-Curtis dissimilarity among samples using the proportional abundance of OTUs in each sample to account for unequal sampling depth (McMurdie & Holmes 2014). We initially tested alternative dissimilarity metrics, including the binary Jaccard distance, the probabilistic Raup-Crick distance, and the information- theoretic based Jensen-Shannon Divergence, and found that all produced qualitatively similar results to those obtained with Bray-Curtis dissimilarity, which we will focus on here. Dissimilarities were visualized using non-metric multidimensional scaling (NMDS) implemented in the R-package vegan (Oksanen et al. 2017). Contours
53 representing increasing site age were fit to the 2-dimensional NMDS ordination, using the vegan function ordisurf, to aid interpretation of species turnover across the chronosequence. The significance of changes in community composition were tested with a permutational multivariate analysis of variance (perMANOVA). Both log10- transformed site age and site identity as a factor were used as predictors in the perMANOVA to account for variation due to turnover along the gradient in addition to variation among sites. OTUs occurring in individual LSAG sites with greater abundance and more often than expected by chance alone were identified with indicator species analysis (Dufrêne & Legendre 1997) using the R-package labdsv (Roberts 2016), and visualized on a bipartite graph of the 50 most abundant OTUs constructed with the R-package bipartite (Dormann et al. 2008).
Statistical analysis of fertilizer plot data
To test the effects of fertilization on fungal composition and diversity, we analyzed both the complete data set and the subset of putative ErMF OTUs from the fertilizer addition plots at the 300 yr and 4.1 myr LSAG sites. Preliminary analyses indicated that pairs of plants from the same plot were not more similar than pairs from different plots with the same fertilization treatment at the same sites, so analyses were conducted at the plant level (n = 8 for each treatment). We used perMANOVAs of Bray-Curtis dissimilarity calculated on proportional OTU abundance to test for an effect of fertilization at each site. Separate pairwise tests were conducted on subsets of the data contrasting each fertilization treatment to the unfertilized control plots. Results of these separate analyses were corrected for multiple testing using the procedure of Holm (1979). To test whether fertilization affected fungal richness or diversity we fit linear models for each site with fertilizer treatment as a predictor and Hill numbers, either as OTU richness (scaling 0) or Shannon diversity (scaling 1), as the response. When a significant effect of fertilization was detected, we used independent contrasts between each fertilizer treatment and the non-fertilized control samples to determine the significance effect of each treatment.
54 All statistical analyses were conducted in the R statistical computing environment (R Core Development Team 2016), using the R-package, phyloseq (McMurdie & Holmes 2013) to manipulate OTU tables and associated metadata.
Results
Identification of fungal OTUs
We retained 2.2 million high-quality sequences after bioinformatic processing, from which we identified 464 fungal OTUs. The subset of putative ErMF included 38% of the total OTUs, and accounted for 81.6% of all sequences. Taxonomic predictions and direct searches of the UNITE database indicated that the fungal order Helotiales accounted for the largest fraction of the data set (45% of all sequences and 22% of unique OTUs). The lack of high similarity matches for many OTUs suggest the presence of many novel taxa (Fig. 2). For example, the most abundant OTU (OTU.1) shares only 81.6% sequence similarity with the nearest UNITE representative sequence and could not be confidently assigned beyond the order Helotiales. The second most abundant OTU (OTU.2), Trechisporales sp., shares 99% sequence similarity with a UNITE species hypothesis that contains only a few of sequences isolated from non-ericaceous roots (Orchidaceae and Clustaceae), originating from Eastern Africa and Taiwan. However, the next closest species hypothesis shares only 75% sequence similarity. Notably, the widespread ErMF species, Rhizoscyphus ericae, was not identified in our sequence data, though we did identify OTUs with affinity to the broader R. ericae aggregate. Other abundant OTUs had affinities with taxa previously reported from ErM and ecto-mycorrhizal roots, including Capnodiales spp., Chaetothyriales spp., Cryptosporiopsis ericae, Dothidiomycete spp., Oidiodendron maius and Rhizoderma veluwensis.
Site age effects on OTU composition and diversity
Patterns of richness and diversity across the chronosequence were different when evaluated for all OTUs and for the subset of putative ErMF OTUs (Fig. 3). For all
55 OTUs, richness displayed a concave relationship with site age, increasing in young and old sites relative to middle age sites, while Shannon diversity increased only at the
2 2 oldest site, though both trends were only marginally significant (χ (2) = 11.2, r = 0.17,
2 2 p = 0.051 & χ (2) = 10.9, r = 0.17, p = 0.068, respectively). For the putative ErMF
2 OTUs, richness and diversity increased linearly with log-transformed site age (χ (1) =
2 2 2 9.71, r = 0.15, p = 0.012 & χ (1) = 12.4, r = 0.19, p = 0.006, respectively).
The composition of fungal OTUs associated with V. calycinum differed among LSAG sites for both the complete data set (F = 5.27, r2 = 0.28, p < 0.001) and the subset of putative ErMF (F = 6.57, r2 = 0.32, p < 0.001). NMDS ordinations (Fig. 4 & S1) revealed a pattern of species turnover across the chronosequence, with similarly aged sites tending to have more similar OTU compositions, though site age explained less than a third of the overall variation in OTU composition in the perMANOVA analyses (r2 = 0.08, for all OTUs and putative ErMF). However, the distributions of sites in the NMDS ordinations (Figs. 4 & S1) suggest that the OTU composition of the 2100 yr old site may be an outlier relative to the pattern of species turnover among the other sites. When this site was excluded from the perMANOVA analyses, site age explained the majority of the variation in community composition among sites, both for all OTUs (r2 = 0.15 vs. 0.09, for site age and residual variation among sites, respectively) and for the putative ErMF OTUs (r2 = 0.18 vs. 0.10). Indicator species analysis identified many of the most abundant OTUs as being significantly associated with individual LSAG sites (Fig. 1).
Fertilization effects on fungal OTU composition and diversity
At the 300 yr site, fertilization affected the overall composition of fungal OTUs (F = 1.62, r2 = 0.15, p = 0.03) but not the overall composition of putative ErMF OTUs (F = 1.17, r2 = 0.11, p = 0.25). However, individual fertilizer treatments at the 300 yr site were not significantly different from control plots for either data set (Table 1). At the 4.1 myr site, the overall compositions of all fungal OTUs and putative ErMF OTUs were affected by fertilization (F = 1.47, r2 = 0.14, p = 0.003 & F = 2.0, r2 = 0.18, p <
56 0.001, respectively). In this old site, fertilization with N and P together resulted in the greatest changes in OTU composition, relative to the unfertilized control, for both all OTUs and putative ErMF OTUs. Fertilization with P alone affected all OTUs but not the subset of putative ErMF OTUs and there was no effect of fertilization with N alone (Table 1).
The effect of fertilization on fungal OTU richness and diversity also depended on which site was considered (Fig. 5). At the 300 yr site, the richness and diversity of fungal OTUs was lowest in samples fertilized with N (or N and P), though the overall effect of fertilization was only marginally significant for richness (F3,28 = 2.32, p =
0.09) and not statistically significant for Shannon diversity (F3,28 = 1.20, p = 0.33). At the 4.1 myr site, fertilization with P (or N and P) reduced both richness (F3,28 = 3.33, p
= 0.03) and Shannon diversity (F3,28 = 4.57, p = 0.009). The diversity of putative ErMF OTUs was not affected by fertilization at either site.
Discussion
Consistent with our initial hypothesis, we found that plants associate with an increasing number of putative ErMF OTUs as site age increases. This pattern was not simply the result of increasing richness, but also greater evenness, which could reflect increased niche partitioning in response to soil development. The linear relationship between ErMF diversity and log-transformed site age suggests a more rapid increase during the early stages of ecosystem development. This is consistent with a response to the rapid accumulation of soil organic matter and development of a structured organic soil horizon during the earlier phases of soil development (Goh et al. 1976; Torn et al. 1997). However, we cannot rule out dispersal limitation (Cline & Zak 2014) or in-situ diversification (Gillespie 2016) as additional factors responsible for increasing diversity across the chronosequence.
Differences in the richness and diversity patterns for putative ErMF OTUs and all OTUs across the chronosequence suggest that different components of the RAF community may be structured by different factors (Sun et al. 2012). For example, as a
57 result of the curvilinear age-richness relationship for all OTUs, the difference in the number of putative ErMF OTUs and all OTUs increased towards both ends of the chronosequence (Fig. 3a). Plant growth is limited by N in the 300 yr site and P in the 4.1 myr site (Vitousek 2004), and P limits rates of litter decomposition and soil organic matter processing in the 4.1 myr site (Hobbie & Vitousek 2000; Reed et al. 2011). Moreover, there is some evidence that mycorrhizal fungi associating with non- ericaceous plants in this system are N-limited at the 300 yr and P-limited at the 4.1 myr sites, but not at the 20 kyr site (Treseder & Allen 2002). If major nutrient limitation of ErMF follows a similar pattern, it is possible that ErMF are better able to competitively exclude opportunistic colonization by non-mycorrhizal species at the nutrient-rich sites. This hypothesis is consistent with our observation that fertilization by N and P at the young and old sites, respectively, resulted in lower richness for all OTUs, but not the putative ErMF OTUs (Fig. 5a,b). This pattern could have emerged as a sampling artifact if the absolute abundance of ErMF, and not non-mycorrhizal RAF, increased in roots when limiting nutrients were added (Amend et al. 2010). However, the proportion of putative ErMF reads in each sample remained consistent across samples despite changes in diversity, which suggests that difference in diversity patterns between putative ErMF and other taxa were not the result of an increase in the relative abundance of ErMF.
Our results revealed a clear pattern of fungal species turnover across the chronosequence, both for all OTUs (Fig. 4) and for the subset of putative ErMF OTUs (Fig. S1). It is not clear why the 2100 yr site was an outlier in respect to this pattern, but one possibility is that stochastic dispersal processes play a role. Although dispersal mechanisms are largely unknown for most ericaceous RAF fungi, there is some evidence that ErMF may be dispersal limited (Hutton et al. 1997; but see, Bergero et al. 2003), which would increase the probability that local species composition is determined by species arrival order (i.e., priority effects; Fukami 2015). The distinction between the species composition of the two youngest sites is particularly striking considering that these sites are physically closer than any other sites sampled
58 (ca. 4.5 km vs. ca. 43 km between the 20 kyr and 150 kyr sites, the next closest). These sites were also both covered by a layer of volcanic tephra during the 1790 Kilauea eruption, though the age classifications of both sites take into account the older substrates beneath this relatively recent deposition. Records show that vegetation at the 300 yr site was completely killed as a result of the 1790 eruption and it is believed that only the largest trees survived at the 2100 yr site (Vitousek 2004). This disturbance likely “reset” plant and fungal community assembly, though some ericaceous RAF may have persisted at the 2100 yr site. As a result of this disturbance, large differences in community composition after the 1790 eruption may be explained by priority effects rather than edaphic conditions. However, alternative explanations, are also possible. For example, V. calycinum density was very low in the areas within and directly adjacent the 2100 yr site (personal observation). This could limit the availability of fungal inoculum, particularly for poorly dispersing ErMF species, and affect the composition of local RAF communities (Genney et al. 2001; Rudgers et al. 2009).
The mechanisms driving patterns of diversity and species composition across the chronosequence remain uncertain. However, results from the experimental fertilization plots suggest that nutrient limitations related to substrate age influence these communities (Fig. 5). Specifically, evidence for ecosystem-level P limitation at the 4.1 myr site (Herbert & Fownes 1995) is consistent with the fact that fertilization with P or both P and N, but not N alone, affected both the structure (Fig. 5b, d) and composition (Table 1) of V. calycinum RAF communities. These results are in contrast with the common characterization of ErM symbioses as being primarily N driven and highlight the relative lack of data on ericaceous RAF communities from P-limited tropical systems. The effect of N limitation on V. calycinum RAF at the 300 yr site was less clear than the effect of P limitation at the 4.1 myr site. Previous work in this system has shown that fertilization with P at the 4.1 myr site significantly affects litter chemistry, whereas fertilization with N at the 300 yr site does not (Harrington et al. 2001). Because ericaceous root systems occur predominantly in the upper, organic soil
59 layers, where ErMF directly access recalcitrant litter-nutrient inputs (Read et al. 2004), changes in litter chemistry may explain the stronger fertilization effects we observed at the 4.1 myr site. However, it is also possible that communities at the 300 yr old site are more resistant to change in response to fine scale modification of abiotic conditions because the local propagule pool is dominated by fewer species (Fig. 2), or that additions of N (unlike those of P) do not create persistent changes in N supply between fertilizations.
We do not have the histological data to determine the morphology of interactions between V. calycinum and the OTUs we identified. However, our results suggest that many novel ErMF species occur here. For example, the two most abundant OTUs in our study, OTU.1 and OTU.2, which accounted for 32% of all reads across the chronosequence and 64% of the reads at the youngest site (Fig. 2), were not closely related to any well-described species or known ErMF. The taxonomic predictions for these OTUs, Helotiales (OTU.1) and Trechisporales (OTU.2), are both fungal orders that may include undescribed ErMF taxa (Leopold 2016). The prevalence of these OTUs suggests that they may be abundant in V. calycinum roots at some sites, as would be expected for ErMF, although biases introduced by our Illumina metabarcoding approach may have affected their abundance in the sequencing data (Nguyen et al. 2015). It is also possible that these species are non-mycorrhizal endophytes, though this does not preclude a role in mediating plant-soil interactions.
Soil chronosequences and their inherent edaphic gradients represent an important, but underutilized, tool for understanding of the factors affecting the composition and structure of RAF communities (Dickie et al. 2013). In this study we have shown that diversity in an ericaceous RAF community increases throughout long-term ecosystem development largely due to increasing richness and evenness of putative ErMF taxa. Functional differences among types of mycorrhizal symbioses and recent reports of a hump-shaped diversity pattern for arbuscular mycorrhizal fungi (Krüger et al. 2015), suggest that mycorrhizal type may determine RAF community response to long-term ecosystem development. However, less is known about potential differences in the
60 function of different mycorrhizal types throughout long-term ecosystem development. Future efforts to link changes in RAF communities with functional attributes of plant- fungal symbioses may be critical for interpreting the relationship between plant communities and soil nutrients across chronosequences.
Acknowledgments
We thank Michael Shintaku and Anne Veillet for access to laboratory space and equipment at the University of Hawaii, Hilo, College of Agriculture, Forestry and Natural Resource Management, and in the Evolutionary Genomics Core Facility. We also thank Matt Knope for feedback on earlier drafts of this manuscript. Site access was granted by the Hawaii Division of Land and Natural Resources, the Hawaii Experimental Tropical Forest, and Parker Ranch. This research was supported by student research grants from the American Society of Naturalists and the Mycological Society of America.
61 References cited
Adamczyk, B., Ahvenainen, A., Sietiö, O.-M., Kanerva, S., Kieloaho, A.-J., Smolander, A., et al. (2016). The contribution of ericoid plants to soil nitrogen chemistry and organic matter decomposition in boreal forest soil. Soil Biol. Biochem., 103, 394–404. Amend, A.S., Seifert, K.A. & Bruns, T.D. (2010). Quantifying microbial communities with 454 pyrosequencing: does read abundance count? Mol. Ecol., 19, 5555– 5565. Bartoń, K. (2016). MuMIn: Multi-Model Inference. R package version 1.15.6. https://CRAN.R-project.org/package=MuMIn. Bates, D., Mächler, M., Bolker, B. & Walker, S. (2015). Fitting linear mixed-effects models using lme4. Journal of Statistical Software, 67, 1–48. Bergero, R., Girlanda, M., Bello, F., Luppi, A. & Perotto, S. (2003). Soil persistence and biodiversity of ericoid mycorrhizal fungi in the absence of the host plant in a Mediterranean ecosystem. Mycorrhiza, 13, 69–75. Cairney, J.W.G. & Meharg, A.A. (2003). Ericoid mycorrhiza: a partnership that exploits harsh edaphic conditions. Eur. J. Soil Sci., 54, 735–740. Cairney, J.W.G., Sawyer, N.A., Sharples, J.M. & Meharg, A.A. (2000). Intraspecific variation in nitrogen source utilisation by isolates of the ericoid mycorrhizal fungus Hymenoscyphus Ericae (Read) Korf and Kernan. Soil Biol. Biochem., 32, 1319–1322. Chadwick, O.A., Derry, L.A., Vitousek, P.M., Huebert, B.J. & Hedin, L.O. (1999). Changing sources of nutrients during four million years of ecosystem development. Nature, 397, 491–497. Chao, A., Gotelli, N.J., Hsieh, T.C., Sander, E.L., Ma, K.H., Colwell, R.K., et al. (2014). Rarefaction and extrapolation with Hill numbers: a framework for sampling and estimation in species diversity studies. Ecol. Monogr., 84, 45–67.
62 Cline, L.C. & Zak, D.R. (2014). Dispersal limitation structures fungal community assembly in a long-term glacial chronosequence. Environ. Microbiol., 16, 1538–1548. Crews, T.E., Kitayama, K., Fownes, J.H., Riley, R.H., Darrell, A., Mueller-dombois, D., et al. (1995). Changes in soil phosphorus fractions and ecosystem dynamics across a long chronosequence in Hawaii. Ecology, 76, 1407–1424. Dickie, I.A., Martínez-García, L.B., Koele, N., Grelet, G.-A., Tylianakis, J.M., Peltzer, D.A., et al. (2013). Mycorrhizas and mycorrhizal fungal communities throughout ecosystem development. Plant Soil, 367, 11–39. Dormann, C.F., Gruber, B. & Fründ, J. (2008). Introducing the bipartite package: analysing ecological networks. R News, 8/2, 8–11. Dufrêne, M. & Legendre, P. (1997). Species assemblages and indicator species: the need for a flexible asymmetrical approach. Ecol. Monogr., 67, 345–366. Edgar, R. (2016a). SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences. bioRxiv, dio: 10.1101/074161. Edgar, R.C. (2013). UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat. Methods, 10, 996–899. Edgar, R.C. (2016b). UNCROSS: Filtering of high-frequency cross-talk in 16S amplicon reads. bioRxiv, doi: 10.1101/088666. Fukami, T. (2015). Historical contingency in community assembly: integrating niches, species pools, and priority effects. Annu. Rev. Ecol. Evol. Syst., 46, 1–23. Genney, D.R., Hartley, S.H. & Alexander, I.J. (2001). Arbuscular mycorrhizal colonization increases with host density in a heathland community. New Phytol., 152, 355–363. Gillespie, R.G. (2016). Island time and the interplay between ecology and evolution in species diversification. Evol. Appl., 9, 53–73. Goh, K.M., Rafter, T.A., Stout, J.D. & Walker, T.W. (1976). The accumulation of soil organic matter and its carbon isotope content in a chronosequence of soils developed on aeolian sand in New Zealand. J. Soil Sci., 27, 89–100.
63 Grelet, G.A., Meharg, A.A., Duff, E.I., Anderson, I.C. & Alexander, I.J. (2009). Small genetic differences between ericoid mycorrhizal fungi affect nitrogen uptake by Vaccinium. New Phytol., 181, 708–718. Halekoh, U. & Højsgaard, S. (2014). A Kenward-Roger approximation and parametric bootstrap methods for tests in linear mixed models–the R package pbkrtest. J. Stat. Softw., 59, 1–32. Harrington, R.A., Fownes, J.H. & Vitousek, P.M. (2001). Production and resource use efficiencies in N- and P-limited tropical forests: a comparison of responses to long-term fertilization. Ecosystems, 4, 646–657. Hazard, C., Gosling, P., Mitchell, D.T., Doohan, F.M. & Bending, G.D. (2014). Diversity of fungi associated with hair roots of ericaceous plants is affected by land use. FEMS Microbiol. Ecol., 87, 586–600. van der Heijden, M.G.A., Bardgett, R.D. & Van Straalen, N.M. (2008). The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol. Lett., 11, 296–310. Herbert, D. a. & Fownes, J.H. (1995). Phosphorus limitation of forest leaf area and net primary production on a highly weathered soil. Biogeochemistry, 29, 223–235. Hobbie, S.E. & Vitousek, P.M. (2000). Nutrient limitation of decomposition in Hawaiian forests. Ecology, 81, 1867–1877. Högberg, P. & Read, D.J. (2006). Towards a more plant physiological perspective on soil ecology. Trends Ecol. Evol., 21, 548–554. Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scand. J. Stat., 6, 65–70. Hsieh, T.C., Ma, K.H. & Chao, A. (2016). iNEXT: iNterpolation and EXTrapolation for Species Diversity. R package version 2.0.12. http://chao.stat.nthu.edu.tw/blog/software-download/ Hutton, B.J., Dixon, K.W., Sivasithamparam, K. & Pate, J.S. (1997). Effect of habitat disturbance on inoculum potential of ericoid endophytes of Western Australian heaths (Epacridaceae). New Phytol., 135, 739–744.
64 Ishida, T.A. & Nordin, A. (2010). No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types. Soil Biol. Biochem., 42, 234–243. Johnson, N.C., Wilson, G.W.T., Bowker, M.A., Wilson, J.A. & Miller, R.M. (2010). Resource limitation is a driver of local adaptation in mycorrhizal symbioses. Proc. Natl. Acad. Sci., 107, 2093–2098. Kitayama, K. & Mueller-Dombois, D. (1995). Vegetation changes along gradients of long-term soil development in the Hawaiian montane rainforest zone. Vegetatio, 120, 1–20. Kõljalg, U., Nilsson, R.H., Abarenkov, K., Tedersoo, L., Taylor, A.F.S., Bahram, M., et al. (2013). Towards a unified paradigm for sequence-based identification of fungi. Mol. Ecol., 22, 5271–5277. Koske, R.E., Gemma, J.N. & Flynn, T. (1992). Mycorrhizae in Hawaiian angiosperms: a survey with implications for the origin of the native flora. Am. J. Bot., 79, 853–862. Krüger, M., Teste, F.P., Laliberté, E., Lambers, H., Coghlan, M., Zemunik, G., et al. (2015). The rise and fall of arbuscular mycorrhizal fungal diversity during ecosystem retrogression. Mol. Ecol., 24, 4912–4930. Lambers, H., Raven, J.A., Shaver, G.R. & Smith, S.E. (2008). Plant nutrient- acquisition strategies change with soil age. Trends Ecol. Evol., 23, 95–103. Leopold, D.R. (2016). Ericoid fungal diversity: challenges and opportunities for mycorrhizal research. Fungal Ecol., 24, 114–123. Martínez-García, L.B., Richardson, S.J., Tylianakis, J.M., Peltzer, D.A. & Dickie, I.A. (2015). Host identity is a dominant driver of mycorrhizal fungal community composition during ecosystem development. New Phytol., 205, 1565–1576. McMurdie, P.J. & Holmes, S. (2013). phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One, 8, e61217. McMurdie, P.J. & Holmes, S. (2014). Waste not, want not: why rarefying microbiome data is inadmissible. PLoS Comput. Biol., 10, e1003531.
65 Myers, M.D. & Leake, J.R. (1996). Phosphodiesters as mycorrhizal P sources. II. Ericoid mycorrhiza and the utilization of nuclei as a phosphorus and nitrogen source by Vaccinium macrocarpon. New Phytol., 132, 445–451. Nasholm, T., Ekblad, A., Nordin, A., Giesler, R., Högberg, M. & Högberg, P. (1998). Boreal forest plants take up organic nitrogen. Nature, 392, 914–916. Newsham, K.K. (2011). A meta-analysis of plant responses to dark septate root endophytes. New Phytol., 190, 783–793. Nguyen, N.H., Smith, D., Peay, K. & Kennedy, P. (2015). Parsing ecological signal from noise in next generation amplicon sequencing. New Phytol., 205, 1389– 1393. Oksanen, J., Blanchet, F.G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., et al. (2017). vegan: Community Ecology Package. R package version 2.4-2. https://CRAN.R-project.org/package=vegan. Pearson, V. & Read, D.J. (1973). Biology of mycorrhiza in the Ericaceae. II. The transport of carbon and phosphorus by the endophyte and the mycorrhiza. New Phytol., 72, 1325–1331. Peltzer, D.A., Wardle, D.A., Allison, V.J., Baisden, W.T., Bardgett, R.D., Chadwick, O.A., et al. (2010). Understanding ecosystem retrogression. Ecol. Monogr., 80, 509–529. Perotto, S., Martino, E., Abba, S. & Vallino, M. (2012). Genetic diversity and functional aspects of ericoid mycorrhizal fungi. In: Fungal Associations 2nd (ed. Hock, B.). Springer Berlin Heidelberg, pp. 255–285. R Development Core Team. (2016). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Read, D.J., Leake, J.R. & Perez-Moreno, J. (2004). Mycorrhizal fungi as drivers of ecosystem processes in heathland and boreal forest biomes. Can. J. Bot., 82, 1243–1263. Read, D.J. & Perez-Moreno, J. (2003). Mycorrhizas and nutrient cycling in ecosystems - A journey towards relevance? New Phytol., 157, 475–492.
66 Reed, S.C., Vitousek, P.M. & Cleveland, C.C. (2011). Are patterns in nutrient limitation belowground consistent with those aboveground: results from a 4 million year chronosequence. Biogeochemistry, 106, 323–336. Richardson, S.J., Peltzer, D.A., Allen, R.B., McGlone, M.S. & Parfitt, R.L. (2004). Rapid development of phosphorus limitation in temperate rainforest along the Franz Josef soil chronosequence. Oecologia, 139, 267–276. Roberts, D.W. (2016). labdsv: Ordination and Multivariate Analysis for Ecology. R package version 1.8-0. https://CRAN.R-project.org/package=labdsv. Rudgers, J.A., Afkhami, M.E., Rúa, M.A., Davitt, A.J., Hammer, S. & Huguet, V.M. (2009). A fungus among us: broad patterns of endophyte distribution in the grasses. Ecology, 90, 1531–1539. Smith, D.P. & Peay, K.G. (2014). Sequence depth, not PCR replication, improves ecological inference from next generation DNA sequencing. PLoS One, 9, e90234. Smith, S. & Read, D. (2006). Mycorrhizal Symbiosis. 3rd edn. Academic Press. Sun, L., Pei, K., Wang, F., Ding, Q., Bing, Y., Gao, B., et al. (2012). Different distribution patterns between putative ericoid mycorrhizal and other fungal assemblages in roots of Rhododendron decorum in the southwest of China. PLoS One, 7, e49867. Tamm, C.O. (1991). Nitrogen in terrestrial ecosystems, questions of productivity, vegetational changes, and ecosystem stability. Ecological Studies, 81, 1-115. Tisserant, E., Malbreil, M., Kuo, A., Kohler, A., Symeonidi, A., Balestrini, R., et al. (2013). Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis. Proc. Natl. Acad. Sci. U. S. A., 110, 20117–20122. Torn, M.S., Trumbore, S.E., Chadwick, O.A., Vitousek, P.M. & Hendricks, D.M. (1997). Mineral control of soil organic carbon storage and turnover. Nature, 389, 170–173. Treseder, K. & Allen, M. (2002). Direct nitrogen and phosphorus limitation of arbuscular mycorrhizal fungi: a model and field test. New Phytol., 155, 507– 515.
67 Vitousek, P. (2004). Nutrient cycling and limitations: Hawaii as a model system. Princeton University Press. Vitousek, P.M. & Farrington, H. (1997). Nutrient limitation and soil development: experimental test of a biogeochemical theory. Biogeochemistry, 37, 63–75. Vitousek, P.M. & Howarth, R.W. (1991). Nitrogen limitation on land and in the sea: how can it occur? Biogeochemistry, 13, 87–115. Vitousek, P.M., Walker, L.R., Whiteaker, L.D. & Matson, P. a. (1993). Nutrient limitations to plant growth during primary succession in Hawaii Volcanoes National Park. Biogeochemistry, 23, 197–215. Walker, L.R., Wardle, D.A., Bardgett, R.D. & Clarkson, B.D. (2010). The use of chronosequences in studies of ecological succession and soil development. J. Ecol., 98, 725–736. Walker, T.W. & Syers, J.K. (1976). The fate of phosphorus during pedogenesis. Geoderma, 15, 1–19. Wardle, D.A. (2004). Ecosystem properties and forest decline in contrasting long-term chronosequences. Science (80-. )., 305, 509–513. Wurzburger, N., Higgins, B.P. & Hendrick, R.L. (2012). Ericoid mycorrhizal root fungi and their multicopper oxidases from a temperate forest shrub. Ecol. Evol., 2, 65–79. Zemunik, G., Turner, B.L., Lambers, H. & Laliberté, E. (2015). Diversity of plant nutrient-acquisition strategies increases during long-term ecosystem development. Nat. Plants, 1, 1–4.
68 Tables
Table 1: Results of perMANOVA analyses examining the effect of fertilizer treatments on the composition of (a) all fungal OTUs or (b) putative ErMF OTUs associated with V. calycinum at the 300 yr and 4.1 myr sites in the Long Substrate Age Gradient chronosequence. For each site and data set results are shown for the overall effect of fertilization treatments and for contrasts between the unfertilized controls (C) and each individual fertilizer treatment, nitrogen (N), phosphorus (P), or both (NP). For the independent contrasts, p-values were adjusted for multiple testing and significance is indicated as + p < 0.1 , * p < 0.05, ** p < 0.01, *** p < 0.001. a) All OTUs
Site Levels F r 2 p (adjusted) Young (300 yr) All 1.615 0.148 0.03* C vs. N 1.238 0.081 0.24 (0.47) C vs. P 1.587 0.102 0.087 (0.26) C vs. NP 0.632 0.043 0.90 (0.90) Old (4.1 myr) All 1.465 0.136 0.003* C vs. N 1.422 0.092 0.061 (0.06)+ C vs. P 1.464 0.095 0.02 (0.04)* C vs. NP 2.088 0.130 < 0.001 (< 0.001)*** b) Putative ErMF
Site Levels F r 2 p (adjusted) Young (300 yr) All 1.172 0.112 0.25 C vs. N 0.965 0.064 0.39 (1) C vs. P 0.934 0.063 0.41 (1) C vs. NP 0.499 0.034 0.92 (1) Old (4.1 myr) All 2.001 0.177 < 0.013*** C vs. N 1.578 0.101 0.069 (0.14) C vs. P 1.403 0.091 0.15 (0.15) C vs. NP 3.351 0.193 < 0.001 (< 0.001)***
69 Figures
Figure 1: Map of the main Hawaiian Islands showing the locations and ages of the 5 sites in the Long Substrate Age Gradient chronosequence used in the current study.
70 Figure 2: Bipartite graph showing the relative abundances of the 50 most common OTUs. The size of nodes representing OTUs (right) is proportional to their mean abundance and the width of connections between OTUs and sites (left) is proportional to their relative abundance at each site. Colored OTU nodes and connections identify significant site associations (indicator species analysis). Taxonomic predictions (bootstrap support) for each OTU were obtained with SINTAX (Edgar 2016a), using the UNITE database (Kõljalg et al. 2013) as a reference, and are shown at the order level, or higher if bootstrap support for order was less than 50%. The taxonomic assignment of the closest matching representative sequence in the UNITE species hypothesis database is shown for each OTU.
71 Figure 2 (continued)
72 Figure 3: Variation in the richness (a) and diversity (b) of fungal OTUs associated with V. calycinum roots across the Long Substrate Age Gradient. Filled points represent all OTUs and open points represent the subset of putative ericoid mycorrhizal fungi (ErMF), with larger bold points indicating site means for each data set. Lines indicate the best-fit model of the relationship between OTU richness or Shannon diversity for all OTUs (solid; p < 0.1) or putative ErMF (dashed; p < 0.05). The position of points on the x-axes have been adjusted to ovoid overlap of the two data sets.
73 Figure 4: Non-metric multidimensional scaling (stress = 0.24) using Bray-Curtis dissimilarity showing fungal communities associated with V. calycinum roots collected across a 4.1 myr chronosequence. Points represent individual plants, with color indicating the age of the site where the sample was collected. Contours indicate a smooth surface of log-transformed site age fit to the ordination space, where increasing site age is indicated by darker contours.
74 Figure 5: The effect of fertilization with nitrogen (N), phosphorus (P), or both (NP) on the number of fungal OTUs associated with the roots of V. calycinum at the 300 yr (a, c) and the 4.1 myr (b, d) sites in the Long Substrate Age Gradient chronosequence. Panels show the mean (± se) OTU richness (a, b), or Shannon diversity (c, d). Shaded bars show results for all OTUs and open bars show results for the subset of putative ericoid mycorrhizal fungi (ErMF). Significant changes in the effective number of OTUs in fertilized plots, relative to the corresponding unfertilized control plots (C), are indicated, where + P < 0.1 , * P < 0.05, ** P < 0.01.
75 Supplemental materials
Figure S1: Non-metric multidimensional scaling (stress = 0.25) using Bray-Curtis dissimilarity showing variation in the composition of putative ericoid mycorrhizal fungi communities associated with V. calycinum roots collected across a 4.1 myr chronosequence. Points represent individual plants, with color indicating the age of the site where the sample was collected. Contours indicate a smooth surface of log- transformed site age fit to the ordination space, where increasing site age is indicated by darker contours.
76 Chapter 4 Local richness increases more with regional richness under older species pools: evidence from root-associated fungi
77 Abstract
Regional species richness and local diversity are correlated across the globe. However, mechanisms underlying this relationship remain unresolved. In this study, we test whether older species pools increase local diversity not simply because they are more species rich, but also because the constituent species are more likely to coexist. We assembled experimental species pools of root-associated fungi in plant-fungal microcosms, using fungi isolated from the roots of an ericaceous plant across a 4.1 myr soil chronosequence. We found that increasing species pool richness resulted in greater local diversity when species were from older ecosystems. Older species pools also had lower phylogenetic and functional diversity, consistent with an increasing role of equalizing fitness differences, rather than greater niche differentiation, in the coexistence of ericaceous root-associated fungi from older ecosystems. Our results suggest ecosystem age is linked to local diversity not just through increased regional richness but also the composition of the species pool.
78 Introduction
It is widely recognized that historical processes occurring across broad spatial scales, such as vicariance and allopatric speciation, are linked to local patterns of species distributions (Terborgh & Faaborg 1980; Ricklefs 1987; Cornell & Lawton 1992; Harrison & Cornell 2008). Because of this relationship, regional and local diversity are positively correlated across the globe and local diversity can often be explained by factors that affect regional species richness (Hillebrand & Blenckner 2002). For example, greater regional richness in geologically older habitats, or habitats that have been historically more abundant, results in increased local species richness (Zobel et al. 2011) and phylogenetic diversity (Lososová et al. 2015). However, processes operating at the local scale, such as abiotic filtering or competitive exclusion, also feed back to influence the composition of the regional species pool (Leibold et al. 2004). If niche filling through diversification and immigration (Purschke et al. 2013; Gillespie 2016) contributes to greater richness in older regions, greater functional diversity in the regional species pool is also expected. As a result, ecosystem age may increase local diversity not simply due to greater regional richness, but also by facilitating species coexistence through the composition of the regional species pool, though this possibility remains untested.
Empirical research on the relationship between regional species richness and local diversity is complicated by the difficulty of attempting to infer process from pattern in natural communities (Loreau 2000). Historically, a diminishing gain in local richness with increasing regional richness, often described as “saturation” of local diversity, was interpreted as indicating dominance of local processes in community assembly (i.e., competition for niche space; Terborgh & Faaborg 1980; Cornell 1985). Alternatively, a linear local-regional richness relationship was interpreted as indicating the dominance of regional processes in determining local diversity (Ricklefs 1987). However, both linear and saturating patterns can arise independent of species interactions simply as an emergent property of regional species abundance distributions (Fox & Srivastava 2006) or local extinction and colonization dynamics
79 (He et al. 2005). In addition, the local-regional richness relationship is highly dependent on the spatial scale used to define the local community relative to the scale at which species interact (Caley & Dolph Schluter 1997), the rate of local disturbance (Caswell & Cohen 1993), and the definition of ecologically relevant regional species pools (Carstensen et al. 2013). Determining whether ecosystem age affects the relationship between regional richness and local diversity therefore requires comparisons that can account for these potentially confounding factors (Lessard et al. 2012).
One approach that has been used to address similarly difficult problems in community ecology is to use microbial model communities, which have rapid temporal dynamics and can be easily manipulated and maintained under controlled conditions (Jessup et al. 2004). Plant-associated fungal symbionts, which are phylogenetically and functionally diverse and are linked to the diversity and productivity of plant communities worldwide (van der Heijden et al. 1998; Vandenkoornhuyse et al. 2002), are one such microbial system (e.g., Maherali & Klironomos 2007). Plant-fungal systems that can be manipulated in controlled microcosm experiments present an underutilized, yet tractable and ecologically relevant model system to test hypotheses linking regional species pools to local community assembly.
In this study, we test the hypothesis that the accumulation of fungal diversity within a host plant will saturate with increasing species pool richness, but that greater local diversity will be realized when the region contributing to the species pool is older. To this end, we assembled experimental species pools of plant-root-associated fungi in plant-fungal microcosms, manipulating fungal species pool richness and the geologic age of the sites contributing to each species pool. In addition, we quantified functional and phylogenetic relationships among species to test the hypothesis that species pools from older ecosystems will have greater functional diversity and that these species pools promote greater local diversity through niche partitioning and the coexistence of less similar species.
80 Materials and methods
Overview and experimental design
We isolated fungi from the roots of an ericaceous plant, Vaccinium calycinum, collected from 5 sites across a 4.1-million-year soil chronosequence in Hawaii, known as the Long Substrate Age Gradient (LSAG; Vitousek 2004). We then assembled experimental species pools of 2-30 fungi, manipulating geologic age by systematically varying the mean of the log10-transformed ages of the LSAG sites where individual species were collected. We also manipulated the variance of the log10-transformed site ages to account for the potential confounding effects of edaphic variation among LSAG sites on species pool functional diversity and assembly outcomes. We used site age variance as a proxy for edaphic variation because the LSAG chronosequence represents a progression of long-term soil development, resulting in greater differences in soil properties (e.g., physical structure, nutrient availability, etc.) with increasing differences in site age (Vitousek 2004). A total of 80 experimental species pools (including one control treatment with no fungi) were inoculated onto sterile V. calycinum seedlings in individual microcosms, each replicated 4 times (320 total microcosms), and community assembly outcomes assessed through Illumina metabarcoding after 5 months.
Fungal culture collection
At each LSAG site, we sampled fungi from the roots of 12 randomly selected V. calycinum plants. A portion of the root system of each plant and the adhering soil (ca. 25 x 25 x 10 cm) was excavated with a hand trowel and transported immediately to the University of Hawaii, Hilo campus, where samples were refrigerated (4 ºC) and processed within 24 hours. Fine terminal roots were manually separated from the soil and rinsed in tap water to remove all visible soil particles and 4 segments (ca. 2 cm) were haphazardly selected and pooled for each plant. Pooled root samples were surface-sterilized by sequential vortexing for 1 minute in sterile water, 70% EtOH, and 50% household bleach (4.5% available chlorine), followed by 3 rinses in sterile water.
81 From each surface-sterilized root fragment, 2 segments (ca. 2 mm) were excised and aseptically transferred to separate petri dishes containing modified Melin-Norkans (MMN) agar, amended with the antibiotics gentamycin (15 mg l-1), streptomycin (15 mg l-1), and tetracycline hydrochloride (12 mg l-1) to retard bacterial growth. For each pair of excised root segments, one was placed on media that also contained benomyl (4 mg l-1), a fungicide that suppresses many ascomycetous fungi, facilitating the isolation of basidiomycetous fungi from roots (Vohník et al. 2012; Bruzone et al. 2015). Plates were monitored regularly, and hyphae growing from root segments immediately transferred to new MMN plates and then maintained by serial transfer every 4 to 8 weeks, depending on growth rate.
Identifying fungal isolates
One representative of each fungal morphotype from each site was selected for possible use in the microcosm study and identified using molecular methods. Fungal DNA was extracted by scraping hyphae from the surface of a colonized agar plate and lysing cells with Extract-N-Amp Plant (Sigma-Aldrich) extraction and neutralization solutions. The complete nrDNA ITS gene and partial large subunit (28S) gene were PCR amplified using the primer pairs ITS1f-ITS4 (White et al. 1990; Gardes & Bruns 1993) and LROR-LR5 (Vilgalys & Hester 1990), respectively, in 30 µl PCR reactions using GoTaq2 Green Master Mix (Promega), forward and reverse primers (10 µM) and 2 µl template DNA. The PCR protocol consisted of an initial denaturation at 95 ºC (2 min), followed by 26 cycles of 95 ºC (1 min), 55 ºC (30 sec), and 72 ºC (1 min), with a final elongation at 72 ºC (5 min). PCR products were sequenced using single pass Sanger sequencing (Beckman Coulter Genomics, Danvers, MA, USA) and sequences were deposited in GenBank under accession numbers [accession numbers TBD]. Putative taxonomy was assigned by searching the full ITS sequence and the complete ITS and partial LSU sequence against the UNITE species hypothesis database v7.1 (Kõljalg et al. 2013), comparing all pair-wise semi-global alignments using USEARCH v9.2.64 (Edgar 2010), with search heuristics disabled to ensure that the best matches were returned.
82 We tentatively identified the fungal isolates used in the microcosm experiment as belonging to a range of ascomycetous taxa commonly associated with ericaceous plants, predominantly in the order Helotiales (Fig. 1). Many of the closest matching sequences in the UNITE database originated from the roots of other ericaceous species and were taxonomically affiliated with ericoid mycorrhizal fungi, including Meliniomyces bicolor and Oidiodendron maius and the genera Rhizoscyphus and Hymenoscyphus. Other taxa had affinities to groups of common endophytic fungi associated ericaceous species, including the genera Chaetosphaeria, Cladophialophora and Hyloscyphaceae. There is considerable taxonomic overlap between these two functional groups and the interactions between our isolates and the host plants are not well-documented (Leopold 2016). However, given the aggressive surface sterilization protocol used and the taxonomic affiliations, we expect all taxa to be either ericoid mycorrhizal fungi or non-mycorrhizal endophytes.
Functional / phylogenetic characterization of fungi
Two complementary approaches were used to characterize the functional similarity among fungal isolates, direct measurements of functional traits and estimation of phylogenetic relationships. For functional traits, we quantified the relative abilities of each isolate to grow on 95 different carbon substrates using phenotypic microarrays (Biolog, FF microplate). Hyphal suspensions were prepared for inoculating microplates by blending hyphae scraped from the surface of an agar plate in 60 ml sterile water. The hyphal suspension was then centrifuged at 3500 rpm for 5 minutes, decanted and the hyphae were resuspended in 10 ml of a sterile inoculation solution of 0.25% Phytagel and 0.03% Tween 40 in water. This concentrated hyphal suspension was then diluted with additional inoculation solution to total volume of ca. 50 ml and an optical density of 0.06 at 590 nm. Using a multichannel pipette, 100 µl of this solution was transferred to each well of three replicate FF microplates for each fungal isolate. Plates were sealed with Parafilm to prevent desiccation and incubated at room temperature for 6 weeks. Fungal growth was monitored by reading optical density at 740 nm, using a microplate reader (Spectramax 190, Molecular Devices), and
83 measurements were collected at 1, 3 and 6 days, and weekly thereafter. Substrate use profiles for each species were generated by fitting a loess smoother to the median optical density value in each well at each time point and integrating the area under the curve as a cumulative measure of substrate use. Values for the no-substrate control well were subtracted from all other wells to account for growth not attributable to the available carbon substrates.
To estimate phylogenetic relationships, we use the previously sequenced 740-bp region of the nrDNA large ribosomal subunit (28S), which allows better resolution of phylogenetic relationships in ascomycetous fungi than the ITS region (Liu et al. 2012). We also retrieved a sequence of the matching gene region of Saccharomyces cerevisiae from GenBank [accession # KY109285.1] to use as an outgroup for phylogenetic analyses. Sequences were aligned on the T-Coffee web-server (Di Tommaso et al. 2011) and alignment columns were weighted using transitive consistency scoring (Chang et al. 2014) before phylogenetic tree construction using maximum likelihood and non-parametric, approximate likelihood-ratio tests of branch support with PhyML (Guindon et al. 2010; Fig. 1).
Fungal species pools
Experimental species pools were constructed using a subset of the total culture library that excluded replicate isolates from the same site with identical nrDNA sequences. Initial Sanger sequencing revealed only 4 Basidiomycete taxa, which we excluded from the assembly experiment to avoid excessive influence of phylogenetic outliers in our analyses. Preliminary tests during methods development also revealed 4 pathogenic isolates (all Cryptosporiopsis spp.) that rapidly killed seedlings in vitro; these isolates were also excluded from the experiment.
In total, 54 fungal isolates were used in the construction of the experimental species pools. To ensure that individual isolates could be identified in the mixed communities at the end of the experiment, isolates with > 97% similarity in their ITS2 nrDNA gene sequences (the targeted barcoding sequence, see below) were never included in the
84 same species pool. The compositions of species pools were selected using a stratified random sampling of the possible parameter space defined by the species pool richness
(Richness; 2-30 species) and the range of possible values of mean log10-transformed site age (Age) and variance of log10-transformed site age (Variance), given the available species (Fig. S1). Briefly, 20 million random possible pools were generated and the parameter space was divided into 79 equal area bins, from which one species pool was randomly selected. This approach assured that more extreme parameter values would be sampled and allowed us to disentangle the effects of Age and Variance.
Microcosms
Microcosms were assembled in 2.5 cm x 15 cm cylindrical glass culture tubes. Each tube received 3 g (dry) of a mixture of sphagnum peat, fine vermiculite and perlite (1:5:1) and 7 ml of a low-carbon mineral nutrient solution, following Grelet et al.
(2009), containing 0.600 mM NH4NO3, 0.599 mM (NH4)2HPO4, 0.662 mM KH2PO4,
0.170 mM MgSO4.7H2O, 0.300 mM NaH2PO4, 0.080 mM K2SO4, 0.034 mM
FeNaEDTA, 0.006 mM ZnSO4.7H2O, 3.69 µM MnSO4.H2O, 0.04 µM
-1 Na2MoO4.2H2O, 9.55µM H3BO3, 1 µM CuSO4.5H2O and 100 mg l glucose. Culture tubes were fitted with polypropylene closures, modified to include 3 2-mm-diamter vent holes which were covered with 2 layers of micro-pore tape to allow gas exchange. Assembled microcosms were autoclaved twice for 30 minutes at 121 ºC with a 24 hour interval and allowed to cool in a sterile-airflow hood.
Seedlings
V. calycinum fruits were collected at the 300-year LSAG site and were cleaned, dried and refrigerated before use (Zee et al. 2008). Prior to assembling the microcosms, seeds were surface-sterilized by gentle vortexing in a 10% solution of H2O2 for 5 minutes, rinsing in sterile water, and then germinated in petri plates on 0.8% water agar in a lighted growth chamber. Individual 6-week-old seedlings (ca. 1 cm tall) were aseptically transferred into the sterile microcosms, which were sealed with parafilm.
85 Microcosms were then placed in a lighted growth chamber providing 150 µmol m-1 s- 1 of photosynthetically active radiation with lighting cycle of 16 h light (25 ºC) and 8 h dark (20 ºC). The positions of the microcosms within the growth chamber were randomized once a week throughout the experiment and seedlings were allowed to acclimate after being transferred to the microcosms for 3 weeks before fungi were added.
Fungal inoculation
Fungal inocula consisted of hyphal suspensions prepared in individual 1-pint canning jars, each fitted with a stainless steel blender assembly, filled with 80 ml distilled water and autoclaved for 30 minutes at 121 ºC. Once cool, each sterile jar received 4 agar plugs (1 cm) taken from the growing edge of a 4 week old fungal culture, and was blended at high speed for 60 seconds. Inoculum for each species pool was prepared in a sterile 15 ml centrifuge tune by combining 150 µl of the hyphal suspension of each individual isolate in the pool and adding sterile water for a total of 5 ml. Microcosms were inoculated under a sterile air-flow hood and received 1 ml of a mixed-species inoculum (or sterile water) which was applied at the base of the seedling using an extra-long (15 cm) pipette tip.
Microcosm harvest
Microcosms were harvested 5 months after the introduction of the fungal species pools. Roots were separated from shoots and were gently cleaned of growth media in molecular biology grade water using flame sterilized forceps and briefly dried on sterile filter paper. The fresh weight of roots and shoots were recorded. Shoots were dried at 60 ºC and analyzed for C and N concentration using an elemental analyzer (Carlo Erba NA 1500). After weighing, roots were transferred to sterile 2-ml screw cap tubes and stored at -80 ºC. Empty 2-ml tubes (4) were opened while harvesting roots and carried through all stages of the metabarcoding procedure as a negative control. We did not find any evidence for an effect of species pool composition on
86 seedling biomass or leaf chemistry and present a graphical summary of these results as supplemental material (Fig. S2).
Fungal species composition
Fungal species composition within seedlings at the end of the experiment was determined using Illumina metabarcoding, targeting the ITS2 region of the nrDNA gene. This region was identified by Sanger sequencing as being variable enough to discriminate between isolates while having minimal length variation to minimize amplicon sequencing bias (Lindahl et al. 2013). In addition to the microcosm samples, we included samples that consisted of either DNA from pure cultures of each fungal isolate or mock communities consisting of fungal DNA mixed at known concentrations (for a description of the mock community design, see Appendix A). Including samples with DNA from individual isolates allowed verification of the Sanger sequencing of the target barcoding region while inclusion of the mock communities allowed us to validate the semi-quantitative nature of the sequencing data.
DNA was extracted from the entire root system of each seedling using bead beating and the Nucleomag 96 Plant Kit (Macherey-Nagel) on a KingFisher Flex (ThermoFisher) automated magnetic particle processor. A dual indexed, two-stage fusion-PCR procedure was used for Illumina library preparation, following the primer design of Toju et al. (2016). We modified the stage-one primers to include the gene primers 58A1F (Martin & Rygiewicz 2005) and ITS4. Because these primers amplify both fungal and host ITS2 nrDNA, we designed an amplification-blocking oligo (Vestheim & Jarman 2008) that overlapped with 2-bp at the 3' end of the forward primer, extended into a host specific sequence, and terminated with a 3' C3 spacer to block elongation during PCR (5'-GTA GCG AAA TGC GAT ACT TGG-3SpC3-3'). Mismatches between the blocking oligo and fungal DNA were consistent across all species, limiting the probability of introducing amplification bias. Stage-one PCR reactions (25 µl) were carried out using MyTaq Hot-Start Red Mix (Bioline), 10 µM
87 forward and reverse primers, 100 µM blocking oligo, 1 µl template DNA, and a thermocycler program consisting of an initial cycle of 95 °C (3 min), 32 cycles of 95 °C (20 sec), 60 °C (10 sec), and 72 °C (10 sec), followed by a final elongation cycle at 72 °C (5 min). Stage-two PCR reactions used 2 µl of the stage-one PCR product as template, 10 µM primers (Toju et al. 2016), and a thermocycler program consisting of an initial cycle of 95 °C (3 min), 6 cycles of 95 °C (20 sec), 55 °C (10 sec), and 72 °C (10 sec), followed by a final elongation cycle at 72 °C (5 min). Thermocycler ramp rates for both PCR stages were slowed to 1 °C / sec to minimize chimera formation (Stevens et al. 2013). Final PCR products were cleaned and normalized to 2.5 ng µl-1 using Just-a-Plate, 96-well normalization and purification plates (Charm Biotech), then pooled and sent to the Stanford Functional Genomics Facility for 250-bp paired- end sequencing on an Illumina MiSeq.
Bioinformatics
Bioinformatic processing of the Illumina sequence data involved first removing primer sequences and low-quality tails with cutadapt v1.12 (Martin 2011). Paired reads were then merged with USEARCH (Edgar & Flyvbjerg 2015) and denoised following the UNOISE2 pipeline (Edgar 2016b), which clusters sequences without an identity threshold using read abundance skew. Data were processed individually for each sample to ensure that noisy reads were assigned to clusters actually present in the sample. Representative sequences for each cluster in each sample were matched against the pool of expected sequences in each sample first and then unexpected sequences were either matched to the library of amplicons expected in the experiment (including the host ITS2 sequence), but not expected in the sample, or identified as likely contamination. Unexpected reads in microcosm samples were predominantly host DNA or likely contaminants from the sample processing and library preparation procedure, which were present in very low abundance and often also identified in the no-DNA negative control samples. These reads were filtered from the data before analysis. Read counts for each denoised amplicon in each sample were merged into a species-by-sample matrix for analyses, discarding samples with low sequencing depth
88 (< 5000 reads) and rarefying the remaining samples to an equal number of reads (6107). After bioinformatic processing of the metabarcoding data, 286 samples remained, leaving 3 or 4 replicates of 77 unique species pools.
Statistical Analyses
To test for the relationship between phylogenetic or functional diversity and species pool composition, we first quantified the mean pairwise phylogenetic and functional distances among isolates for each species pool. Phylogenetic distances were square- root transformed to account for non-linear scaling between evolutionary and functional distances (Letten & Cornwell 2015). Carbon substrate use data was reduced to the first 16 principle components with PCA (accounting for 90% of total variation) to collapse highly correlated substrates. Euclidean distance of the PCA coordinates was then used to calculate mean pairwise functional distances among isolates for each species pool. Linear regression was used to test the hypothesis that phylogenetic and functional diversity increases with Age.
Estimates of alpha diversity in microbial communities are complicated by sample “cross-talk” during sequencing (Edgar 2016a) and amplification of DNA from non- living tissue (Carini et al. 2016). In addition, abundance based measures of microbial diversity can also be confounded by amplification and sequencing bias (Nguyen et al. 2015). To address these limitations, we used two approaches for estimating alpha diversity. First, we excluded very low abundance species (0.05% of total reads in a sample) and calculated observed species richness using binary species presence data.
Second, based on the results of the mock community analysis (Fig. S3), we used log2- transformed sequence counts as a quantitative measure of species abundance. We then calculated the inverse Simpson’s diversity index, using Hill numbers, as an abundance-weighted metric of the “effective number of species” (Haegeman et al. 2013). Analyses using either measure of alpha diversity yielded qualitatively identical patterns. We present results for the observed richness in the main text and for the inverse Simpson’s diversity as supplemental materials (Table S2 & Fig. S4).
89 To determine if the relationship between local alpha diversity and Richness was linear or curvilinear (i.e., saturating), we used linear mixed-models in the R-package nlme (Pinheiro et al. 2017), including species pool composition as a random effect and allowing variance to increase with Richness. Support for a curvilinear model was tested by comparing models with and without a polynomial term for using likelihood- ratio tests. We used two approaches to test whether the relationship between Richness and local alpha diversity was affected by Age or Variance. First, we used polynomial mixed-models, as above, testing for a significant interaction between Richness and Age or Variance. In the second approach, we fit a generalized non-linear least squares model with nlme, using a Michaelis-Mentin function, which takes the form,
D = (a * Richness) / (b + Richness) where D is local alpha diversity, a is the predicted asymptotic value of D, and b is the value of Richness at which half of the asymptotic value of D is reached. As with the mixed-model approach, we allowed variance to increase with Richness. We also used alternative functional forms, including an asymptotic regression through the origin and a Gompertz model, which yielded equivalent results. We then modeled the standardized residuals (Pearson residuals) of the nonlinear model as a function of Age and Variance using linear mixed-models. To determine if Age or Variance affected the accumulation of local diversity with increasing Richness we tested for significance of the interaction terms Richness:Age or Richness:Variance. Conclusions from the polynomial mixed-model and the Michaelis-Menten approaches were identical. We present the results of the Michaelis-Menten model here because the functional form of this model more closely approximates the expected saturating relationship between regional richness and local diversity.
To test whether the phylogenetic or functional structure of realized local communities was related to Age or Variance we calculated the standardized effect sizes for mean pairwise phylogenetic and functional distances, using the R-package picante (Kembel et al. 2010). We weighted species abundances by the log2-transformed read counts and
90 used a null model where taxa names were shuffled on the phylogenetic or functional distance matrix 999 times. The standardized effect sizes were modeled as a response using linear mixed-effects models with Richness and Age or Variance as predictors and species pool composition as a random effect.
Results
In contrast to our predictions, we found that both the phylogenetic and functional diversity of our experimental species pools decreased with increasing Age (F1,77 =
8.86, p = 0.004 & F1,77 = 33.28, p < 0.001, respectively; Fig. 2). This relationship was not affected by including Variance in the model and there was no significant relationship between Variance and phylogenetic or functional diversity (F1,77 = 2.3, p
= 0.13 & F1,77 = 1.21, p = 0.27, respectively; Fig. S5).
Fungal communities within V. calycinum seedlings accumulated diversity at a
2 decreasing rate with increasing species pool richness (χ (1) = 10.89, p = 0.001; Figs 3 and S4). As hypothesized, the relationship between Richness and realized local diversity varied with Age, with older species pools accumulating more diversity (t = 2.54, df = 73, p = 0.01; Figs. 3 and S4; Tables 1 & S1). The relationship between species pool richness and local diversity was not affected by Variance (t = 1.52, df = 73, p = 0.13).
We did not detect significant phylogenetic or functional clustering (standardized effect size > 1.96) or dispersion (standardized effect size > 1.96). However, communities assembled with species pools from younger sites tended to become more phylogenetically dispersed during community assembly than communities associated with species pools from older sites (t = -3.81, df = 73, p < 0.001; Fig. 4). Phylogenetic structure was not affected by Variance (t = 1.43, df = 73, p = 0.15) and functional structure was not affected by either Age or Variance (t = -1.11, df = 73, p =0.27 & t = 0.23, df = 73, p = 0.82, respectively).
91 Discussion
In this study, increasing species pool richness resulted in higher local diversity when species pools were comprised of species from older ecosystems. Our results show that local diversity increases with the geologic age of a region, not just as a result of increased regional diversity, but also because the regional species pool contains species that are more likely to coexist in local communities. Although the mechanisms responsible for fine-scale species coexistence in our experimental microcosms are unknown, our results are consistent with evidence from molecular profiling of fungi in field-collected V. calycinum roots, which showed that the diversity and evenness of the fungal symbiont community associated with an individual plant increase with ecosystem age (Leopold et al. unpublished).
Contrary to our expectations, however, we found that older-age species pools had lower phylogenetic and functional diversity (Fig. 2). This suggests that increased coexistence of species from older sites may be the result of convergence on similar ecological strategies (i.e., equalizing fitness differences), as opposed to niche differentiation (Chesson 2000; Mayfield & Levine 2010; but see Kraft et al. 2015). Although we do not know which traits determine the outcomes of species interactions during community assembly in this system, both evolutionary and ecological mechanisms could contribute to trait convergence in older ecosystems. For example, a competitive trait hierarchy may lead to evolutionary convergence on similar root colonization strategies over geologic time-scales (TerHorst et al. 2010; Kunstler et al, 2012; Tobias et al. 2013). It is also possible that changing conditions with long-term soil development only lead to trait convergence in older sites. In plants, progressive phosphorus limitation with increasing soil age has been shown to result in a shift from niche differentiation to trait convergence as the primary mechanism of coexistence (Mason et al. 2012). Root-associated fungi may also coexist through niche differentiation in younger soils, but converge on a common resource-retentive strategy with declining phosphorus availability (Lambers et al. 2008), resulting in coexistence through functional convergence and reduced fitness differences.
92 Although site age variance did not affect community assembly outcomes, we cannot rule out niche partitioning due to environmental heterogeneity as a mechanism facilitating diversity in this system. For example, the accumulation of complex organic layers and recalcitrant nutrient pools during pedogenesis could promote diversity in ericaceous root-associated fungi through niche partitioning along nutrient resource axes (Wurzburger et al. 2012). This fine-grained resource heterogeneity in older soils may facilitate coexistence among species in the regional species pool more than the broad scale environmental heterogeneity present across the LSAG chronosequence (Tamme et al. 2010). However, soil nutrient resource partitioning is unlikely to explain the patterns we observed in the experimental microcosms, where nutrients were supplied in mineral form.
We did not detect a significant signal of clustering or dispersion in the phylogenetic or functional structure of the assembled communities (Fig. 4). This lack of pattern could be due to the relatively small number of species used in our experiment and biases introduced by our focus on ascomycetous fungi that could be easily cultured (Allen et al. 2003). In addition, spatial resource partitioning within roots between mycorrhizal fungi and non-mycorrhizal endophytes may not result in a strong phylogenetic signal due to the phylogenetic overlap among these functional groups. Nonetheless, we did find that species pools from younger sites tended to increase in phylogenetic dispersion during assembly more than species pools from old sites (Fig. 4). This is consistent with a shift from niche differentiation to equalizing fitness differences as the primary mechanism of species coexistence (Mason et al. 2012). However, functional trait data did not show the same increase in dispersion in younger pools relative to older pools. There are two possible explanations for this discrepancy. First, we provided microcosms with mineral nutrients, limiting opportunities for niche partitioning along nutrient resource axes. Second, traits important for niche differentiation in younger species pools may not have been reflected in the functional trait data we collected using carbon-substrate microarrays. It is possible that measurements of nitrogen substrate use would better reflect niche partitioning in
93 younger pools because younger soils tend to be nitrogen limited (Vitousek et al. 1993) and ericaceous root-associated fungi have been shown to vary in their affinity for different nitrogen sources (Cairney et al. 2000; Grelet et al. 2009).
Although we did not detect functional consequences of fungal community assembly for the host plant, this may be due to the artificial conditions of the microcosm environment. Specifically, the mineral nutrients we provided were directly available to both the fungi and the host plant. This universally available resource pool may explain why seedling growth and leaf tissue chemistry were unaffected by species pool composition and why control seedlings had more shoot biomass and a higher concentration of leaf nitrogen (Fig. S2). In addition, root-associated fungi affect plants through mechanisms other than nutrient uptake in natural communities, including pathogen protection (Newsham et al. 1995) and drought tolerance (Augé 2001), both of which would be unimportant in the microcosms.
Greater local diversity in geologically older habitats has been explained by increased richness of the regional species pool (Zobel et al. 2011; Lososová et al. 2015). In this study, we have shown that older species pools may also be comprised of species that are more likely to coexist. Our results are consistent with an increasing role of equalizing fitness differences in older species pools and a shift from niche partitioning in younger ecosystems to convergence on similar ecological strategies in older ecosystems, possibly in response to progressive phosphorus limitation. However, further study would be needed to conclusively demonstrate coexistence mechanisms in this system. Nonetheless, the results of our microcosm experiment are consistent with diversity patterns in field collected roots of the same species (Leopold et al. unpublished). We suggest that increased local diversity as a result of strengthened species coexistence mechanisms may be a general feature of older species pools and that this possibility should be explored in a variety of systems to advance the general understanding of the link between regional and local diversity.
94 References cited
Allen, T.R., Millar, T., Berch, S.M. & Berbee, M.L. (2003). Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytol., 160, 255–272. Augé, R.M. (2001). Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza, 11, 3–42. Bruzone, M.C., Fontenla, S.B. & Vohník, M. (2015). Is the prominent ericoid mycorrhizal fungus Rhizoscyphus ericae absent in the Southern Hemisphere’s Ericaceae? A case study on the diversity of root mycobionts in Gaultheria spp. from northwest Patagonia, Argentina. Mycorrhiza, 25, 25–40. Cairney, J.W.G., Sawyer, N.A., Sharples, J.M. & Meharg, A.A. (2000). Intraspecific variation in nitrogen source utilisation by isolates of the ericoid mycorrhizal fungus Hymenoscyphus Ericae (Read) Korf and Kernan. Soil Biol. Biochem., 32, 1319–1322. Caley, J. & Dolph Schluter. (1997). The relationship between local and regional diversity. Ecology, 78, 70–80. Carini, P., Marsden, P.J., Leff, J.W., Morgan, E.E., Strickland, M.S. & Fierer, N. (2016). Relic DNA is abundant in soil and obscures estimates of soil microbial diversity. Nat. Microbiol., 2, 16242. Carstensen, D.W., Lessard, J.P., Holt, B.G., Krabbe Borregaard, M. & Rahbek, C. (2013). Introducing the biogeographic species pool. Ecography, 36, 1310– 1318. Caswell, H. & Cohen, J.E. (1993). Local and regional regulation of species-area relations: a patch-occupancy model. In: Species diversity in ecological communities. pp. 99–107. Chang, J.M., Di Tommaso, P. & Notredame, C. (2014). TCS: a new multiple sequence alignment reliability measure to estimate alignment accuracy and improve phylogenetic tree reconstruction. Mol. Biol. Evol., 31, 1625–1637.
95 Chesson, P. (2000). Mechanisms of maintenance of species diversity. Annu. Rev. Ecol. Syst., 31, 343–366. Cornell, H. V. (1985). Local and regional richness of cynipine gall wasps on California oaks. Ecology, 66, 1247–1260. Cornell, H. V. & Lawton, J.H. (1992). Species interactions, local and regional processes, and limits to the richness of ecological communities: a theoretical perspective. J. Anim. Ecol., 61, 1–12. Edgar, R.C. (2010). Search and clustering orders of magnitude faster than BLAST. Bioinformatics, 26, 2460–2461. Edgar, R.C. (2016a). UNCROSS: filtering of high-frequency cross-talk in 16S amplicon reads. bioRxiv, doi: 10.1101/088666. Edgar, R.C. (2016b). UNOISE2: improved error-correction for Illumina 16S and ITS amplicon sequencing. bioRxiv, doi: 10.1101/081257. Edgar, R.C. & Flyvbjerg, H. (2015). Error filtering, pair assembly and error correction for next-generation sequencing reads. Bioinformatics, 31, 3476-3482. Fox, J.W. & Srivastava, D. (2006). Predicting local-regional richness relationships using island biogeography models. Oikos, 113, 376–382. Gardes, M. & Bruns, T.D. (1993). ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol. Ecol., 2, 113–118. Gillespie, R.G. (2016). Island time and the interplay between ecology and evolution in species diversification. Evol. Appl., 9, 53–73. Grelet, G.-A., Johnson, D., Paterson, E., Anderson, I.C. & Alexander, I.J. (2009). Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas. New Phytol., 182, 359–366. Grelet, G.A., Meharg, A.A., Duff, E.I., Anderson, I.C. & Alexander, I.J. (2009). Small genetic differences between ericoid mycorrhizal fungi affect nitrogen uptake by Vaccinium. New Phytol., 181, 708–718.
96 Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 2.0. Syst. Biol., 59, 307– 321. Haegeman, B., Hamelin, J., Moriarty, J., Neal, P., Dushoff, J. & Weitz, J.S. (2013). Robust estimation of microbial diversity in theory and in practice. ISME J., 7, 1092–1101. Harrison, S. & Cornell, H. (2008). Toward a better understanding of the regional causes of local community richness. Ecol. Lett., 11, 969–979. He, F., Gaston, K.J., Connor, E.F. & Srivastava, D.S. (2005). The local-regional relationship: immigration, extinction, and scale. Ecology, 86, 360–365. van der Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis, P., Streitwolf- Engel, R., Boller, T., et al. (1998). Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature, 396, 69–72. Hillebrand, H. & Blenckner, T. (2002). Regional and local impact on species diversity–from pattern to processes. Oecologia, 132, 479–491. Jessup, C.M., Kassen, R., Forde, S.E., Kerr, B., Buckling, A., Rainey, P.B., et al. (2004). Big questions, small worlds: microbial model systems in ecology. Trends Ecol. Evol., 19, 189–197. Kembel, S.W., Cowan, P.D., Helmus, M.R., Cornwell, W.K., Morlon, H., Ackerly, D.D., et al. (2010). Picante: R tools for integrating phylogenies and ecology. Bioinformatics, 26, 1463–1464. Kõljalg, U., Nilsson, R.H., Abarenkov, K., Tedersoo, L., Taylor, A.F.S., Bahram, M., et al. (2013). Towards a unified paradigm for sequence-based identification of fungi. Mol. Ecol., 22, 5271–5277. Kraft, N.J.B., Godoy, O. & Levine, J.M. (2015). Plant functional traits and the multidimensional nature of species coexistence. Proc. Natl. Acad. Sci., 112, 797–802. Kunstler, G., Lavergne, S., Courbaud, B., Thuiller, W., Vieilledent, G., Zimmermann, N.E., et al. (2012). Competitive interactions between forest trees are driven by
97 species’ trait hierarchy, not phylogenetic or functional similarity: implications for forest community assembly. Ecol. Lett., 15, 831–840. Lambers, H., Raven, J.A., Shaver, G.R. & Smith, S.E. (2008). Plant nutrient- acquisition strategies change with soil age. Trends Ecol. Evol., 23, 95–103. Leibold, M.A., Holyoak, M., Mouquet, N., Amarasekare, P., Chase, J.M., Hoopes, M.F., et al. (2004). The metacommunity concept: a framework for multi-scale community ecology. Ecol. Lett., 7, 601–613. Leopold, D.R. (2016). Ericoid fungal diversity: challenges and opportunities for mycorrhizal research. Fungal Ecol., 24, 114–123. Leopold, D.R., Peay, K.G., Vitousek, P.M., Fukami, T. (unpublished). Diversity of Vaccinium calycinum (Ericaceae) root-associated fungi increases throughout long-term ecosystem development. Lessard, J.P., Belmaker, J., Myers, J. a., Chase, J.M. & Rahbek, C. (2012). Inferring local ecological processes amid species pool influences. Trends Ecol. Evol., 27, 600–607. Letten, A.D. & Cornwell, W.K. (2015). Trees, branches and (square) roots: why evolutionary relatedness is not linearly related to functional distance. Methods Ecol. Evol., 6, 439–444. Lindahl, B.D., Nilsson, R.H., Tedersoo, L., Abarenkov, K., Carlsen, T., Kjøller, R., et al. (2013). Fungal community analysis by high-throughput sequencing of amplified markers–a user’s guide. New Phytol., 199, 288–299. Liu, K.-L., Porras-Alfaro, A., Kuske, C.R., Eichorst, S. a & Xie, G. (2012). Accurate, rapid taxonomic classification of fungal large-subunit rRNA genes. Appl. Environ. Microbiol., 78, 1523–33. Loreau, M. (2000). Are communities saturated? On the relationship between alpha, beta and gamma diversity. Ecol. Lett., 3, 73–76. Lososová, Z., Šmarda, P., Chytrý, M., Purschke, O., Pyšek, P., Sádlo, J., et al. (2015). Phylogenetic structure of plant species pools reflects habitat age on the geological time scale. J. Veg. Sci., 26, 1080–1089.
98 Maherali, H. & Klironomos, J.N. (2007). Influence of phylogeny on fungal community assembly and ecosystem functioning. Science (80-. )., 316, 1746– 1748. Martin, K.J. & Rygiewicz, P.T. (2005). Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol., 5, 28. Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal, 17, 10–12. Mason, N.W.H., Richardson, S.J., Peltzer, D.A., de Bello, F., Wardle, D.A. & Allen, R.B. (2012). Changes in coexistence mechanisms along a long-term soil chronosequence revealed by functional trait diversity. J. Ecol., 100, 678–689. Mayfield, M.M. & Levine, J.M. (2010). Opposing effects of competitive exclusion on the phylogenetic structure of communities. Ecol. Lett., 13, 1085–1093. Newsham, K.K., Fitter, A.H. & Watkinson, A.R. (1995). Arbuscular Mycorrhiza Protect an Annual Grass from Root Pathogenic Fungi in the Field. J. Ecol., 83, 991–1000. Nguyen, N.H., Smith, D., Peay, K. & Kennedy, P. (2015). Parsing ecological signal from noise in next generation amplicon sequencing. New Phytol., 205, 1389– 1393. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team. (2017). nlme: Linear and Non-linear Mixed Effects Models. R package version 3.1-130, https://CRAN.R-project.org/package=nlme. Purschke, O., Schmid, B.C., Sykes, M.T., Poschlod, P., Michalski, S.G., Durka, W., et al. (2013). Contrasting changes in taxonomic, phylogenetic and functional diversity during a long-term succession: insights into assembly processes. J. Ecol., 101, 857–866. Ricklefs, R.E. (1987). Community diversity: relative roles of local and regional processes. Science, 235, 167–171.
99 Stevens, J.L., Jackson, R.L. & Olson, J.B. (2013). Slowing PCR ramp speed reduces chimera formation from environmental samples. J. Microbiol. Methods, 93, 203–205. Tamme, R., Hiiesalu, I., Laanisto, L., Szava-Kovats, R. & Pärtel, M. (2010). Environmental heterogeneity, species diversity and co-existence at different spatial scales. J. Veg. Sci., 21, 796–801. Terborgh, J.W. & Faaborg, J. (1980). Saturation of bird communities in the West Indies. Am. Nat., 116, 178–195. TerHorst, C.P., Miller, T.E. & Powell, E. (2010). When can competition for resources lead to ecological equivalence? Evol. Ecol. Res., 12, 843–854. Tobias, J. a, Cornwallis, C.K., Derryberry, E.P., Claramunt, S., Brumfield, R.T. & Seddon, N. (2013). Species coexistence and the dynamics of phenotypic evolution in adaptive radiation. Nature, 506, 359–363. Toju, H., Tanabe, A.S. & Ishii, H.S. (2016). Ericaceous plant-fungus network in a harsh alpine-subalpine environment. Mol. Ecol., 1–16. Di Tommaso, P., Moretti, S., Xenarios, I., Orobitg, M., Montanyola, A., Chang, J.M., et al. (2011). T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res., 39, 13–17. Vandenkoornhuyse, P., Baldauf, S.L., Leyval, C., Straczek, J. & Young, J.P.W. (2002). Extensive fungal diversity in plant roots. Science, 295, 2051. Vestheim, H. & Jarman, S.N. (2008). Blocking primers to enhance PCR amplification of rare sequences in mixed samples–a case study on prey DNA in Antarctic krill stomachs. Front. Zool., 5, 12. Vilgalys, R. & Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol., 172, 4238–46. Vitousek, P. (2004). Nutrient cycling and limitations: Hawaii as a model system. Princeton University Press.
100 Vitousek, P.M., Walker, L.R., Whiteaker, L.D. & Matson, P. a. (1993). Nutrient limitations to plant growth during primary succession in Hawaii Volcanoes National Park. Biogeochemistry, 23, 197–215. Vohník, M., Sadowsky, J.J., Kohout, P., Lhotáková, Z., Nestby, R. & Kolařík, M. (2012). Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed basidiomycete with affinities to Trechisporales. PLoS One, 7, e39524. White, T.J., Bruns, T., Lee, S. & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications (eds. Innis, M.A., Gelfand, D.H., Sninsky, J.. & T.J, W.). Academic Press, San Diego, CA, pp. 315–322. Wurzburger, N., Higgins, B.P. & Hendrick, R.L. (2012). Ericoid mycorrhizal root fungi and their multicopper oxidases from a temperate forest shrub. Ecol. Evol., 2, 65–79. Zee, F., Strauss, A. & Arakawa, C. (2008). Propagation and cultivation of ‘Öhelo. Zobel, M., Otto, R., Laanisto, L., Naranjo-Cigala, A., Pärtel, M. & Fernández- Palacios, J.M. (2011). The formation of species pools: historical habitat abundance affects current local diversity. Glob. Ecol. Biogeogr., 20, 251–259.
101 Tables
Table 1: Analysis of the relationship between species pool richness and local alpha diversity using observed species richness, showing (a) the estimated parameters of the Michaelis-Menten model and (b) the effects of species pool richness (Richness), ecosystem age (Age), and their interaction on the standardized residuals from the Michaelis-Menten model.
a) Parameters of Michaelis-Menten model
Parameter Estimate LCI UCI a (asymptote) 25.3 22.3 28.4 b (half-max) 22.1 18.0 26.2 b) Analysis of model residuals
Parameter Estimate se df t p Richness -0.229 0.092 73 -2.481 0.015 Age -0.343 0.193 73 -1.772 0.081 Richness:Age 0.050 0.019 73 2.538 0.013
102 Figures
Figure 1: Estimated phylogenetic relationships of the 54 fungal isolates used in the current study. The maximum likelihood tree was constructed with PhyML, using the first 740 bp of the nrDNA large ribosomal subunit (28S). Colored points indicate the age of the chronosequence site where each isolate was collected. Following the isolated ID is the closest matching sequence in the UNITE database (v7.1), with its assigned taxonomy and percent similarity.
103 Figure 1 (continued)
104 Figure 2: The relationships between the mean site age and mean pairwise (a) phylogenetic or (b) functional distance for the experimental species pools. Point colors indicate species pool richness and trend lines (± se) indicate significant linear regressions (p < 0.05).
105 Figure 3: The relationship between fungal species pool richness and local alpha diversity (species richness) in experimental microcosms, showing the theoretical maximum local diversity (dotted gray line) and a non-linear saturating model (Michaelis-Menten) fit to the data (dashed black line). Point colors indicate the mean of the log10-transformed age of the sites represented in each species pool. Colored lines show the predicted effect of ecosystem age on the accumulation of diversity with increasing species pool richness.
106 Figure 4: Effect of species pool age on the standardized effects size of mean pairwise phylogenetic distance for fungal communities at harvest. Trend line shows the significant (p > 0.05) effect of species pool age on phylogenetic structure after assembly. Dotted lines indicate the threshold for significant clustering (< -1.96) or dispersion (> 1.96).
107 Supplementary materials
Table S1: Analysis of the relationship between species pool richness and local alpha diversity, using inverse Simpson’s diversity, showing (a) the estimated parameters of the Michaelis-Menten function and (b) the effects of species pool richness (Richness), ecosystem age (Age), and their interaction on the standardized residuals from the Michaelis-Menten model. a) Parameters of Michaelis-Menten model
Parameter Estimate LCI UCI a (asymptote) 23.1 20.4 25.8 b (half-max) 22.6 18.5 26.6 b) Analysis of model residuals
Parameter Estimate se df t p Richness -0.282 0.096 73 -2.946 0.004 Age -0.545 0.200 73 -2.721 0.008 Richness:Age 0.061 0.020 73 3.01 0.004
108 Figure S1: Graphical summary of experimental pool design, showing the pairwise relationships between species pool richness (a & b), and the mean (a & c) and variance
(b & c) of the log10-transformed ages of the sites included. Light gray points indicate the parameter values for 20 million randomly generated possible species pools and black points show the values for the 79 species pools used in the experiment.
109 Figure S2: Relationships between experimental manipulations of species pool composition and seedling (a-c) shoot and (d-f) root biomass and leaf tissue (g-i) nitrogen and (j-l) carbon concentrations. Horizontal lines indicate the median values for the four control seedling that were grown without fungi.
110 Figure S3: Results of the mock community samples showing (a) the strong linear relationship between the amount of DNA added and the number of reads for each fungal isolate and (b) the consistency of this relationship across isolates when using log2-transformed sequence counts. Isolate 86 was an outlier that did not sequence well on the Illumina platform, despite presenting no problems for Sanger sequencing. This isolate was never observed in the mock communities, possibly due to this unexplained bias. However, this isolate was only included in 2 species pools and exclusion of these pools did not affect the conclusions of the study.
111 Figure S4: The relationship between fungal species pool richness and local alpha diversity (inverse Simpson’s diversity) in experimental microcosms, showing the theoretical maximum local diversity (dotted gray line) and a non-linear saturating model (Michaelis-Menten) fit to the data (dashed black line). Point colors indicate the mean of the log10-transformed age of the sites represented in each species pool. Colored lines show the predicted effect of ecosystem age on the accumulation of diversity with increasing species pool richness.
112 Figure S5: The relationships between the variance of site age and mean pairwise (a) phylogenetic or (b) functional distance for the experimental species pools. Point colors indicate species pool richness. Relationships were not significant at p < 0.05.
113 Appendix A: Mock community sample preparation
To determine whether read counts from the Illumina Miseq data could be reasonably used as a proxy for species abundance within roots, we assembled mock community samples with known concentrations of DNA. Mock community samples consisted of DNA from 35 fungal isolates, including one representative isolate for each group with < 97% ITS2 sequence similarity. DNA for each isolated was purified from hyphae scraped from the surface of agar plates using the Nucleospin Plant II kit (Macherey- Nagel), quantified using a Qubit 2.0 Fluorometer, and diluted to a standardized concentration of 10 ng µl-1. For each isolate, the standardized DNA was serially diluted 1:4, 6 times, resulting in 7 solutions ranging in concentration from 10 to 0.00244 ng µl-1. Isolates were assigned to mock communities semi-randomly to ensure that each of the 14 mock community samples included 5 different species at each of the 7 concentrations. Samples were assembled by combining 5 ul of the assigned solution from the dilution series for each isolate, with 25 µl molecular grade water for a total of 200 µl and a concentration of 1.67 ng ul-1. These samples were then used as template DNA for Illumina Miseq amplicon library preparation, following the procedure described for the mock community samples described in the main text.
114 Chapter 5 Priority effects are interactively regulated by top-down and bottom-up forces: evidence from wood decomposer communities
115 Abstract
Both top-down (grazing) and bottom-up (resource availability) forces can determine the strength of priority effects, or the effects of species arrival history on the structure and function of communities, but their combined influences remain unresolved. To test for such influences, we assembled experimental communities of wood- decomposing fungi using a factorial manipulation of fungivore presence, nitrogen availability, and fungal assembly history. We found interactive effects of all three factors on fungal species composition and wood decomposition one year after the fungi were introduced. The strength of priority effects on community structure was affected primarily by nitrogen availability, whereas the strength of priority effects on decomposition rate was interactively regulated by nitrogen and fungivores. These results demonstrate that top-down and bottom-up forces jointly determine how strongly assembly history affects community structure and function.
Keywords: assembly history, fungivore grazing, historical contingency, priority effects, resource availability, saprotrophic fungi, wood decomposition.
116 Introduction
The order and timing of species arrival during community assembly can affect species composition (Palmgren 1926; Sutherland 1974; Drake 1991) and ecosystem functioning (Körner et al. 2008; Tan et al. 2012), the phenomenon known as priority effects. Because species arrival order is often highly stochastic and unknown, priority effects introduce historical contingency in community assembly, which complicates the search for general patterns in communities (Diamond 1975; Lawton 1999). However, the strength of priority effects is known to vary with ecological context (e.g., community size, Orrock & Fletcher Jr. 2005; phylogenetic diversity, Tan et al. 2012; disturbance, Tucker & Fukami 2014). Progress towards a better understanding of community composition and ecosystem function can therefore be made by identifying conditions that make assembly history important (Chase 2003; Fukami 2015).
Factors affecting the strength of priority effects include both top-down forces (e.g., grazing) and bottom-up forces (e.g., resource availability). Grazing by consumers can weaken priority effects by reducing population growth of early-arriving species (Morin 1984) or by altering the competitive hierarchy among species (Louette & De Meester 2007; Chase et al. 2009). It is also possible, however, for consumers to strengthen priority effects if they limit the effective size of a local community (Chase et al. 2009) or equalize fitness differences among species by preferentially consuming a competitively dominant species (Fukami et al. 2016). Greater resource availability generally strengthens priority effects by increasing the growth of early-arriving species (Chase 2003; Fukami 2015; but see Fukami et al. 2010; Dickie et al. 2012), as shown in plants (Kardol et al. 2013), animals (Chase 2010), and microbes (Vannette & Fukami 2014).
Although both top-down and bottom-up forces determine assembly history effects, their potential interactive effects remain unclear. In a pioneering study of grassland plant communities, Ejrnæs et al. (2006) found that interactions between species
117 introduction order, nutrient availability, and simulated herbivory (manual defoliation) determined species richness and composition. In that study, however, introduction order was limited to two treatments that were confounded with plant type (specialist vs generalist) and the simulated herbivory manipulation was applied equally to all species. So far, no studies, to our knowledge, have addressed the possibility that the relationship between assembly history and emergent functional properties of communities depend upon interactions between bottom-up and top-down forces.
In this paper, we experimentally test for the joint effects of top-down and bottom-up forces on priority effects. To this end, we assembled communities of wood- decomposing fungi in laboratory microcosms, manipulating species arrival order, initial nitrogen availability, and the presence of fungivorous springtails (Collembola). Both increased nitrogen availability and fungivore grazing are known to influence the composition and function of fungal communities (Wardle & Yeates 1993; Crowther et al. 2012; Morrison et al. 2016), but their potential to interactively regulate priority effects is unexplored. Because resource availability affects fungal communities more strongly than grazing (Zimmerman et al. 1995; Wardle 2002; Moore et al. 2003), we predicted that nitrogen addition would influence the strength of priority effects more than fungivore presence. However, because the Collembola may also be resource limited initially (Booth & Anderson 1979; Larsen et al. 2011), we also predicted that the influence of fungivores on assembly processes will be greater with, rather than without, nitrogen addition due to more rapid population growth in response to increased fungal resource availability. This study builds on earlier experimental work linking priority effects to functional properties of wood-decomposing fungal communities (i.e., decomposition rate and carbon and nutrient dynamics; Fukami et al. 2010; Dickie et al. 2012). The novelty of the present work is that, unlike any previous research we are aware of, manipulations of both top-down and bottom-up factors were combined with manipulations of species arrival order.
118 Materials and Methods
Overview and experimental design
Fungal communities, consisting of 10 species, were assembled on sterile wood disks in individual microcosms at one of two initial nitrogen levels and in the presence or absence of Collembola. Assembly history was manipulated by introducing one of 4 randomly selected initial fungal species 4 weeks prior to introducing the remaining 9 species. A control treatment, where no fungi were introduced, was also included. The experimental design was fully factorial, with 5 replicates of each treatment destructively harvested at 6 and 12 months, resulting in 200 samples (i.e., 2 nitrogen levels x 2 fungivore treatments x 5 assembly histories (including control) x 5 replicates x 2 harvests, as detailed below). To aid interpretation of the fungal community-level responses to the experimental treatments, a second set of microcosms were inoculated with only one fungal species, resulting in 400 additional, single-species replicates (i.e., 10 species x 2 nitrogen levels x 2 fungivore treatments x 5 replicates x 2 harvests).
Microcosms
Microcosms were prepared as in Fukami et al. (2010). Briefly, mineral soil was collected from a monospecific stand of old-growth Nothofagus solandri (Allen et al. 2000; Clinton et al. 2002) at Craigieburn Forest Park, South Island, New Zealand (43º8.556 S, 171º42.825 E, 1000 m elevation) and added to 2 L glass jars. Each jar received 800 g of dry, well-mixed soil and 400 ml water or water with NH 4NO3 (see below). Wood disks (approximately 8 cm diameter by 1 cm thick) were prepared from freshly felled N. solandri trees, dried to constant weight to determine their initial dry mass. The wood disks were then soaked in water for 48 hrs and placed on top of the soil in the microcosms. Before inoculation with fungi, the assembled microcosms were capped with loosely fitting lids and autoclaved twice at 121 ºC with a 24 hr interval. Experimental treatments were randomly assigned to jars, which were then randomly
119 arranged on laboratory shelves and maintained in the dark at approximately 20 ºC and 60% humidity throughout the course of the experiment.
Assembly history
The fungi used in this study were isolated from the same site as soils (above). The 10 species used in this study largely mirrored those used by Fukami et al. (2010) and Dickie et al. (2012), which were selected from the initial collection of 96 species to maximize ease of handling, phylogenetic diversity, and adequate molecular discrimination using nrDNA internal transcribed spacer (ITS) length heterogeneity. In preliminary experiments, we found that one species used in the previous studies, Phlebia nothofagi, caused rapid and complete mortality of Collembola in vitro. Because P. nothofagi was a final dominant species in previous assembly experiments (Fukami et al. 2010) we excluded it from the current study to ensure that we could effectively manipulate fungivore presence. Instead of this species, we used Helicogloea alba (New Zealand Fungarium, PDD 91620; ICMP 17042; GenBank accession GQ411522.1) in the current study.
Assembly history treatments were established by first inoculating sterilized wood disks with one of four randomly selected species, either Ascocoryne sarcoides, Bisporella citrina, Daldinia novae-zelandiae, or Trametes versicolor. The number of possible assembly history treatments was limited due to practical constraints on the number of samples needed for the factorial design. Inoculation consisted of aseptically transferring a 5 mm plug from a colonized agar plate to a predetermined location on each wood disk. The remaining 9 species were aseptically transferred to the wood disk 4 weeks later. The inoculation positions for each species, relative to the other species, was invariant across all treatments to avoid potential confounding effects of spatial arrangement (Fig. S1).
120 Nitrogen availability
We manipulated nitrogen availability within the microcosms. The mineral soil collected from the field site and used in the microcosms was nutrient poor and primary productivity at the site was nitrogen limited (Clinton et al. 2002; Davis et al. 2004; Smaill et al. 2011). Microcosms assigned to the low nitrogen treatment received no supplemental nitrogen and reflected the natural nutrient availability in the wood disks and mineral soil. The high nitrogen treatment received 1 g NH4NO3 per microcosm. The resulting range of nitrogen availability in the experimental treatments reflected the range of values naturally present in soils where the fungi were collected, including the relatively nutrient rich organic soil layers, and coincides with the high and low nitrogen treatments used by Fukami et al. (2010).
Fungivore presence
We manipulated the presence of fungivorous mesofauna in the experimental microcosms by adding a species of Collembola, Folsomia candida. This species was chosen because it is primarily mycophagous and could be easily reared under laboratory conditions, which helped to avoid unintended introduction of contaminant fungi to the microcosms (Fountain & Hopkin 2005). In addition, F. candida has been previously used in studies of the ecological impacts of grazing by soil fungivores (e.g., Lussenhop 1996; Cragg & Bardgett 2001). To ensure that the initial fungal species had an opportunity to establish on the wood disk and that there would be sufficient resources for the F. candida population, approximately 30 individuals of the fungivores were aseptically transferred into each of half of the microcosms belonging to each assembly and nutrient treatment 4 weeks after the final fungal inoculation.
Harvest and data collection
Microcosms were destructively harvested at one of two time points, 6 or 12 months after the introduction of the initial fungal species, to determine if priority effects were persisting or attenuating over time. Specifically, each wood disk was removed within a
121 laminar flow hood and split along 8 radial lines at predetermined locations relative to the initial inoculation points (Fig. S1). A custom, sterile wood-splitting device was used to avoid contamination of the interior wood, as detailed in Fukami et al. (2010) and Dickie et al. (2012). Approximately 5.5 mg of sawdust was removed from the interior wood at each radial split using a flame-sterilized 1.5-mm drill bit, and collected in a sterile 0.2 ml tube.
To characterize fungal species composition, sawdust samples were analyzed using a modified length-heterogeneity polymerase chain reaction (LH-PCH) assay. First, total fungal DNA was extracted and PCR amplified using the Sigma REDExtract-N-amp Plant PCR kit and the labeled primers ITS1F-6FAM and ITS4-VIC (Dickie et al. 2009). Fungi present in each PCR product were then identified by analyzing ITS fragment lengths with 50-cm capillary electrophoresis in a 3130xl Genetic Analyzer (Applied Biosystems, USA). Peak profiles were compared against a database of the known ITS sequence lengths and converted to species presence data for each sawdust sample using TRAMPR (Fitzjohn & Dickie 2007).
Functional outcomes of fungal community assembly were characterized by measuring the wood mass lost to decomposition and the final carbon and nitrogen concentrations in the wood disks. Following sampling for molecular analysis, the wood disks were dried at 40 ºC to a constant weight and the mass was recorded. The change in dry mass for each disk was used as a proxy for decomposition, but also includes fungal tissue. The mass of sawdust removed for molecular analysis was not accounted for in the final dry weights, but was negligible relative to mass lost to decomposition and was consistent across treatments. Carbon and nitrogen were analyzed after determining the final dry mass of each disk by collecting a composite sawdust sample from new locations along each of the 8 split edges using a 2-mm drill bit.
Statistical analysis
To determine whether the effects of assembly history were regulated interactively by nitrogen availability and fungivore grazing, we tested for significant three-way
122 interactions of these experimental treatments as predictors of fungal community composition and function. To quantify fungal species composition, we used, as a measure of species prevalence in the wood, the numbers of subsamples from each wood disk in which each species was observed to construct a species by sample matrix of relative abundance. The relationships between species composition and experimental treatments were visualized using non-metric multidimensional scaling (NMDS) and the significance of these relationships were tested with a permutational multivariate analysis of variance (perMANOVA) using the adonis function in the R- package vegan (Oksanen et al. 2017). For both the NMDS and perMANOVA analyses, community dissimilarity was calculated using the modified Gower dissimilarity index proposed by Anderson et al. (2006). In addition, to examine how experimental treatments affected individual species, the proportion of subsamples per microcosm where a species was detected was modeled as a binomial response variable using generalized linear models. The full model for each species was simplified using reverse model selection and likelihood-ratio chi square tests.
To test whether the strength of priority effects on species composition was influenced by our experimental treatments, we compared the compositional dissimilarity among assembly history treatments (i.e., a measure of the variation in species composition that can be attributed to species arrival order) for each factorial combination of nitrogen availability and fungivore presence. To quantify compositional dissimilarity, we first calculated the beta-dispersion among samples with the same nitrogen and fungivore treatment using the modified Gower dissimilarity index and the function betadisper in the R-package vegan. We then extracted the distance of each sample to its group centroid and tested whether they were correlated with the nitrogen and fungivore treatments and their interaction, including the assembly history treatment as a random effect, using the function lme in the R-package nlme (Pinheiro et al. 2016).
The effect of the experimental treatments on wood decomposition were tested using analyses of variance (ANOVAs), with the proportion of dry wood mass lost, the percent nitrogen, and the carbon-to-nitrogen ratio as response variables. The carbon-
123 to-nitrogen ratio was log-transformed to better meet the assumption of normality. The full models, including all experimental treatments and interactions, were simplified for each response variable using F tests. In order to link the properties of the wood substrate to fungal community composition, the mass lost, percent nitrogen, and carbon-to-nitrogen ratio for each sample were correlated with the NMDS ordination by vector fitting, using the envfit function in the R-package vegan.
Analyses of species relative abundance and functional outcomes were repeated for the single-species microcosms using the same statistical methods, with the exception that assembly history was not included as a factor. All statistical analyses were conducted in R v3.3.1 (R Development Core Team 2016).
Results
Patterns of fungal community composition and wood decomposition were qualitatively similar at both harvest time points and statistical support for treatment effects after 12 months were as strong as, or stronger than, those observed at 6 months. We will focus here on the 12-month results and report the 6-month results as supplemental materials (Figs. S2-S4).
Fungal community composition
The composition of the fungal community after 12 months varied with assembly history, initial nitrogen availability, fungivore presence, and their interactions
(perMANOVA, F15,64 = 9.32, p < 0.001; Fig. 1a), including a significant effect of the three-way interaction (F3,64 = 2.246, p = 0.019). The greatest amount of variation in community composition was explained by assembly history (R2 = 0.36), followed by nitrogen availability (R2 = 0.21) and fungivore presence (R2 = 0.11). Beta-dispersion among assembly history treatments (i.e., the strength of priority effects; Fig. 1b) was reduced with nitrogen addition (F1,73 = 16.59, p < 0.001), but was unaffected by fungivore presence (F1,73 = 0.153, p = 0.69) or the interaction between nitrogen and fungivores (F1,73 = 0.038, p = 0.85).
124 Species varied in complex ways in their response to the experimental treatments, both in the mixed communities (Fig. 2) and in the single species microcosms (Fig. 3). For example, the proportion of wood disk subsamples where T. versicolor was observed was strongly influenced by assembly history, averaging 86.1% when arriving ahead of other species and only 3.9% otherwise, but was not significantly influenced by fungivore presence or nitrogen availability. The abundance of Sistotrema brinkmannii also responded to assembly history, but only under low nitrogen conditions and did not appear to be influenced strongly by fungivore grazing. For A. sarcoides, B. citrina, Calocera sinensis, and D. novae-zelandiae, the effect of assembly history on abundance varied with both nitrogen availability and fungivore presence. For example, B. citrina was ubiquitous in the wood disk when arriving first under low N conditions, irrespective of grazing. However, under high nitrogen conditions, B. citrina colonized 40% and 4.4% of the wood disk in the absence and presence of fungivores, respectively. When arriving after another species, the prevalence of B. citrina depended on the initial species identity, nitrogen availability and fungivores. For example, the prevalence of B. citrina when preceded by T. versicolor, relative to its prevalence when arriving after T. versicolor, was dependent on the nitrogen and fungivore treatments. Under low nitrogen conditions, the prevalence of B. citrina was reduced when T. versicolor arrived first, irrespective of fungivore presence. Under high nitrogen conditions, initial colonization by T. versicolor had no effect on the prevalence of B. citrina when fungivores were absent, but facilitated growth when fungivores were present. Three species, Armillaria sp., H. alba and Pleurotus purpureoolivaceus, were not detected at all and one species, Artomyces (≡ Clavicorona) candelabrum, was only detected twice at 12 months and once at 6 months.
Wood decomposition
The physical and chemical properties of the decomposing wood disk were correlated with assembly history, initial nitrogen availability, fungivore presence and their interactions (Fig. 4). Significant three-way interactions were observed for wood mass
125 loss (F3,64 = 8.99, p < 0.001), final nitrogen concentration (F3,64 = 11.36, p < 0.001), and the carbon-to-nitrogen ratio (F3,64 = 4.96, p = 0.004). Fungal species composition in NMDS was correlated with these functional outcomes, based on envfit (mass lost, R2 = 0.44, p < 0.001; % N, R2 = 0.26, p < 0.001; C:N, R2 = 0.46, p < 0.001).
The effect of assembly history on decomposition rate depended on nitrogen availability and fungivore presence (Fig. 4a-d). Under low nitrogen conditions, decomposition rate varied among assembly history treatments to a greater degree when fungivores were not present. However, under high nitrogen conditions decomposition rate was only minimally influenced by assembly history or fungivores. This pattern can be largely explained by the response of T. versicolor to the experimental treatments. In the single-species microcosms, decomposition by T. versicolor was approximately 3-fold quicker than by any other species under low nitrogen conditions without fungivores, 2-fold quicker with the addition of fungivores, but only slightly quicker than the across-species average under high nitrogen conditions, with or without fungivores (Fig. 3g-l). Because the prevalence of T. versicolor was strongly influenced by introduction order (Fig. 2u-x) and T. versicolor function was strongly influenced by the nitrogen and fungivore treatments (Fig. 3l, r, x), decomposition rate was much higher when T. versicolor arrived first to colonize a large portion of the wood, but this effect was reduced by nitrogen addition and fungivore presence. The introduction order of T. versicolor was also largely responsible for the effect of assembly history on nitrogen concentration and carbon-to- nitrogen ratio during decomposition (Fig. 4e-l). However, in contrast to decomposition rate, the patterns in the single species microcosms (Fig. 3r, x) could not be directly mapped to the mixed species treatments (Fig. 4e-l).
Discussion
Our results provide experimental evidence that nutrient availability and fungivore presence interact with fungal inoculation history to determine species composition (Fig. 1) and wood decomposition (Fig. 3). Prior research has shown that fungivore
126 grazing can alter competitive interactions among fungi by suppressing or stimulating hyphal growth, depending on the species involved (Crowther et al. 2011a). Our results suggest that these effects of fungivores may depend on resource availability. For species composition, the joint effects of nitrogen availability and fungivore presence can be illustrated by considering the prevalence of B. citrina. Under low nitrogen conditions, this species occupied the smallest volume of wood when arriving after T. versicolor, but at high nitrogen and in the presence of fungivores, had its highest occupancy when arriving after the same species (Fig. 2e-h). While the exact mechanisms remain uncertain, it is possible that increased nitrogen availability altered the effects of fungivores by simply altering the composition of the fungal community that the Collembola were interacting with (Morrison et al. 2016); by facilitating defensive responses to fungivore grazing, such as the production of nitrogen-rich secondary metabolites (Crowther et al. 2011b); or by altering hyphal growth rate and morphology (Dickie et al. 1998).
Although greater resource availability is thought to generally strengthen priority effects (Chase 2003; Fukami 2015), we found that nitrogen addition weakened priority effects for species composition. In this system, nitrogen addition appears to have reduced the inhibition of late-arriving species by early-arriving ones by allowing more species to occupy a larger portion of the wood substrate (Fig. 2). There are several possible explanations for this observation. For example, if priority effects are primarily the result of niche preemption, nitrogen addition may have modified hyphal morphology by increasing local hyphal density while reducing the spatial extent of initial resource exploration (Dickie et al. 1998). Alternatively, priority effects can be the result of niche modification by early-arriving species. There is some evidence that the strength of priority effects for wood-decomposing fungi is linked to their ability to degrade lignocellulose (Cline & Zak 2015; Hiscox et al. 2015a, b), presumably as a result of biochemical modification of the substrate material. In our study, initial colonization by T. versicolor, a ligninolytic, white-rot fungus, led to a distinct species composition, but this effect was less apparent when nitrogen was added (Fig. 1a).
127 Cline & Zak (2015) observed a similar phenomenon, reporting that priority effects exerted by ligninolytic fungi were stronger on low-nutrient, high-lignin leaf litter and weaker on more nutrient-rich litter. However, these observations may reflect either a change in the relative impact of the initial colonizer, due to a change in extracellular enzyme production (Leatham & Kirk 1983), or greater nutrient availability facilitating colonization by later arriving species.
For wood decomposition, we found that the strength of priority effects was modified by the interactive effects of nitrogen availability and fungivore presence, primarily due to the response of T. versicolor to the experimental treatments. This species induced a relatively high decomposition rate compared to the other species, but this distinction was diminished by fungivore presence and nearly eliminated by nitrogen addition (Fig 3l). For some wood-decomposing fungi, nitrogen supplementation can inhibit or delay ligninolytic enzyme production, resulting in lower decomposition rates (Keyser et al. 1978; Leatham & Kirk 1983). Grazing by fungivores has also been shown to affect extracellular enzyme production and decomposition rate (Crowther et al. 2011c, d). Both nitrogen and fungivores may have affected decomposition by modifying enzyme production in T. versicolor, but the stronger effect of nitrogen addition may have precluded any additional effect of fungivory. Alternatively, if fungivores reduced decomposition rate by requiring that the fungus reallocate resources to defense or to repair damage caused by grazing, nitrogen supplementation may simply satisfy the additional demand imposed by fungivore grazing.
The ligninolytic capabilities of T. versicolor may largely explain the differences in decomposition rate among assembly history treatments, but our results suggest that this species was also more adept at translocating available nitrogen than the other species: highest mean nitrogen concentrations at 6 months were observed when T. versicolor arrived first (Fig. S5e-h), but at 12 months these treatments had the lowest mean nitrogen concentrations (Fig. 4e-h). These changes in nitrogen concentration are not consistent with mass loss to respiration and suggest that T. versicolor transported nitrogen from the soil to the wood substrate during the early stages of decomposition
128 and away during the later stages. However, these patterns varied with nitrogen addition and fungivore presence and were not directly correlated with decomposition rate. In addition, patterns of nitrogen concentration in the mixed-species treatments did not directly reflect the patterns observed in the single species treatments (Fig. 3m-x). These discrepancies suggest that nitrogen dynamics during decomposition were influenced by interactions between initial nitrogen availability, fungivore presence and interspecific fungal interactions, possibly involving the production of nitrogen-rich secondary defense compounds or modified, combative hyphae (Hedlund et al. 1991; Heilmann-Clausen & Boddy 2005; Crowther et al. 2012).
Our study used a small number of fungal species and assembly history treatments so that the strength of priority effects was primarily attributed to the influence of the experimental treatments on one species, T. versicolor. A larger species pool is likely to include a broader spectrum of functionally diverse species and should also increase the potential for priority effects by increasing the probability that some species will have high niche overlap (Fukami 2015). Similarly, we used only one fungivore species to introduce top-down pressure, but fungi are likely to be consumed by a wide range of organisms in natural systems. Recent evidence suggests that fungivorous macrofauna in soils, such as isopods or millipedes, can have greater effects on fungal community assembly than Collembola and other mesofauna (Crowther et al. 2011a, 2013). Our use of Collembola can be viewed as a conservative test of top-down factors in fungal community assembly. Future efforts to extend our results could incorporate mesh bags to exclude different size-classes of soil fauna in field based experimental manipulations (Dickie et al. 2012).
Although we have focused here on a simple community of wood-decomposing fungi, interactive regulation of priority effects by top-down and bottom-up forces may be common in natural systems. For example, Bakker et al. (2006) demonstrated that the effects of herbivores on plant diversity in grasslands can vary from negative to positive with increasing resource availability, suggesting that the joint effects of top- down and bottom-up factors regulate assembly processes in these communities. In
129 addition, if herbivores preferentially consume more productive species, or certain plant functional groups, they could modify the effects of assembly history and local resource availability on ecosystem function (Ejrnæs et al. 2006). Similarly, in aquatic invertebrate communities the strength of priority effects on community composition is also known to vary with resources (Chase 2010) and predation (Louette & De Meester 2007; Chase et al. 2009). If resource availability also influences the abundance of predators in these systems, interactive control of the strength of priority effects would be likely. Interactive effects could also arise if predator presence limits prey species’ access to resources, indirectly modifying resource availability. We suggest that further efforts to understand how multiple factors interact to regulate priority effects will contribute to identifying the conditions that make assembly history important to community structure and function.
Acknowledgments
We thank Duckchul Park, Karyn Hoksbergen, and Barbara Paulus for laboratory assistance, Peter Johnston for advice, and Shenandoah Forest for donation of wood. This work was supported by the Marsden Fund using New Zealand Government funding administered by the Royal Society of New Zealand (contract LCR0503) and the former New Zealand Foundation for Research, Science and Technology (Ecosystem Resilience Outcome-Based Investment; contract C09X0502). DRL was supported by the Mycological Society of America Graduate Research Fellowship.
130 References cited
Allen, R.B., Buchanan, P.K., Clinton, P.W. & Cone, A.J. (2000). Composition and diversity of fungi on decaying logs in a New Zealand temperate beech (Nothofagus) forest. Can. J. For. Res., 30, 1025–1033. Anderson, M.J., Ellingsen, K.E. & McArdle, B.H. (2006). Multivariate dispersion as a measure of beta diversity. Ecol. Lett., 9, 683–693. Bakker, E.S., Ritchie, M.E., Olff, H., Milchunas, D.G. & Knops, J.M.H. (2006). Herbivore impact on grassland plant diversity depends on habitat productivity and herbivore size. Ecol. Lett., 9, 780–788. Booth, R.G. & Anderson, J.M. (1979). The influence of fungal food quality on the growth and fecundity of Folsomia candida (Collembola: Isotomidae). Oecologia, 38, 317–323. Chase, J.M. (2003). Community assembly: when should history matter? Oecologia, 136, 489–98. Chase, J.M. (2010). Stochastic community assembly causes higher biodiversity in more productive environments. Science, 328, 1388–1391. Chase, J.M., Biro, E.G., Ryberg, W.A. & Smith, K.G. (2009). Predators temper the relative importance of stochastic processes in the assembly of prey metacommunities. Ecol. Lett., 12, 1210–1218. Cline, L.C. & Zak, D.R. (2015). Initial colonization, community assembly and ecosystem function: fungal colonist traits and litter biochemistry mediate decay rate. Mol. Ecol., 24, 5045–5058. Clinton, P.W., Allen, R.B. & Davis, M.R. (2002). Nitrogen storage and availability during stand development in a New Zealand Nothofagus forest. Can. J. For. Res., 32, 344–352. Cragg, R.G. & Bardgett, R.D. (2001). How changes in soil faunal diversity and composition within a trophic group influence decomposition processes. Soil Biol. Biochem., 33, 2073–2081.
131 Crowther, T., Stanton, D. & Thomas, S. (2013). Top-down control of soil fungal community composition by a globally distributed keystone consumer. Ecology, 94, 2518–2528. Crowther, T.W., Boddy, L. & Hefin Jones, T. (2012). Functional and ecological consequences of saprotrophic fungus-grazer interactions. ISME J., 6, 1992– 2001. Crowther, T.W., Boddy, L. & Jones, T.H. (2011a). Outcomes of fungal interactions are determined by soil invertebrate grazers. Ecol. Lett., 14, 1134–1142. Crowther, T.W., Boddy, L. & Jones, T.H. (2011b). Species-specific effects of soil fauna on fungal foraging and decomposition. Oecologia, 167, 535–545. Crowther, T.W., Jones, T.H. & Boddy, L. (2011c). Species-specific effects of grazing invertebrates on mycelial emergence and growth from woody resources into soil. Fungal Ecol., 4, 333–341. Crowther, T.W., Jones, T.H., Boddy, L. & Baldrian, P. (2011d). Invertebrate grazing determines enzyme production by basidiomycete fungi. Soil Biol. Biochem., 43, 2060–2068. Davis, M.R., Allen, R.B. & Clinton, P.W. (2004). The influence of N addition on nutrient content, leaf carbon isotope ratio, and productivity in a Nothofagus forest during stand development. Can. J. For. Res., 34, 2037–2048. Diamond, J.M. (1975). Assembly of species communities. In: Ecology and Evolution in Communities (eds. Cody, M.L. & Diamond, J.M.). Harvard University Press, Cambridge, MA, pp. 342–444. Dickie, I.A., Fukami, T., Wilkie, J.P., Allen, R.B. & Buchanan, P.K. (2012). Do assembly history effects attenuate from species to ecosystem properties? A field test with wood-inhabiting fungi. Ecol. Lett., 15, 133–141. Dickie, I.A., Koide, R.T. & Stevens, C.M. (1998). Tissue density and growth response of ectomycorrhizal fungi to nitrogen source and concentration. Mycorrhiza, 8, 145–148.
132 Dickie, I.A., Richardson, S.J. & Wiser, S.K. (2009). Ectomycorrhizal fungal communities and soil chemistry in harvested and unharvested temperate Nothofagus rainforests. Can. J. For. Res., 39, 1069–1079. Drake, J. (1991). Community-assembly mechanics and the structure of an experimental species ensemble. Am. Nat., 137, 1–26. Ejrnæs, R., Bruun, H.H. & Graae, B.J. (2006). Community assembly in experimental grasslands: suitable or timely arrival? Ecology, 87, 1225–1233. Fitzjohn, R.G. & Dickie, I.A. (2007). TRAMPR: an R package for analysis and matching of terminal-restriction fragment length polymorphism (TRFLP) profiles. Mol. Ecol. Notes, 7, 583–587. Fountain, M.T. & Hopkin, S.P. (2005). Folsomia candida (Collembola): a “standard” soil arthropod. Annu. Rev. Entomol., 50, 201–222. Fukami, T. (2015). Historical contingency in community assembly: integrating niches, species pools, and priority effects. Annu. Rev. Ecol. Evol. Syst., 46, 1–23. Fukami, T., Dickie, I.A., Wilkie, J.P., Paulus, B.C., Park, D., Roberts, A., et al. (2010). Assembly history dictates ecosystem functioning: evidence from wood decomposer communities. Ecol. Lett., 13, 675–84. Fukami, T., Mordecai, E.A. & Ostling, A. (2016). A framework for priority effects. J. Veg. Sci., 27, 655–657. Hedlund, K., Boddy, L. & Preston, C.M. (1991). Mycelial responses of the soil fungus, Mortierella isabellina, to grazing by Onychiurus armatus (Collembola). Soil Biol. Biochem., 23, 361–366. Heilmann-Clausen, J. & Boddy, L. (2005). Inhibition and stimulation effects in communities of wood decay fungi: exudates from colonized wood influence growth by other species. Microb. Ecol., 49, 399–406. Hiscox, J., Savoury, M., Müller, C.T., Lindahl, B.D., Rogers, H.J. & Boddy, L. (2015a). Priority effects during fungal community establishment in beech wood. ISME J., 1–15.
133 Hiscox, J., Savoury, M., Vaughan, I.P., Müller, C.T. & Boddy, L. (2015b). Antagonistic fungal interactions influence carbon dioxide evolution from decomposing wood. Fungal Ecol., 14, 24–32. Kardol, P., Souza, L. & Classen, A.T. (2013). Resource availability mediates the importance of priority effects in plant community assembly and ecosystem function. Oikos, 122, 84–94. Keyser, P., Kirk, T.K. & Zeikus, J.G. (1978). Ligninolytic enzyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J. Bacteriol., 135, 790–797. Körner, C., Stöcklin, J., Reuther-Thiébaud, L. & Pelaez-Riedl, S. (2008). Small differences in arrival time influence composition and productivity of plant communities. New Phytol., 177, 698–705. Larsen, T., Ventura, M., O’Brien, D.M., Magid, J., Lomstein, B.A. & Larsen, J. (2011). Contrasting effects of nitrogen limitation and amino acid imbalance on carbon and nitrogen turnover in three species of Collembola. Soil Biol. Biochem., 43, 749–759. Lawton, J.H. (1999). Are there general laws in ecology? Oikos, 84, 177–192. Leatham, G.F. & Kirk, T.K. (1983). Regulation of ligninolytic activity by nutrient nitrogen in white-rot basidiomycetes. FEMS Microbiol. Lett., 16, 65–67. Louette, G. & De Meester, L. (2007). Predation and priority effects in experimental zooplankton communities. Oikos, 116, 419–426. Lussenhop, J. (1996). Collembola as mediators of microbial symbiont effects upon soybean. Soil Biol. Biochem., 28, 363–369. Moore, J.C., McCann, K., Setälä, H. & De Ruiter, P.C. (2003). Top-down is bottom- up: does predation in the rhizosphere regulate aboveground dynamics? Ecology, 84, 846–857. Morin, P.J. (1984). Odonate guild composition: experiments with colonization history and fish predation. Ecology, 65, 1866–1873.
134 Morrison, E.W., Frey, S.D., Sadowsky, J.J., van Diepen, L.T.A., Thomas, W.K. & Pringle, A. (2016). Chronic nitrogen additions fundamentally restructure the soil fungal community in a temperate forest. Fungal Ecol., 23, 48–57. Oksanen, J., Blanchet, F.G., Friendly, M., Roeland Kindt, Legendre, P., McGlinn, D., et al. (2017). vegan: Community Ecology Package. R package version 2.4-2. Available at: https://CRAN.R-project.org/package=vegan. Orrock, J.L. & Fletcher Jr., R.J. (2005). Changes in community size affect the outcome of competition. Am. Nat., 166, 107–111. Palmgren, A. (1926). Chance as an element in plant geography. In: Proceedings of the International Congress of Plant Sciences (ed. Duggar, B.). Ithica, New York, pp. 591–602. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team. (2016). nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1-130. Available at: https://CRAN.R-project.org/package=nlme. R Development Core Team. (2016). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Smaill, S.J., Clinton, P.W., Allen, R.B. & Davis, M.R. (2011). Climate cues and resources interact to determine seed production by a masting species. J. Ecol., 99, 870–877. Sutherland, J.P. (1974). Multiple stable points in natural communities. Am. Nat., 108, 859–873. Tan, J., Pu, Z., Ryberg, W.A. & Jiang, L. (2012). Species phylogenetic relatedness, priority effects, and ecosystem functioning. Ecology, 93, 1164–1172. Tucker, C.M. & Fukami, T. (2014). Environmental variability counteracts priority effects to facilitate species coexistence: evidence from nectar microbes. Proc. R. Soc. B Biol. Sci., 281, 20132637. Vannette, R.L. & Fukami, T. (2014). Historical contingency in species interactions: towards niche-based predictions. Ecol. Lett., 17, 115–24.
135 Wardle, D.A. (2002). Communities and Ecosystems: Linking the Aboveground and Belowground Components. Princeton University Press, Princeton, New Jersey, USA. Wardle, D.A. & Yeates, G.W. (1993). The dual importance of competition and predation as regulatory forces in terrestrial ecosystems: evidence from decomposer food-webs. Oecologia, 93, 303–306. Zimmerman, J.K., Pulliam, W.M., Lodge, D.J., Quinonesorfila, V., Fetcher, N., Guzmangrajales, S., et al. (1995). Nitrogen immobilization by decomposing woody debris and the recovery of tropical wet forest from hurricane damage. Oikos, 72, 314–322.
136 Figures
Figure 1: (a) Non-metric multidimensional scaling (NMDS) of fungal community composition (stress = 0.17), using the modified Gower dissimilarity index proposed by Anderson et al. (2006). Colors indicate assembly history treatment, fill (empty or solid) indicates the omission or addition of supplemental nitrogen and shape (square vs. circle) indicates the presence or absence of fungivores. (b) Compositional dissimilarity (modified Gower) for each combination of nitrogen and fungivore treatments, calculated using the betadisper function in the R-package vegan. Large points indicate the mean distance to the multivariate group centroid and small points (colored by initial species treatment) indicate the distance for individual samples.
137 Figure 2: Effect of assembly history (H), nitrogen addition (N) and fungivore presence (F) on the abundance (mean % sub-samples occupied +/- SE) of individual fungal species, excluding species that were not observed in more than one sample. Predictors remaining after model selection and their significance levels are shown for individual binomial generalized linear models, where * P < 0.05, ** P < 0.01, *** P < 0.001.
138 Figure 3: Effect of nitrogen addition (N) and fungivore presence (F) on wood volume occupied (a-f), % wood mass loss (g-l), % nitrogen (m-r), and carbon-to-nitrogen ratio (s-x) for single species inoculations. Significant predictors are indicated for each response, where + P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001.
139 Figure 4: Effect of assembly history (H), nitrogen addition (N) and fungivore presence (F) on wood substrate characteristics 12 months after inoculation. Bar heights for all response variables (dry mass [a-d], % nitrogen [e-h] and carbon-to- nitrogen ratio [i-l]) indicate mean +/- SE. Horizontal dotted lines indicate the mean % nitrogen or carbon-to-nitrogen ratio of the non-inoculated controls. Significant predictors are indicated for each response, where * P < 0.05, ** P < 0.01, *** P < 0.001.
140 Supplemental materials
Figure S1: Layout of each wood disk (indicated by larger circle; 8 cm diameter and 1 cm thick) showing the relative locations of inoculation points for each species, the radial lines where the wood disks were split (dashed line) and the sawdust sampling locations (solid points).
141 Figure S2: Effect of assembly history (H), nitrogen addition (N) and fungivore presence (F) on the abundance (mean % sub-samples occupied +/- SE) of individual fungal species, excluding species that were not observed in more than 2 samples after 6 months. Predictors remaining after model selection and their significance levels are shown for individual binomial generalized linear models, where + P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001.
142 Figure S3: Effect of nitrogen addition (N) and fungivore presence (F) on wood volume occupied (a-f), % wood mass loss (g-l), % nitrogen (m-r), and carbon-to- nitrogen ratio (s-x) for single species inoculations after 6 months. Significant predictors are indicated for each response, where + P < 0.1, * P < 0.05, ** P < 0.01, *** P < 0.001.
143 Figure S4: Effect of assembly history (H), nitrogen addition (N) and fungivore presence (F) on wood substrate characteristics 6 months after inoculation. Bar heights for all response variables (dry mass [a-d], % nitrogen [e-h] and carbon-to-nitrogen ratio [i-l]) indicate mean +/- SE. Horizontal dotted lines indicate the mean % nitrogen or carbon-to-nitrogen ratio of the non-inoculated controls. Significant predictors are indicated for each response, where * P < 0.05, ** P < 0.01, *** P < 0.001.
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