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. 2025 Mar 11;115:43–66. doi: 10.3897/mycokeys.115.139757

Morphology and multi-gene phylogeny reveal three new species of Clonostachys and two combinations of Sesquicillium (Bionectriaceae, Hypocreales) from Xizang, China

Shucheng He 1,2, Vinodhini Thiyagaraja 1, Chitrabhanu S Bhunjun 2, Putarak Chomnunti 2, Lakmali S Dissanayake 3,1, Ruvishika S Jayawardena 2, Hongde Yang 1,4, Yun Wei Zhao 1,5, Fatimah Al-Otibi 6, Qi Zhao 1, Kevin D Hyde 1,2,6,✉
PMCID: PMC11920821  PMID: 40109774

Abstract

Clonostachys and Sesquicillium are genera in Bionectriaceae, and known in sexual perithecial ascomata and hyphomycetous asexual morphs. In their asexual morph, both genera share similar morphology in conidiophores and conidiogenous cell characteristics but differ in the development of conidiophores. The members of Clonostachys are distributed worldwide with the majority occurring in the tropics and the species are commonly reported as soil-borne fungi but also reported as endophytes, epiphytes, and saprotrophs. During a microfungi survey in Xizang, China, six collections of fresh and healthy Ageratinaadenophora and Houttuyniacordata leaves were obtained. The taxonomy of these collections was investigated through a combination of morphological analysis and multigene phylogenetic analysis using Maximum likelihood and Bayesian inference. The newly generated sequences were clustered within Clonostachys and Sesquicillium, showing hyphomycetes asexual morph. The results revealed three new Clonostachys species viz, Clonostachyslinzhiensis, C.motuoensis, and C.yadongensis. This research sheds light on the overlooked fungal diversity in Xizang, China, expanding the known fungal biodiversity in the region. Additionally, two new combinations, Sesquicilliumaquaticum and S.shanghaiense for C.aquatica and C.shanghaiensis, and one synonymy, C.viticola for C.swietenia are established, respectively.

Key words: Asexual morph, endophytes, Hyphomycetes, new taxa, taxonomy

Introduction

Clonostachys (Bionectriaceae, Hypocreales) was established by Corda (1839). The genus was typified by C.araucaria (Corda 1839), which was later synonymized under C.rosea (Rossman et al. 2013). The genus was considered as the asexual morph of Bionectria and both genera were also considered as conspecific in several studies (Luo and Zhuang 2007, 2010; Dong et al. 2023). Bionectria was described by Spegazzini (1918). Based on the One Fungus = One Name (1F = 1N) concept, mycologists propose the protection of the older asexual morph-typified name Clonostachys for this genus (Rossman et al. 2013; Dong et al. 2023). Members of Clonostachys occur as endophytes, entomopathogens, epiphytes, plant pathogens, soil-borne fungi, and saprotrophs, typically found on herbicolous, corticolous, lichenicolous, fungicolous, coprophilous habitats as well as on nematodes and insects (Mazen et al. 2022; Dong et al. 2023; Wang et al. 2023; Zhao et al. 2023). They are distributed globally and commonly occur in tropical regions (Schroers 2001). The sexual morph is characterized by ascomata that do not change colour in 3% Potassium Hydroxide (KOH) or 100% Lactic Acid (LA) (Luo and Zhuang 2007, 2010), perithecial or cleistothecial ascomata that are superficial on the substrate or embedded in the stroma. Ascomata are solitary or densely aggregated, subglobose to pyriform; clavate or cylindrical, sessile or short pedicellate asci, smooth or striated, aseptate to multi-septate, globose, fusiform, ellipsoid or broadly ellipsoid ascospores (Hyde et al. 2020a). The asexual members are characterized by penicillate, sporodochial and dimorphic conidiophores (primary and secondary conidiophores) with phialidic conidiogenous cells, hyaline, smooth, broadly ellipsoidal conidia with ends that are broadly rounded (Bao et al. 2023; Chen et al. 2023; Dong et al. 2023; He et al. 2023; Liu et al. 2023; Perera et al. 2023). Primary conidiophores are mononematous, either verticillium-like or narrowly penicillate, whereas the secondary conidiophores produce imbricate conidial chains that can collapse to slimy masses, particularly on sporodochia (Zhao et al. 2023).

Morphology-based identification of Clonostachys is challenging (Schroers et al. 1999; Abreu et al. 2014) and many species were previously placed in various genera such as Acrostalagmus, Clonostachyopsis, Dendrodochium, Gliocladium, Gliocladochium, Myrothecium, Sesquicillium, Spicaria, Verticilliodochium, or Verticillium (Schroers 2001). Rossman et al. (2001) first conducted the initial molecular investigation of Clonostachys/Bionectria, employing large subunit rDNA sequences, and proposed the monophyletic status. Subsequently, DNA sequences from multi-genes including ITS, 28S, rpb1, rpb2, and tef1 have been extensively employed to address the taxonomy of Clonostachys (Bao et al. 2023; Chen et al. 2023; Perera et al. 2023; Zhao et al. 2023). Wijayawardene et al. (2022) accepted 78 species under Clonostachys, while this was 50 species in Hyde et al. (2024). Zhao et al. (2023) investigated the species diversity within a collection of 420 strains of Clonostachys from the culture collection and personal collections at the Westerdijk Fungal Biodiversity Institute in Utrecht, the Netherlands, and identified 19 species based on phylogenetic and morphological analyses. In China, 19 Clonostachys species have been reported from different hosts and substrates (Bao et al. 2023; Dong et al. 2023; Perera et al. 2023; Piombo et al. 2023; Wang et al. 2023).

During the microfungi survey in China (He et al. 2024a, b, c; Thiyagaraja et al. 2024), we investigated several isolates from the leaves of Ageratinaadenophora and Houttuyniacordata from Xizang, China. Multigene phylogenetic analyses combining 28S, tef1, rpb2, ITS, and tub2 sequences, along with morphological analyses, support the establishment of three new species: Clonostachyslinzhiensis, C.motuoensis and C.yadongensis. The introduction of these new species follows the protocols outlined in Chethana et al. (2021) and Maharachchikumbura et al. (2021). The new species are established based on detailed morphological characterization, and illustrations, along with multigene analyses of maximum likelihood (ML) and Bayesian inference (BI). In addition, through phylogenetic analysis of Clonostachys, we suggest that C.aquatica, C.shanghaiensis, and C.swieteniae be synonymous with Sesquicilliumaquaticum, S.shanghaiense, and C.viticola, respectively.

Materials and methods

Sample collection, isolation, and morphological characterization

Fresh and healthy leaves of Ageratinaadenophora and Houttuyniacordata were collected from Medog County, Linzhi City, Xizang Autonomous Region, China from October 2021 to July 2023, and information on collection was recorded according to the Rathnayaka et al. (2024). The healthy part of the leaves was initially cleaned and cut into small pieces (5 × 5 mm). The leaf fragments were briefly soaked in a 75% ethanol solution for 30 s, followed by a 2.5% sodium hypochlorite solution for the same duration (Bhunjun et al. 2021). Afterward, they were washed thrice with sterile distilled water for 30 s. Once sterilized, the tissue fragments were allowed to air-dry on sterile filter paper and then transferred to potato dextrose agar (PDA) (Senanayake et al. 2020). The PDA plates were cultured at 25 °C for 2–5 days. Single hyphae were carefully selected from the periphery of the growing colonies and inoculated onto new PDA plates. Following 1–2 weeks of purification, a pure culture was obtained. Sporulation was induced on water agar (WA) medium. The mycelia were mounted on a slide in water using a sterile needle. A NIKON ECLIPSE Ni-U compound microscope was used to examine conidiophores and conidia of a small mass of mycelia. Micro-morphological images were captured with a DS-Ri2 camera attached to the compound microscope. The photoplates used for the figure were processed with Adobe Photoshop. The pure cultures were deposited in the Kunming Institute of Botany, the Chinese Academy of Sciences (KUNCC), Kunming, China. Specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Academia Sinica (KUN-HKAS), Kunming, China. Facesoffungi and Index Fungorum numbers were registered following the protocols outlined in Jayasiri et al. (2015) and Index Fungorum, respectively.

DNA extraction, PCR amplification and sequencing

The mycelia growing on a PDA plate were used to extract DNA using the TriliefTM Plant Genomic DNA Kit (Tsingke Biological Technology Co., Ltd in Beijing, China), following the manufacturer’s instructions. The primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Vilgalys and Hester 1990), T1/T22 (Research & Service 1997), EF1-983F/EF1-2218R (Carbone and Kohn 1999), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999) were used for amplification of the internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), large subunit rDNA (28S), beta-tubulin (tub2), translation elongation factor 1-α (tef1) gene and RNA polymerase II second-largest subunit (rpb2), respectively. The PCR was performed in a 25 μL reaction volume, comprising 21 μL PCR Mix (2 × Rapid Taq Master Mix, Vazyme Biotech Co., Ltd., Nanjing, China), 1 μL of each primer, 2 μL of DNA template. For PCR amplification conditions see Table 1. The PCR products were visualized using agarose gel electrophoresis, and those with the targeted bands were sent to Sangon Biotech Co. Ltd., Kunming, China, for sequencing. The newly generated sequences were submitted to GenBank to obtain accession numbers.

Table 1.

Loci, primers, and PCR amplification conditions used in this study.

Locus Primers PCR amplification conditions Reference
ITS ITS5/ITS4 95 °C: 5 min, (95 °C: 15s, 55 °C: 15s, 72 °C: 15s) × 40 cycles White et al. (1990); Vilgalys and Hester (1990)
28S LR0R/LR5
tef1 EF1-983F/EF1-2218R 95 °C: 5 min, (95 °C: 45s, 52 °C: 45s, 72 °C: 70s) × 35 cycles Carbone and Kohn (1999)
tub2 T1/T22 95 °C: 5 min, (95 °C: 45s, 50 °C: 45s, 72 °C: 90s) × 35 cycles Research and Service (1997)
rpb2 fRPB2-5F/fRPB2-7cR 95 °C: 5 min, (95 °C: 45s, 55 °C: 120s, 72 °C: 50s) × 35 cycles Liu et al. (1999)
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Sequence alignment and phylogenetic analyses

The sequences were assembled using Sequencing Project Management (SeqMan) software (Clewley 1995). The assembled sequences were compared with the data in GenBank to determine their close relatives. The results indicate that our specimens were closely related to species of Clonostachys. Reference sequences for Clonostachys were obtained following recent studies (Bao et al. 2023; Liu et al. 2023; Perera et al. 2023; Piombo et al. 2023; Wang et al. 2023; Zhang et al. 2023; Zhao et al. 2023) (Table 2). Each gene matrix was separately aligned using MAFFT v. 6.8 (Katoh et al. 2018). The aligned datasets were manually edited using BioEdit v. 7.0.9 (Hall 1999) and then combined using SequenceMatrix v1.7.8 (Vaidya et al. 2011). The combined alignment was utilized for ML and BI analyses.

Table 2.

Names, voucher numbers, and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses in this study.

Taxa Voucher no. GenBank accession numbers Reference
ITS 28S tub2 tef1 rpb2
Clonostachysagrawalii CBS 533.81 AF358241 N/A AF358187 N/A N/A Schroers (2001)
C.ambigua PAD S00003 MT554898 N/A N/A N/A N/A Forin et al. (2020)
C.apocyni CBS 130.87 AF210688 N/A AF358168 N/A N/A Schroers (2001)
C.aranearum QLS 0625 NR_164542 N/A KU212400 N/A N/A Chen et al. (2016)
C.artemisiae MHZU 23-0116 OR365451 N/A OR700206 N/A N/A Dong et al. (2023)
C.aurantiaca CBS:124757 OQ910531 OQ910890 N/A OQ944545 OQ927609 Zhao et al. (2023)
C.aureofilvella CBS 195.93 AF358226 N/A AF358181 N/A N/A Schroers (2001)
C.australiana CBS:102421 OQ910540 OQ910899 OQ982584 OQ944554 OQ927618 Zhao et al. (2023)
C.bambusae CBS:139411 OQ910542 OQ910901 OQ982586 OQ944556 OQ927620 Zhao et al. (2023)
C.buxicola CBS:102419 OQ910544 OQ910903 OQ982588 OQ944558 OQ927622 Zhao et al. (2023)
C.byssicola CBS 364.78 MH861151 MH872912 AF358153 N/A N/A Vu et al. (2019)
C.capitata CBS 218.93 AF358240 MH874054 AF358188 N/A N/A Schroers (2001)
C.catenulata CBS 154.27 NR_165993 NG_063969 N/A OQ944810 OQ927866 Zhao et al. (2023)
C.chlorina CBS 287.90 NR_137651 MH873895 OQ982590 OQ944560 OQ927624 Schroers (2001)
C.chloroleuca CBS:141588 OQ910549 OQ910908 N/A OQ944563 OQ927627 Zhao et al. (2023)
C.chongqingensis HMAS 290894 OP205475 N/A OP205324 N/A N/A Zeng and Zhuang (2022)
C.compactiuscula CBS:123759 OQ910563 OQ910922 OQ982603 OQ944576 OQ927640 Zhao et al. (2023)
C.compactiuscula CBS 913.97 AF358245 N/A AF358194 N/A N/A Schroers (2001)
C.cylindrica CBS:101113 OQ910569 OQ910928 N/A OQ944582 OQ927646 Zhao et al. (2023)
C.divergens CBS 967.73 NR_137532 OQ910934 AF358191 OQ944587 N/A Schroers (2001)
C.ellipsoidea CBS 175.76 OQ910580 OQ910939 OQ982617 OQ944592 OQ927655 Zhao et al. (2023)
C.epichloe CBS 101037 AF210675 OQ910940 AF358209 OQ944593 OQ927656 Schroers (2001)
C.eriocamporesiana MFLU 18-2713 MN699132 N/A MN699965 MN699964 N/A Hyde et al. (2020b)
C.eriocamporesii MFLU 19-0486 MN699133 NG_068919 OQ982619 N/A N/A Hyde et al. (2020b)
C.farinosa CBS 914.97 AF358252 N/A AF358151 N/A N/A Schroers (2001)
C.flava CBS 915.97 OQ910619 OQ910978 OQ982654 OQ944631 OQ927690 Zhao et al. (2023)
C.fujianensis CBS:127474 OQ910620 OQ910979 OQ982655 OQ944632 OQ927691 Zhao et al. (2023)
C.fusca CBS 207.93 OQ910622 OQ910981 OQ982657 OQ944634 OQ927693 Zhao et al. (2023)
C.garysamuelsii CBS:123964 OQ910624 OQ910983 OQ982658 OQ944636 OQ927695 Zhao et al. (2023)
C.grammicospora CBS 209.93 NR_137650 NG_064165 AF358206 OQ944637 N/A Forin et al. (2020)
C.grammicosporopsis CBS 102834 AF358256 OQ910985 OQ982660 OQ944638 OQ927697 Vu et al. (2019)
C.granuligera PAD S00011 MT554904 N/A N/A N/A N/A Forin et al. (2020)
C.hongkongensis CBS:115291 OQ910630 OQ910989 OQ982663 OQ944642 OQ927700 Zhao et al. (2023)
C.impariphialis HMAS 275560 KX096609 KX096606 N/A N/A N/A Zeng and Zhuang (2022)
C.indica RKV2015 KT291441 N/A N/A N/A N/A Prasher and Chauhan (2017)
C.intermedia CBS 508.82 NR_137652 OQ910991 AF358205 OQ944644 N/A Schroers (2001)
C.kowhai CBS 461.95 NR_154748 OQ910992 AF358170 OQ944645 OQ927702 Schroers (2001)
C.krabiensis MFLU 16-0254 NR168189 MH376707 N/A N/A N/A Tibpromma et al. (2018)
C.krabiensis CBS 192.96 OQ910634 OQ910993 OQ982666 OQ944646 OQ927703 Zhao et al. (2023)
C.kunmingensis YFCC: 898 MW199069 MW199058 MW201676 MW295969 N/A Wang et al. (2023)
C.leptoderma HMAS 255834 OP205474 N/A OP205323 N/A N/A Zeng and Zhuang (2022)
C.leucaenae MFLU 20-0008 ON230050 ON230058 N/A N/A N/A Perera et al. (2023)
C.levigata CBS 948.97 AF210680 N/A AF358196 N/A N/A Schroers (2001)
C.linzhiensis HKAS 133179 PQ522504 PQ634391 PQ650459 PQ650477 N/A present study
C.linzhiensis HKAS 133180 PQ522505 PQ634392 PQ650460 PQ650478 N/A present study
C.longiphialidica CBS 112.87 OQ910643 OQ911002 N/A OQ944655 OQ927712 Zhao et al. (2023)
C.lucifer CBS 100008 AF210683 OQ911003 AF358208 OQ944656 OQ927713 Schroers (2001)
C.miodochialis CBS 997.69 NR_137649 NG_064076 AF358210 OQ944658 OQ927715 Schroers (2001)
C.moreaui CBS:127881 OQ910647 OQ911006 OQ982678 OQ944659 OQ927716 Zhao et al. (2023)
C.motuoensis HKAS 133181 PQ522506 PQ634393 PQ650461 PQ650479 N/A present study
C.motuoensis HKAS 133182 PQ522507 PQ634394 PQ650462 PQ650480 N/A present study
C.oblongispora CBS 100285 AF358248 OQ911007 AF358169 OQ944660 OQ927717 Schroers (2001)
C.obovatispora CBS:118752 OQ910649 OQ911008 OQ982680 OQ944661 OQ927718 Zhao et al. (2023)
C.oligospora HMAS 290895 OP205473 N/A OP205322 N/A N/A Zeng and Zhuang (2022)
C.pallens PAD S00004 MT554899 N/A N/A N/A N/A Forin et al. (2020)
C.palmae CBS 119.87 OQ910650 OQ911009 OQ982681 OQ944662 OQ927719 Zhao et al. (2023)
C.parasporodochialis CBS 192.93 OQ910651 OQ911010 OQ982682 OQ944663 OQ927720 Zhao et al. (2023)
C.penicillata CBS 729.87 OQ910654 OQ911013 OQ982685 OQ944666 OQ927722 Zhao et al. (2023)
C.pilosella CLLG19028 N/A NG_153902 N/A N/A N/A Lechat et al. (2020)
C.pityrodes CBS 102033 AF210672 OQ911014 AF358212 N/A OQ927723 Schroers (2001)
C.pnagiana CLLG19041 N/A NG_153903 N/A N/A N/A Lechat et al. (2020)
C.pseudochroleuca CBS 192.94 AF358238 N/A AF358171 N/A N/A Schroers (2001)
C.pseudostriata CBS 309.96 OQ910673 OQ911032 OQ982704 OQ944685 OQ927741 Zhao et al. (2023)
C.pseudostriatopsis h116 N/A N/A AB237465 N/A N/A Hirooka and Kobayashi (2007)
C.ralfsii CBS 129.87 AF210676 N/A AF358195 N/A N/A Schroers (2001)
C.reniformis CBS 695.86 OQ910685 OQ911044 OQ982714 OQ944697 OQ927753 Zhao et al. (2023)
C.rhinolophicola KUMC 21-0438 ON426841 N/A OR025936 N/A N/A Liu et al. (2023)
C.rhinolophicola HKAS122257 ON426840 N/A OR025937 N/A N/A Liu et al. (2023)
C.rhizophaga CBS 202.37 AF358225 MH867396 AF358156 N/A N/A Schroers (2001)
C.rogersoniana CBS 582.89 AF210691 N/A AF358189 N/A N/A Schroers (2001)
C.rosea CBS 1221.71 DQ674381 OQ911077 OQ982747 OQ944730 OQ927786 Zhao et al. (2023)
C.samuelsii CBS 699.97 OQ910812 N/A AF358190 N/A N/A Zhao et al. (2023)
C.setosa CBS 834.91 AF210670 N/A AF358211 N/A N/A Schroers (2001)
C.solani CBS 101924 AF358232 OQ911196 AF358180 OQ944847 OQ927902 Schroers (2001)
C.spinulosa MFLU 17-0131 ON230049 N/A ON238009 N/A N/A Perera et al. (2023)
C.sporodochialis CBS 101921 AF210685 N/A AF358149 N/A N/A Schroers (2001)
C.squamuligera PAD S00020 MT554908 N/A N/A N/A N/A Forin et al. (2020)
C.squamuligera PAD S00021 MT554909 N/A N/A N/A N/A Forin et al. (2020)
C.subquaternata CBS 100003 MT537603 N/A N/A N/A N/A Forin et al. (2020)
C.vacuolata CBS 191.93 OQ910868 OQ911227 N/A OQ944876 OQ927931 Zhao et al. (2023)
C.venezuelae CBS 107.87 OQ910869 OQ911228 OQ982884 OQ944877 OQ927932 Zhao et al. (2023)
C.vesiculosa HMAS 183151 NR_119828 HM050302 N/A N/A N/A Luo and Zhuang (2010)
C.viticola CAA 944 MK156282 N/A MK156290 MK156286 N/A Torcato et al. (2020)
C.viticola MFLU 18-2770 MT215573 MT396164 N/A MT212204 N/A Perera et al. (2020)
C.wenpingii HMAS 172156 NR_119651 MH874867 N/A N/A N/A Luo and Zhuang (2007)
C.yadongensis HKAS 133183 PQ522508 PQ634395 PQ650463 PQ650481 PQ538524 present study
C.yadongensis HKAS 133184 PQ522509 PQ634396 PQ650464 PQ650482 PQ538525 present study
C.zelandiaenovae CBS 100979 AF358229 OQ911231 N/A OQ944880 OQ927935 Schroers (2001)
C.zelandiaenovae CBS 232.80 AF210684 N/A AF358185 N/A N/A Schroers (2001)
Mycocitruscoccicola HD 2016 KU720552 KU720545 N/A N/A N/A Dao et al. (2016)
M.coxeniae BRIP 49559a OQ629341 N/A N/A N/A N/A Zhao et al. (2023)
Sesquicilliumaquaticum HKAS 125804 OP876724 OP875077 N/A N/A N/A Bao et al. (2023)
S.buxi CBS 696.93 AF210667 KM231721 AF358215 KM231977 KM232416 Schroers (2001)
S.candelabrum CBS 504.67 AF210668 N/A N/A N/A N/A Schroers (2001)
S.candelabrum YFCC 896 MW199067 N/A MW201674 N/A N/A Wang et al. (2023)
S.essexcoheniae BRIP 75170a OQ629342 N/A N/A OQ944511 OQ914830 Zhao et al. (2023)
S.phyllophila CBS 921.97 NR_137531 N/A N/A N/A N/A Schroers (2001)
S.rossmaniae CBS 210.93 AF358227 N/A AF358213 N/A N/A Vu et al. (2019)
S.saulense BRFM 2782 MK635054 N/A N/A N/A N/A Lechat et al. (2020)
S.sesquicillii CBS 180.88 AF210666 NG_228796 AF358214 OQ944535 N/A Schroers (2001)
S.shanghaiense HMAS 351878 OL897002 OL897044 N/A N/A N/A Zhang et al. (2023)
S.shanghaiense GZUIFR 21.916 OL897003 OL897045 N/A N/A N/A Zhang et al. (2023)
Fusariumacutatum CBS 402.97 NR_111142 N/A MT011051 N/A N/A Luo and Zhuang (2007)
Nectriacinnabarina CBS 279.48 AF163025 HM484754 HM484802 HM484649 N/A Hirooka et al. (2011)
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The newly generated sequences are in red. The type strains are indicated in bold. The synonymizing are indicated in green. N/A denotes the unavailable data in GenBank.

A rapid phylogenetic analysis was performed utilizing OFPT (Zeng et al. 2023) according to its standard protocol. The final phylogenetic analyses were carried out on the CIPRES Science Gateway platform (https://www.phylo.org), employing RAxML-HPC v.8 on XSEDE (8.2.12) for maximum likelihood (ML) estimation and MrBayes on XSEDE (3.2.7a) for Bayesian inference (BI). Phylogenetic results were represented by ML bootstrap values (MLB) equal to or greater than 70% and a posterior probability in Bayesian statistics (BYPP) equal to or exceeding 0.90. These values were displayed above each node in all resulting trees. For visualization purposes, the resulting phylograms were displayed using the FigTree v1.4.0 program. The final reorganization was accomplished using Adobe Illustrator 2020.

Results

Phylogenetic analyses

The combined 28S, tef1, rpb2, ITS, and tub2 dataset comprised 104 taxa. Fusariumacutatum (CBS 402.97) and Nectriacinnabarina (CBS 279.48) were selected as outgroup taxa (Prasher and Chauhan 2017; Lechat et al. 2020). The dataset consisted of 3146 total characters, including gaps (28S: 1–784 bp; tef1: 785–1596; rpb2: 1597–2349; ITS: 2350–2826; tub2: 2827–3828). The matrix had 1079 distinct alignment patterns, with 41.89% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.229764, C = 0.268281, G = 0.268313, T = 0.233642; substitution rates: AC = 1.37920, AG = 4.09491, AT = 1.37920, CG = 0.794178, CT = 8.784537, GT = 1.00000; gamma distribution shape parameter α = 0.494958. The best-scoring RAxML tree with a final likelihood value of -23046.167770 is presented in (Fig. 1). Our specimens Clonostachyslinzhiensis (HKAS 133179 & HKAS 133180) and C.motuoensis (HKAS 133181 & HKAS 133182) formed distinct monophyletic clades with C.aranearum with support value of (75% ML) and (85% ML), indicating they are closely related. The two specimens HKAS 133183 and HKAS 133184 formed a sister clade to C.krabiensis with high support (100 ML/0.91 PP).

Figure 1.

Figure 1.

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Phylogenetic tree generated from maximum likelihood analysis based on a combined 28Stef1, rpb2, ITS and tub2 sequence dataset. Bootstrap support values for ML equal to or greater than 70% and PP equal to greater than 0.90 are indicated at the nodes as MLB/BYPP. The ex-type strains are in bold, while the new isolates are in red, and the synonymizing taxa are indicated in green.

Taxonomy

Clonostachys

. Clonostachys

Corda, Pracht-Fl. Eur. Schimmelbild: 31 (1839)

FBDBE180-C198-50DB-A046-6009B883BB13

Index Fungorum: IF7701

Facesoffungi Number: FoF02102

Classification.

Bionectriaceae, Hypocreales, Sordariomycetes.

Morphological characteristics.

Sexual morph: Ascomata perithecial. Perithecia superficial, solitary to gregarious, subglobose to globose, papillate or non-papillate, no colour change in 3% KOH or 100% LA. Asci clavate to subcylindrical, 6–8-spored. Ascospores ellipsoidal to oblong ellipsoidal, uniseptate, hyaline, smooth-walled, uniseriate or irregular biseriate. Asexual morph: Hyphomycetous. Conidiophores dimorphic or monomorphic, sporodochial, synnematous, hyaline, brown or blackish brown. Phialides phialidic, cylindrical to flask-shaped. Conidia aseptate, hyaline, smooth, ovoid to ellipsoid.

Type species.

Clonostachysaraucaria Corda, Pracht-Fl. Eur. Schimmelbild.: 31 (1839)

Notes.

Clonostachys is the second largest genus in Bionectriaceae, with 130 epithets (Index Fungorum 2025). Several members of Clonostachys are ecologically and economically important (Abeywickrama et al. 2023). Some Clonostachys spp. are destructive, including parasitic in myxomycetes, nematodes, ticks, molluscs, and leafhoppers (Schroers 2001; Toledo et al. 2006; Perera et al. 2023). Clonostachysrosea and C.catenulata are reported as destructive to ascomycetes and basidiomycetes (Schroers 2001; Chatterton et al. 2008) and C.chuyangsinensis and C.aranearum have been reported as spider-pathogenic fungi (Wan et al. 2016; Wang et al. 2023).

Clonostachysrosea has been studied as a potential biological control agent for various plant diseases and pests such as strawberry gray mold (Cota et al. 2008), Fusarium head blight of wheat (Xue et al. 2008), and Pythiumtracheiphilum in Chinese cabbage (Møller et al. 2003). Several closely related species to Clonostachysrosea, such as C.byssicola, C.chloroleuca, C.rhizophaga, and C.solani also possess biocontrol properties (Mendoza García et al. 2003; Krauss et al. 2013; Sun et al. 2017; Broberg et al. 2021).

. Clonostachys linzhiensis

S.C. He, K.D. Hyde & Q. Zhao sp. nov.

1CE081EA-EA63-5FC6-B3C9-947F82B9A3F3

Index Fungorum: IF902917

Facesoffungi Number: FoF16789

Fig. 2

Figure 2.

Figure 2.

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Clonostachyslinzhiensis (HKAS 133179, Holotype) a, b culture on PDA (a above b below) c colonies on WAd–g conidiophores h, i phialides j–q conidia. Scale bars: 50 μm (d–g); 50 μm (h, i); 5 μm (j–q).

Etymology.

The species epithet is derived from Linzhi City, where the holotype was collected.

Typification.

China • Xizang Autonomous Region, Linzhi City, Motuo County (29°11'N, 95°8'E, 1561 m), on the lower part of the leaves of Houttuyniacordata, July 27, 2022, collected by Hong-De Yang, YHD691 (holotype: KUN-HKAS 133179); ex-type living culture: KUNCC24-18528). GenBank: ITS: PQ522504, 28S: PQ634391, tef1: PQ650477, tub2: PQ650459.

Description.

Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies on the WA, raised, medium sparse, rough, white at apex. Conidiophores mononematous, erect, simple, verticillium-like, straight or flexuous, branched, smooth-walled, thin-walled, septate, hyaline, produce globose cells at the apex, terminal branches developing into phialides, 110–232 × 2.5–3.9 μm (x̄ = 170 × 3.2 μm, n = 20). Phialides polytretic, terminal on branches, phialides cylindrical but slightly tapering towards the tips, aseptate, hyaline, smooth, thin-walled, terminal developing into conidia, 15.3–23.8 × 1.5–3.3 μm (x̄ = 19.8 × 2.2 μm, n = 20). Conidia amerospores, solitary, acrogenous, simple, doliiform to ellipsoidal, smooth, thin-walled, aseptate, hyaline, 3.9–5.7 × 2.2–3.2 μm (x̄ = 4.7 × 2.6 μm, n = 30).

Culture characteristics.

Colonies on PDA reaching 5.0–5.5 cm after 20 days of incubation at 25 °C, white above, pale yellow reverse, medium spare, concave in the center, convex around, hairy, lobate, velvety, ciliate, not pigment produced,

Habitat.

Leaves of Houttuyniacordata.

Additional material examined.

China • Xizang Autonomous Region, Linzhi City, Motuo County (29°11'N, 95°8'E, 1561 m), on the lower part of the leaves of Houttuyniacordata, July 27, 2022, collected by Hong-De Yang, HSC983 (isotype: KUN-HKAS 133180); ex-isotype living culture: KUNCC24-18529). GenBank: ITS: PQ522505, 28S: PQ634392, tef1: PQ650478, tub2: PQ650460.

Notes.

In the phylogenetic analysis, Clonostachyslinzhiensis shared a close phylogenetic relationship with C.aranearum and C.motuoensis (Fig. 1). Clonostachyslinzhiensis shares similar morphology to C.aranearum and C.motuoensis in having mononematous, erect, verticillium-like conidiophores that are straight or flexuous, smooth-walled, hyaline, phialides are polytretic, terminal, flask-shaped, aseptate, hyaline, smooth and the conidia are amerospores, acrogenous, ellipsoidal, aseptate, hyaline (Wan et al. 2016). However, Clonostachyslinzhiensis (HKAS 133179 and HKAS 133180) has larger conidiophores (L/W ratio: 53 vs 12 and L/W ratio: 53 vs 35) and longer phialides (L/W ratio: 9 vs 6.7 and L/W ratio: 9 vs 4.7) in comparison to C.aranearum and C.motuoensis. Furthermore, the ITS and tub2 sequence of Clonostachyslinzhiensis differs from C.aranearum which revealed 13/510 (2.5%) and 7/291 (2.4%) base pair differences, respectively. Based on the differences in morphology (larger conidiophores and longer phialides) and phylogeny, along with the guidelines of Maharachchimbukura et al. (2021), we identify our specimen as a new species, C.linzhiensis.

. Clonostachys motuoensis

S.C. He, K.D. Hyde & Q. Zhao sp. nov.

B6037BCA-496F-5A18-AAE3-6BE6055D2D80

Index Fungorum: IF902918

Facesoffungi Number: FoF16790

Fig. 3

Figure 3.

Figure 3.

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Clonostachysmotuoensis (HKAS 133181, Holotype) a, b culture on PDA (a above b below) c, d colonies on WAe–h conidiophores and conidiophores apex i–l phialides m conidia. Scale bars: 50 μm (e, g, h); 25 μm (f, i, k, l); 10 μm (j, m).

Etymology.

The species epithet is derived from the location “Motuo County”, from where the holotype was collected.

Typification.

China • Xizang Autonomous Region, Linzhi City, Motuo County (29°11'N, 95°8'E, 1561 m), on the lower part of the leaves of Houttuyniacordata, July 27, 2022, collected by Hong-De Yang, YHD669-1 (holotype: KUN-HKAS HKAS 133181); ex-type living culture: KUNCC24-18530). GenBank:ITS: PQ522506, 28S: PQ634393, tef1: PQ650479, tub2: PQ650461.

Description.

Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies on the WA, solitary or gregarious, white to pale yellow, raised, dense, rough. Conidiophores mononematous, penicillate, straight or flexuous, branched at the apex, smooth, thin-walled, septate, hyaline, conidiophores produce globose cells at the apex, from globose to elongated or continue to differentiate, terminal branches developing into phialides, 94–146 × 2.5–4.7 μm (x̄ = 125 × 3.5 μm, n = 20). Phialides monophialidic, terminal, flask-shaped, aseptate, hyaline, smooth, thin-walled, terminal developing into conidia, 9.1–18.7 × 2.3–3.5 μm (x̄ = 13.2 × 2.8 μm, n = 20). Conidia amerospores, solitary, acrogenous, simple, ellipsoidal to oblong with obtuse ends, smooth, thin-walled, aseptate, hyaline, minutely guttulate, 3.9–5.6 × 2.5–3.3 μm (x̄ = 4.6 × 2.9 μm, n = 30).

Culture characteristics.

Colonies on PDA reaching 3.5–4 cm after 20 days of incubation at 25 °C, white both above and reverse, medium spare, raised, smooth, fimbriate, velvety, ciliate, not pigment produced.

Habitat.

Leaves of Houttuyniacordata.

Additional material examined.

China • Xizang Autonomous Region, Linzhi City, Motuo County (29°11'N, 95°8'E, 1561 m), on the lower part of the leaves of Houttuyniacordata, July 27, 2022, collected by Hong-De Yang, HSC986 (isotype: KUN-HKAS 133182); ex-isotype living culture: KUNCC24-18531). GenBank: ITS: PQ522507, 28S: PQ634394, tef1: PQ650480, tub2: PQ650462.

Notes.

In the phylogenetic analysis, Clonostachysmotuoensis clustered sister to C.linzhiensis and C.aranearum (Fig. 1). Morphologically, our specimen (HKAS 133181 and HKAS 133182) has larger conidiophores (L/W ratio: 35 vs 12) and longer phialides (L/W ratio: 4.7 vs 6.7) in comparison to C.aranearum. Clonostachysmotuoensis differs from C.aranearum by 6/544 (1%) ITS and 4/294 (1.3%) tub2 differences in the nucleotides. It is worth noting that C.aranearum is parasitic on spiders, while C.motuoensis is endophytic on Houttuyniacordata leaves. In addition, C.aranearum was collected from Qian Ling Shan Park, Guiyang City, Guizhou Province, China, with an altitude of 1100–1369 m, belonging to a plateau subtropical climate (Wan et al. 2016). Clonostachysmotuoensis was collected from Motuo County, Linzhi City, Xizang Autonomous Region, China, with an altitude of 1561 m, belonging to a tropical rainforest climate. Based on these distinctions and following the guidelines of Maharachchimbukura et al. (2021), we identified our specimen as a new species, C.motuoensis.

. Clonostachys yadongensis

S.C. He, K.D. Hyde & Q. Zhao sp. nov.

47BAC712-6287-5A66-978F-A4B9754C9ACD

Index Fungorum: IF902919

Facesoffungi Number: FoF16791

Fig. 4

Figure 4.

Figure 4.

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Clonostachysyadongensis (HKAS 133183, Holotype) a, b culture on PDA (a above b below); c colonies on WAd–h conidiophores f–j phialides k, l conidia. Scale bars: 50 μm (d–f); 20 μm (g–l).

Etymology.

The species epithet is derived from Yadong County, where the holotype was collected.

Typification.

China • Xizang Autonomous Region, Linzhi City, Yadong County (27°48'N, 88°83'E, 3894 m), on the lower part of the leaves of Ageratinaadenophora leaves, July 24, 2023, collected by Shu-Cheng He, HSC1025 (holotype: KUN-HKAS 133183); ex-type living culture: KUNCC24-18532). GenBank:ITS: PQ522508, 28S: PQ634395, tef1: PQ650481, tub2: PQ650463, rpb2: PQ538524.

Description.

Sexual morph: Not observed. Asexual morph: Hyphomycetous. Colonies on the WA, solitary or gregarious, white to pale yellow, raised, medium sparse, rough. Conidiophores mononematous, penicillate, straight or flexuous, branched, smooth-walled, thin-walled, septate, hyaline, produce globose cells at the apex, terminal branches developing into phialides, 80–118 × 2.4–3.4 μm (x̄ = 97 × 2.8 μm, n = 20). Phialides polyblastic, terminal, flask-shaped, aseptate, hyaline, smooth, thin-walled, minutely guttulate, terminal developing into conidia, 9.6–15.6 × 1.7–2.3 μm (x̄ = 13.1 × 2 μm, n = 20). Conidia amerospores, solitary, acrogenous, simple, oval to ellipsoidal, smooth, thin-walled, aseptate, hyaline, minutely guttulate, 3.6–5.4 × 2.6–3.3 μm (x̄ = 4.5 × 2.9 μm, n = 30).

Culture characteristics.

Colonies on PDA reaching 5.5–6 cm after 20 days of incubation at 25 °C, white above, pale yellow reverse, medium spare, raised, hairy, fimbriate, velvety, ciliate, not pigment produced.

Habitat.

Leaves of Ageratinaadenophora.

Additional material examined.

China • Xizang Autonomous Region, Linzhi City, Yadong County (27°48'N, 88°83'E, 3894 m), on the lower part of the leaves of Ageratinaadenophora, July 24, 2023, collected by Shu-Cheng He, HSC1025A (isotype: KUN-HKAS 133184; ex-isotype living culture: KUNCC24-18533). GenBank:ITS: PQ522509, 28S: PQ634391, tef1: PQ650482, tub2: PQ650464, rpb2: PQ538525.

Notes.

In the phylogenetic analysis, Clonostachysyadongensis clustered with C.krabiensis with 100% MLB and 0.91 BYPP support (Fig. 1). Clonostachyskrabiensis was introduced by Tibpromma et al. (2018) and is characterized by solitary, superficial, globose to subglobose, orange to brownish orange ascomata, 6–8-spored, cylindrical to clavate asci; fusoid to ellipsoidal, hyaline, with longitudinal striations, granulate ascospores. Its morphology fits well with the generic concept of Clonostachys sexual morph (Bao et al. 2023; Perera et al. 2023; Zhao et al. 2023). Our specimen (HKAS 133183) exhibited an asexual morph that is characterized by mononematous, penicillate, erect conidiophores; flask-shaped or cylindrical, aseptate, hyaline phialides; acrogenous, ellipsoidal or oblong with obtuse ends, hyaline conidia. The 28S and ITS sequences of Clonostachysyadongensis differ from that of C.krabiensis which showed base pair differences, 3/825 (0.35%), 11/513) and (2.1%) respectively. Clonostachyskrabiensis was reported in Papua New Guinea and Thailand as a saprobe on Pandanus sp. and wood litter, while C.yadongensis was reported in the Xizang Autonomous Region, China, mainly as an endophyte on Ageratinaadenophora. Clonostachyskrabiensis has been reported to have a sexual morph, but C.yadongensis has only been observed in its asexual morph. Based on base pair differences and following the guidelines of Maharachchimbukura et al. (2021), we identified our specimen as a new species, Clonostachysyadongensis.

. Clonostachys viticola

C. Torcato & A. Alves, Int. J. Syst. Evol. Microbiol, 6 (2020)

A51DC5C8-09AC-52B0-9255-010571E3BCF4

Index Fungorum: IF835021

Facesoffungi Number: FoF16792

Basionym.

Clonostachysswieteniae R.H. Perera, E.B.G. Jones & K.D. Hyde, Mycosphere 11(1): 2135 (2020)

Description and illustration.

Perera et al. 2020 and Torcato et al. 2020.

Notes.

In the multigene phylogenetic analyses, Clonostachysviticola with C.swieteniae, forms a monophyletic clade in Clonostachys. The taxa in this clade show low genetic differences. Thus, we recommend treating C.viticola and C.swieteniae as conspecific. Clonostachysviticola was established by Torcato et al. (2020) from the root of Vitisvinifera in a terrestrial habitat of Peru (Torcato et al. 2020) and Clonostachysswieteniae was established by Perera et al. (2020) from decaying fruits of Swieteniamahagoni in a terrestrial habitat of Thailand (Perera et al. 2020). Morphologically, C.viticola with C.swieteniae are highly similar, but there are minor differences in phialides (13.1 × 2.1 μm vs 11.4 × 2.6 μm), and conidia (5.6 × 2.9 μm vs 6 × 2.2 μm). Through base pair comparison, the ITS and tef1 sequence of Clonostachysviticola differs from that of C.swieteniae in 0/500 (0%) and 3/406 (0.7%), respectively. The results indicate that different environments have shaped the morphology (Bhunjun et al. 2022; Hyde et al. 2020b; Phukhamsakda et al. 2022). Clonostachysviticola was published prior to C.swieteniae. Therefore, we propose C.swieteniae as a synonym of C.viticola.

New combinations of Sesquicillium

. Sesquicillium

W. Gams, Acta bot. neerl. 17(6): 455 (1968)

1A0F5493-7137-5C5F-A4EF-7B21BBDF4DB4

Index Fungorum: IF9906

Facesoffungi Number: FoF16793

Classification.

Bionectriaceae, Hypocreales, Sordariomycetes

Morphological characteristics.

Sexual morph: Ascomycetous. Perithecia solitary, gregarious or loosely aggregated, globose to subglobose, 200–400 μm diam, pale yellow or pale to light orange, not papillate, Perithecial wall either consisting of two or one major wall regions. Asci clavate, 8-spored, with flat or rounded apex. Ascospores aseptate or 1-septate, hyaline, spinulose, warted, with short striae, ellipsoidal to fusiform. Asexual morph. Hyphomycetous. Conidiophores macronematous, mononematous, monomorphic or dimorphic, penicillate, verticillate; branches at apex. Phialides one or two successive intercalary phialides, terminal, terminal whorls consisting of narrowly flask-shaped, hyaline. Conidia obovoid, ellipsoid, or fusoid, slightly curved or straight, hyaline, aseptate, smooth-walled, thin-walled.

Type species.

Sesquicilliumbuxi (J.C. Schmidt ex Link) W. Gams, Acta bot. neerl. 17(6): 455 (1968)

Notes.

Sesquicillium was established by Gams (1968). Morphologically, Sesquicillium shares similar characteristics with Clonostachys in that the conidiophores are macronematous, monomorphic or dimorphic, penicillate, verticillate-like, branched, flask-shaped conidiogenous cells (Preedanon et al. 2023; Zhao et al. 2023). Zhao et al. (2023) revealed the close relationship between Clonostachys and Sesquicillium and reclassified eight species of Clonostachys to Sesquicillium. The difference between Sesquicillium and Clonostachys lies in the development of their conidiophores. In Sesquicillium, the conidiophore will form a lateral conidia process after bifurcation, leading to the production of conidia. In Clonostachys, the conidiophore will not form lateral conidia protrusions after bifurcation. It continues to differentiate into terminal phialides (Gams 1968; Schroers 2001). Based on the research of Chen et al. (2023), and Zhao et al. (2023), we used ITS, 28S, tef1, tub2, and rpb2 to reconstruct a phylogenetic tree to investigate the relationship of Clonostachys species. The results show that Clonostachysaquatica and C.shanghaiensis are far from Clonostachys and more closely related to Sesquicillium. Therefore, based on morphological and phylogenetic analysis, we propose C.aquatica and C.shanghaiensis are synonyms of S.aquaticum and S.shanghaiense.

. Sesquicillium aquaticum

(D.F. Bao, K.D. Hyde & Z.L. Luo) S.C. He, K.D. Hyde & Jayaward, [as ‘aquatica’] comb. nov.

4E2F58A7-F517-54B1-9252-8C1819871A51

Index Fungorum: IF903022

Facesoffungi Number: FoF16794

Basionym.

Clonostachysaquatica D.F. Bao, K.D. Hyde & Z.L. Luo, Fungal Diversity, (2023).

Holotype.

HKAS 125804.

Description and illustration.

See Bao et al. 2023.

Notes.

Clonostachysaquatica was established by Bao et al. (2023) based on ITS and tub2 sequence data (holotype HKAS 125804). Through the study of Bao et al. (2023), C.aquatica clustered as a clade sister to C.rossmaniae with strong support (94% MLB, 98% MYPP). Following Bao et al. (2023), we added 28S, tef1 and rpb2 sequence data, and the results showed that C.aquatica clustered with Sesquicilliumessexcoheniae (100% MLB, 0.97 BYPP), forming a successive sister clade with S.rossmaniae (99% MLB,/1.00 BYPP) (Fig. 1). Clonostachysaquatica shows a closer relationship with Sesquicillium in phylogenetic analysis. Therefore, based on phylogenetic analysis, we propose C.aquatica as a synonym of S.aquaticum.

. Sesquicillium shanghaiense

(Zhi Yuan Zhang, Y.F. Han & Z.Q. Liang) S. C. He, K.D. Hyde & Jayaward, [as ‘shanghaiensis’] comb. nov.

65F6ECAE-541C-5621-A893-BF016B004401

Index Fungorum: IF903023

Facesoffungi Number: FoF16795

Basionym.

Clonostachysshanghaiensis Zhi Yuan Zhang, Y.F. Han & Z.Q. Liang, MycoKeys 98: 198 (2023).

Holotype.

HMAS 351878.

Description and illustration.

Zhang et al. (2023).

Notes.

Clonostachysshanghaiensis was established by Zhang et al. (2023), based on ITS and tub2 sequence data (HMAS 351878). Clonostachysshanghaiensis clustered as a sister clade to C.rossmaniae (95% MLB, 0.99 BYPP) (Zhang et al. 2023). In this study, phylogenetic analysis showed that Clonostachysshanghaiensis formed a successive sister clade with S.phyllophila, S.saulensis, and S.candelabrum (Fig. 1). It is worth noting that S.phyllophila, S.saulense, and S.candelabrum were renamed by Zhao et al. (2023) as C.phyllophila (Schroers 2001), C.saulensis (Lechat et al. 2020), C.candelabrum (Schroers 2001) and C.chuyangsinensis (Wang et al. 2023) based on morphology and phylogenetic analysis. Therefore, based on phylogenetic analysis, we propose C.shanghaiensis as a synonym of S.shanghaiense.

Discussion

Rossman et al. (2001) studied the asexual species in 15 genera of Bionectriaceae (Hypocreales) using 28S sequence data and showed that Bionectriaceae formed a monophyletic group. Recently, additional DNA gene sequences such as acl1, tub2, rpb1, and tef1 have been used to enhance the precision of phylogenetic trees within the Clonostachys/Bionectria species (Moreira et al. 2016). However, available sequence data for these four protein-encoding gene regions is lacking in GenBank (Moreira et al. 2016). Wang et al. (2023), stated that tef1 sequence data showed the highest resolution for distinguishing Clonostachys species (tef1>tub2>ITS) based on the investigation conducted for genetic divergence comparisons of Clonostachys. Zhao et al. (2023) investigated the generic delineation with broad taxon sampling with morphology and multi-gene (ITS, 28S, tef1, tub2, rpb2) phylogenetic analysis and found a close relationship to Sesquicillium. Further, Sesquicillium was resurrected to accommodate the former subgenera Epiphloea and Uniparietina (Zhao et al. 2023). We constructed a phylogenetic tree (Fig. 1) of Clonostachys based on five genes (28S, tef1, rpb2, ITS, and tub2) and show that Clonostachys/Bionectria form a similar topology with Perera et al. (2023). However, as with other studies, we did not achieve a well-supported clade, as some but not all subgenera are mono- or paraphyletic (Moreira et al. 2016; Bao et al. 2023; Perera et al. 2023; Wang et al. 2023; Zhao et al. 2023). Morphologically, the asexual morphs of Clonostachys exhibit similarities with those of Sesquicillium (Preedanon et al. 2023), Penicillium (Crous et al. 2023), Verticillium (Crous et al. 2022), Gliocladium (Rehner and Samuels 1994) acremonium-like (Preedanon et al. 2023). They typically feature macronematous, monomorphic penicillate, or dimorphic penicillate conidiophore. Based on recent studies by Bao et al. (2023), Wang et al. (2023), and Zhao et al. (2023), we have clarified the relationships within the Clonostachys and proposed that C.aquatica, C.shanghaiensis, and C.swieteniae be considered synonyms of S.aquaticum, S.shanghaiense, and C.viticola, respectively. Clonostachysaquatica and C.shanghaiensis were positioned in a distantly related clade (Clade II) to Clonostachyssensu stricto.Mycocitrus and Sesquicillium, were positioned between Clade I and II (Fig. 1). Thus, further studies are required for the phylogenetic resolution of Clonostachys.

Clonostachys is reported in various plant hosts: Apocynaceae, Arecaceae, Asteraceae, Boraginaceae, Buxaceae, Ericaceae, Fagaceae, Leguminosae, Melampsoraceae, Nelumbonaceae, Pandanaceae, Rosaceae, and Rutaceae (Wang et al. 2023; Jayawardena et al. 2025). Our study reported three new species from Eupatorieae (C.yadongensis) and Saururaceae (C.linzhiensis and C.motuoensis). Clonostachys species exhibit a saprobic or endophytic lifestyle, playing crucial roles in nutrient cycling and plant health (Zeng and Zhuang 2022). Clonostachys species are significant for their adaptability and potential as biological control agents against plant pathogens (Wang et al. 2023; Zhao et al. 2023).

Supplementary Material

XML Treatment for Clonostachys
XML Treatment for Clonostachys linzhiensis
XML Treatment for Clonostachys motuoensis
XML Treatment for Clonostachys yadongensis
XML Treatment for Clonostachys viticola
XML Treatment for Sesquicillium
XML Treatment for Sesquicillium aquaticum
XML Treatment for Sesquicillium shanghaiense

Acknowledgements

This study is supported by the Second Tibetan Plateau Scientific Expedition and Research (STEP) Program (Grant No. 2019QZKK0503), the Yunnan Revitalization Talent Support Program: Science & Technology Champion Project (202305AB350004), the Major Science and Technology projects and key R&D plans/programs, Yunnan Province (202202AE090001) and the Survey of Wildlife Resources in Key Areas of Tibet (ZL202303601), the grant number E1644111K1, titled “Flexible introduction of high-level expert program, Kunming Institute of Botany, Chinese academy of sciences” for its financial support. Vinodhini Thiyagaraja thanks Yunnan Province “Caiyun Postdoctoral Program” in 2023, Choi Wan Postdoctoral Program in 2023, and National Postdoctoral funding, China. Chitrabhanu S. Bhunjun would like to thank the National Research Council of Thailand (NRCT) grant “Total fungal diversity in a given forest area with implications towards species numbers, chemical diversity, and biotechnology” (grant no. N42A650547). The authors extend their appreciation to the Researchers Supporting Project number (RSP2025R114), King Saud University, Riyadh, Saudi Arabia. Shu-cheng He thanks Mae Fah Luang University for the basic research scholar 2567 grant.

Citation

He S, Thiyagaraja V, Bhunjun CS, Chomnunti P, Dissanayake LS, Jayawardena RS, Yang H, Zhao YW, Al-Otibi F, Zhao Q, Hyde KD (2025) Morphology and multi-gene phylogeny reveal three new species of Clonostachys and two combinations of Sesquicillium (Bionectriaceae, Hypocreales) from Xizang, China. MycoKeys 115: 43–66. https://doi.org/10.3897/mycokeys.115.139757

Funding Statement

Kunming Institute of Botany, Chinese Academy of Sciences

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the Chinese Research Fund, grant number E1644111K1, titled “Flexible introduction of the high-level expert program, Kunming Institute of Botany, Chinese Academy of Sciences.

Author contributions

S.-C.H and V.T. conceived and designed the study. H.-D. Y provided two new species. Y.-W.Z make two plates. S.-C.H and Y.-W.Z. generated the DNA sequence data. S.-C.H analyzed the data. S.-C.H. wrote the manuscript draft. V.T., C.S.B., P.C., L.S.D., R.S.J., Q.Z., K.D.H. revised the manuscript. FO provided financial support. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Shucheng He https://orcid.org/0009-0008-7364-4727

Vinodhini Thiyagaraja https://orcid.org/0000-0002-8091-4579

Chitrabhanu S. Bhunjun https://orcid.org/0000-0001-8098-3390

Putarak Chomnunti https://orcid.org/0000-0003-2989-1735

Lakmali S. Dissanayake https://orcid.org/0000-0003-2933-3127

Ruvishika S. Jayawardena https://orcid.org/0000-0001-7702-4885

Yun Wei Zhao https://orcid.org/0009-0006-8211-5232

Fatimah Al-Otibi https://orcid.org/0000-0003-3629-5755

Qi Zhao https://orcid.org/0000-0001-8169-0573

Kevin D. Hyde https://orcid.org/0000-0002-2191-0762

Data availability

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Clonostachys
mycokeys.115.139757-treatment1.xml (29.8KB, xml)
XML Treatment for Clonostachys linzhiensis
mycokeys.115.139757-treatment2.xml (18.1KB, xml)
XML Treatment for Clonostachys motuoensis
mycokeys.115.139757-treatment3.xml (18.1KB, xml)
XML Treatment for Clonostachys yadongensis
mycokeys.115.139757-treatment4.xml (25.6KB, xml)
XML Treatment for Clonostachys viticola
mycokeys.115.139757-treatment5.xml (21.7KB, xml)
XML Treatment for Sesquicillium
mycokeys.115.139757-treatment6.xml (16KB, xml)
XML Treatment for Sesquicillium aquaticum
mycokeys.115.139757-treatment7.xml (11.7KB, xml)
XML Treatment for Sesquicillium shanghaiense
mycokeys.115.139757-treatment8.xml (17.6KB, xml)

Data Availability Statement

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

Articles from MycoKeys are provided here courtesy of Pensoft Publishers

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