A Contribution to the Phylogeny and Taxonomy of Hydnum (Cantharellales, Basidiomycota) from China

Hydnum is a well-characterized genus in the family Hydnaceae of Cantharellales and is characterized by spinose hymenophores. In this study, an ITS phylogenetic overview and a multilocus (ITS-nrLSU-tef1) phylogenetic tree of Hydnum were carried out. On the basis of morphological characteristics and phylogenetic results, seven species from China were confirmed, described, illustrated, and compared with similar species, including three new species, i.e., H. longipes, H. microcarpum, and H. sinorepandum, and four known species, i.e., H. cremeoalbum, H. melitosarxm, H. orientalbidum, and H. pinicola were recorded for the first time in China. A key to the species of Hydnum in China was provided.

Studies on the species diversity of Hydnum in China have accelerated in recent years.Previously, only three species (H.repandum, H. repandum var.album, and H. rufescens) were recorded in China before 2016 [5,8,9].Feng et al. [5] conducted a molecular phylogenetic study on Hydnum in China, and the results showed that Hydnum species are very diverse in China.Cao et al. [2] reported twelve species of Hydnum in China, including ten new species.Until now, about 18 species have been reported from China [2][3][4][5][8][9][10].In recent years, we have carried out investigations on the species diversity of Hydnaceae in southern China and found 11 new species of Cantharellus [11,12].In this study, three new species of Hydnum are described and discussed, and four species are described in China for the first time.

Morphological Studies
Photographs and field notes of the fresh basidiomata were taken in the field or laboratory.Specimens were dried and deposited in the Fungarium of Guangdong Institute of Microbiology (GDGM).Descriptions of macro-morphological characteristics and habitats were obtained from field notes and photographs.Color codes followed Kornerup and Wanscher [13].Micro-morphological observations were carried out on tissues stained with 5% KOH and 1% aqueous Congo red under a light microscope (Olympus BX51, Tokyo, Japan).For basidiospore size descriptions, the notation (a-)b-c(-d) is used, where the range b-c represented 90% or more of the measured values and 'a' and 'd' represented the extreme values; 'av.' represents the mean range of basidiospore length or width; Q referred to the length/width ratio of an individual basidiospore; and Q m referred to the average Q value of all basidiospores ± sample standard deviation.All line drawings of microstructures were made based on rehydrated materials.

Phylogenetic Analyses
Sequences generated in this study and those downloaded from GenBank were combined and used for phylogenetic reconstruction.The sequence matrix of ITS, nrLSU, and tef 1 were separately aligned with MAFFT v7 software using the E-INS-i strategy [17] and manually adjusted in MEGA 6 [18].Phylogenetic analyses were performed in Phy-loSuite [19].Maximum likelihood phylogenies were inferred using IQ-TREE [20].Best models for ITS datasets were searched via ModelFinder [21], and the ITS-LSU-tef 1 combined datasets were searched via PartitionFinder 2 [22].The phylogenetic trees were visualized using FigTree v1.4.23.

Molecular Phylogeny
In the ITS dataset, 410 sequences were included for phylogenetic analysis.The alignment had 843 characters including gaps, and K80 + G4 was selected as the best model for maximum likelihood phylogenies.Sistotrema muscicola (Pers.)S. Lundell was selected as an outgroup based on recent studies [2,6].The ITS phylogenetic tree is shown in Figure 1.In the concatenated (ITS + LSU + tef 1) dataset, the alignment has 274 samples with 3348 columns, 1224 distinct patterns, 764 parsimony-informative, 160 singleton sites, and 2424 constant sites.The best-fit models HKY + F + R3 and TIM3 + F + R2 were selected for the ITS + tef 1 and LSU regions, respectively.The multilocus (ITS + LSU + tef 1) phylogenetic tree is shown in Figure 2. In total, 51 sequences were newly generated and deposited in GenBank in present study.
Habitat and distribution: solitary or gregarious in broad-leaved or mixed forests mainly dominated by Fagaceae trees (such as Castanopsis eyrei, C. fissa, C. lamontii, and Cyclobalanopis chungii).Known from southern China and Japan.
Notes-Hydnum cremeoalbum, recently described from Japan [6], is firstly recorded in China in this study.The morphological description is short in Niskanen et al. [6].Sugawara et al. [24] gave a detailed description of H. cremeoalbum after re-studying the type specimen and examining a large number of specimens collected from Japan.Morphologically, H. cremeoalbum is mainly characterized by the cream-white pileus which first changes to pale ocher when bruised and finally staining greenish to bluish green, the pale cream to pale orange spines, and the broadly ellipsoid basidiospores measuring 5-6.5 × 4-5.5 µm [24].It is worth noting that the discoloration of basidiocarps from greenish to bluish green is rare in Hydnum species [24].
Phylogenetic analyses (Figures 1 and 2) shows that two specimens from China are nested into the H. cremeoalbum clade with well-supported evidence.Morphological comparisons also showed that most features of our specimens are consistent with the description of H. cremeoalbum, except that the discoloration was not observed in the present study.
Notes-Hydnum cremeoalbum, recently described from Japan [6], is firstly recorded in China in this study.The morphological description is short in Niskanen et al. [6].Sugawara et al. [24] gave a detailed description of H. cremeoalbum after re-studying the type specimen and examining a large number of specimens collected from Japan.Morphologically, H. cremeoalbum is mainly characterized by the cream-white pileus which first changes to pale ocher when bruised and finally staining greenish to bluish green, the pale cream to pale orange spines, and the broadly ellipsoid basidiospores measuring 5-6.5 × 4-5.5 µm [24].It is worth noting that the discoloration of basidiocarps from greenish to bluish green is rare in Hydnum species [24].
Phylogenetic analyses (Figures 1 and 2) shows that two specimens from China are nested into the H. cremeoalbum clade with well-supported evidence.Morphological comparisons also showed that most features of our specimens are consistent with the description of H. cremeoalbum, except that the discoloration was not observed in the present study.
Habitat and distribution-Growing solitarily or gregariously in subalpine mixed forests mainly dominated by Quercus aquifolioides and Pinus densata.So far known from southwestern China.
Notes-Phylogentic analysis showed that H. longipes formed a distinct lineage in subg.Rufescentia, and is closely related to H. canadense, H. mulsicolor, and H. submulsicolor.Based on the morphological characteristics given above and the phylogentic results, H. longipes should be a member within subsect.Mulsicoloria.
In the present study (Figures 1 and 2), two specimens (GDGM81873 and GDGM84518), together with several sequences labelled as "Hydnum rufescens" from China, are well nested into the H. melitosarx clade.Although the distribution of this species in China was found by previous analysis [6], no detailed morphology studies have been conducted.This study is the first to describe this species in detail based on Chinese materials.It is worth mentioning that H. melitosarx is a pan-Arctic distributed species, so far known from Andorra, China, Estonia, Finland, Germany, Solenia, Sweden, and the USA [6].
materials.It is worth mentioning that H. melitosarx is a pan-Arctic distributed species, so far known from Andorra, China, Estonia, Finland, Germany, Solenia, Sweden, and the USA [6].
Fungal Name: FN571762 Etymology-microcarpum refers to the species with a smaller basidiocarp.Diagnosis-This species is characterized by its small basidiocarps, yellowish white to orange-white pileus changing from brownish orange to brownish red when bruised, yellowish white spines, and relatively small basidiospores.
Habitat and distribution-Growing in solitary or gregarious conditions under mixed forests in the subtropical region of southern China, which is mainly dominated by Castanopsis trees and Lithocarpus trees mixed with a small number of Pinus massoniana.So far only known from southern China.
Notes-In both the ITS and multilocus phylogenetic analyses (Figures 1 and 2), the new species H. microcarpum formed an independent lineage, close to H. alboluteum R. Sugaw.& N. Endo, H. brevispinum T. Cao & H.S. Yuan, and H. tenuistipitum T. Cao & H.S. Yuan.Based on the morphological features given above and the phylogenetic evidence, the new species H. microcarpum is a member of subg.Brevispina, which has been recently established in Cao et al. [2].
Habitat and distribution-Growing in solitary or gregarious conditions under mixed forests mainly dominated by Fagaceae trees and mixed with Pinus trees.Currently known from China and Japan.
Notes-Hydnum pinicola, originally described from Japan, is characterized by its whitish to cream basidiocarps, pale yellow or pale orange spines, and relatively smaller basidiospores (4.5-5.5 × 4-5 µm) [24].Our specimens generally matched the original description of H. pinicola in morphology and existed in similar habitats.In both the ITS and multilocus phylogenetic analyses (Figures 1 and 2), the new Chinese samples fell into the H. pinicola clade with strong support (BS = 100%).In short, the specimens from southern China are identified as H. pinicola, which was first recorded in China.
Hydnum Diagnosis-This species is characterized by its relatively large basidiocarps, yellowish white to light orange pileus, bitter context changing to orange-white when bruised, yellowish white spines up to 7 mm long, robust stipe, broadly ellipsoid basidiospores, and sub-alpine distribution.
description of H. pinicola in morphology and existed in similar habitats.In both the ITS and multilocus phylogenetic analyses (Figures 1 and 2), the new Chinese samples fell into the H. pinicola clade with strong support (BS = 100%).In short, the specimens from southern China are identified as H. pinicola, which was first recorded in China.
Habitat and distribution-Growing in solitary or gregarious conditions under broadleaved forests of Quercus aquifolioides, Betula albosinensis, Populus tremula, and Rhododendron spp.; and in mixed forests of Quercus, Betula, Rhododendron and Abies georgei in subalpine regions.Currently only known from southwestern China.
Both the ITS and multilocus phylogenetic analyses showed that the four Chinese specimens of H. sinorepandum in this study clustered together with several sequences labelled as "Hydnum sp.8", and that they formed a distinct lineage close to H. sphaericum within the subg.Hydnum.Hydnum sphaericum, also originally described from China, can be easily distinguished from H. sinorepandum by its smaller basidiocarps (pileus 20-35 mm broad in diam.) with subglobose pileus when young, shorter spines, and the distribution in subtropical regions of central China [2].

Discussion
In this study, seven Hydnum species, including three new species (i.e., H. longipes, H. microcarpum, and H. sionrepandum), were confirmed and described on the basis of morphological and molecular analyses in China.Up to now, 25 Hydnum species have been recorded in China, including 15 species originally reported from China [2].A morphological comparison of Hydnum species in China is given in Table 1, including the main morphological characteristics and ecological information.Compared with previous morphological results, the number of Hydnum species now recorded in China has increased dramatically.This is mainly due to the overlapping morphological characteristics between Hydnum species (such as the size, shape, and color of the cap, tooth, and stipe); thus, recognition of Hydnum species based solely upon morphology is very difficult or impossible.
In some species, it is noticeable that some morphology characteristics have intraspecific variations after observing and studying more specimens.For instance, the spore size of H. melitosarx from the northern and subtropical alpine areas of China is larger than that from southern China.The reason for this may be that there is more pressure to survive in the southern regions, and the fungi need to mature as quickly as possible to release more spores to maintain the population.In addition, discoloration (context changing from pale yellowish green to bluish green when bruised) was observed in some Japanese H. cremeoalbum samples [24] but not observed in Chinese specimens in the present study.Many Hydnum species will undergo color changes after injury or handling, but this is not a stable characteristic and it is not a wise choice to distinguish Hydnum species based on color change.
Recently, molecular sequences appeared to be very effective in distinguishing species of this genus, and some cryptic species have been discovered.For example, "Hydnum repandum" had been widely reported in China [8][9][10]30]; however, recent phylogenetic studies proved that the Chinese "H.repandum" is a species complex, containing at least four species, such as, H. berkeleyanum, H. melitosarx, H. orientalbidum, and H. sinorepandum.In addition, H. repandum is so far only known from Europe.
In the multilocus phylogenetic tree (Figure 2), the genus Hydnum is an independent monophyletic group, in which four subgenera (subg.Pallidi, subg.Hydnum, subg.Parvispina, and subg.Rufescentes) are highly supported, and the subg.Alba is not monophyletic, which is similar to the results of previous studies [2,23,24].However, no stable morphological characteristics have been found to distinguish each subgenus.For example, traditionally, subg.Alba is characterized by whitish to cream-colored pileus, a whitish stipe which stains yellowish when handled, and the features also seen in some species of subg.Hydnum, subg.Pallidi and subg.Rufescentes.In addition, the presence of relatively small basidiospores (usually less than 7 mm long) is another characteristic to distinguish subg.Alba from others, while these characteristics are also present in subg.Parvispina.
Most species of Hydnum have restricted distribution areas according to previous studies [5,24,27] and this study.For example, H. subalpinum and H. sinorepandum are only distributed in sub-alpine regions of Asia; H. repandum is only known in Europe [6]; and H. alboaurantiacum, H. cuspidatum, H. ferruginescens, and H. subtilior appeared endemic to North America [25].Currently, only H. melitosarx has been confirmed with a holarctic distribution, so far known from Andorra, China, Estonia, Finland, Germany, Solenia, Sweden, and the USA.It is worth mentioning that Hydnum species can be found in temperate to subtropical regions in China, but it is very rare in tropical regions.
In recent years, a large number of Hydnum species have been found in Asia, making Asia the continent with the highest species diversity of Hydnum species [2,23,24,26,27], and most known Hydnum species are endemic to Asia.China, as one of the mega-biodiverse countries, has numerous plants that can symbiosis with Hydnum, and it can be predicted that with the deepening of investigations and research, many more Hydnum species will be discovered and reported from this country.
Key to species of Hydnum in China 1.

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree of Hydnum inferred from the ITS dataset.Bootstrap values (>70%) are shown above or below supported branches.Specimens from the present study are indicated in bold.

Figure 1 . 25 Figure 2 . 25 Figure 2 .
Figure 1.Maximum likelihood phylogenetic tree of Hydnum inferred from the ITS dataset.Bootstrap values (>70%) are shown above or below supported branches.Specimens from the present study are indicated in bold.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree of Hydnum inferred from the ITS-LSU-tef1 dataset.Bootstrap values (>70%) are shown above or below supported branches.Specimens from this study are indicated in bold.

Figure 2 .
Figure 2. Maximum likelihood phylogenetic tree of Hydnum inferred from the ITS-LSU-tef 1 dataset.Bootstrap values (>70%) are shown above or below supported branches.Specimens from this study are indicated in bold.
sinorepandum Ming Zhang & C.Q. Wang sp.nov.Figures 3g,h and 10.Fungal Name: FN571763 Etymology: Refers to the species reported from China, similar to H. repandum.

Table 1 .
A morphological comparison of Hydnum species in China.