Taxonomy and Phylogenetic Relationships of Clavulinopsis (Clavariaceae, Agaricales): Description of Six New Species and One Newly Recorded Species from China

Specimens of Clavulinopsis (Clavariaceae, Agaricales) collected in China were studied using morphological and molecular methods. Six species—C. aspersa, C. bicolor, C. bispora, C. erubescens, C. incarnata, and C. tropicalis—are described as new to science, and C. trigonospora is a newly recorded species in China. Phylogenetic analysis was conducted based on a combined dataset of internal transcribed spacer and nuclear ribosomal RNA large subunit sequences. The phylogenetic reconstruction revealed that the six new species each formed an independent lineage, and the samples of C. trigonospora from China were nested with accessions of C. trigonospora collected from Italy. The morphology of the seven Chinese species is described in detail, and is illustrated with line drawings and photographs. A key to the known Clavulinopsis species in China is provided.


Introduction
The genus Clavulinopsis Overeem comprises 34 species of coral fungi distributed worldwide [1] and classified in the Clavariaceae (Agaricales, Basidiomycota). Among the 165 taxon records for the genus currently listed in the Index Fungorum database (http://www.indexfungorum.org; accessed on 15 April 2023), approximately 70 species names are legitimately published. However, the majority of taxa lack molecular evidence and some even lack adequate micromorphological data. For example, Clavulinopsis coliformis (Boud.) Corner was transferred to Clavulinopsis by E.J.H. Corner in 1950, but E.J.H. Corner considered that C. coliformis might only be an unusual state of a well-known species, such as C. corniculata (Schaeff.) Corner or C. fusiformis (Sowerby) Corner [2]. Similarly, the taxonomic placement of other Clavulinopsis species, such as C. pusilla (Coker) Corner, C. subflava (Britzelm.) Corner, C. sulphurascens (Schwein.) Corner, and C. tenella (Boud.) Corner, is dubious [2]. In addition, the relationships among Clavaria Vaill. ex L., Clavulinopsis, and Ramariopsis (Donk) Corner, which are classified in the same family, have historically been controversial. Considering macromorphological evidence, the basidiome of the species placed in Clavaria and Clavulinopsis are simple or branched, clavarioid, and variously colored, whereas those of Ramariopsis species are rarely simple [2,3]. Based on micromorphological data, R.H. Petersen noted that Clavaria and Clavulinopsis are not separable at the genus rank because clamp connections are present on the basidia and hyphae in Clavulinopsis, but this is the sole character used to distinguish the genus from Clavaria. With regard to Ramariopsis, R.H. Petersen suggested that the genus must be segregated from Clavaria and Clavulinopsis on account of its branched fruit bodies and echinulate spores [4].
With the increase in taxonomic means, the boundaries between these genera are gradually being resolved. D.N. Pegler and T.W.K. Young [5] used scanning electron microscopy to examine the basidiospore structure in Clavulinopsis and Ramariopsis and observed that,

Specimens
Seventeen specimens of Clavulinopsis were collected from Hainan, Hunan, Jilin, Shaanxi, Sichuan, and Yunnan provinces in China from 2014 to 2022. The fresh specimens were dried using heat or indicating silica gel. The dried vouchers were deposited in the Mycological Herbarium of Hunan Normal University (MHHNU), Changsha, China. Detailed information on the 17 specimens and other samples used in the phylogenetic analysis are provided in Table 1, including GenBank accession numbers and locality information.

DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted from the dried samples using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) in accordance with the manufacturer's instructions. The primer pair ITS4/ITS5 [10] was used to amplify the internal transcribed spacer (ITS) region, and the universal primers LR0R, LR3, and LR5 [11,12] were used to amplify the nuclear ribosomal large subunit (nrLSU) region. Each PCR amplification was performed with an Eppendorf Mastercycler thermal cycler (Eppendorf Inc., Hamburg, Germany) in a 25 µL reaction mixture, which contained 1× PCR buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 0.4 µM of each primer, 1.25 U Taq polymerase (Sangon Biotech, Shanghai, China), and 1 µL DNA template. The thermal cycling was performed as follows: initial denaturation at 94 • C for 4 min, then 34 cycles of 94 • C for 40 s, annealing at 55 • C for 40 s, extension at 72 • C for 1 min, and a final extension at 72 • C for 8 min [13]. All amplified PCR products were electrophoresed on 1% agarose gel, and the purified PCR products were sequenced by Sangon Biotech (Shanghai, China). The specified primers were also used for sequencing reactions. All sequences newly generated in this study were submitted to GenBank.

Morphological Studies
The macromorphological characters of species were based on field notes and habitat photographs. Color codes used in descriptions follow Kornerup and Wanscher [14], and color terms are from Ridgway [15]. The micromorphological characters were observed with a light microscope. Tissue sections of dried materials were first rehydrated with 5% KOH solution or distilled water, and then stained with 1% Congo Red when necessary. The notation [n/m/p] means that n basidiospores were measured from m basidiomes of p specimens. The basidiospore dimensions are described using the notation (a-)b-c(-d). The range b-c contains a minimum of 90% of the measured values, and a and d are extreme values, provided in parentheses. The Q value is the length/width ratio of each basidiospore, and the Q value is the average Q ± standard deviation.

Alignment and Phylogenetic Analyses
Sequence data for the legitimate species of the genus Clavulinopsis and two Mucronella species were downloaded from GenBank. The downloaded and newly generated sequences constituted the dataset for the present analysis. The ITS and nrLSU sequences were, respectively, aligned using the default settings for gap openings and gap extension penalties with MAFFT v7.471 [16], and then manually adjusted where necessary in BIOEDIT v7.2.5 [17]. The concatenated ITS-nrLSU data matrix containing 79 sequences (39 ITS and 40 nrLSU) was assembled with SEQUENCEMATRIX 1.7.8 [18]. A maximum likelihood (ML) analysis was conducted using RAXML v8.0.20 [19] with 1000 bootstrap replicates and the GTR + Gamma evolutionary model. Bayesian inference (BI) was performed with MRBAYES v3.2.7 [20] and run for 1,000,000 generations with the GTR + I + G optimal evolutionary model selected with MRMODELTEST v2.4 [21] for each partition, using four Markov Chain Monte Carlo (MCMC) chains to calculate posterior probabilities. The tree files were merged and edited with FIGTREE v1.4.2 [22].
Habitat and distribution: Solitary or scattered to gregarious, on soil rich in humus, in broadleaved forest, known only in subtropical areas of China; June to July.
Etymology: bicolor (Lat.) refers to the two different colors of the basidiomata, namely, yellow to brown at the base and white or creamy white to pale green-white above.
Habitat and distribution: Scattered to gregarious, on soil rich in humus, in tropical broadleaved forest, known only in a tropical area of China; June.
Etymology: erubescens (Lat.) alludes to the basidiomata becoming red with age.  Comments: Within the genus Clavulinopsis, C. corallinorosacea, C. depokensis (Overeem) Corner, C. miyabeana (S. Ito) S. Ito, and C. sulcata are similar to C. erubescens in having a simple red basidioma. However, compared with C. erubescens, C. corallinorosacea has a taller, occasionally branched basidioma and narrower basidiospores; C. depokensis has four-spored basidia and is specialized among Clavulinopsis species in occurring on dead leaves and twigs in woods rather than on soil. Additionally, C. sulcata has a variably colored basidioma and globose or subglobose basidiospores [2]. Consistent with the morphological differences, the present phylogenetic analysis confirmed that C. erubescens has only a distant affinity with, and is placed in a separate lineage to, C. corallinorosacea, C. miyabeana, and C. sulcata. Comments: Within the genus Clavulinopsis, C. corallinorosacea, C. depokensis (Overeem) Corner, C. miyabeana (S. Ito) S. Ito, and C. sulcata are similar to C. erubescens in having a simple red basidioma. However, compared with C. erubescens, C. corallinorosacea has a taller, occasionally branched basidioma and narrower basidiospores; C. depokensis has four-spored basidia and is specialized among Clavulinopsis species in occurring on dead leaves and twigs in woods rather than on soil. Additionally, C. sulcata has a variably colored basidioma and globose or subglobose basidiospores [2]. Consistent with the morphological differences, the present phylogenetic analysis confirmed that C. erubescens has only a distant affinity with, and is placed in a separate lineage to, C. corallinorosacea, C. miyabeana, and C. sulcata. Clavulinopsis incarnata P. Zhang & Jun Yan, sp. nov.: Figures 9 and 10.   Description: Basidiomata fragile, simple, 30-120 mm tall, 1-7 mm wide, solitary or scattered to gregarious. Fertile part subcylindric to fusiform, sometimes conspicuously twisted and with a distinct longitudinal depression, pinkish [11A2-3, 12A4-5, Hermosa Pink, Eosine Pink Thulite Pink]. Apex rounded or obtuse-acute, obviously paler than the lower part, becoming pale yellow or brown with age. Sterile part narrow, indistinct, concolorous or subtly different in color, without tomentum or mycelial patch at the base. Context fragile, hymenium concolorous or slightly paler. Taste, odor, and macrochemical reactions were not recorded. Description: Basidiomata fragile, simple, 30-120 mm tall, 1-7 mm wide, solitary or scattered to gregarious. Fertile part subcylindric to fusiform, sometimes conspicuously twisted and with a distinct longitudinal depression, pinkish [11A2-3, 12A4-5, Hermosa Pink, Eosine Pink Thulite Pink]. Apex rounded or obtuse-acute, obviously paler than the lower part, becoming pale yellow or brown with age. Sterile part narrow, indistinct, concolorous or subtly different in color, without tomentum or mycelial patch at the base. Context fragile, hymenium concolorous or slightly paler. Taste, odor, and macrochemical reactions were not recorded.
Habitat and distribution: Solitary or scattered to gregarious, on soil rich in humus, in broadleaved forest or mixed coniferous-broadleaved forest, known only in subtropical areas of China; August to September.
Habitat and distribution: Solitary or scattered to gregarious, on soil rich in humus broadleaved forest or mixed coniferous-broadleaved forest, known only in subtropi areas of China; August to September.
Additional  [2,3]. C. corallinorosacea (Cleland) Corner and C. sulc Overeem are more similar to C. incarnata in basidiomata size, but the basidiomata of the two species often grows more densely. In addition, the former species has a narrow fusiform or amygdaliform basidiospore, and the basidiomata of the latter species is thin white and villous at the whitish base [2,3].    Diagnosis: Distinguished from other taxa in the genus by its smooth, thin-walled, subtriangular, quadrilateral to subglobose basidiospores.
Specimens Comments: Based on the records of Franchi and M. Marchetti [9], C. trigonospora has a simple, bright yellow to light yellow-orange, small basidioma (25-45 mm tall, 1-2 mm wide), and its most distinctive character is subtriangular spores. Compared with the type specimen, our specimens collected in China show a larger range in size, but all possess subtriangular spores. The distribution of this species in China was confirmed by phylogenetic analysis of molecular data, and thus the present account is the first record of this species outside of Italy.   MycoBank: 848963 Diagnosis: The species has a red basidioma and ellipsoid to broadly ellipsoid basidiospores, and differs from C. erubescens in having only four-spored basidia and growing in the tropics.
Etymology: tropicalis (Lat.) refers to the climate in which the species was discovered.   Comments: Compared with C. erubescens, C. tropicalis is distributed in the tropics and only has four-spored basidia. Based on the present multi-loci phylogenetic analysis, although C. erubescens and C. tropicalis are placed on sister branches, there was a genetic distance of 0.0400 between the two taxa.

Molecular Phylogenetic Analysis
In total, 79 sequences (39 ITS and 40 nrLSU) from 50 samples (Table 1) were assembled into a multi-gene dataset for the molecular phylogenetic analysis, of which 34 sequences (17 ITS and 17 nrLSU) were newly generated in the present study. The combined ITS-nrLSU dataset consisted of 1612 sites and represented 23 species of Clavulinopsis and two species of Mucronella as the outgroup. The ML and BI (not shown) phylogenies ( Figure 15) were extremely similar in topology and included two resolved clades (A and B) among the species of Clavulinopsis. The monophyly of clade A was strongly supported (PP = 1.00, BP = 100%) and included C. appalachiensis (PP = 1.00, BP = 100%), C. incarnata (PP = 1.00, BP = 100%), one unnamed Clavulinopsis species (PP = 1.00, BP = 100%), C. miyabeana, Figure 15. Phylogenetic relationships of Clavulinopsis species inferred from the combined dataset (ITS and nrLSU) using Bayesian posterior probabilities ≥ 0.90; ML Bootstrap values ≥ 50% are reported on the branches; the sign "-" means under the reported level. Six new species and one newly recorded species are shown in boldface text.

Discussion
In this study, six Clavulinopsis species new to science and one Clavulinopsis species newly recorded in China were identified. The specimens of Clavulinopsis trigonospora from Sichuan and Gansu, as a newly recorded species in China, conformed with the type specimen from Italy in molecular phylogenetic placement and morphological characteristics, and represent the first discovery of this species outside of Italy. Morphology and molecular data verified that the other six species collected were new species. The six new Clavulinopsis species are herein formally named and described in detail, and are illustrated with line drawings and photographs.

Discussion
In this study, six Clavulinopsis species new to science and one Clavulinopsis species newly recorded in China were identified. The specimens of Clavulinopsis trigonospora from Sichuan and Gansu, as a newly recorded species in China, conformed with the type speci-men from Italy in molecular phylogenetic placement and morphological characteristics, and represent the first discovery of this species outside of Italy. Morphology and molecular data verified that the other six species collected were new species. The six new Clavulinopsis species are herein formally named and described in detail, and are illustrated with line drawings and photographs.
In the process of conducting the phylogenetic analysis, we found that available sequences of Clavulinopsis species are still extremely limited in GenBank and many species are represented by only one or two accessions with sequences. After adding the newly generated sequences in this study, we obtained a more stable topology for Clavulinopsis, with two clades resolved, in comparison with the previous works by Birkebak et al. [6] and Hyde et al. [8]. However, the common synapomorphic traits for species within clade A and clade B currently remain unclear and, in any case, samples of additional taxa are needed for reconstruction of a taxonomically complete phylogeny.
Prior to the present study, no new Clavulinopsis species had been described in China. Most Clavulinopsis specimens previously collected in China were assigned to species originating in Europe, Oceania, or the Americas. Based on our field investigations and molecular analyses, it is apparent that the main difficulties hindering the classification of Clavulinopsis are the currently limited availability of molecular data and the paucity of reliable, taxonomically important morphological characters. The present study has provided additional molecular data, provided a key to the known Clavulinopsis species in China (Table A1) and enhanced knowledge of the biological diversity in Clavulinopsis. However, the diversity of the genus in China is much richer than expected and additional research is needed to further explore this diversity in the future.