MYCOBIOLOGY 2019, VOL. 47, NO. 3, 280–291 https://doi.org/10.1080/12298093.2019.1641062 RESEARCH ARTICLE Two New Species and a New Chinese Record of Hypocreaceae as Evidenced by Morphological and Molecular Data Zhao Qing Zeng and Wen Ying Zhuang State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, P.R. China ABSTRACT ARTICLE HISTORY To explore species diversity of Hypocreaceae, collections from Guangdong, Hubei, and Tibet Received 13 February 2019 of China were examined and two new species and a new Chinese record were discovered. Revised 27 June 2019 Morphological characteristics and DNA sequence analyses of the ITS, LSU, EF-1a, and RPB2 Accepted 4 July 2019 regions support their placements in Hypocreaceae and the establishments of the new spe- Hypomyces hubeiensis Agaricus KEYWORDS cies. sp. nov. is characterized by occurrence on fruitbody of Hypomyces hubeiensis; sp., concentric rings formed on MEA medium, verticillium-like conidiophores, subulate phia- morphology; phylogeny; lides, rod-shaped to narrowly ellipsoidal conidia, and absence of chlamydospores. Trichoderma subiculoides Trichoderma subiculoides sp. nov. is distinguished by effuse to confluent rudimentary stro- mata lacking of a well-developed flank and not changing color in KOH, subcylindrical asci containing eight ascospores that disarticulate into 16 dimorphic part-ascospores, verticillium- like conidiophores, subcylindrical phialides, and subellipsoidal to rod-shaped conidia. Morphological distinctions between the new species and their close relatives are discussed. Hypomyces orthosporus is found for the first time from China. 1. Introduction Members of the genus are mainly distributed in temperate and tropical regions and economically The family Hypocreaceae typified by Hypocrea Fr. important in biomedicine and agriculture [32,33]. was established by Saccardo [1] and was redefined Host specificity, color of subicula and perithecia, by Rossman et al. [2] who treated it in a narrow shape, size, septation, surface ornamentation, and sense and recognized 12 genera. Approximate 17 apiculus of ascospores, and type of asexual states are genera are currently accepted [3–6], including main characters used for identifications of Hypomyces (Fr.) Tul. & C. Tul and Trichoderma Hypomyces species. The genus grows on Agaricales, Pers., two major genera encompassing the majority Boletales, Helotiales, and Pezizales are highly host- of species of the family. The phylogenetic relation- specific, while those occurring on Polyporales may ship among genera of the group was first revealed have a slightly wider host range [2,12–16]. For by Spatafora and Blackwell [7]. example, H. lithuanicus Heinr.-Norm. lives only on This study is focused on two major genera of the Lactariustor minosus (Schaeff.) Gray, H. hyalinus family. For a long time, many fungicolous fungi (Schwein.) Tul. & C. Tul. is restricted to Amanita with light- or bright-colored perithecia produced Adans, and H. melanocarpus Rogerson & Mazzeris within subiculum were described as Hypomyces is on Tylopilus P. Karst. However, H. australis which is typified by H. lactifluorum (Schwein.) Tul. (Mont.) Hohn.,€ H. rosellus (Alb. & Schwein.) Tul. & & C. Tul. Morphological characteristics and phylo- C. Tul. and H. tegillum Berk. & M.A. Curtis show genetic analysis based on sequence of nuclear ribo- the least specialized parasites and even being found somal large subunit (LSU) rDNA in the previous on non-fungus substrates, like rotten bark and work indicated that the genus is not a monophyletic wood [14]. group [8–11]. After the comprehensive studies by Trichoderma, the largest genus in the family Rogerson and Samuels [12–15] and Poldmaa~ and Hypocreaceae, was originally established by Persoon collaborators [11,16–22], the generic concept of [34] and typified with T. viride Pers. Since then, Hypomyces became clear. Among the 212 names number of Trichoderma species increased dramatic- listed in Index Fungorum database, about 77 species ally. Bissett et al. [35] provided a list of 254 are commonly accepted [11,23–29]. Twenty-seven of Trichoderma species with DNA sequences or living them have been known from China [27,29–31]. cultures available. There are more than 340 species CONTACT Wen Ying Zhuang [email protected] ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. MYCOBIOLOGY 281 currently recognized in the genus. They occur on Zeiss Axio Imager A2 microscope (Carl Zeiss) for rotten wood, bark and leaves, fruitbodies of other anatomic structures. Measurements of individual fungi, in soil, or within healthy plant tissues as structures were based on 30 units, except when endophytes [36–39]. They are renewable natural otherwise noted. Cultures were obtained from coni- resources and play important roles in production of dia on subiculum or from fresh ascomata using sin- industrial enzymes and antibiotics [40], biological gle ascospore isolation. To determine colony control of soil-borne plant pathogens [41,42], plant features and growth rates, strains were grown on growth promotion [43], induction of plant resistance cornmeal dextrose agar (CMD; Yuanye Bio- [44], production of bioactive secondary metabolites Technology Co. Ltd., Shanghai, China), malt extract [36] and remediation of soil contaminated by heavy agar (MEA; Oxoid Ltd., Basingstoke, UK), potato metals [45]. Taxonomy of Trichoderma species is dextrose agar (PDA; HuiXing Biochemistry Reagent mainly based on anatomy of stromata and perithe- Ltd Co., Shanghai, China) and synthetic nutrient- cia, color, shape, size of ascospores, conidia and poor agar (SNA) [51] in 90 mm plastic Petri dishes chlamydospores, type of conidiophores, colony at 25 C for 7 or 14 d. For observation of conidio- morphology and growth rate, and DNA sequence phores and microconidia, cultures were grown on data [46,47]. The phylogenetic analyses based on SNA at 25 C with alternating periods of light and translation elongation factor 1-a encoding (EF-1a) darkness (12 h/12 h). and RNA polymerase II subunit 2 (RPB2) regions indicated that the hyaline ascospored species are divided into 11 clades and those of green ones are 2.2. DNA extraction, PCR amplification, separated into 7 clades [48–50]. and sequencing During our survey of hypocrealean species on The genomic DNA was extracted from fresh myce- fungi and plant debris in China, two undescribed lium following the methods of Wang and Zhuang taxa are found based on morphological characteris- [52]. Primer pairs, ITS5/ITS4 [53], LR0R/LR5 [9, tics and DNA sequence analyses of the internal 54], and EF1-728F/EF1567R [55,56] were used to transcribed spacer (ITS), LSU, EF-1a, and RPB2. amplify the sequences of ITS, LSU, and EF-1a Differences between the new species and their close regions for Hypomyces species, while EF1-728F/ relatives are discussed. Hypomyces orthosporus is TEF1LLErev [55,57] and fRPB2-5F/fRPB2-7cR [58] reported for the first time from China. were applied to amplify the sequences of EF-1a and RPB2 regions for Trichoderma species. PCR reac- 2. Materials and methods tions were performed on an ABI 2720 Thermal Cycler (Applied Biosciences, Foster City, CA) with a 2.1. Collections and morphological study 25 ml reaction system consisting of 12.5 ml Taq Specimens were collected from Shennongjia Forestry MasterMix, 1 ml each primer (10 mM), 1 ml template m District of Hubei and Chebaling National Nature DNA and 9.5 l ddH2O, based on the procedures Reserve of Guangdong and Mainling of Tibet, and detailed in White et al. [53], Chaverri and Samuels deposited in the Herbarium Mycologicum [59], Rehner and Buckley [56], and Liu et al. [58]. Academiae Sinicae (HMAS). Cultures are kept in DNA sequencing was carried out in both directions the State Key Laboratory of Mycology, Institute of on an ABI 3730XL DNA Sequencer (Applied Microbiology, Chinese Academy of Sciences. Biosciences). Methods used by Jaklitsch [46] and Poldmaa~ [19] were followed. Test for color change of perithecial 2.3. Sequence alignment and phylogenetic wall was made with 3% potassium hydroxide analyses (KOH). To observe anatomic structures of perithe- cia, longitudinal sections of ascomata were made Newly generated sequences and those retrieved from with a freezing microtome (YD-1508-III; Jinhua GenBank are listed in Table 1 (Hypomyces) and Yidi Medical Appliance Co., Jinhua, China) at a Table 2 (Trichoderma). Nectria eustromatica thickness of 6–8 mm. Microscopic examinations and Jaklitsch & Voglmayr and Thyronectria berolinensis measurements were taken from the sections and (Sacc.) Seaver were used as outgroup taxa. squash mounts in lactophenol cotton blue solution Sequences were assembled, aligned, and the primer using an Olympus BH-2 microscope (Tokyo, Japan). sequences were trimmed with BioEdit version 7.0.5 Photographs were taken with a Leica DFC450 digital (Ibis Biosciences, Carlsbad, CA) [60] and converted camera (Leica Camera, Wetzlar, Germany) attached to NEXUS files by ClustalX version 1.83 (EMBL, to a Leica M125 stereomicroscope (Leica) for gross Heidelberg, Germany) [61]. Sequences were first morphology and a Zeiss AxioCamMRc 5 digital subjected