Research Article |
Corresponding author: Tai-Hui Li ( mycolab@263.net ) Academic editor: María P. Martín
© 2020 Chao-Qun Wang, Ming Zhang, Tai-Hui Li.
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:
Wang C-Q, Zhang M, Li T-H (2020) Three new species from Guangdong Province of China, and a molecular assessment of Hygrocybe subsection Hygrocybe. MycoKeys 75: 145-161. https://doi.org/10.3897/mycokeys.75.59600
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Blackening waxcaps (Hygrocybe subsect. Hygrocybe) are a group of colorful and attractive mushrooms. However, the species diversity of subsect. Hygrocybe in China is still poorly known due to the limited sampling. In this study, three new species of this group from Guangdong Province, China are described and illustrated based on their morphological characteristics and molecular analyses of the internal transcribed spacer and large subunit ribosomal DNA regions. Hygrocybe debilipes from grasslands of South China Sea islands is mainly characterized by its orange red to vivid red pileus, fragile stipe, and ellipsoid to oblong basidiospores; H. griseonigricans from woodlands is characterized by its whitish to dull yellow pileus, quick black discoloration and the globose, subglobose to broadly ovoid basidiospores; H. rubroconica from woodlands is characterized by the hemispheric to plano-convex pileus when mature, semitranslucent fibrose stipe, and globose to ellipsoid basidiospores.
Asia, black discoloration, new taxa, phylogeny, waxcaps
The type species of genus Hygrocybe (Fr.) P. Kumm., H. conica (Schaeff.) P. Kumm., belongs to subgen. Hygrocybe (Fr.) P. Kumm., sect. Hygrocybe (Fr.) P. Kumm., subsect. Hygrocybe (Fr.) P. Kumm. (
To date, there are 25 taxa in the world that fit the morphological characteristics of subsect. Hygrocybe. Ten taxa under the group have been originally described from Europe: H. cinereifolia Courtec. & Priou and H. nigrescens (Quél.) Kühner from France, H. conica from Germany, H. conica var. aurantiolutea T. Borgen & Arnolds from Greenland, H. conica var. conicopalustris (Bon) Arnolds and H. conica var. minor Monthoux & Röllin from Switzerland, H. conicoides (P.D. Orton) P.D. Orton & Watling and H. olivaceonigra (P.D. Orton) M.M. Moser from the UK (England), H. pseudoconica J.E. Lange from Denmark, and H. veselskyi Singer & Kuthan from Czech Republic (
The existing knowledge about blackening Hygrocybe in China is still rather limited. There have been no phylogenetic studies based on Chinese specimens until now. No new species of subsect. Hygrocybe has been described from China, while the Chinese samples of this group are commonly treated as European species, such as H. conica and H. nigrescens (
Guangdong Province, which is located in South China, belongs to the East Asian monsoon region. The climate can be divided into the middle subtropical, the southern subtropical, and the tropical climate zones, from north to south. The annual average temperature is 19–24 °C and the average annual precipitation is 1300–2500 mm. During the authors’ field trips over the past ten years, numerous samples of blackening waxcaps with diverse morphological characteristics have been found. Obviously, the species diversity of subsect. Hygrocybe in Guangdong Province has been underestimated in the past.
In this study, a new worldwide phylogenetic framework of subsect. Hygrocybe is reconstructed using the ITS and LSU regions. Three new species from Guangdong Province are described based on the morphological characteristics, molecular phylogenetic evidence, and ecological data.
For each collection, fresh specimens were observed, photographed, and collected in situ; the date, location, collector, elevation, habitat and macroscopic characteristics were documented, and then, the specimens were dried below 50 °C overnight in an electric dryer. Macroscopic descriptions were based on the field records and colored photos. Color descriptions and codes refer to
Genomic DNAs were extracted from a small amount of dry lamellar tissue using the HiPure Fungus DNA Mini kit (Magen Biotech, Guangzhou, China). ITS and LSU gene regions were amplified using Polymerase chain reactions (PCR) with primers ITS1/ITS1F/ITS5 and ITS4 (
To elucidate the phylogenetic position of the fungal samples with new sequences within the genus Hygrocybe, the newly obtained and all the available Hygrocybe LSU sequences in NCBI were included. In addition, the sequences of Hygroaster albellus Singer (EF551314), H. nodulisporus (Dennis) Singer (EF561625), and “H. andersonii Cibula & N.S. Weber” (KF291171) were selected as the outgroup, according to
The LSU alignment contains 138 sequences with 1067 columns, of which 611 are constant sites and 333 are parsimony informative sites. The model of substitution is TIM3e+R4 according to Bayesian Information Criterion using ModelFinder. The ITS alignment has 103 sequences with 772 columns, of which 380 are constant sites and 294 are parsimony informative sites. K3Pu+F+G4 is selected as the best-fit model for the ITS dataset.
Taxa, vouchers, geographic origins and sequence accession numbers of newly generated sequences.
Taxon | Voucher | Geographic origin | ITS | LSU |
---|---|---|---|---|
H. debilipes | GDGM59314 | China: Guangdong | MW001783 | MW007877 |
GDGM57013 | China: Guangdong | MW001782 | MW007875 | |
GDGM59131 | China: Guangdong | MW001785 | – | |
GDGM59133 | China: Guangdong | MW001784 | MW007876 | |
H. griseonigricans | GDGM73527 | China: Guangdong | MW001790 | MW007883 |
GDGM81385 | China: Guangdong | MW001791 | MW007882 | |
GDGM81394 | China: Guangdong | MW001792 | MW007884 | |
H. rubroconica | GDGM45213 | China: Guangdong | MW001786 | MW007881 |
GDGM44706 | China: Hunan | MW001788 | MW007878 | |
GDGM45209 | China: Guangdong | MW001787 | MW007879 | |
GDGM45214 | China: Guangdong | MW001789 | MW007880 |
In the LSU phylogram (Fig.
Maximum Likelihood phylogram of genus Hygrocybe based on LSU matrix, rooted with Hygroaster albellus (EF551314), H. nodulisporus (EF561625) and H. andersonii (KF291171). The newly generated sequences in this study are showed in bold. GenBank accession numbers of downloaded sequences or voucher numbers of new sequences are given after the species names. Bootstrap values more than 50% are shown around the branches.
In the ITS phylogram (Fig.
Maximum-likelihood tree of sect. Hygrocybe based on ITS matrix, rooted with members of subsect. Macrosporae. The newly generated sequences in this study are indicated in bold face. GenBank accession number of downloaded sequence or voucher number of newly generated sequence and place of origin are given after the species name of each sequence. Bootstrap values more than 50% are shown around the branches.
China. Guangdong Province, Taishan City, Chuandao Town, Xiachuan Island, on a grassland, elev. ca. 50 m, 21°37'36"N, 112°34'30"E, 17 July 2017, H. Huang, Q.J. Huang & X.R. Zhong (GDGM59314, holotype!).
“debili-”: not strong, “-pes”: stipe. The species epithet “debilipes” (Lat.) refers to the fragile stipe of the new species.
Hygrocybe debilipes differs from H. singeri in having a smaller pileus, orange red to vivid red pileus before discoloration, pale yellow to light orange lamellae, a fragile and less sticky stipe, ellipsoid to oblong basidiospores, and the distribution in South China Sea islands.
Pileus 5–12 mm diam., conical with an acute umbo when young, convex to hemispherical in age, inrolled at margin, orange red (8A7–8), red to vivid red (9A7–8, 9B7–8), blackening with injury or aging, sticky when moist, hygrophanous. Lamellae free, pale yellow (4A3) to light orange (5A4) when mature, blackening with injury or aging, up to 4 mm broad, distant, waxy, with 1–3 unequal lamellulae between two entire lamellae, with lighter color at lamellar edge. Stipe 22–45 × 2–5 mm, central, cylindrical, equal or slightly tapered at apex, fistulous, semitranslucent, usually too fragile to obtain a complete stipe base, pale yellow (4A3) to light orange (5A4) with white base, blackening with injury or aging, glabrous to fibrillose, moist.
Basidiospores (7.5)8–11.5(12) × (4.5)5–7(7.5) μm [mean length = 9.4 μm, mean width = 6 μm], Q = 1.3–1.8, Qm = 1.6, ellipsoid to oblong, smooth, thin-walled, hyaline. Basidia 32–53 × 8–13 μm, 4-spored, clavate, with sterigmata up to 7 μm long. Pileipellis a cutis of cylindrical hyphae 2–11 μm diam., thin-walled, hyaline. Stipitipellis a cutis or ixocutis of repent, thin-walled, clamped hyphae 2.5–16 μm diam. Hymenophoral trama regular to subregular, consisting of parallel hyphal cells 4–17 μm diam., thin-walled, hyaline.
Scattered on grasslands in summer. Known from South China Sea islands.
China. Guangdong Province, Taishan City, Chuandao Town, Xiachuan Island, elev. ca. 50 m, 17 July 2017, Q.J. Huang, H. Huang & X.R. Zhong (GDGM59131, GDGM59133); Zhuhai City, Dawanshan Island, 28 July 2013, T.H. Li, H. Huang & Y.W. Xia (GDGM57013).
Hygrocybe debilipes is morphologically and genetically a distinct species. Hygrocybe debilipes is characterized by its tiny basidioma, orange red to vivid red pileus, pale yellow to light orange lamellae when mature, fragile and semitranslucent stipe, and ellipsoid to oblong basidiospores measuring (7.5)8–11.5(12) × (4.5)5–7(7.5) μm. Hygrocybe debilipes forms a strongly supported independent clade in both ITS and LSU phylogeny trees (Figs
Hygrocybe cinereifolia, originally described from France, is morphologically similar to H. debilipes in its general appearance. However, H. cinereifolia has larger basidiomata and grayish to gray lamellae (
China. Guangdong Province, Nanxiong City, Qingzhang Mountain, elev. ca. 340 m, 15 May 2018, M. Zhang & X.N. Chen (GDGM73527, holotype!).
“griseo-”: gray, “-nigricans”: black. The species epithet “griseonigricans” refers to its gray pileus with obvious blackening reaction.
Hygrocybe griseonigricans differs from H. astatogala by having a duller pilus color, variable (subglobose to elongate) basidiopore shapes, 1-, 2- and 4-spored basidia and differences in molecular sequences.
Pileus 25–70 mm diam., broad conical to umbonate disc when young, expanded to umbonate, convex, even to almost plane when mature, white, pale yellow (3A3) to dull yellow (3B3), densely covered with radially arranged black hairy fibrils with appressed or uplifted ends on surface, nigrescent when bruised or mature; margin incurved when young, expanded to straight when mature. Lamellae free, white at first, turning black when bruised or mature, up to 7 mm wide, waxy, fragile, with 1–3 lamelluate between two entire lamellae, edge usually eroded. Stipe 50–150 × 5–12 mm, central, cylindrical, sometimes slightly curve, usually wider at base, hollow, white to yellow at the upper part, usually white at the base, changing to black with age or when bruised, covered with clustered black longitudinal fibrils.
Basidiospores 9–10.5 × (6)6.5–9.5(10) μm [mean length = 9.7 μm, mean width = 8 μm], Q = (0.95)1.1–1.6, Qm = 1.23, globose, subglobose to broadly ovoid, smooth, thin-walled. Basidia 32–44.5 × 8–11.5 μm, 1-, 2- or 4-spored, clavate, with sterigmata up to 10 μm long, sterigmata of 2-spored basidia usually longer than those of 4-spored basidia. Pileipellis a cutis or trichoderm, hyphae 2–15 μm in diam. Hymenophoral trama regular, hyphae 2.5–21 μm broad, translucent, mainly thin-walled rarely thick-walled.
Solitary to scattered on soil; basidiomata occurring from April to September. So far known from Southern China.
China. Guangdong Province, Nanxiong City, Qingzhang Mountain, elev. ca. 340 m, 15 May 2018, M. Zhang & X.N. Chen (GDGM73528); Renhua County, Danxia Mountain, elev. ca. 100 m, 12 June 2020, M. Zhang & L.Q. Wu (GDGM81385).
The distinctive morphological features of Hygrocybe griseonigricans are the following: white to dull yellow-colored pileus with black discoloration, white lamellae, white stipe base, and variable basidiospore shapes, from globose, subglobose to broadly ovoid. The ITS and LSU phylogenetic analyses support H. griseonigricans as a distinct species within a well-supported clade that includes H. astatogala (Figs
Hygrocybe conica, originally described from Germany, is similar to H. griseonigricans. However, H. conica has a larger (up to 100 mm diameter) and somewhat fibrillose pileus, and usually grayish lamellae (
China. Guangdong Province, Shaoguan City, Ruyang County, Nanling Natural Reserve, elev. ca 1180 m, 15 May 2015, Ming Zhang, Hao Huang and Jiang Xu (GDGM45213, Holotype!).
“rubro-”: red, “-conica”: conical. The species epithet “rubroconica” refers to its red pileus surface.
Hygrocybe rubroconica differs from H. conica by having redder pileus, white to light reddish white and ventricose to broadly ventricose lamellae, simitranslucent stipe covered with white fibrils at first, and mainly 2-spored basidia.
Pileus 5–25 mm broad, conical, conical when young, broadly conical, hemisphere to plano-convex when mature, sometimes with an umbo in center, red to vivid red (10A7–8, 10B7–8), usually darker at disc, translucently striate from the margin to about a half of radius, blackening when bruised or mature; margin incurved, white to light yellow to reddish yellow (4A5–6). Lamellae free, ventricose to broadly ventricose, white to yellowish white, blackening when bruised or mature, up to 4 mm broad, distinct, waxy, fragile, with 1–3 lamellula between two entire lamellae. Stipe 20–40 mm long, 1–4 mm thick, central, cylindrical, slightly enlarged towards the base, hollow, simitranslucent, pale yellow (4A3) to brownish orange (5C3–5), nigrescent when bruised or old, usually covered with white longitudinal fine fibrils when young.
Basidiospores (7.5)8–10.5(11) × (6.5)7–8.5 μm [mean length = 9 μm, mean width = 7.6 μm], Q = 1–1.47, Qm = 1.2, globose, subglobose, broadly ellipsoid to ellipsoid, smooth, thin-walled. Basidia 25.5–45 × 8.5–12 μm, mainly 2-spored, rarely 4-spored, clavate, containing some black endochrome in KOH, with sterigmata up to 10.5 μm long. Pseudocystidia present. Pileipellis as a cutis, comprised of parallel hyphae 4–10 μm wide, occasionally with bend hyphae, with some black inclusions. Stipipellis made up of parallel hyphae, occasionally with some thick-walled haphae. Hymenophoral trama regular, of hyaline and thin-walled hyphae 8.5–17 μm wide, sometimes containing blackish brown materials.
Scattered to clustered on moist soil in forests; occurring from April to September. So far only known from Southern China.
China. Guangdong Province, Ruyang County, Nanling Natural Reserve, elev. ca. 1400 m, 24°89'N, 113°01'E, 16 June 2014, M. Zhang & T. Li (GDGM45209); same location, 15 May 2015, M. Zhang, H. Huang & J. Xu (GDGM45214).
Hygrocye rubroconica is characterized by the red pileus, ventricose to broadly ventricose lamellae, nearly translucent stipe covered with white fibrils at first, globose to ellipsoid basidiospores, mainly 2-spored basidia, and the presence of pseudocystidia.
Hygrocybe veselskyi Singer & Kuthan, originally described from Czechoslovakia, resembles H. rubroconica in its general appearance due to the red pileus and black staining reaction when touched or mature; however, H. veselskyi differs from H. rubroconica since it has yellow lamellae and bigger basidiospores measuring 10–12.5 × 5.3–6 μm (
In this study, three new species of Hygrocybe subsect. Hygrocybe from Guangdong Province, China are described and compared with similar species based on morphological and molecular data. More comprehensive phylogeny of genus Hygrocybe (focusing on subg. Hygrocybe) based on the LSU sequences and of sect. Hygrocybe based on ITS sequences are provided, including almost all the representatives in GenBank database and newly generated sequences. This study not only provides new species and genetic information of subsect. Hygrocybe from East Asia, but also provide some ideas on the species delimitation of subsect. Hygrocybe based on morphological and phylogenetic evidences.
Morphologically, the sizes of both fresh and dried basidiomata of H. griseonigricans, H. rubroconica and H. debilipes decrease in turns. The stipe of H. debilipes is especially more fragile than that of H. griseonigricans or H. rubroconica. Although the pileus color is variable within a species, the pileus color range, the pileus blackening rate and degree, and the lamellar color can be used as distinguishing features for distinguishing the three new species in southern China. For example, H. debilipes and H. rubroconica have a red to orange pileus, while H. griseonigricans has a white to yellow pileus. The pileus of H. griseonigricans mostly becomes black when mature, while the pileus of H. debilipes and H. rubroconica seldom becomes black when mature. The lamellar color of H. debilipes is orange, while H. griseonigricans and H. rubroconica have almost white lamellae. In contrast with the macro-morphology, limited micro-morphological features can be used to discriminate the species within subsect. Hygrocybe. The intraspecific variations in the basidiospore shape and in the number of basidum sterigmata are small in H. debilipes, but large in H. griseonigricans and H. rubroconica.
Molecular analyses seems to provide more stable information for species identification of subsect. Hygrocybe. Taking H. singeri as an example (Fig.
To reach a point in which all validly published species names of blackening waxcaps are represented by pertinent DNA sequences, it is necessary to obtain sequences from well-identified specimens of existing species. These tasks may not be completed in a short time since specimens from the type location need to be found by the researchers, amateurs, or society citizens, and then carefully identified by fungal taxonomists with relevant research experience. In addition, new species should be published based on both morphological and molecular data.
The authors thank the editors and reviewers for improving the manuscript. All our colleagues who contributed collections are warmly thanked, especially X.N. Chen, H. Huang, Q.J. Huang, T. Li, L.Q. Wu, Y.W Xia, J. Xu and X.R. Zhong. The authors thank Editage (www.editage.cn) for English language editing. This study is supported by the National Natural Foundation of China (Nos. 31800013, 31700021, 31770014, 31800014, 31970016), the GDAS’ Special 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), and the Science and Technology Planning Project of Guangdong Province, China (Nos. 2018B030324001, 2018B020205001).