Amanita heishidingensis, a new species of Amanita sect. Lepidella from China

A new species of Amanita sect. Lepidella, A. heishidingensis, is described based on both morphological and molecular evidences. It was compared with similar species and illustrated with line drawings and photographs. This species was found in Heishiding National Nature Reserve, Guangdong Province, South China.


Introduction
The genus Amanita Pers. in China has been studied extensively in the last twenty years or so and over eighty species including several lethal ones caused many troubles and losses have been reported from China (Yang 1994(Yang , 1997(Yang , 2005Zhang et al. 2010;Chen et al. 2014;Deng et al. 2014). Since November 2008, the first author has collected mushrooms in Heishiding National Nature Reserve (111°49″09″-111°55′01″E, 23°25′15″-23°30′02″N), Guangdong Province, China, and found more than 30 taxa of Amanita there. One of them, found in forests dominated by fagaceous trees, is described and illustrated herein as a new species.

Morphology
The description of the new species is based on morphological studies of fresh material and exsiccata. The photographs ( Fig. 1) depict the holotype. Color code follows Kornerup and Wanscher (1978). Tissues were mounted in 5 % KOH and 1 % Congo red for microscopic examination and making of line drawings. Spores were mounted in Melzer's reagent to test for amyloidity. The abbreviation (n/m/p) means n basidiospores measured from m basidiomata of p collections. Dimensions for basidiospores are given using a range notation of the form (a) b-c (d). The range b-c contains a minimum of 90 % of the measured values. Extreme values (a and d) are given in parentheses. Q = length/width ratio of a basidiospore in side view; Q = average Q of all basidiospores measured ± sample standard deviation. The holotype collection of the new species was deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), Kunming, China. Additional Collections were deposited in HKAS or in the Tottori Mycological Institute (TMI), Tottori, Japan.
DNA extraction, PCR amplification and DNA sequencing Genomic DNA was extracted from fruiting bodies dried in silica gel according the modified CTAB protocol (Doyle and Doyle 1987). The large nuclear ribosomal RNA subunit of the nuclear ribosomal RNA (nrLSU) was amplified with primers LROR and LR5 (Vilgalys and Hester 1990). The PCR amplification followed those in Zeng et al. (2013) and references therein. The PCR products were then purified using a Gel Extraction and PCR Purification Combo Kit (Spin-colum) (Bioteke, Beijing, China). Sequencing was performed with an ABI 3730 DNA analyzer (Applied Biosystem, Foster City, CA, USA) using the same primer pairs used for the PCR.

Sequence alignment and phylogenetic analyses
Four nrLSU sequences of the new species and one nrLSU sequences of Amanita japonica extracted in this study were compared with 26 nrLSU sequences retrieved from GenBank (NCBI; http://blast.ncbi.nlm.nih. gov/). These sequences were aligned with MUSCLE v3. 8.31 (Edgar 2004), and then manually optimised on BioEdit v7.0.5 (Hall 1999). Gaps were treated as missing data. The maximum likelihood analysis (ML) conducted on RAxML v7.2.6 (Stamatakis 2006) and Bayesian inference (BI) executed on MrBayes V3.2 (Ronquist and Huelsenbeck 2003) were implemented for the phylogenetic analyses. The optimal substitution model for ML and BI analyses was determined using the Akaike Information Criterion (AIC) as implemented in MrModeltest v2.3 (Nylander 2004). The statistical branch support values were evaluated using rapid nonparametric bootstrapping with 1000 replicates in RAxML and using posterior probabilities from BI analysis. The MrBayes analysis was automatically terminated using the stoprul and stopval commands when the standard deviation of the split frequencies fell below 0. 01. Chains convergence was further verified using Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/) to confirm sufficiently large ESS values (>200). Subsequently, the sampled trees were summarized after omitting the first 25 % of trees as burn-in using the 'sump' and 'sumt' command implemented in MrBayes.

Morphological analyses
Three collections with over thirty basidiomata of the new species were morphologically examined. For comparison, four collections of Amanita japonica, collected from Japan were examined. Our data indicated that the new species is morphologically different from A. japonica (see discussion below).
Habitat and distribution: gregarious or scattered on soil in forests dominated by Fagaceae, at 190-600 m alt. Presently known only from Heishiding National Nature Reserve, Guangdong Province, China.

Discussion
Amanita heishidingensis, a member of Amanita sect. Lepidella (Bas 1969), is characterized by its medium-sized to large basidioma with a dirty white to whitish viscid pileus covered with grey to brownish grey pyramidal to verrucose volval remnants, light cream lamellae, a whitish stipe covered with white to pale brownish grey floccose to farinose recurving squamules, usually with a big napiform, subclavate to ventricose bulb covered with pale yellow to pale brownish grey subfelted to subtomentose volval remnants, a fugacious annulus, ellipsoid amyloid spores, and abundant clamps in all tissues.
Amanita heishidingensis keyed out in Amanita subsect. Solitaria Bas stirps Solitaria (Bas 1969). It resembles A. japonica Hongo ex Bas. Amanita japonica, originally described from Japan (Bas 1969;Imazeki and Hongo 1987), resembles A. heishidingensis by its similarly shaped basidioma, the greyish voval warts on the cap, and the similar basidiospores. But the main morphologial and anatomical differences in basidioma can distinguish A. japonica from A. heishidingensis. On the morphologial features, A. japonica generally has a medium-sized basidioma (cap 55-80 mm wide, stipe 80-170× 7-15 mm); a small fusiform-rooting to subclavate bulb (up to 25 mm wide); a dry, moderately dark grey to pale buffy grey, felted-subflocculose pileus; a non-gelatinized pileipellis; small (about 2 mm wide and 1.5 mm high) subpyramidal warts adnate on the mature pileus; subflocculose edged lamellae (Bas 1969). While A. heishidingensis usually has a big sized basidioma (cap 70-155 mm wide, stipe 80-205×11-25 mm) with a big napiform to subclavate bulb (20-47 mm wide), with the base always round, not rooting; a viscid, white to whitish (without greyish tint when young), glabrous pileus with the pileipellis always strongly gelatinized; the warts on the pileus are much larger (up to 6 mm in wide and high); the pileipellis under the warts is often strongly gelatinized-making the warts detersile and easily washed off in rainy weather; lamellar edge is always smooth. On the anatomical features, the pileipellis of A. japonica is difficult to delimited from the greyish volval remnants over it, not or very slightly gelatinized, while A. heishidingensis has a clear delimited, gelatinized to strongly gelatinized pileipellis; hyphae and inflated cells in the warts of A. japonica are much more darker colored than those in A. heishidingensis; the annulus of A. japonica has abundant to nearly abundant hyphae, with inflated cells mostly clavate shaped, and with yellowish vacuolar pigmentation in both hyphae and inflated cells, while A. heishidingensis has fewer hyphae in its annulus, with inflated cells mostly globose, subglobose to sphaeropedunculate, and has rare vacuolar pigments in both hyphae and inflated cells. Furthermore, A. heishidingensis grows in the early spring, when the temperature is average 5-12°C, never over 20°C, and can't be found in late spring, summer or autumn; while A. japonica grows in late summer to early autumn, when the weather is much warmer. Our molecular phylogenetic analysis also suggested that A. heishidingensis and A. japonica are different species (Fig. 3).
Amanita cokeri (E.J. Gilb. & Kühner) E. J. Gilb., originally described from North America (Bas 1969), resembles A. heishidingensis in similar shape of basidioma and white pileus covered with rather larger white to brownish pyramidal warts. But A. cokeri can easily be differentiated from A. heishidingensis by its distinctive ample double ring, and somewhat larger basidiospores (11-13.5 ×7-9 μm) (Bas 1969). Additionally, warts of A. heishidingensis are never white at any stage of development, and always having a greyish tint; moreover the bases of warts are not radially fibrillose; and the gills are never pinkish at any stage of development. The molecular phylogenetic analysis also indicated that the two species are distinct.
Amanita miculifera Bas & Hatanaka originally described from Japan (Bas and Hatanaka 1984), resembles A. heishidingensis in its similar sized and shaped basidioma, whitish-greyish (between 1A1 and 1B1) pileus covered with moderately grey subpyramidal warts, and similar sized and shaped basidiospores; but its pileus is conical with obtuse apex to plano-conical; its lamellae are rather narrow; its bulb is strongly rooting; its warts are much smaller and the arrangement of the inflated cells in its volval warts is quite irregular (Bas and Hatanaka 1984).