Multilocus phylogenetic and nested clade analyses of Alaskan and global samples show that Amanita muscaria sensu lato comprises three cryptic phylogenetic species that occur sympatrically in Alaska rather than as allopatric groups. The results point to a Siberian–Beringian origin with fragmentation, speciation and subsequent range expansions across Eurasia and North America, while ancestral pileus and wart colour polymorphisms persisted and Beringian populations adapted to climatic cooling.
Amanita muscaria sensu lato has a wide geographic distribution, occurring in Europe, Asia, Africa, Australia, New Zealand, and North, Central and South America. Previous phylogenetic work by others indicates three geographic clades (i.e. ‘Eurasian’, ‘Eurasian‐alpine’ and ‘North American’ groups) within A. muscaria. However, the historical dispersal patterns of A. muscaria remained unclear. In our project, we collected specimens from arctic, boreal and humid temperate regions in Alaska, and generated DNA sequence data from the protein‐coding beta‐tubulin gene and the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA repeat. Homologous sequences from additional A. muscaria isolates were downloaded from GenBank. We conducted phylogenetic and nested clade analyses (NCA) to reveal the phylogeographic history of the species complex. Although phylogenetic analyses confirmed the existence of the three above‐mentioned clades, representatives of all three groups were found to occur sympatrically in Alaska, suggesting that they represent cryptic phylogenetic species with partially overlapping geographic distributions rather than being allopatric populations. All phylogenetic species share at least two morphological varieties with other species, suggesting ancestral polymorphism in pileus and wart colour pre‐dating their speciations. The ancestral population of A. muscaria likely evolved in the Siberian–Beringian region and underwent fragmentation as inferred from NCA and the coalescent analyses. The data suggest that these populations later evolved into species, expanded their range in North America and Eurasia. In addition to range expansions, populations of all three species remained in Beringia and adapted to the cooling climate.
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Michelot, D., Melendez-Howell, L. M. · The British Mycological Society (2003)
Amanita muscaria, the fly agaric, is a widely distributed ectomycorrhizal fungus known for marked morphological variation (notably pileus and wart colour) and occurrence with a broad range of host trees. Earlier molecular work identified three major geographic groups within A. muscaria that were interpreted as Eurasian, Eurasian subalpine (alpine) and North American clades, but the historical dispersal and population-structure processes that produced these groups remained unclear. Beringia (Alaska and northeastern Siberia) is biogeographically important because much of it remained ice-free during glacial maxima and because it served as a major terrestrial connection between Asia and North America; however, no A. muscaria specimens from Alaska had been included in previous phylogeographic studies. G E M L and colleagues set out to clarify the phylogenetic and phylogeographic structure of the A. muscaria species complex by sampling across Arctic, boreal and humid temperate regions of Alaska and combining newly generated sequences with homologous sequences from GenBank. They generated sequence data from three loci (protein-coding beta-tubulin, and the ribosomal ITS and LSU regions), applied multiple phylogenetic approaches (maximum parsimony, Bayesian inference, and a matrix-representation parsimony supertree), and used nested clade analysis (NCA), coalescent simulations and molecular-clock methods to infer historical demography, divergence times and the likely centre of origin for the complex.
Twenty A. muscaria specimens were collected from various regions of Alaska and deposited in the University of Alaska Fairbanks herbarium. DNA was extracted from dried sporocarps; ITS and beta-tubulin sequences from additional isolates were obtained from GenBank. Amanita pantherina sequences were used to root phylogenetic trees. PCR amplification and Sanger sequencing were performed for a portion of the beta-tubulin gene and for the ITS plus partial LSU regions using established primers and protocols. Sequence reads were edited and assembled; alignments were initiated with Clustal W and manually corrected. A hypervariable region in the beta-tubulin alignment (positions 60–86) contained a 21-bp deletion and other indels; this region was recoded to retain phylogenetic information without over-weighting deletions. Phylogenetic inference used both maximum-parsimony (MP, paup*) and Bayesian methods (MrBayes). The partition homogeneity test examined combinability of loci. Model selection for Bayesian analyses used the Akaike information criterion. MP searches used tree-bisection-reconnection (TBR) with bootstrap assessment (1000 replicates for single-locus, 100 for combined); Bayesian runs sampled trees across many generations with burn-in discarded and convergence assessed by stabilisation of likelihood scores. Kishino-Hasegawa tests compared alternative topologies. A supertree was constructed using matrix representation with parsimony (MRP). The researchers sampled 100 random Bayesian trees per locus after convergence, encoded source-tree nodes as binary characters, combined them into an MRP matrix and carried out MP analyses; node stability was assessed by bootstrap. Phylogeographic inference used nested clade analysis (NCA). Maximum-parsimony haplotype networks (tcs) defined nested clades that were analysed in geodis to compute clade distances and test genotype–geography associations via permutations (10 000 replicates). Nucleotide diversity (π) was estimated with Arlequin. Coalescent-based methods collapsed identical sequences to haplotypes and tested neutrality (Tajima’s D, Fu and Li’s D* and F*), examined recombination blocks (site compatibility matrices), and used mdiv (an MCMC coalescent method) to estimate mutation rate, divergence time, migration rate and time to most recent common ancestor (TMRCA). Genealogies, mutation ages and TMRCA were further explored with genetree. Molecular-clock analyses used ML on LSU sequences with and without clock enforcement; the Ustilaginomycetes/Hymenomycetes split was fixed at 430 Ma to estimate absolute node ages and paml was used to estimate branch lengths and standard errors.
Sequence data sets comprised ITS (717 characters; 36 parsimony-informative), beta-tubulin (468; 14 informative), LSU (625; 12 informative) and the combined alignment (1810; 62 informative). The Tamura–Nei + I model was selected as best fit for individual loci. Bayesian and MP analyses of ITS, LSU and the combined data sets consistently recovered three major clades, labelled Clades I–III; relationships among those clades were unresolved. The beta-tubulin locus provided good support for Clades I and III but did not robustly resolve Clade II’s monophyly; nevertheless, no strong topological conflicts were detected between beta-tubulin and the ribosomal loci. Each clade carried a distinctive signature in the hypervariable beta-tubulin region: Clade I showed a 21-bp deletion, Clade III had several small indels, and Clade II shared a GT motif at positions 82–83 that was absent in the other clades. In the combined data set the three clades received strong support (MP bootstrap 96%, 99% and 100% and Bayesian posterior probabilities of 1.0 for Clades I, II and III, respectively). A southeast Alaskan subclade within Clade II (II/A) was also strongly supported. Supertree (MRP) analyses—using 100 Bayesian trees per locus recoded as node characters—produced a most parsimonious tree that recovered the major clades and subclade II/A with good resolution. Tests of morphological monophyly showed that commonly recognised colour varieties (var. alba, var. formosa, var. regalis) are not monophyletic: Kishino–Hasegawa constrained-tree tests indicated that forcing each variety to be monophyletic produced significantly worse trees (all P < 0.01), consistent with ancestral polymorphism of pileus and wart colour. Phylogeographic analyses identified 25 haplotypes in Northern Hemisphere isolates; the networks for the three clades formed separate 95% connection networks but could be connected at a 92% limit. NCA results varied by clade: the total network for Clade I was consistent with contiguous range expansion (CRE). Clade II showed significant genotype–geography associations, with evidence of CRE in one nested clade (2-2) and either allopatric fragmentation (AF) or isolation by distance (IBD) in another (2-1); overall Clade II’s total cladogram supported CRE. Clade III was represented by few haplotypes and NCA could not distinguish between AF and IBD. Across the combined cladogram, NCA detected patterns consistent with ancient allopatric fragmentation explaining deep divergence among the major clades. Coalescent analyses (after removing indels) produced 18 distinct ITS haplotypes for genealogy reconstruction. The coalescent-based genealogy suggested Clade III is sister to Clade I and provided within-clade radiation age estimates expressed in coalescent units (2N): Clade I mean age 0.128, Clade II 0.276, Clade III 0.507, implying the oldest within-clade radiation in Clade III and the youngest in Clade I. The authors noted lower apparent mutation rate in Clade III. Molecular-clock ML comparisons on LSU with and without enforcing a clock did not differ significantly (χ2 test). Using the calibration constraint, the first separation within A. muscaria (between Clades I and II) was estimated at 7.48 ± 4.53 million years ago (Ma). Nucleotide diversity (π) was highest in Alaskan samples (π = 0.013094 ± 0.00702, n = 20), followed by Eurasia (π = 0.011446 ± 0.006216, n = 18) and other North American samples (π = 0.009614 ± 0.005676, n = 9). The data provided no evidence of postglacial migration of southern North American haplotypes back into Alaska; instead, Alaskan populations appear to have persisted in refugia and given rise to eastern and western North American lineages prior to the Quaternary.
Phylogenetic, supertree and coalescent results concordantly identify three distinct phylogenetic species within the Amanita muscaria complex. Rather than being strictly allopatric, representatives of all three clades occur sympatrically in Alaska; the congruent gene genealogies indicate little or no gene flow among these clades, supporting recognition as separate phylogenetic species. Multiple morphological varieties are shared across clades, and Kishino–Hasegawa tests rejected monophyly of key colour varieties; the authors interpret this pattern as ancestral polymorphism for pileus and wart colour that pre-dated the clade divergences, although environmental factors may also influence expression of colour traits. G E M L and colleagues argue that the combined phylogeographic, coalescent and nucleotide-diversity evidence points to a Beringian (Siberian–Beringian) centre of origin for the A. muscaria complex. They hypothesise that an ancestral A. muscaria population occupied humid temperate forests across Beringia in the late Tertiary, underwent fragmentation (consistent with the opening of the Bering Strait and subsequent cooling) and later diversified into the three clades. NCA and coalescent inferences indicate southward range expansions from Beringia: in North America Clade I expanded along western and eastern routes of the Rocky Mountains to give rise to western US and eastern US populations, respectively. The authors find no evidence in their sample for migration from Eurasia into Alaska; instead, Alaskan populations apparently persisted through glacial maxima in local refugia and contributed lineages to postglacial North American populations. Key uncertainties acknowledged by the investigators include incomplete geographic sampling—particularly in Asia and Siberia—which limits resolution of some phylogeographic patterns, and wide confidence around molecular-clock estimates (the LSU-based estimate for the initial split is 7.48 ± 4.53 Ma). They note limited haplotype sampling within Clade III reduced the power of NCA to distinguish among historical processes for that clade. The authors also highlight the need to verify coalescent assumptions (neutrality, absence of recombination), steps they took to test neutrality and recombination but which nevertheless constrain interpretation. Implications discussed include that Beringia may have been not only a centre of origin but a long-term refugium and biodiversity hotspot for high-latitude ectomycorrhizal fungi. The phylogeographic patterns documented for A. muscaria could be paralleled in other boreal ECM fungi. The authors recommend further sampling, especially from Siberia and other unsampled Asian regions, to refine the historical biogeography and to test the hypotheses of Beringian origin and subsequent southward expansions.
Phylogenies inferred from the individual and combined data sets, and the supertree concordantly suggested three distinct clades in the Amanita muscaria species complex. These clades were first detected byand were referred to as geographic groups (i.e. allopatric populations). However, our data suggest that these groups are not entirely allopatric, but have geographic ranges that overlap in Alaska. We found representatives of all three clades in interior Alaska, and specimens from Clades II and III in western arctic Alaska. Because the nonconflicting gene genealogies indicate the lack of gene flow among the clades, we conclude that these groups represent distinct phylogenetic species with sympatric populations in Alaska (Fig.). Interestingly, all detected phylogenetic species within A. muscaria share at least two morphological varieties with other species. Clades I and II both contain at least fourmuscaria and/or var. flavivolvata), while at least two (var. regalis, var. muscaria and/or var. flavivolvata) have been found to date in Clade III. The most parsimonious explanation for the evolution of these morphological varieties is the presence of ancestral polymorphism in pileus and wart colour that pre-dated the separation of the phylogenetic species. In addition, the pileus colour may be influenced by unknown biotic or abiotic environmental factors. Although different colour varieties generally were found in all sampled climatic zones (temperate, boreal, and arctic-subalpine), eight of the nine A. muscaria var. regalis specimens were from regions with cold climate (either boreal, arctic or subalpine). The only A. muscaria var. regalis found in a more temperate climate was the one from the rainforests of southeastern Alaska, only a few miles from the subalpine zone. This finding confirms its rather limited distribution that is restricted to coniferous forests, low arctic and subalpine regions of northern and central Europe, and Alaska. It is a widely held assumption that low genetic variation is indicative of recent colonization and that the greatest TablePolymorphic sites in the ITS haplotypes collapsed after removing indels from the original ITS data set for the subsequent coalescent analyses. Position refers to that in the original alignment, site number is the designation of the given mutation as shown on Fig.genetic diversity should be found among isolates from regions that have been inhabited for the longest period.hypothesized that the ancestral group of A. muscaria evolved in Eurasia and migrated to North America via land bridges. In our sample, we found the greatest genetic diversity in Alaskan populations (π = 0.013094 ± 0.00702, n = 20 specimens), followed by Eurasia (π = 0.011446 ± 0.006216, n = 18), and by North America (π = 0.009614 ± 0.005676, n = 9). High genetic diversity in Beringia has also been reported in surveys of populations of the Columbian ground squirrel, Spermophilus columbianus, the swallowtail butterfly, Papilio machaon, and the ground beetle Amara alpina. The nucleotide diversity estimates and the results of the phylogenetic, phylogeographic, and coalescent analyses Fig.Coalescent-based genealogy with the highest root probability (L = 6.4693 × 10 -54 , SD = 6.3319 × 10 -51 ) showing the distribution of mutations for the ITS region in the major clades. The inferred genealogy is based on 2 million simulations of the coalescent with a Watterson's estimate of θ = 4.0. The timescale is in coalescent units of 2N, where N is the population size. Mutations and bifurcations are time ordered from the top (past) to the bottom (present). Mutation designations correspond to the site numbers in Table. The numbers below the tree designate the distinct haplotypes, their observed frequencies in total and in the different geographic regions. concordantly suggest that the centre of origin of A. muscaria likely is in Beringia (Fig.). We hypothesize that the ancestral A. muscaria population evolved in the humid, temperate forests that covered much of Beringia in the late Tertiary. Although it is difficult to estimate the divergence times of the major clades due to the wide range of time estimates of our molecular clock analyses (7.48 ± 4.53 Ma), the fragmentation of the ancestral population into at least two major clades might have taken place as a consequence of the opening of the Bering Strait about 12 Ma. Clade III likely is a sister group of Clade I, as inferred from the phylogenetic and coalescent analyses, and it is safe to conclude that the ancestral population was divided into Eurasian and Alaskan populations. With the cooling climate, some populations of Clades I and II likely migrated southward in North America and Eurasia (Fig.), respectively, as is supported by the contiguous range expansion inferred in both clades by NCA. However, coalescent mutation age estimates suggest that the radiation and southward expansion may have happened more recently in North America (Clade I) than in Eurasia (Clades II and III). In North America, the expansion of Clade I took two main directions: (i) southward along the western side the Rocky Mountains which resulted in the extant populations in the western United States, represented by samples from California and Idaho; and (ii) southeastward along the eastern slopes of the Rocky Mountains which allowed the establishment of populations in the eastern United States, represented by samples from Massachusetts and New York. This latter route was shared by numerous plant taxa that originated in Alaska and replaced many species along their migration to the southeast). Interestingly, we did not find any haplotype in Alaska that descended from other North American haplotypes. This suggests that populations of A. muscaria survived the glacial maxima in Alaskan refugia and there was no significant postglacial migration from southern populations back to Alaska. On the contrary, Alaskan populations likely gave rise to both eastern and western North American lineages before the Quaternary period. Range expansion patterns in Clades II and III are more difficult to interpret, partly because of large unsampled areas in Asia. The NCA results in clade 2-1, which is the interior clade and the only one containing both Alaskan and Eurasian samples, indicate allopatric fragmentation or isolation by distance. Isolates from unsampled areas in Asia are needed to clarify this question. However, a more basal bifurcation, separating the southeast Alaskan group (II/A, ITS haplotype IX) from the rest of Clade II, can be observed in the combined phylogeny and the coalescent-based genealogy. It is somewhat surprising that no evidence was found for migrations of A. muscaria from Eurasia to North America/Alaska, despite what had been suggested by. This question should be addressed by further phylogeographic studies with increased sample size. Beside the southward range expansions detailed above, populations of all three species clades have continuously inhabited Beringia. In the Quaternary, the Illinoian and Wisconsinian glaciations likely restricted A. muscaria to isolated refugia of boreal forest and shrub tundra along the Yukon and Tanana rivers in interior Alaska that remained unglaciated. While it is unclear whether conifers were present in the region at glacial maxima, it is very likely that Betula, Dryas, Populus and Salix inhabited at least some parts of the regionand likely were able to maintain refugia of A. muscaria. The ecological plasticity of A. muscaria, i.e. the broad range of potential mycorrhizal hosts, including Betula, Dryas and Salix spp. in subalpine tundra, supports the hypothesis of glacial refugia in Alaska. In addition, although earlier pollen data did not indicate the presence of Picea in Beringia at the last glacial maximum, recent pollen dataand phylogeographic analyses based on DNA sequences (F.S. Hu, personal communication) suggest the existence of glacial refugia of P. glauca and P. mariana in eastern Beringia. In this study, we documented the existence of three distinct phylogenetic species in the A. muscaria species complex. Furthermore, we hypothesized evolutionary and phylogeographic processes leading to speciation and intraspecific population structures. Future studies should include specimens from unsampled regions to further elucidate the phylogeography of the species complex. Among these, Siberia is of particular interest, because it might possess genetically diverse populations, including putatively ancestral Beringian elements. The implications of our results are not restricted to A. muscaria. The phylogeographic patterns seen here might be shared, at least in part, by many boreal ECM fungi in the Northern Hemisphere, particularly in North America. It is certain that many plant lineages contributing to the recent boreal and temperate flora evolved within high-latitude forests of Beringia during the Tertiary and migrated southward as the climate cooled. Furthermore, because there is increasing evidence for boreal forest glacial refugia in Alaska, Holocene migrations of boreal plants and ECM fungi likely occurred not only northward from southern refugia, but southeastward from Alaskan refugia. This is supported by the rapid postglacial colonization of the present boreal regions by Picea, and the fact that no recent migration of A. muscaria from more southern regions of North America to Alaska was detected in our analyses. As a consequence, we propose that Beringia is not only the original and longest inhabited region for many plant and animal taxa, but may represent a biodiversity 'hotspot' for high-latitude ECM fungi as well.
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