Front. Microbiol. Frontiers in Microbiology Front. Microbiol. 1664-302X Frontiers Media S.A. 10.3389/fmicb.2023.1117753 Microbiology Original Research Phylogeny and systematics of the genus Clonostachys Wang Yao 1 2 Tang De-Xiang 1 2 Luo Run 1 2 Wang Yuan-Bing 1 2 Thanarut Chinnapan 3 Dao Van-Minh 4 Yu Hong 1 2 * 1Yunnan Herbal Laboratory, College of Ecology and Environmental Sciences, Yunnan University, Kunming, China 2The International Joint Research Center for Sustainable Utilization of Cordyceps Bioresources in China and Southeast Asia, Yunnan University, Kunming, China 3Faculty of Agricultural Production, Maejo University, Chiang Mai, Thailand 4Institute of Regional Research and Development, Ministry of Science and Technology, Hanoi, Vietnam

Edited by: Dimitris G. Hatzinikolaou, National and Kapodistrian University of Athens, Greece

Reviewed by: Nalin Nilusha Wijayawardene, Qujing Normal University, China; Stefania Mirela Mang, University of Basilicata, Italy

 *Correspondence: Hong Yu, hongyu@ynu.edu.cn

This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology

 03 03 2023 2023 14 1117753 06 12 2022 10 02 2023 Copyright © 2023 Wang, Tang, Luo, Wang, Thanarut, Dao and Yu. 2023 Wang, Tang, Luo, Wang, Thanarut, Dao and Yu

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 Introduction

Clonostachys, a genus with rich morphological and ecological diversity in Bionectriaceae, has a wide distribution among diverse habitats.

 Methods and Results

In the present study, a phylogenetic framework is reconstructed for the family Bionectriaceae focusing on Clonostachys through increased taxon-sampling using the nrLSU sequence. Through surveying Clonostachys in China, Vietnam, and Thailand over the past 3 years, seven Clonostachys spp. were found and identified. Two new species, C. chuyangsinensis and C. kunmingensis, are described and illustrated based on morphological characteristics and molecular data. The phylogenetic positions of the seven species were evaluated based on four genomic loci (ITS, nrLSU, TUB2, and TEF1).

 Discussion

Moreover, the genetic divergence comparisons of Clonostachys species for three markers (ITS, TUB2, and TEF1) are also provided. The results indicated that the TEF1 sequence data provided the best resolution for distinguishing species of Clonostachys, followed by sequence data for the TUB2 and ITS regions.

 Bionectriaceae molecular systematics multi-gene phylogeny morphology new species 32200013 and 32160005 National Natural Science Foundation of China10.13039/501100001809 Introduction

The asexual morph-typified genus Clonostachys was established by Corda (1839) on the basis of the type species, C. araucaria, which possessed penicillate conidiophores and imbricate conidia held in columns. This species is now considered a synonym of C. rosea (Link) Schroers et al. (basionym Penicillium roseum Link) (Schroers et al., 1999). Clonostachys (Bionectriaceae, Hypocreales) is characterized by penicillate, sporodochial, or dimorphic conidiophores and phialidic conidiogenous cells producing hyaline conidia (Schroers, 2001). Teleomorph, is originially described Bionectria (Spegazzini, 1919), and characterized by ascomata typically seated on a pseudoparenchymatous stroma or arising directly on the substrate, being white, pale yellow, or orange to dark brownish-orange, not changing color in 3% KOH or lactic acid, not collapsing or laterally pinched when dry; warted or smooth; an ascomatal wall composed of 1–3 regions with the outer region composed of subglobose to globose, thick-walled cells; ascospores smooth; spinulose, striate or warted (Schroers, 2001; Lechat and Fournier, 2018). Based on the monograph of Bionectria and Clonostachys by Schroers (2001), this connection was confirmed by DNA sequences. Because Clonostachys was described earlier than Bionectria, Rossman et al. (2013) recommended Clonostachys as the name of this genus.

It is generally agreed that distinguishing individual species of Clonostachys using only morphological characteristics can be difficult (Schroers et al., 1999; Abreu et al., 2014). The members of the genus Clonostachys were accommodated in Acrostalagmus, Clonostachyopsis, Dendrodochium, Gliocladium, Gliocladochium, Myrothecium, Sesquicillium, Spicaria, Verticilliodochium, or Verticillium (Schroers, 2001). It is the huge diversification of morphs of closely related Clonostachys species, what did not allow recognition that they all may belong to a single genus, Clonostachys. Given the problems with species delimitation in Clonostachys using morphology, molecular data are essential to establish robust species boundaries. The first molecular study of Clonostachys/Bionectria was carried out by Rossman et al. (2001) using large subunit rDNA sequences. The results showed that the genus represents a well-resolved monophyletic lineage. Subsequently, DNA sequences of the internal transcribed spacer regions of the rDNA (ITS rDNA) and a portion of the β-tubulin (TUB2) gene were widely used to resolve taxonomic questions for Clonostachys/Bionectria (Schroers, 2001; Hirooka and Kobayashi, 2007; Luo and Zhuang, 2010; Chen et al., 2016; Prasher and Chauhan, 2017). Regrettably, not all recognized species inside this group formed well-supported clades in these two-gene phylogenies (Moreira et al., 2016). Other DNA sequences recently employed to improve the resolution of phylogenetic trees for the species of Clonostachys/Bionectria include ATP citrate lyase (ACL1), TUB2, the large subunit of RNA polymerase II (RPB1), and the translation elongation factor 1-α (TEF1) gene regions (Moreira et al., 2016). However, sequence data of the above-mentioned four protein-encoding gene regions in GenBank1 are incomplete for the group.

There is no doubt that Clonostachys belongs to the family Bionectriaceae, but its taxonomic position in relation to other genera is debated within Bionectriaceae (Rossman et al., 2001; Hyde et al., 2020; Schoch et al., 2020). In more recent studies, Clonostachys was suggested as a close relative of the genus Stephanonectria that was confirmed as a member of Bionectriaceae (Hyde et al., 2020). However, Schoch et al. (2020) reported that Stephanonectria was a genus of ascomycetes in the family Nectriaceae (2accessed on 1 July 2022). Rossman et al. (2001) found that the genera Emericellopsis and Stanjemonium belonged to Bionectriaceae in spite of the distant relation to Clonostachys, whereas Schoch et al. (2020) placed their taxonomic positions in the Hypocreales genera, incertae sedis genera (see Footnote 2 accessed on 1 July 2022). Therefore, it is imperative to reconstruct the phylogenetic framework for the Bionectriaceae focusing on Clonostachys through increased taxon sampling.

In the current study, we aimed to: (1) consider the identity of previously unidentified Clonostachys isolates collected over a 3-year period from China, Vietnam, and Thailand and (2) re-evaluate the taxonomic stability of Clonostachys among related genera within Bionectriaceae and phylogenetic relationships between Clonostachys species.

 Materials and methods Soil and specimen collection and fungus isolation

Soil samples and fungus-infected spider specimens were collected from 11 locations in 2017 and 2019, including eight different locations within Yunnan Province, China, two locations within Dak Lak Province, Vietnam, and one location in Chiang Mai, Thailand.

Clonostachys strains were isolated from the soil samples according to methods described in our previous publication (Wang et al., 2015). Briefly, 2 g of soil was added to a flask containing 20 ml sterilized water and glass beads. The soil suspension was shaken for about 10 min and then diluted 100 times. Subsequently, 200 μL of the diluted soil suspension was spread on Petri dishes with solidified onion garlic agar (OGA: 20 g of grated garlic and 20 g of onion were boiled in 1 l of distilled water for 1 h; the boiled biomass was then filtered-off, and 2% agar was added). Czapek yeast extract agar (CYA, Advanced Technology and Industrial Co., Ltd., China) and potato dextrose agar (PDA, Difco, United States) were used, and all media had 50 mg/L rose Bengal and 100 mg/L kanamycin added. Conidia developing on spider cadavers were transplanted onto plates of PDA and cultured at 25°C. Colonies of the isolated filamentous fungi appearing in the culture were transferred onto fresh PDA media. The purified fungal strain was transferred to PDA slants and cultured at 25°C until its hyphae spread across the entire slope. The emerging fungal spores were washed with sterile physiological saline and made into a spore suspension of 1 × 103 cells/mL. To obtain monospore cultures, a part of the spore suspension was placed on PDA using a sterile micropipette, and then a Petri dish was incubated at 25°C. Specimens and type material were deposited in the Yunnan Herbal Herbarium (YHH) at the Institute of Herb Biotic Resources of Yunnan University, China. Cultures were stored in the Yunnan Fungal Culture Collection (YFCC) at the Institute of Herb Biotic Resources of Yunnan University.

 Morphological observations

Macroscopic characters were collected from colonies grown on PDA and corn meal agar (CMA, Shanghai yiyan bio-technology Co., Ltd., China). Cultures on PDA slants were transferred to PDA and CMA plates and incubated at 25°C for 7 days. Reverse colony pigmentation of strains grown on PDA and CMA was assessed according to Kornerup and Wanscher (1978). For morphological evaluation, microscope slides were prepared by placing mycelia from the cultures on PDA and CMA blocks (5 mm diameter) and then overlaid with a coverslip. The sizes and shapes of the microcharacteristics (e.g., ascomata, asci, ascospores, conidiogenous cells, and conidia) were determined using a light microscope (CX40, Olympus Corporation, Tokyo, Japan) and a scanning electron microscope (Quanta 200 FEG, FEI Company, Hillsboro, United States). Individual length and width measurements were taken for 30–100 replicates, including the absolute minima and maxima.

 DNA extraction, polymerase chain reaction, and sequencing

Specimens and live axenic cultures were prepared for DNA extraction. Genomic DNA was extracted using the Genomic DNA Purification Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The primer pair ITS5 and ITS4 was used to amplify the nuclear ribosomal internal transcribed spacer region (ITS) (White et al., 1990). For amplification of the nuclear ribosomal large subunit (nrLSU) and the β-tubulin (TUB2) gene, PCR primer pairs LR5/LR0R and T1/Bt2b (Vilgalys and Hester, 1990; Rehner and Samuels, 1994; Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997) were employed. The translation elongation factor 1α (TEF1) gene was amplified using the primer pair EF1-688F/EF1-1251R (Alves et al., 2008). All of the PCR reactions were performed in a final volume of 50 μL containing 25 μL 2 × Taq PCR Master Mix (Tiangen Biotech Co., LTD, China), 0.5 μL of each primer (10 μM), 1 μL of genomic DNA, and 23 μL of RNase-Free water. PCR products were sequenced by Beijing Sinogenomax Co. Ltd., China.

 Phylogenetic analyses

Phylogenetic analyses were based on the nrLSU and combined ITS+nrLSU + TUB2 + TEF1 sequences. Sequences of ITS, nrLSU, TUB2, and TEF1 were retrieved from GenBank and combined with those generated in our study. The taxonomic information and GenBank accession numbers are provided in Table 1. Sequences were aligned using Clustal X 2.0 and MEGA v6.06 software (Larkin et al., 2007; Tamura et al., 2013). After alignment, the sequences of the genes were concatenated. Conflicts among the six genes were tested using PAUP* 4.0b10 (Swofford, 2002). The results showed that the phylogenetic signals for the four loci were congruent (p = 0.03). Phylogenetic analyses were conducted using the Bayesian Inference (BI) and the Maximum Likelihood (ML) methods employing MrBayes v3.1.2 and RAxML 7.0.3 (Ronquist and Huelsenbeck, 2003; Stamatakis et al., 2008). Models of sequence evolution were estimated using jModelTest version 2.1.4 (Darriba et al., 2012). The following models were implemented in the Bayesian phylogenetic analyses: GTR + I + G for ITS and nrLSU, K80 + G for TUB, SYM + G for TEF1. The BI analysis was run on MrBayes v3.1.2 for 5 million generations. GTR + I was selected as the optimal model for ML analysis, and 1,000 rapid bootstrap replicates were performed on the dataset. Furthermore, ML analysis was applied to single-locus genealogies for ITS, nrLSU, TUB2, and TEF1.

Specimen information and GenBank accession numbers for sequences used in this study.

Taxon Voucher Info.1 Host/substrate Locality GenBank accession number References
ITS nrLSU TUB2 TEF1
Clonostachys agrawalii CBS 533.81 Decomposing buffalo horn India AF358241 AF358187 Schroers (2001)
Clonostachys apocyni CBS 130.87T Dead stem of Apocynum cannabinum United States AF210688 AF358168 Schroers (2001)
Clonostachys aranearum GZAC QLS0625cloT Spider China KU173835 KU212401 Chen et al. (2016)
Clonostachys aureofulvella CBS 195.93 Root of tree New Zealand AF358226 AF358181 Schroers (2001)
Clonostachys aureofulvella CBS 200.93 Bark of Polylepis sericea Venezuela AF358182 Schroers (2001)
Clonostachys buxi CBS 696.93 Leaves of Buxus sempervirens France KM231840 KM231721 KM232111 KM231977 Lombard et al. (2015)
Clonostachys byssicola CBS 364.78T Wood Venezuela MH861151 MH872912 AF358153 KX184967 Schroers (2001), Moreira et al. (2016), andVu et al. (2019)
Clonostachys byssicola CML 2309 Fragaria ananassa Brazil KC806269 KC806269 KF871149 KX184966 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys candelabrum CBS 504.67 Soil Netherlands AF210668 KF871189 KX185029 Schroers (2001), Abreu et al. (2014), and Moreira et al. (2016)
Clonostachys candelabrum CML 2313 Soil Brazil KC806296 KC806296 KF871186 Abreu et al. (2014)
Clonostachys capitata CBS 218.93 Bark Japan MH862394 MH874054 AF358188 Schroers (2001) and Vu et al. (2019)
Clonostachys chlorina CBS 287.90T Brazil Soil MH862212 MH873895 Vu et al. (2019)
Clonostachys chloroleuca CBS 141588T Native soil from Cerrado Brazil KC806286 KC806286 KF871172 KX184988 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys chloroleuca CBS 141589 Native soil from Cerrado Brazil KC806277 KC806277 KF871173 KX184978 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys chuyangsinensis YFCC 895 Soil China MW199068 MW199057 MW201675 MW295968 This work
Clonostachys chuyangsinensis YHH 896 Spider Vietnam MW199066 MW199055 MW201673 MW295966 This work
Clonostachys chuyangsinensis YFCC 896T Spider Vietnam MW199067 MW199056 MW201674 MW295967 This work
Clonostachys coccicola BUcCo Unaspis citri Australia KU720552 KU720550 Dao et al. (2016)
Clonostachys coccicola BUcS Unaspis citri Australia KU720551 KU720549 Dao et al. (2016)
Clonostachys compactiuscula CBS 913.97 Bark of dead Fagus sp United States AF358245 AF358194 Schroers (2001)
Clonostachys compactiuscula CBS 919.97 Twigs of Acer sp United States AF210690 AF210690 Schroers (2001)
Clonostachys compactiuscula YFCC 894 Soil China MW291598 MW291602 MW295976 MW295971 This work
Clonostachys compactiuscula YFCC 897 Soil China MW199071 MW199060 MW201678 MW295972 This work
Clonostachys divergens CBS 967.73bT Soil Germany AF210677 AF210677 AF358191 Schroers (2001)
Clonostachys epichloë CBS 101037 Sasa sp Japan AF210675 AF210675 AF358209 Schroers (2001)
Clonostachys eriocamporesiana MFLUCC 17-2620T Dead stems of Chromolaena odorata Thailand MN699132 MN699965 MN699964 Hyde et al. (2020)
Clonostachys eriocamporesii MFLUCC 19-0486T Dead stems of Pennisetum polystachion Thailand MN699133 MN699128 Hyde et al. (2020)
Clonostachys grammicospora CBS 209.93T Standing dead tree French Guiana AF210678 MH874052 AF358206 Schroers (2001) and Vu et al. (2019)
Clonostachys grammicosporopsis CBS 115.87 Bark of Metrosideros sp New Zealand AF210679 AF210679 AF358204 Schroers (2001)
Clonostachys impariphialis HMAS 275560 Rotten bark China KX096609 KX096606 Zeng and Zhuang (2017)
Clonostachys indicus IBP 2 Dead twigs of Ficus virens India KT291441 Prasher and Chauhan (2017)
Clonostachys intermedia CBS 508.82T Agricultural soil Netherlands AF210682 AF358205 Schroers (2001)
Clonostachys intermedia KUC21274 Soil South Korea MH168099 Unpublished
Clonostachys kowhai CBS 461.95T Bark of Sophora microphylla New Zealand AF358250 AF358170 Schroers (2001)
Clonostachys krabiensis MFLUCC 16-0254T Dead leaves of Pandanus sp Thailand MH388335 MH376707 Tibpromma et al. (2018)
Clonostachys kunmingensis YFCC 898T Soil China MW199069 MW199058 MW201676 MW295969 This work
Clonostachys kunmingensis YFCC 892 Soil China MW199070 MW199059 MW201677 MW295970 This work
Clonostachys kunmingensis YFCC 967 Soil China OP023125 OP023116 This work
Clonostachys levigata CBS 948.97 Branch of dead Buxus sempervirens France AF210680 AF210680 AF358196 Schroers (2001)
Clonostachys lucifer CBS 100008 Bark of dead Casearia arborea United States AF210683 AF358208 Schroers (2001)
Clonostachys moreaui CLL19024T Bark of Laurus novocanariensis Portugal MT160524 Lechat et al. (2020)
Clonostachys oblongispora CBS 100285T Bark of dying tree of Orixa japonica Japan AF358248 AF358169 Schroers (2001)
Clonostachys parva CBS 997.69T Agricultural soil Netherlands AF210674 AF210674 AF358210 Schroers (2001)
Clonostachys phyllophila CBS 921.97T Leaves, Fallen plant France AF210664 AF210664 Schroers (2001)
Clonostachys pilosella BRFM 3113T Bark French Guiana MT248415 Lechat and Fournier (2020)
Clonostachys pityrodes CBS 102033 Bark Mauritius AF210672 AF210672 AF358212 Schroers (2001)
Clonostachys pityrodes CBS 126394 Small, standing dead tree Sri Lanka MH864280 MH875729 Vu et al. (2019)
Clonostachys pnagiana BRFM 3057T Bark French Guiana MT248416 Lechat and Fournier (2020)
Clonostachys pseudochroleuca CBS 187.94T Base of decaying palm frond French Guiana KJ499909 KJ499909 KF871188 KX185003 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys pseudochroleuca CML 1982 Soil Brazil KC806263 KC806263 KF871165 KX185002 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys pseudostriata CBS 119.87 Bark Indonesia AF358251 AF358183 Schroers (2001)
Clonostachys pseudostriatopsis MAFF 239827 Bark of fallen twigs Japan AB237465 Hirooka and Kobayashi (2007)
Clonostachys ralfsii CBS 129.87 Bark New Zealand AF210676 AF210676 AF358195 Schroers (2001)
Clonostachys rhizophaga CBS 202.37 Root of Ulmus americana United States AF358225 MH867396 AF358156 Schroers (2001) and Vu et al. (2019)
Clonostachys rhizophaga CBS 361.77 Culture contaminant Switzerland AF358228 AF358158 KX184993 Schroers (2001)
Clonostachys rhizophaga CML 2312 Culture contaminant Brazil KC806275 KC806275 KF871157 KX184992 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys rhizophaga YFCC 900 Soil China MW199074 MW199063 MW201681 MW295974 This work
Clonostachys rogersoniana CBS 582.89 Rain forest soil Brazil AF210691 AF358189 Schroers (2001)
Clonostachys rogersoniana CML 1216 Soil Brazil KC806287 KC806287 KF871178 KX185017 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys rogersoniana YFCC 899 Soil China MW199073 MW199062 MW201680 MW295973 This work
Clonostachys rosea CBS 154.27 Soil United States MH854911 MH866405 AF358160 KX184995 Schroers (2001), Moreira et al. (2016), and Vu et al. (2019)
Clonostachys rosea CBS 406.95 Bark of Salix sp France AF358249 AF358167 Schroers (2001)
Clonostachys rosea CBS 710.86T Soil Netherlands AF358235 MH873700 AF358161 KX184999 Schroers (2001), Moreira et al. (2016), and Vu et al. (2019)
Clonostachys rosea CML 2310 Fragaria ananassa Brazil KC806257 KC806257 KF871146 KX184998 Abreu et al. (2014) and Moreira et al. (2016)
Clonostachys rosea YFCC 893 Soil Thailand ON287194 ON303656 ON314171 ON314172 This work
Clonostachys rossmaniae CBS 210.93 Bark of twigs French Guiana AF358227 AF358213 Schroers (2001)
Clonostachys samuelsii CBS 699.97 Bark Venezuela AF358236 AF358190 Schroers (2001)
Clonostachys samuelsii CBS 700.97 Bark United States AF210689 Schroers (2001)
Clonostachys saulensis BRFM 2782T Bark of dead liana French Guiana MK635054 Lechat et al. (2019)
Clonostachys setosa CBS 834.91 Twig Cuba AF210670 AF210670 AF358211 Schroers (2001)
Clonostachys sesquicillii CBS 180.88 Twigs and lichen Guyana AF210666 AF210666 AF358214 Schroers (2001)
Clonostachys solani CBS 183.30 Garden soil Netherlands MH855105 MH866555 AF358222 Schroers (2001), Vu et al. (2019)
Clonostachys solani CBS 223.72b Wheat field soil Germany MH860460 MH872186 AF358223 Schroers (2001), Vu et al. (2019)
Clonostachys solani CBS 697.88 Bark Germany MH862150 MH873842 AF358216 Schroers (2001), Vu et al. (2019)
Clonostachys solani CBS 752.68 Wood of angiosperm tree Germany MH859224 MH870947 AF358221 Schroers (2001), Vu et al. (2019)
Clonostachys solani YFCC 901 Soil China MW199072 MW199061 MW201679 MW295975 This work
Clonostachys spinulosispora CBS 133762T Leaves French Guiana MH634702 KY006568 Lechat and Fournier (2018)
Clonostachys sporodochialis CBS 101921T Bark United States AF210685 AF358149 Schroers (2001)
Clonostachys sporodochialis CLL-GUY-12-046 Bark French Guiana KJ802125 Crous et al. (2014)
Clonostachys subquaternata CBS 107.87 Wood Venezuela AF358207 Schroers (2001)
Clonostachys vesiculosa HMAS 183151T Decaying leaves of a dicotyledonous plant China HM050304 HM050302 Luo and Zhuang (2010)
Clonostachys viticola MUM 18.51T root of Vitis vinifera Peru MK156282 MK156290 MK156286 Torcato et al. (2020)
Clonostachys viticola CAA 945 root of Vitis vinifera Peru MK156283 MK156291 MK156287 Torcato et al. (2020)
Clonostachys wenpingii HMAS 172156T Dead leaves China EF612465 HM042410 HM054127 HM054097 Zhao et al. (2011)
Clonostachys zelandiaenovae CBS 232.80 Wood New Zealand AF210684 AF210684 AF358185 Schroers (2001)
Stanjemonium grisellum CBS 655.79T Soil United States AY632671 MH873004 AY632687 Zuccaro et al. (2004) and Vu et al. (2019)
Stanjemonium ochroroseum CBS 656.79T Soil United States AY632672 AF049172 AY632688 AF049194 Zuccaro et al. (2004)

CAA, Culture collection of Artur Alves, housed at Department of Biology, University of Aveiro, Portugal; CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CML, Coleção Micológica de Lavras, Universidade Federal de Lavras, Lavras, Minas Gerais, Brazil; GZAC, Institute of Fungus Resources, Guizhou University, Guiyang, China; HMAS, Herbarium of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MUM, Culture collection hosted at Center for Biological Engineering of University of Minho, Braga, Portugal; YFCC, Yunnan Fungal Culture Collection, Yunnan University, Kunming, China; YHH, Yunnan Herbal Herbarium, Yunnan University, Kunming, China. Tex-type strain. Boldface: data generated in this study.

We applied a (phylo-) genetic distance matrix calculation for the markers (ITS, TUB2, and TEF1) to assess species boundaries of 14 Clonostachys spp. (Supplementary Tables S1–S3), because their sequence data for the three loci were complete. The pairwise genetic distances of the 14 Clonostachys lineages were measured based on the Kimura two-parameter model using MEGA v6.06 software (Tamura et al., 2013).

 Results Sequencing and phylogenetic analyses

Phylogenetic analyses based on nrLSU data consisting of 107 fungal taxa confirmed the presence and positions of Clonostachys and related genera within Bionectriaceae. Eighteen well-supported clades were recognized based on both BI and ML analyses of the 107 taxa from Bionectriaceae and Flammocladiella (Flammocladiaceae, Hypocreales) that accommodate species of the genera Bryocentria, Clonostachys, Emericellopsis, Gliomastix, Heleococcum, Hydropisphaera, Ijuhya, Lasionectria, Nectriopsis, Paracylindrocarpon, Roumegueriella, Selinia, Stanjemonium, Stephanonectria, Stilbocrea, Stromatonectria, Verrucostoma, and Flammocladiella (Figure 1). The genus Clonostachys was phylogenetically clustered with Stephanonectria, and Emericellopsis had a close genetic relationship with Stanjemonium, but they were clearly distinguished from their allied genera by forming four separate clades in the family Bionectriaceae (Figure 1). The combined dataset included sequences from 86 fungal taxa (Table 1). The final dataset consisted of 2,900 bp of sequence data, including gaps (ITS, 654 bp; nrLSU, 903 bp; TUB2, 711 bp; and TEF1, 632 bp). Both BI and ML analyses produced trees with similar topologies that resolved most Clonostachys lineages in separate terminal branches (Figure 2). Phylogenetic trees inferred from analyses of combined data divided Clonostachys into six distinguished clades, designated as Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella clades (Figure 2). The phylogenetic analyses suggested the existence of distinct species in the Bionectria and Epiphloea clade that we accordingly propose as new species: C. chuyangsinensis, which was found in the Epiphloea clade, and C. kunmingensis, which was found in the Bionectria clade (Figure 2).

Phylogenetic reconstruction of Clonostachys and related genera in Bionectriaceae obtained from the nrLSU sequences based on Bayesian inference and Maximum Likelihood analyses. Statistical support values (≥0.9/90%) are shown at the nodes for BI posterior probabilities/ML bootstrap support. Materials in bold type are those analyzed in this study.

Phylogenetic tree of Clonostachys based on Bayesian inference and Maximum Likelihood analyses of a 4-locus (ITS, nrLSU, TUB2, and TEF1) dataset. Statistical support values (≥ 0.9/90%) are shown at the nodes for BI posterior probabilities/ML bootstrap support. Materials in bold type are those analyzed in this study. Isolates representing ex-type material are marked with “T.”

The tree topologies for the individual loci (ITS, nrLSU, TUB2, and TEF1) did not show congruence (Supplementary Figures S1–S4). However, in all analyses C. chuyangsinensis had a close genetic relationship with C. candelabrum. Clonostachys chloroleuca and C. rhizophaga were sisters to the newly discovered species C. kunmingensis, although this relationship received significant bootstrap support only from ITS and TUB2. Phylogenetic analyses based on nrLSU data revealed that C. kunmingensis was closely related to C. rosea (Figure 1; Supplementary Figure S2). And the nrLSU sequences cannot distinguish the two species. But they were regarded as different species with strong support from ITS, TUB2 and TEF1 (Supplementary Figures S1, S3, S4).

The genetic divergence comparisons showed that: (1) the minimum thresholds (p-distances) to distinguish genetic species in the Clonostachys lineages were 0.005, 0.017, and 0.026 for ITS, TUB2, and TEF1, respectively (Supplementary Tables S1–S3); (2) the TEF1 sequence data provided the best resolution distinguishing Clonostachys spp., followed by TUB2 and ITS sequences (Supplementary Tables S1–S3); and (3) the genetic distances strongly supported recognition of C. chuyangsinensis and C. kunmingensis as two new taxa (Table 2).

Genetic distance (p-distances) of the two new Clonostachys species with their related species.

Subgenus Taxa Marker
ITS TUB2 TEF1
Epiphloea Clonostachys chuyangsinensis – Clonostachys candelabrum 0.029 0.097 0.083
Bionectria Clonostachys kunmingensis – Clonostachys chloroleuca 0.009 0.042 0.036
Clonostachys kunmingensis – Clonostachys rhizophaga 0.012 0.023 0.058
Taxonomy

In this study, a collection of 23 isolates of unknown identity were shown to represent five known species and two new species of Clonostachys. The phylogenetic positions of the five known species were evaluated according to phylogenetic inferences based on four loci (ITS, nrLSU, TUB2, and TEF1), including C. compactiuscula, C. rhizophaga, C. rogersoniana, and C. solani from China, and C. rosea from Thailand (see Table 1; Figure 2). The two new species, provided with the names C. chuyangsinensis from Vietnam and China and C. kunmingensis from China, were recognized based on morphological characteristics and molecular data.

Clonostachys chuyangsinensis H. Yu & Y. Wang, sp. nov. Figure 3.

Morphology of Clonostachys chuyangsinensis. (A) Infected spider. (B) Ascomata on the host. (C) Front view of perithecium. (D, E) Conidiogenous structures on the host. (F, G) Colony obverse and reverse on PDA. (H–J) Conidiophores, conidiogenous cells, and conidia on PDA. (K) Colony obverse on CMA. (L) Conidiophores, conidiogenous cells, and conidia on CMA. (M) Conidia on CMA. Scale bars: (A) = 3 mm; (B,D) = 1 mm; (C) = 100 μm; (E,J,L) = 20 μm; (F,G,K) = 30 mm; (H,I) = 50 μm; (M) = 5 μm.

MycoBank number 843885.

Etymology: named after Chu Yang Sin National Park, where this species was first discovered.

Type: Vietnam, Dak Lak Province, Chu Yang Sin National Park (12°29’N, 108°43′E, 1659 m above sea level), on a spider on the underside of a leaf, October 22, 2017, collected by Yuan-Bing Wang (holotype: YHH 896; ex-type: YFCC 896).

Description: Sexual morph: Ascomata on a brown spider, perithecial, solitary or densely crowded in groups, subglobose to oval, (280–)290–380(−400) × (240–)260–330(−340) μm (n = 30), collapsing laterally when dry, pale brown when fresh, becoming dark brown to nearly black when dry, not changing color in 3% KOH or in lactic acid; surface smooth. Asci and ascospores not observed. Asexual morph: Infected spider host covered with a dense brown mycelial mat. Hyphae branched, septate, hyaline, smooth. Conidiophores verticillium-like; phialides divergent in whorls of 2–5 or single from lower levels, generally slightly tapering toward the tip, (5–)5.6–28.3(−36) × (1–)1.4–3.6(−4) μm (n = 30). Conidia smooth-walled, hyaline, subglobose to ellipsoid, (2–)2.5–4.6(−4.8) × (2–)2.4–3.5(−4) μm (n = 30). Colonies on PDA reached 28–32 mm in diameter after 7 days at 25°C, white, circular; reverse pale to light orange (5–6A3–4). Colony surface white powdery to granulose because of the conidiophores and conidial masses; aerial mycelium sparsely produced or absent. Conidiophores monomorphic, verticillate, arising from the agar surface or from the sparse aerial mycelium; stipes (20–)40–130(−150) μm long, (2–)2.5–4(−5) μm wide at the base (n = 50); primary branches divergent, forming independent side-branches; terminal branches and phialides divergent or adpressed; terminal phialides flask-shaped, or cylindrical but narrowing in the upper part, (4.5–)5.5–44.2(−60) × (1.2–)1.5–3.8(−4) μm (n = 50). Conidia in white imbricate columns, smooth-walled, hyaline, subglobose to ellipsoid, (2.2–)2.4–4.7(−5) × (1.5–)1.8–3.5(−3.8) μm (n = 50). Setae not observed. Colonies on CMA reached 25–30 mm in diameter after 7 days at 25°C, white, circular; reverse pale yellowish (1-2A3). Colony surface white powdery due to conidial masses, cottony to felty due to aerial mycelium. Conidiophores monomorphic, verticillate, arising from the agar surface or from the sparse aerial mycelium; stipes (20–)30–145(−160) μm long, (2–)2.5–4(−4.5) μm wide at the base (n = 50); primary branches divergent, forming independent side-branches; terminal branches and phialides divergent or adpressed; terminal phialides flask-shaped, or cylindrical but narrowing in the upper part, (4.5–)5.5–44.2(−50) × (1.2–)1.5–3.8(−4.2) μm (n = 50). Conidia in white imbricate columns, smooth-walled, hyaline, subglobose to ellipsoid, ovoid, (2–)2.2–5(−5.5) × (1.5–)2–3.5(−4) μm (n = 50). Setae not observed.

Distribution: Chu Yang Sin National Park, Dak Lak Province, Vietnam; Kunming City, Yunnan Province, China.

Additional materials examined: China, Yunnan Province, Kunming City, Wild Duck Forest Park (25°13’N, 102°87′E, 2100 m above sea level), from soil on the forest floor, August 20, 2018, Yao Wang (living culture: YFCC 895); China, Yunnan Province, Kunming City, Songming County, Dashao Village (25°24’N, 102°55′E, 2697 m above sea level), from Ophiocordyceps highlandensis, August 25, 2018, De-Xiang Tang (living culture: YFCC 8591) (Zhao et al., 2021).

Notes: Morphologically, C. chuyangsinensis resembles the phylogenetically sister species C. candelabrum. The shape and size of the conidia and the colony color of C. chuyangsinensis among other morphological features have been observed in C. candelabrum. However, C. chuyangsinensis can be distinguished from C. candelabrum by its long phialides ((4.5–)5.5–44.2(−50) × (1.2–)1.5–3.8(−4.2) μm). Both morphological study and phylogenetic analyses of combined ITS, nrLSU, TUB2, and TEF1 sequence data support that this fungus is a distinct species in the genus Clonostachys.

Clonostachys kunmingensis H. Yu & Y. Wang, sp. nov. Figure 4.

Morphology of Clonostachys kunmingensis. (A,B) Colony obverse and reverse on PDA. (C,D) Verticillium-like primary conidiophores on PDA. (E) Conidia from secondary conidiophores. (F,G) Colony obverse and reverse on CMA. (H) Verticillium-like primary conidiophores on CMA. (I) Secondary conidiophores on CMA. (J) Conidia from primary conidiophores. Scale bars: (A,B,F,G) = 20 mm; (C,D,H,I) = 20 μm; (E,J) = 10 μm.

MycoBank number 843886.

Etymology: named after the location Kunming City where the species was collected.

Type: China, Yunnan Province, Kunming City, Wild Duck Forest Park (25°13’N, 102°87′E, 2100 m above sea level), from soil on the forest floor, August 10, 2019, Yao Wang (holotype: YHH 898, dried specimen; ex-type: YFCC 898).

Description: Sexual morph: Undetermined. Asexual morph: Colonies on PDA reaching 32–35 mm in diameter after 7 days at 25°C, pale yellow (4A2–3), circular; reverse pale orange (5A2–3). Colony surface cottony to felty due to aerial mycelium. Conidiophores dimorphic. Primary conidiophores verticillium-like, arising from the agar surface or from the sparse aerial mycelium; (80–)120–260(−380) μm high, stipes (20–)60–140(−230) μm long, (2–)3.5–5(−5.5) μm wide at the base (n = 50), sometimes with short side branches arising from the upper part; phialides divergent, in whorls of 2–6, sometimes singly from lower levels, (14.2–)19.1–36.4(−52.6) × (2–)2.5–3.5(−3.9) μm (n = 50), straight, cylindrical, slightly tapering toward the tip. Secondary conidiophores penicillate, solitary to gregarious, with divergent branching penicilli; bi-to quarter-verticillate, (15–)30–100(−125) μm long, (3–)3.5–5(−5.5) μm wide at the base (n = 50); penicillus 90–145 μm high, typically with two primary branches, divergent, terminating in moderately divergent metulae and adpressed phialides; phialides divergent or adpressed, in whorls of 2–6, almost cylindrical tapering in the upper part, straight to slightly curved, (5.6–)8.0–17.5(−25) μm long, (2–)2.5–3.2(−4) μm wide at the base, (1–)1.2–1.4(−1.6) μm wide near the aperture (n = 50); intercalary phialides rarely observed. Conidial masses on verticillium-like conidiophores small and round collapsing to form whitish, watery masses; conidial masses on penicillate conidiophores inconspicuous, short, and rather thick, columnar, white. Conidia from secondary conidiophores slightly curved, with one slightly flattened side, distally broadly rounded, with laterally displaced hila, (4–)4.2–8.5(−9) × (2.2–)2.5–4(−4.5) μm (n = 100), held in imbricate; conidia from primary conidiophores larger, oblong to cylindrical, frequently less curved, sometimes without a visible hilum, (6–)6.7–11.2(−14) × (2–)2.3–4.5(−4.8) μm (n = 100). Colonies on CMA reaching 25–32 mm in diameter after 7 days at 25°C, white, circular; reverse yellowish white to light yellow (4A2–5). Colony surface white powdery due to conidial masses. Aerial mycelium on CMA not thick, on PDA strongly developed in thick, often erect hyphal strands. Size and shape of Conidiophores, phialides and conidia similar on PDA and CMA.

Additional materials examined: China, Yunnan Province, Kunming City, Songming County, Dashao Village (25°24’N, 102°55′E, 2750 m above sea level), from soil on the forest floor, August 24, 2019, Yao Wang (living culture: YFCC 892, 967).

Notes: Regarding phylogenetic relationships, C. kunmingensis is closely related to C. rhizophaga and C. chloroleuca and further grouped with C. oblongispora (Figure 2). However, C. kunmingensis can be distinguished from C. rhizophaga and C. chloroleuca by its oblong to cylindrical conidia ((6–)6.7–11.2(−14) × (2–)2.3–4.5(−4.8) μm). Clonostachys kunmingensis consistently showed unpigmented conidial masses, while conidial masses of C. rhizophaga and C. chloroleuca can be greenish or weakly greenish (Moreira et al., 2016). Clonostachys oblongispora differs from C. kunmingensis by its longer conidia ((9–)12.6–13.6–14(−19.8) × (2.6–)3.2–3.6–3.8(−4.2) μm) (Schroers, 2001). Morphologically, C. kunmingensis is similar to C. rosea in terms of the shape and size of the conidiogenous cells and the shape of the conidia (Schroers, 2001). However, our morphological observation revealed some differences between them. Colonies of C. kunmingensis on PDA are pale yellow whereas those of C. rosea are white. Furthermore, conidia from secondary conidiophores of C. kunmingensis ((4–)4.2–8.5(−9) × (2.2–)2.5–4(−4.5) μm) are larger than those of C. rosea ((4.2–)4.8–5.2–5.6(−6.6) × (2–)2.4–2.8–3(−3.4) μm).

 Discussion

Clonostachys species are widely distributed and occupy diverse habitats, with various host/substrate associations (see Table 1). The species distribution is cosmopolitan, with the height of known species diversity occurring in tropical regions; the habitat diversity is complicated, with most of the known species having unspecific saprotrophic ability (Schroers, 2001). These known species are commonly found in soils, litter, and dead plant substrata as saprotrophs. They have also been reported as endophytes and epiphytes of living plants (Torcato et al., 2020). Another aspect of the biology of Clonostachys species is their unspecific parasitic ability. Some Clonostachys spp. are known as destructive mycoparasites, with C. rosea and C. rosea f. catenulata being used as biocontrol agents against various ascomycetes, soil-borne hyphomycetes, and basidiomycetes (Schroers, 2001; Chatterton et al., 2008). They are also parasitic to myxomycetes, nematodes, ticks, mollusks, and leafhoppers (Schroers, 2001; Toledo et al., 2006). In this study, we described a novel species, C. chuyangsinensis, which was isolated from a large spider. In fact, Clonostachys species parasitic on spiders have rarely been reported, apart from C. aranearum (Chen et al., 2016). The present study provides new evidence for Clonostachys sp. as an araneopathogenic fungus, thus extending our knowledge of the occurrence and distribution of spider-pathogenic fungi.

Compared with the anamorph of Clonostachys with simple morphological architectures, the teleomorph provided more valuable morphological information to recognize individual Clonostachys species. Schroers (2001) classified the teleomorph in the six distinguished subgenera Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella based on stroma morphology, stroma-perithecium wall interface structure, perithecial wall anatomy, habit of the perithecia on the natural substratum, and ascospore ornamentation and septation. Our phylogenetic analyses based on the combined ITS+nrLSU + TUB2 + TEF1 sequences provide additional evidence supporting these morphologically delimited subgenera (Figure 2). It seems that the divisions of six subgenera do not contradict the unity of the entire genus Clonostachys. All taxa of six subgenera are united by the phenotypic characteristics of the anamorph such as penicillate conidiophores, conidia held in imbricate columns, and predominantly more or less curved conidia with mostly laterally displaced hila (Schroers, 2001). Some intraspecific variations in conidiomata, intercalary phialides, conidiophore dimorphism, and conidial mass color have hampered species identification in Clonostachys, but to a certain extent these may reflect subgeneric affinities (Schroers, 2001). In the current study, it should be noted that the phylogenetic trees inferred from the analyses of combined data excluded C. setosa and C. vesiculosa from the six subgenera (Figure 2). The two species should belong to the subgenus Epiphloea based on diagnostic features (Schroers, 2001; Luo and Zhuang, 2010). However, they are distant relatives of Epiphloea spp. from our results (Figure 2). The phenotypic similarities among non-sister species may result from convergent morphological evolution, perhaps due to occupation of similar ecological niches (Bischoff et al., 2009). Therefore, we propose to protect Clonostachys as the genus name for the entire clade, while acknowledging that future studies including more data and taxonomic sampling may introduce new genera to accommodate these subgenera.

The multilocus phylogenetic approach taken in this study of the genus Clonostachys has shed considerable light on this important group of fungi. The results of the present work indicate that the nrLSU sequences provided little valuable information to separate Clonostachys spp., although they were conducive to determining the phylogenomic relationships between Clonostachys and its related genera. In contrast, sequence data for the ITS and protein-coding gene region TUB2 provided good resolution of Clonostachys spp., confirming the results of previous studies (Schroers, 2001; Hirooka and Kobayashi, 2007; Luo and Zhuang, 2010; Chen et al., 2016; Prasher and Chauhan, 2017). Our study also introduced sequence data for the TEF1 gene region. This region requires only two primers and is easily amplified. Although the sequence length of the TEF1 fragment was the shortest among the four loci analyzed in this study, the introns within TEF1 provided the greatest concentration of informative nucleotide variation and degree of phylogenetic resolution for terminal clades in Clonostachys. Additionally, the genetic distances of Clonostachys species for TEF1 were significantly higher than those for ITS and TUB2 (Supplementary Tables S1–S3). Future studies will determine the use of this single locus for the recognition and identification of phylogenetic species in Clonostachys and other fungal species.

 Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

 Author contributions

YW: conceptualization. YW: methodology, writing—original draft preparation, and formal analysis. YW and RL: software. D-XT and RL: validation. YW, D-XT, Y-BW, CT, and HY: investigation. YW, D-XT, and V-MD: resources. HY: writing—review and editing and funding acquisition. All authors reviewed and approved the final manuscript.

 Funding

This work was supported by the National Natural Science Foundation of China (Nos 32200013 and 32160005).

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

 Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1117753/full#supplementary-material

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1www.ncbi.nlm.nih.gov

2https://www.ncbi.nlm.nih.gov/taxonomy