Additions to Lyophyllaceae s.l. from China

Four new species, viz. Calocybe coacta, C. fulvipes, C. vinacea and Clitolyophyllum umbilicatum, are described in northern China. Comparisons are made of macro- and micromorphological features among the new species and closely related species within the genus. The new species feature unique morphological characteristics that separate them from the previously described species. Calocybe coacta is characterized by medium- to large-sized basidiocarps, greyish cream, felty pileus and non-cellular epicutis. The key characteristics of C. fulvipes are rose-brown to greyish-brown pileus, stone-brown stipe and non-cellular epicutis. The unique morphological characteristics of C. vinacea that distinguish it from its closely related species are pastel red to dull-red pileus and stipe surface with densely white pruina. The main characteristics of Clitolyophyllum umbilicatum are deeply depressed dark orange to light-brown pileus, central stipe and subglobose-ellipsoid spores. Phylogenetic analyses based on the ITS and 28S regions indicated that the four new species are distinct and monophyletic. Full descriptions, color images, illustrations and a phylogenetic tree that show the placement of the four new species are provided. A key to the Calocybe species reported from China is also given.


Introduction
Lyophyllaceae Jülich was suggested by recent molecular phylogenetic analyses to be a putative wider concept that includes several lineages that seem more distantly related, and the generic concepts should be reconsidered [1][2][3][4][5]. Several new genera established in the past ten years were expected to reorganize the system, viz.  [2,[6][7][8][9][10]; however, a few of them show a poor species diversity worldwide. Clitolyophyllum was discovered in 2016, a Turkish species fruiting on the dead bark of Picea orientalis. It is mainly characterized by fan-shaped, translucent-striate pileus; decurrent lamellae; lateral, cylindrical to flattened stipe; smooth, inamyloid spores; nonsiderophilous basidia and irregular pileipellis [7]. Until now, Clitolyophyllum has remained monotypic, and so far, it is known to have only one species in Turkey.
Calocybe Kühner ex Donk, typified by C. gambosa (Fr.) Donk, was a traditional genus of Lyophyllaceae first discovered by Kühner [11]. Singer's concept of Calocybe included characteristics such as mostly bright-colored pileus, vacuolar pigment, small spores, pileipellis of a cutis or cellular [12]. The taxonomic status of Calocybe used to be confused with 2 of 13 Lyophyllum P. Karst. and Rugosomyces Raithelh. [13,14]. Several recent molecular phylogenetic analyses have confirmed Calocybe as an independent genus with the merging of Rugosomyces [2,3,[15][16][17]. At least 57 species can be assigned to Calocybe according to Xu et al. [17]; however, the molecular phylogenetic relationships among members of the Calocybe were inconsistent with the morphological classification made by Singer [12]. Therefore, the infrageneric classification of Calocybe remains in a redelineated position.
During the survey of macrofungi in northern China, four unique species of the family Lyophyllaceae were newly discovered in the forests dominated by Acer sp., Quercus mongolica or Picea crassifolia. The aim of this study was to describe four new lyophylloid species: Calocybe fulvipes, C. coacta, C. vinacea and Clitolyophyllum umbilicatum based on morphological features and molecular data. In addition, we explored the species diversity of Calocybe based on the previous studies and compiled the information on the Calocybe species reported in China.

Specimen Sampling
All the specimens were collected in 2012-2021 from northern China. The collected samples were dried overnight using an electric drying oven at 45 • C and deposited in the Herbarium Mycology of Jilin Agricultural Science and Technology University (HMJU).

Morphological Observation
The macromorphological descriptions were recorded in the field, and images of the basidiocarps were taken in the field with a Canon 80D camera. The Kornerup and Wanscher color description was used to describe color terms, where a specific code is assigned to an observed color [28]. Micromorphology of the specimens was studied with the help of an Olympus BX 53 (Tokyo, Japan) light microscope at 40×, 100×, 400×, 600× and 1000× magnifications (measurements were performed under oil immersion at 1000× magnification). Sections of the dried specimens were mounted in 3% KOH, 1% Congo red and Melzer's reagent for observations. Cotton blue and iron acetocarmine solutions were used to highlight the siderophilous granulation in the basidia. Factor Q is the value of its length divided by width and Qm is the mean of the Q values. The procedure for scanning electron microscopy (SEM) followed that of Xu et al. [29], and an FEI Quanta 200FE-SEM (JEOL Ltd., Akishima, Japan) was used at an accelerating voltage of 5-10 kV.

DNA Extraction, PCR, Sequencing and Phylogenetic Analyses
Total genomic DNA was extracted using an M5 Fungal Genomic DNA Kit (Mei5 Biotechnology Co., Ltd., Beijing, China) and an Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturers' instructions. For the polymerase chain reaction (PCR) amplification, primers ITS1 and ITS4 [30] were used for the ITS region while primer LR0R was paired with LR6 and LR7 in order to obtain sequences for the 28S region [31]. The reactions were performed with the following program: initial denaturation at 94 • C for 5 min (ITS) or 4 min (28S), 33 cycles at 94 • C for 30 s, 46 • C (28S) or 53 • C (ITS) for 30 s or 45 s (28S), and 72 • C for 30 s (ITS) or 40 s (28S), and for terminal elongation, the reaction batches were incubated at 72 • C for 10 min. The PCR products were examined on a 1% agarose gel detected by a JY 600 electrophoresis apparatus (Beijing JUNYI Electrophoresis Co., Ltd., Beijing, China) and then sent to BGI Co., Ltd. (Beijing, China) for sequencing.

Phylogenetic Analyses
The newly generated sequences were compared with the representative ITS and 28S sequences retrieved from GenBank. Species of other genera in Lyophyllaceae were also included according to former phylogenetic studies [2,3,7,10,32], while Entoloma sericeonitidum and Entoloma sericeum were included as the outgroup. Alignments were generated for the ITS and 28S datasets using MAFFT 7.0 [33] under default conditions and then edited with MEGA 7.0 [34]. The selection of the best-fitting model was completed by ModelFinder [35] based on the Bayesian information criterion (BIC) to provide a substitution model. The GTR + F + I + G4 model was chosen for this purpose. Maximum likelihood (ML) and Bayesian inference (BI) analyses were used to infer the phylogenetic position of the new species. ML estimation was performed through IQ-TREE [36] with 1000 bootstrap replicates [37]. BI phylogeny using Markov chain Monte Carlo (MCMC) methods was carried out with MrBayes 3.2.2 [38]. Two parallel runs were conducted with one cold and four heated chains each for 1,000,000 generations, starting with a random tree. Trees were saved every 1000 generations resulting in broad sampling of 10,001 trees. The initial burn-in was set to 25% (2500 trees). A 50% majority-rule consensus cladogram was computed from the remaining trees to obtain estimates for Bayesian posterior probabilities. The significance threshold was set to >0.95 for Bayesian posterior probability (PP) and >70% for ML bootstrap proportions (BP). All the sequences used in this study are listed in Table 1.
Basidiospores  Note: The key characteristics of C. fulvipes are its rose-brown to greyish-brown pileus, stone-brown stipe and non-cellular epicutis, which suggest that it should be in sect. Carneoviolaceae [12]. In that section, C. carnea resembles C. fulvipes due to the light carneous pileus and similar spore size. They can be differentiated by the color of stipe and the shape of the pileus. Calocybe carnea produces a convex pileus and a white stipe, while C. fulvipes has a plane pileus and a brown stipe [41].
Basidiospores Notes: The pastel red to dull-red pileus, smooth spores and pileipellis of the cutis type suggests that C. vinacea should be in sect. Carneoviolaceae [12]. The unique pileus color, deeply plurilobed to subpetaloid margin in age, longitudinally striate stipe with densely white pruina overall and clavate or subcapitate terminal elements in the pileipellis apparently separate it from other species within the section. C. rubra Rick ex Redhead & Singer and C. bipigmentata Singer which used to be placed in sect. Pseudoflammulae by Singer resemble C. vinacea in the pileus color; however, C. rubra differs in a carnea-red and narrow stipe of only 3 mm wide [42,43]. C. bipigmentata is mainly different from C. vinacea in having ochraceous lamellae, a glabrous, ochraceous stipe and narrow-fusoid cheilocystidia, which lacking in C. vinacea [44]. decurrent lamellae, stipe with densely mycelium-like appendages, oblong spores and clavate or subcapitate terminal elements in the pileipellis.
Basidiospores  Note: Clitolyophyllum akcaabatense shares a few characteristics with C. umbilicatum such as the similarly sized pileus, decurrent lamellae, hollow stipe, similar smooth, inamyloid basidiospores, non-siderophilous basidia, absence of hymenial cystidia and the pileocystidia-like terminal elements in the pileipellis. However, C. akcaabatense is distinguished from C. umbilicatum in having a fan-shaped pileus with a small depression where it is connected to the stipe, a lateral stipe that tapers towards the base, with lower two thirds covered with a typical white to creamy, woolly mycelium [7]. Microscopically, C. akcaabatense is mainly different from C. umbilicatum in having ellipsoid-fusoid, sublacrymoid spores with Q = 1.58-1.78 [spores subglobose-ellipsoid, Q = (1.14) 1.20-1.54 (1.62) in C. umbilicatum] and irregular pileipellis [7].

Discussion
In the combined ITS and LSU phylogenetic analysis (Figure 1), Lyophyllaceae s.l. appear to be polyphyletic, which is consistent with previous studies [2,3,5,10]. The new species described in this study occupy independent positions. Clitolyophyllum umbilicatum formed a distinct clade with Clitolyophyllum akcaabatense, but morphologically, C. akcaabatense can be distinguished from C. umbilicatum by a fan-shaped pileus with a small depression, a lateral stipe that tapers to the base and ellipsoid-fusoid, sublacrymoid spores. Furthermore, Clitolyophyllum umbilicatum grows on the moss under forests dominated by Picea crassifolia, while C. akcaabatense grows on the dead bark of Picea orientalis together with mosses.
Phylogenetically, Calocybe fulvipes clusters in a single clade sister to a clade of C. decurrens; however, they can be easily distinguished from one another by morphology. Calocybe fulvipes has a glabrous pileus, adnexed to slightly emarginate lamellae and a stone-brown stipe which does not change with age while C. decurrens has a carneous, slightly pruinose pileus, decurrent lamellae and a pastel stipe that turns brownish-red or greyish-brown when mature [17]. In addition, C. fulvipes grows on soil under mixed forests dominated by Acer sp. and Quercus mongolica, while C. decurrens grows on soil under mixed forests dominated by Salix sp. Calocybe vinacea is a group in clade II and is related to C. carnea and C. persicolor. The combination of the pastel red to dull-red pileus with densely spaced white pruina and white stipe with the same surface are unique to C. vinacea, and are unknown in other Calocybe species. Calocybe coacta is related to C. gangraenosa (Lyophyllum leucophaeatum), while C. coacta is distinct from C. gangraenosa in having a greyish-cream felty pileus and smooth spores, while C. gangraenosa produces a whitish to brownish-grey (when matured) pileus and spores ornamented with warts [27,43].
Calocybe buxea (Maire) Raithelh. and C. hypoxantha (Joss. & Riousset) Bon. were placed in clade I in single-gene analysis based on ITS and 28S by Li et al. but were not present in the combined analysis [15]. In the present study, they were located in clade II. Hence, the location of the two species may require more material, mainly, both taxa sampled and other gene fragments, to determine.
In our research on the species of Calocybe in China, we failed to get the type specimens and corresponding morphological description of C. hebelomoides, and could not confirm the existence of this species in China. The taxonomic treatments of C. hebelomoides from China should be performed on the basis of additional detailed morphological investigations in later studies. A key for the other 16 Calocybe species reported from China is provided below: