Discovery from a large-scaled survey of Trichoderma in soil of China

The first large-scaled survey of soil-inhabiting Trichoderma is conducted in 23 provinces of China. Twenty-three new species belonging to the green-ascospored clades are discovered. Their phylogenetic positions are determined by sequence analyses of the combined partial sequences of translation elongation factor 1-alpha and the second largest RNA polymerase subunit encoding genes. Morphology and culture characteristics are observed, described and illustrated in detail. Distinctions between the new species and their close relatives are compared and discussed. They are named as: T. aggregatum, T. alpinum, T. bannaense, T. breve, T. brevicrassum, T. byssinum, T. chlamydosporicum, T. concentricum, T. ganodermatis, T. hainanense, T. hengshanicum, T. hirsutum, T. hunanense, T. ingratum, T. liberatum, T. linzhiense, T. longisporum, T. polypori, T. pseudodensum, T. simplex, T. solum, T. undatipile and T. zayuense.

On SNA after 72 h colony radius 28-30 mm and mycelium covering the plate after 7 d at 25 °C. Colony hyaline, mycelium loose, margin not well defined, aerial hyphae common. Conidiation starting after 2 d, effuse in aerial hyphae or in small granules, denser around the original inoculum. No chlamydospores observed. No distinct odour, no diffusing pigment observed.  Notes: The collecting sites of T. alpinum are all located at an elevitude above 2000 m, which indicates that the fungus might be adaptable to cool and mountainous areas. Morphologically T. alpinum is characterized by producing loose shrubs on CMD, lageniform phialides and conidia with several minute guttules. Phylogenetically, T. alpinum is closely related to T. alni, but the latter species differs in distributing in low-elevation, slower growth on CMD and PDA and smaller conidia (2.5-3.5 × 2.5-2.7 μm) 21 .  On CMD after 72 h colony radius 30-31 mm, mycelium covering the plate after 6 d at 25 °C. Colony hyaline, radial, mycelium loose, aerial hyphae nearly lacking. Conidial pustules formed after 4 d, common, spreading along the colony margin, hemispherical, compact, remaining discrete, 1-3 mm diam, first white, turning green after 5 d, with short hairs extending beyond the surface. No chlamydospores observed. No distinct odour, no diffusing pigment observed.
On SNA after 72 h colony radius 2-5 mm, mycelium covering the plate after 10-12 d at 25 °C. Colony hyaline, margin irregular, not well defined, mycelium loose, aerial hyphae long, not common. Conidiation starting after 5 d, effused on short erect conidiophores and aerial hyphae near the original inoculum. Conidial pustules noted after 6 d, common, distributed around the colony center, hemispherical, remaining discrete, 0.5-1.5 mm diam, first white, turning green after 7 d, with hairs protruding beyond the surface, hairs straight, tips infrequently branched, fertile. No chlamydospores observed. No distinct odour, no diffusing pigment observed.
Notes: Trichoderma bannaense is distinctive by its elongated fertile tips of conidiophores. Although its greenish discrete pustules on CMD resembles those produced by T. hamatum, the latter has short and wide phialides 14 . Phylogenetically, T. bannaense is closely related to T. breve (see below for description and illustration), but the latter gives rise to numerous chlamydospores on PDA, and grows much faster on all three media.
On SNA after 72 h colony radius 35-36 mm, mycelium covering the plate after 5 d at 25 °C. Colony hyaline, mycelium loose, aerial hyphae nearly lacking. Conidiation starting after 3 d, first formed on short erect conidiophores near the original inoculum, conidial pustules noted after 6 d, appearing around the periphery of the colony, hemispherical, 0.5-1 mm diam, white, turning green after 7 d, with hairs protruding beyond the surface. Chlamydospores rare. No distinct odour, no diffusing pigment observed.
Notes: Trichoderma chlamydosporicum is featured by its numerous chlamydospores produced on PDA. This species also forms numerous compact, hemispherical conidial pustules on PDA that resembles T. hamatum. But it differs obviously from T. hamatum in the elongated conidiophores, which are straight and frequently fertile at the tips 14 . Phylogenetically, T. chlamydosporicum is closely related to T. tibetense, but the latter species differs in colony characteristics, slow growth on PDA, trichoderma-like conidiophores and smaller conidia (3.3-5.6 × 2.5-3.3) 18 .  pustules, pustules abundant, spreading in 3-4 concentric rings, aggregated, up to 5 mm in width of the concentric rings, with white droplets on the surface, first white, turning green after 4 d. Conidiophores symmetry, trichoderma-like, often with a main axis, side branches in acute or straight angles with main axis, typically paired, less commonly in whorls of 3, rebranching 1-3 times. Phialides formed solitary or paired, rarely in whorls of 3, lageniform to narrowly lageniform, 8.6-11.7(−13.6) × 2.5-3.6 μm, l/w 2. On SNA after 72 h colony radius 15-16 mm, mycelium covering the plate after 7 d at 25 °C. Colony hyaline, margin slightly lobed, not well defined, mycelium loose, aerial hyphae inconspicuous. Conidiation starting after 4 d, first formed on short erect conidiophores and aerial hyphae, pustules noted after 5 d, uniformly distributed Notes: Trichoderma concentricum is characterized by the presence of distinct concentric pustules on CMD and PDA, globose conidia and lageniform phialides. Phylogenetically, T. concentricum is closely related to T. corneum and T. ingratum. Compared with the new species, T. ingratum differs in its colony morphology, unpleasant odour on PDA and slightly larger conidia (2.6-3.6 × 2.6-3.2 μm). Trichoderma corneum can be easily separated by its verticillium-like conidiophores, much longer phialides (8-24 × 1.5-3.0 μm) and ellipsoid conidia 24 .  On CMD after 72 h colony radius 24-34 mm, mycelium covering the plate after 5 d at 25 °C. Colony hyaline, radial, mycelium loose, aerial hyphae inconspicuous. Conidiation starting after 3 d, formed in pustules, pustules appearing around the periphery of the colony, hemispherical, loose, 0.5-2 mm diam, white, turning green after 5 d, with hairs protruding beyond the surface, straight, tips often branched and fertile. No chlamydospores observed. No distinct odour, no diffusing pigment observed.

Trichoderma concentricum
On PDA after 72 h colony radius 22-27 mm, mycelium covering the plate after 6-7 d at 25 °C. Colony radial, not finely zonate, mycelium dense, aerial hyphae numerous, short, forming a dense downy mat throughout the colony. Conidiation noted after 2 d, formed on aerial hyphae, first near the original inoculum, spreading throughout the colony after 7 d. Conidiophores trichoderma-like, irregularly branched, often rebranching one time, main axis often ending in branched or unbranched elongations, tips sterile or fertile. Phialides typically formed in whorls of 3-4, not commonly solitary or paired, lageniform, sometimes subulate in terminal position on the axis, (5. Notes: Trichoderma ganodermatis might be fungicolous since it was isolated from a fresh Ganoderma fruitbody. Several Trichoderma species have been reported as fungicolous, e.g. T. hypoxylon, T. pleuroti, T. pleuroticola, T. songyi and T. stromaticum [25][26][27][28] . These species are either causing agents of green mold diseases in mushroom cultivation or mycoparasitic fungi on plant pathogens and have the potential in biocontrol. Phylogenetically, T. ganodermatis is closely related to T. phyllostachydis ( Fig. 1). But the latter fungus grows faster on SNA at 25 °C (34-37 mm) and produces much shorter phialides (6.5-7.0 μm long) and conidia (2.3-3.0 μm long) 7 .
On PDA after 72 h colony radius 68-70 mm, mycelium covering the plate after 3 d at 25 °C. Colony green, mycelium dense, aerial hyphae abundant. Conidiation starting after 8 d, formed in pustules, pustules abundant, uniformly distributed throughout the colony, hemispherical or pulvinate, compact, white, turning green after 9 d, with hairs extending beyond the surface. No chlamydospores observed. No distinct odour, no diffusing pigment observed.
On SNA after 72 h colony radius 32-37 mm, mycelium covering the plate after 7 d at 25 °C. Colony similar to CMD, but aerial hyphae not common. Conidiation starting after 3 d, formed on aerial hyphae and in pustules, Notes: Trichoderma ingratum inhabits in high attitude forests of central and southwestern China, which is similar to T. alpinum. But T. alpinum differs in shorter phialides and larger conidia, and do not produce unpleasant odour in culture. Phylogenetically, T. ingratum is closely related to T. corneum, but the latter species can be easily separated by verticillium-like conidiophores and longer phialides (8-24 × 1.5-3.0 μm) 24 . K. Chen, TC253 (HMAS 248831). Ex-type culture CGMCC 3.18388. On CMD after 72 h colony radius 37-38 mm, mycelium covering the plate after 5 d at 25 °C. Colony hyaline, radial, circular, mycelium dense, aerial hyphae common, more abundant with distance from the original inoculum. Conidiation starting after 3 d, effused on aerial hyphae or in small granules, denser along the colony margin. No chlamydospores observed. No distinct odour, no diffusing pigment observed.
On PDA after 72 h colony radius 31-33 mm, mycelium covering the plate after 7 d at 25 °C. Colony dense, not finely zonate, circular, margin not well defined and slightly wavy. Aerial hyphae numerous, forming a loose floccose mat, denser in the colony center. Conidiation starting after 2 d, on aerial hyphae around the original inoculum, numerous, white, turning green after 3 d. Conidiophores simple, asymmetry, verticillium-like, often comprising a main axis, side branches loosely disposed, paired or unpaired, in right or acute angles with the main axis, not or rebranching one time. Phialides solitary, less commonly paired or in whorls of 3, narrowly lageniform, often hooked, 8.3-13.9 × 2.8-3.9(−4.5) μm, l/w 2.4-4.3(−5.0), 1.9-2.5 μm wide at the base (n = 40). Conidia green, smooth, with several minute guttules, variable in shape, ellipsoid, less commonly globose, oval or oblong, Notes: Trichoderma liberatum well-located in the Harzianum Clade forms verticillium-like conidiophores with the side branches loosely disposed. The conidiophore branching pattern is somewhat similar to T. pararogersonii in the Viride Clade. But the latter fungus produces hyaline ascospores and is remotely related. Phylogenetically, T. liberatum is associated with T. pseudodensum and T. zayuense, but T. pseudodensum can be easily separated by its densely disposed conidiophore branches, while T. zayuense differs in its slender and longer phialides (11.1-15.3 μm).
On PDA after 72 h colony radius 62-63 mm, mycelium covering the plate after 4 d at 25 °C. Colony radial, dense, aerial hyphae numerous, long and wooly. Conidiation starting after 2 d, abundant, effuse in aerial hyphae or densely disposed granules, denser at the colony center. Conidiophores simple, asymmetry, verticillium-like, branches often unpaired, not or rebranching once. Phialides solitary, paired or in whorls of 3-4, inclined upwards with the metula, narrowly lageniform, sometimes subulate, (8. Notes: The colony morphology of T. linzhiense on PDA is very similar to that of T. pseudodensum. Both of them produce dense granules or shrubs on aerial hyphae. In comparison with the new species, T. pseudodensum is distinguishable in forming shorter phialides and conidiophores with dense side branches. Phylogenetically, T. linzhiense is closely related to T. cerinum. But the latter species has much shorter phialides (less than 7.6 μm long) and smaller conidia (2.4-3.5 × 2.0-2.5 μm) 23 . On PDA after 72 h colony radius 46-47 mm, mycelium covering the plate after 5 d at 25 °C. Colony radial, not finely zonate, mycelium dense, aerial hyphae short, inconspicuous. Conidiation noted in pustules after 9 d, pustules spreading in concentric areas, hemispherical to pulvinate, outline not well defined, 1-4 mm diam, first white, turning green after 10 d. Conidiophores pachybasium-like, with a distinct main axis, up to 160 μm long, elongations straight or sinuous, tips sterile, fertile braches arising from the base, paired or solitary, not or rebranching once. Phialides ampulliform, 5.6-9.4(−11.4) × 3.6-5.0 μm, l/w 1.2-2.4, 1.9-3.6 μm wide at the base  On SNA after 72 h colony radius 27-28 mm, mycelium covering the plate after 7 d at 25 °C. Colony hyaline, margin not well defined, aerial hyphae nearly lacking. Conidiation noted in pustules after 10 d, not common, appearing at the colony margin, hemispherical, compact, remaining discrete, 1-2 mm diam, first white, turning green after 11 d, with hairs extending beyond the surface, hairs short, sinuous, sterile at the tips. No chlamydospores, no distinct odour observed. Light yellowish pigment noted.
On PDA after 72 h colony radius 52-53 mm, mycelium covering the plate after 4 d at 25 °C. Colony radial, dense, circular. Aerial hyphae abundant, forming a flat, dense, whitish mat, coilings common. Conidiation starting after 3 d, effuse in aerial hyphae or in densely disposed granules, more abundant at colony center, yellowish green. Conidiophores numerous, symmetry, trichoderma-like, often with a main axis, side branches densely disposed, in right or acute angles with main axis, typically in whorls of 3, less commonly solitary or paired. Phialides densely disposed, paired or in whorls of 3-4, ampulliform to lageniform, (6.  Figure 22 Fungal Names FN570389 Etymology: The specific epithet refers to the phylogeny position of the species which is not closely related to other species in the same clade. Holotype: CHINA, HUBEI: Shennongjia Natural Reserve, elev. 1720 m, isolated from soil, August 2015, K. Chen, TC781 (HMAS 248848). Ex-type culture CGMCC 3.18400. On CMD after 72 h colony radius 53-56 mm, mycelium covering the plate after 4 d at 25 °C. Colony green, radial, not finely zonate, mycelium loose, aerial hyphae common. Conidiation starting after 2 d, effuse in aerial hyphae or in shrubs, shrubs densely disposed on aerial hyphae, often with green drops. No chlamydospores observed. No distinct odour, no diffusing pigment observed.
On PDA after 72 h colony radius 41-47 mm, mycelium covering the plate after 7 d at 25 °C. Colony green, circular, not zonate, margin not well defined, slightly lobed, substrate mycelium abundant, aerial hyphae common, denser at the colony center. Conidiation starting after 2 d, abundant, formed on aerial hyphae, more abundant at the colony center. Conidiophores short and simple, trichoderma-like, often with a short main axis, side branches  Notes: Trichoderma solum is distinguishable by the very dark green granules or tumor-like structures on aerial hyphae, which are uncommon in Trichoderma. Phylogenetically, the three strains of T. solum with identical sequences form a separate terminal branch which is not close related to any other species in the same clade. Trichoderma linzhiense is similar in conidiophore branch patterns and phialides, but differs in colony characteristic, forming yellow pigments on PDA and chlamydospores on CMD and SNA.
Trichoderma undatipile K. Chen & W.Y. Zhuang, sp. nov. Figure 23 Fungal Names: FN570390 Etymology: The specific epithet refers to the curved hairs extending beyond the pustules surface produced in culture. Holotype: CHINA, HUNAN: Hengyang, Hengshan National Nature Reserve, elev. 500 m, isolated from rotten twig, October 2015, K. Chen, TC873 (HMAS 248854). Ex-type culture CGMCC 3.18403. On CMD after 72 h colony radius 57-59 mm, mycelium covering the plate after 4 d at 25 °C. Colony hyaline, radial, mycelium loose, aerial hyphae inconspicuous. Conidiation noted after 4 d, first formed on short erect conidiophores and aerial hyphae, conidial pustules noted after 5 d, not common, spreading along the margin of the colony, pulvinate, compact, 1-4 mm diam, first white, turning green after 6 d, with hairs extending beyond the surface, hairs curved or sinuous, tips sterile. Conidiophores in pustules pachybasium-like, with a distinct main axis, up to 150 μm, tips sterile, fertile branches arising from the base, short and simple, paired or solitary. Phialides typically formed in whorls of 3-4, ampulliform, 4.8-9.7 × 2.  Notes: Trichoderma zayuense is featured by its conidiophore branching pattern, in which the side branches are symmetrically disposed at a right angle to the main axis. This pattern is also found in T. ingratum, but the conidia of the latter species are smaller (2.6-3.6 × 2.6-3.2 μm) and regularly globose in shape. Phylogenetically, T. zayuense is closely related to T. pseudodensum but not conspecific (see Notes under T. pseudodensum).

Discussion
Methods. Elad et al. 33 reported a Trichoderma selective medium (TSM) as a tool for isolating Trichoderma species from soil. However, the complicated components make TSM unsuitable for isolating soil samples in great numbers. In our study, PDA with chloramphenicol was chosen as the selective medium, and it turns out to be effective. Among the three dilution gradients (10 −1 , 10 −2 and 10 −3 ), 10 −2 seems to be most appropriate as it forms moderate colonies that can be easily separated from each other. Ecology. Geographically, soil samples were mainly collected from mountains or nature reserves in different regions of China, which are relatively undisturbed by humans. Compared with those from farmlands 17, 34-37 , our soil samples show a high species diversity that 23 new species are discovered among the 85 Trichoderma species identified. Most of the new species were isolated from southern China (Guangdong, Guangxi, Hainan, Hubei, Hunan, Sichuan, Tibet, and Yunnan). Only two of them (T. breve and T. chlamydosporicum) were from the north (Beijing and Heilongjiang). It is clear that climates between south and north of the country are significantly different and there are more rains in the south bringing high humidity. This result is in accordance with some earlier studies that southern China are of higher Trichoderma species diversity than the northern areas 16,17 . For example, seven new species were found from Hubei Province in addition to the previously described T. hubeiense and T. shennongjianum 18,38 . Hubei is relatively rich in Trichoderma species.
All the new species recorded in this study were isolated from soil except for T. ganodermatis, T. polypori and T. undatipile, which were from fungi or decaying wood. Among soil-inhabiting species, T. alpinum and T. ingratum are also found on decaying wood (unpublished data). Similarly, T. ganodermatis occurs on decaying wood as well. These fungi are obviously not substrate-specific. They may have flexible nutrition modes (fungicolous, saprophytic), or live on different substrata as indicated by Chaverri and Samuels 39 . This makes Trichoderma one of the most successful fungi in various ecosystems. The so-called soil-inhibiting species might become well-adopted in diverse environmental conditions. Phylogeny. With the addition of the 23 new species, phylogenetic relationships among species of the genus are renewed based on the combined sequence analyses of RPB2 and TEF1. A new clade, the Spirale Clade, is established to accommodate three Trichoderma species, T. hunanense, T. longisporum and T. spirale. In the previous studies, phylogenetic position of T. spirale was variable. For example, the fungus was reported closely related to T. polysporum in the Polysporum clade based on analyses of the combined RPB2 and TEF1 7 . However, the TEF1 phylogeny draws a different picture that it belonged to the Strictipile Clade and was closely related to T. longipile and T. strictipile 8 . Jaklitsch and Voglmayr 14 showed that T. spirale was a separate terminal branch, whereas, our results show that T. spirale has close relationship with T. hunanense and T. longisporum receiving very high statistic supports (Fig. 1, MPBP/BIPP = 100%/100%). Fungi in the Spirale Clade share the following morphological similarities: forming hairy pustules, producing yellow pigments in culture, and having more or less oblong conidia.
The Helicum Clade was previously comprised only two species 14 . And the clade appeared to be a separate group distantly related to any other species of the genus. In this study, T. byssinum, T. hainanense and T. undatipile are added. The updated Helicum Clade is associated with T. pseudonigrovirens receiving very low statistic supports (Fig. 1).
The Harzianum Clade formerly containing 41 species is the largest among the green-spored groups. As the 15 new species joining the clade, the relationships among members of the clade are altered. Trichoderma alni previously related to T. christiani 14 is now associated with T. alpinum (Fig. 1, MPBP/BIPP = 97%/100%); and T. christiani is clustered with T. concentricum, T. corneum and T. ingratum with very low supports (Fig. 1). Two of the new species, T. liberatum and T. solum, form lone lineages which are distantly related to any other species of the clade.
Future prospects. Identifications of Trichoderma species are not always easy. It is impossible to define or recognize a species solely based on morphology, especially when sexual state is absent. Many DNA fragments are available for Trichoderma identifications and phylogeny, e.g. ITS, RPB2, TEF1, CAL1 and ACT. ACL1 was recently introduced to study of the genus, which turns out to be efficient 40 . The species concepts may be firmly established with the application of phylogenetic analyses at genomic level.
In summary of the recent studies including this work 11,12,18,32,38,[41][42][43][44][45] , China is undoubtedly a hotspot of Trichoderma species diversity because more than 60 new species have been established and typified by the Chinese materials. However, our knowledge of the group in the country is far from sufficient compared with that in Europe 8,14,19 . Further extensive surveys in unexplored areas are surely required.

Materials and Methods
Soil samples. A total of 750 soil samples were collected from 23 provinces of China (some were generously provided by colleagues and students) during a three-year-investigation to assess the biodiversity of Trichoderma in soil. Most of the samples were from undisturbed areas, e.g. mountains and nature reserves. The soil samples are geographically widely distributed: the northernmost samples are from Heilongjiang Province (53°33′N), and the southernmost are from the Xisha Islands in Hainan Province (15°40′N). The sampled regions comprise diverse climate types: temperate, tropical, subtropical, and alpine climates. The attitudes are ranged from 0 m (Xisha) to 4800 m (Tibet). The various geographic conditions, attitudes and climates makes the samples representative. All the samples were stored at 4 °C or −20 °C before use.
Strains. Gradient dilution and spread plate method were used to isolate the strains: three dilutions (10 −1 , 10 −2 and 10 −3 ) were prepared with 1 g soil and sterile water, 0.2 mL of each dilution was spread on a 9-cm-diam PDA Petri dish (200 g potato + 2% (w/v) dextrose + 2% (w/v) agar, with 300 mg/L chloramphenicol added). Plates were incubated at 25 °C for 1-3 d. Individual colonies were transferred to a 6-cm-diam PDA Petri dish and cultured at 25 °C. Strains were stored in China General Microbiological Culture Collection Center (CGMCC).
Morphology observations. Growth rates and culture characteristics were determined on three different media: CMD (40 g cornmeal + 2% (w/v) dextrose + 2% (w/v) agar), PDA and SNA (synthetic low nutrient agar) (Nirenberg 1976). Strains were first cultured on PDA plates for 2-4 d, and then small agar plugs (0.5 cm diam) were cut from actively growing edges of colonies, and placed on 9-cm-diam Petri dishes (CMD, PDA and SNA) 1-1.5 cm away from plate edge. Strains were incubated at 25 °C in 12 h light and 12 h dark. Microscopic structures were observed with 3% KOH solution as mounting medium. Nikon D3300 was used for photographs of colony morphology, and AxioCam MRc5 connected to a Zeiss Imager A2-M2 microscope (Carl Zeiss AG, Göttingen) for microscopic features. Figures were edited and combined using Adobe Photoshop CS5. DNA extraction and sequencing. Strains were incubated on PDA plates at 25 °C for 3-7 d. Approximately 1 cm 2 agar plug with mycelium was cut and transferred to a 1.5 mL Eppendorf tube. Agar plug was grinded with a glass grinding rod and 600 μL CTAB (Hexadecyl trimethyl ammonium bromide) buffer [Tris-HCl 100 mM, EDTA (Ethylenediaminetetraacetic acid) 20 mM, NaCl 1.4 M, CTAB 2% (w/v), β-mercaptoethanol 0.2% (v/v), pH 8.0] was added. Then the Eppendorf tube was incubated at 65 °C for 30 min. Subsequently, 600 μL chloroform-isoamylalcohol (24:1) was added to the solution, mixed and centrifuged for 10 min at 12,000 rpm. Supernatant was transferred to a new tube and 600 μL chloroform-isoamylalcohol (24:1) was added, repeat the previous step. 300 μL isopropanol was added to the supernatant and kept at −20 °C for 30 min. Then the mixture was centrifuged for 10 min at 12,000 rpm and sediment was washed with ice cold 70% ethanol and dried at room temperature. Sediment was re-suspended with 150 μL double distilled water and stored at −20 °C.
The following gene sections were amplified: ITS was amplified using the primer pair ITS 4 and ITS 5 46 ; RNA polymerase II subunit B (RPB2) was amplified using the primer pair fRPB2-5f and fRPB2-7cr 47 or tRPB2-5F and tRPB2-7R 18 ; translation elongation factor 1 alpha (TEF1) was amplified using the primers EF1-728F 48 and TEF1LLErev 49 . PCR products were purified with the PCR product purification kit (Biocolor BioScience & Technology Co., Shanghai, China) and sequenced by the ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, California) with the same primers as in PCR or internal primers for RPB2 and TEF1 8 .
Phylogenetic analyses. Sequences were assembled and manually adjusted using the DNAStar Seqman program 7.1.0 (DNASTAR. Inc., Madison). ITS sequences were subjected to TrichOKey 50 to give a preliminary identification of the Trichoderma strains. RPB2 and TEF1 sequences were aligned using ClustalX 1.83 51 . Alignment was desposited in TreeBASE under the submission number 20730. All species of the related taxa were included in the analyses. Trichoderma rossicum and T. stromaticum in the Stromaticum Clade were chosen as outgroup taxa. Sequences used in the analyses are listed in Supplementary Table S1. MP analyses were performed with PAUP* 4.0b 10 52 , using 1000 replicates of heuristic search with random addition of sequences and tree bisection-reconnection (TBR) as the branch-swapping algorithm (steepest descent option not in effect, MULTREES option not in effect). All characters were weighted equally and gaps were treated as missing characters. Maximum parsimony bootstrap proportions (MPBP) were calculated from 1000 replicates, each with 10 replicates of random addition of taxa.
For BI analyses, MrBayes 3.1.2 53 was used and the substitution model for each locus was estimated by MrModeltest 2.3 54 . HKY + I + G was selected as the best-fit likelihood model under the output strategy of Akaike Information Criterion (AIC). Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 2000000 generations sampling every 100 generation. The first 2500 trees were discarded as the burn-in phase, and Bayesian inference posterior probability (BIPP) was determined from the remaining trees.
Trees were viewed and examined by TreeView 1.6.6 55 with MPBP above 50% and BIPP above 90% shown at the nodes.