FEMS Microbiology Ecology, 97, 2021, fiab016 doi: 10.1093/femsec/fiab016 Advance Access Publication Date: 29 January 2021 Research Article RESEARCH ARTICLE Downloaded from https://academic.oup.com/femsec/article/97/3/fiab016/6123715 by Stanford Libraries user on 19 April 2021 Diversity of putative ericoid mycorrhizal fungi increases with soil age and progressive phosphorus limitation across a 4.1-million-year chronosequence Devin R. Leopold1,2,*,†, Kabir G. Peay1, Peter M. Vitousek1 and Tadashi Fukami1 1Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA 94305–5020, USA and 2Department of Botany and Plant Pathology, Oregon State University, 4575 SW Research Way, Corvallis, OR 97331, USA ∗Corresponding author: OSU Botany and Plant Pathology, 4575 SW Research Way, Corvallis, OR 97333, USA. Tel: 603-702-1203; E-mail: [email protected] One sentence summary: The diversity and species composition of fungal symbionts associated with the roots of ericaceous plants depends on soil age and nutrient availability. Editor: Ian Anderson †Devin R. Leopold, http://orcid.org/0000-0003-2124-2116 ABSTRACT Ericaceous plants rely on ericoid mycorrhizal fungi for nutrient acquisition. However, the factors that affect the composition and structure of fungal communities associated with the roots of ericaceous plants remain largely unknown. Here, we use a 4.1-million-year (myr) soil chronosequence in Hawaii to test the hypothesis that changes in nutrient availability with soil age determine the diversity and species composition of fungi associated with ericoid roots. We sampled roots of a native Hawaiian plant, Vaccinium calycinum, and used DNA metabarcoding to quantify changes in fungal diversity and community composition. We also used a fertilization experiment at the youngest and oldest sites to assess the importance of nutrient limitation. We found an increase in diversity and a clear pattern of species turnover across the chronosequence, driven largely by putative ericoid mycorrhizal fungi. Fertilization with nitrogen at the youngest site and phosphorus at the oldest site reduced fungal diversity, suggesting a direct role of nutrient limitation. Our results also reveal the presence of novel fungal species associated with Hawaiian Ericaceae and suggest a greater importance of phosphorus availability for communities of ericoid mycorrhizal fungi than is generally assumed. Keywords: chronosequence; ericoid mycorrhizae; mycorrhizal diversity; nutrient limitation; pedogenesis; root-associated fungi INTRODUCTION harsh environments where acidic soils, low temperatures, or excessive soil moisture slow the degradation of organic mat- The plant family Ericaceae includes clades that are largely ter and limit mineral nutrient availability (Read 1991;Cairney defined by morphologically and functionally distinct mycor- and Meharg 2003; Mitchell and Gibson 2006). In these habi- rhizal symbioses, with the largest group forming a unique tats, which can be found on every continent except Antarctica endomycorrhizal association known as an ericoid mycorrhiza (Kohout 2017), ericoid mycorrhizal fungi (ErMF) facilitate nutri- (ErM; Pearson and Read 1973a; Perotto et al. 2012; Leopold 2016). ent cycling by degrading complex organic matter and providing The ErM symbiosis allows ericaceous plants to proliferate in Received: 27 August 2020; Accepted: 27 January 2021 C The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Ecology, 2021, Vol. 97, No. 3 their hosts with access to nutrient pools that would be unavail- availability, studies of ErMF from retrogressive, P-limited habi- able otherwise (Wurzburger, Higgins and Hendrick 2012; Adam- tats are lacking (Dickie et al. 2013). czyk et al. 2016; Perotto, Daghino and Martino 2018). Ericaceous To explore the effects of long-term soil development on ErMF, plants and ErMF also contribute to carbon sequestration in these and the broader community of ericaceous root-associated fungi, environments through the production of recalcitrant plant lit- we sampled roots of a single ericaceous plant species, Vac- ter and hyphal necromass (Clemmensen et al. 2013, 2015). How- cinium calycinum, across a 4.1-million-year (myr) soil chronose- ever, despite the ecological importance of the ErM symbiosis, quence in the Hawaiian Islands, known as the Long Substrate and significant functional variation within and among ErMF Age Gradient (LSAG; Vitousek 2004). The LSAG is a useful sys- species (Cairney et al. 2000; Whittaker and Cairney 2001;Grelet tem to study ErMF for three reasons. First, V. calycinum,acom- et al. 2009b; Wurzburger, Higgins and Hendrick 2012), little is mon host plant species, is present at all LSAG sites. The preva- known about how factors such as soil nutrient availability affect lence of a common host species across the chronosequence Downloaded from https://academic.oup.com/femsec/article/97/3/fiab016/6123715 by Stanford Libraries user on 19 April 2021 the community composition of ErMF and other fungi associated controls for changes in host identity, which can influence the with ericoid roots. composition of mycorrhizal symbiont communities (Mart´ınez- Currently, most data on ErMF originates from temperate and Garc´ıa et al. 2015), though intraspecific variation in host traits boreal regions, where cooler temperatures and repeated glacia- affecting plant–fungal interactions are also possible (Johnson tion throughout the Pleistocene have maintained widespread et al. 2010). Second, vegetation at LSAG sites is dominated by nitrogen (N) limitation, even in late-successional ecosystems a single canopy tree species and a common suite of under- (Tamm 1991; Vitousek and Howarth 1991). As a result, research story plants (Kitayama and Mueller-Dombois 1995), which all on ErMF has focused on their ability to utilize various organic form mycorrhizal associations with AMF (Koske, Gemma and N sources and influence host N status (Leake and Read 1991; Flynn 1992). The only other ericaceous species present at any of Nasholm et al. 1998; Xiao and Berch 1999;Cairneyet al. 2000; the LSAG sites, Vaccinium dentatum and Leptecophylla tameiameiae, Grelet et al. 2009b; Ishida and Nordin 2010). However, the distri- occur very sparsely at all sites, which further limits the poten- bution of ericaceous plants is not limited by these habitat con- tial impact of changes in host vegetation along the chronose- ditions (Leopold 2016; Kohout 2017) and there is evidence that quence. Third, changes in nutrient limitation across the LSAG ErMF also facilitate the uptake of phosphorus (P) and other min- have been experimentally demonstrated through N and P fer- eral nutrients for host plants (Pearson and Read 1973b; Read tilizer addition experiments (Vitousek 2004). Because these fer- 1983; Myers and Leake 1996). This observation suggests that the tilization experiments are long-term (>20 years) and ongoing relative availability of limiting nutrients in soil, P as well as N, at the oldest and youngest LSAG sites, this system presents a may influence ericaceous plant-fungal interactions and ErMF unique opportunity to experimentally test whether ecosystem- community composition (Hazard et al. 2014; Van Geel et al. 2020). level nutrient limitation (i.e. N and P availability in young and One source of natural variation in the availability of N and P old sites, respectively) plays a role in structuring communities in soil is pedogenesis, or long-term soil development. Over hun- of ericaceous root-associated fungi. dreds of thousands to millions of years, and in the absence of We began with the hypothesis that the diversity of fungi rejuvenating disturbance (Peltzer et al. 2010), pedogenesis causes associated with ericaceous roots increases throughout long- predictable changes in ecosystem-level nutrient limitation, pri- term soil development, with ErMF diversity increasing owing to marily through the accumulation of N from biological inputs the accumulation of complex organic nutrient pools. We pre- and the progressive loss and occlusion of rock-derived nutrients, dicted that diversity would increase most rapidly during the especially P (Walker and Syers 1976; Chadwick et al. 1999). These early stages of pedogenesis due to the formation and develop- changes result in an initial, progressive phase, defined by N lim- ment of an organic soil horizon and that an increase would con- itation of primary productivity and the rapid accumulation of tinue into the retrogressive stages as soil weathering and occlu- organic matter; a mature phase, defined by maximal produc- sion of mineral nutrients promotes niche partitioning in the rhi- tivity and co-limitation by N and P; and a retrogressive phase, zosphere (Turner 2008; DeForest and Scott 2010). We also used defined by P limitation and reduced rates of nutrient cycling long-term fertilization experiments to test the hypothesis that (Vitousek and Farrington 1997; Richardson et al. 2004; Wardle nutrient limitation of primary productivity, N at the youngest 2004; Peltzer et al. 2010). site and P at the oldest site, shapes the species composition of Because the direct observation of long-term soil develop- ErMF and other ericaceous root-associated fungi. ment 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 rea- MATERIALS AND METHODS sonably