Phytotaxa 332 (2): 172–180 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2017 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.332.2.4

Gerhardtia sinensis (Agaricales, Lyophyllaceae), a new species and a newly recorded genus for China

TING LI, TAIHUI LI*, CHAOQUN WANG, WANGQIU DENG & BIN SONG State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou, 510070, China * Corresponding author: [email protected]

Abstract

Gerhardtia sinensis, a new species discovered from southern China, is described, illustrated and compared with morphologi- cally similar and phylogenetically related species. Morphologically, it is characterized by its white to yellowish or partially pale yellow pileus with faint striae, subdistant or nearly subdistant intervenose lamellae, and slightly verruculose basidio- spores that are (4.7–)5.2–6.2(–6.6) × (2.5–)3.0–3.4(–3.8) μm in size. Molecular phylogenetic analyses of the new species and related taxa were performed based on the sequences of nuclear ribosomal large subunit (nrLSU) and nuclear ribosomal internal transcribed spacer (ITS).

Key words: Lyophyllaceae, phylogenetics, subtropical fungus, taxonomy

Introduction

Gerhardtia Bon (1994: 66) is a fungal genus in the family Lyophyllaceae, with G. borealis (Fr.) Contu & A. Ortega (2001: 176) [= G. incarnatobrunnea (Ew. Gerhardt) Bon (1994: 66)] as the type species (Bon 1994). It was once regarded as a subgenus Lyophyllopsis Sathe & J.T. Daniel (1981: 87) under Lyophyllum P. Karst. (1881: 29)(Gerhardt 1982), and then was raised to an independent genus with the new name Gerhardtia by Bon (1994) who emphasized the presence of minutely verruculose basidiospores, siderophilous granulation in the basidia, a pileipellis organized as a cutis, trichoderm or hymeniderm and an absence of clamp-connections as the generic characterizing features which distinguish Gerhardtia from other genera in Lyophyllaceae. Vizzini et al. (2017) redefined the characters of this genus, with a particular emphasis on basidiospores that were irregular and undulate to nodulose but not verrucose in outline. To date, only nine species in Gerhardtia have been validated excluding synonyms and varieties (Baroni 1981; Bon 1994; Contu & Ortega 2001; Contu & Consiglio 2004; Kalamees 2008; Cooper 2014; Vizzini et al. 2015, 2017; Matheny et al. 2017). Recently, a new species of Gerhardtia was found in southern China, which was the first discovery of the genus in China and even in Asia. Therefore, full description and illustration of the new species are formally presented as follows.

Materials and methods

Morphological methods Fresh basidiomata were collected from several field sites in southern China. All samples were dried with a food dehydrator and deposited in the Fungal Herbarium of Guangdong Institute of Microbiology (GDGM), Guangzhou, China, except for some small pieces of basidiomata which were dried with silica gel for molecular study. The photographs of the basidiomata were taken in the field with digital cameras in natural light. Microscopic characters were observed with a light microscope (Zeiss Axio Lab. A1). After rehydrating and CO2 critical point drying processes, several tiny pieces of hymenophore were coated with gold/palladium, and basidiospore images were taken with a

172 Accepted by Genevieve Gates: 30 Nov. 2017; published: 19 Dec. 2017 scanning electron microscope (SEM) (HITACHIS-3000N) at 15kV with a working distance of 10 mm. The observed macro-morphological features were based on fresh materials and field photographs; and the microscopic structures were measured and drawn from revived sections of dried specimens, which were hand-sectioned and mounted in 5% KOH solution, 1% Congo Red or Melzer’s reagent, and 0.05% Cotton Blue in lactic acid solution following Kühner (1938), Grund & Marr (1965) and Matheny et al. (2017) in order to examine siderophilous granulation in the basidia. Colour descriptions were made according to Kornerup & Wanscher (1978). The notations of basidiospores (n/m/p) indicate that the measurements were made on ‘n’ randomly selected basidiospores from ‘m’ basidiomata of ‘p’ collections. Basidiospore dimensions are given as: (minimum–) average minus standard deviation–average plus standard deviation (–maximum) of length × (minimum–) average minus standard deviation-average plus standard deviation (–maximum) of width. Q refers to the length/width ratio of an individual basidiospore; Qm refers to the average Q value of all basidiospores ± sample standard deviation.

Molecular methods

Total DNA was isolated from 15 mg of voucher specimen dried with silica gel using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). Primers LROR & LR5 (Vilgalys & Hester 1990, Vilgalys lab. http://www.botany.duke.edu/fungi/mycolab) were used for the LSU rDNA amplification and primers ITS1 & ITS4 for the ITS region amplification (White et al. 1990; Gardes & Bruns 1993). Amplification reactions were performed in 25 μL reaction mixtures with the following total amounts: 5 ng DNA, 2×PCR EasyTaq SuperMix (TransGen Biotech,

Beijing) 10 μL, 1 mM of each primer and added to 25 μL with ddH2O. The cycling parameters were as follows: 5 min at 94℃ for 1 cycle; 30 s at 94℃, 30 s at 55℃, 60 s at 72℃ for 35 cycles, 8 min at 72℃ for 1 cycle, pause at 22℃. PCR products were electrophoresed on a 1% agarose gel and visualized by staining with ethidium bromide. The PCR products were sequenced by the Beijing Genomics Institute (BGI, Shenzhen). The obtained sequences were submitted to GenBank; and the other referred sequences of related taxa were downloaded from GenBank. The nrLSU data matrix includes 19 fungal sequences and consists of 8 taxa with 904 nucleotide sites (gaps included); Tricholoma sinoacerbum T.H. Li, Iqbal Hosen & Ting Li (2016: 234) served as the outgroup. The ITS dataset includes 21 fungal sequences vesting in 8 taxa with 655 nucleotide sites (gaps included), and the sequence of T. sinoacerbum was rooted as the outgroup. Sequence alignment was carried out with ClustalX (Thompson et al. 1997). Alignments were edited with Bioedit software (Hall 1999). Maximum Likelihood (ML) analyses were performed with MEGA version 5.2 (Hall 2013). Bootstrap analysis was implemented with 1000 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. All positions containing gaps and missing data were implemented from the dataset.

Results

Taxonomy

Gerhardtia sinensis T.H. Li, T. Li, C.Q. Wang & W.Q. Deng, sp. nov. Figs. 1–3 MycoBank: MB 819692

Diagnosis:—Differs from G. highlandensis (Hesler & A.H. Sm.) Consiglio & Contu (2004: 158) in having white to yellowish pileus with faint striae, more distant, intervenose lamellae, more lamellulae between two complete lamellae, and slightly larger basidiospores measuring (4.7–)5.2–6.2(–6.6) × (2.5–)3.0–3.4(–3.8) μm. Etymology:—The epithet “sinensis” refers to the type locality in China. Typification:—CHINA. Guangdong Province, Zhaoqing, Dinghushan Biosphere Reserve, at 112°34′ E, 23°10′ N; 8 August 2010, coll. Chunying Deng & Taihui Li (Holotype, GDGM 29981).

Gerhardtia sinensis (Agaricales, Lyophyllaceae) Phytotaxa 332 (2) © 2017 Magnolia Press • 173 FIGURE 1. Gerhardtia sinensis. a. Basidiomata of GDGM 29981 (holotype) b. Basidiomata of GDGM 46394 c. Basidiomata of GDGM 42158 d. Basidiomata of GDGM 45221 Scale bars: a–d = 2 cm. Photos by: Ting Li & Chaoqun Wang.

FIGURE 2. Gerhardtia sinensis. a. Cyanophilous bodies in basidia of GDGM 29981 (holotype). b. Cyanophilic basidiospores of GDGM 29981. c–d. Basidiospores of GDGM 29981 under SEM. e–f. Basidiospores of GDGM 46393 under SEM. Scale bars: a–b = 10 µm; c = 2 µm; d–f = 1 µm. Photos by: Ting Li.

174 • Phytotaxa 332 (2) © 2017 Magnolia Press LI et al. FIGURE 3. Microscopic features of Gerhardtia sinensis (GDGM 29981, holotype). a. Basidiospores. b. Basidia. c. Pileipellis. Scale bars: a–c 10 µm. Drawings by: Ting Li.

Description:—Basidiomata small to medium. Pileus 32–56 mm broad, convex at first, expanding to plano-convex or applanate, depressed over disc when matured, sometimes slightly broadly umbonate or umbilicate at centre; margin involute when young, expanding to inflexed or at times uplifted in places when mature, sometimes flexuous or even somewhat lobed; surface nearly white, yellowish white to pale yellow (1A2–3, 3A3) at disc, snow white, satin white to yellowish white (1A1, 2A1–2, 3A2) near margin, dry, dull, somewhat hygrophanous with moisture loss, glabrous, usually with faint to moderately obvious striation; context 2.5–4.5 mm thick at stipe, less than 1 mm thick at near pileus margin, white to yellowish white (2A1–2), unchanged when cut or injured. Lamellae adnate to slightly sinuate,

Gerhardtia sinensis (Agaricales, Lyophyllaceae) Phytotaxa 332 (2) © 2017 Magnolia Press • 175 subdistant or close to subdistant, with 24–32 complete lamellae and 5–8 lamellulae of different lengths between two complete lamellae, 4–7 mm broad, concolorous with pileus to somewhat yellowish white (1A2–3, 2A2–3), slightly intervenose, not or occasionally forked; edge entire or eroded, concolorous. Stipe 20–60 × 5–12 mm, central or slightly eccentric, cylindrical to subcylindrical, often curved, white to milk white (1A1–2) or yellowish white (2A2), glabrous to finely tomentose, often tomentose at base, dry, somewhat hollow; stipe context white, yellowish white to similar to pileus colour, unchanged when cut or injured. Odour and taste not distinctive.

Basidiospores [50/5/5] (4.7–)5.2–6.2(–6.6) × (2.5–)3.0–3.4(–3.8) μm, [Q = (1.73–)1.81–1.86(–1.93), Qm = 1.84 ± 0.10, elongate-subellipsoid to oblong, thin-walled, guttulate, smooth under light microscope but slightly undulate and with some slightly angled at the distal end under SEM (Fig. 2f), cyanophilous, inamyloid; apiculus small, about 0.5 μm long. Basidia 26–32 × 5–7 μm, narrowly clavate or cylindrical, thin-walled, with cyanophilous and siderophilous granulations, hyaline in H2O and 5% KOH, 4-spored, with sterigmata up to 2–3 μm long, clampless; subhymenium thin, with ramose hyphae. Hymenophoral trama composed of regular, densely parallel hyaline hyphae 4–9 μm wide. Cheilocystidia and pleurocystidia not observed or absent. Pileipellis 85–100 μm thick, a tightly bound cutis, composed of parallel hyphae; hyphae 4–7 μm wide, hyaline, thin-walled. Clamp connections absent. Known distribution:—Known only from Guangdong and Guangxi provinces in southern China. Habit and habitat:—Scattered on soil, in broad-leaved forests or coniferous and broad-leaved mixed forests dominated by Castanopsis chinensis (Spreng.) Hance, Litsea pungens Hemsl., Schima superba Gardner & Champ and Pinus massoniana Lamb., July to September. Additional specimens examined:—CHINA. Guangdong Province, Zhaoqing, Dinghushan Biosphere Reserve, at 112°33′ E, 23°10′ N, 9 July 2012, coll. Taihui Li & Ming Zhang (GDGM 42435); Shaoguan, Chebaling National Nature Reserve, at 114°16′ E, 24°43′ N, 3 September 2013, coll. Chaoqun Wang (GDGM 45221); Guangzhou, Baiyunshan Mountain, at 113°17′ E, 23°12′ N, 8 July 2016, coll. Yaheng Shen (GDGM 46394); Guangxi Province, Qinzhou, Fengliangshan Mountain, at 109°15′ E, 22°19′ N, 13 July 2012, coll. Ming Zhang (GDGM 42158).

Molecular phylogenetic results

Ten sequences including 5 nrLSU and 5 ITS sequences were derived from the new species of Gerhardtia from China (Table 1). The sequences were used to compare the known sequences deposited in GenBank with BLASTn program and to construct the molecular phylogenetic trees with the other known sequences of the genus. The nrLSU sequence identity of G. sinensis (KU563152) and North American G. highlandensis (EF535275) is 98%, with the highest similarity based on BLASTn; while the ITS sequence of G. sinensis (KU563151) is 97% identical to that of G. highlandensis (GU734744). Comparison of the newly generated nrLSU and ITS sequences of G. sinensis with those sequences available in the GenBank indicated that G. sinensis should be distinct (Figs. 4–5). The nrLSU sequences of G. sinensis nest in a clade with those of G. highlandensis, and is 100% supported by ML analyses. The ITS sequences of G. sinensis formed a single clade with 100% support rate, which was closest to that of G. highlandensis with a 96% bootstrap value. The aligned nrLSU and ITS datasets are deposited in TreeBASE (S20691).

FIGURE 4. ML tree and phylogenetic relationships of Gerhardtia inferred from nrLSU sequences analysis. ML bootstrap support values (>50%) are indicated on branches. Tricholoma sinoacerbum is rooted as outgroup; representative members of close genera Calocybella pudica, Lyophyllum moncalvoanum and Lyophyllum shimeji are available in GenBank. Newly generated sequences for G. sinensis is highlighted in bold. GenBank accession numbers are provided after the species name.

176 • Phytotaxa 332 (2) © 2017 Magnolia Press LI et al. FIGURE 5. ML tree and phylogenetic relationships of Gerhardtia analysis based on ITS sequences. ML bootstrap support values (>50%) are indicated on branches. Tricholoma sinoacerbum is rooted as outgroup; representative members of close genera Calocybella pudica, Lyophyllum moncalvoanum and Lyophyllum shimeji are available in GenBank. Newly generated sequences for G. sinensis are highlighted in bold. GenBank accession numbers are provided after the species name.

Discussion

Gerhardtia sinensis matches well the concept of Gerhardtia, sharing slightly undulate and cyanophilc basidiospores, cyanophilous and siderophilous granulations within the basidia, clampless hyphae, and a pileipellis formed as a cutis, trichoderm or hymeniderm. However, it differs obviously from the other species of Gerhardtia in the following ways. Morphologically, G. highlandensis described from North America is the most similar to G. sinensis. However, the former has a faintly pale pinkish cinnamon pileus without striae, close to nearly subdistant lamellae (with more than 50 complete lamellae per pileus) without any intervenosing, fewer lamellulae (usually only 1–3 lamellulae) between two complete lamellae and slightly smaller basidiospores measuring 4–6 × 2.5–3 μm (Bigelow 1985; Contu & Consiglio 2004). Among the other taxa of the genus, G. borealis is characterized by its dark brown pileus and larger basidiospores (6–8.5 × 4–5 μm) (Bigelow 1985; Contu & Consiglio 2004); G. citrinolobata Angelini & Vizzini (2017: 244) has a collybioid habit, and a yellow-green pileus with a strongly wavy and lobate margin (Vizzini et al. 2017); G. leucopaxilloides (H.E. Bigelow & A.H. Sm.) Consiglio & Contu (2004: 158) has a reddish brown pileus, although it has similar sized basidiospores (4–6 × 2–3 μm) (Bigelow 1985; Contu & Consiglio 2004). It is worth emphasizing that G. marasmioides (Singer) Consiglio & Contu (2004: 159) formerly described as Rhodocybe marasmioides Singer (1952: 424), was placed in or near Lyophyllum (Baroni 1981) and was recombined into Gerhardtia after its cyanophilic and siderophilic basidia and cyanophilic basidiospores were recognized (Contu & Consiglio 2004). Unfortunately Co-David et al. (2009), based on limited molecular analyses, considered that all Rhodocybe Maire (1924: 298) species should be transferred to Clitopilus (Fr. ex Rabenh.) P. Kumm.(1871: 23) But there is no morphological or molecular evidence to support the transfer of R. marasmioides to Clitopilus (Co-David et al. 2009), a view currently accepted in Index Fungorum. The present authors agree that R. marasmioides belongs in genus Gerhardtia based on morphological evidence. Gerhardtia marasmioides is characterized by its ochraceous pileus, distant lamellae and narrower basidiospores (4.8–5.5 × 2.7 μm) (Baroni 1981; Contu & Consiglio 2004). Gerhardtia piperata (A.H. Sm.) Bon (1994: 67) has a pale avellaneous to dull yellowish pileus and longer but narrower basidiospores (6–7 × 2–2.5 μm) (Contu & Consiglio 2004; Armin & Zdenko 2009); G. pseudosaponacea J.A. Cooper & P. Leonard (2014: 269) has a straw to buff pileus and smooth or more or less undulate basidiospores (4.9–6.3 × 2.6–3.2 μm) (Cooper 2014; Vizzini et al. 2017); and G. suburens (Clémençon) Consiglio & Contu (2004: 159) has a greyish wax-yellow pileus and longer but narrower basidiospores (6–7.5 × 2–2.5 μm) (Bigelow 1985; Contu & Consiglio 2004). A recent study found that G. cibaria (Singer) Matheny, Sánchez-García & T.J. Baroni (2017: 209) formerly placed in the genus Pleurocollybia Singer (1947: 80), has a brownish-fuscous to fuscous pileus (Matheny et al. 2017). Their differences from the new species are apparently more obvious than that of G. highlandensis. In the genus Gerhardtia, the macroscopic differences

Gerhardtia sinensis (Agaricales, Lyophyllaceae) Phytotaxa 332 (2) © 2017 Magnolia Press • 177 are usually more distinct, and can help to distinguish each species, but the microscopic characteristics are often very similar or even are “in common” as mentioned by Bigelow (1985) and thus not so helpful for species separation. The phylogenetic relationships among the species of Gerhardtia remained unclear, since most reports for Gerhardtia were based on morphological studies. Until now, only four species of Gerhardtia (G. borealis, G. cibaria, G. highlandensis and G. pseudosaponacea) sequences are available in GenBank. Molecular phylogenetic studies showed that Gerhardtia is a strongly supported genus under Lyophyllaceae (Frøslev et al. 2003; Saar et al. 2009; Cooper 2014; Matheny et al. 2017), having a sister group Calocybella Vizzini, Consiglio & Setti (2015: 5) with clamp-connections (Vizzini et al. 2015). Therefore, phylogenetic trees consisting of the members of genus Gerhardtia, Calocybella and Lyophyllum were constructed in this study, including the most similar species G. highlandensis from North America, with an ITS sequence (FJ601808) from the isotype collected from North Carolina, a nrLSU sequence (EF535275) and an ITS (GU734744) from a collection from Massachusetts, the state from which a paratype was collected (Table 1).

TABLE 1. Species used in the molecular phylogenetic analyses, taxa information, vouchers, and GenBank accession numbers. Taxon Voucher Locality nrLSU ITS Reference Calocybella pudica AMB 15995 Italy KP858006 KP858001 Vizzini et al. (2015) C. pudica AMB 15996 Italy KP858007 KP858002 Vizzini et al. (2015) C. pudica AMB 15997 Italy KP858008 KP858003 Vizzini et al. (2015) Gerhardtia borealis AMB 15993 Italy KP858009 KP858004 Vizzini et al. (2015) G. borealis FR 2013194 France N/A KP192534 Bellanger et al. (2015) G. borealis HC01/025 EF421091 EF421103 GenBank G. borealis U.Soderholm 1593 Finland AM946449 N/A Saar et al. (2009) (H) G. cibaria AS s.n. (TENN) Peru N/A KX981985 Matheny et al. (2017) G. highlandensis PBM 2086 USA EF535275 GU734744 Matheny et al. (2017) G. highlandensis TENN 16020 USA N/A FJ601808 Matheny et al. (2017) G. pseudosaponacea PDD 96650 New Zealand KJ461911 N/A Cooper (2014) G. sinensis GDGM 29981 China KU563152 KU563151 This study G. sinensis GDGM 42158 China KY465427 KY465426 This study G. sinensis GDGM 42435 China KX882032 KX882031 This study G. sinensis GDGM 45221 China KY465429 KY465428 This study G. sinensis GDGM 46394 China KX882034 KX882033 This study Lyophyllum PDD 96332 New Zealand N/A KJ461906 Cooper (2014) moncalvoanum L. moncalvoanum PDD 72796 New Zealand KJ461891 KJ461890 Cooper (2014) L. moncalvoanum PDD 96328 New Zealand KJ461905 KJ461904 Cooper (2014) L. shimeji GDGM 47721 China KY684032 KY684031 This study L. shimeji Anttila 20090921 Finland N/A HM572526 Larsson et al. (2011) L. shimeji Domeij 20090913 Sweden N/A HM572525 Larsson et al. (2011) L. shimeji Lipovac090911 Sweden HM572534 N/A Larsson et al. (2011) L. shimeji Sundberg 20090813c Sweden HM572535 N/A Larsson et al. (2011) Tricholoma sinoacerbum GDGM 44680 China KT160221 KT160219 Hosen et al. (2016)

In the phylogenetic trees, all the Gerhardtia sequences are nested in a clade, and the sequences of Calocybella and Lyophyllum form more distant clades (Figs. 4–5). All sequences of the new species are grouped in a well-supported subclade, and most closely related to G. highlandensis with strong support in both nrLSU and ITS trees (Figs. 4–5), which also indicates that G. sinensis is the closest relative to G. highlandensis among the known taxa. Ecologically, G. sinensis grows on humus soil of broad-leaved or coniferous forests and broad-leaved mixed forests, as do G. piperata and G. pseudosaponacea (Armin & Zdenko 2009; Cooper 2014). However, G. borealis and G. leucopaxilloides grow on sandy or clay soil of coniferous forests (Bigelow 1985; Contu & Consiglio 2004); G. cibaria grows in grasslands near Stipa ichu and Puna grass (Matheny et al. 2017); G. citrinolobata grows on wet deciduous forest litter (Vizzini et al. 2017); G. suburens grows on the ground under hardwoods but is lacking details of vegetation type in the descriptions (Bigelow 1985; Contu & Consiglio 2004); G. highlandensis grows on needles under white pine (Bigelow 1985; Contu & Consiglio 2004), or on the soil near rotting wood in mixed woods under Fagus, Quercus and Pinus (https://www.ncbi.nlm.nih.gov/nuccore/EF535275). Although both G. sinensis and G. highlandensis can grow in similar habitats with pine trees, they occur on different continents.

178 • Phytotaxa 332 (2) © 2017 Magnolia Press LI et al. Acknowledgements

The authors express sincere gratitude to Prof. YH Shen, Dr. CY Deng, Dr. M Zhang and Mr. H Huang for collecting the specimens, and also to Dr. Md. Iqbal Hosen for his helpful discussions concerning the morphological and molecular phylogenetic characteristics, illustrated figures and references. This work was financed by the Science and Technology Key Program of Guangzhou, China (201607020017), the National Natural Science Foundation of China (31370072), Special Foundation for State Science and technology Basic Research Program of China (2013FY111500) and Field Station Foundation of Guangdong Academy of Sciences (Sytz201512).

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180 • Phytotaxa 332 (2) © 2017 Magnolia Press LI et al.