Morphological and Phylogenetic Evidences Reveal Four New Species of Cantharellus Subgenus Cantharellus (Hydnaceae, Cantharellales) From China

Species of Cantharellus subgenus Cantharellus are interesting and important for their mycorrhizal properties, medicinal values, and edibility. In China, there are many undescribed species of the subgenus. In this study, four new species of subg. Cantharellus, viz. Cantharellus albopileatus, Cantharellus chuiweifanii, Cantharellus pinetorus, and Cantharellus ravus from Hainan and Hunan Provinces, respectively, were described based on morphological and phylogenetic evidence as a contribution to the knowledge of the species diversity in China. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microstructures of these four new species are presented as well as comparisons with related species.


Morphological Studies
Field notes and digital photographs were made from fresh specimens. Specimens examined in this study were deposited in the Fungal Herbarium of Hainan Medical University (FHMU), Haikou City, Hainan Province of China. Macroscopic descriptions are based on the detailed notes and photographs taken from fresh basidiomata. Color codes follow Kornerup and Wanscher (1981). Samples were hand-sectioned and mounted in 5% KOH solution and 1% congo red. Sections of the pileipellis were cut radial-perpendicularly and halfway between the center and the margin of the pileus. The following notations [n/m/p] indicate that n basidiospores measured m basidiomata of p collections. Dimensions of basidiospores were presented in the form (a-)b-e-c(-d), where the range bc contains at least 90% of the measured values, "a" and "d" were the extreme values, and "e" refers to the average length/width of basidiospores. Q refers to the length/width ratio of basidiospores; Q m refers to the average Q of basidiospores and is given with standard deviation. For basidiospore shape, Q m = 1.15-1.3 describes "broadly ellipsoid, " Q m = 1.3-1.6 "ellipsoid, " and Q m = 1.6-2.0 "elongate" (Yang, 2005). The terms referring to the size of basidioma are based on Bas (1969).

Molecular Procedures
Total genomic DNA was obtained with the Plant Genomic DNA Kit (CWBIO, Beijing, China) according to the manufacturer's instructions from collections dried with silica gel. Primer pairs used for amplification were as follows: nuc 28S rDNA D1-D2 domains (28S) with LR0R/LR5 (Vilgalys and Hester, 1990;James et al., 2006) and the translation elongation factor 1-α gene (TEF1) with EF1-α-F/EF1-α-R (Mikheyev et al., 2006). Polymerase chain reaction (PCR) conditions followed the program of Zhang Y. Z. et al. (2021). PCR products were checked in 1% (w/v) agarose gels. Amplified PCR products were sequenced using an ABI 3730 DNA Analyzer (Guangzhou Branch of BGI, China) with the same primers. Forward or reverse sequences were compiled with BioEdit (Hall, 1999). All sequences newly obtained in this study were deposited to GenBank 1 .

Dataset Assembly
A total of sixty-nine DNA sequences (34 of 28S, and 35 of TEF1) from 36 collections were newly generated. Edited sequences were deposited in GenBank; the GenBank accession numbers are listed in Table 1. For the concatenated dataset, the sequences of 28S and TEF1 from new collections were aligned with selected sequences of subg. Cantharellus from previous studies and GenBank (Table 1); Cantharellus ibityensis Buyck, Randrianj. and V. Hofst. was chosen as an outgroup inferred from Buyck et al. (2014). To test for phylogenetic conflict among 28S and TEF1, single-gene phylogenetic trees based on each of these two fragments were analyzed. The results of analyses showed that 28S and TEF1 were not in conflict. Thus, two datasets (28S and TEF1) were aligned with MUSCLE v3.6 (Edgar, 2004) and manually optimized on BioEdit v7.0.9 (Hall, 1999); then, the two datasets were concatenated using Phyutility v2.2 for further analyses (Smith and Dunn, 2008).

Phylogenetic Analyses
The combined nuclear dataset (28S + TEF1) was analyzed using maximum likelihood (ML) and Bayesian inference (BI). Maximum likelihood tree generation and bootstrap analyses were performed with the program RAxML7.2.6 (Stamatakis, 2006) running 1,000 replicates combined with an ML search. Bayesian analysis with MrBayes 3.1 (Huelsenbeck and Ronquist, 2005) implementing the Markov chain Monto Carlo (MCMC) technique and parameters predetermined with MrModeltes 2.3 (Nylander, 2004) was performed. The best-fit likelihood model of 28S and TEF1 was GTR + I + G and SYM + I + G, respectively. Bayesian analysis of the combined nuclear dataset (28S + TEF1) was repeated for 20 million generations and sampled every 100 generations. Trees sampled from the first 25% of the generations were discarded as burn-in, and Bayesian posterior probabilities (PP) were then calculated for a majority consensus tree of the retained Bayesian trees. Runs were terminated once the average standard deviation of split frequencies went below 0.01.

Molecular Data
The combined dataset (28S + TEF1) of subg. Cantharellus consisted of 129 taxa and 1,707 nucleotide sites, and the alignment was submitted to TreeBase (S29413). The phylogram with branch lengths generated from RAxML and support values is shown in Figure 2. The topologies of the phylogenetic trees based on the combined dataset generated from ML and BI analyses were almost identical, but there was a slight variation in statistical support.
The present molecular data indicate that the Chinese species of subg. Cantharellus were grouped into fourteen independent lineages (Figure 2). A total of seven new lineages were identified in this study (Lineages 1-7 of Figure 2). Lineage 1 was comprised of one collection (FHMU6845) from central China; lineage 2 was comprised of one material (FHMU4592) from southern China; lineage 3, with strong statistical support (BS = 100%, PP = 1.0), was comprised of eleven collections (FHMU5335, FHMU6846, FHMU6847, FHMU1703, FHMU5260, FHMU6848, FHMU6849, FHMU2412, FHMU2839, FHM3236, and FHMU5268) from southern China; lineage 4, with high statistical support (BS = 98%, PP = 1.0), was comprised of two specimens (FHMU3749 and FHMU3759) both from central China; lineage 5, with strong statistical support (BS = 100%, PP = 1.0), was comprised of two materials (FHMU1987 and FHMU6850) from southern China and eastern China, respectively; lineage 6 was comprised of one collection (FHMU5266) from southern China; and lineage 7 was comprised of one specimen (FHMU3834) also from southern China (Figure 2 Basidiomata are very small to small-sized. Pileus is 2.5-5 cm in diameter and convex with a depressed center; the margin was strongly incurved, irregular, often wavy, and lobed; the surface is smooth, slightly greasy, and cream (3A1) to off-white (4A1) in color; the context above stipe was 0.3 cm in thickness, whitish (4A1), unchanging in color when injured. Hymenophore is composed of relatively welldeveloped, decurrent gill folds, branched to furcate, which becomes strongly intervened with age; these folds are about 0.1 cm broad and are yellowish (2A2) to white (4A1) in color. Stipe is 2-3 × 0.4-0.6 cm, central, subcylindrical, young solid, hollowing with age, and curved at the base; the surface was dry, whitish (4A1) to very pale cream (3A1), and nearly concolorous with hymenophore; the context is fleshy, firm, and whitish (3A1). Taste and odor is not distinctive. Spore print is not obtained.
In the phylogenetic analyses, Cantharellus albopileatus forms a well-supported (BS = 100%, PP = 1.0) monophyletic clade, together with the newly described Cantharellus chuiweifanii, equally a tropical species, and Cantharellus pinetorus from central China (Figure 2), which is quite different from any of the other northern hemisphere species in subg. Cantharellus. Basidiomata are very small to medium-sized. Pileus is 2-5 cm in diameter and plano-convex at first but soon depressed in the center; the margin was first very regular and strongly incurved and then became more wavy, the surface is smooth, pale yellow, egg-yolk yellow (1A5) to bright yellow (1A7), sometimes tinged with brownish, and shiny in color; the context above stipe  was 0.05-0.15 cm in thickness, yellowish (3A3) to orange yellow (3A8), but unchanging in color when injured. Hymenophore is composed of well-developed, decurrent, well-spaced, and unequal gill folds, especially near the extreme cap margin with many very short lamellulae or also often forked; these folds are 0.1-0.2 cm broad and are pale yellow, egg-yolk yellow (1A4) to orange yellow in color (2A7). Stipe is 1.2-4 × 0.4-1.0 cm, central, cylindrical; surface dry, whitish (5A1), or yellowish-brown (1A2) in color; the context is fleshy, firm, and yellowish (2A2). Taste and odor is not distinctive. Spore print is not obtained.
Cantharellus pinetorus N.K. Zeng, Y.Z. Zhang and Zhi Q. Liang, sp. nov. Figures 3F,G, 6. MycoBank: MB843064 Diagnosis: It differs from other species of subg. Cantharellus by a bright yellow to orange-yellow pileus, a cream to grayish yellow stipe, a well-developed hymenophore, broadly ellipsoid to ellipsoid basidiospores, and it is associated with pine trees. Etymology: Latin "pinetorus" refers to the association of the new species with pine forests.
Basidiomata are small to medium-sized. Pileus is 3.5-5 cm in diameter and convex when young and then applanate with depressed center; the surface is smooth, bright yellow (3A5) to orange-yellow in color (3A6); the margin is incurved and irregularly wavy; the context above stipe is 0.35-0.55 cm in thickness, yellow (3A7), but unchanging in color when injured. Hymenophore is veined and decurrent; these folds are 0.05-0.2 cm broad and are lemon yellow (1A6) to pale yellow in color (4A2). Stipe is 2.5-3 × 0.5-0.6 cm, cylindrical, central, and hollow; the surface is dry and cream (4A1) to grayish yellow (4A2) in color; the context is white (3A2). Taste and odor is not distinctive. Spore print is not obtained.
Habitat: Solitary, scattered, or gregarious on the ground, in forests dominated by Pinus massoniana Lamb.
Known distribution: central China.
Notes: Malaysian Cantharellus ianthinus Corner and Cantharellus subcibarius are morphologically similar to Cantharellus pinetorus. However, Cantharellus ianthinus has purple fibrils on the surfaces of the pileus and stipe, and larger basidiospores measuring 8-10.5 × 5.5-7 µm (Corner, 1966); Cantharellus subcibarius has a context turning yellow to orange-brown when bruised, and it is not associated with trees of Pinaceae (Corner, 1966;Buyck et al., 2021). In the phylogenetic analyses, Cantharellus pinetorus is allied with Cantharellus albopileatus and Cantharellus chuiweifanii (Figure 2)  Basidiomata are small to medium-sized. Pileus is 3.5-8 cm in diameter and plano-convex to infundibuliform; the surface is smooth, yellowish (1A2) to grayish yellow (2A4) in color, and dull; the margin is incurved or downward; the context above stipe is about 0.3 cm in thickness, yellowish (3A3), unchanging in color when injured. Hymenophore is veined and decurrent; these folds are about 0.1 cm high, forking, creamy yellow (1A4) to yellowish (3A2) in color. Stipe is 3-4 × 0.6-0.9 cm, central, cylindrical; surface dry, grayish yellow to fulvous (4A4), but whitish (5A1) at base. Taste and odor is not distinctive. Spore print is not obtained.
Habitat: Gregarious on the ground in forests dominated by fagaceous trees such as Lithocarpus spp.

Morphological Features and Hosts of Cantharellus Subgenus Cantharellus
In agreement with the previous hypotheses , most species in China have nearly glabrous pileus, with the exception of Cantharellus vaginatus S. C. Shao, X. F. Tian, and P. G. Liu and Cantharellus versicolor S. C. Shao and P. G. Liu having a squamulose cap (Shao et al., 2011(Shao et al., , 2016b. As noted by Buyck et al. (2014) (Shao et al., 2011), and sandy brown to dark brown in Cantharellus versicolor was observed (Shao et al., 2016b).
Besides macro-morphology, some micro-morphological features can also be used to discriminate subg. Cantharellus species. For example, species of the subgenus usually have abundant clamps, and hyphal endings in pileipellis are mostly thick-walled . Our four new species and previous taxa of subg. Cantharellus in China also possess clamp connections and thick-walled hyphal endings in pileipellis (Shao et al., 2011(Shao et al., , 2016b(Shao et al., , 2021An et al., 2017;Cao et al., 2021;Zhang Y. Z. et al., 2021).
Most species of subg. Cantharellus sections Cantharellus and Amethystini Buyck and V. Hofstetter have well-developed hymenophore, section Sublaeves Buyck and V. Hofstetter harbors taxa with smooth hymenophore, and section Amethystini Buyck and V. Hofstetter usually has a pileus with appressed squama. Judging from the positions of our new species and Chinese previous taxa in the molecular phylogenetic tree (Figure 2), plus the morphological features, Cantharellus cibarius, Cantharellus macrocarpus, Cantharellus ravus, Cantharellus versicolor, and Cantharellus yunnanensis are the members of section Cantharellus, Cantharellus hainanensis, and Cantharellus laevihymeninus, also have ill-developed hymenophore Cao et al., 2021), and belong to the section Sublaeves. Cantharellus vaginatus is a member of section Amethystini, and Cantharellus albopileatus, Cantharellus chuiweifanii, and Cantharellus pinetorus probably represent a new section, which will be further studied in the future.
Up to now, taxa in subg. Cantharellus are all from northern hemisphere . Most species of the subgenus included in the present dataset are from temperature areas of northern hemisphere including North America and Europe (Figure 2). In China, with the exception of Cantharellus cibarius and Cantharellus versicolor in temperate areas, most species, viz. Cantharellus albopileatus, Cantharellus chuiweifanii, Cantharellus hainanensis, Cantharellus macrocarpus, Cantharellus pinetorus, Cantharellus ravus, Cantharellus vaginatus, and Cantharellus yunnanensis, occur in subtropical or tropical China. With more field investigations, more taxa of the subgenus will be uncovered in the subtropical and tropical regions.

Phylogenetic Relationships and Geographic Divergence of Chinese Cantharellus Subgenus Cantharellus
Our molecular data based on two-locus DNA sequences (28S + TEF1) with many new specimens from China have contributed to our knowledge of subg. Cantharellus.
It is clear that there are several clades having taxa from both sides of the Pacific, and allied species from East Asia and North America are obviously inferred from this molecular phylogenetic tree (Figure 2). For example, Chinese Cantharellus versicolor is related to North American Cantharellus camphoratus R. H. Petersen and Cantharellus formosus Corner; our new species Cantharellus ravus is affiliated with Cantharellus californicus and Cantharellus velutinus, two species both described from United States; one collection tentatively named C. sp. (FHMU3834) appears closely related to North Americantype collection of Cantharellus spectaculus Foltz and T. J. Volk (Figure 2), which is an earlier synonym of Cantharellus persicinus Petersen (Buyck et al., 2016c). Our study did not identify disjunct populations of the same purported taxon in the two areas (Figure 2). Similar scenarios have been documented for many other fungi (Zhang et al., 2004;Zeng et al., 2013Zeng et al., , 2016. Biogeographic connections between East Asia and Europe have also been discussed in other fungi such as Amanita Pers., Phylloporus Quél. and Rhodotus Maire (Zhang et al., 2004;Zeng et al., 2013;Tang et al., 2014). In this study, we found that Cantharellus cibarius occurs in northeastern China and Europe (Figure 2).
In addition, we also noted that Cantharellus hainanensis is associated with one specimen labeled as Cantharellus lateritius from India (Figure 2). Besides northeastern China and Europe, the geographical distribution range of Cantharellus cibarius also extends to Japan (Figure 2).

AUTHOR CONTRIBUTIONS
Z-QL and N-KZ contributed to the conceptualization. Y-ZZ performed the methodology, wrote the original draft preparation, and carried out the formal analysis. Y-ZZ and D-YA performed the experiment. N-KZ, W-FL, M-SS, Z-HC, PZ, and SJ carried out the resources. N-KZ, BB, and Z-QL wrote, reviewed, and edited the manuscript. N-KZ and Z-QL supervised the data. N-KZ carried out the project administration and funding acquisition. All authors contributed to the article and approved the submitted version.

FUNDING
This study was supported by the National Natural Science Foundation of China (No. 32160001), which includes funds for open access publication fees.