Volvopluteus asiaticus (Agaricales, Basidiomycota

Summary . The specimens collected in the Russian Far East were identified as Volvopluteus asiaticus , based on morphology and nrITS+nrLSU sequences analyses. The detailed description and illustration of the studied collection are provided. This is the first record of the species in Russia and the second find in the world


Introduction
The genus Volvopluteus Vizzini, Contu et Justo was established in 2011 (Justo et al., 2011b) with Volvopluteus gloiocephalus (DC.)Vizzini, Contu et Justo as the type species.The genus comprises four species described using polyphasic approach (V.gloiocephalus, V. earlei (Murrill) Vizzini, Contu et Justo, V. michiganensis (A.H.Sm.) Justo et Minnis and V. asiaticus Justo et Minnis).According to the Index Fungorum (Index Fungorum.URL: https:// indexfungorum.org/),there are two more species of Volvopluteus (namely V. diversisporus M. Kaur et Yadw.Singh and V. shafferi M. Kaur et Yadw.Singh), but latter were described based on morphology only, and additional molecular investigations of the type specimens are needed to confirm their identity.Representatives of the genus are characterized by medium or even large-sized basidiomata with volva at stipe base, viscid pileus, numerous free lamellae and pink or pinkish brown spore print.Volvopluteus asiaticus was described based on molecular data and micromorphological differences of the dried specimen that was previously identified as Volvariella gloiocephala (Justo et al., 2011a).
During the mycobiota survey of the Far East, we found specimens of Volvopluteus sp. with conspicuously brown pileus that turned out to be Volvopluteus asiaticus.This collection represents second finding of the species in the world after the type specimen Volvopluteus asiaticus (Agaricales, Basidiomycota) -the second finding in the world according to GBIF (Global Biodiversity Information Facility.URL: https://www.gbif.org).Here we offer an updated description of the species based on fresh material and accompanied with photos of basidiomata and micromorphological characters as well as with supporting evidence based on nrITS and nrLSU sequences analyses.

Study site
The studied specimens were collected in vicinities of Gorno-Taezhnoye settlement, Primorye Territory (43°42'N, 132°09'E), on gentle northern slope in wet deciduous forest with Tilia spp., Quercus mongolica, Acer mono, Juglans mandshurica, and Fraxinus spp., on soil in proximity to fallen and decayed trunk of unidentifiable deciduous tree.

Sampling and morphological study
Basidiomata were photographed in situ and transported to the field station, where they were examined according to standard techniques applied to fungal taxonomy (Clémençon, 2009).Microscopic observations were made from dried material mounted in 5 % KOH and 10 % Congo Red in NH 4 OH using a Zeiss Axio Scope.A1 and Axio Imager.A1 light microscope with differential interference contrast (DIC).Basidiospore dimensions were based on at least 30 basidiospores from each basidioma; (n = 60, s = 2) indicates measurements based on 60 basidiospores from two basidiomata in one collection.Spore dimensions (without hilar appendage) are provided as (a-)b-c(-d), with b-c containing at least 90 % of all values and a and d representing extreme values.Q indicates the basidiospore length/width ratio, Q* represents the mean length/width quotient of the total basidiospores measured.The measurements of the other morphological structures are based on at least 20 elements.
The studied collection was deposited in the Mycological Herbarium of the Komarov Botanical Institute (LE).

DNA extraction, PCR amplification, and sequencing
We performed DNA extraction from small part of dried basidioma using FitoSORB DNA extraction kit according to the manufacturer's protocol (Syntol, Russia).The following primers were used for amplification and sequencing: ITS1F-ITS4B (Gardes, Bruns, 1993) for internal transcribed spacer (ITS1-5.8S-ITS2);LROR-LR5 (Vilgalys, Hester, 1990;White et al., 1990) for part of the nrLSU region.We visualized product of PCR reaction using agarose gel electrophoresis and SYBR Green staining and subsequently purified it with CleanMag DNA kit (Evrogen, Russia).Sequencing was performed with an ABI model 3500 Genetic Analyzer (Applied Biosystems, CA, USA).Raw data were edited and assembled in MEGA X (Kumar et al., 2018).
All microscopic and molecular studies of specimens were carried out at the Center for collective use of scientific equipment "Cellular and molecular technology of studying plants and fungi" (Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg).

Sequence alignment and phylogenetic analyses
For this study one new nrITS and one nrLSU sequences were generated.In addition, 21 nrITS and 14 nrLSU sequences, including an outgroup, were retrieved from the GenBank database (www.ncbi.nlm.nih.gov/genbank/), using the BLAST application and taxonomic considerations (Justo et al., 2011a;Kaygusuz et al., 2021).The taxonomic identities of these sequences with GenBank accession numbers are given in Table .The sequences were aligned with the Muscle tool (Edgar, 2004) incorporated into MEGA X program separately for each genetic marker and then combined into a single dataset.
Phylogenetic reconstructions were performed with Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.Before the analyses, the bestfit substitution model was estimated based on the Akaike Information Criterion (AIC) using Find-Model web server (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html).For each dataset (nrITS and nrLSU) the model turned out to be the same, thereby GTR+G model was chosen for concatenated dataset (nrITS+nrLSU) and used for further analyses.Maximum likelihood analysis was run on RAxML servers, v.1.0.0 (https://raxmlng.vital-it.ch/#/)with one hundred rapid bootstrap replicates.BI analysis was performed with MrBayes 3.2.5 software (Ronquist et al., 2012), for two independent runs, each with 10 million generations under described model and four chains with sampling every 100 generations.To check for convergence of MCMC analyses and to get estimates of the posterior distribution of parameter values, Tracer v1.7.1 was used (Rambaut et al., 2018).We accepted the result where the ESS (Effective Sample Size) was above 200 and the PSRF (Potential Scale Reduction Factor) was close to 1.
Newly generated sequences were deposited in GenBank with corresponding accession numbers (Table ).

Phylogenetic analyses
We choose the available nrITS and nrLSU sequences of all Volvopluteus species presented in research dealing with its' taxonomy (Justo et al., 2011b;Kaygusuz et al., 2021) to perform phylogenetic analysis with Pluteus heteromarginatus Justo as an outgroup.The final combined nrITS+nrLSU dataset contained 22 nrITS and 15 nrLSU sequences and consisted of 1623 characters (with gaps).Tree topologies were almost congruent while using ML and BI methods for phylogenetic analyses, thus only the BI tree is presented in Fig. 1.Our nrITS+nrLSU phylogeny supports four clades in the genus.These highly supported lineages correspond to four morphological species: V. gloiocephalus, V. earlei, V. michiganensis, and V. asiaticus.In the tree, V. asiaticus is sister to V. michiganensis, and those are closer to V. gloiocephalus than to V. earlei.
The studied specimen from the Russian Far East fits a clade containing V. asiaticus holotype with strong support value (100 % ML BS, PP = 1.0).

Discussion
The pluteoid basidiomata with universal veil remnants present as volva at stipe base, viscid pileus, free and pink lamellae, a stipe that is not viscid, ellipsoid to oblong basidiospores without germ-pore, pileipellis as ixocutis, and terrestrial Volvopluteus asiaticus (Agaricales, Basidiomycota) -the second finding in the world habitat support the placement of our collection in Volvopluteus (Justo et al., 2011a(Justo et al., , 2011b)).The species delimitation within a genus is based primarily on colors of basidiomata, basidiospore size and shape, and morphology of hymenial cystidia.V. asiaticus is characterized by large basidiomata, with dark colored pileus, white volva, large basidiospores and pleurocystidia with apical excrescence.It differs from the closely related species V. gloiocephalus in the presence of rostrate pleurocystidia.
The studied collection from the Russian Far East differs slightly from the type description (Justo et al., 2011b) in the following features: darker pileus coloring with distinct olive shades, slightly smaller spore size (vs 12.0-14.5× 7.0-8.5(9.0)μm in type collection), and predominance of clavate cheilocystidia.Thus, fresh material allows us to clarify and expand the morphological characteristics of the species.
Volvariella asiaticus was originally described solely on herbarium material (TNSF 15191, from Japan) that was previously identified as common and widely distributed species Volvariella gloiocephala (≡ V. gloiocephalus).Volvariella gloiocephalus is known to be characterized by a variety of coloration of basidiomata, as well as a variety of microscopic characters.The strong variability of morphological features can mislead researchers regarding the boundaries of the species.Therefore, specimens of the latter species with brown pileus collected in the Asian region may turn out to be V. asiaticus as a result of further molecular studies.

Fig. 1 .
Fig. 1.Phylogenetic tree of Volvopluteus asiaticus and allies derived from the nrITS+nrLSU dataset using Bayesian analysis.The Bayesian PP/ML bootstrap support are shown above branches.For all taxa the GenBank accession numbers (nrITS/nrLSU) are presented.The specimen studied for this article is highlighted in bold.Scale bar indicates the mean number of nucleotide substitutions per site.

Table
Sequences used for molecular analyses.Newly generated sequences are marked with *, sequences from type specimens are given in bold