Gamma diversity and outlines of Alnus-associated ECM communities
A large diversity of ecological conditions and five different host species and subspecies were investigated. A relatively high diversity of fungal MOTUs was expected; however, only 86 MOTUs were detected. This is twice to four times higher than former studies of Alnus-associated ECM communities from Estonia (Tedersoo et al., 2009 (40 taxa)) and North America (Kennedy & Hill, 2010 (14 taxa); Kennedy et al., 2011 (23 taxa)). Chao estimates suggested that nearly half of the potential MOTUs were detected (Table 2), and singletons were relatively few (27 MOTUs), supposing that the sampling effort was sufficient to detect most Alnus-associated ECM fungi present in France. Only five Alnus-associated ECM species reported from the myco-ecological literature were not found: Amanita friabilis, Alnicola badia, Alnicola suavis, Alpova corsicus, and Cortinarius kuehneri (Brunner & Horak, 1990). Moreover, some species thriving on several sampling sites, such as Gyrodon lividus, Paxillus spp., Alpova alpestris, and Alnicola salabertii, were only detected on a few mycorrhizas. It is likely that additional sampling would better detect some of these ‘specific’ species, possibly overlooked as a result of irregular spatial or temporal distribution.
Among the singletons, wide-ranged species such as Clavulina spp. were also recorded (e.g. from the Sainte Croix Volvestre site, where A. glutinosa is growing along with conifers and other broad-leaved trees). Most singletons were not documented by sporocarps (Table S3) and do not belong to usual alder-associated genera. Moerover, a few clues from experimental in vitro studies also show that Alnus spp. can form ectomycorrhizas with generalist fungi (e.g. A. glutinosa with Pisolithus tinctorius; Molina, 1981). Reciprocally, Alnus ECM fungi can colonize Betula occidentalis in experimental plots (Bogar & Kennedy, 2013). However, such associations rarely occur in nature, supporting the hypothesis of a high specificity of the whole alder–ECM community.
All sites were dominated by a few species of Tomentella and Alnicola, as already revealed in previous studies of alder-associated ECM communities in both Europe and America (Becerra et al., 2005; Tedersoo et al., 2009; Kennedy & Hill, 2010; Kennedy et al., 2011). Phylogenetic studies by Rochet et al. (2011) of Alnicola, as well as by Kennedy & Hill (2010) of Tomentella, have already demonstrated that ECM species found on alders do not occur on other host trees. This observed dominance of a few specific ECM fungi, belonging to few lineages, is also a unique feature of the alder lineage worldwide. Hence, the dominance of such fungi shows that Alnus–ECM fungi mutualism is an isolated system, highly conserved at a large geographical scale and probably not connected to common mycorrhizal networks involving other local tree species.
The drivers of Alnus-associated ECM community diversity and specificity
In Europe, Alnus populations often grow in patches. Thus, Alnus associated ECM fungi might be highly dependent on the Alnus host, and isolated between sites. If dispersal is not limited for these ECM fungi, then community alpha diversity may be shaped either by neutral processes or by a combination of host, habitat and competition. Our sample, covering a broad range of habitats and hosts at a regional scale, was adequate for testing these alternative hypotheses, although competition and other interspecific interactions would require experimental settings.
At the regional scale (southern France), spatial autocorrelation tests and correlation between Bray–Curtis distances and geographic distance were nonsignificant. At the MOTU level, dominant Tomentella spp. and most Alnicola spp. occurred nearly everywhere, such as A. citrinella, present in the Alps, southwest France and Corsica. Interestingly, sequences from A. citrinella were 100% identical to Genbank sequences recorded for fungi found in other Alnus populations, in Europe and the Middle East (e.g. JN198094). Even though gene flow should be measured with markers other than ITS, these results suggest that distance itself might not limit the dispersal of alder–ECM fungi.
Furthermore, the community evenness was lower for high longitude and in Corsica, and changes of beta diversity correlated with longitude (Table 1). Corsica as an island was shown to host some endemic hypogeous species such as A. corsicus (Moreau et al., 2013), but no (or a few) ectomycorrhizas were found for these species. Moreover, communities were slightly less diverse in Corsica, and characterized by a lower evenness. Therefore, the geographic position of Corsica may explain the observed effect of longitude and shows that geography, more than distance itself, shapes these communities.
The topography also played an important role, as altitude influenced both evenness and beta diversity variation. Altitude appeared to be a key determinant of the distribution of several ECM fungi, such as Lactarius and Alnicola spp. (Fig. 2). While Bahram et al. (2012) found a lower species richness at high altitude in the Hyrcanian forest, our study rather suggests a shift of ECM communities over 1400 m, with the occurrence of the subgenus Alnobetula, whatever the region. Therefore, the gap between habitats naturally colonized by the two Alnus subgenera in western Europe is also illustrated by fungal species distribution (Fig. 2).
Among sites, the ECM species richness was variable, but not determined by the tree species. The alpha diversity was low (maximum of 16 MOTUs per site, 8.4 on average), as previously reported (Tedersoo et al., 2009). This common trait of alder species suggests a similar constraint on ECM communities, which should be investigated by comparing alders with other host trees. The differences between host subgenera and species were revealed by community composition. However, most MOTUs were shared among hosts, at least within a subgenus, such as Alnicola spp. and Lactarius spp. (Fig. 6). Then Alnus ‘specific’ ECM fungi showed preferences towards the host rather than strict host-species specificity. This pattern is relatively common for ECM fungi (Dickie, 2007) and is also suggested by meta-analyses on several host trees (Ishida et al., 2007; Tedersoo et al., 2012). In this study, focused on an exceptional model of specific fungi community interaction, by comparing several congeneric host species at a regional scale, both the ‘host species’ effect on communities and the ‘preferences’ of ECM fungi were confirmed, as for other host trees. These results show that the host-species effect cannot result only from host-species specificity, possibly because, at a regional scale, long-distance dispersal may limit the selection for a higher specificity.
Habitat was described both qualitatively and quantitatively, but none of the tested climate and soil parameters correlated with the alpha diversity variations. A larger number of species were generally found on organic soils (e.g. in peat bogs) than in mineral sites (e.g. stream sites), as already reported in the published literature (Tedersoo et al., 2009). Evenness was also higher on organic soils in forest and peat bogs than in mineral substrates (effect of site, Table 1). Thus, other relevant parameters should be measured to identify those that may explain alpha diversity variations between organic and mineral soils, such as soil water content or anoxia. Focusing on factors favoring a high alpha diversity, interpretable as a local patrimonial richness, is of interest in nature conservancy programs, such as riparian forest conservancy in the European Habitat Directive (e.g. Council Directive 92/43/EEC Art. 17 (910E)).
Beta diversity was partly explained by several soil parameters such as organic matter content (Fig. 3, Table 1). However, for each host species, driving soil factors were different. The A. glutinosa communities were shaped by pH, carbon, nitrogen, phosphorus and organic matter content, as shown by Tedersoo et al. (2009), whereas A. alnobetula communities were partly explained by high potassium concentration on most sampled sites (Fig. 4). The distribution of Tomentella species, shown to be indifferent to host identity (Fig. S1b), appeared to be shaped by nitrogen, sulfur, carbon and soil organic matter content. The distribution of Alnicola spp. was mainly influenced by host identity (23% of variance, Fig. 5), but also by soil factors such as pH, organic matter, nitrogen, carbon and sulfur concentration (Figs 5, S1b). Curiously, climatic parameters, although variable in our sample, failed to explain alpha and beta diversity variations. The minor impact of climate in our study stresses the major role of host identity and soil parameters as drivers of these communities at a regional scale.
All in all, host identity, soil parameters, and geographical position accounted for 13, 13 and 9% of the beta diversity variance, respectively (Fig. 5). Interestingly, these factors all explained beta diversity variation in similar proportions, and do not exclude each other. In the future, competition or neutral processes should also be considered, as a large part of beta diversity variation remained unexplained. Moreover, by comparing Alnicola and Tomentella, two dominant genera, our study shows that different processes may explain ECM fungi distribution and that specificity of Alnicola spp. may rely not only on host but also on habitat preferences.
Disentangling host and habitat effects on alders
To confirm host effect independently of altitude and soil in situ, different alder species should be sampled in naturally mixed populations. In the Alps, A. alnobetula and A. incana grow together in some sites, but these situations are often located at the limits of their respective distributional range. A better situation is found in Corsica where the ecology of A. cordata matches that of A. glutinosa along rivers or roadsides. Eight MOTUs were found on both A. cordata and A. glutinosa in Corsica – four were shared locally and four were shared among sites (differences between hosts were still significant; Adonis test on Bray–Curtis distance, F = 2.52, P = 0.004). The shared fungi were dominant MOTUs without host preference (Tomentella spp.) or associated with the Alnus subgenus (Lactarius cyathuliformis, A. citrinella). To extrapolate these few cases, when co-occurring, each Alnus sp. seems to share dominant MOTUs such as Tomentella spp., but keeps a few specific MOTUs.
Interestingly, except for Alnicola salabertii and Clavulina spp., the ‘nonshared’ MOTUs of A. cordata are Pezizales, that is, fungi not considered as host-specific to date (Tedersoo et al., 2006, 2009). The occurrence of these Pezizales suggests that A. cordata, which exhibited a low diversity of MOTUs, hosts less specific associates than other alders. To understand this particularity, further investigations on A. cordata in its native areas (Corsica and Italy) are required, as well as in its increasingly numerous plantations in continental Europe.
By investigating one of the most specific plant–ECM fungal interactions, at a regional scale, this study demonstrates that: host-species specificity is rare and ECM fungi exhibit preferences for a host subgenus; host identity, altitude and soil drive ECM community diversity and structure; specific ECM fungi exhibit a habitat preference within host subgenus distribution range; climate has a minor influence at a regional scale; and still a large part of the variability remains unexplained. These findings can potentially be applied to most ECM fungi, but a few aspects are particular to Alnus, such as a low alpha diversity and high similarity between distant communities. The conserved dominance of a few specific ECM fungi across continents may suggest high gene flow between populations, a constant and uniform selective pressure, or a drastic bottleneck at the beginning of the ECM–Alnus association. These hypotheses have to be tested to refine the comprehension of processes favoring or limiting the selection for more specificity in ECM mutualism at a regional scale.