In the ectomycorrhizal (ECM) symbiosis, the root system of an individual tree is typically colonized by several members of different ECM fungal species, and individual fungi can normally associate with several plants . Ectomycorrhizal fungi display a great range of host specificity [2, 3]. This varies from extremes such as Suilloideae that are almost exclusively associated with Pinaceae  with the example of the very host-specific Suillus pungens colonizing one or few related pine species , to species as Laccaria amethystina that demonstrate a true multihost ability . The tight affinity between many ECM fungal species and their hosts has led to host-based taxonomic treatments in certain genera, e.g. Leccinum , and implicitly suggests that the phylogeny of the fungi follows that of their host plants, a process commonly known as coevolution . Two main categories of events can be proposed to explain highly specialized associations between a plant and a fungal symbiont: (i) cospeciation/codivergence events where a symbiont speciates in response to the speciation of its host (association by descent), the phylogenetic outcome is congruent phylogenies of interacting taxa, and (ii) speciation through host shifts where the symbiont switches from the ancestral host to a new, unrelated host species (association by colonization); the phylogeny of the symbiont is influenced by the host evolution, but it is not reciprocal. Both processes suggest that host diversity, if not necessarily the sole cause of reproductive isolation and speciation, may force diversification and speciation of their symbionts. Host specificity is generally expressed as a symbiont's adaptation to a particular host species or higher taxa. The model investigated in the present study is the ectomycorrhizal symbiosis between the alder genus Alnus Mill. (Betulaceae) and three genera of Basidiomycota.
Based on fossil pollen evidence, the plant genus Alnus would have originated from tropical Eastern Asia around the Late Cretaceous , and it is likely that it reached Europe during early Oligocene. It was diversified in central Europe tropical forests by the end of Oligocene - early Miocene , and adapted to temperate and subarctic environments at the Miocene period . Species of Alnus are now distributed in temperate and arctic regions of the Northern Hemisphere, except A. acuminata Kunth (sensu lato, including A. jorullensis Kunth) that extends as far South as South America. There are 29-35 species of Alnus is the current flora, with 4-5 species in Europe, 9 in the New World, and 18-23 in Asia [8, 10, 11]. Alnus consists of three clades [11, 12] including one subgenus not represented in Europe (subgen. Clethropsis), and two widely distributed subgenera Alnobetula and Alnus. The subgenus Alnobetula (also described as genus Duschekia Opiz) is sister to subgenera Clethropsis and Alnus, and likely the most primitive one [11–13]. It is represented, according to authors, either by a single thicket-forming circumpolar species: Alnus alnobetula (Ehrh.) K.Koch [also known as A. viridis (Chaix) DC.] divided into geographical subspecies [8, 10], or by several allopatric or parapatric species [11, 12]); A. alnobetula was already present in France at the late Miocene, about 5.34 My ago . The second main lineage (subgenus Alnus) is represented by numerous species in Eastern and Central Asia, with radiations towards North and South America, Europe, the Mediterranean basin and Eastern Asia, differentiated during Pleistocene. In subgen. Alnus, A. glutinosa (L.) Gaertn is a European and North African endemic species, present from W Europe and N Maghreb to Fennoscandia; pollen records and molecular data have revealed distinct major southern refuges in the last ice age, including W France, Corsica, S Italy, N Africa, Carpathians, and Turkey [15, 16]. Alnus incana (L.) Moench is distributed across the cooler parts of Europe, mainly in Northern Europe and high elevation mountains in the Alps, the Carpathians and the Caucasus. Alnus cordata (Loisel) Duby is a Tyrrhenian endemic species that was isolated during the Pleistocene in Corsica and in a few other Mediterranean ice-free areas (Southern Italy, Albany) [17, 18] where it remained confined since its massive introduction for forestry all over Europe during the late 20th century.
After the Quaternary glaciations the circumpolar species Alnus alnobetula and A. incana expanded in continental Europe (throughout Alps and Carpathians for A. alnobetula, up to Eastern France and Scandinavia for A. incana) from several refugia located in Central and Eastern Europe [10, 19], while A. alnobetula subsp. suaveolens (Req.) Lambinon & Kerguélen likely evolved isolated as an endemic subspecies in Corsica since the Pleistocene [20, 21].
From analysis of mycorrhizae [22–27] alder trees have revealed an exceptional species-poor assemblage of ECM fungi compared to the other tree species, with less than 50 fungal species (including unidentified taxa) reported worldwide in the literature on A. acuminata, A. alnobetula s. lat., A. glutinosa, A. incana s. lat., and A. rubra. The fungal communities are dominated by six Basidiomycete genera, whatever the species of Alnus considered: Tomentella (12-15 spp.), Alnicola (15-20 spp.), Lactarius (5-8 spp.), Cortinarius (6-10 spp.), Alpova/Melanogaster  (6 spp.), and Russula (2-4 spp.). The fruiting community is also composed of two locally abundant taxa, Paxillus (1-2 spp.) and Gyrodon (1 sp.), rarely found from mycorrhizae analysis (Rochet et al., unpublished results). Other occasionally reported genera are Amanita (A. friabilis), Hebeloma, Inocybe, Pachyphloeus, Pseudotomentella, and unidentified Helotiales (possibly root endophytes). The Alnus-ECM fungi association is considered the most specialized ECM symbiosis. Except Tomentella spp. (for which precise taxonomic information is lacking) and several species of Ascomycota , all species are known, or strongly suspected, to be exclusive to the genus Alnus since they have never been found on any other trees than alders at present. There are reports of species-poor and specialized ECM fungal community for the ectomycorrhizal larch tree Larix spp. and five-needle pines (e.g. Pinus cembra, P. strobus) [29–31]. High specificity patterns are also observed for many orchids and monotrope plants associated with ectomycorrhizal fungi .
For the mycorrhizal symbiosis, it is still an open question how associations between plants and fungi arise and how specificity occurs. One way would be geographical isolation of populations leading to a narrowing of host range and allopatric speciation. However, we hypothesize than in highly specialized associations such as the alder-ECM symbiosis, plants exert considerable selective pressure on their fungal symbionts and are major drivers of diversification. This hypothesis was evaluated here by documenting the history of Alnus-ECM fungi association and specificity through molecular phylogenetic reconstructions.
Strong host-specificity patterns in mutualistic as well as parasitic relationships suggest intuitively a narrow coevolutionary history between symbionts . The degree of parallel speciation and host switching between alders and three ECM taxa of basidiomycetes are explored as well as patterns of biogeography, with aims to develop more specific hypotheses on the processes that contribute to the diversification of the fungal lineages. Our predictions were that (i) the association with subgenus Alnus, more "modern" than subgenus. Alnobetula, is a derived character (the speciation of the fungi is linked to the speciation of the host), or (ii) evolution of a fungal symbiont would lead to increased specialization (there is an ongoing process of speciation by adaptation to new hosts).
The fungal lineages, Alnicola sect. Alnicola , Alpova, and Lactarius, were selected on the following criteria: 1) are present with all species of Alnus (excluding Paxillus and Gyrodon for this reason, never reported with subgen. Alnobetula); 2) have well-defined species concept (excluding Cortinarius, Tomentella and Alnicola sect. Submelinoides  for this reason); 3) contain enough species to obtain an informative phylogenetic tree (excluding Russula and Amanita for this reason). Sequences from one to four DNA regions were analyzed, including the nuclear rDNA ITS, parts of the nuclear genes rpb2 and gpd, and the V9 domain of the mitochondrial SSU-rDNA. Phylogenetic relationships among the European alders (five species and subspecies) were investigated using the ITS and chloroplast MatK sequences.