409 Mycoscience 38: 409-420, 1997 Phylogenetic relationships of the hyphomycete genera Chaetopsina and Kionochaeta based on 18S rDNA sequences Gen Okada, Akiko T a k e m a t s u and Yukiko T a k a m u r a ~ Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), 2-1, Hirosawa, Wako-shi, Saitama 351-01, Japan Accepted for publication 16 October 1997 A setiform dematiaceous hyphomycete, Kionochaeta spissa was newly collected and isolated from evergreen broadleaved forests in the southern parts of Japan. Except for its dematiaceous nature, the species is morphologically similar to a nectriaceous hyphomycete, Chaetopsina fulva. The morphology and cultural properties of the Japanese isolates of K. spissa were described, and the phylogenetic relationships between Chaetopsina (C. fulva (type species)) and Kionochaeta (K. ramifera (type species), K. spissa and K. ivoriensis) were inferred based on nuclear encoded small subunit (18S) rDNA sequences using the neighbor-joining method. Chaetopsina and Kionochaeta were found to be phylogenetically related to the Hypocreales and Sordariales, respectively. Both should be maintained as separate genus for phylogenetic classification. The morphological resemblance especially between C. fulva and K. spissa is an example of the convergent evolution. Key Words Chaetopsina fulva; Kionochaeta spissa; mitosporic fungi; phylogenetic relationship; 18S rDNA sequence. Rambelli (1956) established the anamorph genus Chaetopsina Rambelli typified by C. fulva Rambelli. The type species is characterized by pale reddish brown setiform conidiophores and compact phialides producing mucoid hyaline ameroconidia. The generic circumscription was gradually modified to accommodate some other species with dark brown conidiophores or lateral branches on the conidiophore (Matsushima, 1971; Rambelli and Lunghini, 1976; Sutton and Hodges, 1976; Persiani et al., 1984). More than 15 species were subsequently ascribed to Chaetopsina (Rambelli, 1956; Matsushima, 1971; Rambelli and Lunghini, 1976, 1979; Sutton and Hodges, 1976; Morgan-Jones, 1979; Crane and Schoknecht, 1982; Kirk, 1985; Samuels, 1985; Castafieda, 1986; Wingfield, 1987; Merli et al., 1992; Zucconi and Rambelli, 1993), including moniliaceous and dematiaceous species. Sutton and Hodges (1976) mentioned the heterogeneity among the Chaetopsina species with reddish brown (moniliaceous) and dark brown (dematiaceous) conidiophores. Samuels (1985) described 4 new species of Nectria Fr. with Chaetopsina anamorphs. He tentatively treated N. chaetopsinae Samuels as the teleomorph of C. fulva and restricted the genus Chaetopsina s. str. to anamorphs of the Nectriaceae (Hypocreaceae in Hawksworth et al., 1995) based on the anamorph morphology of these Nectria species and the pigment color Present address: Novo Nordisk Bioindustry Ltd., Makuhari Techno Garden CB-6, Nakase 1-3, Mihama-ku, Chiba-shi 261-01, Japan. change reaction of conidiophores. Kirk (1985), moreover, speculated that the teleomorphs for the dematiaceous Chaetopsina species were likely to be found in Chaetosphaeria Tul. & C. Tul. As a solution to the heterogeneous nature of Chaetopsina s. Int., Kirk and Sutton (1985) introduced the anamorph genus Kionochaeta Kirk & Sutton for dematiaceous species assumed to be anamorphs of the Sphaeriaceae. They proposed the following new species or new combinations for Kionochaeta: K. aristata Kirk, K. malaysiana Kirk, K. spissa Kirk & Sutton, K. ivoriensis (Rambelli & Lunghini) Kirk & Sutton, K. keniensis (Kirk) Kirk & Sutton, K. ramifera (Matsushima) Kirk & Sutton, and K. virtuosa (Rambelli & Lunghini) Kirk & Sutton. Kuthubutheen and Nawawi (1988) and Crous et al. (1994) later described another new species of Kionochaeta. Rambelli (1987), on the other hand, conducted a bibliographic reassessment of the genus Chaetopsina s. Int. in which he rejected the distinction of the genus Kionochaeta. He included Chaetopsina s. Int. in the Dematiaceae, as Ellis (1971) treated this genus in his book "Dematiaceous Hyphomycetes." During a survey of microfungi in evergreen broadleaved forests in Japan, we collected and isolated a Chaetopsina-like fungus from plant debris at Kagoshima and Okinawa in Japan. A literature survey and comparison with the type materials allowed us to identify the hyphomycete as K. spissa. The species is morphologically very similar to C. fulva, but can be distinguished mainly by dematiaceous conidiophores. Although Kirk and Sutton (1985) speculated on the sphaeriaceous 410 G. Okada et al. affinity of the genus Kionochaeta, no teleomorphs have been found for Kionochaeta species. Using new Japanese isolates of K. spissa and authentic strains of K. ramifera, K. ivoriensis and C. fulva, we carried out a phylogenetic analysis of Chaetopsina and Kionochaeta based on nuclear encoded small subunit (18S) rDNA sequences by the neighbor-joining method. Materials and Methods Strains examined The following strains were used for sequencing or for morphological observation: K. ramifera (type species) JCM 9756 (=IFO 9947), K. spissa JCM 9817 and JCM 9818, K. ivoriensis JCM 9876 ( : C B S 374.76, ex-type strain) and C. fulva (type species) JCM 9754 (=IFO 8919, ex-type strain) and JCM 9755 (=IFO 8843). Isolation of DNA To obtain genomic DNA, the strains were cultivated in Difco YM broth at 20-25~ for approximately 7 d. The centrifuged wet hyphae/cells were packed with aluminum foil, frozen at - 8 0 ~ then crushed mechanically with a hammer. The genomic DNA was extracted and purified as described by Yotsumoto et al. (1995). PCR amplification, cloning and sequencing of genomic DNA The DNA of 18S rRNA coding regions from the strains was amplified by the polymerase chain reaction (PCR) method with Taq DNA polymerase (Takara Taq) and oligodeoxynucleotides 5'-dATCTGGTTGATCCTGCCAGTAG-3' (designated primer 2F) and 5'-dTTTCACACAGGAAACAGCTATGAC-3' (designated primer 1794R), which were synthesized on the basis of conserved regions at the 5' and 3' termini of eukaryotic 18S rRNAs (Takara). The PCR was performed with a DNA thermal cycler PJ2000 (Perkin-Elmer Cetus) by 25 amplification cycles consisting of denaturation at 94~ for 30s, primer annealing at 55~ for 30s, primer extension at 72~ for 2 rain, and the final 7 min extension step necessary to make all DNAs double-stranded with 3'A-overhangs. Amplified 18S rDNA was directly ligated into the plasmid vector pCRTMII or pCRTM2.1 (3.9kb), then transformed into One Shot TM INV~F' competent cells using the Original TA Cloning Kit (Invitrogen). The plasmid DNA was extracted and purified from Escherichia coficultures using the alkaline method of Flexi Prep Kit (Pharmacia Biotech), and the presence of the cloned insert was confirmed by restriction enzyme digestion. The purity and concentration of plasmid DNA solutions were determined by agarose gel electrophoresis and by measuring optical densities of solutions in a capillary cell of 0.5 mm in inside diameter (Shimadzu) with a UV-Visible Recording Spectrophotometer (Shimadzu) at the wavelengths of 280, 260 and 230nm. For sequencing the total 18S rDNA, denaturated plasmid DNA and eight deoxyoligonucleotides (Table 1 ) were respectively used as templates and primers in each strain for chain elongation by the dideoxy method (Sanger et al., 1977) with Cy5 TM AutoCycle TM Sequencing Kit (Pharmacia Biotech). Sequencing reactions were then carried out with a GeneAmp PCR System 9600 (Perkin-Elmer Cetus). The conditions of the elongation reaction were 17 cycles consisting of denaturation at 95~ for 36s, annealing at 50~ for 36s, and extension at 72~ for 84s; then 13 cycles consisting of denaturation at 95~ for 36s and extension at 72~ for 84s; and finally extension for 5rain at 72~ The sequences of genomic DNA base Table 1. Primers used in each strain of the Kionochaeta and Chaetopsina species for amplifying and sequencing 18S rDNA. Primer PCR primer for amplification of 18S rDNA 2F: 5'-dATCTGGTTGATCCTGCCAGTAG-3' 1794R: 5"-dGATCCTTCCGCAGGTTCACC-3' Primer for sequencing Universal primer contained in the AutoCycle Sequencing Kit M13(-40): 5'-dCGCCAGGGTTTTCCCAGTCACGAC-3' M13Reverse: 5'-dTTTCACACAGGAAACAGCTATGAC-3" Synthesized forward primer 404F: 5"-dGCTACCACATCCAAGGAAGG-3' 573F: 5"-dCGCGGTAATTCCAGCTCCA-3' 1270F: 5'-dCATGGCCGTTCTTAGTTGG-3' Synthesized reverse primer 581 R: 5'-dATTACCGCGGCTGCTGGC-3" 1332R: 5"-dAAGGTCTCGTTCGTTATCG-3' 1641 R: 5'-dACGGGCGGTGTGTAC-3' a) Modified primer of Nishida and Sugiyama (1993). b) Nishida and Sugiyama (1993). c) Hendriks et al. (1991 ). d)Lane et al. (1985). Corresponding position in the 18S rDNA sequence of Saccharomyces cerevisiae 2-23 a) 1794-1775 b) 404-423 573-591 c) 1270-1289 581-564 c= 1332-1314 1641-1637c,d)