Looking for Lepiotapsalion Huijser & Vellinga (Agaricales, Agaricaceae)

Abstract Lepiotapsalion is fully described based on a recent collection from Sardinia (Italy) and the holotype. NrITS- and nrLSU-based phylogeny demonstrates that sequences deposited in GenBank as “L.psalion” and generated from two Dutch and one Chinese collections are not conspecific with the holotype and represent two distinct, undescribed species. These species are here proposed as Lepiotarecondita sp. nov. and Lepiotasinorecondita ad int.


Introduction
Blue, Cresyl Blue, and Melzer's reagent. Measurements of the microscopic features of Lepiota psalion and L. recondita were made by photographing all the elements occurring in the visual field of an Optika B-383 PLi light microscope. Measurements were performed using the Piximètre 5.9 R 1530 software (http://ach.log.free.fr/Piximetre/) at 1000× magnification. The microphotographs were taken by an Optikam B5, 5 MP× camera.
When possible, dimensions of the microscopic elements are given as: (minimum-) average minus standard deviation -average plus standard deviation (-maximum) of length × (minimum-) average minus standard deviation -average plus standard deviation (-maximum) of width. Spore dimensions do not include the hilar appendix. The width of each basidium was measured at the widest part, and the length was measured from the apex (sterigmata excluded) to the basal septum. The DNA fluorescent dye 4′,6-diamidino-2-phenyl-indoldihydrochloride (DAPI) was used to stain nuclei in spores following Horton (2006). The number of nuclei in spores were then determined using a Leica TCS-SP2 confocal microscope. Samples were excited with 405 nm light and fluorescence was recorded at 440-500 nm. The following abbreviations are used: l = number of lamellulae between each pair of lamellae reaching the stipe; the notation [X, Y, Z] indicates that measurements were made on X randomly selected spores (taken from spore-prints), in Y samples from Z collections; Q = the spore quotient (length/ width ratio); Qav = the average spore quotient. Terminology for descriptive terms is according to Vellinga (1988Vellinga ( , 2001. Herbarium abbreviations follow Thiers (2019, continuously updated). Author citations follow the Index Fungorum -Authors of Fungal Names (http://www.indexfungorum.org/authorsoffungalnames.htm).

DNA extraction, PCR amplification and DNA sequencing
Total DNA was extracted from seven dry basidiomes (Tab. 1): two basidiomes (labelled as "a" and "b") from the same L. psalion CAG P.11_9/7.68 collection, one basidiome from the L. psalion holotype (WU 5152), two basidiomes from two collections of the new species L. recondita, and two basidiomes from two collections of L. sanguineofracta Vizzini (TO-HG2916, holotype and TO-HG2917). DNA extraction and PCR amplifications were performed as described by Alvarado et al. (2015). Primers ITS1F and ITS4 (White et al. 1990; Gardes and Bruns 1993) were used for the nrITS region; primers LR0R and LR5 (Vilgalys and Hester 1990) were used for the nrLSU (28S) rDNA, and finally EF1-983F and EF1-1567R (Rehner and Buckley 2005) for the translation elongation factor 1-α (tef1-α) gene. Chromatograms were checked searching for putative reading errors, and these were corrected. The PCR products were purified with the Wizard SV Gel and PCR Clean-UP System (Promega) following manufacturer's instructions and sequenced forward and reverse by MACROGEN Inc. (Seoul, Republic of Korea). Sequences were checked and assembled using Geneious v. 5.3 (Drummond et al. 2010) and submitted to GenBank (http://www.ncbi.nlm.nih. gov/genbank/). Accession numbers are reported in Table 1.

Sequence alignment, dataset assembly and phylogenetic analysis
Sequences obtained in this study were compared to those available in the GenBank (http://www.ncbi.nlm.nih.gov/) and UNITE (http://unite.ut.ee/) databases by using the Blastn algorithm (Altschul et al. 1990). Based on the BLASTn results (sequences were selected based on the greatest similarity) and outcomes of recent phylogenetic studies incorporating Lepiota sequences (Vellinga 2003(Vellinga , 2004(Vellinga , 2010Vizzini et al. 2014a, b;Justo et al. 2015;Qasim et al. 2015;Hosen et al. 2016) sequences were retrieved from GenBank for the comparative phylogenetic analysis. The nrITS and nrLSU datasets were analysed separately. The combined nrITS/nrLSU phylogeny was not inferred as most Lepiota collections in GenBank are not provided with both molecular markers (Table 1). Although tef1-α sequences were generated for L. psalion, they were not included in phylogenetic analyses because comparable sequences for most Lepiota taxa are currently unavailable in public databases, and, in this case, only the Blastn results were provided in the Results. In the nrITS dataset, besides Lepiota species with a hymeniform pileus covering, eight species (indicated by an asterisk in Fig. 1) representative of the major clades in Lepiota as delimited by Vellinga (2003) were chosen for comparison. The nrLSU dataset consists of all the Lepiota s.l. collections determined at species level present in GenBank. Alignments were generated for each nrITS and nrLSU dataset using MAFFT (Katoh et al. 2002) with default conditions for gap openings and gap extension penalties. The two alignments were imported into MEGA v. 6.0 (Tamura et al. 2013) for manual adjustment. The best-fit substitution model for each single alignment was estimated by the Bayesian information criterion (BIC) with jModelTest 2 (Darriba et al. 2012). The GTR + G model was chosen for the nrITS alignment and the TrN+I+G for the nrLSU alignment. The nrITS dataset was partitioned into ITS1, 5.8S and ITS2 subsets. Chamaemyces fracidus (AY176343 and AY176344) was used as an outgroup taxon in both the nrITS and nrLSU analyses because it is basal in the Agaricaceae (Vellinga 2004(Vellinga , 2010. Phylogenetic hypotheses were constructed with Bayesian inference (BI) and Maximum likelihood (ML) criteria. The BI was performed with MrBayes v. 3.2.6 (Ronquist et al. 2012) with one cold and three incrementally heated simultaneous Monte Carlo Markov chains (MCMC) run for 10 million generations, under the selected evolutionary model. Two simultaneous runs were performed independently. Trees were sampled every 1,000 generations, resulting in overall sampling of 10,001 trees per single run; the first 2,500 trees (25%) were discarded as burn-in. For the remaining trees of the two independent runs, a majority rule consensus tree showing all compatible partitions was computed to obtain estimates for Bayesian posterior probabilities (BPP).
ML estimation was performed with RAxML v. 7.3.2 (Stamatakis 2006), with 1,000 bootstrap replicates (Felsenstein 1985) using the GTRGAMMA algorithm to perform a tree inference and search for a good topology. Support values from bootstrapping runs (MLB) were mapped on the globally best tree using the "-f a" option of RAxML and "-x 12345" as a random seed to invoke the novel rapid bootstrapping algorithm. BI and ML analyses were run on the CIPRES Science Gateway web server (Miller et al. 2010). Only BPP and MLB values over 0.70 and 50%, respectively, are reported in the resulting trees (Figs 1, 2). Pairwise % identity values (P%IV) of the sequences were calculated using MEGA v. 6.0 (Tamura et al. 2013). Alignments and phylogenetic trees are available at TreeBASE (www.treebase. org) under ID S22021.
As both Bayesian and Maximum likelihood analyses produced a consistent topology, only the Bayesian trees with both BPP and MLB values are shown (Figs 1, 2).
Ecology and distribution. Gregarious on bare soil, in gardens and parks; so far known only from the type locality (Austria) and Sardinia (Italy).

Lepiota sinorecondita ad interim
Basidiomata small (Fig. 10a). Pileus 9-17 mm wide, expanding to convex with obtuse umbo; at centre on umbo smooth, dark yellowish brown to dark brown, around umbo split up into pale brown concentrically arranged patches on dirty white to cream background, paler and smaller towards margin. Stipe 35-37 × 1-4 mm, subcylindrical or attenuate, slightly inflated at base; hollow, dirty white and glabrous at the apical part, surface whitish, covered white, tomentose at lower part, with white mycelial cords at base; annulus membranous, superior, whitish on upper surface, with small yellowish brown to brownish squamules on lower whitish surface. Lamellae free, cream, yellow to brown when dry, crowded with lamellulae, edge wavy.
Ecology and distribution. Solitary, terrestrial, on the ground in a larch forest in summer and autumn. So far known only from China.
The phylogenetically closest species are L. coloratipes (= L. rufipes ss. Auct. europ. non ss. orig.) and L. sanguineofracta (Fig. 1). Lepiota coloratipes differs from L. psalion in having a usually smooth pileus surface, a very evanescent partial veil not forming an annulus but leaving fibrillose remnants on stipe surface, a stipe with reddish tinges at base, the presence of oil droplets in all tissues (including spore surface), the hymeniform pileus covering consisting of very tightly arranged clavate to sphaeropedunculate elements, the presence of uninucleate spores which are often verruculose in Melzer's reagent, versiform cheilocystidia (mostly lageniform or lecythiform), and the presence of caulocystidia (Bon 1981(Bon , 1993Candusso and Lanzoni 1990;Vellinga and Huijser 1999;Vellinga 2001;Vizzini et al. 2014b). Lepiota sanguineofracta, recently described from Italy, is characterized by a micaceous but not squamulose pileus surface with distinct green tinges when mature, a fugacious partial veil not forming an annulus, a stipe with reddish tinges towards the base, the context smelling of dried rose petals, basidiome surfaces and context strongly reddening on handling, binucleate spores, and versiform cheilocystidia (clavate to subutriform, subfusiform) (Vizzini et al. 2014a).
The Lepiota psalion complex Lepiota psalion was established by Vellinga and Huijser (1999) based on an Austrian collection made by A. Hausknecht on 23 August 1985 (WU 5152) and determined by M. Bon as L. rufipes fo. annulata ined. (Fig. 4a). The extended description they provided is heterogeneous: the macromorphology was taken from Krieglsteiner (1991) who described a German collection as L. rufipes, collection considered by Vellinga and Huijser as L. psalion, while the micromorphology was based on the analysis of the holotype made by the same Dutch mycologists. NrITS and nrLSU sequences later deposited in GenBank as L. psalion were generated by Vellinga (2004Vellinga ( , 2010) not from the holotype, but from three Dutch collections (vouchers 23-VIII-1999, 15-IX-1999, andhah6177, H.A. Huijser, herb. Huijser).
When the Sardinian specimens were collected, they were morphologically attributed to L. psalion, but when they were sequenced to obtain molecular evidence, they did not cluster either with the Dutch collections or with a collection named L. psalion from China (herb. HMJAU3799; Liang et al. 2011) (tree not shown). Consequently, we decided to request the holotype collection from WU and sequenced it. Phylogenetic analyses highlighted that Sardinian collection and the holotype are conspecific (Figs 1, 2) and sister to L. coloratipes (Fig. 1). Molecular data so confirm L. psalion as independent species in the genus Lepiota; Dutch and Chinese collections are two distinct and yet undescribed new species, phylogenetically close (BPP = 0.97; MLB = 91%) to L. thiersii (Fig. 1). Unfortunately, the collections of the Dutch taxon whose sequences are deposited in GenBank were subsequently lost (Vellinga, pers. comm.) but, based on two newly sequenced additional collections from the same original area of the Dutch taxon, the new species L. recondita is here described. As only one collection (consisting of three basidiomes) is available for the Chinese taxon, it was decided to propose it only as an ad interim species. Further collections will be necessary to describe it as a new species.