Amanitatullossiana, a new species, and two new records of AmanitasectionLepidella from north-western Himalaya, India

Abstract Amanitatullossiana, a new species of Amanita [subgenus Lepidella] section Lepidella from India is described. The species is characterised by its ash grey to brownish-grey pileus covered with dark grey to greyish-black universal veil remnants, the upper part of its rooting stipe base covered by several rows of recurved scales, broadly ellipsoid to ellipsoid basidiospores, absence of basidial clamp connections and pileal remnants of universal veil comprising abundant, disordered inflated cells intermixed with scattered filamentous hyphae. Molecular phylogenetic analysis and morphology both support the association of A.tullossiana with species of Bas’ stirps Cinereoconia – A.cinereoconia and A.griseoverrucosa. Two species, A.griseoverrucosa and A.virgineoides are reported here as new records for India.


Introduction
The Amanitaceae is one of the most dominant and species-rich families of Basidiomycota. Traditionally, this family is divided into three genera, namely Amanita Pers., Limacella Earle and Catatrama Franco-Mol. However, a recent study by Redhead et al. (2016) divided Amanita into two genera, Amanita and Saproamanita Redhead, Vizzini, Drehmel & Contu, the former genus including species which are mycorrhizal in nature and the latter genus including only amycorrhizal/free-living species within Amanita. Subsequent to their establishment of the new genus, Tulloss et al. (2016) argued against the separation of Saproamanita from Amanita because the amycorrhizal species do not form a well-supported clade and are arguably the "mother" of the genus Amanita rather than a sister group within it. In this study, we follow the interpretation of Tulloss et al. (2016).
Species within Amanita sect. Lepidella are recognised by the combination of the following features: non-striate and appendiculate pileus margin and a volva that is friable, not forming an entire membranous sac (with the rare exception of a thin submembranous or membranous exterior layer). Approximately 200 taxa are listed for this section in the Amanitaceae website (http://www.amanitaceae.org/), of which 185 have been validly published (Corner and Bas 1962, Bas 1969, Tulloss and Jenkins 1985, Tulloss et al. 1992, Yang 1997, Wolfe et al. 2012, Deng et al. 2014, Li and Cai 2014, Hosen et al. 2015, Tulloss and Yang 2018. However, only four species, namely A. albofloccosa A.V. Sathe & S.D. Deshp., A. berkeleyi (Hooker f.) Bas, A. eriophora (Berk.) E.-J. Gilbert and A. konkanensis P.G. Sathe & S.M. Kulk. of Amanita sect. Lepidella have been reported from India so far (Bas 1969, Sathe and Daniel 1981, Kulkarni 1992. During the course of macrofungal forays into different parts of the state of Uttarakhand, India, the second author (TM) collected several specimens of Amanita in broad-leaved forests. Morphological examination and molecular data indicated that the new collections herein reported represent one species new to science and two new records for India.

Morphological study
Macromorphological characteristics were documented in the forest or base camp from fresh and dissected young to mature basidiomata. Photography was accomplished using a digital camera (Sony cyber-shot W730 and Cannon Power Shot SX 50). Colour codes follow Kornerup and Wanscher (1978). Samples were dried using an electric drier. Herbarium codes follow Index Herbariorum (Thiers 2018).
Micromorphological characteristics were observed with a compound microscope (Olympus CH20i) with dried material mounted in 5% KOH, 1% Phloxin, Melzer's reagent and 1% Congo red. To present basidiospore measurements, the following notation was used: "[n/m/p]" indicating n basidiospores were measured from m basidiomata of p collections with a minimum of 20 basidiospores from each collection. Biometric variables followed those in Tulloss and Lindgren (2005): L = the range of the average spore length computed per specimen examined. L' = the average spore length computed for all spores measured. W = the range of the average spore width computed per specimen examined. W' = the average spore width computed for all spores measured. Q = the ratio of length/breadth for a single spore and the range of the ratio of length/breadth for all spores measured. Q = the average value of Q computed for one specimen examined and the range of such averages. Q' = average value of Q computed for all spores measured. w cs = the width of the central stratum of a lamella. w st -near = the distance from an outer margin of the central stratum to the nearest base of a basidium. w st -far = the distance from an outer margin of the central stratum to the furthest base of a basidium on the same side of the central stratum. Drawings of microscopic features were made free hand.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from dry basidiomata following the modified CTAB method of Doyle and Doyle (1987). PCR was performed to amplify the partial sequence of the nuclear ribosomal large subunit (nrLSU) using universal primer pairs LR0R (GTACCCGCTGAACTTAAGC) and LR5 (ATCCTGAGGGAAACTTC) LR7 (TACTACCACCAAGATCT) (Vilgalys and Hester 1990) and the second largest subunit of RNA polymerase II (rpb2) using primer pair fRPB2-5F (GAY-GAYMGWGATCAYTTYGG) (Liu et al. 1999) and bRPB2-7.1R (GCHATGGG-KAARCARGCYATGGG) (Matheny 2005). Sequencing was performed on ABI 3730 XL DNA Analyzer (Applied Biosystems). PCR amplification (both nrLSU and rpb2) was conducted on a thermal cycler (Eppendorf, Germany) programmed for 3 min at 94 °C, followed by 35 cycles of 30 sec at 94 °C, 1 min at 55 °C, 1 min at 72 °C and a final stage of 8 min at 72 °C. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN, Germany). Both strands of the PCR fragment were sequenced on the 3730xl DNA Analyzer (Applied Biosystems, USA) using the same primer pair.

Phylogenetic analyses
In this study, a dataset of 49 nrLSU sequences of Amanita subg. Lepidella and one nrLSU sequence of Limacella bangladeshana Iqbal Hosen were used for phylogenetic analysis. The nrLSU sequences of Amanitaceae were selected based on BLASTn search results (Altschul et al. 1997) and availability of sequences of Amanitaceae in GenBank (Clark et al. 2016). The nrLSU dataset was then aligned with Mafft v.6.8 (Katoh et al. 2005) and manually adjusted with BioEdit v.7.0.9 (Hall 1999) using default settings. Maximum Likelihood (ML) phylogenetic analysis inferred from nrLSU sequences was performed using RAxML v.7.2.6 (Stamatakis 2006). Default settings were used for all parameters in the ML analysis and statistical support values were obtained using nonparametric bootstrapping with 1,000 replicates. Gaps in the alignment were treated as missing data in the phylogenetic analysis. Limacella bangladeshana was selected as the outgroup for the molecular phylogenetic analysis.

Molecular phylogenetic results
In this study, five sequences (three for nrLSU and two for rpb2) were generated from three separate collections (RET 717-4, RET 717-9 and TM 16-1228) of Amanita and deposited in GenBank (Table 1). Only nrLSU sequences were used in this study to delimit the Indian Amanita species. The rpb2 sequences were not used for reconstruction of molecular phylogeny because rpb2 sequences for most of the Amanita species (included in the nrLSU phylogeny) are currently unavailable in GenBank for inclusion in this study. The aligned nrLSU dataset consisted of 50 sample sequences of Amanitaceae (Table 1) with 934 nucleotide sites for each sample (gaps included), of which 238 were parsimony informative characters. The resulting dataset was deposited in TreeBASE (S21668). Initial BLASTn search result of the nrLSU sequence of the Indian collection (RET 717-4) against the NCBI database exhibited 98% identity with A. cinereopannosa Bas (GenBank HQ539678) and 97% with A. cinereoconia G.F. Atk. (GenBank HQ593118). Phylogenetically, the collection RET 717-4 is grouped together with A. cinereopannosa, A. cinereoconia and A. griseoverrucosa Zhu L. Yang with strong bootstrap (BS) support ( Fig. 1). Morpho- Diagnosis. Distinct from all the known species of Amanita stirps Cinereoconia by the combination of the following characters: medium-sized to large basidiomata (pileus 90-170 mm wide, stipe 150-185 × 20-25 mm); brownish-grey to dark grey pileus covered with floccose to subfelted, pulverulent patches of universal veil remnants; broadly ellipsoid to ellipsoid basidiospores measuring (8.5-)9-13( -13.5) × (5.8-)6-8(-8.5) µm.
Macrochemical tests on fresh basidiomata. 5% KOH -negative on pileus, 2% phenol -negative and FeSO4 crystals -negative on pileus and in stipe context. Commentary. The grey to brownish-grey universal veil, the absence of clamp connections, disordered inflated cells intermixed with scattered filamentous hyphae, together with broadly ellipsoid to cylindrical basidiospores are the key features of sect. Lepidella stirps Cinereoconia (Bas 1969). Based on the Bas' key, the new taxon could be placed in Amanita [sect. Lepidella subsect. Solitariae] stirps Cinereoconia.
Amanita cinereopannosa, A. cinereoconia and A. griseoverrucosa are the phylogenetically closely related species to the new species (Fig. 1). However, all of them are distinguished morphologically. Amanita cinereopannosa, originally described from USA, has a white to silvery sheen pileus covered with subfelted to subpyramidal warts, abundant filamentous hyphae and ellipsoid to elongated basidiospores (8-)8.8-10(-14.1) × (4.9-)5-6.7(-8.3) µm (Tulloss and Yang 2018). Furthermore, this species is considered endemic to eastern North America and has not been recorded in other parts of the world (Davison et al. 2013). Bas (1969) clearly held A. cinereopannosa to be distinct from the species of stirps Cinereoconia because he placed it in his stirps Strobiliformis. Amanita cinereoconia, originally described from the USA, has a white to greyish pileus covered with grey, pulverulent to small warted universal veil remnants and bears elongate to cylindric basidiospores 7.8-10.9 × 4.7-6.2 µm, with a Q value = 1.72 (Jenkins 1986). In addition, A. cinereoconia has a peculiar smell like "chloride of lime" [meaning the smell of an outdoor pit toilet into which CaCl 2 has been added; hence, an odour of decaying protein] or faintly of "chlorine" (Bas 1969;Jenkins 1986). Bas proposed a variety croceescens of A. cinereoconia; however, Tulloss had the opportunity to observe the transition of a single specimen from the "type variety" to "var. croceescens" and attributed the yellow colouration to the Amanita "yellowing syndrome" (Tulloss, pers. comm.). Amanita griseoverrucosa, originally described from China and reported here from India (see below), has a dirty white to greyish pileus, verrucose to conical universal remnants, a white to greyish-white stipe, a ventricose to clavate bulb and relatively smaller spores measuring 8-11 × 5.5-7 µm (Yang 2004) in comparison to A. tullossiana 9-13 × 6-8 µm.
Habitat and distribution. Solitary to gregarious, with plants of Fagaceae, Pinaceae and Ericaceae (Rhododendron arboretum).
Known distribution. Currently known from China (Yang 2004(Yang , 2015 and now India. Commentary. Morphologically, the Indian collections of A. griseoverrucosa are characterised by a whitish to greyish-white pileus covered with easily detachable greyish-brown to dark grey, felted to verrucose universal veil remnants, a ventricose to clavate stipe base, broadly ellipsoid to ellipsoid basidiospores, universal veil on the pileus with abundant inflated cells and scattered filamentous, undifferentiated hyphae and the absence of clamp connections at bases of basidia. The characteristic features and molecular data from the Indian collections match rather well with the original description of A. griseoverrucosa, reported from China (Yang 2004).