Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 07 Apr 2019 17:47:27 Re-examination of strains formerly assigned to Hyphopichia burtonii, the phylogeny of the genus Hyphopichia, and the description of Hyphopichia pseudoburtonii sp. nov. Marizeth Groenewald and Maudy Th. Smith Correspondence Marizeth Groenewald m.groenewald@cbs.knaw.nl CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands On the basis of the nucleotide divergence in the internal transcribed spacer (ITS) domain of the rRNA gene and the D1/D2 domain of the 26S rRNA gene, species of the genus Hyphopichia can be divided into different phylogenetic groups. In this study, three distinct phylogenetic clusters as well as a novel species, Hyphopichia pseudoburtonii sp. nov. with type strain CBS 2455 T (5JCM 16346 T ), are introduced. Mating studies indicate that strains belonging to Hyphopichia pseudoburtonii are heterothallic. The genus Hyphopichia was introduced by Arx & van der Walt (1976) to accommodate the mycelial yeast species, Pichia burtonii. This heterothallic species is characterized by formation of asci from conjugating yeast cells and by formation of septate hyphae and conidia borne on denticles. Arx & van der Walt (1976) excluded P. burtonii from the genus Pichia by the latter features and from the genus Saccharomycopsis by the former character. Kurtzman & Fell (1998) did not accept the reclassification of P. burtonii as various other yeasts and other dimorphic euascomycetes also share these characters. In a study of the phylogeny of species of five ascomycetous teleomorphic yeast genera, Yamada et al. (1998) showed the isolated position of Hyphopichia in partial 18S and 26S rRNA gene sequence-based phylogenetic trees. Therefore, they proposed to accept this genus. This proposal was supported by phylogenetic analyses using D1/D2 sequences (Kurtzman & Robnett, 1998). Later, accepting Hyphopichia as a genus isolated from other yeast genera, Kurtzman (2005) demonstrated that five Candida species (C. fennica, C. homilentoma, C. rhagii, C. pseudorhagii and C. gotoi) and also Pichia heimii belong to the Hyphopichia clade, con- sequently introducing the new combination Hyphopichia heimii. Hyphopichia burtonii, the type species of the genus, is a food spoilage organism and was shown to produce styrene that results in off-flavours in fermenting baking products with a high sugar content and low water activity (Bonjean & Guillaume, 2003). It has also been isolated from diverse substrates including various food sources (Kurtzman & Fell, 1998), freshwater sites and plants such as cacti, and it is also associated with beetles or beetle larval substrates (Ganter, 2006). In this study, 17 strains previously assigned to H. burtonii, either only on the basis of their molecular characteristics or on the basis of morphological characters, were re-examined phenotypically and phylogenetically. A novel species as well as three distinct groups within the existing Hyphopichia clade are proposed. The strains studied, their origin and original denomination are listed in Table 1. DNA was extracted from cultures grown on GPYA medium (4 % glucose, 0.5 % yeast autolysate, 2 % agar) for three days using the FastDNA kit (Bio 101) with the ‘FastPrep’ Instrument (Q-Biogene). Primers V9G (de Hoog & Gerrits van den Ende, 1998) and LR5 (Vilgalys & Hester, 1990) were used to amplify the partial rRNA gene that includes the D1/D2 domain of the large-subunit rRNA gene as well as the ITS (internal transcribed spacer) domain (ITS1, ITS2 and the interven- ing 5.8S rRNA) as described by Knutsen et al. (2007). The PCR products were separated by electrophoresis at 80 V for 40 min on a 0.8 % (w/v) agarose gel containing 0.1 mg ethidium bromide ml 21 in 16 TAE buffer (0.4 M Tris, 0.05 M sodium acetate and 0.01 M EDTA, pH 7.85) and examined under UV light. The amplicons were sequenced in both directions using the primers LR0R (Vilgalys & Hester, 1990) and LR5 for the D1/D2 domain, and the primers V9G and ITS4 (White et al., 1990) for the ITS domain. The BigDye Terminator version 3.1 Cycle Sequencing kit (Applied Biosystems) was used according to the manufacturer’s recommendations and the products were analysed on an ABI Prism 3730XL DNA Sequencer Abbreviation: ITS, internal transcribed spacer. The GenBank/EMBL/DDBJ accession numbers for the 26S rRNA gene D1/D2 region and ITS region sequences of strain CBS 2455 T are GQ389650 and GU246252, respectively. The Mycobank (http://www.mycobank.org) accession number for H. pseudoburtonii sp. nov. is MB 514824. The single most parsimonious tree of the ITS sequence alignment is available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2010), 60, 2675–2680 DOI 10.1099/ijs.0.018580-0 018580 G 2010 IUMS Printed in Great Britain 2675