A Taxonomic Study of Candolleomyces Specimens from China Revealed Seven New Species

Based on phylogenetic analysis, Candolleomyces (Psathyrellaceae, Agaricales) was established with Psathyrella candolleana as the type species. The basidiomes range from small to large and are typically terrestrial, lignicolous, and rarely fimicolous. We analysed the Candolleomyces species collected during five years in China, and based on morphological and molecular data (nrITS, nrLSU, and tef-1α), we propose seven new Candolleomyces species viz. C. brevisporus, C. gyirongicus, C. lignicola, C. luridus, C. shennongdingicus, C. shennongjianus, and C. sichuanicus. Full descriptions, colour photographs, illustrations, phylogenetic analyses results, and comparisons with related Candolleomyces species of the new taxa are provided. This study enriches the species diversity of Candolleomyces in China.

In the present study, we collected 31 specimens from Beijing City, Guizhou, Hubei, Sichuan, and Yunnan provinces, and the Xizang Autonomous Region in China, and conducted comprehensive phylogenetic analyses using the nrITS, nrLSU, and tef-1α gene regions.Based on the phylogenetic and morphological analyses, seven new species from China are proposed.

Morphological Characteristic Examination
Fresh specimens were collected and photographed (Canon EOS 80D, Tokyo, Japan) in the field from Beijing City, Yunnan, Sichuan, Guizhou, and Hubei provinces, and the Xizang Autonomous Region in China from 2019 to 2023.To avoid mixing or crushing, specimens were packed separately in aluminium foils.Macroscopic characteristics were recorded when fresh, including features of the pileus, veil, context, lamellae, stipe, odour, and chemical reactions.The samples were completely dried with a food drier at 50 • C, sealed in plastic bags, and deposited in the Mycological Herbarium, Institute of Microbiology, Chinese Academy of Sciences (HMAS).
Microscopic characteristics, such as basidia, basidiospores, pileipellis, and cheilocystidia, were observed under an Olympus CX31 light microscope (Olympus, Tokyo, Japan), and at least 30 measurements were made for each character.The description of morphological characteristics followed the protocols of Largent [27].Additionally, 5% KOH and sterilised water were used for microscopic characterisation.Measured values are given as (a)b-c(d), in which a is the lowest value, b-c includes at least 90% of the values, and d is the highest value.The Q value is the ratio of the length and width of a spore [11].The colour designation refers to the Methuen Handbook of Colour.

DNA Extraction, PCR and Sequencing
DNA was extracted from dried specimens using a Broad-spectrum Plant Rapid Genomic DNA Kit (Biomed, Shiyan, China).Primers ITS1 and ITS4 were used for the nuclear internal transcribed spacer (nrITS) of the rDNA region [28], LR7/LR0R were used to amplify the large subunit nuclear ribosomal DNA (nrLSU) region [29], and EF983F/EF2218R were used to amplify the translation elongation factor subunit 1 alpha (tef-1α) region [30].PCR was performed in 25 µL reactions consisting of 2 µL genomic DNA, 1 µL of each forward and reverse primers, 9 µL ddH 2 O, and 12 µL 2 × Es Taq MasterMix (Beijing Cowin Biotech Co., Ltd., Beijing, China).The PCR programmes follow Zhao et al. [31] and Bau and Yan [11].The PCR products were detected by electrophoresis and sent to BGI Genomics Co., Ltd., Shenzhen, China, for purification and sequencing.

Phylogenetic Analyses
Considering the results of BLAST searches against GenBank and previous studies, we analysed the nrITS, nrLSU and tef-1α sequences of 94 taxa.The details are presented in Table 1.The sequences were aligned by Muscle version 3.6 separately [32], then manually adjusted in BioEdit version 7.0.4 to remove the ambiguous areas [33], and assembled in PhyloSuite version 1.2.3 [34].The final alignments were deposited in TreeBASE (study no.31401).Maximum likelihood (ML) analysis of concatenated sequences was carried out using raxmlGUI 1.3 with a GTRGAMMA model and one thousand rapid bootstrap (BS) replicates [35].The best partitioning scheme and evolutionary models for three pre-defined partitions were selected using PartitionFinder2 v2.1.1 [36], with greedy algorithm and AICc criterion: GTR+I+G for nrLSU, GTR+G for nrITS, and GTR+I+G for tef-1α.Bayesian Inference (BI) analysis was performed using MrBayes v3.2.7a [37].Six Markov chains were run for two million generations, and trees were sampled every 100th generation.Burn-ins were determined in Tracer version 1.6 with an ESS value higher than 200, and the remaining trees were used to calculate Bayesian posterior probabilities (PP).The trees were displayed in FigTree version 1.4.0 [38].The sequences generated in this study are marked in bold.

Phylogeny
Eighty-nine specimens from 43 Candolleomyces species were included in the phylogenetic analyses with Hausknechtia floriformis (Hauskn.)D. Wächt.& A. Melzer and H. leucosticta (Pat.)Tkalčec, J.Q. Yan, C.F. Nie & C.K. Pradeep as outgroups.In total, 84 new sequences were generated in this study, which were from 28 specimens from China, all with the nrITS, nrLSU, and tef-1α sequences.The combined dataset with 3033 characters including gaps (679 for nrITS, 1322 for nrLSU, and 1032 for tef-1α) was included in the phylogenetic analyses.The phylogenetic tree of ML and MrBayes were almost identical.The ML tree is shown in Figure 1 with bootstrap values and Bayesian posterior probabilities indicated on the branches.Etymology: 'brevisporus' (Latin) referring to the shorter spores, a distinguishing characteristic of the species.
Habit and habitat: solitary, in pairs, or scattered on the ground with rich humus in broad-leaved forests or broad-leaved shrubs.So far only found in China.
Habit and habitat: solitary, in pairs, or scattered on rotten wood in broad-leaved forests or broad-leaved shrubs.
Etymology: 'luridus' (Latin) refers to the yellowish brown colours of the pileus.
Pileus is 23-63 mm diam, paraboloid when young, obtusely conical, convex, or planoconvex when mature, with or without obtuse umbo, and sometimes cleft or lobed; surface is glabrous, dull, hygrophanous, oak brown (5D6) to bronze (5E5), darker in the centre, and striate up to halfway from the margin or indistinct.Veil is white (5A1), dispersed, fibrillose, and falls off easily.Context is 0.5-1.0mm broad at the centre, the same colour as pileus.Lamellae is moderately close, adnate to slightly adnexed, grey (5C1), brownish orange (5C3) to hair brown (5E4), and the edge becomes white as basidiospores mature.Notes: In the field, Candolleomyces shennongdingicus can be easily confused with C. shennongjianus at first glance, as both species have parabolic pileus when young and convex when mature, and yellowish brown pileus.Additionally, both species exhibit white stipes with pale yellowish brown bases.However, C. shennongdingicus can be distinguished by its slightly smaller basidiospores, longer basidia, as well as fusiform, seldom cylindrical to clavate, and smaller cheilocystidia.Moreover, phylogenetic analysis reveals that C. shennongdingicus is distinct from C. shennongjianus (Figure 3).Based on morphology and phylogenetic analyses, Candolleomyces shennongdingicus is introduced as a new species (Figures 1 and 7).
Etymology: shennongjianus refers to the location Shennongjia National Park, where the type specimen was collected.
Etymology: refers to Sichuan Province, the locality of the type specimen.
Habit and habitat: Scattered or clustered on the ground with rich humus in broadleaved or deciduous coniferous forests.So far only found in China in July/August.and C. subsingeri in China based on morphological characteristics and ITS sequences [21].Subsequently, in 2021, Bau and Yan supplemented these specimens with LSU, tef-1α, and β-Tub sequences [11].Additionally, they identified the sample with the voucher of HFJAU1515 as C. sulcatotuberculosus and provided its ITS, tef-1α, and β-Tub sequences but did not describe its morphology [11].Furthermore, Psathyrella typhae var.bispora was reported as a new variety in China in 2018 [21], and it became synonymised with Candolleomyces typhae [21].However, only its SSU sequence is available in NCBI, lacking the complete sequence data needed to verify its presence in China through phylogenetic analysis.On the other hand, C. albipes has only morphological descriptions in China, with no associated molecular data, leaving its actual presence in the region uncertain and requiring further investigation [50].Furthermore, although C. singeri was previously reported in Hubei, this paper marks the first record of the species in the Shennongjia National Park.In the recorded 43 Candolleomyces species, 36 species have nrITS sequences, 27 species have nrLSU sequences, and 16 species have tef-1α sequences.However, only 14 species have nrITS, nrLSU, and tef-1α sequences.All five new species revealed in this study provided sequences of nrITS, nrLSU, and tef-1α.
Significant progress for Candolleomyces was made in the study, but there are still some challenges.Existing classifications primarily rely on a combination of morphological characters and molecular data [18,20].However, due to the variability of morphological traits and the limited availability of gene sequence data, the identification and classification of certain species remain problematic [9].In recent years, the development of molecular biology techniques, such as tef-1α and β-tub gene sequence analyses, greatly facilitated systematic taxonomic studies of the genus [11,12,19].The ongoing discovery of well-defined boundaries in new taxa, as demonstrated by this study, enhances our understanding of species within this genus.In addition, the edible and medicinal values, as well as the toxicity, of only a few species, such as C. candolleanus, C. tuberculatus, and C. yanshanensis, were clarified, while those of most other species remain unknown.Meanwhile, the ecological functions and distribution ranges of the majority of species are still ambiguous.The diversity of the genus Candolleomyces continues to increase with the discovery of new species, necessitating more comprehensive field investigations, as well as morphological and molecular studies to refine the taxonomic system [12].Future research should focus on integrating morphological, molecular, and ecological methods to further elucidate the phylogenetic relationships and species diversity within the genus.

Figure 1 .
Figure 1.Molecular phylogenetic analyses of Candolleomyces species by the maximum likelihood (ML) method based on combined nrITS, nrLSU, and tef-1α sequences.Maximum likelihood bootstrap support values (ML) ≥ 60% and Bayesian posterior probabilities (PP) ≥ 0.95 are shown at the nodes as ML/PP.Candolleomyces species produced in this study are indicated in bold.

Figure 1 .
Figure 1.Molecular phylogenetic analyses of Candolleomyces species by the maximum likelihood (ML) method based on combined nrITS, nrLSU, and tef-1α sequences.Maximum likelihood bootstrap support values (ML) ≥ 60% and Bayesian posterior probabilities (PP) ≥ 0.95 are shown at the nodes as ML/PP.Candolleomyces species produced in this study are indicated in bold.

Figure 3 .
Figure 3.Nucleotide differences of Candolleomyces brevisporus, C. shennongdingicus, C. gyirongicus, C. lignicola, and C. shennongjianus across ITS, LSU, and tef-1α.The numbers at the top indicate the positions of the polymorphic sites in each fragment.The dashes indicate the lack of data for the respective positions.

Table 1 .
Sequences used in the phylogenetic analysis in this study.Missing sequences are indicated by "-".