Diversity of Cantharellus (Cantharellales, Basidiomycota) in China with Description of Some New Species and New Records

Cantharellus is a well-known genus of edible mushrooms, belonging to the family Hydnaceae in the class Agaricomycetes. In this study, a phylogenetic overview of Cantharellus subg. Cinnabarinus and C. subg. Parvocantharellus in China is carried out with the description of four new species. Species description are based on morphological characters of basidiomata and phylogenetic analyses of multi-locus dataset of 28S + tef1 + rpb2. Among the new species, two species, C. chrysanthus and C. sinocinnabarinus, belong to C. subg. Cinnabarinus and two new species, C. convexus and C. neopersicinus, belong to C. subg. Parvocantharellus. Species delimitation characters of the new taxa are compared with closely related species. In addition, three new records of Cantharellus are reported for China: C. albovenosus and C. citrinus of subg. Cinnabarinus and C. koreanus of subg. Parvocantharellus. A key to the species of subg. Cinnabarinus in China was provided.


Morphological Studies
Photographs of fresh basidiomata were taken in the field. Specimens were dried and deposited in the Fungarium of Guangdong Institute of Microbiology (GDGM). Descriptions of macro-morphological characters and habitats were obtained from photographs and field notes. The color codes followed Kornerup and Wanscher [23]. Microscopic observations were carried out on tissue sections stained with 5% aqueous KOH and 1% aqueous Congo red under a light microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) with a magnification up to 1000×. For basidiospore descriptions, the notation (a-)b-c(-d) describes basidiospore dimensions, where the range b-c represented 90% or more of the measured values and 'a' and 'd' were the extreme values; L m and W m indicated the average length and width (±standard deviation) of the measured basidiospores, respectively; 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.

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA was extracted from the voucher specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co., Ltd., Shanghai, China) according to the manufacturer's instructions. Primer pairs LROR/LR7 [24], tef1F/tef1R and RPB2-5FCanth/RPB2-7cRCanth [3,25] were used to amplify the LSU, tef1 and rpb2 region, respectively. PCR reactions were performed in a total volume of 25 µL containing 0.5 µL template DNA, 11 µL sterile deionized water, 0.5 µL of each primer and 12.5 µL 2 × PCR mix [DreamTaqtm Green PCR Master Mix (2×), Fermentas, MA, USA]. Amplification reactions were performed in a Tprofessional Standard thermocycler (Biometra, Göttingen, Germany) under the following conditions: 95 • C for 4 min; then, 35 cycles of denaturation at 94 • C for 60 s, annealing at 53 • C (LSU)/50 • C (tef 1)/52 • C (rpb2) for 60 s and extension at 72 • C for 60 s; with a final extension at 72 • C for 8 min. The PCR products were electrophoresed on 1% agarose gels and then send for sequencing on an ABI Prism ® 3730 Genetic Analyzer (PE Applied Biosystems, Foster, CA, USA) at the Beijing Genomic Institute (BGI) using the same PCR primers. The raw sequences were assembled and checked with SeqMan implemented in Lasergene v7.1 (DNASTAR Inc., Madison, WI, USA). The newly generated sequences in this study were submitted to GenBank.

Phylogenetic Analyses
Sequences generated in this study and those downloaded from GenBank were combined and used for phylogenetic reconstruction. Detailed information of specimens included in this study was given in Table 1. Three sequence matrices, i.e., nrLSU, tef1 and rpb2, were aligned separately with software MAFFT v6.853 using the E-INS-i strategy [26] and then manually adjusted in MEGA 6 [27]. The ambiguous aligned regions and introns of the two protein-coding genes of tef1 and rpb2 were retained in the final analyses.
Maximum Likelihood (ML) analyses were inferred using RAxML v7.2.6 [28], and all parameters were kept as defaults except for choosing GTRGAMMAI as the model; statistical supports were obtained using rapid non-parametric bootstrapping with 1000 replicates. Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 [29]; the best models of the multi-locus datasets were searched via the PartitionFinder 2 [30] for each locus, i.e., K80 + I + G, SYM + I + G and SYM + I + G for 28S, tef1 and rpb2, respectively. BI analysis using 4 chains were conducted by setting generations to 20 million and stoprul command with the value of stopval set to 0.01; trees were sampled every 1000 generations, the first 25% generations were discarded as burn-in and posterior probabilities (PP) were then calculated from the posterior distribution of the retained Bayesian trees. Cantharellus cibarius Fr. was selected as the outgroup based on recent studies [3,13]. The phylogenetic trees were visualized using FigTree v1.4.23.

Molecular Phylogeny
For phylogenetic analyses, a total of 152 sequences were newly produced in this study, containing 49 nrLSU, 51 tef1 and 52 rpb2, and 185 reliable sequences were downloaded from the GenBank database based on previous studies [3,13]. The combined dataset (LSU + tef1 + rpb2) contained 2892 characters (1311, 707 and 874 for LSU, tef1 and rpb2, respectively), of which 2013 were conserved and 708 were parsimony-informative. ML and BI analyses of the concatenated data set resulted in almost identical topologies, and no strongly-supported conflicts between ML and BI analyses were discovered; thus, only the tree inferred from ML analysis was displayed ( Figure 1). Our phylogenetic analyses indicated that members of C. subg. Cinnabarinus formed a highly support monophyletic group (MLB/BPP = 100%/1.0). Five well-supported clades in the subg. Cinnabarinus were identified based on samples newly collected from China, including two new species, two species newly recorded in China and a known species in China. Besides, three wellsupported clades in the subg. Parvocantharellus were firstly discovered in China, containing two new species and a newly recorded species from China.   Diagnosis-This species is characterized by its orange to orange-yellow pileus, pinkish white to orange white hymenophore, thin-walled pileipellis terminal hyphae, broadly ellipsoid basidiospores (7.5-9 × 5-6.5 µm) and long basidia up to 100 µm.
Basidiospores ( Notes-Cantharellus sinocinnabarinus can be easily recognized in the field by its small reddish orange basidiomata. Morphologically, C. sinocinnabarinus is similar to C. cinnabarinus, C. persicinus R.H. Petersen and C. texensis. However, the latter three species were all originally reported from North America; C. cinnabarinus and C. persicinus differ in their larger basidiomata (pileus up to 40 mm), thicker-walled hyphae of pileipellis terminal cells, and different sizes of basidiospores (6.7-7.57 × 3.82-4.68 µm for C. cinnabarinus, and 10.2-11.9 × 6.3-7.2 µm for C. persicinus) [32]; C. texensis differs in its robust basidiomata and longer but narrower basidiospores (8-8.95 Shao et al. [20] has described a specimen (HKAS58243) under the name C. cinnabarinus on the basis of the LSU sequence, which is geographically close to C. sinocinnabarinus in southwest China. In this study, the specimen (HKAS58243) was re-examined; the morphological features and molecular phylogenetic analyses all demonstrated that it is actually C. sinocinnabarinus.
In the multi-locus phylogentic trees, specimens of C. sinocinnabarinus formed a wellsupported independent terminal branch (BS = 100%, BPP = 1.0) in the subg. Cinnabarinus, and are closely related to C. cinnabarinus. However, they can be easily distinguished by the morphological features and large genetic distance.
Shao et al. [20] has described a specimen (HKAS58243) under the name C. cinnabarinus on the basis of the LSU sequence, which is geographically close to C. sinocinnabarinus in southwest China. In this study, the specimen (HKAS58243) was re-examined; the morphological features and molecular phylogenetic analyses all demonstrated that it is actually C. sinocinnabarinus.
In the multi-locus phylogentic trees, specimens of C. sinocinnabarinus formed a wellsupported independent terminal branch (BS = 100%, BPP = 1.0) in the subg. Cinnabarinus, and are closely related to C. cinnabarinus. However, they can be easily distinguished by the morphological features and large genetic distance.    Basidiomata small-sized. Pileus 20-55 mm broad, convex at first, then broad applanate with a depressed centre, subinfundibuliform when mature or old; margin inflexed to straight when young, then undulate; surface tomentose when young, then glabrescent and radially (innately) fibrillose to finely striate and rugulose, orange, deep orange to reddish orange (5A6-7A6, 5A8-7A8), then pallescent to light orange at margin. Hymenophore decurrent, with a clearly delimitation from stipe surface; lamellate ridges, subdistant to distant, relatively well-developed, 1-1.5 mm high, appropriately bifurcate and interconnected with low veined folds, particularly towards pileus margin, white to orange white (5A2-6A2), unchanging when bruised. Stipe 25-50 × 2.5-9 mm, cylindrical and slightly clavate to bulbose at base, finely tomentose when young, then glabrous or with finely longitudinally fibrillose, concolorous with pileus, orange to reddish orange, sometimes paler to light orange in some specimens. Context white, orangish under pileipellis, solid, becoming hollow-fibrous in stipe. Odor spicy. Taste mild.  Notes-Cantharellus albovenosus, recently reported from South Korea, is characterized by the combined features of the orange to reddish orange pileus, white to orange white and relatively well-developed lamellate hymenophore, the orange to reddish orange stipe, and the ellipsoid to nearly globose basidiospores (7-8.5 × 5-6 µm) [11]. Phylogenetically, C. albovenosus and C. phloginus clustered together in an almost similar phylogenetic position, and cannot be separated in our multi-locus phylogenetic tree (Figure 1). Morphologically, C. phloginus can be distinguished by its pastel red to pastel pink pileus and stipe, pale yellow to yellowish orange hymenophore and large basidiospores [6.8-9.5 (-12) × 5-7 µm] [22]. Ecologically, C. albovenosus is known from subtropical regions of South Korea and eastern China; meanwhile, C. phloginus is currently only known from tropical regions of southwest China. The distinguishable morphological features and different growth habits supported them as two distinct species, but some more effective molecular markers are needed to distinguish the two species. Basidiomata small-sized. Pileus 15-45 mm broad, convex, with involute margin when young, then gradually to broadly infundibuliform with depressed center, irregularly undu-late or slightly cracked margin when old; surface dry or hygrophanous, glabrous or finely subtomentose, greenish yellow, light yellow, yellow to yellowish orange (1A4-4A4, 1A7-4A7). Context yellowish white, 1 mm thick in the center of the pileus, sharply attenuate towards margin, unchanging when exposed. Hymenophore decurrent, subdistant, composed of bifurcate, less than 1 mm high veined folds, particularly towards pileus margin, white to yellowish white (1A2-3A2), unchanging when bruised. Stipe 15-30 × 3-5 mm, central, cylindrical or slightly tapering towards base, hollow, glabrous, concolorous with pileus or paler, unchanging when handled. Odor fruity and pleasant. Taste mild.
Habitat and distribution-Gregarious on soil under mixed forests in southwest China. Known from southwest China and Korea.      Notes-Cantharellus citrinus, recently reported from Korea [11], is characterized by its small basidiomata, greenish yellow to yellowish orange pileus, white to yellowish white hymenophore strongly bifurcate at pileus margin, glabrous and hollow stipe, and broadly elliptical to subglobose basidiospores [7-9 × 5-6 (6.5) µm]. In the multi-locus phylogentic tree, samples of C. citrinus formed a well-supported monophyletic terminal clade, and can be easily distinguished from other Cantharellus species.
Phylogenetically, two specimens of C. convexus formed an isolated lineage in subg. Parvocantharellus, and are closely related to C. tabernensis. A BLAST result of ITS sequence in the GenBank database also demonstrated that the similarity between C. convexus and C. tabernensis (JN944012, O7.064) is 93.7%. However, C. tabernensis, originally reported from North America, differs in its more robust basidiomata, dull orange-yellow to yellowish-brown pileus, vivid orange-yellow hymenophore and stipe and larger basidiospores (6-9 × 4.4-5.9 µm) [40]. Additionally, C. tabernensis, currently only known from Texas, Louisiana and Mississippi in North America, occurs in well-drained (sandy) soil in mixed woods, and near to Pinus elliottii Engelm. Meanwhile, C. convexus was found in broadleaf forests in southern China, close to Fagaceae trees. Another North America species, C. appalachiensis, also demonstrates a close relationship with C. convexus. However, C. appalachiensis differs in its larger and more robust basidiomata, with a drab yellow to dull brown pileus applanate with the center depressed, surface locally dull-grayish due to aggregate minute fibrils and with larger basidiospores (6.6-8.9 × 4.4-5.9 µm) [41,42].
In addition, several species were recently reported from China, and are also similar to C. koreanus in morphology, such as C. galbanus, C. luteolus Ming Zhang, C.Q. Wang & T.H. Li, C. luteovirens and C. sinominor Ming Zhang, C.Q. Wang & T.H. Li [13], but they can be easily separated from each other by the large genetic distances.

Discussion
In this study, the species diversity of C. subg. Cinnabarinus from China were examined. Five species were identified based on morphological characters and multi-locus phylogenetic analyses, containing two new species C. chrysanthus and C. sinocinnabarinus, two newly recorded species C. albovenosus and C. citrinus to China, and a known species, C. phloginus. In addition, three species belonging to the subg. Parvocantharellus were firstly discovered from China, including two new species C. convexus and C. neopersicinus, and a new recorded species, C. koreanus.
In the past, the knowledge of species diversity of Cantharellus in China was poor and the specimens with large and yellow to orange basidiomata were mostly misidentified as the type species of the genus C. cibarius; meanwhile, specimens with small and yellow to orange red basidiomata were often inaccurately treated as C. minor Peck or C. cinnabarinus. However, a recent study proved that the distribution of C. cibarius is limited to northeast China, and the so-called "C. cibarius" reported from southwest China is actually C. yunnanensis W.F. Chiu [8]; meanwhile, the specimens labeled as "C. minor" in China were also proven to be misidentified, several new species with small basidiomata have been reported from China, and the distribution of C. minor with correctly identified specimens has not been found in China [13]. Cantharellus cinnabarinus was widely reported in China [19,21], but those photos of C. cinnabarinus used in the two literatures look like C. albovenosus; the correctly identified specimens of C. cinnabarinus in China have not been found in the present study. However, three morphologically similar species were discovered. The specimen HKAS58243 from southwest China, firstly identified as C. cinnabarinus in Shao et al. [20], was proven to be a native species of C. sinocinnabarinus in the present study. In addition, C. sinocinnabarinus seems to be restricted to subalpine habitats, and prefers symbiosis with Cyclobalanopsis delavayi and Pinus yunnanensis. The other two species, C. albovenosus and C. phloginus, are easily misidentified as C. cinnabarinus by their small basidiomata and reddish pileus color. However, C. albovenosus, recently reported from Korea, has been also found in eastern China, and C. phloginus seems to be restricted to tropical to subtropical regions in southwest China. Thus, we speculate that the specimens of "C. cinnabarinus" in Anhui, Guangdong, Jiangsu and Zhejiang provinces could be C. albovenosus, the distribution of "C. cinnabarinus" from tropical to subtropical regions of southwest China could be C. phloginus and the collections of "C. cinnabarinus" from subalpine regions of southwest China could be C. sinocinnabarinus.
Cantharellus neopersicinus, newly discovered in this study, is a remarkable species in Cantharellus. Morphologically, C. neopersicinus can be easily identified as a member of subg. Cinnabarinus or subg. Cantharellus, due to its pastel red to pink pileus and white to pinkish hymenophore; however, phylogenetic analyses demonstrated that it belongs to the subg. Parvocantharellus, which makes it the first species reported from China with pastel red to pink tinge in the subg. Parvocantharellus. Ecologically, C. neopersicinus is distributed in tropical areas of southern China, and currently, the only known symbiosis is with Eucalyptus robusta.
Cantharellus subg. Parvocantharellus, mainly composed of small-sized species, was suggested to be a monophyletic group, and closely related to the subg. Cinnabarinus [3]. However, in the present study, the subgenus was proven to be paraphyletic or polyphyletic; two species of C. cyanoxanthus R. Heim ex Heinem. and C. subcyanoxanthus Buyck, Randrianj. & Eyssart formed an isolated clade in the multi-locus phylogenetic tree, and could represent a separate generic clade. The result is similar to previous studies [13,16].
Species in the two subgenera are difficult to separate in morphology because most species share similar characteristics of small basidiomata, abundant clamps and thinwalled hyphal ends at the pileus surface. However, they formed two separate clades in the multi-locus phylogenetic trees, and can be easily distinguished by molecular phylogenetic evidence. In addition, the species in subg. Cinnabarinus mostly own distinct orange, pink or red tinge, and can be distinguished from subg. Parvocantharellus. In future work, more detailed morphological observations are needed to provide new evidences for distinguishing the two subgenera.  Data Availability Statement: Publicly available datasets were analyzed in this study. These data can be found here: (https://www.ncbi.nlm.nih.gov/; https://www.mycobank.org/page/Release%20 names; accessed on 20 March 2022).