Plant, fungal, bacterial, and nitrogen interactions in the litter layer of a native Patagonian forest

Introduction

Plant–microbial interactions are increasingly recognized as important drivers of terrestrial ecosystem functioning (van der Putten et al., 2013). These interactions are particularly important aboveground, where microbes drive decomposition of senescent plant material (litter) that lies on the soil surface. Litter decomposition is a key first step in carbon and nutrient turnover in terrestrial ecosystems (Swift, Heal & Anderson, 1979; Schlesinger & Bernhardt, 2013). The effects of plant species identity on litter decomposition have been extensively studied and have contributed greatly to our understanding of the control of litter quality on carbon and nutrient cycling in terrestrial ecosystems (Pérez-Harguindeguy et al., 2000; Vivanco & Austin, 2006). Nevertheless, we still lack a clear understanding of how plant species affect microbial communities, which function as the decomposing engine in many forest ecosystems.

Plant litter is the main source of carbon, energy and nutrients for heterotrophic microbial communities in the litter layer in terrestrial ecosystems. Plant species vary greatly in the physico-chemical characteristics of litter, such as carbon:nitrogen (C:N) ratio, nitrogen (N) and lignin content, and such, the resource quality that they represent for microbial organisms. In addition, plant species also affect the abiotic and the biotic environment in the litter layer through changes in litter biomass, pH, and their interactions with other organisms like herbivores, symbionts and soil fauna (Eviner, Chapin & Vaughn, 2006). Thus, different plant species may give rise to a distinct microbial community in the litter layer through differences in the nature of their litter, and their interactions with other above- and belowground organisms (Austin et al., 2014). This specificity of plant–microbial interactions might change if the availability of resources changes in the environment. Nitrogen (N) is a key resource that microbes obtain from plant litter and from the surrounding environment. Increases in N availability due to N addition have shown important changes in soil bacterial and fungal communities (Leff et al., 2015). It is unclear however, how these changes alter plant–microbial interactions, and if bacteria and fungi are differentially affected.

In most natural ecosystems, plant species co-occur with other plant species creating mixtures of litter from different plant species in the litter layer. Litter mixtures have shown variable effects on decomposition, from additive effects, where litter mixtures decompose at a rate expected from component litter species incubated separately, to strong antagonistic and synergistic effects (Hättenschwiler, Tiunov & Scheu, 2005). Part of this variation has been attributed to the litter chemistry and nutrient transfer among litter types (Handa et al., 2014; Barbe et al., 2017), and the interaction with soil fauna (Hättenschwiler & Gasser, 2005). Litter mixing can also directionally alter the microbial community in the litter layer (Chapman et al., 2013). Yet, the effects of the natural mixing of litter species on bacterial and fungal communities in undisturbed temperate forest have been rarely assessed.

Disentangling plant species effects from other ecosystem variables like climate and soil resources on microbial communities in natural ecosystems is often difficult, mostly because plant species distribution largely reflect plant species interactions with the environment (Chapin et al., 1987). Experimental attempts to overcome this issue include ‘common garden experiments,’ where different plant species are cultivated in a common environment and their effects are evaluated. This approach, however, complicates the exploration of natural long-term plant–microbial interactions, especially with old-growth tree species which may require long time periods to develop sustained plant–microbial interactions.

Patagonian old-growth temperate forests, located at the southern end of South America, offer the opportunity to study long-term plant–microbial interactions in a natural ecosystem with very low historical and present human impact. These Patagonian forests are co-dominated by three tree species of the genus Nothofagus distributed in a well-mixed mosaic (Veblen et al., 1996). Within this mosaic, we identified microsites or ‘tree triangles’ defined by the intersection of three tree-canopies that varied in tree Nothofagus species identity. We identified four types of tree-triangles, three monospecific (Nothofagus dombeyi, N. nervosa, and N. obliqua) and a plurispecific tree triangle (N. dombeyi, N. nervosa, and N. obliqua), that resulted in an appropriate spatial scale to isolate the effects of tree species identity on microbial communities from other ecosystem factors (Vivanco & Austin, 2008) (Fig. S1). In this forest, we previously demonstrated the existence of home field advantage (HFA) for leaf litter decomposition (Vivanco & Austin, 2008). HFA is the observation that leaf litter decomposes faster beneath plants of the same species (home) than beneath different plant species (away), even for slow decomposing litter. We also showed that N addition disrupted HFA and accelerated litter decomposition (Vivanco & Austin, 2011). Our mechanistic understanding of HFA and N effects on litter decomposition is still lacking, but the nature of these phenomena suggests that particular plant–microbial associations in the litter layer might be central to observed changes in carbon turnover due to plant species identity and resource availability (Austin et al., 2014). Resource availability in natural ecosystems can be greatly altered by human activities occurring in neighbour regions. For example, many temperate forests in the Northern Hemisphere have received chronic and increasing inputs of N since the industrial revolution (Dentener et al., 2006). Studies in unpolluted Patagonian forests increase our understanding of the control of N on ecosystem functioning before human domination of natural systems and potential impact in other parts of the world.

Here we characterized the fungal and bacterial community of the litter layer in the Nothofagus tree triangles with and without N addition by amplicon sequencing of the Internal transcribed spacer (ITS) region and the 16s rRNA gene respectively, to evaluate plant species–microbial interactions and their changes due to N addition. We additionally quantified the magnitude of change in litter decomposition due to litter incubation away from the home tree triangle and due to N addition, using data from previous studies (Vivanco & Austin, 2008, 2011). We focused on the following questions: (1) can plant species litter generate specific fungal and bacterial communities in the litter layer? (2) Does exogenous N addition alter or modify microbial communities in the litter layer? (3) What is the magnitude of change in litter decomposition due to HFA and N addition? Results obtained from plant–microbial interactions in the litter layer (questions 1 and 2) allowed us to better interpret changes in decomposition due to HFA and N effects (question 3).

Materials and Methods

Results

Microbial community in the litter layer of Nothofagus forests in Patagonia

Fungi in the litter layer was dominated by the phyla Ascomycota (93% abundance), with high representation of subphyla Pezizomycotina (33% abundance) whose members mostly belonged to the class Leotiomycetes (16% abundance, Fig. 1A). Heliotiales, Pezizales, Orbiliales, and Pleosporales were the most abundant orders, and together represented 23% of fungal abundance (Fig. 1B). Dermae and Mycoarthris, from the order Heliotiales, and Phialea, from the order Pezizales, were the most abundant genus with 7%, 4%, and 5% of abundance respectively. Members of phyla Basidiomycota were present in very low abundance (2%), and most of them belong to the order Agaricales (Fig. 1B). Bacterial communities in the litter layer were dominated by phyla Alphaproteobacteria, Acidobacteria, Verrucomicrobia with 39%, 18%, and 15% abundance respectively (Fig. 2A). The most abundant families (above 5% abundance) were Bradyrhizobiaceae, Acetobacteraceae, Sphingomonadaceae (all Alphaproteobacteria), and Chthoniobacteraceae (Verrucomicrobia) (Fig. 2B).

Stronger fungal than bacterial association with tree species in the litter layer

Plant species identity had a significant effect on the fungal communities of the litter layer (ANOSIM, global R = 1, p = 0.01, Fig. 3A). The relative abundance of the most abundant fungal taxa varied among tree species. The litter layer of N. nervosa trees had two times lower abundance of Helotiales, the most abundant order in the litter layer, than under N. dombeyi and N. obliqua trees (Tukey test, p = 0.01, Fig. 1B). N. nervosa also had three times higher abundance of unclassified sequences (Tukey test, p = 0.006). The litter layer of N. obliqua trees had four times higher abundance of order Orbiliales and two times higher abundance of order Pleosporales than N. dombeyi and N. nervosa trees (Tukey test, p = 0.015, 0.029 respectively, Fig. 1B). Finally, the litter layer of N. dombeyi trees showed higher abundance of class Eurotiomycetes than N. nervosa and N. obliqua although differences were not significant (Fig. 1B). In contrast, plant species identity did not have significant effects on the bacterial communities of the litter layer (ANOSIM, global R = 0.3, p = 0.18, Fig. 3B).

The relative size effect of home field advantage and nitrogen addition on litter decomposition depended on plant species identity

Nitrogen addition stimulated decomposition of N. dombeyi and N. nervosa litter more than HFA (Fig. 4). In contrast, N addition and HFA stimulated decomposition of N. obliqua litter similarly (∼25%, Fig. 4). Interestingly, N addition stimulated litter decomposition at similar rates when added at home or away from the tree species that originated the litter (Fig. 4). The large and consistently positive effects of N addition on decomposition away from home indicates that N stimulation of decomposition surpassed reductions in decomposition due to away from home incubation. Thus, N addition erased the HFA for litter decomposition.

Discussion

Here we show that plant species identity differentially affected fungal and bacterial communities in the litter layer in old-growth Patagonian temperate forests. Plant species significantly affected the composition of the fungal community. Moreover, plant-fungal specific associations persisted even when the availability of resources was altered by exogenous N addition. In contrast, plant species identity and N addition did not have large effects on the bacterial composition of the litter layer. Plant species effects on fungal communities together with the observed HFA for litter decomposition were consistent with the idea that specific microbial communities may drive faster litter decomposition of the litter of the same species identity of the trees above. The non-significant effect of N addition on microbial composition indicated that N stimulation of litter decomposition was not necessarily mediated by alterations in the microbial community composition, and suggest instead an increased activity of decomposers due to increased resource availability.

The litter layer is an important reservoir of carbon and nutrients in terrestrial ecosystems, and habitat for many organisms, especially in temperate forests. The microbial composition of the litter layer in the Patagonian mixed forest showed two unique characteristics compared with the litter layer of temperate forests in the northern hemisphere. First, the fungal communities of the Patagonian mixed species forest were entirely dominated with Ascomycota (93%), with very low representation by Basidiomycota (2%). This contrasts strongly with studies demonstrating abundances of 50–70% Ascomycota and 20% Basidiomycota in European and North American forests (Schneider et al., 2012; Voříšková et al., 2014; Hesse et al., 2015; Urbanová, Šnajdr & Baldrian, 2015), and the dominance of Basidiomycota in the soil of most terrestrial ecosystems, including southern temperate forests (Tedersoo et al., 2014). Second, the bacterial communities of the Patagonian mixed species forest had much lower representation of Actinobacteria (5%, forth position on the racking of abundance), whereas in other temperate forests, it has been shown to be the second most abundant bacterial group (15%) after Proteobacteria (Schneider et al., 2012; Hesse et al., 2015; Urbanová, Šnajdr & Baldrian, 2015). Nothofagus, the southern beech, is a relict flora of Gondwana continent (Veblen et al., 1996). The biogeographic heritage of Nothofagus and other members of the flora and fauna in Patagonia might have contributed to the development of unique microbial community composition. While we cannot rule out differences due to primer biases, the importance of biogeographic patterns of microbial distribution should also be considered when interpreting these patterns (Martiny et al., 2011).

Plant species effects on microbial communities has been established in the rhizosphere and soil (Hartmann et al., 2009) but their effects in the litter layer has received less attention. The most common explanation for plant–microbial specificity is the qualitative and quantitative selective pressure that plant resources exerts on the functioning of the microbial community (Wardle et al., 2004; Fanin, Hättenschwiler & Fromin, 2014). In addition, alternatives to direct effects of litter quality could be plant species alteration of the microclimate, soil resources, and pH (Prescott & Grayston, 2013; Urbanová, Šnajdr & Baldrian, 2015). The Nothofagus tree species, however, do not show large differences in terms of their litter chemistry and the environmental conditions created by their canopies. N. nervosa and N. obliqua were indistinguishable in terms of N and lignin content of their leaf litter (Vivanco & Austin, 2008). Soil carbon, N and phosphate content, humidity, and pH were similar under the influence of the Nothofagus tree triangles (Vivanco & Austin, 2008). The way in which the specificity of the fungal community can arise in the litter layer is still open to question and alternatives, beyond those directly due to plant litter effects, need to be considered in order to fully understand the mechanisms behind this association.

A possible way in which plant species may contribute to microbial assembly is through phyllosphere microbes that reach the ground in falling leaves as it was suggested in a recent review (Austin et al., 2014). Plant species harbour different fungal and bacterial communities in the phyllosphere of their green leaves (Kembel & Mueller, 2014; Kembel et al., 2014). The microbial communities present in green leaves may persist during retranslocation of nutrients, senescence and leaf abscission, providing an effective means of dispersal and a substantial advantage for saprophagous microorganisms for the initial colonization of litter. This mechanism may explain the persistence of plant-fungal association even with N addition. Evidence for this mechanism come from recent studies showing that fungal species in the phyllosphere are also found in early decomposed leaf litter of the same tree species (Peršoh, 2013; Voříšková & Baldrian, 2013).

In contrast with changes often reported for microbial communities in the soil (Allison, Hanson & Treseder, 2007; Ramirez, Craine & Fierer, 2012; Cederlund et al., 2014; Leff et al., 2015), exogenous N addition in our study did not alter plant-fungal association in the litter layer and did not affect the bacterial community. The other few studies examining microbial composition in the litter layer did not find effects of N addition on bacterial communities but they found effects on fungal communities (Blackwood et al., 2007; Hesse et al., 2015). The independent effects of plant species and N addition in studies focused on microbial communities have been difficult to disentangle because N addition can also change both plant species composition and litter quality. Some of those studies, however, indicate that tree species identity has a stronger effect than N addition on soil fungal communities (Frey et al., 2004; Blackwood et al., 2007; Tedersoo et al., 2016), suggesting that the nature of the organic matter rather than the total availability of N may impose a stronger control on fungal communities. In contrast, N addition has shown stronger effects than plant community (or litter carbon quality) on soil bacterial communities in grasslands and agroecosystems (Ramirez et al., 2010). The soil environment is quite different than the litter layer, where microbial communities are subject to more variable conditions of humidity, temperature, carbon, and nutrient sources, as well as interactions with other organisms. Taken together, the contrasting responses between litter and soil microbial communities in forest ecosystems may reflect unique controls in each environment. This is important given that most metagenomic studies in terrestrial ecosystems have focused on soil and highlights the need to better understand the specific nature of the plant–microbial interactions in the litter as these interactions are responsible for a key step in carbon cycling with unique controls such as HFA.

Plant species identity and N addition effects on microbial communities in the litter layer provide insights into the nature of plant–microbial interactions in litter decomposition. While plant-fungal association does not necessarily imply decomposer specialization, it is noteworthy that in this undisturbed temperate forest plant species identity had an important and lasting effect on the fungal composition of the litter layer, with a large fraction of unique fungal species associations in each of the three Nothofagus species. Future studies are needed to bridge this gap between metagenomic information and microbial function in the environment. These results, together with the finding that fungi, not bacteria, are the main producers of extracellular hydrolytic enzymes in decaying leaf litter in forests (Schneider et al., 2012), suggest that fungal communities might be a more important driver of the persistence of plant–microbial affinity relation affecting litter decomposition in forests. At the same time, N stimulation of the decomposition of Nothofagus leaf litter was not reflected in changes in microbial community composition, suggesting that an increase in decomposer activity accelerated litter mass loss with N addition. N addition might have alleviated microbial N limitation imposed by the relatively high C:N ratio of leaf litter (Vivanco & Austin, 2008). Also, N stimulated microbial communities were able to decompose home and away litter at similarly accelerated rates (Fig. 3), suggesting that the advantage to decompose home litter (under no-N added conditions) might arise from different microbial traits to scavenge for litter N.

Microorganisms have traditionally been considered the engine of litter decomposition, with little importance given to their community composition. Here we show that microbial composition is impacted by long-term interactions with plant species that may contribute to HFA for litter decomposition. Plant–microbial controls on the carbon cycle can be particularly important in temperate forests where litter decomposition seems to be driven principally by biotic interactions, as opposed to abiotic processes like photodegradation that dominate decomposition in low rainfall terrestrial ecosystems (Austin & Vivanco, 2006). This study highlights plant–microbial interactions as a currently underappreciated element of the controls on litter decomposition in terrestrial ecosystems, particularly in undisturbed natural forests.

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