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Research Article
Biotrophic and saprophytic fungi from the Rhodocybe-Clitopilus clade (Entolomataceae): two new species and one new record in subtropical China
expand article infoSipeng Jian‡, Xia Chen§, Tianwei Yang‡, Xinjing Xu‡, Feng Gao‡, Yiwei Fang‡, Jing Liu‡, Chunxia Zhang‡
‡ Yunnan Institute of Tropical Crops, Jinghong, China
§ The Administration Bureau of Dr. Sun Yat-sen’s Mausoleum, Nanjing, China

Corresponding author: Chunxia Zhang ( 253394315@qq.com )

Academic editor: Ajay Kumar Gautam
© 2025 Sipeng Jian, Xia Chen, Tianwei Yang, Xinjing Xu, Feng Gao, Yiwei Fang, Jing Liu, Chunxia Zhang.
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: Jian S, Chen X, Yang T, Xu X, Gao F, Fang Y, Liu J, Zhang C (2025) Biotrophic and saprophytic fungi from the Rhodocybe-Clitopilus clade (Entolomataceae): two new species and one new record in subtropical China. MycoKeys 116: 227-254. https://doi.org/10.3897/mycokeys.116.148775
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Abstract

This study proposes two new species and a new record in the Rhodocybe-Clitopilus clade, based on comprehensive morphological and molecular analyses. The nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), the large subunit ribosomal RNA gene (LSU), the RNA polymerase II second largest subunit (RPB2) and the translation elongation factor 1-alpha gene (TEF1), were employed to elucidate the relationships of Clitopilus and Rhodocybe. The first species, Clitopilus parasiticus, is capable of infecting the leaves of host plants in the genera Dryopteris and Oplismenus, exhibiting typical biotrophic behaviour while also demonstrating saprophytic growth on soil. Intraspecific comparisons were conducted, examining environmental factors as well as macro- and microscopic characteristics amongst individuals found on different plant hosts. Furthermore, this study reports the new saprophytic species, Rhodocybe zijinshanensis and provides a detailed description of Clitopilus baronii, a newly-recorded species in China.

Key words:

Biotrophic species, Entolomataceae, morphology, multigene phylogeny, plant pathogens, taxonomy

Introduction

In nature, numerous fungi are well-known for their parasitic relationships, enabling them to thrive in dynamic environments. For example, the ergot (Claviceps purpurea (Fr.) Tul.) and corn smut (Mycosarcoma maydis (DC.) Bref.) are recognised as pathogenic fungi affecting cultivated plants (Triticum aestivum L. and Zea mays L., respectively) (Tudzynski and Scheffer 2004; McTaggart et al. 2016). Additionally, several special form genera, such as Asterophora Ditmar, Squamanita Imbach and Hypomyces (Fr.) Tul. & C. Tul., exhibit fungicolous parasitism or mycoparasitic behaviour (Rogerson and Samuels 2018; Elkhateeb and Daba 2021; Liu et al. 2021). However, parasitic forms are relatively rare in Agaricales Underw., particularly for biotrophic parasitism.

Saprophytic and symbiotic modes of nutrition are predominant amongst fungi in Basidiomycetes, but some fungi also employ parasitic nutrition as a strategy for survival and reproduction (Põlme et al. 2021; Shi et al. 2023). Thereinto, biotrophic parasitism is an intriguing and unique phenomenon in fungi, defined as a nutritional strategy where fungi derive nutrients from a living host while keeping it alive, often causing restricted damage to the host plant (Luttrell 1974; Kemen and Jones 2012). Species within the Agaricales that exhibit distinct biotrophic capabilities often also possess saprophytic abilities, indicating that they are not obligate parasites. For example, Zhang et al. (2022) identified a new species, viz. Crepidotus herbaceus T. Bau & Y.P. Ge, which is not only parasitic on the leaves or stems of Oreocnide frutescens (Thunb.) Miq. and Alpinia japonica (Thunb.) Miq., but is also found on the plant debris or humus.

In the family Entolomataceae Kotl. & Pouzar, there are two main clades: Entoloma (Fr.) P. Kumm. and Rhodocybe-Clitopilus (Co-David et al. 2009; Baroni and Matheny 2011; Kluting et al. 2014). The Rhodocybe-Clitopilus clade differs from the Entoloma clade by its basidiospores, which are characterized by either longitudinal ridges or scattered, finely to distinctly pustulate ornamentations (Baroni 1981; Singer 1986; Kluting et al. 2014). Within the Rhodocybe-Clitopilus clade, most species are primarily regarded as saprophytic (Sanchez-Garcia and Matheny 2017). However, a few species, such as Rhodophana stangliana (Bresinsky & Pfaff) Vizzini, Clitopilus passeckerianus (Pilát) Sing., C. fasciculatus Noordel. and C. daamsii Noordel., also appear mycoparasite (Noordeloos 1984, 1993; Læssøe and Rosendahl 1994; Czederpiltz et al. 2001). Notably, C. hobsonii (Berk.) P.D. Orton has been reported to grow on stumps, fallen logs, twigs and living herbaceous leaves and stems (Orton 1960; Noordeloos 1984), highlighting its saprophytic and biotrophic capacities.

In the current study, several specimens gathered from Jiangsu Province are examined carefully. Three samples closely resembled Pleurotus (Fr.) P. Kumm., Crepidotus (Fr.) Staude and Omphalotus Fayod. Upon microscopic examination, they were all confirmed to the Rhodocybe-Clitopilus clade, respectively. Furthermore, two new species and one new record species were identified, based on the multi-gene phylogenetic tree. Therefore, all three species are described herein.

Materials and methods

Sample collections and morphological observations

The collection information of voucher specimens and the sequences used in phylogenetic analyses are shown in Table 1. The colour codes (hex triplets) from ColorHexa (https://www.colorhexa.com) were employed to depict the colour of basidiomata. These codes consist of characters ranging from a to f and 0 to 9, with each pair corresponding to the red, green and blue components of the colour. The general description of basidiomata, including both macro- and microscopic features, as well as the morphological classification rules in Clitopilus and Rhodocybe Maire were based on the work of Baroni (1981) and Jian et al. (2020a). All voucher specimens have been deposited at the Cryptogamic Herbarium of the Herbaria of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS).

Table 1.
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Sequencing primers and the best annealing temperature for ITS, LSU, RPB2 and TEF1.

Primer name Nucleotide sequence 5’-3’ PCR annealing temperature (°C)
ITS4 TCC TCC GCT TAT TGA TAT GC 52
ITS5 GGA AGT AAA AGT CGT AAC AAG G
LROR ACC CGC TGA ACT TAA GC 52
LR5 TCC TGA GGG AAA CTT CG
EF1-983F GCY CCY GGH CAY CGT GAY TTY AT 56/touchdown*
EF1-1953R CCR GCR ACR GTR TGT CTC AT
bRPB2-6F TGG GGY ATG GTN TGY CCY GC 52
bRPB2-7.1R CCC ATR GCY TGY TTM CCC ATD GC

Notes: * the details of the touchdown method refer to Kluting et al. (2014).

Sections of dried basidiomata were rehydrated in purified water and 5% potassium hydroxide (KOH) and were occasionally stained with 1% Congo Red to enhance visibility. The notation “[n/m/p]” indicates n basidiospores from m basidiomata of p specimens. The measurements of basidiospores are presented in the format (a–)b–c(–d), where the range b–c includes at least 90% of the measured values, while a and d (given in parentheses) represent the extreme values. The average length and width of basidiospore (± standard deviation) are denoted as Lm and Wm, respectively. The term Q refers to the “length/width ratio” of a basidiospore in side view, with Qavg representing the average Q across all specimens (± standard deviation). Fragments isolated from specimens were attached to aluminium stubs using double-sided adhesive tape, and then coated with gold/palladium. Finally, a ZEISS EVO LS10 (Germany) scanning electron microscope (SEM) was used to observe the ornamentation of the basidiospores.

The genetic names appeared in this study are abbreviated as follows: Clitopilus = “C.”, Rhodocybe = “R.”.

Molecular phylogenetic analyses

In this study, we utilised two sequences of non-protein-coding and two protein-coding genes: the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), the large subunit ribosomal RNA gene (LSU), the RNA polymerase II second largest subunit (RPB2) and the translation elongation factor 1-alpha gene (TEF1). The ITS and LSU genes were selected for their availability of universal primers (White et al. 1990), while the RPB2 and TEF1 genes were chosen due to their relatively high number of informative sites and sufficient nucleotide variation, which are essential for inferring evolutionary relationships within the Entolomataceae (Matheny et al. 2007; Co-David et al. 2009; Kluting et al. 2014). All the sequences were submitted to the National Center for Biotechnology Information (NCBI) and detailed information regarding each gene was provided in Table 1.

Genomic DNA was extracted from collected materials and herbarium specimens using the CTAB (cetyltrimethylammonium bromide) procedure outlined by Doyle and Doyle (1987). The PCR protocol followed the touchdown method described by Kluting et al. (2014), with detailed data provided in Table 2. Gel extraction and PCR (polymerase chain reaction) were conducted to purify the PCR products, which were then sequenced on an ABI-3730-XL sequence analyser (Applied Biosystems, Foster City, CA) using the same primers as in the PCR. The new sequences generated from this study are highlighted in bold in Table 1.

Table 2.
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Collection information of voucher specimen and GenBank accession numbers for sequences used in phylogenetic analyses. H in parentheses means the holotype specimen.

Species Collection or collector no. Location and year GenBank accession numbers References
ITS LSU RPB2 TEF1
C. abprunulus KUN-HKAS 107040a Macedonia 2019 NR_172792 NG_074438 MT349666 MT349670 Jian et al. (2020b)
C. abprunulus KUN-HKAS 107041a Macedonia 2019 MT345049 MT345054 MT349667 MT349671 Jian et al. (2020b)
C. abprunulus KUN-HKAS 107042a Macedonia 2019 MT345047 MT345052 MT349665 MT349669 Jian et al. (2020b)
C. abprunulus MEN 2003-09-14b Belgium 2003 KR261096 GQ289149 GQ289221 – Co-David et al. (2009)
C. albidus CAL 1319c Kerala State, India 2001 MF926596 MF926595 MF946579 – Raj and Manimohan (2018)
C. amygdaliformis KUN-HKAS 60406a Yunnan, China 2008 MN061292 – MN148120 – Jian et al. (2020a)
C. amygdaliformis KUN-HKAS 81125a Yunnan, China 2014 NR_172768 MN065681 MN148119 MN166231 Jian et al. (2020a)
C. amygdaliformis KUN-HKAS 87950a Yunnan, China 2014 MN061290 MN065680 MN148118 MN166230 Jian et al. (2020a)
“C. cf. argentinus” MTB 4804/2d Germany 2011 – – KC816907 KC816823 Kluting et al. (2014)
C. austroprunulus MEN2009001e Tahune, Australia 2009 KC139084 – – – Crous et al. (2012)
C. austroprunulus MEN2009062e Tasmania, Australia 2009 KC139085 – – – Crous et al. (2012)
C. baronii KUN-HKAS 145333 Jiangsu, China 2023 PQ793166 PQ781610 PQ788395 PQ788402 This study
C. baronii KUN-HKAS 145334 Jiangsu, China 2023 PQ793167 PQ781611 PQ788396 PQ788403 This study
C. baronii K(M)179703f UK 2012 MN855362 – MN856160 – Consiglio and Setti (2019)
C. baronii AMB 18359g Mantova, Italy 2006 MN855365 – MN856163 MN856174 Consiglio and Setti (2019)
C. baronii AMB 18362g Ferrara, Italy 2007 MN855368 – MN856166 MN856176 Consiglio and Setti (2019)
C. baronii AMB 18363g Mantova, Italy 2007 NR_176131 – MN856167 MN856177 Consiglio and Setti (2019)
C. baronii AMB 18378g Pisa, Italy 2007 MN855370 – MN856168 MN856178 Consiglio and Setti (2019)
C. brunneiceps KUN-HKAS 73123a Yunnan, China 2011 MN061294 MN065683 MN148122 MN166233 Jian et al. (2020a)
C. brunneiceps KUN-HKAS 80211a Hubei, China 2013 MN061293 MN065682 MN148121 MN166232 Jian et al. (2020a)
C. brunneiceps KUN-HKAS 104510a Yunnan, China 2018 NR_172769 MN065684 MN148123 MN166234 Jian et al. (2020a)
C. brunneiceps HMJAU 23509h Neimenggu, China 2013 MN061296 MN065685 MN148115 – Jian et al. (2020a)
C. chichawatniensis LAH37431i Punjab, Pakistan 2019 ON980767 ON980764 – – Fatima et al. (2022)
C. chichawatniensis LAH37432i Punjab, Pakistan 2020 ON980766 ON980763 – – Fatima et al. (2022)
C. chrischonensis TO HG1994j Basilea, Switzerland 2008 HM623128 HM623131 – – Vizzini et al. (2011)
“C. cinerascens” 8024 TJBd Florida, USA 1996 – GU384613 KC816908 KC816824 Kluting et al. (2014)
“C. cinerascens” 8133 TJBd Louisiana, USA 1996 – – KC816909 KC816825 Kluting et al. (2014)
C. cretoalbus LAH37017i Punjab, Pakistan 2020 OM935685 OM934826 – – Izhar et al. (2023)
C. cretoalbus LAH35709i Punjab, Pakistan 2017 ON117610 ON229505 – – Izhar et al. (2023)
C. crispus 9982 TJBd Chiang Mai, Thailand 2006 – – KC816910 KC816826 Kluting et al. (2014)
C. crispus 10027 TJBd Chiang Mai, Thailand 2006 – – KC816911 KC816827 Kluting et al. (2014)
C. crispus KUN-HKAS 84667a Yunnan, China 2014 MN061314 MN065705 MN148142 MN166254 Jian et al. (2020a)
C. crispus KUN-HKAS 87018a Yunnan, China 2014 MN061315 MN065706 MN148143 MN166255 Jian et al. (2020a)
C. crispus KUN-HKAS 90506a Yunnan, China 2015 MN061312 MN065702 MN148139 MN166251 Jian et al. (2020a)
C. crispus KUN-HKAS 90508a Yunnan, China 2015 – MN065703 MN148140 MN166252 Jian et al. (2020a)
C. crispus KUN-HKAS 97509a Yunnan, China 2016 MN061318 MN065708 MN148145 MN166258 Jian et al. (2020a)
C. crispus KUN-HKAS 102670a Yunnan, China 2017 MN061313 MN065704 MN148141 MN166253 Jian et al. (2020a)
C. crispus KUN-HKAS 104507a Yunnan, China 2017 MN061316 MN065707 MN148144 MN166256 Jian et al. (2020a)
C. cystidiatus MEN 200350 Slovakia 2003 – GQ289147 GQ289220 – Co-David et al. (2009)
C. fasciculatus MO#297071 California, USA 2017 MG551863 – – – Direct submission
C. fusiformis SAAS 1038k Yunnan, China 2015 KY385634 – KY385632 – Wang et al. (2017)
C. fusiformis SAAS 1892k Yunnan, China 2015 NR_158328 – KY385633 – Wang et al. (2017)
C. fusiformis KUN-HKAS 104513a Yunnan, China 2018 MN061297 MN065686 MN148124 MN166235 Jian et al. (2020a)
C. fusiformis KUN-HKAS 104514a Yunnan, China 2018 MN061298 MN065687 MN148125 MN166236 Jian et al. (2020a)
C. fusiformis KUN-HKAS 104515a Yunnan, China 2018 MN061300 MN065690 MN148128 MN166239 Jian et al. (2020a)
C. fusiformis KUN-HKAS 104516a Yunnan, China 2018 MN061299 MN065688 MN148126 MN166237 Jian et al. (2020a)
C. fusiformis KUN-HKAS 104517a Yunnan, China 2018 – MN065689 MN148127 MN166238 Jian et al. (2020a)
C. giovanellae S.F.14368l Trento, Italy 1888 EF413030 EF413027 – – Moreno et al. (2007)
C. giovanellae AH 19780m Spain 1998 – EF413026 – – Moreno et al. (2007)
C. highlandensis KUN-HKAS 68389a Yunnan, China 2010 MN061310 MN065700 MN148137 MN166249 Jian et al. (2020a)
C. highlandensis KUN-HKAS 117632a Yunnan, China 2021 ON999061 ON999062 OP006563 OP006564 Jian et al. (2023)
C. hobsonii K(M) 167650f UK 2010 MN855371 – MN856169 – Consiglio and Setti (2019)
C. hobsonii K(M) 122842f UK 2004 NR_182819 – MN856170 – Consiglio and Setti (2019)
C. hobsonii K(M) 199928f UK 2015 MN855373 – MN856171 – Consiglio and Setti (2019)
“C. hobsonii” QYL10 – OK652826 OK655769 MN092372 MN092373 Peng et al. (2021)
“C. hobsonii” DLL 9779 Queensland, Australia 2010 – – KC816916 KC816831 Kluting et al. (2014)
“C. hobsonii” 5967 TJBd New York, USA 1988 – – KC816917 – Kluting et al. (2014)
“C. hobsonii” DLL 9586 Queensland, Australia 2009 – KJ021698 KC816912 KC816828 Kluting et al. (2014)
“C. hobsonii” DLL 9635 Queensland, Australia 2009 – – KC816913 KC816829 Kluting et al. (2014)
“C. hobsonii” DLL 9643 Queensland, Australia 2009 – – KC816914 – Kluting et al. (2014)
“C. hobsonii” DLL 9746 Queensland, Australia 2010 – – KC816915 KC816830 Kluting et al. (2014)
“C. hobsonii grp.” 7051 TJBd North Carolina, USA 1993 – – KC816918 – Kluting et al. (2014)
C. aff. hobsonii K:M195388f UK 2014 MN855375 – MN856172 MN856179 Consiglio and Setti (2019)
“C. aff. hobsonii” UC 1860830n California, USA 2011 – – KC816928 KC816841 Kluting et al. (2014)
C. cf. kamaka KA12-0364° South Korea 2012 KR673433 – – – Kim et al. (2015)
C. kamaka PDD 96106p New Zealand 2010 NR_137867 – – – Cooper (2014)
C. lampangensis SDBR-CMUJK 0147q Lampang, Thailand 2018 NR_175631 MK764935 MK784129 – Kumla et al. (2019)
C. lampangensis SDBR-CMUNK 0047q Lampang, Thailand 2018 MK764934 MK773856 MK784128 – Kumla et al. (2019)
C. orientalis CAL 1613c Kerala State, India 2011 MG345134 MG321558 MG321559 – Raj and Manimohan (2018)
C. parasiticus KUN-HKAS 145335a Jiangsu, China 2023 PQ793168 PQ781612 PQ788397 PQ788404 This study
C. parasiticus (H) KUN-HKAS 145336a Jiangsu, China 2024 PQ793169 PQ781613 PQ788398 – This study
C. parasiticus KUN-HKAS 145337a Jiangsu, China 2024 PQ793170 PQ781614 PQ788399 PQ788405 This study
C. passeckerianus CBS:299.35r Austria – MH855682 MH867198 – – Vu et al. (2019)
C. passeckerianus P73 South Korea 2015 KY962489 KY963073 – – Direct submission
C. passeckerianus P78 South Korea 2015 KY962494 KY963078 – – Direct submission
C. passeckerianus K:M134571f UK 2005 MN855376 – MN856173 – Consiglio and Setti (2019)
C. paxilloides 5809 TJBd California, USA 1987 – – KC816919 KC816832 Kluting et al. (2014)
“C. peri” 10040 TJBd Chiang Mai, Thailand 2006 – – KC816921 KC816834 Kluting et al. (2014)
“C. peri” 10033 TJBd Chiang Mai, Thailand 2006 – – KC816920 KC816833 Kluting et al. (2014)
“C. peri” 10041 TJBd Chiang Mai, Thailand 2006 – – KC816922 KC816835 Kluting et al. (2014)
C. pinsitus CBS 623.70r England, UK – MH859879 MH871665 – – Vu et al. (2019)
C. pinsitus G. Immerzeel 1990-11 Netherlands 1990 – GQ289148 – – Co-David et al. (2009)
“C. prunulus” CORT:11CA012d California, USA 2011 – – KC816926 KC816839 Kluting et al. (2014)
C. prunulus REH8456d Novgorod Region, Russa 2003 – – KC816923 KC816836 Kluting et al. (2014)
“C. prunulus” 6805 TJBd New York, USA 1992 – – KC816924 KC816837 Kluting et al. (2014)
“C. prunulus” TJB 9425d Dominican Republic 2002 – – MN893320 MN893330 Baroni et al. (2020)
“C. prunulus” AFTOL522, TJB6838d USA – DQ202272 AY700181 – – Direct submission
“C. prunulus” TB8229d New York, USA 1996 – GU384615 GU384650 – Baroni et al. (2011)
“C. prunulus” TB9663d – – GU384614 GU384648 – Baroni et al. (2011)
C. prunulus KUN-HKAS 96158a Austria 2016 MN061301 MN065691 MN148129 MN166240 Jian et al. (2020a)
C. prunulus KUN-HKAS 123138a France – OP626992 OP646418 OP939970 OP687894 He et al. (2023)
C. prunulus HMJAU 4521s Kirov, Russia 2006 MN061302 MN065692 MN148117 MN166241 Jian et al. (2020a)
C. cf. prunulus KUN-HKAS 75845a California, USA 2011 MN061303 MN065693 MN148130 MN166242 Jian et al. (2020a)
C. ravus KUN-HKAS 107043a Yunnan, China 2019 MT345050 MT345055 MT349668 MT349672 Jian et al. (2020b)
C. reticulosporus WU27150b Vienna, Austria 2004 KC885966 HM164412 HM164416 – Morgado et al. (2016)
C. rugosiceps KUN-HKAS 57003a Yunnan, China 2009 MN061304 MN065694 MN148131 MN166243 Jian et al. (2020a)
C. rugosiceps KUN-HKAS 59455a Yunnan, China 2009 – MN065696 MN148133 MN166245 Jian et al. (2020a)
C. rugosiceps KUN-HKAS 73232a Yunnan, China 2011 NR_172771 MN065695 MN148132 MN166244 Jian et al. (2020a)
C. rugosiceps KUN-HKAS 107044a Yunnan, China 2019 MT345046 MT345051 – – Jian et al. (2020b)
C. rugosiceps KUN-HKAS 115921a Yunnan, China 2017 MZ855871 MZ853557 MZ826364 MZ826362 He and Yang (2022)
C. scyphoides CBS 127.47r France – MH856181 MH867707 – – Vu et al. (2019)
C. cf. scyphoides KUN-HKAS 104511a Gansu, China 2016 MN061329 MN065720 MN148157 MN166268 Jian et al. (2020a)
C. sinoapalus KUN-HKAS 77037a Jiangxi, China 2012 MN061321 MN065713 MN148149 – Jian et al. (2020a)
C. sinoapalus KUN-HKAS 82230a Guangzhou, China 2013 MN061320 MN065712 MN148148 – Jian et al. (2020a)
C. sinoapalus KUN-HKAS 83831a Yunnan, China 2014 – MN065714 MN148150 – Jian et al. (2020a)
C. sinoapalus KUN-HKAS 101191a Yunnan, China 2017 NR_172773 MN065711 MN148151 MN166261 Jian et al. (2020a)
C. sinoapalus KUN-HKAS 102737a Yunnan, China 2017 – MN065709 MN148146 MN166259 Jian et al. (2020a)
C. sinoapalus KUN-HKAS 102807a Yunnan, China 2017 MN061319 MN065710 MN148147 MN166260 Jian et al. (2020a)
C. subalbidus GDGM 72219t Guangdong, China 2018 NR_198267 NG_243733 ON959185 ON959190 Jian et al. (2023)
C. subalbidus GDGM 72229t Guangdong, China 2018 ON963952 ON963946 ON959186 – Jian et al. (2023)
C. subscyphoides CAL 1325c Kerala State, India 2011 MF927542 MF946580 MF946581 – Raj and Manimohan (2018)
C. subscyphoides GDGM 72195t Guangdong, China 2018 – – ON959188 – Jian et al. (2023)
C. subscyphoides GDGM 72683t Guangdong, China 2018 ON963953 ON963947 – – Jian et al. (2023)
C. subscyphoides GDGM 73056t Guangdong, China 2018 ON963954 ON963948 ON959187 ON959191 Jian et al. (2023)
C. umbilicatus KUN-HKAS 80289a Hunan, China 2013 MN061323 MN065715 MN148152 MN166262 Jian et al. (2020a)
C. umbilicatus KUN-HKAS 80310a Anhui, China 2013 MN061324 MN065716 MN148153 MN166263 Jian et al. (2020a)
C. umbilicatus KUN-HKAS 80370a Fujian, China 2013 MN061325 MN065717 MN148154 MN166264 Jian et al. (2020a)
C. umbilicatus KUN-HKAS 80945a Anhui, China 2013 MN061326 MN065718 MN148155 MN166265 Jian et al. (2020a)
C. umbilicatus KUN-HKAS 104509a Yunnan, China 2017 MN061327 MN065719 MN148156 MN166266 Jian et al. (2020a)
C. velutinus CORT 014618d Dominican Republic 2015 MN784991 – MN893321 MN893331 Baroni et al. (2020)
C. venososulcatus 8111 TJBd Louisiana, USA 1996 – – KC816930 – Kluting et al. (2014)
C. yunnanensis KUN-HKAS 59712a Yunnan, China 2009 MN061307 – MN148135 – Jian et al. (2020a)
C. yunnanensis KUN-HKAS 82076a Yunnan, China 2012 MN061306 MN065697 MN148134 MN166246 Jian et al. (2020a)
C. yunnanensis KUN-HKAS 104518a Yunnan, China 2018 MN061308 MN065698 MN148136 MN166247 Jian et al. (2020a)
C. yunnanensis HMJAU 24677s Sichuan, China 2013 MN061309 MN065699 MN148116 MN166248 Jian et al. (2020a)
“Clitopilus sp.” 7130 TJBd New York, USA 1993 – – KC816929 – Kluting et al. (2014)
Clitopilus sp. TB8067d Florida, USA 1996 – GU384612 GU384649 – Baroni et al. (2011)
Clitopilus sp. KUN-HKAS 104508a Yunnan, China 2017 MN061311 MN065701 MN148138 MN166250 Jian et al. (2020a)
Clitopilus sp. KUN-HKAS 104512a Yunnan, China 2018 MN061330 MN065721 MN148158 MN166269 Jian et al. (2020a)
R. alutacea 5726 TJBd North Carolina, USA 1987 – – KC816931 KC816842 Kluting et al. (2014)
R. asanii KATO 3659u Turkey 2015 KX834263 KX834264 – – Seslİ and Vizzini (2017)
R. asanii KATO 3657u Turkey 2015 KX834265 – – – Seslİ and Vizzini (2017)
R. asanii NA13102020 East Sussex, UK 2020 MW375030 – – – Aplin et al. (2022)
R. asyae KATO 3640u Trabzon, Turkey 2015 KX834266 KX834267 – – Seslİ and Vizzini (2017)
R. asyae KATO 3653u Trabzon, Turkey 2015 KX834268 – – – Seslİ and Vizzini (2017)
R. asyae NA131019v East Sussex, UK 2019 MN840644 – – – Aplin et al. (2022)
R. aureicystidiata PBM 1902w Washington, USA – – AY380407 AY337412 – Matheny (2005)
R. brunneoaurantiaca CAL 1825c West Bengal, India 2019 MW031906 MW031916 – – Dutta et al. (2021)
R. brunneoaurantiaca CUH AM720x West Bengal, India 2019 MW023201 MW023223 – – Dutta et al. (2021)
R. brunnescens TENN 056140y Tennessee, USA 1985 NR_119914 NG_058820 – – Baroni et al. (2011)
R. brunnescens TENN 056140-2y Tennessee, USA 1987 HQ222033 JF706313 – – Baroni et al. (2011)
R. byssisedoides AG 2004-04-27 Jena, Germay 2004 – GQ289212 GQ289279 – Co-David et al. (2009)
R. caelata 511 Germany 2005 – GQ289208 – – Co-David et al. (2009)
“R. caelata” 6919 TJBd North Carolina, USA 1992 – – KC816933 KC816843 Kluting et al. (2014)
R. caelata J. Parkind Ontario, Canada 1988 – – KC816934 – Kluting et al. (2014)
R. caelata REH3569d Jurmala, Latvia 1982 – – KC816932 – Kluting et al. (2014)
R. caelata TB5890d – – AF261282 – – Moncalvo et al. (2002)
“R. caelata” TB6995d – – GU384625 GU384652 – Baroni et al. (2011)
R. cistetorum KATO 4260u Trabzon, Turkey 2019 NR_176724 MT252601 – – Seslİ (2021)
R. collybioides 10417 TJBd Jujuy, Argentina 2011 – – KC816935 KC816844 Kluting et al. (2014)
R. dominicana ANGE 464 Dominican Republic 2014 – – MN893322 MN893332 Baroni et al. (2020)
R. dominicana ANGE 473 Dominican Republic 2014 – – MN893323 MN893333 Baroni et al. (2020)
R. formosa 1061015-6d Catalonia, Spain 2006 KU862856 – KC816939 KC816849 Kluting et al. (2014)
R. formosa 12/198 Latium, Italy 2012 KU862857 – – – Vizzini et al. (2016)
R. formosa 12/208 Latium, Italy 2012 KU862858 – – – Vizzini et al. (2016)
R. formosa 1071101-4d Catalonia, Spain 2007 KU862860 – KC816947 KC816857 Kluting et al. (2014)
R. formosa K(M): 158060f England, UK 2006 MZ159381 – KC816978 KC816885 Direct submission
R. fuliginea E537d Tasmania, Australia 1999 – – KC816940 KC816850 Kluting et al. (2014)
R. fumanellii HFRG_PC200928_1 Buckinghamshire, UK 2020 MW401761 – – – Aplin et al. (2022)
R. fumanellii BOLGH_22122001 Tuscany, Italy 2022 OR831361 – – – Direct submission
R. fumanellii MCVE 29550z Veneto, Italy 2017 MH399225 MH399226 – – Vizzini et al. (2018)
R. fusipes DLK 587aa Amazonas, Brazil 2012 MN306209 – – – Silva-Filho et al. (2020)
R. fusipes DLK 298aa Amazonas, Brazil 2012 MN306210 – – – Silva-Filho et al. (2020)
R. gemina GZ 2003-09-14 Belgium 2003 – – GQ289277 – Co-David et al. (2009)
“R. gemina” MEN 2001119 – 2001 – HM164411 – – Morgado et al. (2016)
R. gemina CBS 604.76r – – AF223168 – – Vu et al. (2019)
R. gemina KATO 2658u Turkey 2009 – KX834269 – – Seslİ and Vizzini (2017)
R. gemina CBS 482.50r – EF421110 AF223167 EF421019 KP255478 Baroni et al. (2011)
R. griseoaurantia CAL 1324c Kerala, India 2011 NR_154435 KX083574 KX083568 – Hyde et al. (2016)
R. griseonigrella 1081204ab Barcelona, Spain 2008 KU862859 – – – Vizzini et al. (2016)
R. hondensis 6103 TJBd California, US 1988 – – KC816941 KC816851 Kluting et al. (2014)
R. incarnata REH5369 Venezuela 1987 MT254071 – – – Silva-Filho et al. (2020)
R. indica CAL 1323c Kerala, India 2013 KX083569 NG_060166 KX083566 – Hyde et al. (2016)
R. lateritia Co-David 418 – – HM164410 – – Morgado et al. (2016)
R. lateritia E1589d Tasmania, Australia 2002 – – KC816942 KC816852 Kluting et al. (2014)
R. luteobrunnea CAL 1322c Kerala, India 2010 NR_154434 NG_060167 KX083567 – Hyde et al. (2016)
R. luteocinnamomea GUA241d Guana Island, UK 1999 – – KC816943 KC816853 Kluting et al. (2014)
R. luteocinnamomea var. fulva ANGE 169 Dominican Republic 2013 – – MN893324 MN893334 Baroni et al. (2020)
R. matesina MCVE 29262z Campania, Italy 2012 KY629961 KY629963 – – Crous et al. (2017)
R. matesina MCVE 29261z Campania, Italy 2016 KY629962 KY629964 – – Crous et al. (2017)
R. matesina F3-2 Fnaydek, Lebanon 2018 MZ088085 – – – Sleiman et al. (2021)
“R. mellea” ANGE 893 Dominican Republic 2016 MN784993 – MN893326 – Baroni et al. (2020)
“R. mellea” TJB 9823d Belize 2004 MN784994 – – – Baroni et al. (2020)
R. mellea NYBG815044 Costa Rica 1986 MN784995 – – – Baroni et al. (2020)
“R. mellea” 6883 TJBd Florida, USA 1992 – MG702608 KC816944 KC816854 Kluting et al. (2014)
“R. mellea” TJB 9805d Dominican Republic 2003 MN784992 – MN893325 – Baroni et al. (2020)
R. mellea var. depressa FW 08/2019 Brazil 2019 MT408926 OL687341 – – Xavier et al. (2022)
R. nuciolens WTU-F-074620 Washington, USA 2017 OP828513 – – – Direct submission
R. nuciolens TENN:076696y Washington, USA 2021 ON478246 – – – Direct submission
R. nuciolens iN147673878 California, USA 2023 OR162504 – – – Direct submission
R. nuciolens iN147466901 California, USA 2023 OR168848 – – – Direct submission
R. pakistanica LAH37947i Punjab, Pakistan 2022 OR606543 OR606541 – – Khan and Khalid (2024)
R. pakistanica LAH37948i Punjab, Pakistan 2022 OR606544 OR606542 – – Khan and Khalid (2024)
R. pallidogrisea CORT 013944d Australia – NR_154437 – – – Direct submission
R. pallidogrisea 118 Tasmania, Australia 2004 – GQ289216 GQ289283 – Co-David et al. (2009)
R. pallidogrisea E652d Tasmania, Australia 1999 – – KC816968 KC816875 Kluting et al. (2014)
R. paurii JM99/233 Uttaranchal, India 1999 – AY286004 – – Moncalvo et al. (2004)
R. paurii JM99/233-2 Uttaranchal, India 1999 – – KC816969 KC816876 Kluting et al. (2014)
R. praesidentialis MCVE 21991z Italy – EF679793 – – – Consiglio et al. (2007)
R. pruinosostipitata MCA1492 Guyana – – GU384627 GU384653 – Baroni et al. (2011)
R. pseudoalutacea TJB 9466d Dominican Republican 2003 – – MN893327 MN893335 Baroni et al. (2020)
R. pseudoalutacea TJB 9507d Dominican Republican 2003 – – MN893328 MN893336 Baroni et al. (2020)
R. pseudopiperita E1159d Tasmania, Australia 2001 – – KC816979 KC816886 Kluting et al. (2014)
R. pseudopiperita 162 Tasmania, Australia 2004 – GQ289217 GQ289284 – Co-David et al. (2009)
R. reticulata E2183d Tasmania, Australia 2005 – – KC816980 KC816887 Kluting et al. (2014)
R. rhizogena 5551 TJBd North Carolina, USA 1987 – – KC816981 KC816888 Kluting et al. (2014)
R. roseiavellanea 8130 TJBd Louisiana, USA 1996 – KR869930 KC816982 KC816889 Kluting et al. (2014)
R. roseiavellanea PBM4056 Tennessee, USA – MF686525 – – – Direct submission
R. roseiavellanea ANGE 947 Dominican Republic 2017 – – MN893329 MN893337 Baroni et al. (2020)
R. rubrobrunnea CAL 1387c Kerala, India 2014 KX951452 – – – Crous et al. (2016)
Rhodocybe sp. DLL9851 New South Wales, Australia 2010 – – KC816986 KC816893 Kluting et al. (2014)
Rhodocybe sp. DLL9846 New South Wales, Australia 2010 – – KC816985 KC816892 Kluting et al. (2014)
Rhodocybe sp. DLL9860 New South Wales, Australia 2010 – – KC816987 KC816894 Kluting et al. (2014)
Rhodocybe sp. DLL9952 New South Wales, Australia 2010 – – KC816988 KC816895 Kluting et al. (2014)
Rhodocybe sp. DLL9957 New South Wales, Australia 2010 – – KC816989 KC816896 Kluting et al. (2014)
Rhodocybe sp. DLL10218 New South Wales, Australia 2011 – – KC816990 KC816897 Kluting et al. (2014)
Rhodocybe sp. DLL10032 Queensland, Australia 2011 – – KC816991 KC816898 Kluting et al. (2014)
Rhodocybe sp. KUN-HKAS 89081a Yunnan, China 2023 MZ675559 MZ675570 MZ681892 MZ681870 He and Yang (2022)
Rhodocybe sp. MEL:2382939 Palmerston, Australia 2014 KP012803 – – – Direct submission
Rhodocybe sp. MEL:2382705 Australia 2014 KP012885 – – – Direct submission
Rhodocybe sp. KS-RE53 New Zealand – – MK277733 – – Varga et al. (2019)
Rhodocybe sp. Buyck 99.152 Madagascar – – MK278564 – – Varga et al. (2019)
Rhodocybe sp. Sulzbacher 340 Brazil – LT594979 – – – Sulzbacher et al. (2017)
Rhodocybe sp. Sulzbacher 413 Brazil – LT594984 – – – Sulzbacher et al. (2017)
Rhodocybe sp. HFRG_EJ171117_1 Hampshire, UK 2017 MW397197 MW397521 – – Aplin et al. (2022)
Rhodocybe sp. iN130319090 Indiana, USA 2022 OP749482 – – – Direct submission
Rhodocybe sp. iN129753148 Indiana, USA 2022 OP749140 – – – Direct submission
Rhodocybe sp. iN130020200 Indiana, USA 2022 OP643320 – – – Direct submission
Rhodocybe sp. AD5 (TENN)y Tennessee, USA 2011 MF773647 – – – Direct submission
R. stipitata 5523 TJBd Tennessee, USA 1987 – – KC816993 – Kluting et al. (2014)
R. spongiosa MCA2129 – GU384628 GU384657 – Baroni et al. (2011)
R. subasyae HMJAU56921-1s Jilin, China 2020 MW298803 – – – Sun and Bau (2023)
R. subasyae HMJAU56921-2s Jilin, China 2020 MW298804 – – – Sun and Bau (2023)
R. subasyae HMJAU56921-3s Jilin, China 2020 MW298805 – – – Sun and Bau (2023)
R. tugrulii KATO 3340u Trabzon, Turkey 2014 KX271751 KX271754 – – Vizzini et al. (2016)
R. tugrulii MSNG3938 Italy – KY945354 – – – Direct submission
R. tugrulii CORT:14755d New York, USA 2018 MZ322093 – – – Direct submission
R. tugrulii IMG-7316d New York, USA 2017 MG050105 MG050111 – – Direct submission
R. tugrulii WU-MYC 0010084b Burgenland, Austria 1991 OP363995 – – – Vizzini et al. (2023)
R. tugrulii WU-MYC 0022202b Niederosterreich, Austria 2002 OP363994 OP363999 OP381082 OP381084 Vizzini et al. (2023)
R. tugrulii WU-MYC 0006178b Niederosterreich, Austria 1987 – OP364000 – – Vizzini et al. (2023)
R. tugrulii WU-MYC 0006320b Niederosterreich, Austria 1987 OP363992 OP363997 OP381080 OP381083 Vizzini et al. (2023)
R. tugrulii WU-MYC 0004222b Niederosterreich, Austria 1984 OP363991 – – – Vizzini et al. (2023)
R. tugrulii WU-MYC 0003753b Niederosterreich, Austria 1984 OP363993 OP363998 OP381081 – Vizzini et al. (2023)
R. tugrulii GB-013 1395 Skaane, Sweden 1983 OP363996 OP364001 – – Vizzini et al. (2023)
R. zijinshanensis (H) KUN-HKAS 145338a Jiangsu, China 2024 PQ793171 PQ781615 PQ788400 PQ788406 This study
R. zijinshanensis KUN-HKAS 145339a Jiangsu, China 2024 PQ793172 PQ781616 PQ788401 PQ788407 This study
Lulesia umbrinomarginata MHHNU 20023-2 Guangdong, China 2023 PP060632 PP059607 PP158704 PP158696 Xiao et al. (2024)
Lulesia orientalis KUN-HKAS 75548a Hubei, China 2012 MN061333 MN065727 MN148164 MN166275 Jian et al. (2020a)
Clitopilopsis albida KUN-HKAS 104520a Yunnan, China 2018 MN061336 MN065731 MN148168 MN166279 Jian et al. (2020a)
Clitopilopsis hirneola MEN 199956 Italy – KC710132 GQ289211 GQ289278 – Co-David et al. (2009)

a The Cryptogamic Herbarium of Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, China (KUN-HKAS) b Herbarium, Institute of Botany, University of Vienna, Austria (WU) c Central National Herbarium, Kolkata, India (CAL) d State University of New York College at Cortland Herbarium, Cortland, New York, USA (CORT) e The Leiden Herbarium of Naturalis Biodiversity Center, Leiden, Netherland (L) f The Royal Herbarium, Royal Botanic Garden, Kew, Richmond, Surrey, England, UK (KEW) g Associazione Micologica Bresadola, Trento, Italy (AMB) h The Herbarium of Mycology, Jilin Agricultural University, Changchun, Jilin, China (HMJAU) i The Lahore, Institute of Botany, University of the Punjab, Pakistan (LAH) j Herbarium generale del Dipartimento di Biologia Vegetale, Università degli Studi di Torino, Italy (TO) k The Herbarium of Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Sichuan, China (SAAS) l The Swedish Museum of Natural History, Stockholm, Sweden (S) m The Herbarium of the University of Alcalá, Madrid, Spain (AH) n Jepson Herbarium, University of California, Berkeley, California, USA (JEPS) ° The Herbarium of Korea National Arboretum, Gyeonggi-do, Republic of Korea (KH) p New Zealand Fungal Herbarium, Auckland, New Zealand (PDD) q The Herbarium of the Sustainable Development of Biological Resources Laboratory, Chiang Mai University, Thailand (SDBR-CMU) r Centraalbureau voor Schimmelcultures, Utrecht, Netherland (CBS) s The Herbarium of Mycology, Jilin Agricultural University, Changchun, Jilin, China (HMJAU) t The Mycological Herbarium of the Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China (GDGM) u Karadeniz Technical University Faculty of Forestry Herbarium, Trabzon, Turkey (KATO) v United States National Arboretum, USDA-ARS, Washington, USA (NA) w The Herbarium of Mahidol University, Nakhon Pathom, Thailand (PBM) x The Calcutta University Herbarium, Kolkata, West Bengal, India (CUH) y The Herbarium of University of Tennessee – Knoxville, Tennessee, USA (TENN) z New Herbarium of Museo di Storia Naturale di Venezia, Venezia, Italy (MCVE) aa Herbário of Instituto Nacional de Pesquisas da Amazônia, Amazonas, Brazil (INPA) ab Herbier of Université de Lille, Lille, France (LIP) (Newly-generated information of specimens and sequences are in bold).

For the sequence alignments, Sequencher 4.1.4 (Gene Code Corp., Ann Arbor, MI) was used to concatenate sequences obtained from both direction (5’–3’ & 3’–5’), to remove regions with heavy peaks and to merge degenerate bases. The sequences were then aligned using MAFFT 7.526 (Katoh et al. 2005) and manually checked in BioEdit 7.7.1 (Hall 1999). Separate single-gene analyses were performed to exclude conflicts amongst topologies using Maximum Likelihood and Bayesian Inference. Subsequently, Phyutility 2.2 (Smith and Dunn 2008) was employed to combine all the separate single-gene datasets. Any deficiencies in the DNA fragment sequences were treated as missing data in the subsequent analyses. A super-matrix was generated by combining sequences of all four loci.

Under the Akaike Information Criterion (AIC), the best-fitted substitution model for each dataset was determined with MrModelTest 2.3 (Nylander 2004). Phylogenetic analyses were conducted using Maximum Likelihood (ML) with RAxML 7.2.6 (Stamatakis 2006) and Bayesian Inference (BI) with MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003). In view of the close kinship amongst Lulesia Singer & Clitopilus, as well as Clitopilopsis Maire & Rhodocybe, Lulesia umbrinomarginata Y.Q. Xiao et al. and Lulesia orientalis (S.P. Jian & Zhu L. Yang) Vizzini et al. were selected as outgroups for the phylogenetic tree of Clitopilus. Similarly, Clitopilopsis albida S.P. Jian & Zhu L. Yang and Clitopilopsis hirneola (Fr.) Kühner were chosen as outgroups for the phylogenetic tree of Rhodocybe.

For ML analyses, the GTRGAMMAI model was applied to the combined dataset, with statistical support for internodes obtained through non-parametric bootstrapping with 1000 replications. For the BI analyses of the combined dataset, a partitioned mixed model was implemented, defining the sequences of ITS, LSU, RPB2 and TEF1 as four independent partitions, with each gene estimated using different model parameters. The best-selected model was employed and the Markov Chain Monte Carlo (MCMC) chain was run for four million generations. The STOPRULE command was set with STOPVAL = 0.01 and trees were sampled every 100 generations. We verified chain convergence using Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer) to ensure sufficiently large effective sample size (ESS) values greater than 200. The combined tree was summarised using the sump and sumt commands with a 25% burn-in.

Results

Phylogenetic analyses

No topological inconsistency was detected between the ML and BI analyses, both for the individual genes and the multigene data. The phylogenetic tree inferred from the ML strategy is presented, with statistical results from both ML (Bootstrap Supports, BS) and BI (Posterior Probabilities, PP) displayed on the branches (see Figs 1, 2). The best-fit model for ML and BI analyses was the GTR+I+R model. In the multigene matrix for Clitopilus, we assembled a total of 131 sequences from four genes: 96 for ITS, 89 for LSU, 110 for RPB2 and 78 for TEF1. Analogously, the multigene matrix for Rhodocybe included 120 sequences derived from the same four genes, with 72 for ITS, 48 for LSU, 55 for RPB2 and 37 for TEF1. Finally, the dataset for Clitopilus included 3664 sites in total, with 788 from ITS, 957 from LSU, 784 from RPB2 and 1135 from TEF1. Similarly, the dataset for Rhodocybe included 3881 sites, with 960 from ITS, 985 from LSU, 799 from RPB2 and 1137 from TEF1.

Figure 1. 

Phylogenetic relationships amongst representative species of Clitopilus were inferred from a multigene dataset (ITS-LSU-RPB2-TEF1) using both ML and BI methods (only shown the ML tree). Supported branches indicate bootstrap supports (BS > 50%) and posterior probabilities (PP > 0.90). Sequences from type specimens (holotype, epitype or isotype) are marked, while new and new record taxa are highlighted in red. The abbreviations ILRT stand for: I = ITS, L = LSU, R = RPB2 and T = TEF1.

Figure 2. 

Phylogenetic relationships amongst representative species of Rhodocybe were inferred from a multigene dataset (ITS-LSU-RPB2-TEF1) using both ML and BI methods (only shown the ML tree). Supported branches indicate bootstrap supports (BS > 50%) and posterior probabilities (PP > 0.90). Sequences from type specimens (holotype, paratype or isotype) are marked, while new and new record taxa are highlighted in red. The abbreviations ILRT stand for: I = ITS, L = LSU, R = RPB2 and T = TEF1.

In the phylogenetic tree of Clitopilus (Fig. 1), species with parasitic abilities are clustered together in Clitopilus sect. Scyphoides Singer, representing a new species. The species collected from rotten wood are positioned close to C. baronii Consiglio & Setti. Furthermore, the phylogenetic tree of Rhodocybe (Fig. 2) shows that other species collected from rotten wood are clustered within Rhodocybe sect. Rufobrunnea Singer ex T.J. Baroni, also signifying a new species.

Morphological observations and SEM

The images of fresh basidiomata, substrate and habitats of the collected specimens are shown in Fig. 3. Scanning Electron Microscopy revealed that the ornamentation of basidiospores provides some extra valuable information (Fig. 4). The basidiospores of Clitopilus exhibit several classical longitudinal ridges (Fig. 4a–f), while those of Rhodocybe are characterised by undulate-pustulate walls (Fig. 4g–h). In addition, crystals on the pileus hyphae of the new species in Clitopilus was also identified (Fig. 4i).

Figure 3. 

Basidiomata of Clitopilus and Rhodocybe a–d Clitopilus baronii (a, b KUN-HKAS 145333; c, d KUN-HKAS 145334) e–h Clitopilus parasiticus (e KUN-HKAS 145336, holotype; f, g KUN-HKAS 145335; h KUN-HKAS 145337) i–k Rhodocybe zijinshanensis (KUN-HKAS 145338, holotype). Scale bar: 5 mm.

Figure 4. 

Basidiospores and crystals of Clitopilus and Rhodocybe reveal by SEM a–c Clitopilus baronii (KUN-HKAS 145334) d–f Clitopilus parasiticus (KUN-HKAS 145336, holotype) g, h Rhodocybe zijinshanensis (KUN-HKAS 145338, holotype) i the crystals around the hyphae in pileipellis of Clitopilus parasiticus (KUN-HKAS 145336, holotype). Scale bar: 2 μm.

Taxonomy

Clitopilus parasiticus S.P. Jian, X. Chen & Z.H. Zhang, sp. nov.

MycoBank No: 857348
Figs 1, 3e–h, 4d–f, 5a–c

Holotype.

China • Jiangsu Province, Nanjing City, Zijinshan, E 118.83, N 32.08, alt. 32 m, scattered on soil, in the mixed broadleaf (i.e. Quercus variabilis, Robinia pseudoacacia, Osmanthus fragrans, Broussonetia papyrifera, Ilex latifolia and Yulania sp.) forest, 15 August 2024, collected by X. Chen and Z.H. Zhang, CX 966 (KUN-HKAS 145336). GenBank: ITS = PQ793169; LSU = PQ781613; RPB2 = PQ788398.

Etymology.

“parasiticus” is proposed by its biotrophic behaviour.

Diagnosis.

Clitopilus parasiticus is similar to C. hobsonii, but differs by the tomentose pileus, explanate margin and smaller basidiospores.

Description.

Basidiomata pleurotoid to conchoid, small size. Pileus 2–8 mm, convex; surface whitish (#b4c4cb) to chalk white (#e3edf3), with fine tomentose texture usually extending beyond the margin and densely woolly-tomentose at the base; margin typically applanate; context less than 1 mm thick. Lamellae meeting at an excentric point, whitish (#c6d4d3) to yellowish-white (#d3dad4) to yellowish (#dac7ac), slightly dense or crowded, edges entire and concolorous, lamellulae numerous. Stipe absent or very short, eccentric to lateral, measuring 1–2 × 0.2–0.5 mm, concolorous with lamellae. The base with white (#dddddf) mycelium. Odour none.

Basidiospores (5) 5.5–8.5 × 3.5–5.0 (5.5) μm, Lm × Wm = 6.6 (± 0.63) × 4.2 (± 0.34) μm, Q = 1.20–1.90 (Qavg = 1.55 ± 0.13) [186/9/3], hyaline, ellipsoid to broadly fusiform, subovoid in profile and face view, slightly angled in polar view, with 7–9 inconspicuous or obscure longitudinal ridges in total. Basidia 16–23 × 6–9.5 μm, clavate, hyaline, 4-spored, rarely 2-spored; sterigmata 2–3 μm. Lamellar trama subregular, composed of thin-walled, hyaline, cylindrical hyphae with a diameter of 2.5–9 μm. Lamellae edges fertile. Pleurocystidia and cheilocystidia absent. Pileipellis a cutis composed of sparsely arranged, thin-walled, hyaline, smooth, interwoven, cylindrical hyphae with a diameter of 3–5 μm, sometimes featuring erect hyphae; crystals present around the hyphae, square to subsquare, measuring 3 × 3 μm to 14 × 15 μm in area; pileal trama subregular, composed of hyaline, filamentous, thin-walled hyphae, with a diameter of 3–7.5 μm. Clamp connections absent.

Figure 5. 

Microscopic features of Clitopilus parasiticus (KUN-HKAS 145336, holotype) a basidiospores b hymenium and subhymenium c pileipellis. Scale bars: 5 μm (a); 10 μm (b); 20 μm (c).

Ecology and distribution.

Solitary, scattered on soil, lignicolous or gregariously living on leaves of plants (Dryopteris sp. and Oplismenus undulatifolius) in the mixed broadleaf forest, distributed in Jiangsu Province, China, in August.

Additional specimens examined.

China • Jiangsu Province, Nanjing City, Zijinshan, alt. 48 m, dispersedly or gregariously lignicolous or living on twigs or leaves of Oplismenus undulatifolius, in the mixed broadleaf (i.e. Quercus variabilis, Quercus aliena, Cunninghamia lanceolata, Symplocos tanakana, Celtis sinensis and Ilex cornuta) forest, 16 August 2023, collected by X. Chen and Z.H. Zhang, CX 628 (KUN-HKAS 145335); same places, alt. 48 m, dispersedly or gregariously living on leaves of Dryopteris sp., 16 August 2024, collected by X. Chen and Z.H. Zhang, CX 967 (KUN-HKAS 145337).

Notes.

Clitopilus parasiticus belongs to Clitopilus sect. Scyphoides (Fig. 1). This new taxon is similar to C. hobsonii, C. daamsii, C. passeckerianus, C. pinsitus and C. baronii. Clitopilus hobsonii was originally described from Britain and exhibits both saprophytic and parasitic abilities. It resembles C. parasiticus in its living habits and the shape of its basidiomata, but differs from the latter by its involute or inflexed margins of the pileus and larger basidiospores (Lm × Wm = 7.5 × 5 μm) (Orton 1960; Noordeloos 1984; Noordeloos 1988). Meanwhile, C. daamsii was also similar to C. parasiticus in outline; however, it differs due to its xylogenous or mycoparasitic behaviour, involute margin of pileus and larger basidiospores (8–11.5 × 4.8–6.6 μm) (Noordeloos 1984). Another closely-related species is C. passeckerianus, which has sessile basidiomata and a white pileus. However, the habit of growing on mushroom-beds, basidiomata size (8–40 mm), the reniform to spathulate shape of the pileus and larger basidiospores (7–9 × 4–5 μm) of C. passeckerianus significantly differs from C. parasiticus (Pilát 1935; Noordeloos 1993). Clitopilus pinsitus was first collected from Sweden and was found growing on the trunk of Quercus. This species is characterised by its spatulate, white pileus (15–40 mm) and ellipsoidal to amygdaliform basidiospores (7.5–9 × 4.6–5.3 μm) with 7–8 obscure longitudinal ridges (Josserand 1937; Singer 1946a). Lastly, C. baronii, recently described by Consiglio and Setti (2019) in Marmirolo, Italy, resembles C. parasiticus, but can be differentiated by its larger pileus (5–40 mm) and basidiospores (Lm × Wm = 7.6 × 5.0 μm), as well as its lageniform cheilocystidia.

Clitopilus baronii Consiglio & Setti, Index Fungorum 427: 1. 2019.

Figs 3a–d, 4a–c, 6a–c

Description.

Basidiomata pleurotoid to crepidotoid, small size. Pileus 3–15 mm wide, convex then expanded; surface yellowish-white (#9a8a7a), greyish (#a6a39f) to bluish-grey (#6a757b), usually subtly woolly-tomentose at the base then reduced to border; margin slightly incurved, even, sometimes faintly striated; context less than 1 mm thick. Lamellae whitish (#a9a7a8) to yellowish (#9d896d), sometimes hygrophanous, slightly dense or crowded, edges entire and concolorous, lamellulae numerous. Stipe absent; the base with white (#e9ebed) mycelium. Odour none.

Basidiospores (6) 6.5–9.5 (11) × 4–5 (5.5) μm, Lm × Wm = 7.5 (± 1.01) × 4.5 (± 0.35) μm, Q = 1.4–1.98 (Qavg = 1.66 ± 0.14) [43/2/2], hyaline, ellipsoid to fusiform, subovoid in profile and face view, slightly angled in polar view with 8–10 inconspicuous or obscure longitudinal ridges in total. Basidia 17.5–24 × 6.5–9 μm, clavate, hyaline, 2- or 4-spored; sterigmata 3–5.5 μm. Lamellar trama subregular, composed of thin-walled, hyaline, cylindrical hyphae with a diameter of 2.5–9 μm. Lamellae edges fertile. Pleurocystidia and cheilocystidia absent, but occasionally forming a few cylindrical tramal hyphae with a diameter of 2–3 μm over the edge. Pileus context about 150–200 μm thick. Pileipellis a cutis composed of compactly arranged, thin-walled, hyaline, smooth, cylindrical hyphae with a diameter of 3.5–9 μm, featuring sparely arranged and erect hyphae with a diameter of 2–3 μm; pileal trama subregular or irregular, composed of hyaline, filamentous, thin-walled hyphae, with a diameter of 2.5–8.5 μm. Clamp connections absent.

Figure 6. 

Microscopic features of Clitopilus baronii (KUN-HKAS 145334) a hymenium and subhymenium b basidiospores c pileipellis. Scale bars: 5 μm (a); 10 μm (b); 20 μm (c).

Ecology and distribution.

Lignicolous, scattered or gregarious on rotten wood in the mixed broadleaf forest, distributed in Jiangsu Province, China, in May.

Additional specimens examined.

China • Jiangsu Province, Nanjing City, Zijinshan, alt. 42 m, scattered or gregarious on rotten wood (Quercus sp.), in the mixed broadleaf (i.e. Quercus acutissima, Quercus aliena, Celtis sinensis, Liquidambar formosana and Cunninghamia lanceolata) forest, 7 May 2023, collected by X. Chen, CX 119 (KUN-HKAS 145333); same places, alt. 38 m, scattered on rotten wood (Quercus sp.), in the mixed broadleaf (Quercus glauca, Pterocarya stenoptera, Ilex chinensis, Cunninghamia lanceolata, Ilex cornuta, Liquidambar formosana and Ligustrum lucidum) forest, 9 May 2023, collected by X. Chen, CX 134 (KUN-HKAS 145334).

Notes.

Clitopilus baronii belongs to C. sect. Scyphoides (Fig. 1). In the original description, this species was found growing on a decaying trunk of Quercus sp. It is characterised by its sessile basidiomata, orbicular to conchate white pileus, cream-rose lamellae, ellipsoidal to subamygdaliform basidiospores with 8–10 obscure longitudinal ridges and lageniform cheilocystidia. The macro- and microscopic features of our specimens (KUN-HKAS 145333 & 145334) closely match those described in the primary literature (Consiglio and Setti 2019). However, we did not observe any lageniform cheilocystidia in our specimens; we only identified a few thin cylindrical tramal hyphae over the edge. This observation aligns with findings by Noordeloos (1984) regarding C. daamsii, particularly in some older specimens. In our previous study, we also noted this phenomenon of thin cylindrical tramal hyphae at the edge in C. crispus Pat. However, this occurrence was generally casual and rare.

In the phylogenetic tree of Clitopilus, we could discover some unusual results regarding C. baronii. In the combined multigene analyses (ITS-LSU-RPB2-TEF1), our specimens were found to separate from the clades of C. baronii and grouped (BS/PP = 69/1) closer to C. pinstus (G. Immerzeel 1990-11). When we compared the different genes separately between our samples and holotype of C. baronii (AMB 18363), we found that our samples exhibited over 99% similarity in ITS region. However, the similarity was only about 90% for both RPB2 and TEF1. For ITS, we have tested them several times in different companies, but all yielded consistent results. Regarding RPB2 and TEF1, we did not detect any issues with the original data; all sequences were bidirectionally sequenced to ensure unimodality and were matched by hand in software. Considering the macro- and microscopic features, we tentatively classified our specimens as Clitopilus baronii. More samples are needed to resolve our uncertainties regarding both the presence of cheilocystidia and the phylogenetic relationship.

Rhodocybe zijinshanensis S.P. Jian & X. Chen, sp. nov.

MycoBank No: 857349
Figs 2, 3i–k, 4g–h, 7a–c

Holotype.

China • Jiangsu Province, Nanjing City, Zijinshan, E 118.87, N 32.06, alt. 99 m, solitary on rotten wood, in mixed broadleaf (i.e. Quercus acutissima, Quercus aliena, Aphananthe aspera, Osmanthus fragrans, Liquidambar formosana, Photinia serratifolia and Ilex chinensis) forest, 30 August 2024, collected by X. Chen, CX 664 (KUN-HKAS 145338). GenBank: ITS = PQ793171; LSU = PQ781615; RPB2 = PQ PQ788400; TEF1 = PQ788406.

Etymology.

“zijinshanensis” indicates the source place, where it was located in Nanjing City, China.

Diagnosis.

Rhodocybe zijinshanensis is similar to R. subasyae, but differs by its smaller yellow pileus, shorter and more slender stipes and the absence of cheilocystidia.

Description.

Basidiomata omphalioid, small size. Pileus 10–15 mm wide, applanate to plano-concave; surface yellow (#eac7a2) over edge and brownish-yellow (#6c3620) over disc, distributing some radiate fibrillose, sometimes hygrophanous; margin slightly inflexed, even or undate; context about 1 mm thick. Lamellae adnate to subdecurrent, yellowish (#cdbead) to greyish-pink (#d1b4a2), dense or crowded, edges entire or undate, sometimes with transverse intervenose, concolorous with lamellae, lamellulae numerous. Stipe 7–19 × 1–2 mm, central to eccentric, cylindrical to tapering downwards, usually concolorous with pileus, densely fine scales dispersed around the top. Odour none.

Figure 7. 

Microscopic features of Rhodocybe zijinshanensis (KUN-HKAS 145338, holotype) a hymenium and subhymenium b basidiospores. Scale bars: 5 μm (a); 10 μm (b); 20 μm (c).

Basidiospores (4.5) 5–6.5 × 3.5–5 μm, Lm × Wm = 5.5 (± 0.54) × 4.3 (± 0.31) μm, Q = 1.09–1.55 (Qavg = 1.28 ± 0.11) [41/2/2], hyaline, subglobose, subamygdaliform to broadly ellipsoid in profile view, ellipsoid in face view and minutely, but obviously angular in polar view (7–9 facets in total), undulate-pustulate in all views. Basidia 18.5–32 × 5.5–7.5 μm, clavate, hyaline, 2- or 4-spored; sterigmata up to 5 μm long. Lamellar trama regular, composed of 2.5–10.5 μm in diam., thin-walled, hyaline hyphae. Lamellae edges fertile. Pleurocystidia and cheilocystidia absent. Pileipellis a cutis composed of radially arranged, subregular hyphae, hyphae thin-walled, yellowish, smooth, cylindrical, 3.5–11.5 μm in diam., sometimes with oleiferous hyphae; pileal trama regular, composed of hyaline, thin-walled, cylindrical hyphae with a diameter of 2–11 μm. Stipitipellis a cutis composed of compactly arranged, regular, thin-walled and hyaline hyphae with a diameter of 3.5–9.5 μm; Stipe trama regular, composed of thin-walled and hyaline hyphae with a diameter of 4–10.5 μm. Caulocystidia absent. Clamp connections absent.

Ecology and distribution.

Solitary on rotten wood in broad-leaved forest, only found in Jiangsu Province, China, August to October.

Additional specimens examined.

China • Jiangsu Province, Nanjing City, Zijinshan, E 118.87, N 32.06, alt. 99 m, solitary on rotten wood, in mixed broadleaf (i.e. Quercus acutissima, Quercus aliena, Aphananthe aspera, Osmanthus fragrans, Liquidambar formosana, Photinia serratifolia and Ilex chinensis) forest, 30 August 2024, collected by X. Chen, CX 665 (KUN-HKAS 145339).

Notes.

Rhodocybe zijinshanensis belongs to R. sect. Rufobrunnea (Fig. 2). Species in this section are characterised by centrally stipitate basidiomata, pilei ranged from pinkish, reddish, brown, tan to fulvous (but never greyish or white), lamellae that are adnexed to adnate or decurrent, the absence of hymenial pseudocystidia and clamp connections (Baroni 1981). Rhodocybe zijinshanensis is similar to several other species, including R. asyae Seslı & Vizzini, R. subasyae T. Bau & Y.L. Sun, R. pseudoalutacea T.J. Baroni et al. and R. alutacea Singer. Rhodocybe asyae, first recorded in Turkey, can be differentiated from R. zijinshanensis by its relatively larger, smooth pileus (10–30 mm), longer and thicker stipe (25–30 × 2–5 mm) and flexuous cheilocystidia (20–30 × 4–6 μm) (Seslİ and Vizzini 2017). Rhodocybe subasyae, a recently described species from Jilin, China, is also similar to R. zijinshanensis, but differs in having a smooth pileus, a longer and thicker stipe (22–37 × 5–7 mm), slightly larger basidiospores (Qavg = 1.4), and cheilocystidia measuring 22.4–28.2 × 3.9–6.8 μm (Sun and Bau 2023). For R. pseudoalutacea, it was reported from the Dominican Republic, featured by its slightly larger pileus (10–35 mm), slender yet thick stipe (15–50 × 2–6 mm) and pileipellis composed of finely encrusted cylindrical hyphae (Baroni et al. 2020). The last species which resembled R. zijinshanensis was R. alutacea, found in Florida, USA. It is characterised by the greater pileus size (25–35 mm), a longer stipe (23–35 × 2.5–5.5 mm), and septate, flexuous cheilocystidia (20–35 × 6.5–7 μm) (Singer 1946b; Baroni 1981).

Discussion

In this study, we described two new species and documented a new record species in China: C. parasiticus, R. zijinshanensis and C. baronii. For the phylogenetic analysis, we utilised nearly all available sequences for the genera Clitopilus and Rhodocybe, uploaded by classified references or expert researchers (see Fig. 1). The phylogenetic tree indicates that C. parasiticus is closely related to C. velutinus T. J. Baroni & Angelini, which was discovered in the Dominican Republic. However, it can be distinguished by its larger pileus (10–25 mm), the existence of an eccentric stipe and larger basidiospores (Lm × Wm = 8.0 × 5.0 μm) with more longitudinal ridges (10–14) (Baroni et al. 2020). Similarly, C. baronii is closer to C. venososulcatus Singer, which is occurring only in Florida, USA. Nonetheless, the latter typically exhibits a larger, venose and sulcate pileus (12–23 mm) and slightly larger basidiospores (8–8.5 × 4.5–5 μm) with 6–8 obscurely longitudinal ridges (Singer 1946a). Finally, R. zijinshanensis approaches to R. nuciolens (Murrill) Singer and R. gemina (Paulet) Kuyper & Noordel., but the larger size of their basidiomata (particularly in the pileus and stipe) and the presence of cheilocystidia make them easy to distinguish from the former (Baroni 1981; Seslİ and Vizzini 2017; Vizzini et al. 2018). The similar species of above species are compared in Table 3.

Table 3.
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The comparison of morphological characters amongst C. parasiticus, C. baronii, R. zijinshanensis and similar species.

Taxa Badisiomata Pileus Basidiospores (ridges) Hymenial cystidia Habitat Locality References
Clitopilus sect. Scyphoides
C. baronii (Holotype) Orbicular to conchate or spatulate, sessile 5–40 mm, white to greyish 6.9–8.4 × 4.4–5.5 μm (8–10), Q = 1.68–1.71 Cheilocystidia lageniform On a decaying trunk of Quercus sp. Italy Consiglio and Setti (2019)
C. baronii Conchate, sessile 3–15 mm, white to greyish 6.5–9.5 × 4–5 μm (8–10), Q = 1.4–1.98 None On rotten wood China This study
C. daamsii (Holotype) Orbicular to conchate, sessile 2–8 mm, white 8–11.5 × 4.8–6.6 μm (6–9), Q = 1.4–2 None On wood or other fungi Netherlands Noordeloos (1984)
C. fasciculatus (Holotype) Fasiculata, sessile Individual 24 × 20 mm, pale brown 4.7–6.3 × 3.0–3.5 μm (3–6), Q = 1.2–1.85 None On beds of cultivated mushrooms Netherlands Noordeloos (1984)
C. hobsonii (Holotype) Orbicular or slightly reniform, sessile 5–18 mm, white to pale greyish 6.5–9 × 4–5.5 μm (7–12), Q = 1.2–2 None On plant debris or herbaceous stems Britain Orton (1960)
C. parasiticus (Holotype) Conchate, sessile 2–8 mm, whitish to chalk white 5.5–8.5 × 3.5–5 μm (7–9), Q = 1.2–1.9 None On soil, rotten wood and leaves of plants China This study
C. passeckerianus (Holotype) Reniform or resembling an ear, sessile 8–40 mm, white 7–9 × 4–5 μm (7–12), Q = 1.45–2.25 None On mushroom-beds Europe Pilát (1935)
C. pinsitus (Holotype) Spatulate, semi-cicular, sessile 15–40 mm, white to pale ochre 7–9 × 4.6–5.3 μm (7–8) None On trunk of Quercus sp. Sweden Josserand (1937)
C. velutinus (Holotype) Clitocyboid 10–25 mm, pure white 7–9 × 5–6 μm (7–8), Q = 1.27–1.8 None On soil Dominican Republic Baroni et al. (2020)
C. venososulcatus (Holotype) Pleurotoid, sessile or sub sessile 12–23 mm, pallid white 8–8.5 × 4.5–5 μm (6–8) None On trunks or logs of Ficus aurea USA Singer (1946a)
Rhodocybe sect. Rufobrunnea
R. alutacea (Holotype) 25–35 mm, yellowish, hygrophanous 23–35 × 2.5–5.5 mm, subequal 5.8–7.5 × 3.5–5 μm (7–9) Cheilocystidia On sandy soil and fallen leaves USA Singer (1946b)
R. asyae (Holotype) 10–30 mm, salmon pink 25–30 × 2–5 mm, tapering 5–7 × 4–5 μm, Q = 1.1–1.4 Cheilocystidia On the grass Turkey Seslİ and Vizzini (2017)
R. gemina 15–80 mm, reddish incarnate 25–50 × 3–15 mm, subequal 5–6.5 × 4–5 μm Cheilocystidia On humus Europe Baroni (1981)
R. nuciolens 10–60 mm, pinkish cinnamon, hygrophanous 35–80 × 2–9 mm, equal 5.5–8 × 4–5 μm Cheilocystidia On humus, sandy soil or decaying wood USA Baroni (1981)
R. pseudoalutacea (Holotype) 10–35 mm, brown or brownish orange, hygrophanous 15–50 × 2–6 mm, equal or enlarged downwards 5.5–7 × 4–5 μm (7–10), Q = 1.2–1.6 None On decaying humus or woody debris Dominican Republic Baroni et al. (2020)
R. subasyae (Holotype) 19–25 mm, beige red 22–37 × 5–7 mm, cylindrical 5.4–6.8 × 3.9–4.9 μm (6–8), Q = 1.2–1.6 Cheilocystidia On sandy soil China Sun and Bau (2023)
R. zijinshanensis (Holotype) 10–15 mm, yellow, hygrophanous 7–19 × 1–2 mm, cylindrical to tapering 5–6.5 × 3.5–5 μm (7–9), Q = 1.09–1.55 None On rotten wood China This study

In the family Entolomataceae Kotl. & Pouzar, there are over 1500 described species worldwide (Co-David et al. 2009; Baroni and Matheny 2011; Karstedt et al. 2019). However, only a few species exhibit mycoparasitic capabilities, like Entoloma abortivum (Berk. & M.A. Curtis) Donk, E. parasiticum (Quél.) Kreisel, E. pseudoparasiticum Noordel. and Rhodophana stangliana (Bresinsky & Pfaff) Vizzini (Noordeloos 1988; Læssøe and Rosendahl 1994; Czederpiltz et al. 2001). Thereinto, Entoloma abortivum is frequently reported to co-occur with Armillaria (Fr.) Staude, leading to the hypothesis that Armillaria attacks and parasitises the basidiomata of Entoloma abortivum (Watling 1974). On the contrary, Czederpiltz et al. (2001) demonstrated that E. abortivum can actually abort the growth of Armillaria in culture media. Furthermore, Koch and Herr (2021) explained this phenomenon using transcriptomics.

Notably, some species, such as E. clypeatum (L.) P. Kumm., E. niphoides Romagn. ex Noordel., E. saepium (Noulet & Dass.) Richon & Roze and E. sericeoides (J.E. Lange) Noordel., have been reported to associate with rosaceous woody plants. However, these species are more likely to be detrimental to roots rather than forming typical mycorrhizae (Agerer and Waller 1993; Gryndler et al. 2010; Shishikura et al. 2020). In the Rhodocybe-Clitopilus clade, C. daamsii has been observed growing on Hydnoporia tabacina (Sowerby) Spirin et al. (previously classified as Hymenochaete tabacina (Sowerby) Lév.), while C. passeckerianu and C. fasciculatus have been associated with the growing-beds of cultivated Agaricus L. (Noordeloos 1984; Noordeloos 1988; Noordeloos 1993), although Singer questioned the mycoparasitic behaviour of C. passeckerianu (Singer and Harris 1987).

To investigate the saprophytic and biotrophic abilities of C. parasiticus, we carefully examined different specimens to identify the discrepancies between various hosts and growth on soil. The results are presented in Table 4. We found that the basidiospores from specimens KUN-HKAS 145335 and 145337, which were collected from the leaves of Oplismenus sp. and Dryopteris sp., respectively, showed no significant differences. However, there was an obvious difference with specimen KUN-HKAS 145336, where the basidiospores of C. parasiticus growing on soil were larger than those from specimens growing on plant leaves. Larger basidiospores often indicate more robust growth of basidiomata (Kauserud et al. 2011; Halbwachs et al. 2017), suggesting that this species may be better suited to a soil habitat than to a biotrophic lifestyle.

Table 4.
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The intraspecies comparison of C. parasiticus in morphological characters and microenvironment.

Taxa Voucher specimen Pileus Basidiospores (ridges) Crystals in pileipellis Habitate Temp. (°C) Prec. (mm/d)
C. parasiticus KUN-HKAS145335 (CX628) 2–8 mm 5.5–7.0 × 4–5.5 μm, Lm × Wm = 6.3 (± 0.47) × 4.24 (± 0.35) μm, Q = 1.20–1.84 (Qavg = 1.49 ± 0.13) (8–9) [63/3/1] None On leaves of Oplismenus undulatifolius 29.04 5.58
C. parasiticus (Holotype) KUN-HKAS145336 (CX966) 3–7 mm 6.0–8.5 × 4–5 μm, Lm × Wm = 7.06 (± 0.6) × 4.40 (± 0.30) μm, Q = 1.40–1.81 (Qavg = 1.61 ± 0.10) (7–8) [62/3/1] Present On soil 31.25 4.43
C. parasiticus KUN-HKAS145337 (CX967) 3–5.5 mm 5.5–7.5 × 3.5–5 μm, Lm × Wm = 6.33 (± 0.50) × 4.09 (± 0.28) μm, Q = 1.20–1.90 (Qavg = 1.55 ± 0.13) (7–9) [61/3/1] Present On leaves of Dryopteris sp. 31 4.42

Notes: Temp./Prec. means the average temperatures/precipitations one week before and one week after the date of collection.

Furthermore, the average temperature over a fortnight in 2024 was slightly higher than in 2023, while the average precipitation during the same period was slightly lower in 2024 compared to 2023. These subtle discrepancies could influence the nutritional mode and even the choice of parasitic host. Admittedly, our judgement that this species is biotrophic on the basis of only two collections from different plant leaves, is not entirely rigorous. More experiments, including physiological and genomic analyses, are necessary for a comprehensive assessment.

Acknowledgements

The authors are very grateful to Drs. Bang Feng, Xiang-Hua Wang (Kunming Institute of Botany, Chinese Academy of Sciences), Yu-Peng Ge (School of Horticulture, Ludong University) and Ms. Ya-Jun Hou for providing some constructive information and suggestions. The authors thank the herbaria KUN-HKAS for providing materials and pictures. The authors thank Mr. Ting Tang for supplying scanning electron microscope (SEM) images.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was funded by the National Natural Science Foundation of China (No. 32060707) and the Funds of Sci-Tech Innovation System Construction for Tropical Crops of Yunnan Province (No. 655-4-3).

Author contributions

Sipeng Jian conceived, designed and completed the experiments under the guidance of Chunxia Zhang. Xia Chen, Yiwei Fang and Tianwei Yang helped to collect samples, use and adjust the microscope, with some photographs. Xinjing Xu, Jing Liu and Feng Gao assisted with extracting DNA and PCR amplification. Sipeng Jian wrote the manuscript and Chunxia Zhang revised it.

Author ORCIDs

Sipeng Jian https://orcid.org/0000-0002-2055-3169

Xia Chen https://orcid.org/0009-0002-1239-1865

Data availability

In this study, DNA sequences have been deposited in GenBank. Specimens were placed at Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS).

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