Recircumscription of A.sect. Phalloideae

In our analyses, some unexpected relationships within the section Phalloideae were indicated. Amanita griseoturcosa, A. manginiana, A. modesta, A. pseudoporphyria and A. oberwinklerana, which were previously defined as lethal amanitas based exclusively on morphological studies, are phylogenetically nested in the section Lepidella (Figures 1 and 2), [3, 8, 9, 22, 75]. On the other hand, A. areolata T. Oda et al., A. hesleri Bas and A. zangii are robustly supported to form a monophyletic group with lethal amanitas (Figures 1 and 2), although they were originally allocated to the section Lepidella due to their felty to subfibrillose, adnate volval remnants on pilei, appendiculate pileal margins, and elongated stipes with indistinct bulb with farinose to floccose volval remnants [3, 8, 10, 22]. Our biochemical analyses failed to detect either amatoxins or phallotoxins in A. manginiana (fresh or dried basidiomata of it has been sold as food in free markets in Yunnan for centuries [3]), A. modesta, A. pseudoprphyria (Table 1). We also did not detect any toxins in A. zangii and A. sp. HKAS77321 (Table 1). Consequently, the five species, A. griseoturcosa, A. manginiana, A. modesta, A. pseudoporphyria and A. oberwinklerana, which were previously treated as members of lethal amanitas, should be excluded from the section Phalloideae and then allocated to the section Lepidella. In contrast, A. areolata, A. hesleri and A. zangii should be transferred from the section Lepidella to the section Phalloideae as a basal lineage producing neither amatoxins nor phallotoxins. That kind of similar morphology among distantly related species may have resulted from the evolutionary convergence or from shared plesiomorphies, which is also suggested in other fungi [76, 77]. Moreover, A. zangii and A. areolata are indistinguishable in our molecular phylogenetic analysis (Figure 2), and thus A. areolata should be regarded as a synonym of A. zangii, as suggested by Yang [3] based on morphological studies.

Diversity of lethal amanitas

Nine major lineages comprising 28 phylogenetic species are supported within lethal amanitas (Figures 1 and 2). In the following discussion, we focus on the most significant features circumscribing the major clades and their distribution patterns.

Clade I includes two taxa, the East Asian species A. pallidorosea (C in Figure 4) and the eastern North American species A. bisporigera G. F. Atk. The two taxa share the characteristics of a white basidioma and globose to subglobose basidiospores. Amanita pallidorosea is widely distributed in East Asia from Yunnan, southwestern China to Jilin, northeastern China and Hokkaido, Japan under broad-leaved forests. Although A. pallidorosea is clustered into two subclades in all of the single-gene phylogenetic trees, it is ultimately delimited as a single species following a conservative approach, as the two subclades are not well supported in the phylogenetic trees inferred from both the nrLSU and ITS sequences (Figure 2 and Additional file 3: Figure S1), and the interspecific divergence between them is lower than the cut off values of ITS sequences variations. Zhang et al. [27] suggested that A. pallidorosea, excluding the white forms, could be distinguished from other East Asian white amanitas by its rose pileus with a conspicuous umbo over the disc. However, A. sp. 2 (G in Figure 4) in our study also possesses a pallid rose pileus with a noticeable umbo. Although A. pallidorosea and A. sp. 2 are morphologically similar to each other, they can be distinguished through microscopic analyses, because the spores of A. sp. 2, (7–) 7.5–10 (−11.5) × (7–) 7.5–9 (−10.5) μm, are larger than those of A. pallidorosea (6–) 6.5–8 (−10) × 6–7.5 (−9.5) μm. Amanita bisporigera is the sister species to A. pallidorosea in eastern North America. It is interesting to note that A. pallidorosea has four-spored basidia, while the basidia of A. bisporigera are two-spored.

Figure 4 Representatives of lethal amanitas. (A) Amanita fuliginea; (B) A. fuligineoides; (C) A. pallidorosea; (D) A. subjunquillea; (E) A. rimosa; (F) A. virosa; (G) A. sp. 2; (H) A. sp. 3; (I) A. sp. 4; (J) A. sp. 6; (K) A. sp. 7; and (L) A. sp. 8. Full size image

Clade II consists of two species with a white basidioma, A. rimosa (E in Figure 4) from subtropical East Asia under Fagaceae trees, and A. sp. 13 (‘A. cf. virosa’ AY325830) from eastern North America. Amanita rimosa was initially suggested to be distinct from the other white lethal amanitas in its rimose pileal surface [27]. However, this feature is found to be variable and dependent on environmental conditions in our analyses. Compared with other currently known white lethal amanitas restricted to tropical and subtropical East Asia, A. rimosa has a much smaller and more slender basidioma, with a pileus of 1.5–5 cm in diameter.

Clade III includes seven phylogenetic species ranging from the Palearctic to the Nearctic. All of these species have a white basidioma with the exception of A. arocheae which has a grayish pileus. Amanita virosa (F in Figure 4) was originally described from northern Europe under mixed forests of Fagaceae and Pinaceae [5], and it has often been reported in eastern and southern Asia [27, 78]. Based on our molecular and morphological analyses, A. virosa does occur in East Asia, but is restricted to its northeastern region. Amanita virosa was also reported from North America [21]. However, Tulloss et al. [79] questioned its distribution in North America. In our analyses, all of the North American collections closely related to A. virosa are clustered in their own lineage, A. sp. 1. Although the ITS sequences divergence between them is low, they are ultimately delimited as two different species because their monophyly was not well supported even in the combined analysis (Figures 1, 2 and Additional file 3: Figure S1). Therefore, A. virosa only occurs in the Palearctic regions (Europe and northeastern Asia) and its American counterpart might represent a new taxon. Amanita ocreata under the mixed forest of Fagaceae and Pinaceae is the only western North American species in this clade. Because A. sp. 2, A. sp. 11 and A. sp. 12 are all represented by a single sample, their definitions require further research.

Clade IV includes two European species, A. verna and A. phalloides, and one East Asian species, A. subjunquillea (D in Figure 4), with no close relatives known from North America to date. The three species are associated with mixed forest of Fagaceae, Pinaceae and Betulaceae throughout the broad-leaved forests of Europe and East Asia. Amanita verna, which has a white basidioma, is sister to A. phalloides and A. subjunquillea with moderate support values. Amanita subjunquillea and A. phalloides share the characteristic olive-green to yellow pileus. Amanita phalloides was originally described in Europe, and has been introduced to North America, Australia, New Zealand and South Africa together with its host plants [30, 80]. It has also been reported in China [81] and Japan [75]. However, those collections identified as A. phalloides in China were found to be A. subjunquillea[3, 27]. In East Asia, A. subjunquillea has often been mistaken as A. phalloides due to their high degree of morphological similarities and close phylogenetic relationship, but the spores, basidia and basidiomata of A. subjunquillea are usually smaller than those of A. phalloides[3, 8].

Clade V consists of three sympatric species occurring with Fagaceae restricted to tropical and subtropical East Asia, A. fuliginea, A. sp. 3 and A. sp. 4 (A, H and I in Figure 4, respectively). Taxa of this clade are characterised by their small basidioma with a fuliginous to almost blackish pileus 2–6 cm in diameter. The similarity among them may result from the morphological stasis caused by stable and similar habitats, which has been proposed as a common phenomenon in the evolution of fungi [82–84]. Zhang et al. [27] suggested that there were two subclades of A. fuliginea, and interpreted one of the subclades (A. sp. 4 in Figures 1 and 2) as a different population or a cryptic species of A. fuliginea. However, in our analyses, the two subclades were suggested to be different species based on both the GCPSR criterion and the variation of ITS sequences (Figures 1 and 2). Amanita fuligineoides (B in Figure 4) in Clade VIII is also morphologically similar to A. fuliginea, but it has a larger-sized basidioma [27].

Clade VI includes four phylogenetic species characterised by white basidioma and subglobose, globose to ellipsoid basidiospores. The four species are largely associated with Fagaceae. In this clade, the delimitation of A. suballiacea is controversial. According to the ITS sequences, A. suballiacea AY325837, A. sp. 14 and A. sp. 16 are identified as different phylogenetic species. However, our morphological and microscopic analyses both indicate that the three collections of A. sp. 14 RET478-6, 490-1 and 491-7 are identical to A. suballiacea. Consequently, they are ultimately treated as a single species (Figure 2). Although the sequence divergence between A. sp. 5 and A. suballiacea is low, they have been proved to be different phylogenetic species by both the GCPSR and ITS sequences divergence criteria (Figures 1 and 2). Amanita sp. 15 (‘A. ocreata’ EU909446, GQ250405 and AY918962) from western North America was identified as A. ocreata, but the real A. ocreata is within Clade III in our analyses as proved by both morphological and anatomical evidence. Thus, A. sp. 15 represents an independent species. Amanita exitialis, which is restricted to tropical and subtropical East Asia, is distinct from its North American sister group in its two-spored basidia.

Clade VII contains two East Asian species, A. sp. 6 and A. sp. 7 (J and K in Figure 4), associated with mixed forests of Fagaceae and Pinus. Amanita sp. 6, with a white basidioma and four-spored basidia, was restricted to temperate East Asia. In contrast, A. sp. 7, with a grayish pileus and two-spored basidia, is only known from tropical East Asia.

Clade IX consists of four phylogenetic species occupying the basal position in the phylogenetic tree (Figure 1). Morphologically, the four species are characterised by their brown, grayish or grey-brown basidiomata with the exception of A. sp. 10, which has a white basidioma. Amanita reidii Eicker & Greuning was originally reported in South Africa under the introduced Australian plant Eucalyptus with a citation of the collection PREM 48618 from Sabie [85], which was erroneously recoded as ‘Amanita phalloides var. umbrina’ in GenBank [AY325825]. The Hawaiian A. marmorata subsp. myrtacearum O. K. Mill. et al. was observed in association with exotic trees of Myrtaceae, Casuarinaceae and Araucariaceae [7]. Hallen et al. [29] suggested that A. reidii might be synonymous with the Australian species A. marmorata subsp. marmorata Cleland & E.-J. Gilbert and the Hawaiian subspecies myrtacearum. Miller et al. [7] suggested that the three taxa, A. marmorata subsp. marmorata, A. marmorata subsp myrtacearum and A. reidii, were a complex of closely related taxa that might have originated in eastern Australia and been imported into the Hawaiian islands and South Africa with their host plants, with which we fully agreed. In our analyses, the three collections from Hawaii and South Africa showed intraspecific divergence lower than the cutoff value, and thus they were identified as a single species, A. reidii (Figure 2). Amanita sp. 10 was collected in Tasmania, Australia under Casuarina. Another two phylogenetic species, A. sp. 8 (L in Figure 4) and A. sp. 9, were collected in South Asia and tropical East Asia associated with Dipterocarpaceae and Fagaceae, respectively. The two species or their affinities are likely to be found in other parts of tropical Asia in future studies.

Although the relationships among the deeper clades of lethal amanitas are not well resolved, a few interesting features seem to have evolved convergently (Figures 1 and 2). For example, species with two-spored basidia such as A. exitialis, A. bisporigera and A. sp. 7 do not have close relationships to each other. Instead, they are closely related to taxa with four-spored basidia. Furthermore, species with unpigmented and pigmented pilei do not correspondingly form monophyletic groups but some of them are clustered together, such as A. sp. 7 (with a gray-brown pileus) and A. sp. 6 (with a white pileus) (Clade VII in Figures 1 and 2). It could be speculated that the characteristics of two-spored basidia and unpigmented/pigmented pilei have evolved independently several times within lethal amanitas. Yet, the presence of lethal substances within Amanita is suggested to have a single origin and it seems to be a synapomorphy of lethal amanitas, because our biochemical analyses show that the sister species of lethal amanitas and those samples from the other three sections of the subgenus Lepidella contained no detectable cyclic peptide toxins (Table 1). That is also consistent with the studies of Hallen et al. [86], in which they proposed that the lack of toxin production among other species of Amanita outside of section Phalloideae were due to the absence of encoding genes.

Divergence time within lethal amanitas and their intercontinental distribution patterns

Our analyses show that the divergence times estimated by the two fossils, which should be consistent, are greatly different. The second calibration point (Q. cranhamii) seems to have vastly underestimated the divergence time of Ascomycota/Basidiomycota and Amanita. That might be resulted from Q. cranhamii representing a relatively young taxon of Quatsinoporites, or that the hypothesised position of this fossil taxon within Basidiomycota requires further verification, as the phylogenetic position of the fossil appears to have great influence over the estimation results. For example, the estimated divergence time between Ascomycota and Basidiomycota varied from 452 Mya to 582 Mya with the calibration point P. devonicus placed in different subphyla of Ascomycota [62, 87].

In our biogeographical analyses, those lethal amanitas in the basal and sub-basal groups were collected in tropical East Asia, South Asia, South Africa and Australia, showing a palaeotropical distribution pattern. Furthermore, the sister species of lethal amanitas, A. zangii and A. sp. HKAS77321, were also collected in the palaeotropical areas (tropical East Asia). These findings strongly suggest a possible palaeotropical origin of lethal amanitas, which has also been suggested for other ECF such as Hysterangiales [88], Inocybaceae [89] and Porcini mushrooms [90]. In the basal group, A. sp. 10 and A. reidii were collected in Australia, South Africa and Hawaii associated with Araucariaceae, Casuarinaceae and Mytraceae, and A. sp. 8 was collected in Bangladesh under Shorea robusta[7, 85]. In addition, there are also about seven lethal amanitas reported in Madagascar, the Congo and South America associated with Fabaceae and Nothofagus[25, 91, 92]. Unfortunately, we know of no collections of these species except types which are not suitable for molecular phylogenetic studies. However, according to the coevolution of fungi and host plants, the Gondwana origin can not be rejected because Araucariaceae, Dipterocarpaceae, Nothofagus and Myrtaceae were all suggested to have a Gondwana origin [93–97].

Three independent sister species or sister groups among Eurasia/East Asia and the Americas are indicated in the ancestral area reconstructions analyses (Figures 1, 2 and 3). The first species pair is within Clade I, which exhibits an East Asian–eastern North American disjunct distribution (Graysian distribution, [2]) (Figures 1 and 3), and the estimated divergence time between them is about 11.4 Mya (1.11–13.84 Mya, 95% HPD), in the late Miocene. The second intercontinental distribution among Eurasia and North/Central America is exhibited in Clade III. The dated divergence time of the western American species A. ocreata, which occupies the basal position in the clade, is about 26 Mya (17.74–35.72 Mya, 95% HPD) in the late Oligocene. In addition, the divergence between the Eurasian species A. virosa and its eastern North America counterpart (A. sp. 1) is estimated to occur about 13 Mya (3.75–19.84 Mya, 95% HPD) in the middle Miocene. The other three Central American species in the clade, A. arocheae, A. sp. 11 and A. sp. 12, diversified during 15.7–10 Mya. Due to their close relationships with the North American species and their relatively recent divergence, we speculate that they originated in the northern part of the Americas and then extended into Central America with their host plants oaks, which had a North Temperate origin [2, 98]. The third species group which exhibited the East Asia and North/Central America disjunct distribution pattern is within clade VI, with one East Asian, one western North American and two eastern North American species. The estimated divergence time between the East Asian taxon A. exitialis and its American sister group is around 27 Mya (8.88–46.58 Mya) in the middle Oligocene, and the divergence time between the western North America species A. sp. 15 and its eastern North America sister group is around 19.36 Mya in the early Miocene.

Our results suggest that the intercontinental distribution patterns of the sister species or sister groups among Eurasia/East Asia and the Americas were mainly established during the middle Oligocene to the middle Miocene, which coincides with the paleoclimates. Since the climatic deterioration at 33 Mya, the temperature began to fluctuate from the early Oligocene to the middle Miocene (34–15 Mya) [99]. The fluctuation of temperature, especially the relatively warm climates from the late Oligocene to the middle Miocene, may have allowed temperate elements to migrate between continents via the Bering Land Bridge (BLB). This biogeographic distribution pattern was elucidated in A. muscaria[100], and was also consistent with the diversification of the major host plant family in the Northern Hemisphere, Fagaceae, which appeared to have achieved a continuous distribution spanning Asia, North America, and Europe during the Oligocene through the floristic exchanges via the North Atlantic Land Bridge before the lower Oligocene and later via the BLB, followed by allopatric speciation in the middle Miocene due to the climate change [71].

In Clade IX, the four lethal amanitas from southern East Asia, Hawaii, Australia and South Africa exhibit close relationships. Amanita reidii might have originated from Australia and then been introduced to South Africa with the host plants Eucalyptus[7, 85]. The sister group relationships among the tropical East Asian species A. sp. 9, South Asian species A. sp. 8 and A. reidii are not surprising, as the two continents were connected after the collision of the Australian and Asian plates in the Miocene [101, 102]. However, the estimated divergence time of A. sp. 10 and the other species in the clade, 27 Mya, predates the Miocene collision between Australia and Asia. Amantia sp. 10 might be an old relic of lethal amanitas in the palaeotropical regions because it showed great divergence from other lethal amanitas and occupied a basal position in the phylogenetic tree (Figures 1 and 3). The same may be true for A. rimosa and A. fuligineoides, which showed significant divergence from other lethal amanitas and occupied an isolated position in the phylogenetic tree (Figures 1 and 3).

Our results also confirm an East Asian–European allopatric speciation, viz. A. subjunquillea and A. phalloides. The sequence variations between them are relatively low even in the multi-locus analysis, which indicates a recent divergence, 7 Mya (2.04–12.91 Mya, 95% HPD). It is probable that the divergence of A. subjunquillea and A. phalloides was brought about by the vicariance of a recent common ancestral distribution in the Holarctic region. Later, in the late Miocene, the common ancestor moved southward with their host plants because of the distinct climatic cooling in that period [99, 103], and then diverged into two regional species. These findings indicate that the dispersal-vicariance theory, which has been widely used to explain the disjunctions of plants between the Palearctic and Nearctic regions [104], is applicable in understanding the intercontinental distribution patterns of ECF.

In our molecular phylogenetic analyses, the relationships among the temperate-subtropical clades (Clades I–V, Figure 1) were not well resolved. That could also be explained by the paleoclimatic changes. The gradual global cooling after 50 Mya in the Eocene may have stimulated the early diversification of lethal amanitas into their major extant tropical to temperate clades after origination in the early Paleocene in Palaeotropical areas, whereas the climate deterioration at 33 Mya may have led to an elimination of tropical elements (Figure 3) [103, 105]. That the molecular phylogenetic analyses could not resolve the relationships among those temperate-subtropical clades may be explained by the extinction of tropical species obscuring the relationships among temperate-subtropical clades, or by the rapid speciation of temperate-subtropical species triggered by ecological changes.