Host phylogeny and datation
The phylogeny based on the combined data sets from ITS and MatK (Figure 1; see also Additional file 1: list of tree sequences used in this study) shows that European native species of Alnus are separated into two major clades that correspond to the subgenera Alnobetula and Alnus. Within the subgenus Alnobetula, A. alnobetula subsp. alnobetula and A. alnobetula subsp. suaveolens formed a monophyletic group. The subgenus Alnus clade comprises A. glutinosa and A. incana. Alnus cordata clusters outside this group. The multigene phylogeny confirms the relationship between the two Alnus clades with subgenus Alnobetula in a basal position. The molecular dating method gave the divergence between the two main subgenera of Alnus at the Eocene (around 48,6 My ago), and the original split between A. cordata and the A. glutinosa-incana complex at the Oligocene (around 22,9 My ago; Figure 1). Results also suggest recent lineage diversification at the Pleistocene for both subgenera (around 1,1 and 7,9 My ago for subgenus Alnobetula and the A. glutinosa-incana complex, respectively; Figure 1).
Alnicola phylogeny and reconstruction of the history of associations
The ITS sequences for most Alnicola [abbreviated Alc. below] collections were complete (see Additional file 2: list of fungal sequences used in this study). However, the numbers of variable and informative characters of the ITS molecular dataset were very low (27 on ITS1 and 21 on ITS2 for the whole section, i.e. 14 species). Most species were separated from each other by 2 to 6 nucleotides at best. Only Alnicola pseudosalabertii collections group together in a strongly supported clade that is well separated from the other lineages [32]. However, fourteen infrageneric groups (clusters or phylogroups, interpreted here as phylogenetic species) are identified, that are congruent with existing morphological taxonomy (morphospecies) at least for well-documented taxa [33, 34]. Six of them could not be identified to valid taxa and are here cited under provisional names (Alnicola badiofusca, Alc. citrinella, Alc. longicystis, Alc. pallidifolia, Alc. pseudosalabertii, Alc. xanthophylla).
Because the datasets of the four genes were highly congruent, a combined analysis was conducted on a 2743 bases long alignment, obtained by concatenation of all genes for each sampled morphospecies, with a Bayesian approach (Figure 2, 3, 4). Alnicola geraniolens Courtec., which belongs to Alnicola sect. Amarescens [33] (not associated with Alnus) was taken as an outgroup [32]. Alnicola cholea Kühner, in sect. Cholea P.A. Moreau [35], a more distant species not associated with Alnus, was also tested as outgroup but gave a weaker resolution on some branches (not shown). Although species relationships are still not completely resolved even when data from additional genes are included, the same fourteen species are clearly identified in all the analyses (each gene separately or the concatenated dataset), likely representing independently evolved lineages (Figure 2). The low sequence divergence and lack of phylogenetic resolution including with the combined gene dataset, suggests a fast radiation of these species in a short geological time.
The/pseudosalabertii clade is coming out as basal lineage amongst sect. Alnicola, with strong support in the concatenated analysis (Posterior Probability = 1, Figure 2). It is only represented by a rare species, only known from a few sites under Alnus alnobetula in mineral-rich subalpine situations. It has distinctive morphological characters such as spore features and pileal microstructure (unpublished data) that are also present in the non-alnicolous species of Alnicola sect. Amarescens, but not in other species of sect. Alnicola. These ancestral characters are congruent with its basal position in the phylogeny of Alnicola section Alnicola. Alc. pseudosalabertii, a species strictly associated to subgenus Alnobetula, appears clearly as an ancestral lineage.
The/luteolofibrillosa lineage appears as a collective species (or as a species complex), represented in Europe by at least two genetically distinct lineages. The former, Alnicola luteolofibrillosa s. str., is strictly associated with Alnus alnobetula in the Alps. The later, here named Alc. luteolofibrillosa "2", is collected under Alnus sect. Alnus in Europe and North America. Suprisingly, Alc. luteolofibrillosa "2" is found also with Alnus alnobetula subsp. suaveolens in Corsica, where Alc. luteolofibrillosa s. str would be expected according to host specificity. Alnicola silvae-novae (D.A. Reid) Courtec., a rare species encountered with Alnus glutinosa, comes out in the same cluster as Alc. luteolofibrillosa (Figure 2).
The ancestral state in the/luteolofibrillosa lineage is subgenus Alnus, while the host switches to Alnobetula probably occurred via a generalist species and niche contraction or host-dependent speciation (duplication).
In the/badia lineage, three species, Alnicola badia Kühner with Alnus alnobetula, Alc. longicystis ad int. and Alc. xanthophylla ad int. with Alnus glutinosa and A. incana, group together with very little nucleotide variation (7-8 characteristic positions on ITS1-2). All three are apparently restricted to specific soil conditions, e.g. sands and alluvial substrates.
The/umbrina lineage contains Alnicola subconspersa (P.D. Orton) Bon, Alc. umbrina (Maire) Kühner, and Alc. badiofusca ad int., that are three abundant fruiting species in alder stands. Both Alc. subconspersa and Alc. umbrina are associated with Alnus glutinosa and occasionally with A. cordata and A. incana, while Alc. badiofusca is found under Alnus alnobetula. In the present study, Alnicola dubis P.-A. Moreau & Vidonne is assimilated to Alc. subconspersa. Both Alc. striatula (P.D. Orton) Romagn. and Alc. scolecina (Fr.) Romagn. s. auct. [36] are assimilated to Alc. umbrina. Despite a recognition of Alc. dubis, Alc. striatula and Alc. scolecina in traditional taxonomy [34], these three species are temporarily grouped with the previous ones because of weak morphological support and only 1-2 nucleotide differences in the ITS sequences. These provisional taxonomic choices do not influence the results of our analysis since these taxa share the same host (Alnus glutinosa). However, taxonomy in this lineage obviously remains to be precised.
Finally four distinct species that comprise Alnicola citrinella (Alc. escharioides (Fr.: Fr.) Romagn. s. auct. pl.) with Alnus glutinosa and A. incana, Alc. pallidifolia with A. alnobetula, Alc. salabertii P.-A. Moreau & Guy Garcia with A. cordata [37], and Alc. sphagneti (P.D. Orton) Romagn., are also difficult to position in the phylogeny. However, they are characterized by distinctive morphological and ecological characters as well as nucleotide differences in the ITS and the other gene regions. Alc. sphagneti is associated with Alnus glutinosa, and apparently restricted to North Atlantic acidic localities; in the phylogenetic analyses, it comes out at the basis of the/umbrina lineage (Figure 2).
Alpova phylogeny
The monophyletism of both genera Alpova [abbreviated Alp. below] and Melanogaster [28] is strongly supported by both ITS (PP = 1) and multigene phylogenies (Figure 3), and these results are consistent with previous studies [38, 39]. Melanogaster is here taken as outgroup [40] to root the Alpova phylogeny. The relationships among the Alpova species were also well resolved with both phylogenies (not shown). Because Alpova appears as a genus with a reduced diversity in Europe (three species, Figure 3) the analysis was extended to extra-European species of Alpova, and phylogenetic reconstructions performed using ITS sequences.
Phylogenetic analysis of the ITS dataset clearly splits Alpova into two strongly supported clades, both including North American and European species (Figure 3). The first clade (PP = 0,83) contains an unidentified American taxon, Alpova "diplophloeus 1", in a basal position, while Alp. alpestris, a European species often encountered under Alnus alnobetula in the Alps, Carpathians and Corsica, is placed in a derived position. No significant molecular difference was found among continental collections of Alp. alpestris (represented by 9 sporocarps from 4 localities, all located in the French Alps). The two Corsican collections (3 sporocarps from 2 localities) are separated from the mainland collections by 9 nucleotide positions: two in the ITS1, one in RPB2 and 6 in GPD. The second clade (PP = 1) is rooted by another unidentified American taxon (Alp. "diplophloeus 2") and the South American Alp. austroalnicola associated with Alnus acuminata s.lat. (subgenus Alnus). It comprises three derived taxa that are the Corsican endemic Alp. corsicus (non host-specific but mainly with subgenus Alnus), Alp. "diplophloeus 3" (DQ989497) under Alnus incana, and a European collection (Alp. cf. cinnamomeus) that has been found so far under A. alnobetula.
The case of Alpova corsicus deserves a particular attention as this species, represented here by 8 sporocarp collections from 6 sites in Corsica (see Additional file 2: list of fungal sequences used in this study) and one sequence from mycorrhiza (not shown), appears restricted to the island of Corsica. When found, it fruits abundantly under Alnus glutinosa in riparian and peaty forests, but also under A. cordata at supramediterranean level, and more occasionally under A. alnobetula subsp. suaveolens (one single collection with longer spores) [28]. Remarkably, our numerous prospects under A. glutinosa or A. incana in continental France never showed us any basidiome of Alpova. In addition to the information provided by sporocarp surveys, molecular studies of ECM communities have not revealed so far the presence of Alpova species in alder stands of A. glutinosa or A. incana elsewhere in continental Europe. The few reports of Alpova sporocarps from Alnus incana and A. glutinosa in central Europe are referred to Melanogaster luteus [28, 41].
Lactarius phylogeny
Collections of Alnus-associated Lactarius are grouped in four independent lineages, based on the combined molecular phylogeny of ITS and rpb2 sequences (Figure 4A, 4B) as well as on separated gene phylogenies (not shown). The internal systematics of Lactarius is still insufficiently documented and the nomenclature of subgenera and sections follows the most recent phylogenetic assessment of the whole genus [42, 43]; Alnus-associated species belong to subgen. Piperites and subgen. Russulares which form together a monophyletic lineage. Subgenus Plinthogali is taken as outgroup for rooting the Piperites-Russulares phylogeny [42].
The/lepidotus lineage contains two species that are Lactarius lepidotus Hesler & A.H. Sm. strictly associated with Alnus alnobetula, and L. lilacinus (Lasch: Fr.) Fr. associated with A. glutinosa and A. incana. The two species are closely related despite strong morphological differences. Both are usually classified by taxonomists in subsection Coloratini (including species such as L. helvus, L. glyciosmus, and L. alpinus) due to the presence of typical dry squamulose pileus [44], but here surprisingly, they show genetic affinities with several groups with gelatinized pileus (ixocutis) of subgen. Piperites, represented in the phylogeny by sect. Deliciosi (L. quieticolor) (Figure 4B).
All collections of L. alpinus Peck, from the Alps and Corsica, group together with other species of Lactarius from subgen. Piperites, sect. Glutinosi, surprinsingly without close relationship with L. lepidotus despite morphological affinities (Figure 4B).
L. alpinus is associated to Alnus alnobetula and has been reported worldwide in the distribution range of its host, e.g. in N.E. USA, Alaska, Greenland, Carpathian Mountains [44, 45]. L. alpinus var. mitis Hesler & A.H. Sm. is known from alder trees (subgen. Alnus) in Western U.S.A. [45], and might represent a possible host-shifted vicariant of L. alpinus; unfortunately no DNA sequence or reliable dry material could be obtained.
The two remaining lineages (/omphaliformis and/obscuratus) are more modern than the previous ones, coming out from a species-rich group of small Lactarius (subgen. Russularia) mostly common in temperate-subarctic areas, such as L. tabidus (Fr.: Fr.) Fr., L. subsericatus Bon, or L. aurantiacus Fr., and strongly host-specific such as L. subdulcis (Fr.: Fr.) Fr. (restricted to Fagus) and L. hepaticus Plowr. (restricted to Pinus).
L. omphaliformis Romagn. forms a single clade that is genetically very stable, and surprisingly comprises a sequence obtained from Alnus acuminata in Argentina (GenBank DQ195543). In Europe, L. omphaliformis has been reported from the West under A. glutinosa in typically acidic situations, but it might be a cosmopolitan species associated with more species of Alnus sect. Alnus worldwide. The lack of reports under A. incana could be due to narrow biogeographical and edaphic preferences, not compatible with the natural distribution of the host in Europe. It is also one of the very few symbiotic species of Alnus sect. Alnus for which no relative species is known on A. alnobetula.
The/obscuratus lineage is composed of morphologically highly variable and taxonomically confused taxa (Figure 4A). Six distinct species or varieties are recognized by mycologists [44, 46, 47] and many names are usually cited in literature (e.g. L. obscuratus Lasch: Fr., L. cyathuliformis Bon, L. clethrophilus Romagn., L. brunneohepaticus M.M. Moser, L. obscuratus var. radiatus (J.E. Lange) Romagn., L. obscuratus var. subalpinus Basso, L. radiatus var. alnobetulae Bon). In the present study, three well-separated phylogroups are identified with a clear host specificity (Figure 4A) Both L. obscuratus s. str. (including L. clethrophilus) and L. cyathuliformis are found in association with A. glutinosa, A. incana and A. cordata, while L. brunneohepaticus (including L. obscuratus var. subalpinus) is associated with A. alnobetula in the Alps and Corsica.
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