Two New Lyophyllum Species from Yunnan, China

: Two novel species of Lyophyllum are proposed based on morphological and phylogenetic investigations. Lyophyllum bulborhizum sp. nov. has abundant black scales on the surface, is a dry, dark grey at the center, grey at the margin, has a clear bulbous stipe base, and has a narrowly cylindrical or narrowly clavate pleurocystidia and cheilocystidia. Lyophyllum nigrum sp. nov. is tufted and has a dark grey pileus and subclavate, a greyish green-to-olive stipe, and a narrowly cylindrical or narrowly clavate pleurocystidia and cheilocystidia. In addition, L. rhombisporum is described with morphology and phylogeny and compared with the type species. Molecular analyses employing internal transcribed spacer (ITS) sequences strongly support the two new species to be unique in the genus Lyophyllum . Full descriptions, colour photos, illustrations, and phylogenetic analyses results of the two new taxa and the known taxon are presented in this study.


Introduction
Lyophyllum P. Karst belongs to the Lyophyllaceae family, within the Agaricales order [1].The defining characteristics of this genus include basidiomata with colour that remains constant or darkens upon cutting, occurring in a scattered, gregarious, or solitary fashion.The pileus surface is smooth, while the stipe is solid, cylindrical, or tapers upwards.Lyophyllum species typically exhibit saprophytic and symbiotic habits, and their basidiospores contain colourless membranes and exhibit variable shapes such as globose, ellipsoid, or broadly fusiform and siderophile basidia in acetoferric carmine [2][3][4][5][6][7].
The initial identification of a Lyophyllum species in China was conducted by Dai in 1979 [6], who recognized L. cinerascens (Bull.)Konrad & Maubl., which is now referred to as L. decastes var.fumosum (Pers.)Gminder [8].Subsequently, an additional 22 Lyophyllum species have been documented in China [6,9].These identifications primarily relied on phenotypic resemblances to European Lyophyllum species, lacking comprehensive descriptions and molecular data.
Despite the number of studies performed within the genus, species identification, also by molecular data, remains challenging.Larsson and Sundberg [10] provided ITS and LSU data, which provided more evidence for identifying species.Later, several Lyophyllum species were published based on ITS and LSU data [11][12][13][14].
Commercially cultivated species of Lyophyllum include L. shimeji (Kawam.)Hongo, L. decastes and L. fumosum (Pers.)P.D. Orton.Lyophyllum shimeji naturally forms ectomycorrhizae and has been cultivated in synthetic media.This achievement is credited to the mycelium's capacity for rapid growth in various media, such as potato dextrose agar (PDA) [15][16][17][18].The classification of this genus and the cultivation techniques, particularly for L. shimeji, have been thoroughly investigated in countries such as Switzerland, Sweden, Japan, and Italy [19].
During a survey of Lyophyllum in China, two novel species, L. bulborhizum and L. nigrum, belonging to sec.Lyophyllum, were collected and are described in this paper based on morphological features and molecular data.In addition, L. rhombisporum is described with morphological characteristics and phylogenetic analyses and compared with the type species.

Morphological Studies
Eight specimens of Lyophyllum were gathered from Yunnan, China and immediately preserved in aluminum foil and collection boxes for subsequent examination.Macromorphological characteristics were documented directly from the fresh collections, with colour specifications determined in accordance with the guidelines outlined by Kornerup and Wanscher [20], and morphological descriptions followed Vellinga [21].Acetoferric carmine was also used to check the siderophilous granulations in the basidia [7].Following collection, specimens were subjected to drying at 50 • C using a food drier and then securely stored in sealed plastic bags.These preserved specimens were deposited at the Biotechnology and Germplasm Resources Institute, Yunnan Academy of Agricultural Sciences in Kunming, and the Herbarium of Cryptogams at the Kunming Institute of Botany, Chinese Academy of Sciences.Micro-morphological characteristics were examined using dried material under a light microscope.All tissues underwent revitalization in 5-10% KOH and were mounted in Congo red.Twenty basidia and over 50 basidiospores were measured and photographed utilizing a Nikon Eclipse 80i microscope (Tokyo, Japan) at magnifications up to ×1000.The notation [x/y/z] indicates that measurements were conducted on 'x' basidiospores from 'y' basidiomata and 'z' collections.Basidiospore dimensions are expressed as '(a) b-n-c (d)'.Here, 'a' and 'd' represent the lower and upper limits of all measurements, respectively, 'b-c' denotes the range encompassing 95% of the measured basidiospores, and 'n' signifies the average dimension.Additionally, 'Q' represents the length/width ratio of basidiospores, while 'Q m ' denotes the average Q calculated from all basidiospores.

DNA Extraction, PCR Amplification and Sequencing
Molecular analyses were conducted at the Yunnan Academy of Agricultural Sciences in China.Genomic DNA was extracted from dry specimens using the Ezup Column Fungi genomic DNA extraction kit (Sangon Biotech, Shanghai, China), according to the manufacturer's protocol.The primer pair ITS1/ITS4 [22] was used to amplify the ITS region.PCR was executed on a C1000 thermal cycler (Bio-Rad, Kunming, China) with the following cycling program for ITS: initial denaturation at 95 • C for 5 min, 35 cycles of denaturation at 95 • C for 30 s, annealing at 55 • C for 30 s, extension at 72 • C for 90 s, and a final extension at 72 • C for 10 min [22].
The PCR products were subjected to electrophoresis on 1% agarose gels stained with ethidium bromide and visualized under UV light.Subsequently, the PCR amplicons were sent to Sangon Biotech (Shanghai, China) for Sanger sequencing in both directions, employing the PCR primers.

Sequence Alignment and Phylogenetic Analyses
The Lyophyllum sequences produced in this study were submitted to the NCBI database.The Basic Local Alignment Search Tool (BLAST) was employed on the GenBank database to verify whether the newly generated sequences represented amplified DNA from contaminants and to explore clusters with closely related sequences.DNA sequences were obtained and assembled using SeqMan.The sequence alignments were conducted using MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/accessed on 10 September 2023) [23], and individual gene analyses were performed using BioEdit version 7 [24].The maximum likelihood (ML) analysis was executed using RAxML-HPC2 v. 8.2.12 [25] through the CIPRES v. 1.15 portal (https://www.phylo.org/portal2/login!input.action,(accessed on 10 September 2023)) [26].The analysis utilized the GTR+G+I model, and 1000 rapid bootstrap (BS) replicates were performed for all genes.
To ensure consistency between tree topologies from individual genes, a reciprocal 70% bootstrap support method was employed.As no substantial incongruence in topology was observed among the maximum likelihood (ML) trees, the internal transcribed spacer (ITS) region was partitioned for subsequent phylogenetic analyses.
Bayesian inference (BI) employed the best substitution models for each partition, as determined by MrModeltest 2.2 [27].The recommended models were ITS1: JC+I, 5.8S: GTR+G+I, and ITS2: K80+I+G.The Bayesian analysis was conducted using MrBayes ver.3.2.7athrough the CIPRES portal.To ensure convergence, four parallel runs were executed, each comprising one cold and three heated chains, over 10 million generations with sampling every 100 generations for single gene trees and 50 million generations with sampling every 1000.Parameter convergence (>200) was confirmed using Tracer v. 1.7 [28].Phylogenetic clades were considered strongly supported if the bootstrap support value (BS) was ≥70% and/or the posterior probability (PP) was ≥0.95.

ITS Genetic Distances Calculation
After opening the alignment matrix, BioEdit software was used to check the number of different bases between the two specimens.The different numbers of bases were used to compare the length of the matrix bases to obtain ITS genetic distances.

Phylogenetic Analyses
Based on the BLAST results of the length of the ITS region, two new species were found to share less than 98.5% similarity with the known species of Lyophyllum, respectively: 98.4% with L. sykosporum and 96.99% with L. cf.rhopalopodium.
Eight newly generated sequences and fifty-one sequences from GenBank were used as the ingroup.Three sequences of Calocybe gambosa, C. carnea, and C. persicolor, retrieved from GenBank, were used as the outgroup (Table 1).
Table 1.Lyophyllum taxa included in this study are listed by taxon, specimen number country, and GenBank accession number for DNA sequences used in the phylogenetic analyses.Newly generated sequences are listed in bold and a "*" after the specimen number indicates that the specimen is the holotype of that species.ML and BI analyses generated nearly identical tree topologies with minimal variations in statistical support values.Thus, only the ML tree is displayed (Figure 1).Phylogenetic data and thorough morphological analysis (see below) showed that the two newly described taxa in this study are not conspecific with other known Lyophyllum species.

Taxon
ML and BI analyses generated nearly identical tree topologies with minimal variations in statistical support values.Thus, only the ML tree is displayed (Figure 1).Phylogenetic data and thorough morphological analysis (see below) showed that the two newly described taxa in this study are not conspecific with other known Lyophyllum species.Etymology: bulborhizum refers to the unusually bulbous stipe base.Pileus 3.0-5.0cm diameter, fleshy, fragile, hemispherical, becoming convex with age, abundant black floccus on the surface, dry, dark grey (1F1) at the center, grey (1D1) at the margin, slightly depressed of center, involute of margin; pileus context thick, 0.3-0.5 cm wide, white (1A1).Lamellae moderately close together, triangular, subdecurrent to decurrent, ventricose to broadly ventricose, adnate to narrowly adnate, broad, white (1A1), dark grey when bruised, 3-4 tiers, 0.5-0.6 cm wide, edge even or entire.Stipe 1.0-3.1 × 1.0-1.9cm, cylindrical to clavate, dark grey (1F1) points and lines on the surface, bulbous at the base, smooth, unchanging in colour when injured.Odor and taste not distinctive.
Ecology and distribution: Tufted, growth in forests of Magnolia alba.Summer to Autumn (August to October).Known only from Yunnan Province, China.
Notes: Lyophyllum nigrum is distinguished from other Lyophyllum species by its dark grey pileus surface; lamellae soon changing to grey dark when bruised, greyish green to olive and subclavate stipe; very broadly fusiform to broadly fusiform basidiospores 5.4-8.2 × 4.1-7.0µm; narrowly cylindrical or narrowly clavate cheilocystidia and pleurocystidia.
According to phylogenetic analyses, L. nigrum clusters with L. sykosporum (IFO 30978).However, the original description of L. sykosporum from Japan has whitish pruinose on the stipe apex [33], and the ITS genetic distance between the holotype L. nigrum L5091 and L. sykosporum IFO 30978 is 2.08% (12/577); thus, L. nigrum is introduced as a distinct new species.
Ecology and distribution: Mostly solitary, rarely scattered, growth in forests of Morella rubra.Summer to Autumn (August to October).Known only from Yunnan Province, China.
Ecology and distribution: Mostly solitary, rarely scattered, growth in forests of Morella rubra.Summer to Autumn (August to October).Known only from Yunnan Province, China.Notes: Lyophyllum rhombisporum was originally described from Yunnan, China, by Wang et al. [32].In the original description, specimen L1763 had larger basidiospores (14.5-17 × 10-11.5 µm) and basidia (36.5-46 × 10-11.3µm), but cheilocystidia and pleurocystidia were absent.It was surprising and puzzling due to the difficulty of finding such large basidiospores in the Lyophyllaceae.We rechecked the L. rhombisporum specimen (holotype, L1763) and compared it with our L. rhombisporum specimens L5010 and L5084; we found that the ITS sequences were 100% similar, thus, they belong to the same species.
We report L. rhombisporum based on two new collections (YAAS L5010 and YAAS L5084).Comparing our collections with the L. rhombisporum type specimen (holotype L1763) showed that basidiospores' size varies significantly; thus, we assume that either the microscope calibrations were wrong, or the magnification was measured incorrectly in the original description.
In our study, we present morphological characteristics and phylogenetic data for two new Lyophyllum species and one known Lyophyllum species.Our findings, in line with previous studies, highlight the challenge of distinguishing Lyophyllum species based solely on morphology due to their similarities.Consequently, employing molecular methods becomes essential for accurately delineating species boundaries in Lyophyllum.
Collecting a diverse range of Lyophyllum species from various geographic locations, acquiring additional sequencing data, and thoroughly documenting their morphology are essential to enhance our understanding of their taxonomy and phylogeny.

Figure 1 .
Figure 1.Phylogram generated from maximum likelihood (RAxML) analysis based on a partial ITS1-5.8S-ITS2sequence alignment of Lyophyllum.Calocybe persicolor, C. carnea, and C. gambosa were used as the outgroup.ML bootstrap support values/Bayesian posterior probability values equal to or greater than 70%/0.95are indicated on the nodes.Names of the species in red represent new species, and the holotype of each species is in bold.

Figure 1 .
Figure 1.Phylogram generated from maximum likelihood (RAxML) analysis based on a partial ITS1-5.8S-ITS2sequence alignment of Lyophyllum.Calocybe persicolor, C. carnea, and C. gambosa were used as the outgroup.ML bootstrap support values/Bayesian posterior probability values equal to or greater than 70%/0.95are indicated on the nodes.Names of the species in red represent new species, and the holotype of each species is in bold.
rhombisporum Shu H. Li & Y.C. Zhao from subtropical Yunnan Province, L. atrofuscum S.W. Wei, Q. Wang & Yu Li, and L. subalpinarum S.W. Wei, Q. Wang & Yu Li from Xizang Province [10].Within the clade, in all the species of Lyophyllum, lamellae change to dark grey when bruised, basidiospores are variable, and in most of the species, basidiospores are very broadly fusiform to fusiform, and rarely globose to subglobose (L.moncalvoanum J.A. Cooper & P. Leonard).