Seven wood-inhabiting new species of the genus Trichoderma (Fungi, Ascomycota) in Viride clade

More than 200 recent collections of Trichoderma from China were examined and 16 species belonging to the Viride clade were identified based on integrated studies of phenotypic and molecular data. Among them, seven wood-inhabiting new species, T. albofulvopsis, T. densum, T. laevisporum, T. sinokoningii, T. sparsum, T. sphaerosporum and T. subviride, are found. They form trichoderma- to verticillium-like conidiophores, lageniform to subulate phialides and globose to ellipsoidal conidia, but vary greatly in colony features, growth rates, and sizes of phialides and conidia. To explore their taxonomic positions, the phylogenetic tree including all the known species of the Viride clade is constructed based on sequence analyses of the combined RNA polymerase II subunit b and translation elongation factor 1 alpha exon genes. Our results indicated that the seven new species were well-located in the Koningii, Rogersonii and Neorufum subclades as well as a few independent terminal branches. They are clearly distinguishable from any existing species. Morphological distinctions and sequence divergences between the new species and their close relatives were discussed.


Results
Phylogenetic analyses. The partition homogeneity test (P = 0.01) of RPB2 and TEF1-α sequences indicated that the individual partitions were generally congruent 18 . Phylogenetic positions of the new species were determined by analyses of the combined RPB2 and TEF1-α data set containing 72 taxa and 2479 characters. Bayesian Inference (BI), Maximum likelihood (ML) and Maximum parsimony (MP) trees generated shared the same topology. In MP analyses, 1504 characters were constant, 207 variable characters were parsimony-uninformative, and 768 were parsimony-informative. One of the 76 most-parsimonious trees was shown here as Fig. 1 (Tree length = 3985, Consistency index = 0.3967, Homoplasy index = 0.6033, Rescaled consistency index = 0.2475, Retention index = 0.6238).
On CMD after 72 h 38-40 mm and mycelium covering the plate after 5 d at 25 C. Colony circular, with distinct denser/looser concentric zones and looser zones much wider. Surface downy, floccose or farinose, yellowish green to green. Conidiation noted after 3 d. No distinct odor, no diffusing pigment observed.
On PDA after 72 h 35-41 mm and mycelium covering the plate after 5 d at 25 C. Colony dense and zonate, aerial hyphae frequent, forming radial strands, green to pale green. Conidiation starting after 2 d on and around the plug, effuse, spreading slowly towards the distal margin. A distinct coconut-like odor detected, pigment inconspicuous or pale yellow.
Notes. As the sister of T. albofulvopsis in the Koningii subclade ( Fig. 1), T. albofulvum also forms trichoderma-like conidiophores, but the presence of reddish brown pigments in culture, fast-growth, and green smaller conidia [2.8-3.5 × 1.8-3.0 μ m] apparently separate them. And the conidiophore branches of T. albofulvum are more complex, and phialides are solitary or commonly divergent in whorls of 2-5(-6) 20 . Compared with the sequences from the epitype of T. albofulvum 21   On PDA after 72 h 27-30 mm and mycelium covering the plate after 8 d at 25 C. Colony first hyaline, with coarsely wavy margin, not zonate; aerial hyphae numerous, thick, radially arranged on the margin, gradually forming a thick mat separated into 2-3 broad zones with irregular outline and a whitish to pale yellowish, downy to finely floccose surface. Conidiation noted after 24 h around the plug. Chlamydospores lacking or rare. A distinct coconut-like odor detected, no diffusing pigment observed.
On SNA after 72 h 32-35 mm and mycelium covering the plate after 7 d at 25 C. Colony homogeneous, not zonate, margin radially fan-shaped. Aerial hyphae sparser than that on CMD. Conidiation starting after 24-48 h, no chlamydospores observed. A faint coconut-like odor detected, no diffusing pigment observed.  Etymology. The specific epithet refers to the smooth conidia.
On CMD after 72 h 48-50 mm and mycelium covering the plate after 5 d at 25 C. Colony dense, whitish, surface downy, farinose to floccose, with marginal surface hyphae thicker. Conidiation noted after 4 d. A distinct coconut-like odor detected, no diffusing pigment observed.
On PDA after 72 h 43-45 mm and mycelium covering the plate after 5 d at 25 C. Colony similar to but denser than that on CMD, macroscopically homogeneous, whitish, surface downy, farinose to floccose, covered by abundant aerial hyphae several mm thick. Conidiation noted after 3 d. A distinct coconut-like odor detected, no diffusing pigment observed.
Trichoderma hispanicum and T. samuelsii might be related (Fig. 1, MLBP/MPBP/BIPP = 75%/77%/99%), and produce smooth conidia and similar phialides as T. laevisporum. However, conidiation pustules of T. hispanicum are more compact, and pustules on SNA are formed only at the lateral and distal margin of the Petri dish. Trichoderma samuelsii is unique in pyridine-like odor on PDA 9 , which is lacking in T. laevisporum. Etymology. The specific epithet refers to the similarity between the Chinese material and T. koningii.
On CMD after 72 h mycelium covering the plate at 25 C. Colony hyaline, circular, with distinct concentric zones. Conidiation starting after 3 d, developing abundantly at the edges of the concentric rings; surface On PDA after 72 h mycelium covering the plate at 25 C. Colony whitish, conspicuously dense, typically not zonate with distinct circular outline and well-defined margin. Aerial hyphae numerous, densely disposed in the centre, thick and branched, mostly radially arranged, making surface becoming hairy. Conidiation starting after 2 d on and around the plug in short minute shrubs, spreading, growing to tufts or pustules, green. No distinct odor, no diffusing pigment observed. On SNA after 72 h 55 mm and mycelium covering the plate after 4 d at 25 C. Colony hyaline, thin, aerial hyphae loosely arranged, radially. Conidiation noted after 3 d along the margin. No distinct odor, no diffusing pigment observed.
Notes. Trichoderma sparsum is diagnostic by fast growth, downy, floccose or farinose surface of colonies on CMD and PDA, trichoderma-like conidiophores, and globose or subglobose conidia. It shares common ancestor with T. rogersonii and T. subeffusum (Fig. 1)  Etymology. The specific epithet refers to the globose to subglobose conidia.
On CMD after 72 h 45-46 mm and mycelium covering the plate after 8 d at 25 C. Colony dense, whitish, with a well-defined margin. Aerial hyphae apparent toward the downy or floccose distal margin, becoming fertile. A distinct coconut-like odor detected, no diffusing pigment observed.
On PDA after 72 h 28-30 mm and mycelium covering the plate after 10 d at 25 C. Colony dense, white, macroscopically homogeneous, circle outline. Conidiation noted after 2 d, conidiophores formed widely spaced on abundant aerial hyphae. A distinct coconut-like odor detected, no diffusing pigment observed.
On CMD after 72 h 58-60 mm, mycelium covering the plate after 4 d at 25 C. Colony dense, with well-defined outline, aerial hyphae numerous. Conidiation noted after 2 d in distal areas of the plate, first effuse in minute shrubs, later in numerous minute granules and pustules with granulose or plumose surface, pale green. A distinct coconut-like odor detected, no diffusing pigment observed.
On PDA after 72 h 60-63 mm, mycelium covering the plate after 4 d at 25 C. Colony, dense, aerial hyphae abundant, conidiation noted after 2 d in numerous pale yellow to yellow green squamose granules or tufts and gradually formed two concentric zonate. A distinct coconut-like odor detected, no diffusing pigment observed.

Discussion
More than 100 recent collections of Trichoderma Viride clade from northern and central China were examined, and seven new species are found based on the integrated studies of phenotypic and molecular data. To explore their taxonomic positions, the phylogenetic tree containing all species of the Viride clade was constructed based on analyses of the combined sequences of RPB2 and TEF1-α. The 69 currently known species in this clade clustered together (Fig. 1). Six subclades, Hamatum/Asperellum, Koningii, Neorufum, Rogersonii, Viride and Viridescens, were recognized, which is basically congruent with the results by Jaklitsch and Voglmayr 12 . The seven new species are well-located in the Viride clade, and distributed in Koningii, Neorufum and Rogersonii subclades as well as the unnamed terminal branches; which are clearly distinguishable from any existing species.
Taxonomy of Trichoderma in China dates back to 1895 23 . In more than a century, successive findings have brought the number of the known species of the genus in China up to over 100 17,[24][25][26] . Species of the genus are located throughout the country, among which 75 are wood-inhabiting and have been found in Anhui, Fujian, Guangdong, Guangxi, Guizhou, Hainan, Hebei, Henan, Hubei, Hunan, Jiangsu, Jiangxi, Jilin, Liaoning, Shandong, Sichuan, Taiwan, Xizang, Yunnan, and Zhejiang provinces. As to the Viride clade, 13 species are wood-inhabiting among the 22 taxa reported in the country 10,[27][28][29][30][31][32] . China is one of the world biodiversity hotspots. Trichoderma serves as a good example. The known species of the genus in China occupy 40% of the world records.
Along with increased number of species joining the Viride clade and through the integrated studies on morphology, ecology and phylogeny, our understanding of the group will become more sophisticated and intelligible, reasonable species concepts will be firmly established, and co-relation between morphology and sequence data will be explored. Accumulations of our knowledge of Trichoderma will provide useful information for sufficient utilization of fungal resources.

Materials and Methods
Isolates and specimens. Specimens examined were collected from Beijing, Henan, Hubei and Hunan provinces, China. Strains were obtained from direct isolation from asexual morphs on the substrates or single-ascospore isolation following the method by Jaklitsch 33 . They were deposited in the fungarium of the Institute of Microbiology, Chinese Academy of Sciences (HMAS).
Morphological characterization. Methods and morphology were described basically following counterparts by Jaklitsch 33 and Jaklitsch & Voglmayr 12 . Longitudinal sections of rehydrated stromata were made at a thickness of 8-10 μ m using a freezing microtome (YD-1508-III, Yidi Medical Appliance Factory, Jinhua, Zhejiang, China). Colony radius and characteristics on Cornmeal Dextrose agar (CMD), Potato Dextrose agar (PDA) and synthetic low nutrient agar (SNA) were measured and noted. Photographs were taken with a Leica DFC450 digital camera (Wetzlar, Germany) connected to a Leica M125 stereomicroscope (Milton Keynes, UK) for gross morphology and a Zeiss AxioCam MRc 5 digital camera (Jena, Germany) connected to a Zeiss Imager A2 microscope (Göttingen, Germany) for anatomical structures.
DNA extraction, PCR amplification and sequencing.-Genomic DNAs were extracted following methods by Wang & Zhuang 34 from mycelia prepared previously. Two primer pairs, fRPB2-5f and fRPB2-7cr 35 , EF1-728F 36 and TEF1LLErev 37 were separately used to amplify fragments of RPB2 and TEF1-α. PCR products were purified with the PCR Product Purification Kit (Biocolor BioScience and Technology Co., Shanghai, China) and then cycle-sequenced using the primer pairs reported by Jaklitsch 33

Phylogenetic analyses. To locate the phylogenetic positions of the seven new species, 76 combined
Trichoderma sequences of RPB2 and TEF1-α were used for analyses, among them T. danicum and T. spinulosum were outgroup taxa. The sequences were aligned and assembled with BioEdit 7.0.5.3 38 , NEXUS files were subsequently generated by ClustalX 1.83 39 .
Maximum likelihood analysis was carried out with RaxML 40 with the PHY files generated by Mesquite 2.75 41 . GTRGAMMA was specified as the model. The analysis was run with a rapid bootstrap analysis using a random starting tree. Topological confidence of resulted trees was assessed by bootstrap proportion (BP) with 1000 replicates.
Maximum parsimony analysis was conducted by PAUP 4.0b10 42 using a heuristic search with tree-bisection-reconnection branch swapping. All characters were treated as unordered and unweighted, gaps were treated as missing data, sequences were auto-increased and Maxtrees was 1000. Topological confidence of resulted trees was tested by bootstrap proportion with 1000 replicates, each with 10 replicates of random addition of taxa.
Bayesian Inference analysis was performed with MrBayes 3.1.2 43 using Markov ChainMonte Carlo (MCMC) algorithm. Appropriate nucleotide substitution models was determined by MrModeltest 2.3 44 and the best fit model GTR+ I+ G was selected by Akaike Information Criterion for the investigated data set. Four MCMC chains (one cold and three heated) were run for one million generations with the trees sampled every 100 generations. The first 25% trees were excluded as the burn-in phase of the analyses, and posterior probability (PP) values were estimated by the 75% remaining trees. Trees were viewed in TreeView 1.6.6 45