Persoonia 20, 2008: 53–58 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158508X314732 Morphological and molecular characterisation of a new anamorphic genus Cheirosporium, from freshwater in China L. Cai1, X.Y. Guo2, K.D. Hyde3,4 Key words Abstract Cheirosporium gen. nov. is characterised by the production of sporodochial conidiomata, semi­macrone­ matous to macronematous conidiophores that possess several distinct sterile branches, and cheiroid, smooth­walled ascomycetes conidia with rhexolytic secession. The 28S rDNA and ITS rDNA operon of this taxon were amplified and sequenced. Pleosporales A BLAST search revealed low homology between Cheirosporium triseriale and existing sequences in public data­ systematics bases, supporting the hypothesis that the species is new to science. Phylogenetic analysis showed that C. triseriale taxonomy groups with Dictyosporium and allied species, and nests within the Pleosporales (Dothideomycetes, Ascomycota). Cheirosporium is morphologically distinct from the cheirosporous genera Cheiromyces, Cheiromycina, Dictyosporium, Digitomyces, Digitodesmium and Pseudodictyosporium and these differences are discussed. Article info Received: 28 January 2008; Accepted: 17 April 2008; Published: 23 April 2008. INTRODUCTION ITS rDNA sequences showed that this fungus represents a new ascomycetous anamorph that could not be linked to a teleomor­ Freshwater fungi are taxonomically diverse, with more than phic genus. It is, therefore, described here as Cheirosporium 1 000 documented species, which include representatives from triseriale gen. & sp. nov. almost all major fungal classes (Tsui & Hyde 2003, Vijaykrishna et al. 2006). These fungi colonise many different substrates MATERIALS AND METHODS such as submerged plant litter, and aquatic organisms (Vijay­ krishna & Hyde 2006). Because most plant litter in freshwater is Submerged woody substrata were collected by Cai from a of terrestrial origin, the aquatic origin of most freshwater fungi is small stream in Yunnan, China. Samples were processed and debatable. Many of the known freshwater anamorphic species examined following the methods described in Cai et al. (2003). have also been recorded from terrestrial habitats, although this Observations and photomicrographs were made from material is not generally the case with teleomorphs (Tsui & Hyde 2003, mounted in water. Conidia were measured at their widest point. Vijaykrishna et al. 2006). The range between minimum and maximum values for micro­ Cheirosporous hyphomycetes were studied recently by Tsui scopic measurements is given. Mean values are in brackets et al. (2006) and Kodsueb et al. (2007), while Ho et al. (2000) with ‘n’ being the number of measurements. The holotype is reviewed cheirosporous genera and provided a key based deposited at the Mycological Herbarium of the Chinese Acad­ on morphological characters. Tsui et al. (2006) investigated emy of Science (HMAS) in Beijing, China. the phylogenetic relationships among several cheirosporous genera and found that Dictyosporium and allied genera are DNA extraction, PCR and sequencing closely related and form a strong monophyletic grouping in DNA was extracted from the herbarium specimen HMAS Pleosporales. New species of cheirosporous fungi have also 180703 using E.Z.N.A. Forensic DNA Extraction Kit (Omega recently been described, such as Aquaticheirospora, including product No: D3529­01/02). A sporodochial mass was removed a single species with synnematous conidiomata (Kodsueb et from the specimen for extraction using fine forceps, following the al. 2007). We are studying the freshwater fungi of China (e.g. manufacturer’s protocols. Partial 28S rDNA and ITS rDNA were Cai et al. 2006a, Cai & Hyde 2007), and collected an interesting amplified using fungal specific primers LROR and LR5 (Vilgalys cheirosporous taxon from Lijiang, Yunnan. Critical morphologi­ & Hester 1990) and ITS1 and ITS4 (White et al. 1990), respec­ cal examination and phylogenetic analysis based on 28S and tively. The polymerase chain reaction (PCR) was carried out in a 50 μL reaction volume and the PCR thermal cycles based 1 Centre for Research in Fungal Diversity, School of Biological Sciences, on the profile detailed in Cai et al. (2006b). PCR amplification The University of Hong Kong, Pokfulam Road, Hong Kong; corresponding was confirmed on 1 % agarose electrophoresis gels stained author e­mail: [email protected]. with ethidium bromide. Amplicons were then purified using 2 Longtengwu 8­3­502, Huilongguan, Changping District, Beijing, 102208, minicolumns, purification resin and buffer, following the manu­ PR China. facturer’s protocols (Amersham product code: 27­9602­01). 3 International Fungal Research & Development Centre, The Research Institute of Resource Insects, Chinese Academy of Forestry, Belongsi, DNA sequencing was performed using the primers mentioned Kunming 650224, Yunnan Province, PR China. above in an Applied Biosystem 3730 DNA Analyser at the Ge­ 4 School of Science, Mae Fah Luang University, Tasud, Chiang Rai 57100, nome Research Centre, The University of Hong Kong. Thailand. © 2008 Nationaal Herbarium Nederland Centraalbureau voor Schimmelcultures 54 Persoonia – Volume 20, 2008 Sequence alignment and phylogenetic analysis Yang 1996, Zhaxybayeva & Gogarten 2002) were determined Sequences were aligned using BioEdit (Hall 1999). Two data by Markov Chain Monte Carlo sampling (BMCMC) in MrBayes sets were analysed; the 28S rDNA data set and ITS/5.8S v. 3.0b4 (Huelsenbeck & Ronquist 2001). Six simultaneous rDNA data set. Novel sequences (EU413953, EU413954), Markov chains were run for 1 000 000 generations and trees together with reference sequences obtained from GenBank, were sampled every 100th generation (resulting 10 000 total were aligned using Clustal X (Thomson et al. 1997). Reference trees). The first 2 000 trees, which represented the burn-in sequences used in this study are mainly from Lumbsch et al. phase of the analyses, were discarded and the remaining 8 000 (2005) and Tsui et al. (2006), both studies providing 5 or more trees were used for calculating posterior probabilities (PP) in reference sequences. Alignment was manually adjusted to al­ the majority rule consensus tree. low maximum alignment and minimise gaps. Phylogenetic analyses were performed using maximum par­ 50 simony as implemented in PAUP v. 4.0b10 (Swofford 2002). Dictyosporium digitatum DQ018102 Ambiguously aligned regions, 26 and 49 characters in 28S and Digitodesmium bambusicola DQ018103 ITS data sets respectively, were excluded from all analyses. Dictyosporium toruloides DQ018104 Characters were equally weighted and gaps were treated as Aquaticheirospora lignicola AY736378 missing data. Trees were derived using the heuristic search Dictyosporium alatum DQ018101 option with TBR branch swapping and 1 000 random sequence 79 Pseudodictyosporium wauense DQ018105 additions. Maxtrees were unlimited, branches of zero length 100 Cheiromoniliophora elegans DQ018106 were collapsed and all parsimonious trees were saved. Clade Cheirosporium triseriale stability was assessed using a bootstrap (BT) analysis with Leptosphaeria calvescens AY849944 1 000 replicates, each with 10 replicates of random stepwise 90 addition of taxa. Kishino­Hasegawa tests (KH Test) (Kishino 95 Letendraea eurotioides AY787935 & Hasegawa 1989) were performed in order to determine 60 Letendraea helminthicola AY016362 whether trees were significantly different. Trees were figured 74 Bimuria novae-zelandiae AY016356 63 using Treeview (Page 1996). Munkovalsaria appendiculata AY772016 The model of evolution was estimated by using Mrmodeltest 80 Byssothecium circinans AY016357 2.2 (Nylander 2004). Posterior probabilities (PP) (Rannala & Pleospora herbarum var. herbarum AY382386 84 Lewia infectoria AY154718 72 Curvularia inaequalis AY163990 69 Cochliobolus hawaiiensis AY163979 97 Phaeosphaeria avenaria AY544684 Ophiobolus fulgidus U43472 56 Leptosphaeria doliolum U43475 69 Westerdykella ornata AY853401 99 Westerdykella cylindrica AY004343 Pleosporales 65 Preussia minima AY510391 74 Preussia australis AY510376 Trematosphaeria heterospora AY016369 Melanomma radicans U43479 Delitschia didyma AY853366 Farlowiella carmichaeliana AY541492 Hysteriales 92 Helicomyces roseus AY787932 Pleosporales Thaxteriella helicoma AY787939 85 Dothidea ribesia AY016360 100 Stylodothis puccinioides AY004342 Dothideales 61 92 Dothidea sambuci AY544681 78 Myriangium duriaei AY016365 Myriangiales 100 Microxyphium citri AY004337 Capnodiales Phaeotrichum benjaminii AY004340 Pleosporales Venturia hanliniana AF050290 Aniptodera chesapeakensis U46882 10 Fig. 1 Phylogenetic tree generated from parsimony analysis based on 28S rDNA sequences. Data were analysed with random addition sequence, and treat­ ing gaps as missing data. Bootstrap values ≥ 50 % are shown above or below branches. Thickened branches indicate Bayesian posterior probabilities ≥ 95 %. The tree is rooted with Aniptodera chesapeakensis. L. Cai et al.: Cheirosporium from freshwater in China 55 RESULTS Taxonomy The LSU sequence obtained from the herbarium specimen Cheirosporium triseriale gen. & sp. nov. consisted of 724 nucleotides. A BLAST search in NCBI (www. ncbi.nih.gov/blast) showed that this sequence is most similar to Cheirosporium L. Cai & K.D. Hyde, gen. nov. — MycoBank Aquaticheirospora lignicola (AY736378, 93 % identity), Munko­ MB506570. valsaria appendiculata (AY772016, 89 %), Leptosphaeria Coloniae in substrato naturali sporodochiales, dispersae, punctiformes, brun­ calvescens (AY849944, 89