Sequencing and phylogenetic analyses
Phylogenetic analyses based on nrLSU data consisting of 107 fungal taxa confirmed the presence and positions of Clonostachys and related genera within Bionectriaceae. Eighteen well-supported clades were recognized based on both BI and ML analyses of the 107 taxa from Bionectriaceae and Flammocladiella (Flammocladiaceae, Hypocreales) that accommodate species of the genera Bryocentria, Clonostachys, Emericellopsis, Gliomastix, Heleococcum, Hydropisphaera, Ijuhya, Lasionectria, Nectriopsis, Paracylindrocarpon, Roumegueriella, Selinia, Stanjemonium, Stephanonectria, Stilbocrea, Stromatonectria, Verrucostoma, and Flammocladiella (Fig. 1). The genus Clonostachys was phylogenetically clustered with Stephanonectria, and Emericellopsis had a close genetic relationship with Stanjemonium, but they were clearly distinguished from their allied genera by forming four separate clades in the family Bionectriaceae (Fig. 1). The combined dataset included sequences from 86 fungal taxa (Table 1). The final dataset consisted of 2900 bp of sequence data, including gaps (ITS, 654 bp; nrLSU, 903 bp; TUB2, 711 bp; and TEF1, 632 bp). Both BI and ML analyses produced trees with similar topologies that resolved most Clonostachys lineages in separate terminal branches (Fig. 2). Phylogenetic trees inferred from analyses of combined data divided Clonostachys into six distinguished clades, designated as Astromata, Bionectria, Epiphloea, Myronectria, Uniparietina, and Zebrinella clades (Fig. 2). The phylogenetic analyses suggested the existence of distinct species in the Bionectria and Epiphloea clade that we accordingly propose as new species: C. chuyangsinensis, which was found in the Epiphloea clade, and C. kunmingensis, which was found in the Bionectria clade (Fig. 2).
The tree topologies for the individual loci (ITS, nrLSU, TUB2, and TEF1) did not show congruence (Supplementary Figs. S1–S4). However, in all analyses C. chuyangsinensis had a close genetic relationship with C. candelabrum. Clonostachys chloroleuca and C. rhizophaga were sisters to the newly discovered species C. kunmingensis, although this relationship received significant bootstrap support only from ITS and TUB2. Phylogenetic analyses based on nrLSU data revealed that C. kunmingensis was closely related to C. rosea (Fig. 1 and Supplementary Fig. S2). And the nrLSU sequences cannot distinguish the two species. But they were regarded as different species with strong support from ITS, TUB2 and TEF1 (Supplementary Figs. S1, S3, S4).
The genetic divergence comparisons showed that: (1) the minimum thresholds (p-distances) to distinguish genetic species in the Clonostachys lineages were 0.005, 0.017, and 0.026 for ITS, TUB2, and TEF1, respectively (Supplementary Tables S1–S3); (2) the TEF1 sequence data provided the best resolution distinguishing Clonostachys spp., followed by TUB2 and ITS sequences (Supplementary Tables S1–S3); and (3) the genetic distances strongly supported recognition of C. chuyangsinensis and C. kunmingensis as two new taxa (Table 2).
Table 2
Genetic distance (p-distances) of the two new species with their related species
Subgenus | Taxa | Marker |
ITS | TUB2 | TEF1 |
Epiphloea | Clonostachys chuyangsinensis – Clonostachys candelabrum | 0029 | 0097 | 0083 |
Bionectria | Clonostachys kunmingensis – Clonostachys chloroleuca | 0009 | 0042 | 0036 |
Clonostachys kunmingensis – Clonostachys rhizophaga | 0012 | 0023 | 0058 |
Taxonomy
In this study, a collection of 23 isolates of unknown identity were shown to represent five known species and two new species of Clonostachys. The phylogenetic positions of the five known species were evaluated according to phylogenetic inferences based on four loci (ITS, nrLSU, TUB2, and TEF1), including C. compactiuscula, C. rhizophaga, C. rogersoniana, and C. solani from China, and C. rosea from Thailand (see Table 1 and Fig. 2). The two new species, provided with the names C. chuyangsinensis from Vietnam and China and C. kunmingensis from China, were recognized based on morphological characteristics and molecular data.
Clonostachys chuyangsinensis H. Yu & Y. Wang, sp. nov. (Fig. 3)
MycoBank MB843885
Etymology
named after Chu Yang Sin National Park, where this species was first discovered.
Type: Vietnam, Dak Lak Province, Chu Yang Sin National Park (12°29'N, 108°43'E, 1659 m above sea level), on a spider on the underside of a leaf, October 22, 2017, collected by Yuan-Bing Wang (holotype: YHH 896; ex-type living culture: YFCC 896).
Description: Sexual morph: Ascomata on a brown spider, perithecial, solitary or densely crowded in groups, subglobose to oval, (280–)290–380(–400) × (240–)260–330(–340) µm (n = 30), collapsing laterally when dry, pale brown when fresh, becoming dark brown to nearly black when dry, not changing color in 3% KOH or in lactic acid; surface smooth. Asci and ascospores not observed. Asexual morph: Infected spider host covered with a dense brown mycelial mat. Hyphae branched, septate, hyaline, smooth. Conidiophores verticillium-like; phialides divergent in whorls of 2–5 or single from lower levels, generally slightly tapering toward the tip, (5–)5.6–28.3(–36) × (1–)1.4–3.6(–4) µm (n = 30). Conidia smooth-walled, hyaline, subglobose to ellipsoid, (2–)2.5–4.6(–4.8) × (2–)2.4–3.5(–4) µm (n = 30). Colonies on PDA reached 28–32 mm in diameter after 7 days at 25°C, white, circular; reverse pale to light orange (5–6A3–4). Colony surface white powdery to granulose because of the conidiophores and conidial masses; aerial mycelium sparsely produced or absent. Conidiophores monomorphic, verticillate, arising from the agar surface or from the sparse aerial mycelium; stipes (20–)40–130(–150) µm long, (2–)2.5–4(–5) µm wide at the base (n = 50); primary branches divergent, forming independent side-branches; terminal branches and phialides divergent or adpressed; terminal phialides flask-shaped, or cylindrical but narrowing in the upper part, (4.5–)5.5–44.2(–60) × (1.2–)1.5–3.8(–4) µm (n = 50). Conidia in white imbricate columns, smooth-walled, hyaline, subglobose to ellipsoid, (2.2–)2.4–4.7(–5) × (1.5–)1.8–3.5(–3.8) µm (n = 50). Setae not observed. Colonies on CMA reached 25–30 mm in diameter after 7 days at 25°C, white, circular; reverse pale yellowish (1-2A3). Colony surface white powdery due to conidial masses, cottony to felty due to aerial mycelium. Conidiophores monomorphic, verticillate, arising from the agar surface or from the sparse aerial mycelium; stipes (20–)30–145(–160) µm long, (2–)2.5–4(–4.5) µm wide at the base (n = 50); primary branches divergent, forming independent side-branches; terminal branches and phialides divergent or adpressed; terminal phialides flask-shaped, or cylindrical but narrowing in the upper part, (4.5–)5.5–44.2(–50) × (1.2–)1.5–3.8(–4.2) µm (n = 50). Conidia in white imbricate columns, smooth-walled, hyaline, subglobose to ellipsoid, ovoid, (2–)2.2–5(–5.5) × (1.5–)2–3.5(–4) µm (n = 50). Setae not observed.
Distribution
Chu Yang Sin National Park, Dak Lak Province, Vietnam; Kunming City, Yunnan Province, China.
Additional materials examined: China, Yunnan Province, Kunming City, Wild Duck Forest Park (25°13'N, 102°87'E, 2100 m above sea level), from soil on the forest floor, August 20, 2018, Yao Wang (living culture: YFCC 895); China, Yunnan Province, Kunming City, Songming County, Dashao Village (25°24'N, 102°55'E, 2697 m above sea level), from Ophiocordyceps highlandensis, August 25, 2018, De-Xiang Tang (living culture: YFCC 8591) (Zhao et al. 2021).
Commentary
Morphologically, C. chuyangsinensis resembles the phylogenetically sister species C. candelabrum. The shape and size of the conidia and the colony color of C. chuyangsinensis among other morphological features have been observed in C. candelabrum. However, C. chuyangsinensis can be distinguished from C. candelabrum by its long phialides ((4.5–)5.5–44.2(–50) × (1.2–)1.5–3.8(–4.2) µm). Both morphological study and phylogenetic analyses of combined ITS, nrLSU, TUB2, and TEF1 sequence data support that this fungus is a distinct species in the genus Clonostachys.
Clonostachys kunmingensis H. Yu & Y. Wang, sp. nov. (Fig. 4)
MycoBank MB843886
Etymology
named after the location Kunming City where the species was collected.
Type: China, Yunnan Province, Kunming City, Wild Duck Forest Park (25°13'N, 102°87'E, 2100 m above sea level), from soil on the forest floor, August 10, 2019, Yao Wang (holotype: YHH 898, dried specimen; ex-type living culture: YFCC 898).
Description: Sexual morph: Undetermined. Asexual morph: Colonies on PDA reaching 32–35 mm in diameter after 7 days at 25°C, pale yellow (4A2–3), circular; reverse pale orange (5A2–3). Colony surface cottony to felty due to aerial mycelium. Conidiophores dimorphic. Primary conidiophores verticillium-like, arising from the agar surface or from the sparse aerial mycelium; (80–)120–260(–380) µm high, stipes (20–)60–140(–230) µm long, (2–)3.5–5(–5.5) µm wide at the base (n = 50), sometimes with short side branches arising from the upper part; phialides divergent, in whorls of 2–6, sometimes singly from lower levels, (14.2–)19.1–36.4(–52.6) × (2–)2.5–3.5(–3.9) µm (n = 50), straight, cylindrical, slightly tapering toward the tip. Secondary conidiophores penicillate, solitary to gregarious, with divergent branching penicilli; bi- to quarter-verticillate, (15–)30–100(–125) µm long, (3–)3.5–5(–5.5) µm wide at the base (n = 50); penicillus 90–145 µm high, typically with two primary branches, divergent, terminating in moderately divergent metulae and adpressed phialides; phialides divergent or adpressed, in whorls of 2–6, almost cylindrical tapering in the upper part, straight to slightly curved, (5.6–)8.0–17.5(–25) µm long, (2–)2.5–3.2(–4) µm wide at the base, (1–)1.2–1.4(–1.6) µm wide near the aperture (n = 50); intercalary phialides rarely observed. Conidial masses on verticillium-like conidiophores small and round collapsing to form whitish, watery masses; conidial masses on penicillate conidiophores inconspicuous, short, and rather thick, columnar, white. Conidia from secondary conidiophores slightly curved, with one slightly flattened side, distally broadly rounded, with laterally displaced hila, (4–)4.2–8.5(–9) × (2.2–)2.5–4(–4.5) µm (n = 100), held in imbricate; conidia from primary conidiophores larger, oblong to cylindrical, frequently less curved, sometimes without a visible hilum, (6–)6.7–11.2(–14) × (2–)2.3–4.5(–4.8) µm (n = 100). Colonies on CMA reaching 25–32 mm in diameter after 7 days at 25°C, white, circular; reverse yellowish white to light yellow (4A2–5). Colony surface white powdery due to conidial masses. Aerial mycelium on CMA not thick, on PDA strongly developed in thick, often erect hyphal strands. Size and shape of Conidiophores, phialides and conidia similar on PDA and CMA.
Distribution
at present, known only in Wild Duck Forest Park, Kunming City, Yunnan Province, China.
Additional materials examined: China, Yunnan Province, Kunming City, Wild Duck Forest Park (25°13'N, 102°87'E, 2100 m above sea level), from soil on the forest floor, August 10, 2019, Yao Wang (living culture: YFCC 892, 967).
Commentary
Regarding phylogenetic relationships, C. kunmingensis is closely related to C. rhizophaga and C. chloroleuca and further grouped with C. oblongispora (Fig. 2). However, C. kunmingensis can be distinguished from C. rhizophaga and C. chloroleuca by its oblong to cylindrical conidia ((6–)6.7–11.2(–14) × (2–)2.3–4.5(–4.8) µm). Clonostachys kunmingensis consistently showed unpigmented conidial masses, while conidial masses of C. rhizophaga and C. chloroleuca can be greenish or weakly greenish (Moreira et al. 2016). Clonostachys oblongispora differs from C. kunmingensis by its longer conidia ((9–)12.6–13.6–14(–19.8) × (2.6–)3.2–3.6–3.8(–4.2) µm) (Schroers 2001). Morphologically, C. kunmingensis is similar to C. rosea in terms of the shape and size of the conidiogenous cells and the shape of the conidia (Schroers 2001). However, our morphological observation revealed some differences between them. Colonies of C. kunmingensis on PDA are pale yellow whereas those of C. rosea are white. Furthermore, conidia from secondary conidiophores of C. kunmingensis ((4–)4.2–8.5(–9) × (2.2–)2.5–4(–4.5) µm) are larger than those of C. rosea ((4.2–)4.8–5.2–5.6(–6.6) × (2–)2.4–2.8–3(–3.4) µm).