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Hygrophorus subsection Hygrophorus (Hygrophoraceae, Agaricales) in China
expand article infoChao-Qun Wang, Tai-Hui Li, Ming Zhang, Xiao-Lan He§, Wei-Qiang Qin|, Tie-Zhi Liu, Nian-Kai Zeng#, Xiang-Hua Wang¤, Jian-Wei Liu¤, Tie-Zheng Wei«, Jiang Xu, Yue-Qiu Li, Ya-Heng Shen
‡ Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangdong, China
§ Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences, Chengdu, China
| Jishou University, Hunan, China
¶ Chifeng University, Chifeng, China
# Hainan Medical University, Haikou, China
¤ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
« Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Open Access

Abstract

Hygrophorus subsect. Hygrophorus has been relatively well-studied in Europe and North America, but studies on the taxa in Asia, particularly in China, are still limited. In this study, phylogenetic overviews of genus Hygrophorus, based on the nuclear large subunit (LSU) ribosomal RNA gene and of subsect. Hygrophorus, based on the nuclear ribosomal internal transcribed spacer (ITS) regions were generated. Four new species, i.e. H. brunneodiscus, H. fuscopapillatus, H. glutiniceps and H. griseodiscus are described from southern China; and a rarely reported edible species H. hedrychii is described in detail, based upon the materials from north-eastern China. The main characteristics of the species under subsect. Hygrophorus worldwide are summarised in a table.

Keywords

East Asia, molecular systematics, taxonomy, waxycap, woodwax

Introduction

Hygrophorus Fr. (Hygrophoraceae, Agaricales, Basidiomycota) is a cosmopolitan fungal genus, mainly distributed in the northern hemisphere. The characteristics that distinguish the genus are: the ectomycorrhizal habit, robust basidiomata, usually viscid pileus surface, waxy, thick and distant lamellae, divergent lamellar trama and white or hyaline thin-walled basidiospores (Candusso 1997, Young 2005). The divergent lamellar trama morphologically distinguishes Hygrophorus from the other genera in the family Hygrophoraceae (Singer 1986, Lodge et al. 2014). According to the recent phylogenetic study, Hygrophorus can be divided into three subgenera [subg. Camarophylli Fr., subg. Colorati (Bataille) E. Larss. and subg. Hygrophorus], while subg. Hygrophorus is divided into three sections [sect. Discoidei (Bataille) Konrad & Maubl., sect. Hygrophorus and sect. Picearum E. Larss.] and sect. Hygrophorus consists of subsect. Fulventes E. Larss. and subsect. Hygrophorus, which includes the generic type species H. eburneus (Bull.: Fr.) Fr. (Lodge et al. 2014).

Morphologically, members in subsect. Hygrophorus share the characteristics of glutinous and white or pallid pileus, almost white and sometimes darkening lamellae, glutinous stipe and Cossus-odour, resembling the smell of Cossus cossus (Lepidoptera) (Larsson and Jacobsson 2004, Lodge et al. 2014). Five known species are currently included in subsect. Hygrophorus, i.e. H. cossus (Sow.) Fr., H. discoxanthus (Fr.) Rea, H. eburneus, H. hedrychii (Velen.) K. Kult. and H. scabrellus A. Naseer & A.N. Khalid, according to Larsson and Jacobsson (2004), Lodge et al. (2014) and Naseer et al. (2019).

Phylogenetically, the relationships of subsect. Hygrophorus are still controversial. The ITS-LSU-SSU-RPB2 combined analysis in Lodge et al. (2014) showed that subsect. Hygrophorus was a polyphyletic group; ITS analysis in Endo et al. (2018) showed subsect. Hygrophorus as a polyphyletic group since H. discoxanthus was located at the base of sect. Hygrophorus. ITS analysis in Lodge et al. (2014) showed that subsection was mostly monophyletic with an unstable support, but H. discoideus should belong to subsect. Discoidei. The ITS-LSU combined analysis in Lodge et al. (2014) and ITS analysis in Naseer et al. (2019) indicated subsect. Hygrophorus as a monophyletic group.

During the authors’ study on the diversity of subsect. Hygrophorus in China, five species were discovered. In order to assess their phylogenetic positions, a phylogenetic overview of genus Hygrophorus was conducted, based on all available sequences of the nuclear large subunit (LSU) ribosomal RNA gene from GenBank (Benson et al. 2017) and the newly-obtained Chinese sequences in this study; and a phylogenetic overview of subsect. Hygrophorus was also made with the available sequence data of nuclear ribosomal internal transcribed spacer (ITS) regions from GenBank and UNITE (Nilsson et al. 2019) and the newly-obtained sequences from the Chinese materials. Through morphological comparisons along with the phylogenetic analyses, four new species, i.e. H. brunneodiscus from Central South China, H. fuscopapillatus and H. griseodiscus from south-western China and H. glutiniceps from southern China, are firstly introduced in this paper; and the presence of H. hedrychii in China is confirmed using molecular data, based on fungal collections from north-eastern China and also described and elucidated, based on macro- and micromorphological observations.

Materials and methods

Morphological observation and descriptions

Specimens are deposited in the Mycological Herbarium of Chifeng University (CFSZ), Fungal Herbarium of Hainan Medical University (FHMU), Fungarium of Guangdong Institute of Microbiology (GDGM), Herbarium of Mycology, Academia Sinica (HMAS) and Mycological Herbarium of Soil and Fertilizer Institute, Sichuan Academy of Agricultural Sciences (SAAS). Macro-morphological characteristics are described on the basis of field notes and photos and colour codes followed Kornerup and Wanscher (1978). Micro-morphological characteristics are measured when tissues are mounted in 5% aqueous potassium hydroxide (KOH) and 1% Congo Red solution under an Olympus BX51 microscope (Olympus Co., Tokyo, Japan). Twenty basidiospores and ten basidia from mature lamellae are measured for each specimen, Q refers to the ratio of length to width and Qm refers to the mean value of Q values of all spores; pileipellis and stipitipellis are observed using the hand-sliced tissues approximately at the surface of the mid-radius of the pileus and the middle of the stipe length, respectively. Description terms for morphological characteristics mainly follow Vellinga (1988).

DNA extraction, PCR amplification and sequencing

The total genomic DNA extracted from dry samples using a HiPure Fungus DNA Mini kit (Megen Biotech Co. Ltd., Guangzhou, China) according to the manufacturer’s instructions. Gene regions of the large subunit (LSU) and the internal transcribed spacer (ITS) nuclear ribosomal RNA gene are amplified by Polymerase Chain Reactions with the primers LR5 and LR0R (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/) and ITS1/ITS5 and ITS4 (White et al. 1990, Gardes and Bruns 1993), respectively. Sequencing of both directions was performed on an ABI 3730 sequencer analyser (Applied Biosystems, Foster City, CA, USA) using the same PCR primers at Beijing Liuhe Co. Ltd. Raw sequences are assembled by using SEQMAN version 7.1.0 of LASERGENE software (DNAStar, Madison, WI, USA). Newly-obtained consensus sequences are deposited in GenBank (Table 1).

Table 1.

Taxa, vouchers, geographic origin and GenBank accession numbers of the newly-generated sequences in this study.

Taxon Voucher Geographic origin ITS LSU
H. brunneodiscus GDGM73213 China: Hunan MN378318 MT093623
GDGM75489 China: Hunan MN378317 MT093622
GDGM76934 China: Hunan MT093605 MT093621
H. eburneus GDGM70059 (BHS2011-11) USA MT093608
H. fuscopapillatus GDGM44412 China: Sichuan MN378337 MT093625
LJW1858 China: Yunnan MT093606 MT093626
XHW6609 China: Yunnan MT093607 MT093627
H. griseodiscus FHMU1578 (Zeng2452) China: Hainan MN378311
FHMU2013 (Zeng3052) China: Hainan MN378312 MT093614
GDGM42140 China: Guangdong MN378310 MT093619
GDGM42188 China: Guangdong MN378313 MT093618
GDGM42217 China: Guangdong MN378309 MT093617
GDGM45220 China: Hainan MN378315 MT093620
GDGM53153 China: Jiangxi MT093603 MT093615
GDGM53496 China: Hunan MN378314
HMAS273294 China: Guangdong MT093604
SAAS462 China: Sichuan MN378338 MT093624
H. hedrychii CFSZ2559 China: Inner Mongolia MT093610
CFSZ2851 China: Inner Mongolia MN378306 MT093628
CFSZ4268 China: Inner Mongolia MT093609
CFSZ10761 China: Inner Mongolia MT093611
CFSZ18159 China: Inner Mongolia MN378308 MT093629
CFSZ18269 China: Inner Mongolia MN378307 MT093630

Sequence alignments and phylogenetic analyses

For the LSU dataset, the newly-obtained sequences and all available Hygrophorus sequences longer than 300 bps from GenBank are treated as ingroups and the sequences of Cantharocybe gruberi (A.H. Sm.) H.E. Bigelow & A.H. Sm. are selected as outgroup, based on Razaq et al. (2014) and Naseer et al. (2019); for the ITS dataset, the newly-obtained sequences and the downloaded sequences from GenBank and UNITE longer than 300 bps and of subsect. Hygrophorus are combined as ingroups and the sequences of H. arbustivus Fr. are selected as outgroups, since H. arbustivus is the type species of the sister group subsect. Fulventes, according to Lodge et al. (2014). Both datasets are combined, using GENEIOUS software (Biomatters Ltd.) and aligned by MAFFT (Multiple Alignment using Fast Fourier Transform) online service version 7 (Katoh et al. 2017). Maximum Likelihood analyses are performed by IQ-TREE web server with 1000 rapid bootstrap (BS) replicates (Trifinopoulos et al. 2016). The trees are viewed and edited in ITOL (Interactive Tree of Life) web server (Letunic and Bork 2016).

Results

Molecular phylogenetic results

For the aligned LSU dataset, 121 sequences with 980 sites were included, amongst them 119 Hygrophorus sequences are placed in the ingroups and two sequences of Cantharocybe gruberi are in the outgroups. In the LSU phylogenetic tree (Fig. 1), both sect. Hygrophorus and subsect. Hygrophorus are presented as monophyletic groups with low support values, 61% BS and 54% BS, respectively; in addition, the sequences named as “H. lindtneri” (MK278193), “H. marzuolus” (MK278194) and “H. cf. arbustivus” (MK278183) are present within these clades. Three sequences from three samples (GDGM44412, LJW1858 and XHW6609) of H. fuscopapillatus are present as a monophyletic clade with high support value (100% BS), H. griseodiscus is present as a sister clade to H. fuscopapillatus with 100% BS, and, together, they form a sister clade to H. cossus with 75% BS; three sequences of H. brunneodiscus samples (GDGM73213, GDGM75489 and GDGM76934) and seven sequences of H. glutiniceps samples (GDGM42140, GDGM42154, GDGM42188, GDGM42217, GDGM45220, GDGM53153 and Zeng3052) form two independent monophyletic clades with high support values (100% BS) respectively; in addition, three sequences of H. hedrychii specimens (CFSZ2851, CFSZ18159 and CFSZ18269) form a monophyletic clade with high support value (99% BS).

Figure 1. 

Phylogram showing the interspecific relationships under the genus Hygrophorus inferred from a LSU dataset using IQ-tree. Sequences of Cantharocybe gruberi were selected as outgroups. Bootstrap values greater than 50% are indicated around the branches. For downloaded sequences, specimen names and GenBank accession numbers are presented; for newly-generated sequences, species names and specimen vouchers are listed. Four newly-described species’ sequences are highlighted in colour; sequences with quotation marks are incorrect names.

For the aligned ITS dataset, 88 sequences with 689 sites were included, two sequences of H. arbustivus are treated as the outgroups and 86 sequences of subsect. Hygrophorus are within the ingroups. At least thirteen respectively-supported clades that correspond to different species were recovered in the ITS phylogenetic tree (Fig. 2), five of them coming from China. Two newly-generated sequences of H. griseodiscus from two different fruit-bodies of SAAS462 fall together in a supported clade with two GenBank downloaded sequences (KU836529–30) from Sichuan province in China and an uncultured Hygrophorus sequence (LC175568) from Hokkaido in Japan and the support value of the H. griseodiscus-clade is 91% BS. Three sequences from samples (GDGM44412, LJW1858 and XHW6609) of H. fuscopapillatus form a clade with 85% BS. Three sequences of H. brunneodiscus samples (GDGM73213, GDGM75489 and GDGM76934) form a clade with strong support value (100% BS). Nine sequences of H. glutiniceps specimens (GDGM42140, GDGM42188, GDGM42217, GDGM45220, GDGM53153, GDGM53496, HMAS273294, Zeng2452 and Zeng3052) are also clustered with strong support value (100% BS). Six newly-generated sequences of H. hedrychii samples (CFSZ2559, CFSZ2851, CFSZ4268, CFSZ10761, CFSZ18159 and CFSZ18269) from Inner Mongolia Autonomous Region of China are clustered with other H. hedrychii sequences from China (KX497168) and Sweden (AY242854 and AY463490) with 92% BS; in addition, MK575431 named as “H. eburneus” from Wisconsin in USA and DQ367904, labelled as “H. eburneus var. eburneus” from Canada, form a clade with 98% BS, this North American clade being the sister clade with the Eurasian H. hedrychii-clade with 100% BS.

Figure 2. 

Phylogram of species under subsect. Hygrophorus inferred from an ITS dataset using IQ-tree. Sequences of Hygrophorus arbustivus were selected as outgroups. Bootstrap values greater than 50% are indicated around the branches. For downloaded sequences, specimen names, GenBank accession numbers and locations are presented; for newly-generated sequences, species names, specimen vouchers and locations are listed. Four newly-described species’ sequences are highlighted in colour; sequences with incorrect names are marked with quotation marks.

Species of subsect. Hygrophorus known from China

According to the molecular phylogenetic analyses (Figs 1, 2) and morphological examinations on the Chinese specimens in this study, at least five species of Hygrophorus subsect. Hygrophorus are present in China, including H. brunneodiscus, H. fuscopapillatus, H. glutiniceps, H. griseodiscus and H. hedrychii. Although H. cossus and H. eburneus of subsect. Hygrophorus have been reported in China (Chen and Li 2013), but they are not described in the present study, because no DNA sequences or fresh specimens of these two species have been obtained from China in this study.

Taxonomy

Hygrophorus brunneodiscus C.Q. Wang & T.H. Li, sp. nov.

Fungal Names: FN570664
Figure 3

Typification

China, Hunan Province, Zhangjiajie City, Zhangjiajie Campus of Jishou University, on the ground of a forest dominated by Quercus fabri and Q. serrata, elev. ca. 220 m, 29°8'24"N, 110°27'42"E, 26 May 2019, W.Q. Qin (GDGM73213, Holotype!), ITS MN378318.

Etymology

brunneo-”: brown, “-discus”: pileus disc. The species epithet “brunneodiscus” (Lat.) refers to the brown pileus disc of this new taxon.

Diagnosis

Hygrophorus brunneodiscus differs from H. cossus by the smaller pileus (20–50 mm broad), brownish pileus disc, thinner stipe (4–7 mm wide) and the shorter basidia (32–48 µm long); differs from H. fuscopapillatus and H. griseodiscus by the low elevation distribution and brown pileus disc.

Figure 3. 

Hygrophorus brunneodiscus. a, b Basidiomata (GDGM73213, Holotype) c, d Basidiomata (GDGM75489) e Basidiospores (GDGM73213) f Basidia (GDGM73213) g Elements of pileipellis (GDGM73213). Scale bars: 2 cm (a–d).

Description

Pileus 20–50 mm broad, hemispherical or conical with a slightly involute or slightly revolute margin when young, becoming convex with an expanded margin when mature, whitish to brownish as a whole, brownish-orange, light brown, yellowish-brown (5C4–5, 5D4–5, 5E5–8) at the disc (about one fourth part of the radius from the centre to the margin), becoming paler to greyish-yellow (4B3–4), greyish-white (4B1) or whitish outwards and white at the margin or with a white marginal zone of 1–2 mm wide, viscid, covered with a glutinous layer of transparent materials when wet. Lamellae short decurrent to decurrent, white, waxy, with 36–40 complete lamellae and 1–3 lamellulae between two complete lamellae; lamella edge concolorous, entire. Stipe 40–90 × 4–7 mm, cylindrical, hollow, nearly equal to slightly thinner at apex and tapering towards the base; pale yellow to greyish-yellow (4A3, 4B3), white to yellowish-white (4A2) at apex, sometimes white at base; sticky, covered with a layer of transparent materials when wet, easily-sticking debris on the slime layer, usually fibrillose or with scattered white fibrillose dots at apex. Context thin, white to light brown, with slight fishy odour.

Basidiospores (6.5)7–9(9.5) × 4–5.5(6) μm [mean length = 7.6 µm, mean width = 4.6 µm], Q = (1.3)1.4–2(2.1), Qm = 1.68, ellipsoid to oblong, smooth. Basidia 32–48 × 6–8.5 µm, Q = 4.1–6.6, Qm = 5.3, clavate, thin-walled, 4-spored, with sterigmata 5.5–7(9) µm long. Pileipellis an ixotrichoderm, composed of septate cylindrical hyphae, covered with a gelatinous layer; hyphae thin-walled, 2.5–5 μm wide, slightly yellowish and glutinous in KOH. Hymenophoral trama divergent, composed of septate, thin-walled and cylindrical hyphae; hyphal cells 45–70 × 6–10 μm, hyaline. Clamp connections present.

Habit, habitat and distribution

Solitary to scattered, on the ground of subtropical broad-leaf forest dominated by Quercus, so far only known from Hunan Province in South Central China.

Additional specimens examined

China, Hunan Province, Zhangjiajie City, Zhangjiajie Campus of Jishou University, 26 October 2018, W.Q. Qin (GDGM75489); ibidem, 30 June 2019, W.Q. Qin (GDGM76934).

Remarks

Hygrophorus brunneodiscus is characterised by its brown tint on pileus disc, sticky pileus and stipe surface, basidiospores (6.5)7–9(9.5) × 4–5.5(6) μm, basidia 32–48 × 6–8.5 µm and subtropical and low elevation distribution.

Hygrophorus brunneodiscus can be easily recognised within subsect. Hygrophorus for its brownish colour on the pileus disc. Apart from that, H. cossus differs from H. brunneodiscus by having pale ochraceous grey colour at the pileus centre and more slender basidia (48–60 × 7–8.5 μm) (Larsson and Jacobsson 2004). Hygrophorus discoxanthus differs in the pure white mature basidiomata as young, rusty-brown pileus margin and discolouration of rusty brown at the lamellar edge and different host-association (with Fagus) (Candusso 1997, Larsson and Jacobsson 2004). Hygrophorus eburneus differs from it by having more carnose pileus and thicker stipe (4–10 mm wide), forming an ectomycorrhizal relationship with Fagus (Larsson and Jacobsson 2004). Hygrophorus hedrychii differs from it by having larger and more robust fruit-bodies (pileus 30–80 mm in diam. and stipe 5–10 mm in width), forming an ectomycorrhizal relationship with Betula (Larsson and Jacobsson 2004, Campo 2015). Hygrophorus scabrellus differs from it by owning smaller (24–28 mm in diam.) and off-white with dark green colour pileus, smaller basidiospores (mean length = 6.5 µm, mean width = 3.84 µm) and distribution in temperate forests under Quercus trees (Naseer et al. 2019).

Hygrophorus fuscopapillatus C.Q. Wang & T.H. Li, sp. nov.

Fungal Names: FN570701
Figure 4

Typification

China, Sichuan Province, Panzhihua City, Yanbian County, Gesala Ecotourism Area, elev. ca. 2900 m, 27°08'10"N, 101°11'33"E, 25 August 2013, M. Zhang & C.Q. Wang (GDGM44412, Holotype!), ITS MN378337.

Etymology

fusco-”: dark brown, “-papillatus”: papillate. The species epithet “fuscopalillatus” (Lat.) refers to the dark brown or grey brown papilla on the pileus of the new species.

Diagnosis

Differs from H. griseodiscus by the host association with Fagaceae, solitary habit, adnate to subdecurrent lamellae and slightly smaller basidispores measuring (6)7–9.5(10) × (4)4.5–5.5(6) µm. The ITS sequence is 95% similar to H. griseodiscus.

Figure 4. 

Hygrophorus fuscopapillatus. a, b Basidiomata (GDGM44412, Holotype) c, d Basidiospores Basidiomata (LJW1858) e, f Basidiospores and Basidia (XHW6609) g, h Basidiospores and Basidia (GDGM44412) i, j Basidiospores and Basidia (LJW1858). Scale bars: 2 cm (a–f).

Description

Pileus 20–30 mm broad, convex to hemispherical when young, applanate to plano-concave when mature, with a papilla or small umbo in the centre, white to pale grey (1B1), grey or brownish-grey to olive brown (4E2–3,4F1–3) at papilla, gradually becoming lighter from centre to margin, white to pale grey (1B1) at margin, glutinous when wet; margin even, occasionally split. Lamellae adnate to subdecurrent, white, thick, with 30–36 complete lamellae per pileus and 1–3 lamellulae between two entire lamellae. Stipe 40–60 × 4–5 mm, cylindrical, white to yellowish-grey (4B2), covered by a glutinous layer. Context thin, whitish.

Basidiospores (6)7–9.5(10) × (4)4.5–5.5(6) µm [mean length = 8.2 µm, mean width = 5 µm], Q =1.3–1.9, Qm =1.65, broadly ellipsoid, ellipsoid to oblong, smooth, hyaline. Basidia (32)35–46(48) × (6)6.5–8.5(9) µm, Q = 4.4–6.8, Qm = 5.5, clavate, thin-walled, 4-spored, with sterigmata 5–7.5 µm long. Pileipellis an ixotrichoderm, composed of septate and thin-walled cylindrical hyphae, covered with a gelatinous layer; hyphal cells 3–5 μm broad. Hymenophoral trama divergent, composed of septate, thin-walled and cylindrical hyphae; hyphal elements 13–31.5 μm broad. Clamp connections present.

Habit, habitat and distribution

Solitary, on the ground of Fagaceae-dominated forests, so far only known from Sichuan and Yunnan provinces in Southwest China.

Additional specimens examined

China, Yunnan Province, Yulong County, Jade Dragon Snow Mountain, Lijiang Alpine Botanic Garden, on the ground of Quercus pannosa dominated forest, elev. ca. 3267 m, 27°00'02"N, 100°10'52"E, 31 August 2019, J.W. Liu (LJW1858); Binchuan County, Jizushan, on the ground of Castanopsis and Lithocarpus dominated forest, elev. ca. 2853 m, 25°58'06"N, 100°21'39"E, 18 September 2019, X.H. Wang (XHW6609).

Remarks

Hygrophorus fuscopapillatus is distinguished by the solitary basidiomes, the brownish-grey to olive brown papilla in the pileus centre, the broadly ellipsoid, ellipsoid to oblong basidiospores measuring (6)7–9.5(10) × (4)4.5–5.5(6) µm.

Amongst the members of subsect. Hygrophours, H. griseodiscus closely resembles H. fuscopapillatus; however, H. griseodiscus differs from H. fuscopapillatus by the host association with Pinaceae, the larger pileus (2–4.5 cm broad), the emarginate lamellae with decurrent tooth, the larger basidispores measuring (7)8–10(10.5) × (4)4.5–6(6.5) µm and the broader basidia (7–11 mm broad). In addition, H. brunneodiscus is distinguished from H. fuscopapillatus by the broader pileus (2–5 cm in diam.) and the brownish pileus disc. Hygrophorus cossus is separated by the larger pileus (3–9 cm in diam.), greyish-white lamellae with a cream yellow tint and a thicker stipe (0.6–2 cm broad) (Larsson and Jacobsson 2004). Hygrophorus discoxanthus differs by the rusty brown lamellae when mature and the wider stipe (up to 1.2 cm broad); H. eburneus is distinguished by the gregarious habit, the more robust basidiomes and the white pileus (Candusso 1997, Larsson and Jacobsson 2004). Hygrophorus glutiniceps differs by the subtropical to tropical distribution and smaller basidiospores measuring (5)6–8.5(10) × (3.5)4–6 µm. Hygrophorus hedrychii is separated by the larger basidiomes (pileus up to 8 cm in diam.) and the reddish-yellow pileus centre (Larsson and Jacobsson 2004). Hygrophorus scabrellus is distinguished by the off-white with dark green pileus, the off-white to beige lamellae and the much smaller basidiospores measuring 6.5 × 3.84 µm (Naseer et al. 2019).

Hygrophorus glutiniceps C.Q. Wang & T.H. Li, sp. nov.

Fungal Names: FN570663
Figures 5, 6

Typification

China, Guangdong Province, Guangzhou City, Tianluhu Forest Park, on the ground of a forest dominated by Castanopsis fissa, elev. ca. 250 m, 23°13'39"N, 113°25'53"E, 6 September 2012, M. Zhang (GDGM42188, Holotype!), ITS MN378313.

Etymology

glutini-”: glutinous, “-ceps”: pileus. The species epithet “glutiniceps” (Lat.) refers to the glutinous surface of pileus.

Diagnosis

Hygrophorus glutiniceps differs from H. discoxanthus by the subtropical to tropical distribution, the darker lamellae and the thinner stipe (3–6 mm broad).

Figure 5. 

Basidiomata of Hygrophorus glutiniceps. a, b GDGM42140 c GDGM42154 d GDGM42188 (Holotype) e GDGM42217 f GDGM45220. Scale bars: 2 cm.

Description

Pileus 8–40 mm broad, hemispherical to convex, often with an inconspicuous umbo and a usually involute margin when young, broad-convex to depressed with a slightly incurved to rarely uplifting margin when mature, covered with a layer of gelatine-like or transparent gluey materials when wet, white with cream or light yellow to orange tint (4A2–4) at disc, becoming light to yellowish-brown (5E8) with age (especially at margin and in wounded area), usually brownish-orange (5C5) to brown (6E8) when exsiccated. Lamellae broadly adnate or with decurrent tooth when young, short to moderately decurrent when mature, white when young, then changing to ochraceous or even brown (6E8), waxy when wet, with 25–30 complete lamellae reaching to stipe and 1–3 lamellulae between two entire lamellae. Stipe 25–60 × 3–6 mm, equal but often thinner at the base, cylindrical but usually curved, white, occasionally with yellowish tint (2A2), covered with transparent glutinous materials when wet. Context thin, white when young, changing to brown when old.

Basidiospores (5)6–8.5(10) × (3.5)4–5.5(6) µm [mean length = 7.1 µm, mean width = 4.7 µm], Q = (1.2)1.3–1.77(2), Qm = 1.52, ellipsoid to oblong, smooth, hyaline. Basidia 35–47 × 5–8.5 µm, Q values usually more than 5, clavate, thin-walled, 4-spored, with sterigmata up to 7.5 µm long, hyaline. Pileipellis an ixotrichoderm, composed of septate hyphae, usually covered with a gelatinous layer; hyphae thin-walled, 3–5 μm wide, with yellowish gluten in KOH. Stipitipellis an ixotrichoderm, hyphae 3–5 μm wide, similar to those of pileipellis. Hymenophoral trama divergent, composed of septate, thin-walled and cylindrical hyaline in 4–17 μm diam. Clamp connections present.

Habit, habitat and distribution

Gregarious to scattered, on the ground of subtropical broad-leaf forest dominated by Castanopsis, currently only known from subtropical to tropical areas of China.

Additional specimens examined

China, Guangdong Province, Guangzhou City, Tianluhu Forest Park, 6 September 2012, M. Zhang (GDGM42217); ibidem, 6 September 2012, J. Xu (GDGM42140). Hainan Province, Changjiang County, 3 July 2013, M. Zhang (GDGM45220); Baisha County, Yinggeling National Nature Reserve, elev. ca. 600 m, 1 August 2015, N.K. Zeng 2452 (FHMU1578); Ledong County, Yinggeling National Nature Reserve, elev. ca. 650 m, 4 June 2017, N.K. Zeng 3052 (FHMU2013).

Remarks

Hygrophorus glutiniceps is macro-morphologically characterised by its subtropical-tropical distribution, white and sticky pileus and stipe, darkening lamellae when mature or wounded. The size of basidiospores [(5)6–8.5(10) × (3.5)4–5.5(6) µm] and basidia [35–47 × 5–8.5 µm] can be used to confirm the recognition. The association with Castanopsis fissa is also helpful for its identification.

Hygrophorus glutiniceps can be morphologically distinguished from closely-related species by the following differences. Hygrophorus cossus looks different from H. glutiniceps in the different ectomycorrhizal connection (with Quercus), the temperate distribution, the longer basidiospores [(7–9.5 μm long in Candusso (1997), 7–9 μm long in Larsson and Jacobsson (2004)] and the higher ratio of length to width [Qm = 1.7–1.75 in Candusso (1997)]. Hygrophorus eburneus differs in the different host-connection (with Fagus), thicker stipe (7–10 mm in width) and larger basidiospores (8–10 × 4.5–5.5 μm) with larger Qm (1.78–1.82); H. discoxanthus differs in the different host-connection (with Fagus), the more rusty brown tint on pileus and lamellae and the thicker stipe (5–12 mm in width); H. hedrychii differs in the different host-connection (with Betula) and larger pileus (30–60 mm broad); H. laurae Morgan has a much larger basidioma (with pileus 20–40 mm broad) and a wash of red or brown on the disc; and H. laurae var. decipiens Peck, described from New York, USA, differs in the absence of the pileus discolouration when dry and the nearly unchangeable lamellae (Morgan 1883, Peck 1905, Candusso 1997, Larsson and Jacobsson 2004).

Figure 6. 

Macro- and microscopic features of Hygrophorus glutiniceps. a Basidiomata (GDGM42217) b Basidiospores (GDGM42188) c Basidia (GDGM42188) d Terminal cells of pileipellis (GDGM42188).

Hygrophorus griseodiscus C.Q. Wang & T.H. Li, sp. nov.

Fungal Names: FN570000
Figure 7

Typification

China, Sichuan Province, Jiuzhaigou, elev. ca. 3100 m, 11 September 2012, X.L. He (SAAS462, Holotype!), ITS MN378338.

Etymology

griseo-”: grey, “-discus”: pileus. The species epithet “griseodiscus” (Lat.) refers to the grey disc of the pileus.

Diagnosis

Hygrophorus griseodiscus is distinguished from H. brunneodiscus by the greyish pileus with a darker grey pileus disc and larger basidiospores measuring (7)8–10(10.5) × (4)4.5–6(6.5) µm.

Figure 7. 

Hygrophorus griseodiscus (SAAS462, Holotype). a, b Basidiomata c Basidiospores d Basidia e Elements of pileipellis.

Description

Pileus 20–45 mm broad, convex, obtusely umbonate at disc, grey to light grey (1C1, 1D1), medium to dark grey or olive grey (1E1, 1F1–4) at disc, white to pale grey (1B1) at margin, glutinous when wet; margin even, slightly involuted to extended. Lamellae emarginate with decurrent tooth or subdecurrent, white, thick, subcrowded, unequal, with 1–3 lamellulae between two entire lamellae. Stipe 40–70 × 4–6 mm, cylindrical, white to pale grey (1B1), covered with transparent glutinous materials when wet. Context slightly thick, white.

Basidiospores (7)8–10(10.5) × (4)4.5–6(6.5) µm [mean length = 9 µm, mean width = 5.2 µm], Q = 1.4–2.1, Qm = 1.74, ellipsoid, oblong to subcylindrical, smooth, hyaline. Basidia 29–56.5 × 7–11 µm, Q = 3.05–6(6.9), Qm = 4.39, clavate to cylindrical, thin-walled, 4-spored, with sterigmata up to 6 µm long. Pileipellis an ixotrichoderm, covered with a gelatinous layer; hyphae thin-walled, 2.5–6 μm wide. Hymenophoral trama divergent, composed of septate, thin-walled and cylindrical hyphae; hyphal cells 5.5–20 μm in width, hyaline. Clamp connections present.

Habit, habitat and distribution

Scattered, on the ground of subalpine coniferous forest dominated by Abies and Picea, often surrounded by mosses, so far only known from Sichuan Province in Southwest China.

Remarks

Hygrophorus griseodiscus is characterised by its convex and grey pileus with a dark grey to olive grey disc, emarginate to subdecurrent lamellae. The Asian subalpine coniferous habitat may be a helpful character for its identification.

Morphologically, H. brunneodiscus is distinguished from H. griseodiscus by the brownish pileus disc and smaller basidiospores (6.5–9.5 × 4–5 µm). Hygrophorus cossus differs in the greyish-white lamellae with a cream yellow tint and a thicker stipe (6–20 mm wide) (Candusso 1997, Campo 2015). Hygrophorus discoxanthus can be separated by the pure white pileus when young and rusty brown lamellae when mature (Candusso 1997, Campo 2015). Hygrophorus eburneus is different by the white pileus and the wider basidiospores (8–10 × 4.5–5.5, Qm = 1.78–1.82) (Candusso 1997). Hygrophorus glutiniceps is separated by the white pileus with cream or light yellow to orange tint at the disc, shorter basidiospores [(5)6–8.5(10) × (3.5)4–6 µm] and subtropical to tropical distribution. Hygrophorus hedrychii is distinguished by the presence of a pale orange tint on the pileus disc and an orange-pink tint on the lamellae (Larsson and Jacobsson 2004). Hygrophorus scabrellus is readily distinguished from H. brunneodiscus by its smaller basidiomata (pileus 2.4–2.8 cm broad), dark green tint on pileus and much smaller basidiospores (6.5 × 3.84 µm) (Naseer et al. 2019).

Hygrophorus hedrychii (Helen.) K. Kult, Česká Mykol. 10(4): 232 (1956)

Figure 8

Description

Pileus 10–50 mm broad when dried, subglobose when young, becoming hemispherical, convex to nearly plane when mature; margin incurved when young, even, expanded to sometimes slightly upturned when mature; surface covered with a thick layer of transparent and sticky materials, white, with pinkish (7A2) to yellowish (4A2) tones or cream colour (1A2) on the disc. Lamellae adnate to short decurrent, white at first, changing to pinkish-white (7A2) or pale yellow (4A3) or cream colour (1A2), waxy, with 1–3 lamellulae between two entire lamellae. Stipe 20–85 × 3–10 mm when dried, cylindrical or nearly so, often thinner at apex, usually slightly enlarged at base, uneven, with white short fibrils at apex, sometimes longitudinally lacerated when mature, covered with a layer of transparent sticky materials, white, changing to orange white (5A2, 6A2) where touched. Context thick, white when young, pale to pinkish-yellow when mature, with Cossus smell.

Basidiospores (6)7–8(9) × (3.5)4–4.5(5) µm, Q = 1.5–2(2.3), Qm = 1.8, ellipsoid to subcylindrical, colourless, thin-walled, smooth. Basidia 30–40 × 6–9 µm, Q = 4.1–5.7, clavate, 4-spored; sterigmata 4–5 µm long; basal clamp connections common. Pileipellis an ixotrichoderm, composed of septate hyphae, covered with a gelatinous layer; hyphae thin-walled, 4–5 μm wide. Stipitipellis a trichoderm; hyphae 7–10 μm wide, light yellowish-brown intracellular pigment presented in a few hyphae when immersed in 5% KOH solution. Hymenophoral trama composed of septate, thin-walled and cylindrical hyaline hyphae, size 58–105 × 3–23 μm. Clamp connections present.

Figure 8. 

Hygrophorus hedrychii. a Basidiospores b Basidia c Basidiomata (CFSZ2851) d Basidiomata (CFSZ18159) e Basidiomata (CFSZ18269) f Sold at a local market, named as “baizhenmo”.

Habit, habitat and distribution

Scattered to gregarious in the north temperate forests dominated by Betula, known from north-eastern China (this study), as well as from Europe where the species was firstly discovered.

Specimens examined

China, Inner Mongolia Autonomous Region, Chifeng City, Harqin Banner, Wangyedian village, 30 August 2007, T.Z. Liu (CFSZ2851); ibidem, 8 August 2017, T.Z. Liu, Y.Q. Guang & N. Liu (CFSZ18159); Hexigten Banner, Jingpeng Town, 15 August 2017, T.Z. Liu & G.L. Yu (CFSZ18269).

Remarks

Macroscopically, Hygrophorus hedrychii is a distinctive waxycap, which is relatively easily recognised in the field by its white pileus disc and lamellae changing to pale ochraceous pink when mature and the host association with birch. Microscopically, the Chinese specimens agree with the descriptions by Larsson and Jacobsson (2004) for the Swedish samples and Campo (2015) for the French collections. Molecular phylogenetically, they are also identical to the European species. The Chinese samples from Inner Mongolia are traded as edible fungi at some local markets. The European H. hedrychii was not, however, treated as an edible mushroom (Larsson, personal communication). This difference may indicate different dietary habits in different areas.

Discussion

Phylogeny and circumscription of subsect. Hygrophorus

In recent years, the phylogenetic framework of Hygrophorus has been reconstructed by Lodge et al. (2014), based on morphological characteristics and 1–4 gene regions, based phylogenetic analyses, thus, the subsect. Hygrophorus has been inclusively redefined. In order to assess the monophyly of subsect. Hygrophorus, a LSU-based phylogeny overview of the genus Hygrophorus and an ITS-based phylogeny overview of subsect. Hygrophorus are made in this study. According to the LSU tree, most species’ sequences of subsect. Hygrophorus are well-grouped in a clade, but three sequences with species names outside of subsect. Hygrophorus (MK278183 named as “H. cf. arbustivus”, MK278193 named as “H. lindtneri” and MK278194 named as “H. marzuolus”) are inserted in the clade of subsect. Hygrophorus. It is well-known that H. arbustivus and H. lindtneri are members of subgen. Hygrophorus/sect. Hygrophorus/subsect. Fulventes and H. marzuolus is a member of subgen. Camarophylli/sect. Camarophylli, according to Lodge et al. (2014). If the sequences were well-identified, it is necessary to reassess their taxonomic positions or reconsider the phylogeny framework of subsect. Hygrophorus. However, through careful checking, it was found that all three sequences were submitted by Varga et al. (2019), which mainly focused on the phylogeny reconstruction of various higher agaric taxa in a much larger scale, the species identification of the sequences not being the focus of that paper. Therefore, the correctness of the sequences’ identification is still questionable.

According to the ITS phylogenetic tree in this study, subsect. Hygrophorus is monophyletic. Thus, the concept of subsect. Hygrophorus proposed by Lodge et al. (2014) is acceptable at present, since both LSU and ITS analyses are not strongly in conflict with the circumscription. In the future, analyses, based on more samples and additional genes, are still needed.

The species diversity of subsect. Hygrophorus

Previously, five species were confirmed as members of subsect. Hygrophorus, including H. cossus, H. discoxanthus, H. eburneus, H. hedrychii and H. scabrellus (Larsson and Jacobsson 2004, Lodge et al. 2014, Naseer et al. 2019). In this study, at least 13 phylogenetic species-level clades are presented as members of subsect. Hygrophorus, according to ITS-based analysis (Fig. 2), although some species are still to be verified or to be formally named.

Apart from the four new species from China, proposed in this study (H. brunneodiscus, H. fuscopapillatus, H. glutiniceps and H. griseodiscus), two species with American sequence samples (“H. chrysaspis Métrod” and H. occidentalis A.H. Sm. & Hesler) and two undescribed species, H. sp1 from Europe and H. sp2 from North America, are present as phylogenetic species under subsect. Hygrophorus. Four sequences labelled as “H. chrysaspis” from USA are grouped together. Although the North American samples have not been proved to be conspecific with the European H. chrysaspis (originally described from France) with molecular evidence due to the absence of the French sequence, the USA samples can still present a member of subsect. Hygrophorus; in addition, Hesler and Smith (1963) has also mentioned that the USA H. chrysaspis resembled H. eburneus (the type species of subsect. Hygrophorus). The American species H. occidentalis, originally described from Michigan (USA), is further confirmed as a member of subsect. Hygrophorus in this study (Table 2), since the isotype’s sequence (MH656479) is included; it had been regarded as a grey to fuscous member of H. eburneus complex, based on morphological evidence (Hesler and Smith 1963). Thus, H. occidentalis should be included in subsect. Hygrophours (Table 2).

It is worth mentioning that the species diversity within subsect. Hygrophorus may be underestimated for these two main reasons: 1) some species from the well-studied countries may have been left out. For example, H. occidentalis had been listed in the North American monograph (Hesler and Smith 1963), but it was not included in Lodge et al. (2014) since the absence of sequences at that time. 2) Many areas have been less investigated or not at all. Taking the ITS gene sequences as an example (Fig. 2), the majority sequences come from European and USA samples and very few sequences come from Africa, Oceania or South America, where there might be many taxa, different from those of Europe and North America.

Table 2.

Morphological characteristics of known species of subsect. Hygrophorus.

Species Pileus size (cm) Colour of mature pileus Colour of lamellae Stipe width (cm) Spore size (μm) Host- connection Reference
H. brunneodiscus 2–5 White with brownish-orange to light brown at the disc White 0.4–0.7 6.5–9.5 × 4–5 Quercus This study
H. cossus 3–9 Pale ochraceous grey at the centre of the pileus Greyish-white with a cream yellow tint 0.6–2.0 7–9 × 4–5 Quercus Larsson and Jacobsson 2004
H. discoxanthus 3–7 White with rusty-brown margin upon drying White as young, then rusty brown 0.5–1.2 6.5–9 × 3.5–5.5 Fagus Larsson and Jacobsson 2004
H. eburneus 2–7 White Cream-white as young then cream-yellow 0.4–1.0 7.5–10 × 4–5.5 Fagus Larsson and Jacobsson 2004
H. fuscopapillatus 2–3 White to pale grey at margin, pale grey to olive brown at papilla White 0.4–0.5 (6)7–9.5(10) × (4)4.5–5.5(6) Fagaceae This study
H. glutiniceps 0.8–4 White with cream or light yellow to orange tint at disc White when young, then changing to ochraceous or even brown 0.3–0.6 (5)6–8.5(10) × (3.5)4–6 Castanopsis This study
H. griseodiscus 2–4.5 Grey to light grey with dark grey at disc White 0.4–0.6 (7)8–10(10.5) × (4)4.5–6(6.5) Abies or Picea This study
H. hedrychii 3–8 White with orange-pinkish tint at centre White with orange-pinkish tint 0.5–1.0 6.5–9 × 3.5–5 Betula Larsson and Jacobsson 2004
H. occidentalis 2–8(10) “hair brown” to “fuscous”, at times yellowish or smoky at disc White then tinged cream 0.3–1(1.5) 6–8 ×3.5–5 unclear Hesler and Smith 1963
H. scabrellus 2.4–2.8 Off-white with dark green Off-white to beige 2.1–2.4 6.5 × 3.84 Quercus Naseer et al. 2019

Morphology and sequence misidentification in subsect. Hygrophorus

Some Hygrophorus taxa are difficult to be distinguished with the naked eye by the specific differences of macro-morphological characteristics and it will be harder when considering the infraspecific variations; the microscopic features for distinguishing Hygrophorus species are also limited, since they usually lack cystidia and have slight interspecific differences in the size and shape of basidiospores and basidia.

Due to the morphological similarity, misidentifications are common in subsect. Hygrophorus. For example, 15 samples with the same name as “H. eburneus” are included in the analysis (Fig. 2), but they are nested in different positions in the phylogram, which indicates that at least some of them are misidentified. The sequences of AY242855, AY463484, AY463485 and UDB000555 from Sweden (where the species was originally described) are identified in Larsson and Jacobsson (2004) and they should be the true H. eburneus. Some other European sequences, i.e. MK028422 from Switzerland, MK088116 from Hungry, MK627047 from Austria and UDB000021 labelled as “H. discoxanthus” from Denmark, are clustered with those real H. eburneus sequences from Sweden with strong support (94% BS); they should also be conspecific and treated as H. eburneus. These European sequences form a clade of “H. eburneus (Europe)”. On the other hand, there are eight sequences from USA which are gathered together in the phylogram with 95% BS, forming a sister clade to the European H. eburneus. Due to the close molecular similarity and the lack of adequate morphological comparison and other information, they are still treated as H. eburneus here as a clade of “H. eburneus (North America)”. However, the sequence MK737049, labelled as “H. cf. eburneus” from USA, is isolated from the true H. eburneus; thus, it should represent an unidentified species. In addition, the two other sequences, MK575431 from USA and DQ367904 from Canada, labelled as “H. eburneus” and “H. eburneus var. eburneus” respectively, are grouped as a sister clade of the Europe-Asia H. hedrychii clade with 100% BS. Obviously, MK575431 and DQ367904 are not the true “H. eburneus”, though it is not clear whether they are H. hedrychii or not since the species boundaries of H. hedrychii are not clear enough. The sequence MK564560 from USA, labelled as “H. eburneus”, should also be misidentified since it is clustered with the sequences (MK575247 and MF686502) of “H. chrysaspis”.

It is worthy to mention that, although some samples might have been misidentified, their molecular sequences can still provide some useful information for analysing the relationships amongst the samples or even potentially representing some undescribed taxa and reflecting richer species diversity.

The distribution of Chinese species of subsect. Hygrophorus

Ecologically, according to the authors’ investigation since 2010, Hygrophorus species are relatively rare in the subtropical to tropical areas of Guangdong and Hainan Provinces in South China. The reports of the Chinese Hygrophorus are mainly concentrated in the temperate regions and in the high-elevation areas of subtropical zone (Zeng and Yang 1991, Yuan and Sun 2007, Chen and Li 2013). With the description of H. glutiniceps, it is scientifically confirmed from the genetic level that the distribution of Hygrophorus species can extend to the southernmost province of China (tropical Hainan Province).

With the application of integrative taxonomy, considering the morphology characteristics, molecular data, the symbiotic association of plants etc., it is now easier to distinguish species within subsect. Hygrophorus. Even the study of genus Hygrophorus has entered an era in which a mass of new species have been discovered (Jacobsson and Larsson 2007; Yu et al. 2007; Larsson et al. 2014, 2018; Endo et al. 2018; Moreau et al. 2018; Huang et al. 2018; Naseer et al. 2019). Due to the support of the Chinese government, many mushroom investigations are being carried out. It is foreseeable that, in the next few years, a large number of new species will be reported from China and the distribution of species under subsect. Hygrophorus will be expanded.

Acknowledgements

The authors sincerely thank the editors and reviewers for their valuable comments and suggestions. Thanks are given to Dr David Hibbett for giving specimens to the first author. Thanks are given to Dr Yi-Jian Yao and Ms Liu Yang for their help in loaning specimens. Thanks are given to Mr Jun-Yan Xu for identifying plant species. This study is supported by the National Natural Science Foundation of China (Nos. 31800013, 31760004), the GDAS’ Project of Science and Technology Development (No. 2020GDASYL-20200104013), the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (No. 2019HJ2096001006), the Science and Technology Planning Project of Guangdong Province, China (Nos. 2018B030324001, 2018B020205001) and the Science and Technology Planning Project of Guizhou Province, China (No. Qian Ke He Fu Qi [2019] 4007).

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