Hygrophorus subsection Hygrophorus (Hygrophoraceae, Agaricales) in China

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.


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 (Lodge et al. 2014).
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.

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 Q m 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_prim-ers_for_fungi/) and ITS1/ITS5 and ITS4 (White et al. 1990, Gardes andBruns 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).

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 newlyobtained sequences and the downloaded sequences from GenBank and UNITE longer than 300 bps and of subsect. Hygrophorus are combined as ingroups and the sequences  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).

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) 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.

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, 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. 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.  (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.  Etymology. "brunneo-": brown, "-discus": pileus disc. The species epithet "brunneodiscus" (Lat.) refers to the brown pileus disc of this new taxon.
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, yellowishbrown (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.
Diagnosis. Hygrophorus glutiniceps differs from H. discoxanthus by the subtropical to tropical distribution, the darker lamellae and the thinner stipe (3-6 mm broad).
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 com-plete 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.
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. 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.
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.
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. 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.

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.  (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.
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 Spore size (μm) Hostconnection 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.

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. 2014Larsson et al. , 2018Endo 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.