Alder and the Golden Fleece: high diversity of Frankia and ectomycorrhizal fungi revealed from Alnus glutinosa subsp. barbata roots close to a Tertiary and glacial refugium

Introduction

Glacial refugia are recognized sources of genetic diversity and hot spots of taxonomic diversity (Médail & Diadema, 2009). They are also key regions for studying biogeography, as recently shown for fungi (Geml et al., 2010; Ghobad-Nejhad et al., 2012; Merényi et al., 2014). In Europe, the numerous fossil records from glacial periods and the analysis of allelic diversity have contributed to identification of refugia (Taberlet & Cheddadi, 2002; Gavin et al., 2014), mainly in Italy, the Iberian Peninsula and the Balkans (Médail & Diadema, 2009). Demographic contractions and expansions, and population genetic patterns associated with ice age refugia have been deeply studied in oaks, pines, beech, and alders (Taberlet & Cheddadi, 2002; Hampe & Jump, 2011). Interestingly, all these tree species host mutualistic fungi on their roots, and are ectomycorrhizal (EM; Smith & Read, 2008). However, whether these biogeographic and genetic processes play out similarly in microbes is unknown. In obligatory host-associated microbes, the potential for joint genetic responses to extinction and recolonization is high. On the other hand, they could have also survived outside of refugia, either in spore banks, or associated with other hosts. The existing knowledge about tree biogeography offers a framework to compare their symbiont communities inside and out of refugia, and test the hypothesis that symbionts have survived with their host in glacial refugia,

The recent history of Alnus has been intensely studied in Europe (King & Ferris, 1998; Douda et al., 2014; Havrdová et al., 2015; Mandák et al., 2016a; Mandák et al., 2016b). Compared to timber trees, alders are ideal models for biogeographical studies of historical migrations because there are numerous natural populations with low human impact (Douda et al., 2014). Thanks to molecular markers, southern refugia have been detected for A. glutinosa (L.) Gaertn. in the Iberian, Apennine, Corsica, North Africa, Balkan and Anatolian Peninsulas (King & Ferris, 1998). More recently, the existence of both refugia in Belarus and western Russia has been demonstrated; they were important sources for northward post-glacial expansion of alders (Douda et al., 2014). Today, A. glutinosa populations are still marked by recent isolation in glacial refugia (King & Ferris, 1998), and several unique chloroplast haplotypes have been detected in relict populations from Morocco (Lepais et al., 2013) and Turkey (King & Ferris, 1998; Havrdová et al., 2015). Of interest, the high genetic diversity detected in Turkey concurs with the observed morphological variations of A. glutinosa in this region. Therefore, four subspecies are recognized in this region: A. glutinosa subsp. glutinosa, A. glutinosa subsp. antitaurica Yalt, A.  glutinosa subsp. betuloides Ansin, and A. glutinosa subsp. barbata (CA Meyer) Yalt.

Alnus glutinosa subsp. barbata is distributed from Turkey to Iran, and is considered as a Tertiary relict plant, common in the Colchis forests of Georgia. Pollen records from the Quaternary era and distribution modeling have revealed that Alnus swamps were extended in Colchis floodplains and existed continuously over the last 10,000 years (Connor & Kvavadze, 2009). Today, stands of A. glutinosa subsp. barbata still dominate the Colchis floodplain (Nakhutsrishvili, 2012). Located in the South Caucasus, the Colchis region is one of the three major refugia of Tertiary relict taxa worldwide (Milne & Abbott, 2002; Kikvidze & Ohsawa, 2001; Denk, Frotzler & Davitashvili, 2001). Because a mountain ridge blocks rains coming from the Black Sea, the Colchis floodplain is well separated from arid lowlands and steppes from Eastern Georgia (Nakhutsrishvili, 2012). According to pollen records, alder recolonized Eastern Georgia and the central mountain ridge around 2,000 years BP, and are represented today by isolated populations (Connor & Kvavadze, 2009). Adjacent to the Colchis forests of Georgia, the other major Western Eurasia climate refugium for temperate forests is the Hyrcanian forest, located on the southern coast of the Caspian Sea in the region of Iran and southern Azerbaidjan. The two refugia lie approximately 2,000 km apart, and are now completely isolated from each other by steppe vegetation. However, they still share several Tertiary plant relict species, and are considered to be the two oldest temperate deciduous forests in western Eurasia (Maharramova et al., 2015).

As compared to other tree species, alders host few lineages of EM fungi (Molina, 1981; Moreau, Peintner & Gardes, 2006; Moreau et al., 2011; Rochet et al., 2011) and species-poor EM communities (Põlme et al., 2013; Roy et al., 2013; Kennedy, Walker & Bogar, 2015). Among frequently encountered EM taxa, Alnicola and Alpova are two genera that appear to be strictly associated with alders because they have never been found on any other tree species. Several species of Lactarius, Russula, Amanita and Cortinarius are also exclusive to alders. Alders also associate with nitrogen-fixing actinobacteria, all belonging to the genus Frankia (Weber, Nurmiaho-Lassila & Sundman, 1987) and also with strong host-specificity (Cotin-Galvan et al., 2016). At a worldwide scale, differences among Frankia communities are partly correlated with differences in EM communities (Põlme et al., 2014). From North America to Mexico, the similarities between EM communities also support a co-migration with their host (Kennedy et al., 2011). Together, these results suggest shared histories at broad scales, for the plant, Frankia and EM fungi.

If alder populations, Frankia and EM fungi communities have undergone a shared history, the bacterial and the fungal communities should have both survived in the Colchis, and alders should host more endemic symbionts in this refugium. Moreover, if South Caucasus has been a refugium and a recolonization source for Europe, European specific symbionts should be present in Georgia, together with endemic symbionts, especially in the Colchis. Therefore, the main objectives of the present study are to (1) document the diversity of the microbial symbionts communities of A. glutinosa subsp. barbata in Georgia and determine whether the Colchis floodplain hosts endemic symbionts, (2) test if EM fungi and Frankia communities are both more species rich in the Colchis, (3) examine how recent climatic history has imprinted the distribution of alder symbionts and their communities at a larger scale, by comparing our results to recent studies on the Hyrcanian forest.

Material and Methods

Sampling sites

Eight sites were studied in Georgia, including sites representing both large and isolated Alnus stands located from 0 to 1,800 m elevation. Three sites were located in the Colchis, considered as a refugium (PV, PP, PF). Five sites were located in the recolonized zone, three in central Georgia near the water separation line that splits Western and Eastern Georgia (BA, BO and SV), and two in Eastern Georgia at the Caucasus footstep (TS and TR; Fig. 1A, Table S1 of Appendix S1). Climatic data (19 variables) were retrieved from the Worldclim database (http://www.worldclim.org) through R raster package (Hijmans & Van Etten, 2012).

Diversity patterns of symbiont communities in Georgia

For Frankia and EM fungi, the species richness (alpha diversity) was measured at the site level, and the beta diversity was measured through Bray–Curtis distance, which takes species abundance into account. Species accumulation curves at the scale of Georgia and Chao 1 diversity indices were computed to reflect gamma diversity. The spatial autocorrelation of communities was tested by a multivariate autocorrelation test (multispati r test, ade4 package, Dray & Dufour, 2007). The correlation between beta diversity and distance was tested by a Mantel test (999 permutations). Because these tests were not statistically significant, no vector of spatial weights was integrated in the following tests. The difference in community structure (measured by the Bray– Curtis distance) between the three sampling zones was tested by a permutational multivariate analysis of variance (perMANOVA). Similarities between communities were visualized by a non-metric multidimensional scaling (NMDS). The correlation of NMDS structure with bioclimatic factors, latitude, longitude, and elevation was tested by permutations tests (envfit function, vegan package in R, Oksanen et al., 2007). The same analysis was computed for Frankia and for EM fungi together and separately.

Results

Phylogenetic diversity of Frankia and comparison with European strains

The Frankia strains detected in Georgia did not cluster into a monophyletic clade, and the twelve MOTUs were scattered throughout the phylogeny (clades 1b, 3 and 5 of Pozzi et al., 2015): clade 3 and 5 comprised three and four MOTUs respectively, and “new clade II” gathered two distinct MOTUs. All MOTUs but one belonged to clades commonly associated with A. glutinosa in Europe (Fig. 3). Two sequences from the Eastern population (TR) belonged to a monophyletic clade, frequently associated with A. alnobetula (Erhr.) K Koch (clade 1b, Fig. 3). Three sequences from Western populations were not similar to any other known sequence (new clade II and III, Fig. 3). Finally, two Central Georgia sequences clustered in an isolated lineage (new clade I, Fig. 3).

Phylogenetic diversity of ectomycorrhizal fungi and comparison with European sequences

DNA was extracted and PCR-amplified from a total of 732 individual root tips: 484 of them produced ITS sequences among which 328 met our quality threshold. The four Helotiales and non-EM fungi sequences were not taken into account in our analysis (one Xylariaceae, four Rhizoctonia, one Diaporthales). Twenty-nine MOTUs were recorded among which 21 belonged to Basidiomycota and 8 to Ascomycota (91.2% and 8.8% of ectomycorrhizae, respectively). Seven Alnicola species were detected, all strictly identical to species found in Europe. Of interest, two new ITS variants of Alnicola xanthophylla were detected in Central Georgia, differing by five positions from their European relatives. Among non-Alnicola EM fungi, five new MOTUs were detected in Georgia. Three were Ascomycota (Tuber sp., Tarzetta sp., Peziza sp.) and two Basidiomycota (Inocybe spp.). These five possibly endemic EM fungi were found in Central and Eastern Georgia.

Diversity patterns of symbiont communities in Georgia

Species accumulation curves show that sampling was not saturated, especially for EM fungi. According to Chao 1 estimates, up to 26.7 Frankia and 44.7 EM MOTUs could occur in Georgia (Fig. 4A). Species richness per site varied between 7 and 15 MOTUs for fungi, and from 2 to 4 for Frankia (Fig. 4B). Compared to the recolonized zone, Frankia and EM communities were neither significantly richer (Kruskal-Wallis test, p = 0.48 and p = 0.17 respectively, Fig. 4B) nor different based on community composition (perMANOVA on Bray–Curtis distances, p = 0.40 and p = 0.19 for Frankia and EM fungi, respectively). Communities were not spatially autocorrelated (multivariate spatial autocorrelation test, p-value = 0.338 for EM fungi, p = 0.348 for Frankia), and the spatial distance did not correlate with Bray–Curtis distances at least for EM fungi (Mantel test, p = 0.16). For Frankia, the beta diversity was marginally correlated with distance (Mantel test, p = 0.0594). For the two types of symbionts, Bray–Curtis distances between sites were significantly correlated with longitude and several bioclimatic factors linked with precipitations (see Table S3 of Appendix S1, Fig. 5A).

Discussion

Our first objective was to document the diversity of symbiont communities associated with Alnus glutinosa ssp. barbata in Georgia, especially in the Colchis floodplain, a glacial and Tertiary refugium for alders. In all, 29 EM MOTUs and 12 Frankia MOTUs were uncovered, and Chao 1 estimates showed that twice as many species may occur there. For Frankia, the common European lineages were detected, together with five new lineages, which is an exceptional pattern considering the extensive worldwide Frankia database (Pozzi et al., 2015). Georgia proved to be a region of high Frankia diversity, probably of ancient origin as suggested by the long branches supporting Caucasian lineages (Fig. 2). For EM fungi, most Georgian MOTUs (24 out of 29) were already reported from Europe, on roots of A. glutinosa, A. incana and A. cordata (Loisel.) Duby. No endemic MOTU was detected for the most specialized lineages, but two unique ITS variants of Alnicola xanthophylla were found in Central Georgia. From previous study on the genus Alnicola (Moreau, Peintner & Gardes, 2006; Rochet et al., 2011), the ITS is known to be often not variable within species, and 1.1% genetic variability is exceptional for this genus. Although a deeper sampling may reveal more fungi, these results reveal that endemism is relatively rare in the Caucasus region for EM fungi, even the more specialized ones associated with alders. Interestingly, a similar analysis on corticioid fungi in South Caucasus has also pointed out the low number of endemics, and the high similarity with European communities (Ghobad-Nejhad et al., 2012).

The presence of five endemic Frankia, but no endemic specialized EM fungi was not expected but could be explained by differences in dispersal and survival abilities between the two types of symbionts. A free-living stage (outside the host) has been reported for the Frankia strains with low host specificity (Maunuksela et al., 1999), but aside from this case, most uncultured Frankia are highly dependent of their host (Pozzi et al., 2015). As with Frankia, most EM fungi cannot survive in the soil without their host (Lindahl & Tunlid, 2015). Frankia produces spores belowground and is dispersed by water or animals (Chaia, Wall & Huss-Danell, 2010) and sometimes by wind (Dawson, 2007) leading to short-distance dispersal, and perhaps greater endemism whereas Alnicola are wind-dispersed and potentially disperse over large distances. Among fungi, two potential endemic species belong to a wind-dispersed genus (Inocybe). Although species of Inocybe are regularly detected on alder roots (Bogar & Kennedy, 2013; Roy et al., 2013; Põlme et al., 2013), current knowledge on this genus is still too limited to determine if these Inocybe sp. are specific to Alnus or if their occurrence reflects a local adaptation to a particular habitat. More generally, Ryberg et al. (2008) have shown that the ITS evolves faster in the genus Inocybe, and that environmental sequencing often reveals new species in this genus. Among the EM fungi lineages that form hypogeous (below-ground) fruitbodies, three potential endemic Ascomycota species were detected. The degree of specificity of these species towards Alnus is still unknown, and two genera, Tuber and Tarzetta, are often described as pioneer species with broad host ranges. The presence of endemics for these two genera might be due to their long-term persistence in refugia. Similarly, the persistence of the black truffle Tuber melanosporum Vittad. during glaciations has been shown in Spanish (García-Cunchillos et al., 2014) and Italian refugia (Rubini et al., 2005).

Our second objective was to assess whether the symbiont communities and their host populations follow a shared history at the scale of Georgia. We hypothesized that sites in the refugium would host more endemic and diverse communities of both Frankia and EM fungi, but we did not confirm our hypothesis. First, the host population were genetically more diverse in the recolonized zone, where several unique ITS and matK variants were detected for A. glutinosa subsp. barbata. These variants were clearly distinct from other sequences of European A. glutinosa and this result confirms that Georgia, and not only the Colchis, is a hot spot of genetic diversity for the A. glutinosa complex (pointed out by King & Ferris, 1998). Interestingly, all endemic EM MOTUs were detected in Central and Eastern sites, associated with the most genetically diverse host populations. However, the species richness of Frankia communities showed a different distribution, as three endemic strains were detected in the Colchis, and two in the Central population. These different distributions of genetic diversity and species richness suggest that all symbionts do not necessarily survive with their host in refugia. Moreover, the occurrence of unique ITS and matK variants for alders and endemic EM fungi in Central and Eastern sites show that isolated host populations could also act as refugia, especially in mountain ranges, as highlighted by recent reviews on the Mediterranean basin (Médail & Diadema, 2009; Dobrowski, 2011; Keppel et al., 2012). The differences between the Colchis and Central and Eastern populations could reflect different biogeographic histories, as shown for snails and amphibians (Tarkhnishvili, 1996; Tarkhnishvili, Thorpe & Arntzen, 2000; Pokryszko et al., 2011) but also correlate with changes in annual precipitation and summer temperatures, as pinpointed by our analyses (Fig. 5).

Our third objective was to compare the Colchis and the Hyrcanian communities, associated with two alder species, A. glutinosa subsp. barbata and A. subcordata, that are both considered to be Tertiary relics. The two refugia hosted distinct EM communities, and differences were strongly explained by both host identity and geographic distance. The lack of shared MOTUs, even abundant ones (Table S2 of Appendix S1), explains these differences, and suggests a limited spore flow between the two regions. The scarcity of alder stands between these two regions in the steppe landscapes could indeed limit dispersal, and maintain isolation between the two regions, as observed for plants (Nakhutsrishvili et al., 2015) and corticioid fungi (Ghobad-Nejhad et al., 2012). Interestingly, both A. subcordata and A. glutinosa subsp. barbata associate with several Inocybe, Ascomycota and especially Tuber. Additional Tertiary refugia should be investigated in Asia to determine if these associations are also relic from the Tertiary, or derive from local and independent adaptations in relatively dry regions for alders. Indeed, Tuber and hypogeous fungi are known to be particularly resistant to drought (Richard et al., 2011; Herzog et al., 2013 ) and abundant in dry habitats (Zambonelli et al., 2014).

For microbes, biogeographic studies are relatively recent (Martiny et al., 2006), partly because the “everything is everywhere” idiom was long said to be the rule. Now, Beringia has been pinpointed as a hotspot for high-latitude fungi (Geml et al., 2010; Bellemain et al., 2013), and could have been a glacial refugium for the fungi. In Europe, the Perigord truffle Tuber melanosporum, also an EM species, has more genetically diverse populations in southern-Italy (Rubini et al., 2005) and Spain (García-Cunchillos et al., 2014). Finally, the anther smut fungus Microbotryum sp., an obligate parasite of Caryophyllaceae, has apparently followed its host during recent glaciation (Vercken et al., 2010). Here, we focus on entire communities and show on the contrary, that alpha diversity was not higher in the refugium than in the recolonized zone. The results from analyzing communities versus populations are not necessarily coupled (Vellend & Geber, 2005), but theoretically area size and immigration have parallel effects on species richness and genetic diversity (Vellend & Geber, 2005). The Theory of Island Biogeography (MacArthur & Wilson, 1967) predicts that smaller refugia can favor local extinctions (Kadmon & Allouche, 2007), and moreover, bottleneck effects may also reduce local diversity (Bennett & Provan, 2008). On the other hand, recent experiments on alders have shown that their specificity might be controlled by pH and nitrogen availability (Huggins et al., 2014), which may reduce the influence of host biogeography on symbiont community diversity. In this framework, it is even more puzzling to observe more endemic EM fungi and rare plant ITS variant in the isolated populations from Central and Eastern Georgia. As the endemic Inocybe and Tuber are not usually associated with alders, we suspect that these taxa have only recently colonized alder roots, a host shift probably favored by the actual isolation of alders in this region.

Conclusions

Wiens & Donoghue (2004) have already highlighted the chasm between historical biogeography and community ecology, and suggested that more links should be drawn between these topics. In a second step, studies on symbiont populations may help confirming the lack of isolation in refugia. The recent studies of alder refugia in Europe could be used as a framework to study more in depth alder symbiont biogeography (Douda et al., 2014; Mandák et al., 2016a; Mandák et al., 2016b). Finally, the occurrence of endemic OTUs detected for both EM fungi and Frankia outside of refugia highlight their relevance to biodiversity conservation not only in Tertiary and glacial refugia, but also in isolated host populations.

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