STUDIES IN MYCOLOGY 50: 489–515. 2004. Ribosomal gene phylogeny and species delimitation in Geotrichum and its teleomorphs G. Sybren de Hoog1,2* and Maudy Th. Smith1 1Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; 2Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands *Correspondence: G.S. de Hoog, E-mail: [email protected] Abstract: A taxonomic revision is presented of all filamentous Hemiascomycetes that reproduce with predominantly arthric conidiogenesis. On the basis of SSU rDNA data, two widely divergent groups (1 and 2) are known to exist. Both are distantly related to the Hemiascomycetes, and show remarkable diversity in ITS rDNA, leading to the supposition that phylogenetically ancient fungi are concerned. The teleomorph / anamorph genera occurring in Groups 1 vs. 2 are classified in (1) Galactomy- ces and Dipodascus with Geotrichum anamorphs, vs. (2) Magnusiomyces with Saprochaete anamorphs. Taxonomy at the species level is based on ITS rDNA sequences and nDNA/DNA reassociation data. In total, 32 taxa are recognized. The phenetic data set applied is nutritional physiology. A key to the species is provided. Taxonomic novelties: Saprochaete chiloënsis (Ramírez & González) Kurtzman, Robnett, de Hoog & M.Th. Smith comb. nov., S. clavata (de Hoog et al.) de Hoog & M.Th. Smith comb. nov., S. suaveolens (Krzemecki) de Hoog & M.Th. Smith comb. nov., S. fungicola de Hoog & M.Th. Smith sp. nov., S. ingens (van der Walt & van Kerken) de Hoog & M.Th. Smith sp. nov., S. japonica de Hoog & M.Th. Smith sp. nov., S. ludwigii (Hansen) de Hoog & M.Th. Smith comb. nov., S. psychro- phila de Hoog & M.Th. Smith sp. nov., S. quercus de Hoog & M.Th. Smith sp. nov., S. capitata (Diddens & Lodder) de Hoog & M.Th. Smith comb. nov., S. sericea (Stautz) de Hoog & M.Th. Smith comb. nov., S. gigas (J. Smit & L. Meyer) de Hoog & M.Th. Smith comb. nov., Magnusiomyces capitatus (de Hoog et al.) de Hoog & M.Th. Smith comb. nov., M. mag- nusii (Ludwig) de Hoog & M.Th. Smith comb. nov., M. ovetensis (Pelãez & Ramírez) de Hoog & M.Th. Smith comb. nov., M. spicifer (de Hoog et al.) de Hoog & M.Th. Smith comb. nov., M. starmeri (Phaff et al.) de Hoog & M.Th. Smith comb. nov., M. tetrasperma (Macy & Miller) de Hoog & M.Th. Smith comb. nov., M. ingens de Hoog & M.Th. Smith comb. nov., Galactomyces pseudocandidus de Hoog & M.Th. Smith sp. nov., Gal. candidus de Hoog & M.Th. Smith sp. nov., Geotrichum europaeum de Hoog & M.Th. Smith sp. nov., Geo. restrictum de Hoog & M.Th. Smith sp. nov. Key words: ancient organisms, Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, Saprochaete, Hemiascomycetes, phylogeny, ribosomal genes, taxonomy, yeast-like fungi. INTRODUCTION Galactomyces comprises the generic type species of Geotrichum, Geo. candidum Link : Fr. (de Hoog et The taxonomy of the genus of filamentous yeast-like al. 1986). Geotrichum is morphologically character- fungi, Geotrichum Link : Fr. (Hemiascomycetes) has ized by the presence of arthroconidia that are liberated been studied in depth by Smith and co-workers in a schizolytically in random order. Septal walls are series of publications (de Hoog et al. 1986, Smith et perforated by micropores. In some species, such as al. 1995, 2000, Smith & Poot 1998, 2003, Naumov et Geo. fermentans (Diddens & Lodder) Arx and Geo. al. 1999, Naumova et al. 2001). A review of the capitatum (Diddens & Lodder) Arx, additional ho- taxonomy of the fungi concerned was presented in loblastic conidia are observed. Some species of the Kurtzman & Fell (1998). Techniques used for genus- Geo. capitatum complex are remarkable by having wide comparisons were morphology, physiology, blastic conidia arranged in a profusely branched cultural characteristics, nDNA reassociation, mol % conidial apparatus, each cell producing an elongate, G+C of genomic DNA and properties of the thermal regularly sympodial rachis. For this reason Salkin et denaturation curve, and mating type systems, while al. (1985) classified Geo. capitatum in a separate for smaller species complexes electrophoretic karyo- genus, Blastoschizomyces Salkin et al. However, since typing, PCR-fingerprinting and multilocus enzyme similar though less pronounced structures are found in electrophoresis were applied. At present, a total of 21 numerous Dipodascus species, this classification was species are recognized, of which 13 have a teleomorph not generally accepted (de Hoog et al. 2000). in Dipodascus de Lagerh., three in Galactomyces The bipartition of Geotrichum along lines of their Redhead & Malloch, and for five the teleomorph is teleomorph morphology, either Dipodascus or Galac- unknown. tomyces, recently appeared not to be supported by ribosomal DNA data. Kurtzman & Robnett (1998) 489 DE HOOG & SMITH found a bipartition in 26S rDNA sequence data, which for 5 min at 14000 r.p.m. After transferring the aque- grouped Galactomyces with several Dipodascus ous supernatant to a new Eppendorf tube, 2 volumes species far apart from the remainder of Dipodascus. A (~800 µL) ethanol 96 %, –20 °C were added and similar bipartition was reflected in 18S rDNA se- mixed gently. The DNA was precipitated at –20 °C quence data (Ueda-Nishimura & Mikata 2000), where for at least 30 min. The pellet, obtained by centrifuga- a Galactomyces clustered as Group 1 with six Dipod- tion for 5 min at 14000 r.p.m., was washed twice with ascus species (including the generic type species, D. 500 µL ethanol 70 % at –20 °C. DNA was dried albidus de Lagerh.), while six remaining Dipodascus overnight at room temperature and suspended in 97.5 species (Group 2) were remote from this cluster. In µL TE-buffer (10 mM Tris, 10 mM Na-EDTA, pH addition, the SSU molecule of members of Group 2 8.0) with 2.5 µL RNAse-solution (10 mg pancreatic appeared to be remarkable in the fungal Kingdom by RNAse 20 U/mg was added to 1 mL 0.01 M Na- comprising 1634–1636 bp only, as a result of large acetate, heated at 100 °C during 15 min and cooled deletions in the V2, V3 and V8 variable domains. In slowly to room temperature; the pH was adjusted to both publications (Kurtzman & Robnett 1998, Ueda- 7.4 by adding 100 µL TrisHCl). The sample was Nishimura & Mikata 2000) it was noted that the incubated for 5–30 min at 37 °C and then stored in the distances between species were unexpectedly large, refrigerator. although de Hoog et al. (1986) has stressed the mor- phological and ecological unity of Dipodascus, re- Sequencing garding the pronounced phenotypes as variations on a Amplicons V9G and LS 266 (De Hoog & Gerrits van single, basic theme. These remarkable findings and den Ende 1998) were generated as above and purified conflicts in visions prompted an in depth study of using the Gel Band Purification Kit (Amersham ribosomal sequence comparison comprising 18S, 26S Pharmacia, Roosendaal, The Netherlands). DNA was and ITS regions of the molecule, with the aim to bound to GFX-columns, eluted according to protocols compare the results with those from nDNA reassocia- given by the supplier, and collected with TE-buffer. tion, and morphological and physiological data gener- Concentrations of amplicons were estimated by com- ated during previous studies. parison with SmartLadder markers (Eurogentec, Seraing, Belgium) on 1 % agarose gels. Sequencing primers were ITS1, ITS 4 and ITS 5; reactions (96 °C, MATERIAL AND METHODS 10 min; 50 °C, 5 min; 60 °C, 4 s; 25 cycles) were carried out with 15–50 ng of DNA for a 10 µL reac- List of strains tion mixture including 4 pmol primer and 4 µL Big- All strains examined previously and in this study, are Dye RR Mix (Applied Biosystems, Nieuwerkerk a/d presented in Table 1. The order is according to the IJssel, The Netherlands). Subsequently DNA was precipitated with ethanol and sequenced using an ABI phylogenetic relatedness (Ueda-Nishimura & Mikata TM 2000) and the taxonomy of the present study. Prism 310 Genetic Analyzer (Applied Biosystems). nDNA reassociation Alignment and phylogenetic analysis Strains were grown for 2 d at 25 °C on a rotary shaker Sequences were adjusted using SeqMan of Lasergene at 125 rpm in 2 L YM broth (Wickerham 1951) using software (DNASTAR, Madison, Wisconsin). Align- 1 L flat-bottom flasks. Isolation and purification of ment of the Internal Transcribed Spacer (ITS) region DNA and determination of DNA base composition was done using BioNumerics (Applied Maths, Kort- were done according the procedures cited before rijk, Belgium) guided by iterative production of trees (Smith et al. 1995). DNA-DNA hybridization experi- based on Ward’s averaging algorithm. Distance trees ments were carried out according to the procedures were constructed with neighbour-joining with Ki- described by Seidler & Mandel (1971) and modified mura-2 correction using the TREECON v. 1.3b software by Kurtzman et al. (1980). package (Van de Peer & De Wachter 1993), and phylogenetic trees using PAUP v. 4.0b8 with heuristic DNA extraction search option (data not shown). Bootstrap values were About 1 g of mycelium was transferred to a 2 : 1 calculated from 100 resampled datasets. The 18S mixture of silicagel and Celite 545 with 300 µL cetyl- sequences were aligned with the ARB package devel- trimethylammoniumbromide (CTAB) buffer added oped by W. Ludwig (www.mikro.biologie.tu- [TrisHCl, 200 mM, pH 7.5; Na-EDTA (ethylenedia- muenchen.de/pub/ARB). Small Subunit (SSU) trees minotetraacetic acid sodium salt), 200 mM; NaCl 8.2 were reconstructed using positional variability with %; CTAB 2 %]. The material was ground with a the neighbour-joining (not shown) and parsimony micropestle (Eppendorf). After adding 200 µL CTAB- options in ARB. Alignment was optimized in a sub- buffer and vigorously shaking the sample was incu- file of a database containing about 2500 near- bated for 10 min in a 65 °C water bath; 500 µL chlo- complete fungal SSU sequences available at CBS.