The study showed that in unmanaged boreal forests, fungal community structure in fallen Norway spruce logs is strongly related to stage of decay and wood density. Similar successions have been observed in polypore fruiting body occurrences (e.g. Renvall, 1995; Lindblad, 1998) and in fungal mycelia determined through culture-free DNA/RNA analysis (Rajala et al., 2011). Here, results based on DNA extracted directly from nearly 550 variably decomposed spruce logs in five sites described a more complete turnover of fungal species and functional groups with changing substrate quality.
Quality of decaying spruces and fungal community structure
Decay stage, density, moisture, nitrogen content and C : N ratio had the strongest correspondence to fungal community structure, and trends were similar in all study sites. Lignin and distance from contact with the soil were also important to fungal community composition. Unfortunately, the most important factor is difficult to discern because of the strong correlation among measured variables that change in concert during decomposition.
C : N ratios measured during the early stages of decay and decreasing rations with loss of mass are consistent with values reported elsewhere for Norway spruce (Kostiainen et al., 2004; Palviainen et al., 2008). A lignin concentration of 300 mg g−1 measured here in recently dead logs is similar to that measured from stem wood of Norway spruce (e.g. Anttonen et al., 2002; Raiskila et al., 2007). Our study is among the first to profile changes in lignin content during the decomposition of Norway spruce logs. The observed increase in lignin concentration with loss of mass agrees with the earlier study of Preston et al. (1998) that showed slow changes in early stages and a remarkable increase of lignin in highly decayed western hemlock, red cedar and Douglas fir.
Besides the physico-chemical properties, log diameter and volume affected fungal species composition, but not the number of species. Large logs usually harbour more fruiting bodies than smaller ones (Renvall, 1995; Hottola et al., 2009). This may be due to some species having high nutrient demands during fruiting body formation, and they remain obscure in logs that are beneath a threshold volume (Moore et al., 2008). Accordingly, we observed that white-rot fungi typically forming conspicuous fruiting bodies tend to favour large logs although they occurred in all size classes (data not shown). However, soft-rot fungi and other ascomycetes were typical in small logs, while more detailed information about the fungal species in the logs is needed before detailed conclusions.
Increasing fungal species richness in decaying logs
The number of fungal species increased with loss of mass and peaked in the most decayed logs. An increasing number of species during decomposition was not observed in a similar study of a smaller set of Norway spruce samples (Rajala et al., 2011), emphasizing the importance of large data sets. However, an increasing number of species during decomposition agrees with studies based on directly extracted fungal RNA (Rajala et al., 2011), as well as the culturing of microfungi (Lumley et al., 2001; Fukasawa et al., 2009b).
The number of polypore species bearing fruiting bodies is frequently observed to peak at intermediate decay stages (Bader et al., 1995; Renvall, 1995; Lindblad, 1998). A similar trend was observed in this study although the data were rather scant (not shown). A drop in polypore species number and their fruiting bodies during the late stages of decay can be explained by the fact that fruiting body production typically has a large resource requirement that is difficult to obtain in heavily decayed wood (Stenlid et al., 2008).
We propose the observed increase in fungal diversity during decomposition is because of the following reasons. Recently dead spruces offer a relatively harsh habitat for fungi because of low moisture and high levels of terpene and phenolic compounds (Groot, 1972; Boddy, 1992). At intermediate stages of decay, competition for resources and dominance of effective saprotrophic species may limit fungal diversity temporarily. In the final stages of decay, numerous soil fungi (Buée et al., 2009) begin to colonize heavily decayed logs as the distinction between these two substrates becomes increasingly slight.
Change in fungal species and their functionalities
At the onset of decay, fungal species in logs were mainly ascomycetes, but as decomposition continued, the community became increasingly dominated by basidiomycetes. Accordingly, fungal RNA- and DNA-based surveys have shown that soft-rot and other ascomycetes dominate the early decay stages of Norway spruce (Lindner et al., 2011; Rajala et al., 2011). Many ascomycetes are pioneer species that can rapidly colonize new and competition-free woody substrates (Siitonen, 2001). Moreover, some ascomycetes are already present in living trees, and after death, they may change from latent endophytes to saprotrophs (Menkis et al., 2004; Parfitt et al., 2010). However, ascomycetes are not able to degrade lignin appreciably, and decay remains localized (Boddy & Watkinson, 1995), which may lead to their replacement by fungi better suited to the substrate at this stage.
Functionalities of wood-inhabiting fungi changed so that at the first stage of decay, when no more than 20% of the substrate was decomposed, around one-fifth of the identified sequences were white-rot fungi, and a small proportion were brown-rot fungi. In early decay stages, Renvall (1995) observed a similar relationship between brown-rot and white-rot fungi based on the occurrence of fruiting bodies. However, fungal DNA extracted directly from woody tissue revealed that fungi with unknown life strategies were the most common in the early stages. These were often ascomycetes, which could have been soft-rot, staining or endophytic fungi.
In the middle stages of decay, the proportion of brown-rot fungal taxa peaked and together with white-rot fungi, they constituted at least half the species. An increase in the relative proportion of brown- to white-rot fungi has not been observed in studies based on fruiting bodies (Renvall, 1995), indicating that the role of brown-rot fungi might be more significant than previously thought. Coniophora olivaceae was a generalist brown-rot fungi detected in logs of decay stages 2–5. Accordingly, fruiting bodies of C. olivaceae on Norway spruce have been recorded in decay stages 1–4 (Renvall, 1995). The white-rot fungi Phellinus viticola and P. nigrolimitatus were frequently found generalist species detected in decay stages 1–4, whereas their fruiting bodies occur at intermediate and late stages of decay (Renvall, 1995; Stokland & Kauserud, 2004; Jönsson et al., 2008). This suggests that P. viticola and P. nigrolimitatus can colonize recently dead logs, but their fruiting body formation is delayed.
In addition to decomposer fungi, ECM fungi are an important functional group in boreal forests. Unlike saprotrophs, which acquire carbon from dead organic matter, ECM fungi depend on carbon sources provided by their host plants. ECM fungi utilize organic nutrients through their digestion with extracellular enzymes (Lindahl et al., 2002). Consequently, ECM and saprotrophic fungi may partly share woody habitats although saprotrophs usually dominate in competitive interactions as long as the amount of substrate available to saprotrophs is high (Lindahl et al., 2001).
Species known as ECM fungi were first detected in slightly decayed logs (stage 2), after which they increased to become the most dominant life strategy group at the expense of white- and brown-rot fungi. Species included members of Lactarius, Piloderma, Russula and Tylospora, which are all common ECM fungi associated with boreal forest trees (e.g. Tedersoo et al., 2003; Korkama et al., 2006; Toljander et al., 2006). The detection of ECM fungi in decaying logs is in line with earlier studies (Hashimoto & Yoneda, 2006; Tedersoo et al., 2008) and suggests that they can out-compete white- and brown-rot fungi in the late phases of decay when easily decomposed organic compounds are low. A comparable shift in fungal composition occurs in the boreal forest soil horizon, where saprotrophic fungi inhabit the energy-rich surface litter layers and ECM fungi increase in lower soil horizons with decreasing C : N ratio (Lindahl et al., 2006).
In boreal forest soil, ECM fungi may constitute one-third of the microbial biomass (Högberg & Högberg, 2002) and around 47–84% of fungal biomass (Bååth et al., 2004). As such, the potential colonization of ECM fungi is high for logs that are in contact with the soil. However, the detection of ECM fungi in weakly decayed wood is an intriguing result, and it agrees with results based on RNA of metabolically active fungi (Rajala et al., 2011). Likewise, Olsson et al. (2011) found that ECM fungi quickly colonized wood baits in boreal forests, indicating that suitable substrate availability may restrict their occurrence. ECM fungi can capture nutrients from living or dead mycelia of saprotrophic species (Lindahl et al., 1999; Buée et al., 2007). Although evidence concerning the ability of Lactarius, Piloderma, Russula, Tylospora and many other ECM fungi to degrade lignolytic complexes by themselves is increasing (Chambers et al., 1999; Luis et al., 2005; Bödeker et al., 2009), the notion of their role as facultative saprotrophs remains contentious (Baldrian, 2009).
Effect of fungal community structure on decomposition
Polypores and other white- and brown-rot fungi are the most important wood decomposers (Rayner & Boddy, 1988; Boddy et al., 2008), and the rate of mass loss likely peaks during their dominance. We were not able to determine the rate of decay directly, but based on model predictions (Harmon et al., 2000; Mäkinen et al., 2006; Tuomi et al., 2011), decay rate is highest at intermediate phases of decomposition, which coincides with the period when we observed white- and brown-rot fungi to be at their peak prevalence.
Results also indicated that fungal composition controls substrate quality of spruce logs. Variation in lignin concentration in strongly decaying logs was rather broad, and lower levels were associated with the presence of white-rot fungi. Although colonization of white-rot fungi at earlier phases of decay succession cannot be ruled out, the finding emphasizes their efficacy as lignin degraders. Nitrogen concentration and total amount of N peaked in logs of an advanced stage of decay that were inhabited by ECM fungi. This is likely a result of nitrogen translocated from the soil. Bacteria capable of fixing atmospheric nitrogen associate with certain ECM fungi (Timonen & Hurek, 2006) and could also influence local nitrogen concentrations.
Interpretation of DNA-based identifications
As expected (e.g. Lindner et al., 2011), the DNA approach detected a higher number of wood-inhabiting fungal species than the fruiting body inventory. However, only one-third of species observed as fruiting bodies were detected via sequencing of DGGE bands, probably due to inadequate sampling and sequencing effort. In fact, deeper sequencing is needed for complete exploration and determination of indicator species in each decay stage, because PCR-DGGE and Sanger sequencing reveals only part of the fungal diversity inhabiting wood (Ovaskainen et al., 2010). Nevertheless, DGGE-based approach does not show the actual abundance of each fungus in the community, whereas high throughput pyrosequencing coupled with PCR amplification may not be quantitative method either.
A remaining problem with molecular-based inventories is that their resolving power is dependent on the quality and coverage of reference databases. In addition to the problem of known species that have yet to be sequenced, many sequences in the public domain are undescribed species (Hibbett et al., 2011). Indeed, many sequences in our sample could not be identified based on their comparison with public databases. Recently, Hibbett et al. (2011) analysed nearly 100 000 fungal ITS sequences in GenBank and found that 37% of clusters contained only unidentified environmental sequences. We agree that this problem represents a great opportunity to fungal systematists.