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Research Article
Three new species and a new record of Conocybe section Pilosellae (Bolbitiaceae, Agaricales) from Jilin Province, China
expand article infoHan-bing Song‡, Tolgor Bau‡
‡ Jilin Agricultural University, Changchun, China

Corresponding author: Tolgor Bau ( junwusuo@126.com )

Academic editor: Yupeng Ge
© 2025 Han-bing Song, Tolgor Bau.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Song H-bing, Bau T (2025) Three new species and a new record of Conocybe section Pilosellae (Bolbitiaceae, Agaricales) from Jilin Province, China. MycoKeys 114: 67-94. https://doi.org/10.3897/mycokeys.114.140056
Open Access

Abstract

This study is based on the phylogenetic framework of Conocybe section Pilosellae and incorporates materials from Jilin Province. A systematic phylogenetic tree was constructed using maximum likelihood and Bayesian analyses of internal transcribed spacer region (ITS) and nuclear large subunit ribosomal DNA (nrLSU), and translation elongation factor 1-alpha (tef1-α) sequences. As a result, three new species were discovered in Jilin Province: Conocybe verna, which emerges in broad-leaved forests during spring; C. angulispora, characterized by angular and submitriform or slightly hexagonal basidiospores; and C. rubrocyanea, with basidiomata displaying a reddish hue when fresh and a bluish hue when dry. Additionally, a new record for China, C. hexagonospora was identified, characterized by the lack of distinct pubescence on the pileus and slightly hexagonal basidiospores, increasing the total number of species within sect. Pilosellae to 22. Key for sect. Pilosellae is provided, accompanied by morphological descriptions and line drawings for the new species and a new record for China.

Key words:

Conocybe section Pilosellae, morphology, new taxa, phylogeny

Introduction

Conocybe Fayod belongs to the family Bolbitiaceae Singer and was established by Fayod 1889. Its taxonomic status has undergone multiple revisions and clarifications (Fries 1821; Fayod 1889; Kühner 1935; Singer 1949; Watling 1982). Conocybe sect. Pilosellae Singer, a basal group of Conocybe, was established by Singer in 1962 based on stipes with hairs and non-lecythiform caulocystidia (Singer 1962). The classification history has been summarized by Song and Bau (2023), and we won’t go into further detail here. Please refer to the following references for more information (Kühner 1935; Singer 1949; Singer 1962; Watling 1982; Bon 1992; Arnolds 2005; Hausknecht 2005; Hausknecht and Krisai-Greilhuber 2006). Hausknecht and Krisai-Greilhuber (2006) divided sect. Pilosellae into two subsections, based on the size and shape of basidiospores, the presence of lecythiform caulocystidia, and habitat. The two subsections are subsect. Pilosellae and subsect. Siligineae Hauskn. & Krisai. Subsect. Pilosellae includes seven series: ser. Pilosella, ser. Sienophylla Hauskn. & Krisai, ser. Anthracophila Hauskn. & Krisai, ser. Bispora Hauskn. & Krisai, ser. Microrrhiza Hauskn. & Krisai, ser. Inocybeoides Hauskn. & Krisai, and ser. Cylindracea Hauskn. & Krisai. Subsection Siligineae Hauskn. & Krisai includes four series: ser. Siliginea Hauskn. & Krisai, ser. Fimetaria Hauskn. & Krisai, ser. Murinacea Hauskn. & Krisai, and ser. Lenticulospora Hauskn. & Krisai (Hausknecht and Krisai-Greilhuber 2006; Hausknecht 2009). Tóth et al. (2013) included members of sect. Pilosellae in a molecular phylogenetic analysis of Bolbitiaceae based on combined dataset of ITS, nrLSU, and tef1-α sequences, providing important reference data for subsequent phylogenetic studies of Bolbitiaceae. Species of sect. Pilosellae have a wide distribution and are mostly found in fertile soil and herbivore dung (Hausknecht 2009). They contain toxic substances such as psilocybin, phallotoxins, and amatoxins, and have certain pharmacological value (Griffiths et al. 2016; Johnson et al. 2017; Paul 2021).

In China, research on Conocybe began with Tai (1979). As of 2023, a total of 40 species of Conocybe have been recorded in China, including 17 species in sect. Pilosellae (Tai 1979; Xie et al. 1986; Li et al. 1993; Bi et al. 1994; Yuan and Sun 1995; Zhang and Mao 1995; Li and Bau 2003; Li and Azbukina 2011; Bau et al. 2014; Li et al. 2015; Wang and Tzean 2015; Bau 2016; Liu and Bau 2018; Liu 2018; Zhang 2019; Ye 2021; Song et al. 2023; Song and Bau 2023). However, it is important to note that the majority of these species are distributed in Northeast China.

Jilin Province is characterized by a temperate monsoon climate with distinct seasons. The summers are rainy and warm, while the winters are dry and cold. The region’s main geographical features include mountains and plains. Forested areas are mainly concentrated in the Changbai Mountains, where diverse vegetation types, such as mixed coniferous and broadleaf forests, dominate. The region’s excellent natural environment and abundant vegetation provide favorable conditions for fungal diversity. However, the number of reported species of sect. Pilosellae in Jilin province is significantly lower compared to Europe and North America at the same latitude. This indicates the need for further investigation and research. Based on results from this study, the number of species of sect. Pilosellae species in China is increased to 22 (include C. siliginea, collected from Henan Province).

Materials and methods

Samplings and morphological analyses

The specimens for this study were collected from Jilin Province, China, from 2022 to 2023. Upon discovery, photographs were taken, and information on habitat and morphological features was recorded. Subsequently, the materials were dried using silica gel desiccants, prepared as specimens, and stored at the Fungarium of Jilin Agricultural University (FJAU). To examine the microscopic structures of the specimens, they were treated with a 5% KOH solution and a 1% Congo red solution. Reacting with lamellar structures using a 25% ammonia solution (Hausknecht 2009). The observations were made using a Carl Zeiss Primo Star optical microscope from Jena, Germany. Additionally, the color of fresh or dried basidiomata was described using the color-coding system developed by the German Institute for Quality Assurance and Certification (Reichs-Ausschuss fur Lieferbedingungen und Guetesicherung, available at https://www.ral-guetezeichen.de/), the abbreviation used in the text (RAL).

In this study, the basidiospore measurements do not include the apiculus. They are presented as ‘(a)b–c(d)’, where ‘b–c’ represents the minimum at least 90% of the measured values, and ‘a’ and ‘d’ represent the extreme values. To accurately record their dimensions, the main body (excluding sterigmata or excrescences) of the basidia, cheilocystidia, caulocystidia, and pileipellis were measured if present. At least 20 were measured. The notation (n/m/p) indicates that the measurements were made on “n” randomly selected basidiospores from “m” basidiomata of “p” collections. Twenty basidiospores are measured from each basidioma. This sampling method ensures a representative measurement sample. The ratio of length divided by width, known as Q, provides a measure of the elongation of the spores. The average quotient (length/width and breadth), denoted as Qm, is calculated along with the standard deviation to provide an overall average value with variation.

DNA extraction, PCR amplification, and sequencing

To extract total genomic DNA from the dried specimens, we followed the manufacturer’s instructions and used a NuClean Plant Genomic DNA kit (ComWin Biotech, CW0531M, Taizhou, China). For amplifications, we employed the primer pairs ITS1F/ITS4 (White et al. 1990; Gardes and Bruns 1993), LR0R/LR7 (Vilgalys and Hester 1990; Moncalvo et al. 2000), and EF1-983F/EF1-2218R (Rehner and Buckley 2005) for the ITS, nrLSU, and tef1-α sequences, respectively. Polymerase chain reaction (PCR) amplification was conducted on a Bio-Rad T100TM Thermal cycler (Bio-RAD Inc., Hercules, CA, USA). In a 30 µL reaction mixture, we used the following final concentrations or total amounts: 2 µL of template DNA, 15 µL of 2× SanTaq PCR Master Mix (B532061, Sangon Biotech, Shanghai, China), 1.5 µL of each primer, and 10 µL of double-distilled water (ddH2O).

The PCR protocol for ITS and nrLSU involved the following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C (ITS, nrLSU) for 30 s, and extension at 72 °C for 45 s (ITS)/80 s (nrLSU). The final extension was performed at 72 °C for 10 min, followed by cooling at 4 °C indefinitely. For tef1-α, the touchdown PCR protocol was as follows: initial denaturation at 94 °C for 3 min, followed by 8 cycles of denaturation at 94 °C for 40 s, annealing at 60 °C for 40 s (with the temperature decreasing by 1 °C per cycle), and extension at 72 °C for 2 min. After the initial cycles, the denaturation step was repeated at 94 °C for 45 s, followed by annealing at 60 °C for 40 s and extension at 72 °C for 2 min, repeated for a total of 36 cycles. Finally, a final extension step was performed at 72 °C for 10 min and cooling at 15 °C indefinitely.

Following the PCR amplification, the products were electrophoresed on a 1% agarose gel along with known standard DNA markers. The resulting PCR products were sent for sequencing services to Sangon Biotech (Shanghai) Co., Ltd., and sequence data was obtained. To ensure the quality of the chromatograms, they were checked in BioEdit v7.2.5 (Hall 1999), ensuring that each base was of good quality. Additionally, a BLAST search was conducted using the National Center of Biotechnology Information (NCBI) database to confirm that the sequencing results matched the specimens. Finally, the sequences were submitted to GenBank.

Phylogenetic analyses

Sequences were downloaded from GenBank (Table 1). The ITS, nrLSU, and tef1-α sequences were aligned using the G-INS-i algorithm with two iterative cycles only via the online Mafft tool (Katoh et al. 2019; https://mafft.cbrc.jp/alignment/server/). The resulting alignment was then manually refined and trimmed using MEGA7 (Kumar et al. 2016). To generate the concatenated alignment, PhyloSuite 1.2.2 (Zhang et al. 2020) was employed. The best-fit partition model (edge-unlinked) was selected using the BIC criterion with ModelFinder v2.2.0 (Kalyaanamoorthy et al. 2017). For maximum likelihood phylogenies, IQ-TREE was used under the Edge-linked partition model for 1000 standard bootstraps, along with the Shimodaira-Hasegawa-like approximate likelihood-ratio test, with settings based on the results from ModelFinder (Nguyen et al. 2015; Guindon et al. 2010). Bayesian inference phylogenies were inferred using MrBayes 3.2.7a under the partition model (Ronquist et al. 2012) through two parallel runs (MCMC) and 4,500,000 generations, discarding the initial 25% of the sampled data as burn-in, average standard deviation of split frequencies is 0.009. Finally, the figures were edited using iTOL (Letunic and Bork 2019), Adobe Photoshop 2021, and Adobe Illustrator 2021. The outgroup used was the Psathyrella species (Song and Bau 2023).

Table 1.
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Information on the DNA sequences used to reconstruct phylogenetic trees. Sequences in bold were newly generated in this study. T = holotype.

Taxon Voucher specimen GenBank accession numbers Origin References
ITS nrLSU tef1-α
Bolbitius coprophilus HMJAU64958 OQ780315 OQ758216 – China Song and Bau 2023
B. coprophilus SZMC-NL-2640 JX968253 JX968370 – Hungary Tóth et al. 2013
B. reticulatus WU30001 JX968249 JX968366 JX968455 Hungary Tóth et al. 2013
B. subvolvatus WU28379 JX968248 JX968365 JX968454 Italy Tóth et al. 2013
Conocybe alkovii LE262841 JQ247196 – – Russia Malysheva 2012
C. angulispora FJAU65120 T PP501383 PP501393 PP501651 China This study
C. angulispora FJAU65122 PP501384 PP501394 PP501652 China This study
C. anthracophila var. ovispora WU25461 JX968237 JX968355 – Italy Tóth et al. 2013
C. antipus WU19791 JX968215 JX968332 JX968432 Austria Tóth et al. 2013
C. bispora SZMC-NL-2573 JX968203 JX968320 JX968423 Hungary Tóth et al. 2013
C. bisporigera SZMC-NL-1904 JX968235 JX968353 JX968446 Hungary Tóth et al. 2013
C. brachypodii HMJAU45017 MH141423 – – China Liu 2018
C. brunneidisca HMJAU45069 OQ780317 – – China Song and Bau 2023
C. ceracea HMJAU64951 OQ758110 OQ758218 OQ758305 China Song and Bau 2023
C. coniferarum LE313009 KY614061 – – Russia Malysheva 2017
C. crispella WU27367 JX968208 JX968325 JX968426 Australia Tóth et al. 2013
C. cylindracea WU20796 JX968240 JX968358 JX968449 Italy Tóth et al. 2013
C. cylindrospora HMJAU42440 MG250375 OQ758203 – China Liu and Bau 2018; Song and Bau 2023
C. deliquescens HMJAU61998 OP373403 OQ758204 OQ758292 China Song and Bau 2023
C. elegans SZMC-NL-0908 JX968223 JX968341 JX968437 Sweden Tóth et al. 2013
C. enderlei WU21272 JX968163 JX968279 – Italy Tóth et al. 2013
C. fuscimarginata HMJAU45033 OQ780310 OQ758208 OQ758296 China Song and Bau 2023
C. fuscimarginata SZMC-NL-3668 JX968238 JX968356 JX968448 Sweden Tóth et al. 2013
C. gigasperma SZMC-NL-3972 JX968179 JX968295 JX968403 Slovakia Tóth et al. 2013
C. hausknechtii LE253789 JQ247194 – – Russia Malysheva 2013
C. hexagonospora FJAU71661 PP501385 PP501395 PP501653 China This study
C. hydrophila HMJAU64954 OQ758116 OQ758232 OQ758313 China Song and Bau 2023
C. incarnata FJAU71663 PP501390 PP501400 PP501658 China Song and Bau 2023
C. incarnata WU21897 JX968229 JX968347 JX968441 Finland Tóth et al. 2013
C. incerta LE313017 KY614062 – – Russia Malysheva 2017
C. ingridiae WU28158 JX968244 JX968361 JX968451 Italy Tóth et al. 2013
C. karakensis KTK05 ON392730 – – Pakistan Ullah et al. 2023
C. lenticulospora SZMC-NL-0923 JX968242 JX968359 JX968450 Sweden Tóth et al. 2013
C. mesospora HMJAU45049 MH141419 – – China Liu 2018
C. microrrhiza SZMC-NL-2180 JX968222 JX968340 JX968436 Hungary Tóth et al. 2013
C. moseri GLM-F40421 MK412354 – – Germany Unpublished
C. moseri HMJAU45075 OQ780309 OQ758207 – China Song and Bau 2023
C. muscicola HMJAU64939 OQ758113 OQ758223 OQ758309 China Song and Bau 2023
C. nigrescens WU27557 JX968234 JX968352 JX968445 Italy Tóth et al. 2013
C. nitrophila WANG140019 KR998384 – – China Wang and Tzean 2015
C. nitrophila WU20916 JX968233 JX968351 JX968444 India Tóth et al. 2013
C. ochrostriata var. favrei WU29786 JX968245 JX968362 JX968452 Italy Tóth et al. 2013
C. olivaceopileata LE313106 KY614059 – – Russia Malysheva 2017
C. pallidospora WU7395 JX968239 JX968357 – Austria Tóth et al. 2013
C. parapilosella 90551 MN872706 – – Spain Siquier and Salom 2021
C. pilosella HMJAU45062 OQ780305 OQ758205 OQ758294 China Song and Bau 2023
C. pilosa HMJAU64947 OQ758122 OQ758222 OQ758307 China Song and Bau 2023
C. praticola HMJAU64965 OQ780303 – – China Song and Bau 2023
C. pseudocrispa HMJAU64946 OQ780307 OQ758212 OQ758293 China Song and Bau 2023
C. pseudocrispa WU18009 JX968230 JX968348 JX968442 Austria Tóth et al. 2013
C. pubescens WU20759 JX968170 JX968286 JX968396 Italy Tóth et al. 2013
C. reniformis HMJAU64942 OQ758108 OQ758229 OQ758311 China Song and Bau 2023
C. rickenii AH21067 MF142238 – – Spain Siquier and Salom 2018
C. romagnesii HMJAU64960 OQ780304 – – China Song and Bau 2023
C. rostellata SZMC-NL-2499 JX968162 JX968278 JX968390 Sweden Tóth et al. 2013
C. rubrocyanea HMJAU64964 OQ749742 – – China Song and Bau 2023
C. rubrocyanea FJAU65123 T PP501388 PP501398 PP501656 China This study
C. rubrocyanea FJAU71654 PP501389 PP501399 PP501657 China This study
C. rufostipes HMJAU64937 OQ758120 OQ758227 OQ758317 China Song and Bau 2023
C. semiglobata WU8794 JX968188 JX968304 – Austria Tóth et al. 2013
C. siennophylla HMJAU64966 OQ780312 OQ758210 OQ758297 China Song and Bau 2023
C. siennophylla SZMC-NL-1210 JX968246 JX968363 JX968453 Hungary Tóth et al. 2013
C. siliginea SZMC-NL-2313 JX968225 JX968343 JX968438 Sweden Tóth et al. 2013
C. siliginea FJAU71664 PP501392 PP501402 PP501660 China This study
C. singeriana HMJAU64956 OQ780314 OQ758214 – China Song and Bau 2023
C. singeriana WU22129 JX968166 JX968282 JX968393 Austria Tóth et al. 2013
C. sinobispora HMJAU64949 OQ758118 OQ758230 OQ758315 China Song and Bau 2023
Conocybe sp.1 HMJAU44988 OQ749737 OQ740305 OQ758302 China Song and Bau 2023
Conocybe sp.2 HMJAU64963 OQ749740 OQ740307 OQ758304 China Song and Bau 2023
Conocybe sp.3 HMJAU64967 OQ749741 – – China Song and Bau 2023
C. tetrasporoides WU17385 JX968232 JX968350 – New Zealand Tóth et al. 2013
C. velutinomarginata WU28695 JX968226 JX968344 JX968439 Germany Tóth et al. 2013
C. velutipes FJAU71662 PP501391 PP501401 PP501659 China This study
C. velutipes SZMC-NL-2187 JX968228 JX968346 JX968440 Hungary Tóth et al. 2013
C. verna FJAU65117 T PP501386 PP501396 PP501654 China This study
C. verna FJAU65118 PP501387 PP501397 PP501655 China This study
C. volvicystidiata LIP0001212 KY346827 – – France Hausknecht and Broussal 2016
C. watlingii WU22744 JX968172 JX968288 JX968398 Finland Tóth et al. 2013
C.nocybula. coprophila SZMC-NL-2176 JX968156 JX968273 – Hungary Tóth et al. 2013; Song and Bau 2024
C.. cyanopus WU2134 JX968157 JX968274 JX968388 Austria Tóth et al. 2013; Song and Bau 2024
C.. smithii HMJAU62001 OP373407 OQ758215 OQ758300 China Song and Bau 2023; Song and Bau 2024
Conobolbitina dasypus SZMC-NL-2279 JX968152 JX968269 JX968385 Hungary Tóth et al. 2013; Song and Bau 2024
Descolea antarctica NZ5182 AF325647 – – USA Peintner et al. 2001
D. quercina HMJAU64959 OQ780313 OQ758213 OQ758299 China Song and Bau 2023
Pholiotina arrhenii SZMC-NL-2509 JX968261 JX968377 – Sweden Tóth et al. 2013
Ph. brunnea SZMC-NL-1216 JX968259 JX968375 JX968461 Hungary Tóth et al. 2013
Ph. dentatomarginata SZMC-NL-2921 JX968258 JX968374 JX968460 Hungary Tóth et al. 2013
Ph. serrata HMJAU62006 OP538570 OQ758217 OQ758301 China Song and Bau 2023
Ph. sulcata SZMC-NL-1975 JX968153 JX968270 JX968386 Hungary Tóth et al. 2013
Ph. teneroides SZMC-NL-3501 JX968264 JX968379 JX968465 Slovakia Tóth et al. 2013
Ph. utricystidiata WU20164 JX968262 JX968463 – Germany Tóth et al. 2013
Ph. vexans SZMC-NL-3967 JX968265 JX968380 JX968466 Slovakia Tóth et al. 2013
Psathyrella leucotephra SZMC-NL-1953 FM163226 FM160683 FM897219 Hungary Nagy et al. 2011
P. piluliformis HMJAU37922 MG734716 MW413364 MW411001 China Yan and Bau 2018

Results

Phylogenetic analyses

The Bayesian tree was constructed based on a combined dataset of ITS, nrLSU, and tef1-α, while the ML phylogenetic tree was not presented due to their similar topology. Bootstrap support values were indicated on the tree nodes. Only the data meeting the criteria of Bayesian posterior probabilities (PP ≥ 0.9) and ML bootstrap values (MLbs ≥ 70%) were retained (Fig. 1). The multi-locus dataset (ITS + nrLSU + tef1-α) of Conocybe comprised 826 bp for ITS, 1299 bp for nrLSU, and 1131 bp for tef1-α. The alignment included 94 sequences with 3256 columns, resulting in 1446 distinct patterns, 997 parsimony-informative sites, 316 singleton sites, and 1943 constant sites. During the construction of ML phylogenetic trees, the best-fit models, GTR+F+R4 for ITS, TIM3+F+I+I+R2 for nrLSU, and TIM2e+I+I+R4 for tef1-α based on the BIC. Similarly, for Bayesian phylogenetic trees, the best-fit models according to the BIC were GTR+F+I+G4 for ITS and nrLSU, and SYM+I+G4 for tef1-α.

Figure 1. 

The phylogenetic relationships of Conocybe sect. Pilosellae in Bolbitiaceae using Bayesian inference and maximum likelihood methods based on a multi-locus dataset (ITS, nrLSU, and tef1-α). In the phylogenetic tree, the newly proposed species are indicated in bold red color, while the newly recorded species is indicated in bold black color, the outgroup is Psathyrella species, T = holotype.

In the phylogenetic tree, the newly proposed species are indicated in bold red color, while the newly recorded species is indicated in bold black color (Fig. 1). Notably, specimens FJAU65123, FJAU71654, and HMJAU64964 clustered together, forming a distinct branch and serving as sister taxa to Conocybe muscicola T. Bau & H.B. Song. However, their phylogenetic relationship exhibits low support, with a PP/MLbs value of 0.7/55. Similarly, FJAU65120 and FJAU65122 comprise a separate branch and act as sister taxa to C. hexagonospora Métrod ex Hauskn. & Enderle (FJAU71661), demonstrating a PP/MLbs value of 1/84. Furthermore, FJAU65117 and FJAU65118 form another distinct branch and serve as sister taxa to C. ingridiae Hauskn. and C. ochrostriata var. favrei Hauskn., with a PP/MLbs value of 1/99. We conducted a Standard Nucleotide BLAST of the ITS sequences of Conocybe verna (FJAU65117), C. angulispora (FJAU65120), and C. rubrocyanea (FJAU65123) against the NCBI database. The results, presented in descending order of similarity, showed that C. verna had a similarity of 96.7% with Conocybe cf. rostellata (SMNS-STU-F-0900917), 92.8% with C. ingridiae (WU28158), and 93.1% with C. ochrostriata var. favrei (WU29786). Conocybe angulispora exhibited a similarity of 98.2% with C. lenticulospora (HMJAU45069), 98.6% with C. cylindracea (WU20796), and 88.7% with C. hydrophila. The similarity between C. rubrocyanea and C. muscicola (HMJAU64939) was 95.2%, with C. velutipes (SZMC-NL-2187) was 94.4%, and with C. fuscimarginata (HMJAU45033) was 93.8%. And then based on the phylogenetic tree and morphological findings, three new species are proposed: C. rubrocyanea (for the clade FJAU65123, FJAU71654, and HMJAU64964), C. angulispora (for the clade FJAU65120 and FJAU65122), and C. verna (for the clade FJAU65117 and FJAU65118). While the type specimen of C. hexagonospora lacks sequence data, the identification of this species as a new record for China was accomplished through traditional morphology, and reference sequences have been provided to facilitate future confirmation. Finally, the branch containing C. angulispora, C. hexagonospora, C. cylindracea Maire & Kühner ex Singer, and C. brunneidisca (Murrill) Hauskn. referred to as ser. Lenticulospora, following the viewpoint of Hausknecht and Krisai-Greilhuber (2006) (for more detailed information, please refer to the Discussion section).

Taxonomy

Conocybe verna T. Bau & H. B. Song, sp. nov.

MycoBank No: 852866
Figs 2A–D, 3, 4

Etymology.

“verna” refers to spring-born.

Holotypus.

China, • Jilin Province, Tonghua City, Ji’an City, Yushan Park, 8 May 2023, 41°08'01"N, 126°10'45"E, alt. 280 m, Zheng-Qing Chen, CZQ23050801 (FJAU65117).

Diagnosis.

The main characteristic of Conocybe verna includes a straight to reflexed edge of the pileus after maturity, with no surface pubescence. The basidiospores exhibit a suprahilar depression and have an oblong, subcylindrical shape with a slightly thin wall. The basidia are 2-spored.

Description.

Basidioma mycenoid. Pileus diameter 0.5–2.5 cm, initially paraboloid, nearly hemispherical, margin deflexed, matured obtusely conical, campanulate, margin straight to reflexed. Pileus initially beige (RAL1001) to ivory (RAL1014), matured light ivory (RAL1015), powdery yellow (RAL1034) to ochre brown (RAL8001), surface hygrophanous, pubescence absent, when moist, it exhibits striae, which disappear upon slight drying, margin undulate. Context thin, ivory (RAL1014) to beige (RAL1001), no specific odor or taste. Lamellae adnexed to narrowly adnate, ventricose, crowded, unequal in length, ivory (RAL1014), powdery yellow (RAL1034) to ochre brown (RAL8001), smooth margin. Stipe 2.0–8.0 cm long, 1.0–3.0 mm thick, cylindrical, slightly thicker downward, ivory (RAL1014) to ochre brown (RAL8001), deer brown (RAL8007), surface pruinose and short pubescent, longitudinally fibrous striate, subbulbous at the base.

Figure 2. 

Basidiomata of Conocybe sect. Pilosellae species A C. verna (FJAU65117 T) B C. verna (FJAU65118) C, D C. verna (FJAU65119) E, F C. angulispora (FJAU65120 T) G C. angulispora (FJAU65122) H C. angulispora (FJAU65121) I C. rubrocyanea (FJAU65123 T) J C. rubrocyanea (FJAU71654) K C. rubrocyanea (FJAU71658) L C. rubrocyanea (FJAU71652) M C. rubrocyanea (FJAU71650) N–P C. hexagonospora (FJAU71661), Scale bars: 1 cm, T = holotype.

Basidiospores (60/3/3) (10–)11–15.5(–16) × (5.5–)6–8.5(–9) μm, Q=(1.65–)1.71–2.07(–2.21), Qm = 1.86(±0.10), with a suprahilar depression, oblong, subcylindrical, wall slightly thin, containing oil droplets, germ pore diameter 0.5–2.0 μm. Basidiospores in 5% KOH solution appear ochre brown (RAL8001) to copper brown (RAL8004). Basidia (20–)21–33(–35) × (7–)8–11 μm, clavate, 2-spored, sterigmata 3–7 μm long, basidia with vacuolar contents. Cheilocystidia (16–)17–25(–26) × (6–)7–11(–13) μm, lecythiform, with capitula 3–6 μm wide. Caulocystidia ellipsoid to oblong, lageniform, long-necked lageniform, subcylindrical, clavate, narrowly utriform to utriform, fusiform, conical, nettle hair-shaped, (9–)10–50(–53) × 5–12 μm, with capilliform elements reaching up to 80 μm, among which rare lecythiform cystidia are mixed. Pileipellis hymeniform, composed of (23–)31–63(–65) × (14–)15–22(–23) μm sphaeropedunculate elements, with yellow pigments at the base. Pileocystidia absent. All structures have clamp connections. Weakly positive reaction with ammonia forming rhomboid crystals.

Figure 3. 

Conocybe verna (FJAU65117) A basidiomata B basidiospores in KOH C hymenium and subhymenium D cheilocystidia E stipitipellis F pileipellis. Scale bars: 1 cm (A); 10 μm (B–F).

Habitat.

Found singly or scattered in broad-leaved forests during spring.

Known distribution.

Jilin Province, China.

Additional specimens measured.

China, • Jilin Province, Tonghua City, Ji’an City, Yushan Park, 8 May 2023, 41°08'01"N, 126°10'45"E, alt. 280 m, Qian-Ru Liu, LQR23050801 (FJAU65118); • Tonghua City, Ji’an City, Jiangkou Village, 9 May 2023, 40°59'37"N, 126°03'02"E, alt. 260 m, Mu Liu, LM230509 (FJAU65119).

Figure 4. 

Microscopic structure images of Conocybe verna (FJAU65117) A basidiospores B basidia C cheilocystidia D pileipellis E stipitipellis. Scale bars: 10 μm (A–E).

Notes.

Conocybe verna is classified in sect. Pilosellae primarily due to the presence of non-lecythiform caulocystidia. The distinguishing characteristics of C. verna from other 2-spored species in sect. Pilosellae are as follows: C. verna differs from C. bisporigera (Hausknecht & Krisai) Arnolds in that the latter has a chocolate brown pileus and lentiform basidiospores (Arnolds 2003). The distinction between C. verna and C. caespitosa (Murrill) Watling is that the latter has basidiospores with a suprahilar plage and basidia measuring 19–24 μm in length, which is shorter than the basidia of C. verna (Hausknecht 2009). In contrast to C. bispora (Singer) Hauskn., C. verna has a pileus without distinct striations, while the basidiospores of C. bispora are on average 2 μm shorter (Hausknecht 1998). The distinction between C. verna and C. umbellula var. lednicensis lies in the latter having a striate pileus, and basidia measuring less than 20 μm in length (Hausknecht 2009). Furthermore, C. verna is differentiated from C. leporina (Velen.) Singer and C. microrrhiza Hauskn. by the presence of a pseudorhiza in the latter two, as well as their smaller basidiospores (Singer 1989; Hausknecht 1999). Conocybe verna differs from C. inocybeoides Watling in that the latter has a pileus with radiating striations and possesses pileocystidia (Watling 1980). Additionally, C. verna is distinguished from C. velutinomarginata Hauskn. & Zugna and C. rickenii (Jul. Schäff.) Kühner by the presence of capilliform pileocystidia in the latter two; C. velutinomarginata has a nearly spherical pileus, while C. rickenii has a grayish-brown pileus (Kühner 1935; Hausknecht 2009). Conocybe verna can be differentiated from C. siliginea (Fr.) Kühner by the latter’s lime-colored pileus and lecythiform pileocystidia (Kühner 1935). Finally, the distinction between C. verna and C. gigasperma Enderle & Hauskn. lies in the latter’s basidiospores measuring 18.3–20.1 μm in length, which are larger than those of C. verna, and the presence of pileocystidia (Hausknecht and Enderle 1992). Conocybe verna is also distinguished from C. sinobispora T. Bau & H.B. Song, as the latter has a striate pileus and cylindrical to lageniform pileocystidia (Song and Bau 2023).

In terms of phylogeny, C. verna is closely related to C. ingridiae and C. ochrostriata var. favrei. However, C. ingridiae has a pileus with distinct striations and basidiospores measuring 9.6–10.5 μm in length, while C. ochrostriata var. favrei also has a striate pileus and possesses 4-spored basidia, making them easily distinguishable (Hausknecht 2009). Among these similar species, the following have been sequenced and are clearly separated in the phylogeny: C. bisporigena, C. bispora, C. ingridiae, C. microrrhiza, C. velutinomarginata, C. rickenii, C. siliginea, and C. sinobispora.

Conocybe angulispora T. Bau & H. B. Song, sp. nov.

MycoBank No: 852867
Figs 2E–H, 5, 6

Etymology.

“angulispora” refers to basidiospores that are angular and submitriform or slightly hexagonal in shape.

Holotypus.

China, • Jilin Province, Jilin City, Jiaohe City, Shansongling, 26 August 2023, 43°32'25"N, 127°02'21"E, alt. 550 m, Hong Cheng, C2382612 (FJAU65120).

Diagnosis.

Conocybe angulispora basidiospores are lentiform, frontal view slightly hexagonal or submitriform, side view ellipsoid to oblong, ovoid, amygdaliform, basidia are 4(2)-spored, and pileocystidia are abundant.

Description.

Basidioma mycenoid. Pileus diameter 0.5–2.5 cm, initially paraboloid to obtusely conical, later conical to broadly conical, edge straight, undulate. In early stages, pileus center color ranges from signal brown (RAL8002) to mahogany brown (RAL8016), with slightly lighter color at the edges, brown beige (RAL1011), sandy yellow (RAL1002) to maize yellow (RAL1001). When mature, pileus center color changes to reddish-brown (RAL8012) to mahogany brown (RAL8016), while the edge remains brown beige (RAL1011) and ivory (RAL1014). Pileus hygrophanous, distinctly pubescent, with striations extending to the center. Context thin, ivory (RAL1014) to light ivory (RAL1015), no specific odor or taste. Lamellae adnexed to narrowly adnate, ventricose, slightly crowded, unequal in length, sandy yellow (RAL1002) to ochre brown (RAL8001), with smooth edges. Stipe length 2.5–5.0 cm, thick 1.0–2.0 mm, cylindrical, light ivory (RAL1015), sandy yellow (RAL1002) to signal brown (RAL8002), surface covered with pubescent, longitudinally fibrous striations, subbulbous base.

Figure 5. 

Conocybe angulispora (FJAU65120) A basidiomata B basidiospores in KOH C hymenium and subhymenium D cheilocystidia E stipitipellis F pileipellis. Scale bars: 1 cm (A); 10 μm (B–F).

Basidiospores (60/3/3) 8–10(–10.5) × 5.5–6.5 × (4.5–)5–6 μm, Q=(1.35–)1.39–1.76(–1.83), Qm = 1.57(±0.11), lentiform, angular and submitriform or slightly hexagonal in frontal view, ellipsoid to oblong, ovoid, or amygdaliform in side view, with partially thick walls and containing oil droplets, germ pore diameter 0.5–2.0 μm, basidiospores in 5% KOH solution ochre brown (RAL8001) to copper brown (RAL8004) in KOH. Basidia 14–24(–25) × (8–)9–11(–12) μm, broadly clavate to clavate, 4(2)-spored, sterigmata 2–6 μm long, basidia with vacuolar contents. Cheilocystidia 13–22 × 6–10(–11) μm, lecythiform, with capitula 3–6 μm wide. Caulocystidia ellipsoid to oblong, lageniform, long-necked lageniform, nettle hair-shaped, narrowly conical, fusiform, cylindrical, clavate, narrowly utriform to utriform, (10–)11–42(–45) × (4–)5–9 μm, capilliform cystidia can exceed 100 μm, among which rare lecythiform cystidia are mixed at the apex. Pileipellis hymeniform, composed of (25–)28–62(–66) × 15–34(–36) μm broadly clavate, spheropedunculate, and obpyriform elements, with yellow pigment at the base. Pileocystidia abundant, (22–)23–58(–60) × 5–18(–19) μm, lageniform to long-necked lageniform, lecythiform, tibiiform, and nettle hair-shaped, capilliform cystidia can exceed 100 μm. Clamp connections are rare in all tissues. Shows negative reaction with ammonia solution.

Figure 6. 

Microscopic structure images of Conocybe angulispora (FJAU65120) A basidiospores B basidia C cheilocystidia D pileipellis E stipitipellis. Scale bars: 10 μm (A–E).

Habitat.

In summer, they grow scattered or in groups in the humus layer of mixed forests.

Known distribution.

Jilin Province, China.

Additional specimens measured.

China, • Jilin Province, Jilin City, Jiaohe City, Laoyeling, 28 July 2023, 43°40'57"N, 127°11'58"E, alt. 430 m, Xia Wang, W23072815 (FJAU65121); • Jilin City, Jiaohe City, Shansongling, 26 August 2023, 43°32'09"N, 127°02'23"E, alt. 530 m, Hong Cheng, C2382621 (FJAU65122).

Notes.

In some species of section Pilosellae, the frontal view of basidiospores appears slightly hexagonal, which can be easily confused with C. angulispora. The difference between C. angulispora and C. hexagonospora is that C. hexagonospora lacks distinct pubescence on the pileus and has rare pileocystidia, making it easy to differentiate (Hausknecht 1993). Additionally, the ITS sequence similarity between C. angulispora and C. hexagonospora is 91.2%. Conocybe angulispora can be distinguished from C. brunneidisca by the larger length of basidiospores in C. brunneidisca, which can reach 9.9–12.1 μm, and it is found in fertile grasslands or dung (Hausknecht and Contu 2007). Conocybe angulispora can be differentiated from C. pulchra (Clem.) Hauskn., Krisai & Voglmayr by the length of basidiospores, which measures 11.5–15 μm in C. pulchra, and C. pulchra lacks pileocystidia (Hausknecht et al. 2004). The difference between C. angulispora and C. lentispora Singer is that the basidiospores of C. lentispora are shorter than 7 μm and broadly ellipsoid in shape (Hausknecht 2005). Conocybe angulispora can be differentiated from C. brunneoaurantiaca K.A. Thomas, Hauskn. & Manim. such that C. brunneoaurantiaca lacks pubescence on the pileus and pileocystidia (Hausknecht 2009; Thomas et al. 2001).

Conocybe rubrocyanea T. Bau & H. B. Song, sp. nov.

MycoBank No: 852868
Figs 2I–M, 7, 8

Etymology.

“ rubrocyanea “ refers to basidiomata that have a reddish hue when fresh and a bluish hue when dry.

Holotypus.

China, • Jilin Province, Jilin City, Jiaohe City, Shansongling, 30 July 2023, 43°32'14"N, 127°01'33"E, alt. 610 m, Shi-En Wang, E2307268 (FJAU65123).

Diagnosis.

Conocybe rubrocyanea, when fresh, displays a mainly red color on the pileus, transitioning to blue upon drying. Basidiospores are lentiform, ellipsoid to oblong, frontal view near hexagonal, side view phaseoliform, cheilocystidia clavate, utriform, ellipsoid, or fusiform on one side near the edge of the pileus, and lecythiform on the side near the stipe, and some pileipellis cells contain blue lilac pigment.

Description.

Basidioma mycenoid. Pileus diameter 0.5–2.0 cm, initially hemispherical, conical, later obtusely conical, with straight, undulate margin. When fresh, pileus salmon orange (RAL2012), antique pink (RAL3014) to rose (RAL3017), tomato red (RAL3013) to pearl ruby red (RAL3032), and when dry, it becomes slate gray (RAL7015), brown gray (RAL7013) to cobalt blue (RAL5013). Pileus hygrophanous, covered in distinct pubescence and striations that extend up to one-third towards the center. Context thin, salmon orange (RAL2012) to light ivory (RAL1015), no specific odor or taste. Lamellae ventricose, adnexed to narrowly adnate, moderately crowded, unequally long, initially light ivory (RAL1015) to ivory (RAL1014), later pastel yellow (RAL1034) to ochre-brown (RAL8001), with inconspicuous, slightly eroded edges. Stipe 2.0–8.0 cm long, 1.0–4.0 mm thick, cylindrical, clay brown (RAL8003), rose (RAL3017), antique pink (RAL3014) to pearl ruby red (RAL3032), surface pruinose and pubescent, longitudinally striate, base bulbous.

Figure 7. 

Conocybe rubrocyanea (FJAU65123) A basidiomata B basidiospores in KOH C hymenium and subhymenium D cheilocystidia E stipitipellis F pileipellis. Scale bars: 1 cm (A); 10 μm (B–F).

Basidiospores (60/3/3) 8–11.5(–12.5) × 5–7.5 × 5–6(–6.5) μm, Q=(1.33–)1.42–2.08(–2.14), Qm = 1.76(±0.17), lentiform, ellipsoid to oblong, frontal view near hexagonal, side view phaseoliform, slight constriction at center, with thick walls, containing oil droplets, germ pore diameter 0.5–2.0 μm, basidiospores in KOH solution ochre brown (RAL8001) to copper brown (RAL8004). Basidia (13–)15–26(–27) × 8–11(–12) μm, broadly clavate to clavate, 4(2)-spored, with sterigmata measuring 2–6 μm in length, basidia contain vacuolar contents. Cheilocystidia (14–)15–27(–28) × (6–)7–14(–15) μm, clavate, utriform, ellipsoid, or fusiform on one side near the edge of the pileus, and lecythiform on the side near the stipe, with capitula 3–6 μm wide. Caulocystidia elliptical to oblong, lageniform, long-necked lageniform, nettle hair-shaped, conical, fusiform, cylindrical, clavate, narrowly utriform to utriform, (10–)12–82(–85) × (5–)6–16 μm, capilliform cystidia may exceed 100 μm, with rare occurrences of lecythiform and sub-lecythiform cystidia at the apex. Pileipellis hymeniform, composed of spheropedunculate and fusiform cells (25–)27–53(–54) × (14–)15–28(–29) μm, some containing blue lilac (RAL4005) pigment, with yellow pigment at the base. Pileocystidia (21–)23–55(–60) × 4–23 μm, with long-necked lageniform, lecythiform, cylindrical, and nettle hair-shaped forms, and capilliform cystidia can exceed 100 μm. Clamp connections are rare in all tissues. It shows a positive reaction with ammonia, forming diamond-shaped crystals.

Figure 8. 

Microscopic structure images of Conocybe rubrocyanea (FJAU65123) A basidiospores, B basidia C cheilocystidia D stipitipellis E pileipellis. Scale bars: 10 μm (A–E).

Habitat.

Scattered or grouped in mixed forests during the summer season, on cow dung.

Known distribution.

Jilin Province, China.

Additional specimens measured.

China, • Jilin Province, Jilin City, Jiaohe City, Shansongling, 26 July 2022, 43°32'02"N, 127°02'36"E, alt. 580 m, Han-Bing Song, S22072618 (HMJAU64964); • Jilin City, Jiaohe City, Shansongling, 29 July 2023, 43°32'20"N, 127°03'09"E, alt. 530 m, Shi-En Wang, E2307247 (FJAU71648); • Jilin City, Jiaohe City, Shansongling, 30 July 2023, 43°32'20"N, 127°01'50"E, alt. 550 m, Shi-En Wang, Xia Wang, Si-Ying Li, W23073002 (FJAU71649), W23073003 (FJAU71650), W23073004 (FJAU71651), E2307277 (FJAU71652), L23073033 (FJAU71653); • Jilin City, Jiaohe City, Shansongling, 26 August 2023, 43°32'26"N, 127°02'23"E, alt. 550 m, Zheng-Qing Chen, Mu Liu, Hong Cheng, Q2382626 (FJAU71654), LM230864 (FJAU71655), C2382603 (FJAU71656), C2382605 (FJAU71657), C2382611 (FJAU71658), C2382615 (FJAU71659); • Jilin City, Huadian City, Redstone National Forest Park, 28 August 2023, 42°58'08"N, 127°03'36"E, alt. 430 m, Xian-Yan Zhou, Y2382804 (FJAU71660).

Notes.

Conocybe rubrocyanea can be differentiated from species with near hexagonal basidiospores in sect. Pilosellae, such as C. hexagonospora, C. brunneidisca, C. lentispora, C. brunneoaurantiaca, C. pulchra and C. angulispora, by presence of red color tone on the pileus (Hausknecht 2009). Conocybe rubrocyanea is closely related to C. incarnata (Jul. Schäff.) Hauskn. & Arnolds and C. muscicola, and they are easily confused in macroscopic morphology. However, C. incarnata and C. muscicola basidiospores are not lentiform or hexagonal, and pileipellis cells lack blue lilac pigment (Arnolds and Hausknecht 2003).

Conocybe hexagonospora Métrod ex Hauskn. & Enderle

Figs 2N–P, 9, 10

Description.

Basidioma mycenoid. Pileus diameter 1.0–1.5 cm, obtusely conical, edge straight, undulate, center signal brown (RAL8002) to deer brown (RAL8007), fading towards the edge, brown beige (RAL1011) to ivory (RAL1014), pileus hygrophanous, smooth, striate towards the center. Context thin, ivory (RAL1014) to light ivory (RAL1015), no specific odor or taste. Lamellae adnexed to narrowly adnate, ventricose, slightly loosely, unequal in length, beige (RAL1001) to sandy yellow (RAL1002), with smooth margins. Stipe length 3.5–4.0 cm, width 0.5–1.5 mm, cylindrical, brown beige (RAL1011) to sandy yellow (RAL1002), surface pubescent, longitudinally fibrous striate, subbulbous at the base.

Figure 9. 

Conocybe hexagonospora (FJAU71661) A basidiomata B basidiospores in KOH C hymenium and subhymenium D cheilocystidia E stipitipellis F pileipellis. Scale bars: 1 cm (A); 10 μm (B–F).

Basidiospores (40/1/1) 7.5–9.5(–10) × 5.5–6.5 × 5–6 μm, Q=(1.32–)1.34–1.78(–1.80), Qm = 1.49(±0.11), lentiform, frontal view nearly hexagonal or submitriform, side view ellipsoid to oblong, thick-walled, containing oil droplets, germ pore diameter 0.5–1.5 μm. Basidiospores in 5% KOH solution ochre brown (RAL8001) to copper brown (RAL8004). Basidia (14–)15–21(–22) × 8–10 μm, broadly clavate to clavate, 4-spored, with sterigmata length 3–6 μm, basidia contain vacuolar contents. Cheilocystidia (13–)15–21 × 7–10(–11) μm, lecythiform, with capitula 3–6 μm wide. Caulocystidia are ellipsoid to oblong, lageniform, long-necked lageniform, nettle hair-shaped, narrowly conical, fusiform, cylindrical, clavate, narrowly utriform to utriform, measuring (20–)22–55(–57) × (5–)6–16 μm, capilliform cystidia can reach a length of 100 μm, with rare lecythiform cystidia mixed in. Pileipellis hymeniform, consists of spheropedunculate and obpyriform cells, 29–48(–50) × (18–)19–27(–30) μm, with yellow pigment at the base. Pileocystidia are rare and lageniform in shape. All tissues exhibit clamp connections. It shows a negative reaction to ammonia solution.

Figure 10. 

Microscopic structure images of Conocybe hexagonospora (FJAU71661) A basidiospores B basidia C cheilocystidia D stipitipellis E pileipellis. Scale bars: 10 μm (A–E).

Habitat.

Solitary in mixed forests during autumn.

Known distribution.

Asia: China, Russia; Europe: Sweden, Finland, Latvia, Hungary, Germany, Austria (Holotype), Belgium, United Kingdom, France, Italy (Hausknecht 2009).

Additional specimens measured.

China, • Jilin Province, Siping City, Yitong Manchu Autonomous County, 7 September 2023, 43°35'58"N, 125°12'12"E, alt. 290 m, Han-Bing Song, S23090710 (FJAU71661).

Notes.

Although this species does not have gene sequences in the NCBI database, its macroscopic and microscopic structures are consistent with those of C. hexagonospora, leading to its identification as C. hexagonospora. There are also some species in sect. Pilosellae with basidiospores’ shapes similar to C. hexagonospora, but they are distinct species, differentiated as follows: The difference between C. hexagonospora and C. brunneidisca is that the latter has longer basidiospores, reaching a length of 9.9–12.1 μm, and the pileus color and habitat are also different (Hausknecht and Contu 2007). The difference between C. hexagonospora and C. pulchra is that the latter has basidiospores measuring 11.5–15 μm in length and lacks pileocystidia (Hausknecht et al. 2004). The difference between C. hexagonospora and C. lentispora is that the latter has basidiospores with a length smaller than 7 μm and are broadly ellipsoid (Hausknecht 2005). Meanwhile, the difference between C. hexagonospora and C. brunneoaurantiaca is that C. brunneoaurantiaca has cheilocystidia reaching up to 30 μm and lacks pileocystidia (Thomas et al. 2001). In the phylogenetic tree, C. hexagonospora and C. angulispora are sister taxa to each other, but their ITS sequence similarity is only 91%. Conocybe angulispora has distinct pubescence on its pileus, allowing for differentiation from C. hexagonospora. Of these similar species, the following are sequenced and clearly separate in the phylogeny: C. hexagonospora, C. brunneidisca, C. angulispora.

Key to Chinese Species of Conocybe Sect. Pilosellae

1 2-spored 2
– 4-spored 6
2 Pileus unstriated or not distinct 3
– Pileus striated 4
3 Tibiiform pileocystidia present C. siliginea
– Tibiiform pileocystidia absent C. pseudocrispa
4 Basidiospores with suprahilar depression C. verna
– Basidiospores with suprahilar plage 5
5 Pileus blackish in color C. bisporigera
– Pileus yellowish in color C. sinobispora
6 Basidiospores nearly hexagonal 7
– Basidiospores not hexagonal 10
7 Pileus reddish in color C. rubrocyanea
– Pileus lacking a red color 8
8 Average length of basidiospores can reach 12 μm C. brunneidisca
– Average length of basidiospores is less than 10 μm 9
9 Pileus pubescence distinct C. angulispora
– Pileus pubescence absent or indistinct C. hexagonospora
10 Pseudorhiza present C. incarnata
– Pseudorhiza absent 11
11 Germ pore absent or not distinct C. pilosella
– Germ pore present 12
12 Basidiospores phaseoliform, reniform C. reniformis
– Basidiospores never phaseoliform 13
13 Pileus unstriated 14
– Pileus striated 15
14 Waxy crystals precipitate upon drying C. ceracea
– No crystallization occurs upon drying C. fuscimarginata
15 Pileus densely pubescent 16
– Pileus pubescence absent or slight 17
16 Pileus salmon orange when young C. muscicola
– Pileus blackish-red when young C. pilosa
17 Lamellae edge serrate C. hydrophila
– Lamellae edge not serrate 18
18 Basidiospores lentiform 19
– Basidiospores never lentiform 20
19 Length of basidiospores may exceed 15 μm, 11–16 μm C. nitrophila
– Length of basidiospores may be less than 10 μm, 9–13 μm C. velutipes
20 Grows on cow dung C. rufostipis
– Grows in meadows 21
21 Pileus honey yellow C. siennophylla
– Pileus brown beige C. moseri

Discussion

Building on the phylogenetic framework of Tóth et al. (2013) and Song and Bau (2023), a phylogenetic tree was reconstructed for sect. Pilosellae incorporating materials from Jilin province and using ITS, nrLSU, and tef1-α. The analysis revealed the presence of three new species and a new record for China. The new species are C. verna, C. angulispora, and C. rubrocyanea, while the newly recorded species is C. hexagonospora. Conocybe verna is found in spring in broad-leaved forests and has a campanulate pileus, which lacks pubescence. It has 2-spored basidia and basidiospores are with a suprahilar depression, which distinguishes it from other species in sect. Pilosellae. On the other hand, C. angulispora is found in mixed forests and has an obtusely conical pileus with distinct pubescence, and basidiospores are lentiform, angular, submitriform, or slightly hexagonal in frontal view. Conocybe rubrocyanea grows on cow dung, with macroscopic features similar to C. incarnata, but the basidiospores of C. rubrocyanea are lentiform in shape, and the pileipellis contains blue lilac pigment. Both dried specimens in water and KOH solution secrete blue-purple pigments. Among the three new species, C. rubrocyanea is a particularly unique species. Its caulocystidia predominantly exhibit a non-lecythiform shape, which aligns with the stipe type in sect. Pilosellae. However, its pileipellis is composed of spheropedunculate and fusiform elements, some of which are partly rostellate, characteristics that are consistent with the classification features of sect. Obscurae Hauskn. & Krisai (Hausknecht and Krisai-Greilhuber 2006). Currently, the sect. Obscurae includes only one species, C. obscura Watling, which is found in the Democratic Republic of the Congo in Africa. The caulocystidia of C. obscura also conform to the stipe type in sect. Pilosellae, yet it lacks molecular sequences, leaving its taxonomic position unclear. It is uncertain whether C. obscura clusters with C. rubrocyanea, especially since its basidiospores are neither lentiform nor hexagonal, making them easy to distinguish from those of C. rubrocyanea (Watling 1973). Although the morphological features of C. rubrocyanea are consistent with other species of sect. Obscurae, its position on the basis of the molecular phylogeny is actually within sect. Pilosellae. If C. obscura and C. rubrocyanea do not cluster together, this would suggest that pileipellis characteristics are not used as criteria for distinguishing sections. In this case, C. obscura would also belong to sect. Pilosellae. To resolve this issue, further research on the holotype specimen of C. obscura is necessary before reconsidering the classification of C. obscura and C. rubrocyanea. In this study the holotype of C. hexagonospora lacks a sequence, the macroscopic and microscopic structures of specimen FJAU71661 are consistent with that of C. hexagonospora, thus confirming FJAU71661 as C. hexagonospora (Hausknecht 2009). Additionally, C. siliginea (FJAU71664), collected from Henan Province, grows in greenhouse soil, with a lime-colored pileus and 2-spored basidia. We identified specimen FJAU71662 as C. velutipes based on its macroscopic and microscopic structures. Subsequently, we obtained the ITS, nrLSU, and tef1-α sequences of specimen FJAU71662, which are similar to those of C. velutipes (SZMC-NL-2187), with an ITS similarity of 99.7%, nrLSU similarity of 99.9%, and tef1-α similarity of 99.2%. This further supports the correctness of our traditional taxonomic identification of the species. Hausknecht and Contu (2007) has already stated that C. lenticulospora is a synonym of C. brunneidisca, but since 2007, some have continued to use C. lenticulospora as a species name, and it is still treated as an independent species in MycoBank and Index Fungorum. Liu (2018) described and illustrated specimen HMJAU45069 as C. lenticulospora Watling, with its ITS sequence showing a similarity of 99.7% to C. lenticulospora (SZMC-NL-0923). Therefore, we reexamined HMJAU45069, and its basidiospores measured 9.5–13.5 × 6.5–8.5 × 5.5–7.5 μm, lentiform in shape, with a nearly hexagonal or submitriform frontal view and an ellipsoid to oblong side view. The microscopic features were consistent with those of C. brunneidisca, supporting Hausknecht and Contu’s (2007) viewpoint that C. lenticulospora is a synonym of C. brunneidisca.

Based on morphological classification, Hausknecht and Krisai-Greilhuber (2006) divided sect. Pilosellae into 2 subsections and 11 series, contributing significantly to this field. However, when molecular techniques and phylogenetic methods were applied to taxonomy, the correlation between morphological classification and phylogeny revealed some discrepancies. For instance, C. cylindracea, classified under ser. Cylindracea, clustered with C. brunneidisca, which is the type species of ser. Lenticulospora. Given that C. cylindracea has lentiform and slightly angular basidiospores, we propose placing it in ser. Lenticulospora, a change supported by phylogenetic analysis (Fig. 1). This finding suggests that pileus shape is not a reliable feature for series classification. Similarly, C. angulispora and C. hexagonospora, discovered in Jilin province, fit the definition of ser. Lenticulospora. Consequently, the branch consisting of C. brunneidisca, C. cylindracea, C. angulispora, and C. hexagonospora is now designated as ser. Lenticulospora, based on consistency between morphological and phylogenetic analyses. However, C. rubrocyanea, which possesses lentiform basidiospores and a hexagonal frontal view does not cluster with ser. Lenticulospora. This disparity between morphological and phylogenetic congruence is also observed in other series. For example, C. incarnata, belonging to ser. Microrrhiza due to its pseudorhiza, clusters with C. muscicola and C. rubrocyanea, making it challenging to differentiate it from other series. Although all these species share the common feature of a reddish pileus, we do not introduce it as a new series. This decision is based on the extensive description and recording of nearly 60 species within sect. Pilosellae by Hausknecht (2009), with many species lacking sequences. Consequently, it remains uncertain whether other species can cluster with the branch containing C. rubrocyanea.

This article primarily introduces three new species from Jilin province and a new record for China. Additionally, a key to differentiate the 22 species within sect. Pilosellae in China is provided. However, the phylogenetic positions of the series within sect. Pilosellae are still uncertain. To address this issue, a substantial number of specimens and sequences are required to identify stable shared characteristics for distinguishing different branches. Further in-depth research is needed to investigate this matter.

Acknowledgements

We sincerely thank the team for their help. Thank you for the support of the National Natural Science Foundation of China (32270001).

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was supported by the National Natural Science Foundation of China (32270001).

Author contributions

Conceptualization: Tolgor Bau and Han-Bing Song. Methodology: Han-Bing Song. Software: Han-Bing Song. Validation: Han-Bing Song and Tolgor Bau. Formal analysis: Han-Bing Song. Investigation: Han-Bing Song and Tolgor Bau. Resources: Han-Bing Song and Tolgor Bau. Data curation: Han-Bing Song and Tolgor Bau. Writing original draft preparation: Han-Bing Song. Writing review and editing: Han-Bing Song and Tolgor Bau. Visualization: Han-Bing Song and Tolgor Bau. Supervision: Tolgor Bau. Project administration: Tolgor Bau. Funding acquisition: Tolgor Bau.

Author ORCIDs

Han-bing Song https://orcid.org/0000-0002-7440-5444

Tolgor Bau https://orcid.org/0000-0003-2461-9345

Data availability

All the sequences have been deposited in GenBank (https://www.ncbi.nlm.nih.gov) and Mycobank (https://www.mycobank.org). The data presented in this study are deposited in the Zenodo repository, accession number https://doi.org/10.5281/zenodo.14836573.

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