Beringian origins and cryptic speciation events in the fly agaric (Amanita muscaria)

This phylogenetic study (2006) investigated the common ancestor of three different Amanita Muscaria (Fly Agaric) variants and found evidence that these variants may belong to separate genetic species that are overlapping in their geographic distribution. This suggests that the variance of its alkaloid composition and the pharmacological effects exerted onto organisms may be subject to unique differences across similar-looking species of Fly Agaric.

Abstract

“Introduction: 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.

Methods: 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.

Results: 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.

Discussion: 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.”

Authors: József Geml, G. A. Laursen, Keit O’Neill, H. Chad Nusbaum & D. Lee Taylor

Summary

The phylogenetic history of Amanita muscaria sensu lato was revealed by DNA sequence data from arctic, boreal and humid temperate regions in Alaska. The data suggest that the three phylogenetic species share at least two morphological varieties with other species, suggesting ancestral polymorphism in pileus and wart colour pre-dating their speciations.

Introduction

Amanita muscaria, the fly agaric, is probably the most famous and most illustrated fungus and embodies the concept of ‘mushroom’ in many cultures. It is native to temperate or boreal forest regions of the Northern Hemisphere, but has been introduced to New Zealand, Australia, South America, and South Africa. A. muscaria forms ectomycorrhizal symbioses with a wide range of hosts, including birch, pine, spruce, fir and larch trees, and has also been found associated with Dryas and Salix species in interior Alaska.

Prior research suggests that A. muscaria exhibits substantial variation in morphology and toxin content across different geographic regions, but Oda et al . (2004) found three distinct clades in A. muscaria that correspond to geographic differences (i.e. allopatric populations).

Despite its importance for biogeographic research, no specimen of A. muscaria has been investigated from Alaska. We collected and analysed 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, and conducted comprehensive phylogenetic analyses.

We conducted phylogenetic analyses using logical concordance and nested clade analyses to determine the phylogenetic species boundaries within A. muscaria . We also conducted coalescent-based simulations of genealogical relationships to enhance the precision of our estimates.

Isolates and DNA extraction

Twenty specimens of Amanita muscaria were collected from various geographic regions of Alaska. DNA was extracted from small samples of dried specimens and rooted using homologous sequences of Amanita pantherina.

PCR and DNA sequencing

A portion of the beta-tubulin gene was amplified using polymerase chain reaction (PCR) and cycle sequencing reactions in a PTC-220 thermocycler using primers and settings specified by Oda et al . (2004).

The entire ITS and partial LSU regions were PCR amplified and sequenced using an ABI 3730xl automated capillary DNA sequencer. Two internal primers were used for cycle sequencing, ITS4 and CTB6, in addition to the primers used in the PCRs.

Phylogenetic analysis

Sequence data were edited and assembled for each isolate using codoncode aligner version 1.3.4 (LI-COR). A hypervariable region was observed in the beta-tubulin data set corresponding to positions 60 – 86, which was recoded using inaase 2.3b to retain the phylogenetic information present in the region without overweighing the deletions. The partition homogeneity test was performed on 1000 randomized data sets, and the Akaike information criterion was used to determine the best-fit evolutionary model for Bayesian analyses.

The number of replicates was 1000 for the single-locus data set and 100 for the combined data set. 200 000 generations were run in four chains and 100 trees were sampled every 100th generation.

Supertree construction

We constructed supertrees using the Matrix Representation with Parsimony method (MRP), which is a supertree approach for analysing and combining individual trees derived from multiple data sets. MRP handles conflict by weighing the evidence in different source trees without any tree having the power of veto.

We used the Bayesian trees generated earlier in this study for individual loci to construct supertrees. We weighted the nodes according to their posterior probability values and carried out MRP analyses with the heuristic search option using the TBR algorithm with 100 random sequence additions.

Phylogeographic analyses

NCA was used to infer the phylogeographic history of the A. muscaria species complex. The haplotype representing the sample from New Zealand was removed. Maximum-parsimony haplotype networks were generated using tcs version 1.18 (Clement et al. 2000), and nested clades were created using geodis version 2.0 (Posada et al. 2000). Nucleotide diversity was calculated using arlequin version 2.0 to compare the amount of genetic diversity found in Alaska to that of other geographic groups.

Coalescent analyses

Identical sequences were collapsed into haplotypes using snap map and sites version 1.1, and recombination blocks were detected using mdiv. The genealogy with the highest root probability, the ages of mutations, and the time since the most recent common ancestor was reconstructed using coalescent simulations with population subdivision.

Molecular clock analyses

Maximum-likelihood analyses were conducted using paup * 4b10 based on LSU sequences to estimate the ages of the nodes. The absolute ages of the nodes were estimated by fixing the age of the Ustilaginomycetes/Hymenomycetes separation at 430 million years ago (Ma).

Phylogenetic analyses

The Tamura – Nei model was selected as the best-fit evolutionary model for the ITS, beta-tubulin, LSU and combined data sets.

Bayesian analyses of the ITS, beta-tubulin, LSU, and combined data sets generated 16, 39, 3, and 10 000 equally parsimonious trees, respectively. The ITS phylograms were 95 steps long with CI = 0.874, RI = 0.965, rescaled consistency index (RC) = 0.843, and homoplasy index (HI) = 0.126.

Three major clades were detected within Amanita muscaria based on phylogenetic analyses of the ITS and LSU alignments, but the relationships among Clades I, II, and III were not clear. All three groups had unique ‘signature sequences’ in the hypervariable region corresponding to positions 60 – 86 in the alignment. The beta-tubulin MP tree did not support the monophyly of Clade II, but did not show significant conflict with the ITS and LSU trees. The phylogenetic relationships among Clades I, II, and III remained unclear, however, because none of the groupings were supported by significant MPB and BPP values.

Supertree construction

Matrix representations of the ITS, beta-tubulin, and LSU resulted in 20 500 characters, of which 9800 were parsimony-informative. The single most parsimonious tree was 33 803 steps long.

Evolution of morphological varieties

Representatives of multiple morphologically distinct varieties were found in several clades. Kishino – Hasegawa tests were performed to test whether specimens with shared phenotype were monophyletic, and the constrained trees had significantly more steps and lower likelihood scores than the unconstrained trees.

Phylogeographic analyses

A total of 25 haplotypes were detected in A. muscaria isolates from the Northern Hemisphere. These haplotypes were nested into three separate networks at 95% connection limit, representing Clades I, II, and III, respectively.

The null hypothesis of no association between genotype and geographic origin was rejected in clade 3-2 and the total cladogram, and there was significant association between haplotype and geography in clade 3-2 due to contiguous range expansion.

In Clade II, a statistically significant association between genotype and geographic origin was found in clades 2-1 and 2-2. CRE was inferred as the underlying mechanism in clade 2-2.

The Clade III network contained only a single one-step clade, and it was not possible to discriminate between IBD and AF due to the small number of sampled haplotypes. The NCAs indicated that Clade II was the interior clade.

Coalescent analyses

After removing the indels, seven previously detected haplotypes collapsed, resulting in 18 distinct ITS haplotypes. The coalescent-based ITS genealogy was informative for inferring the mutational history with respect to variation between and within the major clades.

Molecular clock analyses

Analysis of the LSU data set with and without a molecular clock produced identical trees, and the age of the first separation within A. muscaria was estimated at 7.48 4.4 Ma.

Discussion

We inferred three distinct clades in the Amanita muscaria species complex, and found that these clades overlap in Alaska, suggesting that these clades are distinct phylogenetic species with sympatric populations.

All detected species within A. muscaria share at least two morphological varieties with other species, and eight of nine A. muscaria var. regalis specimens were found in regions with cold climate (either boreal, arctic or subalpine).

The greatest genetic diversity was found in Alaskan populations, followed by Eurasia and North America. This was also observed in surveys of populations of the Columbian ground squirrel, the swallowtail butterfly, and the ground beetle Amara alpina.

The nucleotide diversity estimates and the results of the phylogenetic, phylogeographic, and coalescent analyses suggest that the centre of origin of A. muscaria likely is in Beringia. The ancestral population was fragmented into at least two major clades, and some populations migrated southward in North America and Eurasia.

Clade I expanded in two directions in North America: southward along the western side of the Rocky Mountains and southeastward along the eastern slopes of the Rocky Mountains. Alaskan populations likely gave rise to both eastern and western North American lineages before the Quaternary period.

The results indicate allopatric fragmentation or isolation by distance in clade 2-1, but no evidence was found for migrations of A. muscaria from Eurasia to North America/Alaska.

In the Quaternary, populations of all three species clades have continuously inhabited Beringia. It is very likely that Betula, Dryas, Populus and Salix inhabited at least some parts of the region at glacial maxima, and likely were able to maintain refugia of A. muscaria.

This study documented three distinct phylogenetic species in the A. muscaria species complex, and hypothesized evolutionary and phylogeographic processes leading to speciation and intraspecific population structures.

Our results suggest that many boreal ECM fungi evolved in high-latitude forests of Beringia during the Tertiary and migrated southward as the climate cooled. Furthermore, there is increasing evidence for boreal forest glacial refugia in Alaska, which supports the idea that boreal plants and ECM fungi evolved in Beringia.