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Article

Three New Species of Clonostachys (Hypocreales, Ascomycota) from China

by
Zhao-Qing Zeng
* and
Wen-Ying Zhuang
*
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2022, 8(10), 1027; https://doi.org/10.3390/jof8101027
Submission received: 24 August 2022 / Revised: 22 September 2022 / Accepted: 26 September 2022 / Published: 28 September 2022
(This article belongs to the Special Issue Fungal Taxonomy, Phylogeny, and Ecology: A Themed Issue Dedicated to Academician Wen-Ying Zhuang)
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Abstract

:
Three new species of Clonostachys are introduced based on specimens collected from China. Clonostachys chongqingensis sp. nov. is distinguished by pale yellow to pale orange-yellow perithecia with a very low papilla, clavate to subcylindrical asci possessing ellipsoidal to elongate-ellipsoidal spinulose ascospores 13–16 × 4.5–5.5 μm; it has acremonium- to verticillium-like conidiophores and ellipsoidal to rod-shaped conidia. Clonostachys leptoderma sp. nov. has pinkish-white subglobose to globose perithecia on a well-developed stroma and with a thin perithecial wall, clavate to subcylindrical asci with ellipsoidal to elongate-ellipsoidal spinulose ascospores 7.5–11 × 2.5–3.5 μm; it produces verticillium-like conidiophores and ellipsoidal to subellipsoidal conidia. Clonostachys oligospora sp. nov. features solitary to gregarious perithecia with a papilla, clavate asci containing 6–8 smooth-walled ascospores 9–17 × 3–5.5 μm; it forms verticillium-like conidiophores and sparse, subfusiform conidia. The morphological characteristics and phylogenetic analyses of combined nuclear ribosomal DNA ITS1-5.8S-ITS2 and beta-tubulin sequences support their placement in Clonostachys and their classification as new to science. Distinctions between the novel taxa and their close relatives are compared herein.

1. Introduction

Clonostachys Corda, typified by C. araucaria Corda, is characterized by solitary to gregarious, subglobose or globose to ovoid perithecia that are white, yellow, pale orange, tan, or brown; perithecial walls are KOH− and LA−; there are narrowly clavate to clavate asci containing eight ascospores; it produces penicillium-, verticillium-, gliocladium-, or acremonium-like conidiophores, cylindrical to narrowly flask-shaped phialides, and ellipsoidal to subfusiform conidia [1]. Members of the genus usually have a broad range of lifestyles and occur on the bark of recently dead trees, decaying leaves, and less frequently on other fungi, nematodes, and insects [1,2,3]. They are economically important in the fields of pharmaceutics and agriculture [4]. For instance, the secondary metabolites produced by C. byssicola Schroers exhibited antibacterial activities [5], and strains of C. rosea (Link) Schroers, Samuels, Seifert & W. Gams have been widely used as biocontrol agents [6].
About 100 names have been created under the genus Clonostachys (www.indexfungorum.org (accessed on 1 July 2022)), among which 65 species are commonly accepted [2,3,7,8,9,10,11,12,13,14,15,16,17,18,19,20]. Twenty-four species are known from China [2,7,21]. In this study, three additional taxa are introduced based on morphological characteristics and sequence analyses of combined nuclear ribosomal DNA ITS1-5.8S-ITS2 (ITS) and beta-tubulin (BenA) regions. Comparisons between these novel species and their close relatives are performed.

2. Materials and Methods

2.1. Sampling and Morphological Studies

Specimens were collected from Chongqing and Yunnan Province and were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). Cultures were obtained by single ascospore isolation from fresh perithecium and are preserved in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. The methods of Hirooka et al. [22] were generally followed for morphological observations. Perithecial wall reactions were tested in 3% potassium hydroxide (KOH) and 100% lactic acid (LA). Longitudinal sections through the perithecia were made with a freezing microtome (YD-1508-III, Jinhua, China) at a thickness of 6–8 μm. Photographs were taken using a Canon G5 digital camera (Tokyo, Japan) connected to a Zeiss Axioskop 2 plus microscope (Göttingen, Germany). For colony characteristics and growth rates, strains were grown on potato dextrose agar (PDA) (200 g potato + 2% (w/v) dextrose + 2% (w/v) agar) and synthetic low-nutrient agar (SNA) [23] in 90 mm plastic Petri dishes at 25 °C for 2 weeks with alternating periods of light and darkness (12 h/12 h).

2.2. DNA Extraction, PCR Amplification, and Sequencing

The genomic DNA was extracted from fresh mycelium following the method of Wang and Zhuang [24]. Five primer pairs, namely ITS5/ITS4 [25], T1/T22 [26], acl1-230up/acl1-1220low [27], Crpb1a/rpb1c [28], and EF1-728F/EF2 [29,30], were used to amplify the sequences of ITS, BenA, ATP citrate lyase (ACL1), the largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-α (TEF1), respectively. PCR reactions were performed on an ABI 2720 Thermal Cycler (Applied Biosciences, Foster City, CA, USA) with a 25 μL reaction system consisting of 12.5 μL Taq MasterMix, 1 μL of each primer (10 μM), 1 μL template DNA, and 9.5 μL ddH2O. DNA sequencing was carried out in both directions with the same primer pairs using an ABI 3730 XL DNA Sequencer (Applied Biosciences, Foster City, CA, USA). Newly achieved sequences and those retrieved from GenBank are listed in Table 1. Fusarium acutatum Nirenberg & O’Donnell and Nectria cinnabarina (Tode) Fr. were chosen as outgroup taxa.

2.3. Sequence Alignment and Phylogenetic Analyses

Sequences were assembled and aligned with BioEdit 7.0.5 [31] and converted to nexus files by ClustalX 1.8 [32]. To confirm the taxonomic positions of the new species, ITS and BenA sequences were combined and analyzed with Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods. A partition homogeneity test (PHT) was performed with 1000 replicates in PAUP*4.0b10 [33] to evaluate the statistical congruence between the two loci. The BI analysis was conducted by MrBayes 3.1.2 [34] using a Markov chain Monte Carlo (MCMC) algorithm. Nucleotide substitution models were determined by MrModeltest 2.3 [35]. Four Markov chains were run simultaneously for 1,000,000 generations with the trees sampled every 100 generations. A 50% majority rule consensus tree was computed after excluding the first 2500 trees as “burn-in”. Bayesian inference posterior probability (BIPP) was determined from the remaining trees. The ML analysis was performed via IQ-Tree 1.6.12 [36] using the best model for each locus, as chosen by ModelFinder [37]. The MP analysis was performed with PAUP 4.0b10 [33] using heuristic searches with 1000 replicates of random addition of sequences and subsequent TBR (tree bisection and reconnection) branch swapping. The topological confidence of the resulting trees and statistical support of the branches were tested in maximum parsimony bootstrap proportion (MPBP) with 1000 replications and each with 10 replicates of the random addition of taxa. Trees were examined by TreeView 1.6.6 [38]. The maximum likelihood bootstrap (MLBP) values, MPBP values greater than 70%, and BIPP values greater than 90% were shown at the nodes.

3. Results

3.1. Phylogeny

The sequences of ITS and BenA from 50 representative species of Clonostachys were analyzed. The PHT (p = 0.05) indicated that the individual partitions were not highly incongruent [39]; thus, the two loci were combined for phylogenetic analyses. In the MP analysis, the datasets included 1159 nucleotide characters, of which 543 bp were constant, 154 were variable and parsimony-uninformative, and 462 were parsimony-informative. The MP analysis resulted in 123 most parsimonious trees (tree length = 2794, consistency index = 0.4098, homoplasy index = 0.5902, retention index = 0.4677, rescaled consistency index = 0.1917). One of the MP trees generated is shown in Figure 1. The topologies of the BI and ML trees were similar to that of the MP tree. The isolates 12581, 12672, and 11691 were grouped with the other Clonostachys taxa investigated (MPBP/MLBP/BIPP = 100%/100%/100%), which confirmed their taxonomic positions. The isolate 12581 was grouped with C. agrawalii (Kushwaha) Schroers and C. capitata Schroers, with low statistical support. The isolate 12672 clustered with C. zelandiaenovae Schroers (MPBP/MLBP/BIPP = 94%/96%/100%), and 11691 formed a separate lineage.

3.2. Taxonomy

  • Clonostachys chongqingensis Z.Q. Zeng and W.Y. Zhuang, sp. nov. (Figure 2).
Fungal Names: FN571276.
Etymology: The specific epithet refers to the type locality of the fungus.
Typification: China, Chongqing City, Jinfo Mountain, 29°2′50″ N 107°11′0″ E, on rotten bark of Alnus sp., 25 October 2020, Z.Q. Zeng, H.D. Zheng, X.C. Wang, C. Liu 12672 (holotype HMAS 290894).
DNA barcodes: ITS OP205475, BenA OP205324, ACL1 OP493559, TEF1 OP493562.
The mycelium was not visible on the natural substratum. Perithecia were superficial, solitary to gregarious, non-stromatic or with a basal stroma, subglobose to globose, with very low papilla and slightly roughened surface; they mostly did not collapse upon drying, and a few were slightly pinched at the apical portion, colored pale yellow to pale orange-yellow. There was no color change in 3% KOH or 100% LA, and the size was 304–353 × 294–392 μm. Perithecial walls were two-layered, 40–70 μm thick; the outer layer was of textura globulosa to textura angularis, 30–45 μm thick, with cells 5–15 × 3–12 μm and cell walls 0.8–1 μm thick. The inner layer was of textura prismatica, 10–25 μm thick, with cells 8–14 × 2.5–3.5 μm and cell walls 1–1.2 μm thick. Asci were clavate to subcylindrical, eight-spored, with a round and simple apex, and 60–85 × 6–13 μm. Ascospores were ellipsoidal to elongate-ellipsoidal, uniseptate, hyaline, spinulose, and uniseriate or irregular-biseriate, and 13–16 × 4.5–5.5 μm.
Jof 08 01027 g002 550
Figure 2. Clonostachys chongqingensis (holotype). (a,b) Ascomata on natural substratum; (c) colony after 2 weeks at 25 °C on PDA; (d) colony after 2 weeks at 25 °C on SNA; (e) median section through the perithecium; (f) asci with ascospores; (g–k) ascospore; (l,m) conidiophores, phialides, and conidia. Bars: (a,b) = 1 mm; (e) = 50 μm; (f–m) = 10 μm.
Figure 2. Clonostachys chongqingensis (holotype). (a,b) Ascomata on natural substratum; (c) colony after 2 weeks at 25 °C on PDA; (d) colony after 2 weeks at 25 °C on SNA; (e) median section through the perithecium; (f) asci with ascospores; (g–k) ascospore; (l,m) conidiophores, phialides, and conidia. Bars: (a,b) = 1 mm; (e) = 50 μm; (f–m) = 10 μm.
Jof 08 01027 g002
Colonies on PDA were 53 mm in diam. in average after 2 weeks at 25 °C; their surface was cottony, with a dense, whitish aerial mycelium. Colonies on SNA were 50 mm in diam. in average after 2 weeks at 25 °C; their surface was velvet, with a sparse, whitish aerial mycelium. Conidiophores were acremonium- to verticillium-like, arising from aerial hyphae and septate. Phialides were subulate tapered toward the apex, 15–74 μm long, 1.6–2.5 μm wide at the base, and 0.3–0.4 μm wide at the tip. Conidia were ellipsoidal to rod-shaped, unicellular, smooth-walled, hyaline, and 4–10 × 2.5–4 μm.
Notes: Morphologically, the species most resembles C. sesquicillii Schroers in having superficial, solitary to gregarious ascomata and clavate to subcylindrical asci with eight ellipsoidal, single-septate, spinulose ascospores [1]. However, the perithecia of the latter are often laterally or apically pinched when dry and have shorter asci (35–63 μm long) and smaller ascospores (8.2–14.4 × 2.2–4.4 μm) [1]. The two-locus phylogeny indicated the two fungi are remotely related (Figure 1).
Phylogenetically, C. chongqingensis is closely related to C. zelandiaenovae (Figure 1). The latter differs in its well-developed stroma, narrowly clavate asci with an apex ring, and wider ascospores (3.8–7.4 μm wide) [1].
  • Clonostachys leptoderma Z.Q. Zeng and W.Y. Zhuang, sp. nov. (Figure 3)
Fungal Names: FN571277.
Etymology: The specific epithet refers to the thin-walled perithecia.
Typification: China, Chongqing City, Jinyun Mountain, 29°49′46″ N 106°22′49″ E, on rotten bark, 23 October 2020, Z.Q. Zeng, H.D. Zheng, X.C. Wang, C. Liu 12581 (holotype HMAS 255834).
DNA barcodes: ITS OP205474, BenA OP205323, RPB1 OP493564, TEF1 OP493561.
Jof 08 01027 g003 550
Figure 3. Clonostachys leptoderma (holotype). (a,b) Ascomata on natural substratum; (c) colony after 2 weeks at 25 °C on PDA; (d) colony after 2 weeks at 25 °C on SNA; (e) median section through the perithecium; (f–h) asci with ascospores; (i–l) ascospore; (m–q) conidiophores, phialides, and conidia; (r) conidia. Bars: (a,b) = 1 mm; (e) = 50 μm; (f–r) = 10 μm.
Figure 3. Clonostachys leptoderma (holotype). (a,b) Ascomata on natural substratum; (c) colony after 2 weeks at 25 °C on PDA; (d) colony after 2 weeks at 25 °C on SNA; (e) median section through the perithecium; (f–h) asci with ascospores; (i–l) ascospore; (m–q) conidiophores, phialides, and conidia; (r) conidia. Bars: (a,b) = 1 mm; (e) = 50 μm; (f–r) = 10 μm.
Jof 08 01027 g003
The mycelium was not visible on the natural substratum. Perithecia were superficial, solitary to gregarious, with a well-developed stroma, subglobose to globose, non-papillate, with surface slightly roughened, and did not collapse upon drying. They were pinkish-white, did not change color in 3% KOH or 100% LA, and were with a size of 216–284 × 206–265 μm. Perithecial walls were two-layered, 13–45 μm thick; the outer layer was of textura globulosa to textura angularis, 8–23 μm thick, with cells 5–10 × 4–8 μm and cell walls 1–1.2 μm thick; the inner layer was of textura prismatica, 5–22 μm thick, with cells 5–12 × 2–3 μm and cell walls 0.8–1 μm thick. Asci were clavate to subcylindrical, 6–8-spored, with a round and simple apex, and 53–63 × 4.8–7 μm. Ascospores were ellipsoidal to elongate-ellipsoidal, uniseptate, hyaline, spinulose, uniseriate or irregular-biseriate, and 7.5–11 × 2.5–3.5 μm.
Colonies on PDA was 31 mm in diam. in average after 2 weeks at 25 °C; their surface was cottony, with a dense, whitish aerial mycelium, and it produced a yellowish-brown pigment in medium. Colony on SNA were 18 mm in diam. in average after 2 weeks at 25 °C, with a sparse, whitish aerial mycelium. Conidiophores were verticillium-like, arising from aerial hyphae; they were septate, with dense phialides. Phialides were subulate, tapering toward the apex, 9–18 μm long, 1.5–2.5 μm wide at the base, and 0.2–0.3 μm wide at the tip. Conidia were subglobose, ellipsoidal to subellipsoidal, unicellular, smooth-walled, hyaline, and 2–7 × 2–5 μm.
Notes: Morphologically, the fungus is most similar to C. epichloe Schroers in having solitary to gregarious perithecia and ellipsoidal, bi-cellular, spinulose ascospores of a similar size [1]. Nevertheless, the latter differs in its smaller perithecia (140–240 × 140–200 μm) that is pinched when dry, its wider asci (5–10 μm wide) [1], and the presence of 36 bp and 132 bp divergences in the ITS and BenA regions. Obviously, they are not conspecific.
Phylogenetically, C. leptoderma is closely related to C. capitata and C. agrawalii (Figure 1). Clonostachys capitata can be differentiated by its thicker perithecial wall (45–60 μm thick), wider asci (7–12 μm wide), and larger ascospores (11.6–18.8 × 3.6–5.8 μm) [1]. Clonostachys agrawalii, which is known to have only an asexual stage, can be easily distinguished by its bi- to quarter-verticillate conidiophores and its cylindrical to flask-shaped, somewhat larger phialides (7–42 × 1.4–3.4 μm) [1].
  • Clonostachys oligospora Z.Q. Zeng and W.Y. Zhuang, sp. nov. (Figure 4)
Fungal Names: FN571278.
Etymology: The specific epithet refers to the very few conidia produced.
Typification: China, Yunnan Province, Chuxiong Prefecture, Zixi Mountain, Xianrengu, 25°54′0″ N 101°24′46″ E, on a rotten twig, 23 September 2017, Y. Zhang, H.D. Zheng, X.C. Wang, Y.B. Zhang 11691 (holotype HMAS 290895).
DNA barcodes: ITS OP205473, BenA OP205322, ACL1 OP493560, RPB1 OP493563.
The mycelium was not visible on the natural substratum. Perithecia were superficial, solitary to gregarious, either with a basal stroma or non-stromatic. They were subglobose to globose and papillate, with surface slightly warted; the warts were 6–25 μm high. They did not collapse upon drying, were colored pale yellow to light yellow, did not change color in 3% KOH or 100% LA, and were 225–274 × 225–265 μm. The perithecial walls were two-layered, 25–48 μm thick; the outer layer was of textura globulosa to textura angularis, 15–38 μm thick, with cells 8–18 × 9–15 μm and cell walls 0.5–0.8 μm thick; the inner layer was of textura prismatica, 10–15 μm thick, with cells 5–8 × 1.5–2.5 μm and cell walls 0.8–1 μm thick. Asci were clavate, 6–8-spored, with a rounded and simple apex, and 45–65 × 7.5–11 μm. Ascospores were ellipsoidal, uniseptate, hyaline, smooth-walled, uniseriate or irregular biseriate, and 9–17 × 3–5.5 μm.
Colonies on PDA were 50 mm in diam. in average after 2 weeks at 25 °C; their surface was cottony, with a dense, whitish aerial mycelium. Colonies on SNA were 25 mm in diam. in average after 2 weeks at 25 °C, with a sparse, whitish aerial mycelium. Conidiophores were verticillium-like, arising from aerial hyphae, and septate. Phialides were subulate, tapering toward the apex, 9–15 μm long; they were 1.5–2.5 μm wide at the base and 0.2–0.3 μm wide at the tip. Conidia were sparse, subfusiform, unicellular, smooth-walled, hyaline, and 5–13 × 1.8–2.2 μm.
Notes: Among the known species of Clonostachys, this fungus resembles C. setosa (Vittal) Schroers in terms of solitary to gregarious perithecia and ascospores that are ellipsoidal, bi-cellular, smooth-walled, and of a similar size [1]. However, the latter fungus is distinguished by asci with an apical ring, as well as by conidiophores that are penicillium-like and cylindrical conidia that are slightly larger (8.6–19.2 × 2–3.2 μm) [1]. In addition, there are 47 bp and 128 bp divergences in the ITS and BenA regions between HMAS 290895 and CBS 834.91. Both morphology and DNA sequence data support their distinction at the species level.
Jof 08 01027 g004 550
Figure 4. Clonostachysoligospora (holotype). (a,b) Ascomata on natural substratum; (c) colony after 2 weeks at 25 °C on PDA; (d) colony after 2 weeks at 25 °C on SNA; (e) median section through the perithecium; (f,g) asci with ascospores; (h–m) ascospore; (n–p) conidiophores, phialides, and conidia. Bars: (a,b) = 1 mm; (e) = 50 μm; (f–p) = 10 μm.
Figure 4. Clonostachysoligospora (holotype). (a,b) Ascomata on natural substratum; (c) colony after 2 weeks at 25 °C on PDA; (d) colony after 2 weeks at 25 °C on SNA; (e) median section through the perithecium; (f,g) asci with ascospores; (h–m) ascospore; (n–p) conidiophores, phialides, and conidia. Bars: (a,b) = 1 mm; (e) = 50 μm; (f–p) = 10 μm.
Jof 08 01027 g004

4. Discussion

Although Clonostachys was established in 1839, the name was not commonly used until Schroers’ monographic treatment of the genus, from which 44 species were accepted [1]. The generic name Bionectria Speg. was introduced later [40], and the genus was reviewed by Rossman et al. [41]; in that work, the species included those previously placed in the Nectria ochroleuca group, the N. ralfsii group, and the N. muscivora group, as well as those having Sesquicillium W. Gams asexual stages. Clonostachys and Bionectria are of anamorph and teleomorph connections [1,41]. According to the current International Code of Nomenclature for algae, fungi, and plants [42], under the principle that one fungus requires one name, Clonostachys was recommended as the preferable name [43].
The previous phylogenetic overview of Clonostachys that was based on two-locus (ITS and BenA) sequence analyses showed that the genus is monophyletic [19,20]. Our analyses provided a similar tree topology, and species of the genus formed a well-supported clade (MPBP/MLBP/BIPP = 100%/100%/100%), including the three new taxa (Figure 1). Clonostachys oligospora is a well-separated lineage in between C. indica Prasher & R. Chauhan and C. samuelsii Schroers. Clonostachys chongqingensis clustered with C. zelandiaenovae, receiving relatively high statistical support (MPBP/MLBP/BIPP = 94%/96%/100%), and had moderate sequence divergences, i.e., 11/518 bp (2.1%) for ITS and 15/557 bp (2.7%) for BenA. Clonostachys leptoderma was grouped with C. agrawalii and C. capitata, which is poorly supported. Compared with the previously demonstrated phylogenies [19,20], minor changes were detected. For example, C. pseudostriata Schroers formerly constituted a separate lineage by itself [19,20]; whereas, with the joining of the new species, the fungus seemed to be closely related to C. krabiensis Tibpromma & K.D. Hyde and C. viticola C. Torcato & A. Alves, with low statistical support. Comparisons between each new species and closely related taxa are provided in Table 2. Along with the discovery of additional new species, the relationships among the species of the genus will become well-established.
More than 220 secondary metabolites have been reported from species of the genus. For example, C. byssicola, C. candelabrum (Bonord.) Schroers, C. compactiuscula (Sacc.) D. Hawksw. & W. Gams, C. grammicospora Schroers & Samuels, C. pityrodes Schroers, C. rogersoniana Schroers, and C. rosea were demonstrated to have the potential for biocontrol application [4,44,45,46,47]. Meanwhile, strains of C. rosea were occasionally reported as an opportunistic phytopathogen [48,49]. Therefore, studies on the biodiversity of Clonostachys are of theoretical and practical importance and need to be carried out continuously and extensively. China is diverse in climate, vegetation, and geographic structures and has rich niches for organisms [50,51]. Large-scale surveys in unexplored regions will significantly improve our knowledge of fungal species diversity.

5. Conclusions

The species diversity of the genus Clonostachys was investigated, and three new species were discovered. With the joining of the new species, the phylogenetic relationships among species of the genus are updated.

Author Contributions

Conceptualization, W.-Y.Z.; methodology, Z.-Q.Z.; software, Z.-Q.Z.; validation, Z.-Q.Z.; formal analysis, Z.-Q.Z.; investigation, Z.-Q.Z.; resources, W.-Y.Z. and Z.-Q.Z.; data curation, Z.-Q.Z.; writing—original draft preparation, Z.-Q.Z.; writing—review and editing, W.-Y.Z. and Z.-Q.Z.; visualization, Z.-Q.Z.; supervision, Z.-Q.Z. and W.-Y.Z.; project administration, W.-Y.Z.; funding acquisition, W.-Y.Z. and Z.-Q.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31870012, 31750001), the National Project on Scientific Groundwork, Ministry of Science and Technology of China (2019FY100700), and the Frontier Key Program of the Chinese Academy of Sciences (QYZDY-SSWSMC029).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The names of the new species were formally registered in the database Fungal Names (https://nmdc.cn/fungalnames (accessed on 11 August 2022)). Specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). The newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank (accessed on 22 September 2022)).

Acknowledgments

The authors would like to thank H.D. Zheng, X.C. Wang, Y.B. Zhang, Y. Zhang, and C. Liu for collecting specimens jointly for this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Jof 08 01027 g001 550
Figure 1. A maximum parsimony tree generated from analyses of combined ITS and BenA sequences of Clonostachys species. The MP analysis was performed using heuristic searches with 1000 replicates. MPBP (left) and MLBP (middle) values greater than 70% and BIPP (right) values greater than 90% were shown at the nodes. Fusarium acutatum and Nectria cinnabarina were chosen as outgroup taxa.
Figure 1. A maximum parsimony tree generated from analyses of combined ITS and BenA sequences of Clonostachys species. The MP analysis was performed using heuristic searches with 1000 replicates. MPBP (left) and MLBP (middle) values greater than 70% and BIPP (right) values greater than 90% were shown at the nodes. Fusarium acutatum and Nectria cinnabarina were chosen as outgroup taxa.
Jof 08 01027 g001
Table
Table 1. Sequences used in this study.
Table 1. Sequences used in this study.
SpeciesHerbarium/Strain NumbersGenBank Accession Numbers
ITSBenA
C. agrawaliiCBS 53381AF358241AF358187
C. apocyniCBS 13087AF210688AF358168
C. aranearumQLS 0625NR164542KU212400
C. aureofulvellaCBS 19593AF358226AF358181
C. buxiCBS 69693KM231840KM232111
C. byssicolaCBS 36478MH861151AF358153
C. candelabrumCBS 50467/CML 2313MH859044KF871186
C. capitataCBS 21893AF358240AF358188
C. chlorinaCBS 28790NR137651-
C. chloroleucaCML 1941KC806286KF871172
C.chongqingensisHMAS 290894OP205475 aOP205324
C. coccicolaHD 2016KU720552KU720552
C. compactiusculaCBS 72987AF358242AF358193
C. divergensCBS 96773bNR137532AF358191
C. epichloeCBS 101037AF210675AF358209
C. eriocamporesianaMFLUCC 17-2620NR168235MN699965
C. eriocamporesiiMFLUCC 190486NR168236-
C. grammicosporaCBS 20993NR137650AF358206
C. grammicosporopsisCBS 11587AF210679AF358204
C. impariphialisHMAS 275560KX096609-
C. indicaRKV 2015KT291441KT291441
C. intermediaCBS 50882NR137652AF358205
C. kowhaiCBS 46195AF358250AF358170
C. krabiensisMFLUCC 160254NR168189-
C. leptodermaHMAS 255834OP205474OP205323
C. levigataCBS 94897AF210680AF358196
C. luciferCBS 100008AF210683AF358208
C. miodochialisCBS 99769NR137649AF358210
C. oblongisporaCBS 100285AF358248AF358169
C.oligosporaHMAS 290895OP205473OP205322
C. phyllophilaCBS 92197NR137531-
C. pityrodesCBS 102033AF210672AF358212
C. pseudochroleucaCML 2513KJ499909KF871188
C. pseudostriataCBS 12087MH862056AF358184
C. ralfsiiCBS 102845AF358253AF358219
C. rhizophagaCBS 36177AF358228AF358158
C. rogersonianaCBS 58289/CML 2557AF210691KX185047
C. roseaCBS 71086MH862010AF358161
C. rossmaniaeCBS 21093AF358227AF358213
C. samuelsiiCBS 69997AF358236AF358190
C. saulensisBRFM 2782MK635054-
C. sesquicilliiCBS 18088AF210666AF358214
C. setosaCBS 83491AF210670AF358211
C. solaniCBS 101926AF358230AF358179
C. sporondochialisCBS 101921AF210685AF358149
C. subquaternataCBS 100003/CBS 10787MT537603AF358207
C. vesiculosaHMAS 183151NR119828-
C. viticolaCAA 944MK156282MK156290
C. wenpingiiHMAS 172156EF612465HM054127
C. zelandiaenovaeCBS 23280AF210684AF358185
F. acutatumCBS 40297NR111142KU603870
N. cinnabarinaCBS 18987HM484699HM484835
a Numbers in bold indicate the newly provided sequences.
Table
Table 2. Morphological comparisons of the new species and their close relatives.
Table 2. Morphological comparisons of the new species and their close relatives.
SpeciesSexual MorphAsexual MorphReference Source
PeritheciaAsciAscosporesConidiophoresPhialidesConidia
C. chongqingensisSubglobose to globose, 304–353 × 294–392 μm.Clavate to subcylindrical, 60–85 × 6–13 μm.Ellipsoidal to elongate-ellipsoidal, spinulose, 13–16 × 4.5–5.5 μm.Acremonium- to verticillium-like.Subulate, 15–74 × 1.6–2.5 μm.Ellipsoidal to rod-shaped, 4–10 × 2.5–4 μm.This study
C. krabiensisN.A.N.A.N.A.Aggregated into sporodochia.Subulate, 10–13 × 1.5–2.5 μm.Cylindrical to oblong, 5–7 × 1–2 μm.[15]
C. sesquicilliiGlobose, 250–300 μm diam.Narrowly clavate to cylindrical, 35–63 × 5–13 μm.Ellipsoidal, warted, 8.2–14.4 × 2.2–4.4 μm.Penicillate conidiophores bi- to quarter-verticillate, verticillate conidiophores sparsely formed.Cylindrical to flask-shaped, 6.4–18.8 × 1.4–3.6 μm.Ellipsoidal to oblong ellipsoidal, 4.2–9.6 × 1.6–3 μm.[1]
C. viticolaN.A.N.A.N.A.Primary conidiophores verticillate, secondary conidiophores bi- to ter-verticillate, 45.3–64.7 × 2.1–3.7 μm.Cylindrical, 10.4–32.8 × 1.7–2.7 μm.Ellipsoidal to oval, 4.5–6.7 × 2.4–3.4 μm.[3]
C. zelandiaenovaeSubglobose to globose, 290–550 μm diam.Narrowly clavate, 60–104 × 7–15.5 μm.Ellipsoidal, spinulose, rarely smooth, 11.6–21.4 × 3.8–7.4 μm.Primary conidiophores verticillate, secondary conidiophores penicillate, ter- to quinquies-verticillate.Cylindrical to narrowly flask-shaped, 4.8–20.6 × 1.6–3.4 μm.Distally broadly rounded, 4–13.2 × 2.4–4.2 μm.[1]
C. leptodermaSubglobose to globose, 216–284 × 206–265 μm.Clavate to subcylindrical, 53–63 × 4.8–7 μm.Ellipsoidal to elongate-ellipsoidal, spinulose, 7.5–11 × 2.5–3.5 μm.Verticillium-like.Subulate, 9–18 × 1.5–2.5 μm.Subglobose, ellipsoidal to subellipsoidal, 2–7 × 2–5 μm.This study
C. agrawaliiN.A.N.A.N.A.Primary conidiophores irregularly branched to ter-verticillate, secondary conidiophores bi- to quarter-verticillate.Cylindrical to flask-shaped, 7–42 × 1.4–3.4 μm.Ends broadly rounded, 3.8–5.8 × 2.2–3 μm.[1]
C. capitataSubglobose to oval, around 300 μm diam.Narrowly clavate, 50.5–89.5 × 7–12 μm.Ellipsoidal to oblong-ellipsoidal, spinulose to warted, 11.6–18.8 × 3.6–5.8 μm.Primary conidiophores verticillium-like, secondary conidiophores ter- to quinquies-verticillium-like.Cylindrical to narrowly flask-shaped, 8.8–46.6 × 1.4–3.6 μm.Ends broadly rounded, 4.6–12.4 × 2.2–4.2 μm.[1]
C. epichloe140–240 × 140–200 μm.Clavate to narrowly clavate, 32–65 × 5–10 μm.Ellipsoidal, smooth to spinulose, 7.2–13 × 2.4–4.4 μm.Divergently branched to adpressed.Cylindrical to narrowly flask-shaped, 7–29 × 2.2–3.2 μm.Ellipsoidal to narrowly clavate, 4.8–9.6 × 1.6–3.6 μm.[1]
C. oligosporaSubglobose to globose, 225–274 × 225–265 μm.Clavate, 45–65 × 7.5–11 μm.Ellipsoidal, smooth, 9–17 × 3–5.5 μm.Verticillium-like.Subulate, 9–15 × 1.5–2.5 μm.Sparse, subfusiform, 5–13 × 1.8–2.2 μm.This study
C. indicaN.A.N.A.N.A.Primary conidiophores verticillium-like, secondary conidiophores bi- to quarter-verticillate.Cylindrical, 10.5–35.9 × 1.9–3.9 μm.Ovoid to subglobose, 3.9–7.4 × 2–3.7 μm.[12]
C. samuelsiiSubglobose to oval, 250–350 μm diam.Narrowly clavate, 43–71 × 5–11.5 μm.Ellipsoidal to broadly ellipsoidal, warted, 7.8–15.4 × 2.8–5.6 μm.Penicillate or irregularly penicillate, ter- to quarter-verticillate. Phialides cylindrical to narrowly flask-shaped, 3.8–20.6 × 1.8–5 μm. Conidia ellipsoidal, 4.4–11.6 × 2.2–3.8 μm.[1]
C. setosaGlobose, around 200 μm diam.Narrowly clavate, 45–53 × 6.5–9 μm.Ellipsoidal, smooth to striate, 8.8–13 × 2.4–3.8 μm.Penicillate, mono- to ter-verticillate.Cylindrical, 5.4–13.4 × 2.4–4.2 μm.Cylindrical, 8.6–19.2 × 2–3.2 μm.[1]
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Zeng, Z.-Q.; Zhuang, W.-Y. Three New Species of Clonostachys (Hypocreales, Ascomycota) from China. J. Fungi 2022, 8, 1027. https://doi.org/10.3390/jof8101027

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Zeng Z-Q, Zhuang W-Y. Three New Species of Clonostachys (Hypocreales, Ascomycota) from China. Journal of Fungi. 2022; 8(10):1027. https://doi.org/10.3390/jof8101027

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Zeng, Zhao-Qing, and Wen-Ying Zhuang. 2022. "Three New Species of Clonostachys (Hypocreales, Ascomycota) from China" Journal of Fungi 8, no. 10: 1027. https://doi.org/10.3390/jof8101027

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