Hygrophorus russula complex (Hygrophoraceae, Agaricales) in China

The Chinese species complex of Hygrophorus russula in subsection Clitocyboides has been studied. Three new species, H. orientalis, H. qinggangjun, and H. yunnanensis were proposed based on morphology, molecular systematics, and chemical reactions. Hygrophorus qinggangjun and H. yunnanensis occur at high elevations of Yunnan China; H. orientalis widely distributes at low elevations in East Asia. At present, seven taxa have been revealed in the H. russula species complex, and a key to these species is provided.

Hygrophorus russula (Schaeff. ex Fr.) Kauffman was originally described based on material from Europe (Schaeffer 1774), belongs to subsection Clitocyboides (Hesler & A.H. Sm.) E. Larss, and is characterised by a pinkish-red to vinaceous-purple pileus. This name has been widely used for some similar species found in Africa (GenBank sequence KU973852), Asia (Hongo 1982;Chen and Li 2013), North America (Hesler and Smith 1963;Siegel and Schwarz 2016) These results indicated that the species diversity within Hygrophorus is higher than previous estimates. In order to explore the genetic diversity, host preference and geographic distribution of the H. russula complex, we studied more than 80 well-documented specimens mainly from China. Based on morphological characters, chemical reactions and phylogenetic results, along with sequences of this species complex from GenBank, three species are proposed as new: H. qinggangjun and H. yunnanensis from south-western China and H. orientalis widely distributing in East Asia.

Sampling and morphological studies
Most specimens were collected in central, northern, and south-western China during rainy seasons (July-October). Basidiomes were found in broad-leaved forests, mainly Ericaceae and Fagaceae. Herbarium materials, identified as H. russula, were loaned from the University of Alcalá (AH). Other specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), the Mycological Herbarium, Institute of Mycology, Chinese Academy of Sciences (HMAS), the Mycological Herbarium of Kunming Medical University (MHKMU), and the Herbarium of the Royal Botanic Garden, Edinburgh (RBGE).
Macro-morphological descriptions were taken from field notes and images of basidiomes, with colour codes following Kornerup and Wanscher (1981). Micro-morphological characters were observed from dried materials after being sectioned and mounted in 5% potassium hydroxide (KOH) and 1% Congo Red solution (w/v) under a Leica DM2500 microscope. Melzer's reagent was used to test the amyloidy of basidiospores. Basidiospores and basal mycelium of stipe were examined with a ZEISS Sigma 300 scanning electron microscope (SEM) at 7.00 kV. The procedures followed those in Huang et al. (2018Huang et al. ( , 2020 and are referenced therein. The following abbreviations [n/m/p] indicate n basidiospores measured from m basidiomes of p collections. Dimensions for basidiospores are given using (a) b-c (d); the range of b-c contains a minimum of 90% of the measured values and 'a' and 'd' were the extreme values. Q means the quotient of length and width in the side view of basidiospores and Q m indicates average Q of all basidiospores ± sample standard deviation.

Sequence alignment and phylogenetic analyses
Raw sequences were assembled and edited using SeqMan (DNASTAR Lasergene 9) and deposited in GenBank (http:// www. ncbi. nlm. nih. gov) (see Table 1). DNA sequences of ITS, LSU, and TEF1 were independently aligned using MUSCLE 3.6 (Edgar 2004) and manually adjusted where necessary in BioEdit 7.0.9 (Hall 1999); the concatenated datasets were manually constructed. The dataset was analysed with maximum likelihood (ML) and Bayesian inference (BI). ML analyses were performed using RAxML 7.0.3 (Stamatakis et al. 2008); GTRGAMMA was set by default as the selected model; statistical support of clades was obtained with 1000 rapid bootstrap replicates. For BI, concatenated sequences were partitioned into ITS1 (1-136), 5.8S (137-300), ITS2 (301-585), LSU (586-1442), and TEF1 . The best-fit model of nucleotide substitution was obtained in PartitionFinder 2 (Lanfear et al. 2016) based on the Akaike information criteria (AIC). The selected models were GTR + G + I for ITS1 and ITS2, SYM + G for 5.8S, GTR + I for LSU, and SYM + G for TEF1. Bayesian analysis was performed with MrBayes 3.2 (Ronquist et al. 2012) on the CIPRES portal. Four simultaneous Markov chains were run for 6,000,000 generations for ITS and LSU, 3,000,000 generations for TEF1, and 5,000,000 generations for concatenated sequences and sampled every 1000 generations. At the end of the run, the average 1 3   1 3 deviation of split frequencies was below 0.005. Burn-in values were determined in Tracer 1.7 (Rambaut et al. 2018). The first 25% of generations were discarded as burn-in, when the plot generated by the sump command levelled off and effective sample sizes were well over 200 for all sampled parameters for each run.

Phylogenetic analysis
One hundred fifty-six ITS, LSU, and TEF1 sequences were newly generated for the H. russula complex in this study. DNA sequences were edited and aligned. The alignment length is 585 characters for the ITS dataset, 874 characters for the LSU  (Huang et al. 2018); Hygrophorus pudorinus (Fr.) Fr. in subsection Pudorini served as outgroups for the TEF1 dataset. The alignment is available at TreeBASE (Accession 27,296). All sequences acquired from this study are listed in Table 1. The phylogenetic trees from BI and RAxML were almost identical, while statistical support showed slight differences. The ITS and concatenated trees were almost identical. The phylogenetic analysis recovered 13 supported clades from Asia, Europe, North America, and South America, corresponding to unique lineages within the sequence data of H. russula (Figs. 1, 2, and 3, Appendix 1-2). European "H. russula" formed two groups. Sequences of "H. russula" from North America represented five phylogenetic species. The Belize sequence of "H. russula" represented the South American group forming a single branch, which is the basal group of the H. russula species complex in the ITS tree (Fig. 1). The Chinese collections respectively formed five monophyletic clades with strong support, corresponding to five taxa, three new species (H. orientalis, H. qinggangjun, and H. yunnanensis) and two known taxa (H. deliciosus and H. parvirussula). These collections of "H. russula" from the same continent did not cluster together, although some species have close relationships. For example, H. deliciosus exhibited a sister relationship with H. qinggangjun with strong support in the ITS and concatenated trees, while appeared to be sister to H. yunnanensis in the LSU tree. Hygrophorus parvirussula and H. russula were sister species in the ITS and LSU, while appeared to be sister to H. yunnanensis in the TEF1 tree. Hygrophorus orientalis had a close relationship to "H. russula" from Europe, probably as its Europe vicariant; the relationships cannot be resolved according to the present data and needs further research.

Chemical reactions studies
FeSO 4 , NH 4 OH, KOH, and Melzer's reagent were applied to test possible colour changes, which help to delimit sections or distinguish some species in Hygrophorus (Helser and Smith 1963; Larsson and Jacobsson 2004;Moreau et al. 2018). In this study, we used seven reagents based on the previous research (Huang et al. 2018). All species in the H. russula complex from China showed a negative reaction to EtOH, FeCl 3 , FeSO 4 , KOH, NH 4 OH, and Melzer's reagent, while the pileus turning pinkish in C 6 H 5 OH.
The following description is mainly taken from Wang and Li (2020), combined with our field notes including macro-morphology, habit, distribution, host plants, and examination.
Known distribution: South-western China, the subalpine to the alpine belt of Sichuan, Tibet and Yunnan. Comments: Hygrophorus deliciosus is quite common in SW China as a wild commercial fungus known from the local name "Qinggangjun" or "Mitangjun". "Mitang" is a kind of soup made from rice. "Mitangjun" means that this mushroom soup is thick and looks like "Mitang". It is distinguished by its medium-sized basidiomata, thick context (up to 2.4 cm), broad ellipsoid basidiospores, probably associated with Quercus aquifolioides, and scattered to gregarious at varied elevations from 1900 to 3700 m.
This species is likely to be confused with H. qinggangjun; both are sold as edible mushrooms in Yunnan Province markets and have been identified from the samples of Nanhua Wild Mushrooms Market. These two species have a very weak or mild taste when fresh and a little bitter after cooking. Hygrophorus qinggangjun has wavy and uplifted margin, thin context (0.4-0.9 cm), broad basidia (43-49 × 6-9 μm), and varied basidiospores (broad ellipsoid, ellipsoid, and oblong). This taxon prefers to grow in a solitary manner or scattered in mixed forests, probably associated with Quercus serrata, and with narrow distribution (Yunnan Province) and elevation (2000-2780 m).
Known distribution: East Asia: including China, Japan (GenBank accession KF291216), South Korea (GenBank accession KX814449-814,450).  . a, b H. deliciosus. c, d H. orientalis. e, f H. qinggangjun. g, h H. yunnanensis. i-l  H. russula. a, c, e, g, i, j basidia and cheilocystidia, b, d, f, h,  Comments: Hygrophorus orientalis can be recognised from other species of the H. russula complex by its large basidiomata, dense lamellae, small basidiospores and scattered to gregarious (at times in arcs or fairy rings) habit, probably associated with Cyclobalanopsis sp. or Quercus mongolica. This taxon has a wide distribution in East Asia (including China, Japan, and South Korea), usually occurring below elev. 2400 m. It is known as an edible mushroom in NE China, where locals prefer it raw rather than cooked.
Phylogenetically, this species is related to "H. russula" from Europe, probably as its Europe vicariant; the sister relationship of H. orientalis cannot be resolved according to the present data and needs further research.
The morphological and phylogenetic analyses (ITS and concatenated trees) show that H. qinggangjun is closely related to H. deliciosus; the comparison between H. deliciosus and H. qinggangjun can be found in our treatment of H. deliciosus.
The following description is mainly taken from Arnolds (1990) and Candusso (1997), combined with the field notes including macro-morphology, habit, distribution, host plants, and examination.
Description: Basidiomata solitary to sub-gregarious on soil, medium-sized, fleshy, rather fragile. Pileus 4-10 (-15) cm diam., hemispherical, convex, applanate, firstly almost white to pale pink with scattered pink to wine-red spots or appressed, fibrillose scales, gradually darker, becoming pinkish-red to wine-red at the centre, finally dark purplishred, with small to large concolorous spots elsewhere, surface dry to slight viscid. Context white, becoming pale pink to pinkish-purple when damaged, up to 2.5 cm thick. Lamellae adnate to short-decurrent, rather crowded to subdistant, 70-150 pieces of complete lamellae/cap, narrow, up to 0.5 cm wide, at first white to very pale pink, then spotted wine-red, brown-red or purplish-red, finally entirely dark reddish. Stipe 6-10 (-15) cm long, 0.8-2.5 (-4) cm diam., cylindrical, solid, dry, equal or slightly tapering towards base, white with some wine-red to purplish-red spots to almost completely brownish-purple. Odour absent to rather weakly unpleasant, sweetish. Taste mild.
Known distribution: Europe. Habit and habitat: Solitary to sub-gregarious in mixed forests, associated with Quercus spp., elev. below 1100 m; in autumn to winter (September-December).
Materials Comments: Hygrophorus russula was originally described from Germany as Agaricus russula Schaeff. in 1774 (Schaeffer 1774;Fries 1821), but with few macroscopic details and without microscopic details in the original description. Subsequently, it was placed in the genus Tricholoma in 1878 (Gillet 1878), then was placed in the genus Hygrophorus (Kauffman 1918). In 1918, Kauffman CH firstly described H. russula in detail, however, based on material from North America rather than from Europe. For a long time, H. russula was assumed to be a widespread species and has been widely reported from several continents (Hesler and Smith 1963;Hongo 1982;Arnolds 1990;Candusso 1997;Chen and Li 2013;Siegel and Schwarz 2016). Spanish material in this study matches the description based on European collections (Arnolds 1990;Candusso 1997). According to those references and our study, European H. russula is distinguished by medium-sized basidiomata, narrow lamellae, large basidiospores, occurring under Quercus spp. (possibly Q. ilex in the south and Q. robur in the northern part), mainly on basic soil, and solitary to sub-gregarious habit at low elevations (below 1100 m) (Arnolds 1990;Candusso 1997). We propose that H. russula is a strictly European species based on this study.
Odour not distinctive. Taste mild.

Species delimitations of Hygrophorus russula complex
Due to its recognisable characteristics in the field, consisting of a pale flesh pink to reddish-purple pileus, H. russula was once 1 3 considered a species with a wide distribution in the Northern Hemisphere (Hongo 1982;Arnolds 1990;Hesler and Smith 1963;Chen and Li 2013) and South Hemisphere according to the data from GenBank. With the application of molecular methods, seven molecularly distinct species have been revealed under this name, and supported by diagnosable criteria including morphological characters, ecological traits, and geographical distribution (see Table 2). Three species, H. orientalis, H. qinggangjun, and H. yunnanensis from China, are newly described in this study.
Morphologically, H. russula complex has similar colours (pale reddish-purple, dark red, reddish-brown, to brownishvinaceous), which are hard to identify in the field. Still, they are divergent in geographical distribution, host preferences, the size of basidiomata, and microscopic characters.
Three species, H. parvirussula, H. russuliformis and H. yunnanensis, have a similar pileus less than 10 cm diam. However, H. russuliformis, so far restricted to North America, occurs under oak trees at low elevations (less than 500 m) of Florida; the context is unchanged on exposure and has narrower basidiospores measuring 8-10 × 2-3.2 μm (Hesler and Smith 1963). Hygrophorus parvirussula and H. yunnanensis were both found in south-western China; the context became darker on exposure, and probably associated with Ericaceae.

The taxonomic importance of comprehensive data in the Hygrophorus russula complex
Due to similar characteristics, it is quite difficult to identify the species complex. Thus, the application of comprehensive evidence is important for the accurate identification of this species complex. In our study, SEM characteristics are helpful to distinguish the complex species of H. russula. Hygrophorus parvirussula is characterised by bacillate ornamentation of basidiospores under SEM; the other four taxa in this complex are smooth. In fungal taxonomy, the application of characteristics of basal hyphae is rarely seen. Huang et al. (2020) firstly reported abundant morphological characters of the basal hyphae of seven species from Clavariadelphus as taxonomical evidence. In the H. russula complex, the basal hyphae also vary from smooth to a massive nipple-shaped protuberance under SEM (see Fig. 8