Three new species from Guangdong Province of China, and a molecular assessment of Hygrocybe subsection Hygrocybe

Abstract 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.

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 (Chen and Li 2013).
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.

Sampling and morphological studies
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 Kornerup and Wanscher (1978). Sizes and shapes of basidiospores, basidia, pileipellis, stipitipellis, and hymenophoral trama were observed using handmade tissues stained with 5% potassium hydroxide solution and/or 1% Congo red solution under an AX10 light microscope and photographed using ZEN 2 lite software (Zeiss, Oberkochen, Germany). The detailed measurement method has been described by Wang et al. (2020). Specimens are deposited in the Fungarium of Guangdong Institute of Microbiology, Guangzhou, China (GDGM).

DNA extraction, PCR amplification and sequencing
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 (White et al. 1990;Gardes and Bruns 1993), LR0R and LR5/LR7 (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/), respectively. Bidirectional PCR product sequencing was carried out using the same primers that were used in the PCR reactions. The forward and reverse raw sequences were assembled and trimmed using SeqMan version 7.1.0 (DNAStar, Inc.). The consensus sequences were blasted in the National Center for Biotechnology Information (NCBI) to rule out the possibility of contamination, and then the correct consensus sequences were

Phylogenetic analyses
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), Ha. nodulisporus (Dennis) Singer (EF561625), and "H. andersonii Cibula & N.S. Weber" (KF291171) were selected as the outgroup, according to Vizzini et al. (2015). To clarify the phylogenetic position of the new sequences within subsect. Hygrocybe, the newly gained and all usable ITS sequences of sect. Hygrocybe were included, and members of subsect. Macrosporae were treated as the outgroup, according to Lodge et al. (2014) and Wang et al. (2019). To make LSU and ITS matrixes, the new sequences and the downloaded reference sequences were first combined, aligned using MAFFT online service (Katoh et al. 2017) by applying the auto strategy, and then viewed and trimmed (the front and back parts) using MEGA-X software (Kumar et al. 2018). The data alignments have been submitted to TreeBASE, submission ID 27252 (LSU) and 27253 (ITS). The substitution models were selected using the Bayesian information criterion with ModelFinder (Kalyaanamoorthy et al. 2017) in PhyloSuite ). Phylogenetic analysis for each dataset was performed using the maximum likelihood (ML) in IQ-TREE (Nguyen et al. 2015); and the node bootstrap support values (BS) were estimated using an ultrafast bootstrap with 5000 replicates (Minh et al. 2013). Phylograms were viewed and annotated with iTOL version 5.5 (Letunic and Bork 2016).

Phylogenetic analyses
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.
In the ITS phylogram ( Fig. 2), members of subsect. Hygrocybe and subsect. Macrosporae are treated as the ingroups and outgroups, respectively. Although all new species are present in subsect. Hygrocybe, the ITS analysis produces partially different typologies from the LSU analysis regarding the relationships of taxa under subsect. Hygrocybe. In the ITS analysis, four new sequences of H. debilipes, three new sequences of H. griseonigricans, and five sequences [four newly from China and one from Japan labeled as "H. conica" (AB509683)] of H. rubroconica form three independent clades, respectively. Sequences ex holotype. MW001783 (ITS), MW007877 (LSU). Etymology. "debili-": not strong, "-pes": stipe. The species epithet "debilipes" (Lat.) refers to the fragile stipe of the new species.
Diagnosis. 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.
Habit, habitat and distribution. Scattered on grasslands in summer. Known from South China Sea islands.
Description. 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.
Diagnosis. 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.
Habit, habitat and distribution. Scattered to clustered on moist soil in forests; occurring from April to September. So far only known from Southern China.
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 (Candusso 1997).

Discussion
In this study, three new species of Hygrocybe subsect. Hygrocybe from Guangdong Province, China are described and compared with similar species based on mor-phological 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 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. 2), due to the release of the type specimen's ITS sequences in GenBank (NR_119897 and HQ185708), it is clear that the sequences retrieved from several samples that are labeled as "H. singeri" (KM248895, KC581351, MH062990 and MN089502) should be a different species. However, the shortage of released correctly identified sequences is a basic issue that needs to be urgently addressed. Within subsect. Hygrocybe, it is highly urgent to obtain the sequences from a well-identified H. conica specimen from the type location at Bavaria, Germany, since H. conica has undergone various interpretations in different continents (Figs 1-2).
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.
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).