Taxonomic studies of Amanita muscaria ( L . ) Lam ( Amanitaceae , Agaricomycetes ) and its infraspecifi c taxa in Brazil

We analyzed specimens identifi ed as Amanita muscaria, some recently collected and others already deposited in herbaria, in Brazil. We concluded that two subspecies of A. muscaria occur in Brazil: A. muscaria var. muscaria; and A. muscaria var. fl avivolvata. Th e fi rst taxon was found in association with Castanea sativa, and the second (one specimen only) was found in association with Pinus and Eucalyptus spp. Morphologically, A. muscaria var. fl avivolvata is distinguished by a shallower subhymenium and by basidiospores that are more elongated than are those of A. muscaria var. muscaria, which is the more widely known subspecies. We present descriptions, discussions, illustrations and a dichotomous key for these two subspecies.

Initially, A. muscaria was suspected to be a well-defi ned morphospecies with ample geographic distribution, also associated with ectomycorrhizal hosts dispersed across multiple genres of vascular plants (Trappe 1962).However, phylogenetic studies conducted by Oda et al. (2004) showed that A. muscaria occurring in Eurasia and North America correspond to phylogenetically distinct populations.Later, Geml et al. (2006Geml et al. ( , 2009) ) found that it is likely that cryptic, sympatric speciation occurred in the Beringia region of what is now Alaska, and Geml et al. (2008) concluded that A. muscaria sensu lato has a strong inter-and intra-continental phylogeographic structure, mainly in North America, and that several phylogenetic species occur within A. muscaria sensu lato.Th e distribution of the species is listed by Tulloss & Yang (2012).
As a continuation of studies on the genus Amanita conducted by our group (Wartchow & Maia 2007;Wartchow et al., 2007Wartchow et al., , 2009Wartchow et al., , 2013)), the present study raises the possibility of the occurrence of various distinct subspecies of A. muscaria in Brazil.We also address morphological studies, as well as discussing the importance of using morphological and ecological criteria in order to distinguish between subspecies.

Materials and Methods
In May 2009, fresh A. muscaria specimens were collected in the São Francisco de Paula National Forest (29°23'S; 50°23'W), which covers an area of 1606 ha in the state of Rio Grande do Sul, Brazil.Although the composition of this nature reserve is classifi ed as a mixed ombrophilous forest, there are also exotic plantation species of Pinus, Eucalyptus and Castanea sativa Mill.(Dobrovolski et al. 2006;Longhi et al. 2006, Ribeiro et al. 2007).Other materials examined were obtained from the collections of the herbaria of the following institutions (Th iers 2012): the Federal University of Santa Catarina (code, FLOR); the Blumenau Regional University Foundation (code, FURB); the University of Santa Cruz do Sul (code, HCB); the Federal University of Santa Maria (code, SMDB); the São Paulo State Department of the Environment (code, SP); and the Federal University of Pernambuco (code, URM).

Results and Discussion
In comparison with that of the material studied by Tulloss & Yang (2012), the L' value of the material analyzed here was 2% higher (10.0 μm vs. 10.2 μm).It is noteworthy that we measured only 100 basidiospores from a single collection, and one of the basidiomes showed an L of 10.9 μm, which contributed to increasing the L' value.Th at basidiome was probably dehydrated at the beginning of sporulation (Tulloss, personal communication).Geml et al. (2006Geml et al. ( , 2009) ) were the fi rst to suspect a cryptic speciation in A. muscaria sensu lato, with the dispersion center located in Alaska, USA, in a region known as Beringia.In other studies, Geml et al. (2008) analyzed a larger sample and recognized at least six phylogenetically distinct clades (I to VI), which might represent distinct phylogenetic species.A. muscaria var.muscaria probably corresponds to clade II, with distribution from Eurasia to Alaska and the Pacifi c Northeastern region of the United States; in temperate, boreal and coastal forests with various species of conifers and deciduous trees, relatively common in Europe (Beardslee 1905;Jenkins & Petersen 1976;Breitenbach & Kränzlin 1995;Mattock 1995, Castro 1996;Neville & Poumarat 2001, 2004;Vaasma 2009).For other parts of the world, this taxon certainly corresponds to material collected from exotic plantations, growing under Pinus and other tree species (e.g., Quercus, Picea and Pseudoptsuga) imported from Europe to Tanzania (Härkönen et al. 1994, Tulloss personal communication), Australia (Reid 1979;Grgurinovic 1997, Wood 1997;Hawkeswood 2006;Robinson 2010), South Africa (Pearson 1950;Reid & Eicker 1991) and New Zealand (Stevenson 1962;Ridley 1991).In Brazil, this taxon is reportedly found in the plateau region of Rio Grande do Sul among the "European pines" planted there (Homrich 1965).Unfortunately, we could not locate the material needed in order to determine the true identity of this specimen.

Amanita muscaria var. fl avivolvata
Comments: Th is subspecies, fi rst found in San Francisco, California, was originally named for its yellowish volva (Singer 1958), and the name continued to be used by other authors in North America (e.g.Jenkins 1977Jenkins , 1986;;Th iers 1982) and Europe (Poumarat & Neville, 2001, 2004).Nevertheless, the color of the volva does not appear to be crucial to the defi nition of this subspecies.Jenkins & Petersen (1976) described a neotype of A. muscaria var.muscaria as having a cream-colored to yellowish volva, which raised the possibility that the entity A. muscaria var.fl avivolvata occurs in Europe as well (Neville & Poumarat 2004).
Apparently, information about the color of the volva and pileus are of little relevance, because it can be infl uenced by the climatic conditions to which the basidiomes are submitted.Th ose conditions can generate yellow or albino forms of pileus within each population (Geml et al. 2008).Fungal pigments include muscaflavin, which produces yellow; muscarine, which produces an orange-red color; muscapurpurin, which produces purple; and muscaaurin, which produces reddish-brown (Meléndez- Michelot & Howell 2003).Th e mixture or suppression of certain chemical components can determine what color prevails in a fungus, such as the yellow in the pilei of certain populations in the northeastern United States, which must be attributable to an abundance of muscafl avin.
Amanita muscaria var.fl avivolvata is found in proximity to exotic plantations introduced into Australia (Sawyer et al. 2001), Chile (Garrido 1986) and Colombia (Tulloss et al. 1992); in Costa Rica, it is reported as occurring associated with Quercus (Tulloss et al. 2011).Daniele et al. (2005) cited A. muscaria in association with Cedrus Deodara (Roxb.ex D. Don) G. Don in Argentina, although the authors did not mention the infrageneric group to which it belongs.As demonstrated in the present study, A. muscaria var.fl avivolvata occurs in the Brazilian states of Parana, Rio Grande do Sul, Santa Catarina and São Paulo.Specimens collected in Brazil by Guerrero & Homrich (1983), Fusco-Mucci & Yokomizo (1985), Figueiredo et al. (1996), Giachini et al. (2000Giachini et al. ( , 2004)), Meijer (2001Meijer ( , 2006) ) and Sobestiansky (2005) might represent A. muscaria var.fl avivolvata, because the specimens were found primarily in proximity to P. elliottii and P. taeda, two species imported from North America.Th e material F. Karstedt 425 (FURB 840) certainly matches the voucher for the material cited in Karstedt & Stürmer (2008).One of the specimens examined here (V.G.Cortez 097/08; URM 82988, RET) was collected under a Eucalyptus sp.It is of note that A. muscaria sensu lato has oft en been cited in Australia, albeit associated with exotic plantations (Reid 1979;Grgurinovic 1997, Wood 1997;Hawkeswood 2006, Robinson 2010).However, a study conducted by Malajczuk et al. (1982) demonstrated that this species, in its broader sense, is also associated with Eucalyptus.
Th e dichotomous key recently proposed by Menolli et al. (2009) emphasizes the yellow color of the universal veil elements in early development as a means of distinguishing A. muscaria var.fl avivolvata from A. muscaria var.muscaria.Poumarat & Neville (2004) also considered the conditions of the universal veil a major distinguishing feature in A. muscaria.However, we consider the features of the universal veil irrelevant because they can result merely from environmental conditions to which the basidiome was submitted.Th e most important features distinguishing A. muscaria var.muscaria from A. muscaria var.fl avivolvata are the depth of the subhymenium, the size of the basidiospores, and the geographic distribution (Tulloss & Yang 2012;Tulloss, personal communication).We have devised a new dichotomous key that focuses on characteristics that are more appropriate to diff erentiating between the two A. muscaria subspecies in Brazil (Table 1).
In agreement with our interpretation, Geml et al. (2008) suggested that clades corresponding to A. muscaria var.muscaria and A. muscaria var.fl avivolvata belong to distinct phylogenetic species.In addition, Vellinga et al. (2009) called for more detailed taxonomic studies in order to elucidate which A. muscaria sensu lato occurs in the southern hemisphere.* -natural distribution in North America.

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Pileus: 70.0-110.0mm, plano-convex expanding to plano-concave, dark red, becoming paler and eventually orange, shiny surface, slightly viscid, fi nely striated margin, reaching 11.0 mm in length; invariably white context, approximately 10.0 mm thick at the center and gradually tapering toward the margin and eventually tapering more abruptly in the furrows; universal veil invariably with white pyramidal warts that are moderately easy to remove  Lamellae: ranging from closely spaced to completely separated, forming a decurrent line at the apex of the stipe in older individuals, invariably white from edge to edge, 10.0 mm wide, proximal; lamellulae truncate to obtusely truncate, varying in length  Stipe: 80.0-100.0× 25.0-30.0mm, narrowing toward apex, invariably white, longitudinally thinly fi brillose (seen only with a ≥ 10× lens); bulb from 20.0-50.0 mm in length and 25.0-40.0mm in width, fusoid; context white, unchanging, solid, central cylinder 10.0 mm diameter., insect or larva tunnels of reddish brown (salmon colored); partial veil white near the midpoint, smooth with remnants of universal veil on the edge;
Castanea sativa Pinus spp.* w st -near -distance from one side of the central stratum to the base of the nearest basidium; w st -far -distance from one side of central stratum to the base of the farthest basidium; L' -the average length of all basidiospores; W' -the average width of all basidiospores; Q' -the mean ratio of length to width computed for all basidiospores of all basidiomes.

Table 1 .
Dichotomous key to the Amanita muscaria subspecies occurring in Brazil.