Updated taxonomy of Chinese Cantharellus subgenera Afrocantharellus and Magni (Hydnaceae, Cantharellales): Three new taxa and amended descriptions of one previous species

Cantharellus, one of the main genera of Hydnaceae (Cantharellales), is both ecologically and economically important. Although many studies have focused on this genus in China, the taxonomy should be further updated. In the present study, Cantharellus subgenera Afrocantharellus and Magni were investigated based on morphology and molecular phylogenetic analyses with new collections from China. Five phylogenetic species were recognized among the studied collections, three of which were described as new: C. bellus, C. cineraceus, and C. laevigatus; one was previously described taxon: C. hygrophoroides; and the remaining species was not defined due to the paucity of the materials. Among the four described species, both C. bellus and C. laevigatus are members of subgen. Magni, whereas C. cineraceus and C. hygrophoroides belong to subgen. Afrocantharellus.


Morphological studies
The studied specimens were collected from Hainan, Fujian, Hunan, and Zhejiang Provinces of China and deposited in the Fungal Herbarium of Hainan Medical University (FHMU), Haikou city, Hainan Province of China. Based on detailed notes and photographs taken from fresh basidiomata, we obtained the macroscopic descriptions. Color documentation of fresh materials follows Kornerup and Wanscher (1981). Observations and measurements of microscopic features were made in 5% KOH solution and stained with 1% Congo Red (Zhang Y. Z. et al., 2022). The sections of the pileipellis were taken from the pileus between the center and the margin. The number of measured basidiospores is given as n/m/p, where "n" represents the total number of basidiospores measured from "m" basidiomata of "p" collections. The dimensions of basidiospores are presented in the form (a-)b-e-c(−d), where the range b-c contains at least of 90% of the measured values (5th to 95th percentile), "a" and "d" are the extreme values, and "e" refers to the average length/width of basidiospores. "Q" refers to the length/width ratio of basidiospores and "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
The total genomic DNA was extracted from collections and dried with silica gel using the Plant Genomic DNA Kit (CWBIO, Beijing, China) according to the manufacturer's instructions. Primer pairs used for amplification were 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). PCR conditions followed the program of Zhang Y. Z. et al. (2021). PCR products were checked in 1% (w/v) agarose gels. The PCR amplification products were sequenced using an ABI 3730 DNA Analyzer (Guangzhou Branch of BGI, China) with the same primers. DNA sequences were compiled with SeqMan (DNASTAR Lasergene 9) or BioEdit (Hall, 1999) and then deposited in GenBank. 1

Dataset assembly
Forty DNA sequences (22 of 28S and 18 of TEF1) from 23 collections were newly generated. 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 sequences of taxa of subgenera Afrocantharellus and Magni, and representative species of other subgenera of Cantharellus from previous studies and GenBank (Table 1). Craterellus badiogriseus T. Cao & H.S. Yuan was chosen as out-group inferred from Cao et al. (2021). To test for phylogenetic conflict among 28S and TEF1, single-gene phylogenetic trees based on each of these two fragments were analyzed. A conflict was assumed to be significant if two different relationships for the same set of taxa (one being monophyletic and the other non-monophyletic) were observed in rival trees (Vadthanarat et al., 2021). The results of analyses showed that 28S and TEF1 were not in conflict. Thus, the datasets (28S and TEF1) were aligned with MUSCLE v3.6 (Edgar, 2004) and 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). For ML, tree generation and bootstrap analyses were performed with RAxML7.2.6 (Stamatakis, 2006) running 1,000 replicates combined with an ML search. For BI, the best-fit model of substitution among those implementable in MrBayes was estimated separately for each character set using jModelTest (Darriba et al., 2012) on the CIPRES portal, based on the Bayesian information criterion. The best-fit likelihood models for 28S and TEF1 were GTR + I + G and SYM + I + G, respectively. BI was conducted in MrBayes v3.1 (Huelsenbeck and Ronquist, 2005) on the CIPRES Science Gateway portal (Miller et al., 2011). For the BI analyses, four chains were processed with the generation set as 10 million using the selected model for each gene. The trees were sampled every 100 generations. Other parameters were kept at their default setting. The chain convergence was determined using Tracer v 1.5 2 to ensure sufficiently Frontiers in Microbiology 03 frontiersin.org  > 200). Finally, 6.5 million generations were taken to reach the convergence, and the average deviation of split frequencies was 0.009922. The trees were summarized, and statistical values were obtained using the sump and sumt commands with burn-ins (i.e., the first 25% of the samples) discarded. In addition, phylogenetic distances of some Cantharellus species were calculated using MEGA 11 (Tamura et al., 2021).

Molecular data
The combined dataset (28S + TEF1) consisted of 139 taxa and 1862 nucleotide sites (986 of 28S and 876 of TEF1). The phylogram with branch lengths generated from RAxML and support values (BS and PP) is shown in Figure 1. The topologies of phylogenetic trees   Phylogram of genus Cantharellus, with emphasis on subgenera Afrocantharellus and Magni, inferred from a two-locus (rDNA 28S, TEF1) dataset using RAxML. BS (≥70%) and PP (≥0.95) are indicated above the branches.
Habitat: Solitary on the ground of forests dominated by Castanopsis kawakamii Hayata.
Habitat: Gregarious on the ground in forests dominated by  Microbiology  11 frontiersin.org has a yellow cap often with olivaceous or grayish tinges, smaller basidiospores measuring 7.5-9(−10) × 5-5.75(−6) μm, and it is associated with trees of Dipterocarpaceae (Eyssartier et al., 2009); C. cuticulatus, originally described from Malaysia, has a larger basidioma (pileus up to 7.5 cm in diameter), a stipe tinged with yellow, and a trichodermal structure of the pileipellis (Corner, 1966;. In future, more interesting information will be provided with more collections made and more genes investigated. Cantharellus splendens Buyck is also morphologically similar to C. cineraceus. However, the former has narrower basidiospores measuring 8-9.5-11 × 5-5.25-6 μm, a trichodermal structure of the pileipellis, and it is distributed in Africa (Buyck, 2012;.

Discussion
In the present study, five phylogenetic species of Cantharellus were recognized (Figure 1), three lineages were described as new species: C. bellus, C. cineraceus, and C. laevigatus, one was previously described taxon: C. hygrophoroides, and the remaining one was not defined due to the paucity of the materials. Cantharellus bellus and C. laevigatus are both members of subgen. Magni, whereas C. cineraceus and C. hygrophoroides belong to subgen. Afrocantharellus. As mentioned earlier, subgen. Afrocantharellus has been divided into two sections . Our molecular data indicated C. cineraceus and C. hygrophoroides are members of sect. Cutirellus (Figure 1).
In addition to the four described taxa of subgenera Afrocantharellus and Magni, C. cerinoalbus and C. magnus were also described/reported in previous studies (Song et al., 2017;Cao et al., 2021). It is worth noting that the Chinese collections labeled as C. cerinoalbus and our new species C. cineraceus grouped together with high statistical support ( Figure 1); moreover, judging from the descriptions of Chinese specimens identified as C. cerinoalbus (Song et al., 2017), they match well with those of C. cineraceus. Thus, we are sure the specimens identified as C. cerinoalbus from China are really C. cineraceus. Interestingly, the other two new collections (FHMU6948 and FHMU6949) also from the south of China are probably the true C. cerinoalbus for they grouped with the isotype of the Malaysian species with statistical support (Figure 1). Unfortunately, due to the paucity of the two materials, they were not studied thoroughly. Thus, the occurrence of C. cerinoalbus in China will be confirmed with more collections made and more DNA sequences obtained in future.
Earlier studies indicated the taxa of subgen. Afrocantharellus were all described from tropical areas of the world including Africa, Madagascar, and Malaysia (Corner, 1966;, while species of the subgenus, viz. C. hygrophoroides, was uncovered in tropical China (Shao et al., 2014). In the present study, C. cineraceus was described from subtropical China, which extends the range of distribution of subgen. Afrocantharellus.
It is noteworthy that collections identified as C. splendens appear in four parts of the tree; one of them (BB 96.306 and BB 96.199) corresponds to the true C. splendens for isotype of the taxon included in the lineage, the second (ADK 6071, JD 896, and JD968) awaits identification, the third (DDT57) seems to be C. symoensii Heinem. for the specimen and the epitype of C. symoensii group together with statistical support, and the fourth (DDT17) seems to be C. platyphyllus Heinem. for the collection clustered with the epitype of C. platyphyllus (Figure 1). Moreover, one specimen (DDT63) labeled as "C. cyanescens Buyck, " being also clustered with the epitype of C. platyphyllus, is most likely to be C. platyphyllus (Figure 1).
Frontiers in Microbiology 14 frontiersin.org As already noted by previous studies, C. cuticulatus and C. splendens have trichodermal structures in the pileipellis (Corner, 1966;, and C. hygrophoroides were also observed to have intricate trichodermal pileipellis. In addition, the Chinese species classified in the subgen. Afrocantharellus lack bluish context, which was observed on African C. platyphyllus and its Malagasy subspecies bojeriensis . We also noted that most species of Cantharellus with smooth hymenophores belong to subgen. Cantharellus and subgen. Rubrinus, respectively An et al., 2017). The recently erected subgen. Magni was also reported to have a smooth hymenophore , and our new species C. laevigatus is the second species of the subgenus uncovered with a smooth hymenophore. Interestingly, a well-developed hymenophore was observed from our new species C. bellus, also a member of subgen. Magni, which indicates the diagnostic features of subgen. Magni should be revised.
Author contributions Z-QL and N-KZ contributed to the conceptualization. Y-ZZ performed the methodology and conducted the formal analysis. The original draft preparation was written by Y-ZZ and H-ZQ. The experiment was performed by Y-ZZ. N-KZ, Z-HC, and W-FL. SJ carried out the resources. N-KZ and Z-QL wrote, reviewed, and edited the manuscript and directed the data. N-KZ was responsible for project management and funding access. All authors contributed to the article and approved the submitted version.

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