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Research Article
Morphological characteristics and phylogenetic evidence reveal two new species and the first report of Comoclathris (Pleosporaceae, Pleosporales) on dicotyledonous plants from China
expand article infoRong Xu‡§, Wenxin Su§, Yang Wang|, Shangqing Tian§, Yu Li‡, Chayanard Phukhamsakda§
‡ Yangzhou University, Yangzhou, China
§ Jilin Agricultural University, Changchun, China
| Shenyang Agricultural University, Shenyang, China
¶ Mae Fah Luang University, Chiang Rai, Thailand

Corresponding author: Yu Li ( yuli966@126.com )

Corresponding author: Chayanard Phukhamsakda ( chayanard91@gmail.com )

Academic editor: Rungtiwa Phookamsak
© 2024 Rong Xu, Wenxin Su, Yang Wang, Shangqing Tian, Yu Li, Chayanard Phukhamsakda.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Xu R, Su W, Wang Y, Tian S, Li Y, Phukhamsakda C (2024) Morphological characteristics and phylogenetic evidence reveal two new species and the first report of Comoclathris (Pleosporaceae, Pleosporales) on dicotyledonous plants from China. MycoKeys 101: 95-112. https://doi.org/10.3897/mycokeys.101.113040
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Abstract

Two novel Comoclathris species were identified from dicotyledonous plants (Clematis sp. and Xanthoceras sorbifolium) in China. The results were supported by morphological characters and Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Multi-gene phylogenetic analyses of the ITS, LSU, SSU and rpb2 sequences revealed two new species Comoclathris clematidis and C. xanthoceratis, which are phylogenetically distinct. The new species are phylogenetically closely related to C. arrhenatheri. However, they are distinguishable from C. arrhenatheri by having comparatively larger asci and ascospores. This study improves our knowledge of Comoclathris as no species has been previously described from China. This suggests such taxa may be rare and it is likely that new taxa will be discovered from hosts and environments that have not yet been extensively investigated.

Key words

Ascomycota, Clematis, new species, saprobes, taxonomy, Xanthoceras sorbifolium

Introduction

Clements (1909) introduced the genus Comoclathris with C. lanata Clem as the type species. The species was originally assigned to the Diademaceae, based on having ascomata with flat circular lid-like opening (Shoemaker and Babcock 1992). Previously, Comoclathris was considered a synonym of Platyspora (Ariyawansa et al. 2014) and Comoclathris has been associated with an asexual morph resembling Alternaria-like (Simmons 1967); thus, the genus was temporarily referred to Pleosporaceae, based to these morphological characteristics (Zhang et al. 2012; Woudenberg et al. 2013). Two strains of Comoclathris compressa (CBS 157.53 and CBS 156.53) were treated as representative sequences which formed a well-supported clade within the family Pleosporaceae (Ariyawansa et al. 2014). Subsequently, Comoclathris was placed into Pleosporaceae, based on phylogenetic evidence coupled with morphological characteristics (Ariyawansa et al. 2015; Thambugala et al. 2017; Wijayawardene et al. 2017; Wanasinghe et al. 2018).

Comoclathris can be distinguished from Pleospora, Pleoseptum and Clathrospora by its applanate and dark reddish-brown muriform ascospores with a single longitudinal septum and ascomata with circular lid-like opening (versus two or more rows of longitudinal septa of Clathrospora species) (Shoemaker and Babcock 1992; Zhang et al. 2012; Ariyawansa et al. 2014, 2015). Thirty-eight epithets have been recorded as Comoclathris in Species Fungorum (2023); however, most lack molecular data, including the type species C. lanata. Comoclathris has been found from America, Antarctica, Argentina, Austria, Bulgaria, Canada, Central Asia, Finland, Greece, India, Iran, Iraq, Italy, Netherlands, Norway, Pakistan, Portugal, Romania, Russia, Spain, Sweden, Switzerland, Syria, Tunisia, Turkey, Ukraine and Yugoslavia (Ahmad 1978; Shoemaker and Babcock 1992; Chlebicki 2002; Checa 2004; Pande 2008; Woudenberg et al. 2013; Eriksson 2014; Thambugala et al. 2017; Hongsanan et al. 2020). Most Comoclathris species are saprobes, with recent reports from Italy (Hyde et al. 2016; Wanasinghe et al. 2018; Brahmanage et al. 2020).

The aim of this study was to explore the diversity of Comoclathris species from dicotyledonous plants in China. Two new Comoclathris species (C. clematidis and C. xanthoceratis) from Jilin and Yunnan Provinces, China are described. The morphology was compared to other Comoclathris species. Maximum Likelihood and Bayesian Inference phylogenetic analyses were performed to confirm the taxonomic position of the isolates using ITS, LSU, SSU and rpb2 datasets. The results improve our understanding of the occurrence and distribution of Comoclathris species from China, thus expanding the knowledge of fungal biodiversity. This is also the first report of Comoclathris on dicotyledonous plants in China.

Materials and methods

Sample collection, morphological study and isolation

Dried wood samples were collected from Jilin (Temperate zone, 43°10′N, 124°20′E) and Yunnan Provinces (Subtropical region, 25°23′N, 102°42′E) in China. The samples were transferred to the laboratory in plastic bags with labels indicating the details of the collection. The characteristics of specimens were observed using a Zeiss Stemi 2000C stereomicroscope, equipped with a Leica DFC450C digital camera (Leica, Germany). Morphological characteristics of ascomata (n = 5), peridium (n = 10), hamathecium (n = 20), asci (n = 20), ascospores (n = 40) and other microscopic characteristics associated with ascomata were documented using a Zeiss AX10 microscope, equipped with an Axiocam 506 digital camera (ZEISS, Germany). The ZEN 3.4 application (blue edition) was used for microscopic measurements (ZEISS, Germany). The photos were edited using Adobe Photoshop CC2020 (Adobe Systems, USA).

Single spore isolation was used to obtain pure cultures (Senanayake et al. 2020) and germinated spores were cultured at 25 °C on potato dextrose agar (PDA). Type specimens were deposited in the Herbarium of Mycology, Jilin Agricultural University (HMJAU), Changchun, China and isotypes were deposited in Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand. Ex-type cultures were deposited in the International Cooperation Research Center of China for New Germplasm Breeding of Edible Mushrooms Culture Collection (CCMJ). The new taxa were registered in MycoBank (Crous et al. 2004).

DNA extraction, PCR amplification and sequencing

Pure mycelia were harvested after two weeks of incubation at 25 °C on PDA. The internal transcribed spacer regions (ITS), large subunit (LSU), small subunit (SSU) and RNA polymerase II second-largest subunit (rpb2) were amplified by polymerase chain reaction (PCR) using ITS5/ITS4, NS1/NS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999) primers, respectively. The amplification reactions and conditions for ITS, LSU and SSU were performed using the conditions described by Xu et al. (2022). The amplification conditions for rpb2 annealing conditions were different: 94 °C for 5 min, then 35 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 45 s, elongation at 72 °C for 90 s and a final extension at 72 °C for 10 min. The amplification reactions were performed using 20 μl PCR mixtures containing 9 μl ddH2O, 10 μl of 2× EsTaq MasterMix (Dye), 0.4 μl (200 ng/µl) of DNA template and 0.3 μl of 2 μmol/μl of forward and reverse primers. The PCR products were verified on 1% agarose electrophoresis gels stained with 0.5 ml of 10,000X standard DNA dye (Biotium, United States). Purification and sequencing of amplified PCR fragments were performed by Sangon Biotech Co, Shanghai, China.

Sequencing and sequence alignment

Sequences obtained from this study were searched in the GenBank database (http://blast.ncbi.nlm.nih.gov/) using BLAST. The newly-obtained sequences and data from recent publications (Brahmanage et al. 2020; Crous et al. 2021) were used in the analysis (Table 1). Neocamarosporium betae (CBS 523.66) and N. calvescens (CBS 246.79) were used as the outgroup in the phylogenetic analyses. The sequences were edited using BioEdit v. 7.1.3.0 and aligned with MAFFT v. 7 (Hall 1999; Katoh and Standley 2013). The alignments were trimmed using trimAI v. 1.2 under the gappyout option (Capella-Gutierrez et al. 2009). The datasets were combined using SequenceMatrix v. 1.7.8 (Vaidya et al. 2011). The newly-generated sequence data were deposited in GenBank (Benson et al. 2013).

Table 1.
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Taxa used in the phylogenetic analyses and their corresponding GenBank accession numbers. The ex-type strains are indicated in bold and the newly-generated sequences are shown in cells with light grey shading.

Taxa Strain Host/Substrate Country GenBank accession numbers References
ITS LSU SSU rpb2
Comoclathris ambigua CBS 366.52 – USA KY940748 AY787937 – KT216533 (Woudenberg et al. 2017)
C. antarctica WA0000074564 Soil Antarctica MW040594 MW040597 – – (Crous et al. 2021)
C. arrhenatheri MFLUCC 15-0465 Arrhenatherum elatius Italy KX965737 KY000647 KX986348 KX938346 (Thambugala et al. 2017)
C. arrhenatheri MFLUCC 15-0476 Dactylis glomerata Italy KY026595 KY000648 KX986349
C. clematidis CCMJ 13076 Clematis sp. China OQ534243 OQ534239 OQ676454 OQ547800 This study
C. clematidis CCMJ 13077 Clematis sp. China OQ534244 OQ534240 OQ676455 OQ547801
C. compressa CBS 156.53 Castilleja miniata USA – KC584372 KC584630 KC584497 (Woudenberg et al. 2013)
C. compressa CBS 157.53 – USA – MH868679 KC584631 KC584498
europaeae MFLU 20-0391 – Italy MT370396 MT370421 MT370367 MT729650 (Brahmanage et al. 2020)
C. flammulae MFLU 20-0397 Clematis flammula Italy MT370397 MT370422 MT370368 MT729651
C. flammulae MFLU 20-0399 Colutea arborescens Italy MT370395 MT370420 MT370366 –
C. galatellae MFLUCC 18-0773 Galatella villosa Ukraine MN632549 MN632550 MN632551 – (Hongsanan et al. 2020)
C. incompta CBS 467.76 Olea europaea Greece – GU238087 GU238220 KC584504 (Aveskamp et al. 2010)
C. incompta CH-16 Olea europaea Tunisia KU973716 KU973729 – – (Moral et al. 2017)
C. italica MFLUCC 15-0073 Thalictrum sp. Italy KX500109 – – – (Tibpromma et al. 2015,)
C. lini MFLUCC 14-0968 Linum sp. Italy KR049218 KR049219 KT210389 – (Nwanasinghe et al. 2015)
C. lini MFLUCC 14-0561 Ononis spinosa Italy KT591614 KT591615 KT591616 –
C. lonicerae MFLU 20-0385 Lonicera sp. Italy MT370394 MT370419 MT370365 MT729649 (Brahmanage et al. 2020)
C. lonicerae MFLU 18-1236 Colutea arborescens Italy OL744429 OL744433 OL744435 OL771441
C. permunda MFLUCC 14-0974 Phleum sp. Italy KY659561 KY659564 KY659568 – (Vu et al. 2019)
C. pimpinellae MFLUCC 14-1159 Pimpinella tragium Russia KU987665 KU987666 KU987667 – (Li et al. 2016)
C. rosae MFLU 15-0203 Rosa canina Italy MG828876 MG828992 MG829103 MG829249 (Wanasinghe et al. 2018)
C. rosae MFLU 16-0234 Rosa canina Italy MG828877 MG828993 MG829104 MG829250
C. rosarum MFLUCC 14-0962 Rosa canina Italy MG828878 MG828994 MG829105 MG829251
C. rosigena MFLU 16-0229 Rosa canina Italy MG828879 MG828995 MG829106 MG829252
C. sedi MFLUCC 13-0763 Rosa sp. Italy KP334717 KP334707 KP334727 – (Ariyawansa,et al. 2014)
C. sedi MFLUCC 13-0817 Sedum sp. Italy KP334715 KP334705 KP334725 –
C. spartii MFLUCC 13-0214 Spartium junceum Italy KM577159 KM577160 KM577161 – (Cours et al. 2014)
C. typhicola CBS 602.72 – Netherlands MH860592 MH872288 – – (Vu et al. 2019)
C. xanthoceratis CCMJ 13078 Xanthoceras sorbifolium China OQ534245 OQ534241 OQ676456 OQ547802 This study
C. xanthoceratis CCMJ 13079 Xanthoceras sorbifolium China OQ534246 OQ534242 OQ676457 OQ547803
Neocamarosporium betae CBS 523.66 Beta vulgaris Netherlands FJ426981 MH870520 EU754080 KT389670 (Aveskamp et al. 2009)
N. calvescens CBS 246.79 Atriplex calotheca Germany MH861203 EU754131 EU754032 KC584500 (Vu et al. 2019)

Phylogenetic analysis

The phylogenetic analyses were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. RAxML-HPC2 on XSEDE, implemented in the CIPRES web portal (http://www.phylo.org/portal2/), was used for ML analysis, with a rapid bootstrapping algorithm of 1000 replicates (Stamatakis 2014). The suitability of the DNA model was analysed using jModelTest v. 2.1.10 on the CIPRES online portal for posterior probability. The best fit evolutionary models for individual and combined datasets were calculated under the Akaike Information Criterion (AIC) (Nylander 2004) and are as follows: GTR+I+G model for the ITS alignment, K80+I model for the LSU and SSU alignments, GTR+G model for the rpb2 alignment and SYM+I+G model for the combined datasets. Bayesian Inference analyses were carried out by using MrBayes v. 3.2.6 on the CIPRES web platform (Ronquist and Huelsenbeck 2003). Tree samples were taken every 1000th generation while Markov chains were run for 15,000,000 generations. Phylogenetic trees were illustrated in FigTree v. 1.4.4 (Rambaut 2018) and altered in Adobe Illustrator CS v. 6. RAxML bootstrap support values greater than or equal to 98% and Bayesian posterior probabilities equal to 1.00 were considered as strong statistical support. The data used in this study were deposited in the Zenodo repository (accession number doi: 10.5281/zenodo.7675986).

Results

Phylogenetic analyses

The combined multi-loci (ITS, LSU, SSU and rpb2) sequence dataset consisted of 32 taxa and 3,280 characters including gaps (ITS: 1–559 bp, LSU: 560–1,441 bp, SSU: 1,442–2,415 bp and rpb2: 2,416–3,280 bp). The best-scoring RAxML tree had a final log-likelihood value of -10805.548630. There were 691 distinct alignment patterns with 26.80% undetermined characters or gaps in the matrix. Estimated base frequencies were as follows: A = 0.254018, C = 0.226589, G = 0.268862, T = 0.250530; substitution rates AC = 2.459854, AG = 4.593060, AT = 1.418628, CG = 1.005203, CT = 7.378387 and GT = 1.000000. The proportion of invariable sites (I) was estimated to be 0.690973 and the gamma distribution shape parameter (α) was estimated to be 0.927322. A total of 4,592 trees were sampled in the BI analysis after the 20% burn-in with a stop value of 0.009967. The ML and BI trees were similar in topology (Fig. 1). Phylogenetic results demonstrated that Comoclathris clematidis and C. xanthoceratis formed a distinct lineage and clustered with C. arrhenatheri with strong statistical support (98% ML and 1.00 BPP). Comoclathris clematidis (CCMJ13076 and CCMJ 13077) and C. xanthoceratis (CCMJ 13078 and CCMJ 13079) formed a closely-related clade with high statistical support (100% ML and 1.00 BPP).

Figure 1. 

The Bayesian 50% majority-rule consensus phylogram, based on a concatenated ITS, LSU, SSU and rpb2 dataset of Comoclathris. The tree is rooted with Neocamarosporium betae (CBS 523.66) and N. calvescens (CBS 246.79). RAxML bootstrap support values ≥ 70% (ML, left) and Bayesian posterior probabilities ≥ 0.90 (BPP, right) are shown near the nodes. The new isolates are indicated in orange. The type strains are in bold and labelled with T.

Taxonomy

Comoclathris clematidis R. Xu, Phukhams. & Y. Li, sp. nov.

MycoBank No: 847614
Fig. 2

Etymology

Refers to the host genus, Clematis.

Description

Saprobic on dried branches of Clematis species. Sexual morph: Ascomata 150–230 × 120–150 μm (x– = 176 × 138 μm, n = 5), solitary, scattered or aggregated in small groups, immersed to erumpent, subglobose, elongated, black, without a distinct ostiole. Peridium 10–20 μm wide at the base, 15–20 μm wide at the sides, comprising thick-walled cells of textura angularis, dark brown to black. Hamathecium comprising numerous, 1–3.5 μm wide (x–= 2.0 μm, n = 20), filamentous, septate, rarely branched pseudoparaphyses, hyaline, embedded in a gelatinous matrix, extending above the asci. Asci 114–174 × 27–43 μm (x– = 140 × 34 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical-clavate, short pedicellate, apically rounded, with an ocular chamber. Ascospores 22–39 × 8–21 μm (x– = 30 × 14 μm, n = 40), 1–2-seriate, partially overlapping, broadly fusiform, initially 3-septate and yellowish, becoming brown, verrucose or echinulate wall, muriform, with 3 transversely septa and a vertical septum in second and third cells, constricted at the septa, with obtuse ends, smooth-walled, surrounded by a thick mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics

Colonies on PDA reaching 40 mm diam. after three weeks at 25 °C. Cultures from above, circular, flat to umbonate, covered with flocculent aerial mycelium, velvety on the surface, greenish-olivaceous, dense, entire edge; reverse black in the middle, green olivaceous radiating outwardly, white mycelium at the edge.

Figure 2. 

Comoclathris clematidis (HMJAU 64844, holotype) A, B appearance of ascomata on host substrate C vertical section of ascoma D peridium E pseudoparaphyses F–H asci I–O ascospores P, Q culture characteristics on PDA after three weeks at 25 °C. Scale bars: 200 µm (B); 50 µm (C); 20 µm (E–H, O); 10 µm (D, I–N).

Material examined

China. Yunnan Province, Kunming, on the dead aerial branch of Clematis sp. (Ranunculaceae), 24 April 2021, S. Tibpromma, S42, HMJAU 64844 (holotype); ex-type, CCMJ 13076; MFLU 23-0384 (isotype), ex-isotype, CCMJ 13077.

Notes

In the phylogenetic analyses, Comoclathris clematidis (CCMJ 13076 and CCMJ 13077) clustered with C. xanthoceratis (CCMJ 13078 and CCMJ 13079) with 82% ML and 100 BPP within Comoclathris (Fig. 1). Comoclathris clematidis was found on dried stems of Clematis species in the subtropical zone of Yunnan Province, China. The majority of Comoclathris species are found in temperate regions, but only C. incompta (CH-16) has been identified in subtropical regions (Moral et al. 2017). Comoclathris clematidis differs from C. flammulae which was also found on Clematis by its larger asci (114–174 × 27–43 µm vs. 50–55 × 13–17 µm) and larger ascospores (22–39 × 8–21 µm vs. 16–22 × 10–16 µm). In addition, C. clematidis contains fewer transverse septa in ascospores (3 transverse septa vs. 6 transverse septa) (Brahmanage et al. 2020). The new species Comoclathris clematidis is distinguishable from Comoclathris sedi which was also isolated from Clematis by having larger asci (114–174 × 27–43 µm vs. 80–110 × 16–18 µm), larger ascospores (22–39 × 8–21 µm vs. 19–20 × 8–10 µm) and fewer ascospore septa (3 transverse septa vs. 4–5 transverse septa) (Ariyawansa et al. 2015). The ascomata of C. clematidis are immersed to superficial and appear as black spots or convex surfaces, while the ascomata of C. xanthoceratis are immersed to semi-immersed and covered with dark brown setae. Comoclathris clematidis has cylindrical-clavate asci and verrucose or echinulate ascospore walls, while C. xanthoceratis has clavate asci and smooth-walled ascospores. Both C. clematidis and C. xanthoceratis have ascospores with 3 transverse septa and 2 vertical septa. In addition, the two species show different culture characteristics and only C. xanthoceratis produce ascocarps in the culture. The ITS and rpb2 base pair differences between the two species are 0.95% (5/526, no gaps) and 4.69% (34/725, no gaps), respectively.

In the BLASTn search, the rpb2 sequence was 89.53% similar to Comoclathris arrhenatheri (MFLUCC 15-0465) with 100% query cover, translating to 89.53% similarity. The LSU sequence was 98.76% similar to C. permunda (CBS: 127967) with 99% query cover, translating to 97.77% similarity, while the SSU sequence was 98.58% similar to C. lini (MFLUCC 14-0968) with 100% query cover, translating to 98.58% similarity. The ITS region was 97.93% similar to Comoclathris sp. (14APR) with 93% query cover, translating to 91.07% similarity. Therefore, Comoclathris clematidis was introduced as a novel species.

Comoclathris xanthoceratis R. Xu, Phukhams. & Y. Li, sp. nov.

MycoBank No: 847615
Fig. 3

Etymology

Refers to the host genus, Xanthoceras.

Description

Saprobic on dried stems of Xanthoceras sorbifolium. Sexual morph: Ascomata solitary, scattered or aggregated in small groups, 147–221 × 114–130 μm (x– = 187–124 μm, n = 5), immersed to semi-immersed, subglobose, black, elongated, covered with dark brown setae, without a distinct ostiole. Peridium 13–20 μm wide at the base, 20–32 μm wide at the sides, comprising thick-walled cells of textura angularis, dark brown to black; inner layer composed of thin-walled cells of textura angularis, hyaline. Hamathecium comprising 1.5–4.0 μm wide, septate, filiform, embedded in a gelatinous matrix, rarely branched pseudoparaphyses, extending above the asci. Asci 8-spored, bitunicate, fissitunicate, 99–165 × 36–48 μm (x– = 127 × 42 μm, n = 20), clavate, short pedicellate, apically rounded, with an ocular chamber. Ascospores 23–42 × 9–19 μm (x– = 37 × 16 μm, n = 40), 1–2-seriate, muriform, broadly fusiform, with 3 transverse septa and a vertical septum in second and third cells, brown to dark brown, with obtuse ends, smooth-walled, surrounded by a thick mucilaginous sheath. Asexual morph: Undetermined.

Culture characteristics

Colonies on PDA reaching 30 mm diam. after three weeks at 25 °C. Cultures from above, dense, round, umbonate, wrinkled and folded, papillate with white aerial mycelium, radial edge, orange at the margin; reverse reddish, white mycelium present at the margin.

Material examined

China. Jilin Province, Changchun, on dead stem of Xanthoceras sorbifolium Bunge (Sapindaceae), 2 July 2022, Rong Xu, XR71, HMJAU 64846 (holotype); ex-type, CCMJ 13078; MFLU 23-0385 (isotype), ex-isotype, CCMJ 13079.

Notes

Comoclathris xanthoceratis (CCMJ 13078 and CCMJ 13079) is closely related to C. clematidis (CCMJ 13076 and CCMJ 13077) (100% ML and 1.00 BPP). The two species are phylogenetically closely related to C. arrhenatheri (MFLUCC 15-0465). However, there are distinct differences in morphology (Thambugala et al. 2017). The asci of C. arrhenatheri are smaller than C. clematidis and C. xanthoceratis (C. arrhenatheri vs. C. clematidis vs. C. xanthoceratis: 70–95 × 18.5–25 vs. 114–174 × 27–43 vs. 99–165 × 36–48 μm, respectively). Comoclathris arrhenatheri have ascospores with 4 transverse septa and 2–3 vertical septa, while C. clematidis and C. xanthoceratis only have 3 transverse septa and 2 vertical septa. Additionally, the ascospores of C. arrhenatheri are shorter than C. clematidis and C. xanthoceratis (C. arrhenatheri vs. C. clematidis vs. C. xanthoceratis: 16.5–22 × 7.7–10.2 vs. 22–39 × 8–21 vs. 23–42 × 9–19 µm).

A pairwise comparison of the ITS region between C. xanthoceratis and C. arrhenatheri demonstrated 8.95% (46/514, no gaps) base-pairs difference, while there were 74 base-pair difference in the rpb2 gene (10.2%, no gaps). Hence, C. xanthoceratis is introduced as a new species, based on morphological and nucleotide differences. This is also the first report of Comoclathris species found on Xanthoceras sorbifolium.

Figure 3. 

Comoclathris xanthoceratis (HMJAU 64846, holotype) A–C appearance of ascomata on host substrate D vertical section of ascoma E peridium F pseudoparaphyses G–J asci (H, I) asci production from the sterile condition) K–Q ascospores R culture characteristics on PDA after three weeks at 25 °C (black dots indicate the sexual reproduction in culture condition). Scale bars: 200 µm (C); 50 µm (D); 20 µm (G–J); 10 µm (E, K–Q); 5 µm (F).

Discussion

In this study, we described two new Comoclathris species from China, based on morphological and multi-locus phylogenic analyses. The identifying morphological features of Comoclathris include operculate perithecia and muriform, asymmetrical, strongly divided ascospores (Shoemaker and Babcock 1992; Wanasinghe et al. 2018). Phylogenetic analyses, based on four combined loci (ITS, LSU, SSU and rpb2), as well as morphological characters, are important for the identification of Comoclathris species (Table 2). The phylogeny presented here is similar to previous studies (Thambugala et al. 2017; Wijayawardene et al. 2017; Wanasinghe et al. 2018; Brahmanage et al. 2020; Hongsanan et al. 2020), demonstrating a robust backbone tree in this study.

Table 2.
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Synopsis of Comoclathris species with the newly-introduced species in bold.

Taxa Sexual Morph Asexual morph Reference
Ascomata Asci Ascospores Conidiomata Conidia
Comoclathris antarctica 339 (± 103) × 299 (± 97) µm, separate or in groups, dark brown to almost black, strongly enclosed in aerial hyphae, ovoid to spherical, without distinct ostiole, neck very short, operculum semi-spherical, flattened; perithecial hyphae dark; wall of 2–3 cell layers. 72–84 × 18–26 µm, mostly 8-spored, immature asci shorter (~ 60 µm), cylindrical to clavate, bitunicate with a rounded apex. 31 × 13.5 µm, lanceolate to ovoid, clavate, yellow to pale brown, elongated, asymmetrical with a blunt apex, muriform, with 6–8 transvers septa, apical cell not divided. Undetermined (Crous et al. 2021)
C. arrhenatheri 100–150 × 80–120 μm, solitary, scattered or aggregated in small groups, immersed to erumpent, black, elongate, subglobose, covered with pale to dark brown setae, without a distinct ostiole. 65–95 × 18.5–25 μm, 8-spored, cylindrical-clavate, short pedicellate, apically rounded, with an ocular chamber. 16.5–22 × 7.7–10.2 μm, 1–2 seriate, partially overlapping initially yellowish, 1-septate, becoming yellow to pale brown and muriform, with 4 transverse septa and 2–3 vertical septa. Undetermined (Thambugala et al. 2017)
C. clematidis 150–230 × 120–150 μm solitary, scattered or aggregated in small groups, immersed to erumpent, subglobose, elongated, black, without a distinct ostiole. 114–174 × 27–43 μm, 8-spored, cylindrical-clavate, short pedicellate, apically rounded, with an ocular chamber. 22–39 × 8–21 μm, 1–2 seriate, partially overlapping, broadly fusiform, initially 3 septate and yellowish, becoming brown, muriform, with 3 transversely septa and a vertical septum, with a thick mucilaginous sheath. Undetermined This study
C. compressa 200–520 × 150–320 µm, scattered, immersed, sub-epidermal, later superficial, depressed globose, with smooth, straight to bent, tapered, brown hairs. 80–120 × 20–30 µm, numerous, saccate, with tetraserlate to biseriate spores. 24–29 × 10–14 μm, fusoid, straight, transversely 3-septate, with 1 longitudinal septum in central cells, dark reddish-brown, with guttules, smooth, with a uniform sheath 2–3 μm wide. Undetermined (Shoemaker et al. 1992)
C. europaeae 240–250 × 145–165 µm, solitary, scattered, semi-immersed to slightly erumpent, dark brown to black, globose to subglobose, without a distinct ostiole. 60–70 × 15–18 µm, 8-spored, cylindrical-clavate, pedicellate, apex rounded, with an indistinct ocular chamber. 20–22 × 11–13 µm, uni-to biseriate, partially overlapping, muriform, brown, transversely septate or muriform, with 7 transverse septa. Undetermined (Brahmanage et al. 2020)
C. flammulae 105–130 × 80–90 µm, solitary or aggregated, immersed, globose to subglobose, dark brown to black, without a distinct ostiole. 50–55 × 13–17 µm, 8-spored, cylindrical-clavate, short pedicellate, rounded at the apex, with an indistinct ocular chamber. 16–22 × 10–16 µm, overlapping uni-to biseriate, yellowish-brown when immature, becoming dark brown at maturity, clavate, with acute ends, muriform, with 6 transverse septa, 1–2 longitudinal septa. Undetermined (Brahmanage et al. 2020)
C. galatellae 200–550 × 230–340 μm, immersed, erumpent to superficial, broadly to narrowly oblong and flattened, ostiolate. 50–90 × 14–17 µm, 8-spored, cylindrical to clavate, with furcate pedicel and minute ocular chamber. 20–30 × 6–8 µm, uni-seriate or partially overlapping, mostly ellipsoidal, brown or pale brown, muriform, 2–4 transverse septa, 1–2 longitudinal septa, without sheath. None 2–4 × 1–2 µm, oval to ellipsoid, hyaline, aseptate, guttulate. (Hongsanan et al. 2020)
C. italica 180–240 × 200–250 µm, semi-immersed to erumpent, solitary, scattered, broadly oblong to flattened, dark brown to black, coriaceous, cupulate when dry. 100–120 × 30–35 µm, 8-spored, clavate, short pedicellate, thick-walled at the apex, with a minute ocular chamber. 30–35 × 10–15 µm, overlapping 1–3 seriate, initially 1 septate and hyaline, becoming brown at maturity, muriform, mostly ellipsoidal, 6–8 transversely septate, with 1–2 vertical septa. Undetermined (Thambugala et al. 2017)
C. lini 260–290 × 300–350 µm, superficial, solitary, scattered, broadly oblong and flattened, dark brown to black, coriaceous, cupulate when dry, ostiolate. 110–130 × 15–25 µm, 8-spored, cylindrical to cylindrical-clavate, pedicellate, thick walled at the apex, with a minute ocular chamber. 20–25 × 10–12 µm, overlapping, initially hyaline, becoming brown at maturity; mostly ellipsoidal, with upper part widest, muriform, with 4–6 transverse septa and 4–6 vertical septa. Undetermined (Wanasinghe et al. 2015)
C. lonicerae 370–485 × 255–360 µm, solitary or aggregated, scattered, semi-immersed to erumpent, globose to subglobose, dark brown to black, without a distinct ostiole. 180–192 × 60–74 µm, 8-spored, broadly cylindrical to cylindrical clavate, short pedicellate, rounded at the apex, with an indistinct, shallow ocular chamber. 55–70 × 20–30 µm, overlapping uni or biseriate, yellowish-brown, transversely septate or muriform, with 3–5 transverse septa, 1–2 longitudinal septa, with rounded ends. Undetermined (Brahmanage et al. 2020)
C. permunda 150–200 × 150–200 µm, semi-immersed to erumpent, solitary, scattered, broadly oblong to flattened, dark brown to black, coriaceous, cupulate when dry, with brown to reddishbrown, setae. 90–110 × 19–22 µm, 8-spored, cylindrical-clavate, with a 20–30 µm long pedicel, thick-walled at the apex, with a minute ocular chamber. 22–28 × 9–12 µm, overlapping 1–2-seriate,
muriform, mostly ellipsoidal, 2–4 transversely septate, with 1–2 vertical septa, initially hyaline, becoming golden brown at maturity, surrounded by a thick, hyaline, mucilaginous sheath.		 Undetermined (Thambugala et al. 2017)
C. pimpinellae 155–135 × 88–95 µm, solitary or aggregated, semi-immersed or rarely somewhat superficial, globose to subglobose, dark brown to black. 58–75 × 14–16 µm, 8-spored, cylindrical-clavate, short-pedicellate, rounded at the apex, with indistinct, shallow, ocular chamber. 14–16 × 5–8 µm, overlapping biseriate, yellow to light brown, transversely septate or muriform, with 3 transverse septa, central segments with 2 longitudinal septa, end segments with 2 angular septa, surrounded by a thick, hyaline, a mucilaginous sheath. Undetermined (Li et al. 2016)
C. rosae 120–150 × 175–200 µm diam., immersed to erumpent, globose or subglobose, dark brown to black, coriaceous. 70–110 × 15–30 µm, 8-spored, cylindrical-clavate to clavate, pedicellate, thick-walled at the apex, with minute ocular chamber. 20–30 × 8–15 µm, overlapping 1–2 seriate, mostly ellipsoidal, muriform, 4–7 transversely septate, with 1–2 vertical septa, conically rounded at both ends. Undetermined (Wanasinghe et al. 2018)
C. rosarum 200–300 × 300–400 µm diam., immersed to erumpent, globose or subglobose, dark brown to black, coriaceous. 150–200 × 35–50 µm, 8-spored, clavate, pedicellate, thick-walled at the apex, with minute ocular chamber. 40–60 × 20–25 µm, overlapping 1–2 seriate, mostly ellipsoidal, muriform, 6–7 transversely septate, with 2–4 vertical septa, deeply constricted at the middle septum. Undetermined (Wanasinghe et al. 2018)
C. rosigena 180–220 × 300–400 µm, immersed to erumpent, globose or subglobose, dark brown to black, coriaceous. 150–180 × 45–60 µm, 8-spored, cylindrical-clavate to clavate, pedicellate, thick-walled at the apex, with minute ocular chamber. 40–60 × 16–24 µm, overlapping biseriate, mostly ellipsoidal, muriform, 5–7 transversely septate, with 1 vertical septum, slightly constricted at the middle septum. Undetermined (Wanasinghe et al. 2018)
C. sedi 200–250 × 290–350 μm, scattered or aggregated on the host stem, subglobose or nearly globose, superficial, coriaceous, brown to blackish-brown with a blunt ostiole. 80–110 × 16–18 μm, 8-spored, cylindrical to cylindrical-clavate, with a short knob–like pedicel and indistinct shallow ocular chamber. 19–20 × 8–10 μm, 1–2 overlapping seriate, fusiform, muriform, with 4–5 transverse septa and 1–2 longitudinal septa, not constricted at the septa. Undetermined (Ariyawansa et al. 2015)
C. spartii Up to 200 μm diam., solitary, scattered or aggregated in small groups, immersed in host tissue, dark brown to black, globose to subglobose, without a distinct ostiole. 100–180 × 23–28 μm, 8-spored cylindrical-clavate, stipitate, apex rounded, with a small apical chamber. 25–34 × 9–14.5 μm, uni- to biseriate in asci, muriform, yellow to pale brown, broadly fusiform, with obtuse ends, constricted at the primary septum, surrounded by a mucilaginous sheath. Undetermined (Crous et al. 2014)
C. typhicola 350–400 µm diam. Ostiole 100–125 µm diam. 100–125 × 25–30 µm, numerous, clavate, hyaline. 45–50 × 10–12.5 µm, muriform, oval to cylindrical, straight, rounded at one end, slightly tapered at the other, hyaline when immature, light yellow to yellow. Undetermined (Adamska et al. 2012)
C. xanthoceratis 147–221 × 114–130 µm, solitary, scattered or aggregated in small groups, , immersed to semi-immersed, subglobose, black, elongated, covered with dark brown setae, without a distinct ostiole. 99–165 × 36–48 μm, 8-spored, bitunicate, fissitunicate, clavate, short pedicellate, apically rounded, with an ocular chamber. 23–42 × 9–19 μm, 1–2 seriate, muriform, broadly fusiform, with 3 transverse septa and a vertical septum in second and third cells, brown to dark brown, with obtuse ends, smooth-walled, with a thick mucilaginous sheath. Undetermined This study

In the phylogenetic analyses, many species appear to be conspecific and their phylogenetic placement remains to be resolved. It implied that the concept of subdivision, based on molecular phylogeny alone, has been inaccurate. For example, Wanasinghe et al. (2015) introduced C. lini as a new species, although C. lini grouped in a well-supported clade with C. sedi (100% ML and 1.00 BPP). Comoclathris lini is different from C. sedi in having comparatively larger asci and different ascospore septa (4–6 transverse septa, 4–6 longitudinal septa vs. 4–5 transverse septa, 1–2 longitudinal septa). In our study, the base pair differences amongst ITS, LSU and rpb2 of Comoclathris clematidis and C. xanthoceratis were 0.95% (5/526, no gaps), 0.12% (1/803, no gaps) and 4.69% (34/725, no gaps), respectively. There were no differences in the SSU sequences between the two species. The rpb2 can be used as an effective barcode to distinguish Comoclathris species including C. clematidis and C. xanthoceratis as it is phylogenetically informative and reflects interspecific relationships (Woudenberg et al. 2013; Ariyawansa et al. 2015; Thambugala et al. 2017; Wanasinghe et al. 2018; Brahmanage et al. 2020). Thus, we recommend using morphological characters coupled with molecular phylogeny to delineate Comoclathris, especially including rpb2 marker as a protein-coding locus.

The host specificity of Comoclathris remains unclear. A single Comoclathris species can be found colonising more than one host, while various Comoclathris species have also been associated with the same host (Ariyawansa et al. 2015; Thambugala et al. 2017; Wanasinghe et al. 2018; Brahmanage et al. 2020). For example, C. flammulae and C. lonicerae were found on Colutea arborescens (Brahmanage et al. 2020), while C. rosae, C. rosarum and C. rosigena were found on Rosa canina (Wanasinghe et al. 2018). Some Comoclathris species have been associated with different hosts. Comoclathris arrhenatheri was collected from Arrhenatherum elatius and Dactylis glomerata (Thambugala et al. 2017, Italy), while C. flammulae was collected from Clematis flammula and Colutea arborescens in Italy (Brahmanage et al. 2020).

Comoclathris members are mostly distributed in the temperate areas (i.e. Greece, Italy, Netherlands, Russia, Ukraine and USA), while only C. incompta (CH-16) and C. antarctica (WA0000074564) have been reported in the subtropical and Arctic zones, respectively (Moral et al. 2017; Crous et al. 2021). In this study, C. clematidis (CCMJ 13076 and CCMJ 13077) was collected from Clematis species (Ranunculaceae) in Kunming City, which is located in the subtropical region. Comoclathris xanthoceratis (CCMJ 13078 and CCMJ 13079) was isolated from Xanthoceras sorbifolium (Sapindaceae) in Changchun, Jilin Province (temperate zone), which is consistent with many previous studies (Woudenberg et al. 2013; Ariyawansa et al. 2015; Hyde et al. 2016; Li et al. 2016; Thambugala et al. 2017; Wijayawardene et al. 2017; Wanasinghe et al. 2018; Brahmanage et al. 2020; Hongsanan et al. 2020). This study also extends the knowledge of the host range and geographic distribution of Comoclathris species.

Acknowledgements

We thank the Herbarium of Mae Fah Laung University for preservation of fungi specimens.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was financially supported by National Natural Science Foundation of China (NSFC) for granting a Youth Science Fund Project (number 32100007) and the Program of Creation and Utilization of Germplasm of Mushroom Crop of “111” Project (No. D17014).

Author contributions

Data curation, Rong Xu and Wengxin Su; Formal analysis, Shangqing Tian; Funding acquisition, Chayanard Phukhamsakda and Yu Li; Investigation, Rong Xu and Wengxin Su; Project administration, Chayanard Phukhamsakda and Yu Li; Software, Yang Wang; Supervision, Chayanard Phukhamsakda; Writing – original draft, Rong Xu; Writing – review and editing: Chayanard Phukhamsakda and Yu Li. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Rong Xu https://orcid.org/0000-0002-7744-6321

Wenxin Su https://orcid.org/0000-0002-5470-5853

Yang Wang https://orcid.org/0000-0002-5899-3987

Shangqing Tian https://orcid.org/0000-0003-4758-3023

Yu Li https://orcid.org/0000-0003-4966-701X

Chayanard Phukhamsakda https://orcid.org/0000-0002-1033-937X

Data availability

All of the data that support the findings of this study are available in the main text or supplementary information.

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Supplementary material

Supplementary material 1 

Phylogram generated from maximum likelihood analysis based on combined ITS, LSU, SSU, and rpb2 sequnence data

Rong Xu, Wenxin Su, Yang Wang, Shangqing Tian, Yu Li, Chayanard Phukhamsakda

Data type: pdf

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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