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

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.


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].

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).

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.

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. 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  DNA barcodes: ITS OP205475, BenA OP205324, ACL1 OP493559, TEF1 OP493562.
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.
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.
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.

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. muscivoragroup, 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].

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.

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.