Tree species influence on microbial communities in litter and soil: Current knowledge and research needs

Forest Ecology and Management 309 (2013) 19–27

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Tree species influence on microbial communities in litter and soil: Current knowledge and research needs q Cindy E. Prescott ⇑, Sue J. Grayston Belowground Ecosystem Group, Department of Forest and Conservation Sciences, Faculty of Forestry, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T 1Z4

a r t i c l e

i n f o

Article history: Available online 31 March 2013 Keywords: Tree species Litter Forest floor Soil Microorganisms Rhizosphere

a b s t r a c t Microbial communities in litter and soil are the functional link through which the tree species occupying a site may alter rates of soil processes fundamental to nutrient cycling and carbon flux. Through differences in the nature of their litter, their mycorrhizal fungal associates and the exudates they release into the rhizo/mycorrhizosphere, different tree species may give rise to distinct microbial communities in litter and soil. We examine the evidence that tree species influence the composition of the microbial communities in decomposing litter, forest floors, soil and the rhizo/mycorrhizosphere. The microbial communities considered in this review include bacteria, archaea, fungi and both free-living and symbiotic organisms. There is evidence that distinct microbial communities develop on decomposing leaf litters of different tree species, however, given the well-documented succession of microbes on decomposing litter, comparisons amongst tree species of microbial communities in litters and forest floors at the same stage of decay are needed to definitively deduce the influence of tree species. Distinct microbial communities have been reported in forest floors under different tree species; differences are most pronounced in the F layer. Distinctions in microbial communities in mineral soil under different tree species have been determined in several common garden experiments. The main factors associated with differences in microbial communities in litter, forest floors and soil are the pH and base cation content of the litter and whether the trees are broadleaf or coniferous. Identified differences in microbial communities in the rhizospheres of different tree species are more likely to be differences in the mycorrhizospheres and hyphospheres, given the predominance of associations with mycorrhizal fungi. Distinct microbial communities have been identified in the mycorrhizosphere under different tree species; these are influenced both by the tree species and by the mycorrhizal fungi with which the tree associates. Heightened attention during sampling to ensure comparison of ‘like with like’ could improve our ability to distinguish influence of tree species in decomposing litter, forest floors and soil. A significant remaining challenge is characterizing exudates from different tree-mycorrhizal fungi associations and understanding interactions between mycorrhizal fungi and microbial communities in the hyphosphere. A complete tapestry of the linkages between tree species and soil microbial communities requires that we also weave in the effects of soil fauna. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Interactions between aboveground and belowground components of terrestrial ecosystems are receiving much research attention in light of their importance in driving ecosystem processes that govern ecosystem productivity, gas fluxes and carbon sequestration (Wardle et al., 2004). One particularly intriguing question is the degree to which the nature of the plant community influences the composition and activities of the soil microbial community. Feedbacks between these two ecosystem components could lead q Contribution to Special Issue from Eurosoil Session S10.3. ‘Influences of Tree Species on Forest Soils’, 2012, Bari, Italy (Lars Vesterdal and Cindy Prescott). ⇑ Corresponding author. Tel.: +1 604 822 4701; fax: +1 604 822 9102. E-mail address: [email protected] (C.E. Prescott).

0378-1127/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.foreco.2013.02.034

to unpredicted changes in ecosystem function in response to alterations in the nature of the plant community as a consequence of climate change or species selection for forestry. Several lines of evidence indicate that changes in the nature of the plant community could initiate profound changes in cycling of C and N in ecosystems (Hobbie, 1996; Mitchell et al., 2010). As the principal drivers of soil nutrient cycling processes, soil micro-organisms are the critical link between shifts in the composition of the dominant vegetation and fundamental shifts in ecosystem functioning. Characteristics (or functional traits) of the vegetation on a site could influence the composition and functioning of the soil microbial community through alteration of microclimate (through shading, frost protection, throughfall and uptake/transpiration of soil water), production of litter (both aboveground and roots), interactions with herbivores (both above and belowground), production

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of root exudates, and interactions with root symbiotic organisms such as mycorrhizal fungi. Despite this web of interactions, the strength and directness of links between plant and microbial communities are far from clear. This may be due to difficulties in characterizing microbial communities, which has improved extraordinarily with the advent of molecular techniques (Zak et al., 2006). However, it may also be that plant and microbial communities are not directly linked, and respond to a different suite of environmental factors – plants being more sensitive to phenological constraints such as frost, photoperiod and growing degree days, and soil microorganisms being more sensitive to soil factors such as texture, moisture and organic matter content. Recent studies of the biogeography of soil microorganisms have demonstrated strong relationships between the soil microbial community and factors such as pH, texture, organic matter content and C:N ratio of the soil (Fierer and Jackson, 2006; Fierer et al., 2009; Rousk et al., 2010; Brockett et al., 2012). These site factors could quite conceivably overwhelm the influences of tree species in structuring microbial communities. So how compelling is the evidence that tree species exert a substantial influence over soil microbial communities? In this paper we review evidence of the influence of tree species on the soil microbial community. In terms of the microbial community we include bacteria, archaea, fungi and both free-living and symbiotic organisms, but exclude algae and viruses as there is little information about these groups in soil. We include information derived from studies using a variety of techniques for describing soil microbial communities; descriptions of these techniques and their relative scope and merits can be found in Stefanis et al. (2013) and Paul (2007). Given that the mechanisms of plant effects differ above and belowground, and between rhizosphere and bulk soil, we stratify the paper into these sections, reviewing the evidence for effects of tree species on microbial communities in decomposing litter, forest floor, bulk soil and in the rhizosphere.

2. Decomposing litter Interest in the degree to which the nature of the litter produced by plants influences the community of decomposing organisms within it has been stimulated by recent reports of faster decomposition of litter when placed in its native environment (i.e. the ‘home-field advantage’ – HFA; Gholz et al., 2000). Recent meta-analyses of forest litter decomposition studies have reported overall HFA of 8% (Ayres et al., 2009b) and 4% (Wang et al., 2012) indicating that although a HFA is not ubiquitous, litter decomposes (on average) 4–8% faster in its native environment. The HFA has been interpreted as a consequence of the litter microbial community being adapted (physiologically or evolutionarily) to decompose litter that is characteristic of that ecosystem. As such it is expected that the litter microbial community differs among ecosystems in accordance with the nature of the litter (Ayres et al., 2009b). Ayres et al. (2006) tested the hypothesis that soil microbial communities are adapted to decompose native litter by incubating litter of three tree species with a solution of microorganisms extracted from soil under each tree species. Respiration rates were not affected by the origin of the solution, indicating that the soil microbial communities were not better able to decompose the litter from their ‘home’ forest. However shortcomings of this experiment, such as exclusion of soil fauna and of fungi that depend on intact mycelia, prevent it from being a definitive test of the home-field advantage hypothesis (Ayres et al., 2009b). A first step towards understanding the HFA is assessing the evidence for the supposition that the nature of the litter influences the microbial community that colonizes and decomposes it, which we address in this review. Determining the degree to which tree species influence microbial communities in decomposing litter is more challenging than

it appears, for several reasons. First, because microbial communities in litter are affected by temperature and moisture conditions (and potentially influenced by soil properties), the pure effect of species can only be distinguished by comparing litter of different species decomposing on the same site. Studies comparing microbial communities in litters among forests that differ in several factors including tree species are useful in distinguishing the influence of the site-species complex, but not the pure effect of tree species. Secondly, a successional process of fungal and bacterial communities in decomposing litter, associated with changes in the nature of the litter during decomposition, has been well documented through observation/plating and molecular techniques (Kendrick and Burges, 1962; Frankland, 1998; Snajdr et al., 2008; Baldrian et al., 2012). So distinguishing the influence of tree species requires that the microbial communities in different litter types be compared at the same stage of decay. If litters of different species decompose at different rates, they have to be compared at the same state of mass or C loss rather than just the same incubation time. Most early studies of communities of fungi in decaying litter were intensive studies of decomposition of a single litter type (Kendrick and Burges, 1962; Visser and Parkinson, 1975), but a few compared fungal communities amongst different litter types. Using cultivation-dependent techniques, Frankland (1975, 1998) followed the succession of fungi on leaf litter of Quercus, Betula, Corylus and Fraxinus in a mixed-species broadleaf woodland in the UK. The dominant species of fungi identified were similar in all litter types, although the timing of their arrival and peak varied according to the state of decay of each litter type. Likewise Hayes (1966) compared fungal communities in decomposing litters of pine, spruce and fir in a mixed-conifer plantation in Wales. Some fungal species were occasionally missing from one of the litters, but overall, fungal communities were similar in the three litters. The reported similarity in fungal communities may be attributed in part to the culturing method used at that time to study fungal communities, which preferentially isolates rapid spore-forming fungi. It may also simply reflect that these were the common fungi in this particular forest (in which all tree species occurred), making it likely that the four litters would be attacked by similar dominant fungi. In fact, greater divergence of fungi was observed within a single litter type (Pteridium aquilinum) while decomposing in six adjacent but different habitats. Studies in ‘common garden experiments’ with pure plots of each tree species are better indicators of the particular influence of tree species, both directly through the litter being decomposed, and indirectly through the nature of the forest floor created under its canopy. In one such study, Kubartová et al. (2009) applied TGGE to compare fungal communities in decomposing litter of four tree species (Fagus, Quercus, Picea and Pseudotsuga) in pure plots of each species on a single site. The majority of fungal species were detected in several or all litter types, but their relative abundances differed among the litter types. Five of the most common fungal species were detected in all litter types, while five were detected only in one species. Tree species explained almost 50% of the variability in fungal community composition in the litters, with spruce litter showing the most distinct fungal community structure. However remaining litter mass after 4 and 24 months differed by up to 20% among the litter types, so the litters were at different decay stages at the time of sampling, Some experimental designs have allowed distinction between the effects of the litter itself from the effects of the forest floor that develops under each tree species in influencing the microbial community that develops in decomposing litter. This can be accomplished by transplanting litters amongst plots containing trees of the same species as the litter (i.e. the home field) and trees of other species (away field). Aneja et al. (2006) removed green leaves from beech and spruce plants grown for three years in the same soil and

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incubated them in the Ah layer of a beech forest and a mixed beech-spruce forest. After 2 and 8 weeks, DGGE profiles of both bacteria and fungi clustered strongly according to litter type, and distributions of clones of bacterial groups differed among the two litter types. The microbial communities in the two litter types were more similar to each other than to the communities in the soil from the forest. These findings indicate that different litters encourage development of distinct microbial communities regardless of the site of placement (in keeping with the HFA hypothesis). An alternate approach was used by Bray et al. (2012), who incubated litter of 10 plant species together in a common garden which did not contain trees of any the species of litter (so no litter had a HFA). After 1, 2, and 8 months of decay, microbial community composition (PLFA) differed significantly in litters of differing ‘lability’ class, indicating that the nature of the litter itself influenced the organisms that decompose it. However, decomposition constants ranged from < 0.4 to nearly 2.0 yr 1, so some of the differences in microbial communities in the different litters (especially after 8 months) may be attributable to them being at different decay stages of the litters. In contrast, Wallenstein et al. (2010) showed the underlying forest floor also influences the microbial community in decomposing litter. They found evidence of distinct metabolomes in partially decomposed aspen litter that had been incubated for one year in stands dominated by pine, spruce or aspen. In conclusion, it appears that leaf litters of different tree species develop distinct microbial communities during their decomposition, and that these result from influences of the nature of the litter itself and from differences in microbial communities in forest floors under different tree species.

3. Forest floor Several studies have reported differences in microbial communities in bulk forest floor samples (all layers combined) from under different tree species, particularly when conifers and broadleaves are compared. In forest floors beneath five tree species (four broadleaf and one conifer), Weand et al. (2010) found the most distinct microbial communities (PLFA) associated with the conifer (hemlock), which had the lowest abundance of Gram-negative bacteria, greatest abundance of fungi and actinomycetes, and greatest fungal:bacterial ratio. Ushio et al. (2008) found distinct microbial communities (PLFA) in the top 5 cm of organic soil under two conifer species compared to three broadleaf species in a mixed forest in Borneo. Distinct microbial communities have also been reported in forest floors beneath conifers (usually spruce) and broadleaved species in boreal forests (Saetre and Baath, 2000; Priha et al., 2001; Hannam et al., 2006). Given the well-documented succession of fungi during litter decomposition and stratification of microbial communities according to forest floor layer (Grayston and Prescott, 2005; Kubartová et al., 2009; Snajdr et al., 2008; Baldrian et al., 2012), studies that examine forest floor layers separately may be superior in comparing ‘like with like’ under different tree species. Ushio et al. (2010) reported distinct microbial communities (PLFA) in separated upper and lower O layers (and upper and lower A) under a conifer and a broadleaf species in a mixed-species tropical montane forest in Borneo. Kubartová et al. (2009) found different fungal communities (temperature gradient gel electrophoresis; TGGE) in separated L (litter) and F (see below) layers all four tree species (two conifer and two broadleaf) were all distinct from one another. In pure plots of four conifer species, Grayston and Prescott (2005) found the most distinct microbial community (PLFA and CLPP) in F and H (humus) layers under western redcedar, which had more Grampositive bacteria and actinomycetes, lower fungal biomass and lower fungi:bacteria ratio and greater utilization of most C

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substrates than the other species. This was hypothesized to be related to higher calcium concentrations and pH of cedar forest floors. It may be that the defining factor is not whether the litter is broadleaf or needleleaf but rather its calcium content, and that the effect on the microbial community is exerted through the influence of Ca on the soil faunal community (Reich et al., 2005; Hobbie et al., 2006). Explicit examination of relationships among Ca or base cation content of litter and forest floors, communities of soil fauna (especially ecosystem engineers and litter transformers) and microbial communities would probably be instructive in elucidating the mechanisms underlying differences in microbial communities and nutrient cycling in forest floors under different tree species. In studies in which forest floor layers were analyzed separately (Grayston and Prescott, 2005; Kubartová et al., 2009; Ushio et al., 2010) the greatest differences in microbial communities amongst tree species were found in the F layer. The F (formultning; Swedish for ‘decay’ or ‘mold’) is the layer/phase in which most decomposition occurs, when fungi are most abundant and fauna are most active, the litter at this point having been ‘conditioned’ through leaching of phenolics and growth of fungal tissue. In contrast, the L layer is the site of leaching, initial colonization and decay of readily accessible tissues, and the H layer is populated by a less diverse and abundant group of ‘soil fungi’ and has low rates of mass loss. The F layer is also where the greatest differences among tree species in respiration and N mineralization occur (Kanerva and Smolander, 2007). The F layer is also the layer containing the greatest abundance of mycorrhizal fungi, which influence microbial communities directly (see below) and by contributing dead fungal tissue, which may comprise a significant proportion of the dead organic matter in forest floors (Langley and Hungate, 2003). Comparison of microbial communities is F layers of forest floors may be the best way to reduce variability caused by bulking all layers while not incurring the additional costs of assessing each layer separately.

4. Soil Trees influence the soil beneath the forest floor (hereafter referred to as mineral soil) through many mechanisms including leaching of dissolved organic materials and nutrients from the forest floor, permeation by roots which may alter soil physical structure and water flow, input of organic matter in the form of root litter, and exudation of ions and organic compounds. The soil can be expected to be less influenced by tree species than the forest floor, which is largely created from the litter of the trees. Effects of tree species may take longer to become detectable in the mineral soil compared to the forest floor. It is also more challenging in mineral soils to discern if differences arise from the current tree species, or from past vegetation or land uses. The problem of comparing ‘like with like’ may also be significant in mineral soils. The nature of the upper mineral horizon may differ under different tree species (being eluviated under some and enriched with organic matter under others as a consequence of the influence of soil fauna), making it difficult to know if sampling is best stratified by depth or by soil horizon. Finally, given known differences in resource availability and microbial communities between rhizosphere and bulk soil, it is necessary to sample and compare these separately. In this section we discuss studies in which it appears that root-free soil was examined under different tree species. Distinct microbial communities in mineral soil under different tree species have been reported in two common garden experiments. Lejon et al. (2005) found distinct ARISA profiles, indicating distinct genetic structures of both bacterial and fungal communities in mineral soil (0–5, 10–20 cm depth) in pure plots of spruce,

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Douglas-fir, oak and beech in France. Jiang et al. (2012) reported greater catabolic diversity (CLPP) and distinct bacterial and fungal communities (DGGE) in soil (0–10 cm depth, horizons not specified) beneath broadleaf and mixed species plantations compared with soil beneath conifers. Other studies of natural stands are also suggestive of influences of tree species on microbial communities in mineral soils. Within a mixed deciduous forest in Germany, Thoms et al. (2010) compared PLFA profiles in mineral soils in plots dominated by (1) Fagus, (2) Fagus, Tilia and Fraxinus, and (3) these species plus Carpinus and Acer. Microbial communities differed among the three types of plots in samples from 0 to 5 and 10–20 cm depths. Differentiation was related to presence of Tilia and Acer vs Fagus, and to pH, which may indicate that the presence of species which produce base-rich litter exert the greatest influence on microbial communities in soil (consistent with cedar effect, above). Likewise, Ayres et al. (2009a) reported differences in fungal and bacterial communities (TRFLP) in the upper 10 cm of soil among adjacent stands dominated by pine, spruce and aspen in Colorado. Additional studies have reported differences soil microbial communities in forests of different tree species that were not on the same site; i.e. not in common gardens (Myers et al., 2001; Hackl et al., 2005; Selvam et al., 2010). In these studies the pure effect of tree species cannot be distinguished from influences of other factors such as soil and topography, so differences represent the influence of the site-species complex rather than the pure effect of tree species.

5. Mycorrhizae The composition of the mycorrhizal fungal community associated with tree roots is a key way in which tree species can influence the soil microbial community. ECM mycelia, which form symbiotic associations with most temperate tree species, can account for up to 80% of the fungal community and 30% of the total microbial biomass in forest soils (Högberg and Högberg, 2002; Wallander, 2006). There are estimated to be 6000 species of ECM fungi (Smith and Read, 2008) and though many ECM have broad host range (e.g. Lactarius; Kennedy et al., 2003) some have only narrow host range (e.g. Suillus; Bruns et al., 2002). Equally some tree species have wide fungal receptivity, such as Douglas-fir which has more than 2000 known ECM (Molina et al., 1992) but other trees, such as alder have narrow host receptivity, only associating with 50 known ECM (Pritsch et al., 1997). Whole-genome sequencing of both trees and ectomycorrhizal fungi (Eastwood et al., 2011) is now enabling us to understand some of the signaling molecules and mechanisms which are responsible for the specificity of some ectomycorrhizal associations (Podila et al., 2009). A recent global meta-study of the factors influencing richness and community structure of ectomycorrhizal fungi, across all the common biomes containing these symbionts (subarctic tundra to tropical rainforest), revealed that host family explained 34% of the variation in the fungal community composition (Tedersoo et al., 2012). Less is known about the specificity of arbuscular mycorrhizal associations (Brachmann and Parniske, 2006; Bonfante and Genre, 2012), which are less common in temperate forests, but occur on some species such as cedar and maple. Differences in mycorrhizal fungi associated with different tree species probably contribute to the differences in the microbial communities in forest floors and soil under different tree species that were summarized above. Molecular techniques that better identify mycorrhizal and saprophytic fungi will better differentiate effects of tree species on these two distinct fungal communities. One recent study (Buée et al., 2011) found evidence of tree species influencing communities of both mycorrhizal and saprophytic fungi. They collected fungal sporocarps in native forest and

monocultures of conifers (Norway spruce, Nordmann fir, Douglas-fir, Corsican pine) and deciduous trees (oak, beech) 3 times per year over a 7-year period. A total of 331 fungal species were identified, of which 25 ECM and 21 saprophytic fungi were associated with only one tree species. The native forest, and the beech, Norway spruce and Nordmann fir stands had higher fungal diversity than the oak, Douglas-fir and Coriscan pine stands. This study also highlighted the problem of studies conducted outside the natural range of tree species: Douglas-fir, which is not a native of Europe, had low fungal diversity (49 fungal species total), but in its native Pacific Northwest, Douglas-fir has high fungal diversity (86 species, Smith et al., 2002). This may be an issue in comparing the effects of tree species in common garden experiments that are not within their native range.

6. Rhizosphere The rhizosphere is the area of soil under the influence of roots, but given the ubiquity of mycorrhizal symbionts, most studies are probably examining the mycorrhizosphere (the area of soil under the influence of mycorrhizal roots) and the hyphosphere (the area of soil under the influence of mycorrhizal extramatrical hyphae). These areas are highly integrated, which makes it difficult to discuss each area independently. Therefore we will discuss them together in this section. Microbial communities in the rhizosphere of trees differ from those in bulk soil (Shi et al., 2012; Mestre et al., 2011; Pires et al., 2012), which is thought to result from release of a diverse array of exudates as well as growth regulators and inhibitory compounds. Tree exudates are comprised of carbohydrates, amino acids, low molecular weight aliphatic and aromatic acids, fatty acids, enzymes and hormones (Grayston et al., 1997; Kuzyakov and Domanski, 2000; Neumann and Romheld, 2001; Jones et al., 2004). Exudates are also released from mycorrhizal fungal symbionts of trees, either arbuscular or ecto-mycorrhizal, in the mycorrhizosphere (infected fine root tips), or more accurately, the hyphosphere, surrounding extramatrical hyphae. Hyphae excrete carbohydrates and organic acids that are used by bacteria and other microorganisms (Heinonsalo et al., 2004; Jones et al., 2004), and the mycorrhizosphere has been described as a ‘hotspot’ for bacteria in soil (Nazir et al., 2010). Ectomycorrhizal (ECM) hyphal tips of so-called long-distance-exploring, rhizomorphic mycorrhizal fungi (e.g. Boletales; Agerer, 2001) are active sites of exudation and re-adsorption of compounds (Sun et al., 1999). Other ECM (contact explorers, e.g. Lactarius, Russula species), produce little emanating hyphae which exude compounds throughout their hyphae (Agerer, 2001). ECM root tips support diverse populations of bacteria and microfungi (Frey-Klett et al., 2007; Tedersoo et al., 2009; Finlay, 2008). Exudates from arbuscular mycorrhizal (AM) fungi also influence bacteria in their mycorrhizosphere (Toljander et al., 2007). The amount and nature of exudation are influenced by both the tree species, as demonstrated in studies with the same ectomycorrhizal symbiont on different tree species (Martin et al., 2008; Tuason and Arocena, 2009) and the fungal species, verified in studies of a single tree species with different ectomycorrhizal symbionts (van Hees et al., 2005; Sandnes et al., 2005). For example, Douglas-fir and poplar infected with the same ECM (Laccaria bicolor) produced different exudates (Martin et al., 2008). The presence of different ectomycorrhizal symbionts on roots increased exudation of organic acids (Sun et al., 1999; Ahonen-Jonnarth et al., 2000; Casarin et al., 2003; Sandnes et al., 2005; van Hees et al., 2003, 2005; Johansson et al., 2008, 2009), and changed the organic acids released (van Hees et al., 2005; van Scholl et al., 2006; Klugh and Cumming, 2007). Although most of our knowledge about differences in the character of exudates from different tree species is regarding organic acids,

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Liebeke et al. (2009) used GC–MS to reveal differences in the sugar content of soil extracts performed in different forest soils and demonstrated that oak soil contained mannitol and trehalose that was not present in the beech soil. They hypothesized this difference was responsible for differences in the bacterial communities under these tree species. Several studies have reported distinct microbial communities in the rhizospheres of different tree species, using a variety of techniques. Microbial communities (based on their carbon utilization profiles; CLPP) were discriminated in the rhizospheres of hybrid larch and Sitka spruce (Grayston and Campbell, 1996) and in the rhizoplane, but not the rhizosphere soil, under larch, Sitka spruce and sycamore (Grayston, 2000). Likewise, Ross et al. (2006) found distinct physiological profiles (CLPP) of microbial communities in both the hyphosphere and rhizosphere under red beech and radiata pine after growing for 6 years in open-topped chambers. Golin´ska and Dahm (2011) isolated actinomycetes from the rhizosphere and bulk soil of mature stands of silver birch, Scots pine and black alder in Poland. They found the greatest number of eubacteria were associated with the alder (bulk soil, rhizosphere and rhizoplane), and the smallest number with the pine, whereas actinomycetes were the most numerous in the birch rhizosphere. Using pyrosequencing and PCR-DGGE of archaeal 16S rRNA genes, Pires et al. (2012) found differences in archaeal richness in the rhizospheres of two mangrove species. Using PCR-DGGE of rRNA, Shi et al. (2012) found significant differences in the active microbial communities (specifically alpha proteobacteria and pseudomonads) in the rhizosphere of a genetically-modified radiata pine compared to natural radiata pine. Each of these examinations of tree rhizospheres were probably actually studies of the mycorrhizosphere (as mentioned above), but most did not report the mycorrhizal status of the roots or did not identify the mycorrhizae present. There have been two studies which have compared the effect of a known ectomycorrhizosphere relative to microbial communities in bulk soil. Calvaruso et al. (2007) and Frey-Klett et al. (2005) demonstrated that mineral-weathering bacteria and fungi were enhanced in the ectomycorrhizosphere of Scleroderma citrinum under oak in the field and in the ectomycorrhizosphere of Laccaria bicolour under Douglas-fir in a nursery, respectively. There has been one study on the effect of mycorrhizae and tree species on archaeal communities. Bomberg and Timonen (2009) surveyed archaeal community composition in the mycorrhizosphere of pine, spruce, birch and alder trees grown in the same humus taken from a pine forest in Finland. They found an influence of tree species on archaeal communities in the absence of mycorrhizae, but the influence of tree species was minimized by the presence of the mycorrhizal fungus, Paxillus involutus. However, the survey did not include different mycorrhizal fungi on the same tree species. Nurmiaho-Lassila et al. (1997) used transmission electron microscopy to examine the bacterial composition of the mycorrhizospheres of Scots pine infected with either P. involutus or Suillus bovinus, the extramatrical hyphospheres of these two fungi, and non-mycorrhizal fine roots. The Scots-pine mycorrhizospheres formed by two different ectomycorrhizal fungi were clearly dissimilar habitats for mycorrhizosphere-associated bacteria. In addition, the hyphospheres hosted distinct and abundant populations of bacteria. These studies suggest that the mycorrhizal fungi have the dominant influence on microbial populations in the hyphosphere (Frey-Klett et al., 2005). A challenge which is particularly acute for understanding the origins of differences in the microbial communities in the rhizo/ mycorrhizosphere of different tree species is accounting for differences in temporal patterns of belowground C flux among tree species. ECM fungal community structure and activity exhibit considerable temporal variation (Buée et al., 2005; Courty et al.,

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2007, 2008; Pickles et al., 2010; Burke et al., 2011; but see Kluber et al., 2011). In temperate forests, some studies have shown rhizosphere bacterial densities reach their maximum during spring (Kauri, 1982; Rogers and Tate, 2001; Collignon et al., 2011), when the photosynthetic capacity of the trees is high and the amount of carbon increases into the rhizosphere (Esperschütz et al., 2009), while other studies report bacterial densities peaking during winter (Griffiths et al., 1990; Berg et al., 1998). A number of studies have documented seasonal trends in soil microbial communities and activities in a variety of ecosystems (Allison and Treseder, 2008; Björk et al., 2008; Cruz-Martinez et al., 2009; Brant et al., 2006; Moore-Kucera and Dick, 2008), which may be related to seasonal differences in C flow. A recent study using 13CO2 pulse-labeling demonstrated considerably different seasonal patterns in belowground C allocation among different tree species (Epron et al., 2011). Therefore, comparing ‘like with like’ in the rhizo/ mycorrhizosphere microbial communities of different tree species may entail sampling them at the same time relative to belowground C flux, which may occur at different times for each tree species. Another significant challenge is collecting and characterizing the exudates released by different tree species. Most studies have used young seedlings grown hydroponically or in sand or soil systems. Hydroponic systems suffer from the fact that exudates can be re-adsorbed by roots and they lack mechanical impedance for roots, which increases exudation, sand systems lack microorganisms which also affect exudation rates, and in soil systems exudates can be adsorbed or degraded by microbes (reviewed by Grayston et al., 1997). The few field studies of tree root exudation have again mainly studied young seedlings (Smith, 1976; Sandnes et al., 2005; Phillips et al., 2008) using either excavated root tips placed in sterile tubes, or soil extraction with associated problems of adsorption and degradation. Shi et al. (2012) recently demonstrated an improved anion exchange membrane system to collect root exudates in situ. Because the resins rapidly adsorb released exudates there is less opportunity for consumption or adsorption. This technique may allow greater insight into the nature of exudates under different tree species, which may assist in elucidating the mechanism behind the differences in microbial communities in the rhizo/mycorrhizosphere of different tree species.

7. Conclusions There is evidence of differences in microbial communities in litter, forest floors and soil that can be attributed to differences in the tree species occupying a site. There is evidence that leaf litters of different tree species develop distinct microbial communities during their decomposition, and that these result from influences of the nature of the litter itself and from differences in microbial communities in forest floors under different tree species. Most studies of microbial communities in decaying litter have compared litters at the same incubation time, but at different stages of decay (due to differences in decay rate), which exaggerate the differences between tree species. Distinct microbial communities have been reported in forest floors under different tree species in a variety of forest types, in studies comparing the entire forest floor (bulk) and others examining individual forest floor layers. Differences in microbial communities appear to be most pronounced in the F layer and between broadleaf and coniferous species. Differences in microbial communities have been determined in the upper mineral soil under different tree species.

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The factors identified as underlying differences in microbial communities in litter, forest floors and soil are the pH and base cation content of the litter and whether the trees are broadleaf or coniferous (needleleaf). Differences in microbial communities have been demonstrated in the rhizospheres of different tree species, probably reflecting differences in the amount and nature of root exudates. Differences in microbial communities in the rhizospheres of different tree species are more likely to be differences in the mycorrhizospheres and hyphospheres, given the predominance of associations with mycorrhizal fungi. Like roots, mycorrhizal fungi release compounds which influence microorganisms in the mycorrhizosphere and hyphosphere. Metagenomic techniques enabling identification of more of the soil microbial community have demonstrated specificity of tree species and their fungal symbionts and detected influences of both on microbial communities in the mycorrhizosphere. There is evidence that the mycorrhizal fungal associate may override tree species effects in determining the nature of the microbial community in the mycorrhizosphere.

8. Implications for ecosystem function This assessment of the degree to which soil microbial communities are influenced by the tree species under which they occur is a first step to understanding how tree species influence the ecosystem in which they occur. Significant challenges remain both in characterizing the microbial communities and understanding how changes in their composition might affect ecosystem processes. This no longer appears an impossible task, given advances such as metagenomic techniques which allow assessment of whole microbial communities through DNA and RNA analysis and expression of gene sequences for specific enzymes (Baldrian et al., 2012), and metaproteomics which allow specific microbial activities to be linked to defined organisms Schneider et al. 2012). It is not overly optimistic to posit that we may be within a decade of finally being able to demonstrate the functional linkages between plant communities and soil microbial communities, and the soil processes that arise as a consequence of their activities. With respect to litter decomposition, the primary implication of the reported differences in microbial communities in litter and forest floors of different tree species is that microbial communities under different tree species are adapted to decompose the type of litter produced in that environment (i.e. the home-field advantage), so litter decomposes faster than it would were there not this specialization. How important is this for ecosystem function? Although HFAs are detectable, they are quite small (5–8%), and so far have been demonstrated to occur only in the early stages of decay, and not affect the proportion of litter that is humified rather than decomposed. Freschet et al. (2012) explicitly tested the HFA and found that the interactions had substantially less influence on decay rate than did the direct influences of litter quality and site. This is consistent with many studies showing that high-quality litter decomposes faster than low-quality litter, regardless of site, and that any litter type decomposes faster on sites that are richer and moister and are dominated by species that produce higherquality litter. Freschet et al. (2012) further suggest that HFA occurs with extreme litters, for which some specialization of the microflora may exist. At this juncture, it also remains to be determined if the HFA is a consequence of distinct communities of decomposer organisms in decomposing litters of different species. Specialization of microbial communities under different tree species may have more important implications for physiologically narrow ecosystem processes (Schimel, 1995), i.e. those that are performed by a more limited suite of organisms. Indeed, there have

been reports of altered communities of organisms that perform more specialized processes such as ammonia oxidization and weathering of elements from parent materials and methanogenesis. Boyle-Yarwood et al. (2008) found the community composition of ammonia-oxidizing archaea and bacteria (amo A, T-RFLP) differed between red alder and Douglas-fir at both of two sites in Oregon, which provides the biotic link between higher concentrations of N and higher levels of nitrate in alder soils. Calvaruso et al. (2007) and Frey-Klett et al. (2005) demonstrated that mineral weathering bacteria and fungi were enhanced in the ectomycorrhizosphere of Scleroderma citrinum under oak in the field and in the ectomycorrhizosphere of L. bicolour under Douglas-fir in a nursery, respectively. Pires et al. (2012) showed using pyrosequencing and PCR-DGGE of archaeal 16S rRNA genes that the rhizospheres of two different species of mangrove (Rhizophora mangle and Laguncularia racemosa) contained distinct archaeal communities, in particular one class of methanogenic archaea, Methanomicrobia, were only found in the rhizosphere of R. mangle. As methanogenesis is an anaerobic process it is thought that CH4 is only produced in significant quantities in flooded soils. However, there is evidence of very high methane emissions in upland forests (Yavitt et al., 1995; Frankenberg et al., 2005; Carmo et al., 2006; Crutzen et al., 2006) that could be explained by hotspots and hotmoments of CH4 emissions following brief wetting events and explaining the missing source of 10 Tg CH4 per year (Megonigal and Guenther, 2008). It is known in other ecosystems that CH4 production is tightly coupled to plant photosynthesis, therefore it could be predicted that different upland tree species may have varying effects on methanogen communities (Megonigal and Guenther, 2008).

9. Challenges and research needs Despite decades of research interest and remarkable advances in techniques to characterize microbial communities, our understanding of the degree to which and the manner in which tree species influence the microbial communities in litter, humus and soil is far from complete. Part of the challenge lies in separating the effects of tree species from those of the myriad other factors that influence microbial communities, which can be addressed by comparing microbial communities in common garden experiments in which several tree species have been grown in pure plots on the same soil and site for long enough to have influenced the soil. The more intransigent problem in addressing this question is comparing ‘like with like’; given that microbial communities change during the process of litter decomposition, and vary among forest floor layers and soil horizons and with proximity to roots, sampling must be done in such a way that similar materials are being compared in order to distinguish the effects of the tree species. The effects of tree species effects on microbial communities in decomposing litter has not been definitively demonstrated because most studies to date compare litters at the same incubation time, but at different stages of decay, due to differences in their rates of decay. Because the organisms found in decomposing litter change along the decomposition continuum, the microbial communities in different litters must be compared at same stage of decay (rather than incubation time). This will require frequent sampling so that microbial communities can be tracked along the decay continuum, and compared as a function of mass or C loss. More studies with transplanted litter in common garden experiments are also needed to distinguish the influences of litter characteristics versus forest floor microbial communities in determining communities in decomposing litter. Comparing ‘like with like’ in forest floors can be accomplished by sampling each layer separately, although this is challenging

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when there are different humus types (mull/mor) under the different species such that the nature of the substrate is fundamentally different due to different amounts of transformation and mixing of soil fauna. In these situations any differences in microbial communities would represent the tree species’ influence on the humus/ fauna/microbe complex. Considerable effort is needed to weave in the influence of the soil faunal that link tree species and microbial communities. Although not covered in this review, interactions between tree species and soil faunal communities must be better understood before we will have a full picture of the links between tree species and soil microbial communities. Understanding the mechanisms underlying the influences of tree species on microbial communities in forest floors and soil necessitates distinguishing mycorrhizal fungal associates of the tree species from other soil microorganisms. Molecular techniques are making this increasingly possible. Exudate capture and characterization remains a methodological challenge, particularly in field situations. An improved ion exchange resin technique (Shi et al., 2012) for collecting root exudates in situ shows promise for addressing this need. A particularly intriguing challenge is determining the extent to which differences in exudates from different tree species (and/or their mycorrhizal associates) affect the composition of the microbial community in the rhizosphere and mycorrhizosphere, and also potentially in the bulk soil or forest floor. Determination of differences in the microbial communities in rhizospheres or mycorrhizospheres of different tree species has the additional complication of accounting for differences in the timing of belowground C flux. If tree species differ in their phenology the magnitude and nature of root exudation may not be synchronized even if they are on the same site. Assessment of microbial communities in rhizospheres/mycorrhizospheres during peak and trough periods of belowground C flux would provide a better ‘like-for-like’ comparison among different species. Reported seasonal changes in microbial communities in bulk and rhizosphere/mycorrhizosphere soil suggest that comparisons at multiple times during the year would assist in developing a more complete image of the soil microbial communities associated with different tree species. Acknowledgements We wish to thank Shalima Ganesan for initial literature survey and David Levy-Booth, Dr Jesper Christiansen and two anonymous reviewers for constructive comments on the manuscript. This work was supported by NSERC Discovery Grants to both C.E.P and S.J.G. References Agerer, R., 2001. Exploration types of ectomycorrhizae: a proposal to classify ectomycorrhizal mycelial systems according to their patterns of differentiation and putative ecological importance. Mycorrhiza 11, 107–114. Ahonen-Jonnarth, U., Van Hees, P.A.W., Lundstrom, U.S., Finlay, R.D., 2000. Organic acids produced by mycorrhizal Pinus sylvestris exposed to elevated aluminium and heavy metal concentrations. New Phytologist 146, 557–567. Allison, S.D., Treseder, K.K., 2008. Warming and drying suppress microbial activity and carbon cycling in boreal forest soils. Global Change Biology 14, 2898–2909. Aneja, M.K., Sharma, S., Fleischmann, F., Stich, S., Heller, W., Bahnweg, G., Munch, J.C., Schloter, M., 2006. Microbial colonization of beech and spruce litter – influence of decomposition site and plant litter species on the diversity of microbial communities. Microbial Ecology 52, 127–135. Ayres, E., Dromph, K.M., Bardgett, R.D., 2006. Do plant species encourage soil biota that specialize in the rapid decomposition of their litter? Soil Biology & Biochemistry 38, 183–186. Ayres, E., Steltzer, H., Berg, S., Wallenstein, M.D., Simmons, B.L., Wall, D.H., 2009a. Tree species traits influence soil physical, chemical, and biological properties in high elevation forests. PLoS ONE 4 (6), e5964. http://dx.doi.org/10.1371/ journal.pone.0005964. Ayres, E., Steltzer, H., Berg, S., Simmons, B.L., Simpson, R.T., Steinweg, J.M., Wallenstein, M.D., Mellor, N., Parton, W.J., Moore, J.C., Wall, D.H., 2009b.

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