﻿Three new species of the genus Clavulina (Hydnaceae, Cantharellales) from North China based on morphological and phylogenetic analysis

﻿Abstract Clavulina possesses important ecological and economic value and has attracted extensive attention from mycologists. Macrofungal diversity is high in China, but Clavulina species have not been thoroughly studied. In this study, based on morphological evidence and phylogenetic analyses of the nucleotide sequences of three loci (nrITS, nrLSU, and rpb2), three new species of Clavulina from North China were identified. Morphologically, Clavulinachengdeensis is characterized by its white to dirty white basidiomata with somewhat pale orange tips and somewhat wrinkled hymenium. Clavulinagriseoviolacea is characterized by its gray to dark grayish violet basidiomata, with a sometimes-white stipe base, monopodial or irregularly polychotomous toward branch apices. Clavulinapallida is characterized by its white to pale cream white basidiomata with somewhat orange tips. Phylogenetically, the three new species form three independent branches with high support values in the phylogenetic tree.


Collecting and site description
The specimens were collected from 2017 to 2023 in Beijing, Hebei Province, and Tianjin, North China.These areas have a warm temperate continental monsoon climate and a diverse assortment of plants.The dominant forest types are deciduous broad-leaved forest and mixed coniferous and broad-leaved forest.The dominant trees include Quercus mongolica Fisch.ex Ledeb., Betula platyphylla Suk., Abies nephrolepis (Trautv.)Maxim., Populus tomentosa Carrière, and Pinus tabuliformis Carr.(Wang et al. 2021;Zhou et al. 2022).The annual precipitation is approximately 700 mm.The altitude ranges from 200 to 2200 m.The collected specimens were dehydrated using an electric dryer (Dorrex) at 50 °C and kept in the Herbarium of the College of Life Science, Capital Normal University, Beijing, China (BJTC).

Morphological observation
Macroscopic characteristics were documented from dried specimens and photographs, while thin sections of specimens mounted in 3% potassium hydroxide (KOH) or sterilized water were analyzed for microscopic features.The morphology and dimensions of their microscopic structures were observed and recorded using a light microscope [Olympus DP71, Tokyo, Japan].In the description of basidiospores, the abbreviation n/m/p means that n basidiospores were measured from m basidomata of p collections.The measurements and Q values were presented in the form of (a)b-c (d), in which "b-c" contained a minimum of 90% of the measured values, and extreme values (a and d) were given in parentheses.Q represents the ratio of the length to width of basidiospores, and Q m represents the average Q value of all basidiospores measured ± the sample standard deviation (Huang et al. 2023).The nomenclatural details were submitted to MycoBank.Color designation was referred to the website colorhexa (https://www.colorhexa.com).

Molecular phylogenetic analyses
The newly obtained sequences from this study were submitted to NCBI (https:// www.ncbi.nlm.nih.gov).The nrITS, nrLSU, and rpb2 sequences of the concatenated nrITS-nrLSU-rpb2 datasets were aligned with selected sequences from GenBank and previous studies.All sequences are listed in Table 1.The generated raw reads of the DNA sequences were used to obtain consensus sequences using SeqMan v.7.1.0 in the DNASTAR Lasergene Core Suite software (DNAS-TAR Inc., Madison, WI, USA).All sequences were aligned using MAFFT v.6 (Katoh and Toh 2010) and trimmed manually with MEGA 6 (Tamura et al. 2013).For phylogenetic analyses, newly obtained sequences and additional reference sequences of Clavulina species were included in the dataset of the combined nrITS-nrLSU-rpb2 fragment (Table 1), with Hydnum repandum L. and Hydnum rufescens Pers.as the outgroups following He et al. (2016).
To estimate Maximum Likelihood (ML) phylogenetic trees, we utilized RAx-ML 7.4.2Black Box software (Stamatakis 2006;Stamatakis et al. 2008;Zhou and Hou 2019;Zhou et al. 2021) with a GTRGAMMAI site substitution model (Guindon et al. 2010).Branch support was calculated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993).Bayesian Inference (BI) analysis was conducted using MrBayes 3.1.2(Ronquist and Huelsenbeck 2003) with a Markov chain Monte Carlo (MCMC) algorithm (Rannala and Yang 1996).The best model was estimated using MrModeltest 2.3 (Zhou and Hou 2019;Zhou et al. 2021Zhou et al. , 2022)).The models employed for each marker of the nrITS-nrLSU-rpb2 dataset were HKY + G for nrITS, GTR + I + G for nrLSU, and HKY + I + G for rpb2.We ran two MCMC chains for 100,000,000 generations, stopping when the average standard deviation of split frequencies dropped below 0.01.Trees were saved every 1000 generations, and the initial 25% of trees were discarded as the burn-in phase for each analysis.Significant Bayesian posterior probabilities were calculated for branches in the remaining trees, as the analysis yielded relatively stable topologies, and clades with high Bayesian posterior probability (pp) values reflected the relative relationships between species (Posada and Crandall 1998).

Molecular phylogeny
A total of 36 sequences, including 12 for nrITS, 12 for nrLSU and 12 for rpb2, were generated in this study.The nrITS-nrLSU-rpb2 dataset included 320 sequences (140 for nrITS, 109 for nrLSU, and 71 for rpb2), with 161 samples.The concatenated alignment contained 2188 characters, including gaps.ML and Bayesian analyses resulted in highly similar estimates of tree topologies; thus, only the tree inferred from the ML analysis is shown (Fig. 1).
Etymology.The epithet "chengdeensis" refers to the specimens collected from Chengde city.
Habit, habitat, and distribution.Solitary or gregarious caespitose in humus layers on soils in broad-leaved deciduous forests associated with Castanea Mill., Betula L., and Platanus L. Basidiomata generally occur from July to September; currently known from Hebei Province and Beijing, China.
Etymology.The epithet "griseoviolacea" refers to the basidiomata being gray to dark grayish violet.

Clavulina pallida
Etymology.The epithet "pallida" refers to the basidiomata being pale white.

Discussion
In early studies on Clavulina, only brief records of the macroscopic and microscopic features of the species were made.This resulted in difficulties in distinguishing between similar genera or species and led to the occurrence of many synonymous names (Hibbett et al. 1997;Binder et al. 2005;Moncalvo et al. 2006;Larsson 2007;Uehling et al. 2012b).For example, Clavulina paraincrustata Meiras-Ottoni & Gibertoni, described only by morphology from the Brazilian Atlantic rainforest (Tibpromma et al. 2017), is treated as a synonym of C. incrustata.Wartchow found the same region (Wartchow 2012) on the basis of molecular evidence (de Meiras-Ottoni and Gibertoni 2023).
Previous studies have indicated that Clavarioid fungi, including Clavulina, are highly diverse and distributed worldwide (Corner 1950).However, to date, only 11 species of Clavulina have been described in China.China has a vast territory, complex climate, habitat types, and abundant species resources, with extremely high fungal diversity (Mcneely et al. 1990).The lack of records in this case may be due to the limited number of collections and collection areas.In this study, three new species of Clavulina were described on the basis of samples collected from North China, by means of both nrITS-nrLSU-rpb2 three-locus phylogenetic analyses (Fig. 1) and macrofungal morphological examinations, which increase understanding of the species diversity of this genus.The natural growth of Clavulina may be related to precipitation.However, the investigations and specimen collection in this study were carried out in the rainy season from August to September, with no collection in other periods.Therefore, more Clavulina species were likely to be present in the study area.

Figure 1 .
Figure 1.Phylogenetic tree generated from a ML analysis based on combined nrITS-nrLSU-rpb2 sequences.Numbers representing Maximum Likelihood bootstrap support (MLBS ≥ 75%, right) and significant Bayesian posterior probability (BPP ≥ 0.95, left) are indicated above the nodes.Novel sequences are printed in bold.Voucher specimens and localities where the specimens were collected are provided behind the species names.

Table 1 .
Specimens used in phylogenetic analysis and their GenBank accession numbers.The newly generated sequences are shown in bold.