International Journal ofSystematic andEvolutionary Microbiology (2000), 50, 1683-1686 PrInted in Great Bntall1 #8398 Pichia hawaiiensis sp. nov., occurring in decaying bark of Charpentiera trees in the Hawaiian archipelago Herman J. Phaff,l William T. Starmer2 and Cletus P. Kurtzman3 Author for correspondence: Herman J. Phaff. Tel: -'-I 530752 1465. Fax: - \ 530752 4751J. e-mail: hjphafl"" ucdavls.edu 1 Department of Food A description is given for Pichia hawaiiensis sp. nov., a nitrate-utilizing Science and Technology, member of the genus Pichia E. C. Hansen emend. Kurtzman. Seven strains of University of California, Davis, CA 95616, USA the new species were isolated during the years 1972,1973 and 1978 from rotting bark of the Hawaiian tree genera Charpentiera, Pisonia and 2 Department of Biology, Syracuse University, Cheirodendron. P. hawaiiensis is heterothallic but appears to occur in nature Syracuse, NY 13244, USA mainly in the diploid state. Asci are deliquescent and produce up to four hat­ 3 Microbial Properties shaped spores per ascus. Phylogenetic analysis of the 600 nucleotide D1/02 Research Unit, National domain of the 26S rONA showed that P. hawaiiensis is most closely related to Center for Agricultural Pichia populi and Williopsis califomica (syn. Hansenula californica). The type Utilization Research, Agricultural Research strain of P. hawaiiensis, isolated on the island of Hawaii from the rotting bark T Service, US Department of of Charpentiera sp. containing insect larvae, is strain UCD-FST 72-181 ( = ATeC Agriculture, Peoria, MYA-137 T = CBS 8760T = NRRL Y-27270l). IL61604, USA Keywords: Pichia hmraiiellsis sp. nov.. phylogenetic analysis. large-subunit rDNA analysis INTRODUCTION substitutions by I % or more. Comparison of the D I/D2 sequence from a representative strain of the During explorations (in 1972. 1973 and 1978) of the Hawaiian isolates with a database of0 I/D2 sequences_ veast biota associated with native Hawaiian plants. from all currently utilized ascomycetous yeasts seven phenotypically similar strains of a species rep­ (Kurtzman & Robnett. 1997. 1998) revealed that these resentative of the yeast genus Pichia E. C. Hansen isolates represent a new species and also provided an emend. Kurtzman were recovered from rotting bark estimate of phylogenetic placement. samples of several endemic tree species on the islands of Hawaii and Kauai in the Haviaiian archipelago. We propose to name the new species Pichia hmraiicl1sis because of the geographic area of origin. which Attempts to identify these isolates by using standard yeast identification methods that depend on diagnostic includes two ofthe Hawaiian islands. ranging from the responses to physiological tests (Kurtzman. 19R4. northwest island of Kauai to the island of Hawaii at the eastern end of the archipelago. 1998: Barnett C! al.. 1990) failed to give satisfactorY matches with known species. Because~ of the common variability of certain physiological tests (Kurtzman & METHODS Fell. 1998) or tests controlled by single nuclear genes (such as hvdrolases). confirmation of the noveltv of the Strain isolation. Samples of moist. rotting tree bark were Hawaiian- isolates was sought from divergence in the collected in sterile plastic containers from Charpell!iera (Amaranthaceae) sp.. Cheirodelldroll (Araliaceae) sp. and variable DI/D2 domain of the large-subunit (26S) Pisollia sp. (Nyctaginaceae) in Volcanoes National Park on rDNA. Kurtzman & Robnett (1995.1997.1998) have the island of Hawaii. and at Honopu on the island of Kauai. demonstrated that the extent of divergence in this Samples were streaked on acidified (to pH 3·8 with IM Hel) domain appears sufficient to resolve all species of yeast extract/malt extract agar (YM: Difco) and stored at ascomycetous yeasts that differ in terms of nucleotide approximately 25°C. Pure cultures were obtained by restreaking on YM agar. Morphological and physiological characteristics ofthe isolates were determined using methods Abbreviation: L5U, large subunit. currently used in yeast taxonomy (Yarrow. 1998). Among The GenBank accession number for the 265 rDNA D1/D2 sequence of Pichia the many strains isolated from the above sources during hawaiiensis is AFl 53675. three separate collection trips. seven strains were represen- 01447 © 2000 IUM5 1683 H. J. Phaff. W. T. Stanner and C. P. Kurtzman Table 1. Strain numbers, host plants, origins and ploidy of Pichia hawaiiensis isolates Strain* Host plant and location Ploid~' UCD 72-181 T Rotting bark of Charpellliera sp.. Island of Hawaii Diploid UCD 72-170 Rotting bark of Charpenriera sp.. Island of Hawaii Diploid UCD 73-508.1 Rotting bark of Charpellliera sp.. Island of Hawaii Diploid S 78-341.1 Rotting bark of Pisonia sp.. Island of Hawaii Diploid S 78-345.1 Rotting bark of Pisonia sp.. Island of Hawaii Diploid S 78-346.1 Rotting bark of Cheirodendron sp.. Island of Hawaii Diploid S 78-689.1 Rotting bark of Charpe11liera sp.. Island of Kauai Diploid UCD n-181.1 Single-spore isolate from UCD 72-18I T Haploid h­ UCD 72-181.2 Single-spore isolate from UCD n-181 T Haploid h­ UCD 72-181.3 Single-spore isolate from UCD n-181 T Haploid h­ UCD n-181.4 Single-spore isolate from UCD n-181 T Haploid h- * UCD (= UCD-FST). Culture Collection of the Department of Food Science and Technology. University of California. Davis. CA. USA; S prefix. assigned by W. T. Starmer. The four single­ ascospore isolates came from the same ascus. tative ofP. hmraiiensis (Table I). Single ascospores produced ceming the phylogenetic circumscription of Pichia. on dilute (1 :4) V-8 agar were isolated from individual four­ Williopsis and related genera. the new ascosporic spored asci with the aid of a micromanipulator (Fowell. species described here is placed in Pichia as currently 1969). defined from phenotypic characteristics (Kurtzman. DNA isolation, peR, rONA sequencing and sequence analysis. 1998). Methods for nuclear DNA isolation. amplification of the 600-nucleotide 26S rDNA domain D 1/D2 by PCR and P. IzGlraiiensis can be differentiated phenotypically sequencing with the ABI Taq DyeDeoxy Terminator Cycle from P. populi by its lack of growth on D-mannitol. D­ sequencing kit and the ABI model 377 automated DNA glucitol and D-gluconate as sole carbon sources or on seq uencer (A pplied Biosystems) were as described previously cadaverine as the sole source of nitrogen. It differs (Kurtzman & Robnett. 1997). from r-l'. cal(rornica by its lack of growth on D­ mannitol. D-gluconate and citrate as sole carbon Sequence data were visually aligned with QEDIT 2.15 (Sem\Vare). Phylogenetic relationships were calculated with sources. a Power Macintosh 8500/120 using the maximum-par­ simony program of PAL'P* 4.0 (Swofford. 1993; test version Latin diagnosis of Pichia hawaiiensis Phaff, Starmer & distributed by Sinauer Associates) with the heuristic search Kurtzman sp. nov. option and ~andom addition of sequences. Relationships were further analysed using the neighbour-joining program In YM (Difco) liquido pOST dies 5 ad 25°C. cellulae of PAL:P* 4.0 with the Jukes-Cantor distance measure. ovoideae. 2-4 x 5-6·5 ~m. singulae. hinae auT C(/fenis Schi::oSl/ccharo/7/rces pO/7/he was designated outgroup in all hrevis; sedimelllum pallidum. annulus Tenuis: pellicula analyses. Support for the nodes of the phylogenetic tree were nulla. Cultura in agaro malTi pOST dies 21 ad 25°C estimated from bootstrap analysis (1000 replications). The cremea vel ravida. hUTyrosa. plana. niTie/a. margo GenBank accession numbers for reference species have been glahrum vel serrularum. In agarofarinae Zea mays posT reported by Kurtzman & Robnett (1998 l. dies 10 pseudomycelium nullum vel rudimelllarium. CulTura heTerofhallica. Asci formal1lur ex cellulae RESULTS AND DISCUSSION diploideae, habel1lus 2-4 sporos pi/eiformae: asci rumpunTer pOST dies 4--10. FermenraTio glucosi rarda. Phylogenetic placement of P. hmraiicl1sis was deter­ Glucosul1l. cellobiosum. D-xrlosum. L-rlzamnosum mined from analysis of 26S rDNA domain D I /D2 (exigue. rarda), eTlzanolum, glycerolum. salicinum. sequences (approx. 600 nucleotides) from all currently meThyl fJ-D-glucosidum. acidum lacTicum, acie/um recognized ascomycetous yeast species. as reported succinicum assimi/anTur aT non galacrosum. L-sorhosum. in the study by Kurtzman & Robnett (1998). This malrosum. sacclzarosu11l. Trelzalosum. lacTosum. meli­ analysis demonstrated that P. hGlraiiensis is a unique biosum. raffinosum. mele=iTOSU11l. inulinum. amylum species that is closely related to Pichia populi (56 solubile. L-arabinosum. D-arabinosum. D-ribosum. nucleotide differences) and Williopsis cal(romica eryTlzriroluJll, ribifolum. galacTirolum. D-nul1lniTolu111. (63 nucleotide differences) as shown in Fig. I. Weak D-gluciTolum. methyl-rx-D-glucosidum. glucono-J­ basal branch support is evident from the domain lacronum. 2-kerogluconaTum. 5-keTogluconarum. acidum D I ID2 analysis presented in Fig. I. and complete 18S ciTriclim. meso-inosiTolu111. D-gluconatum. methanolum. sequence analyses are often similarly weak (Kurtzman D-glucosaminum, N-aceTyl-D-glucosaminum. nee hexa­ & Robnett. 1998). In view of the uncertainty con- decanum. Kalium niTricum, NaTrium niTrosum. Iysinum, 1684 International journal ofSystematic and Evolutionary Microbiology SO H. J. Phaff. W. T. Starmer and C. P. Kurtzman nose. melezitose. inulin. soluble starch. L-arabinose. REFERENCES D-arabinose. D-ribose. ervthritol. ribitol. g:alactitol. inethyl-O:-D-gluco~de. Barnett, J. A., Payne, R. W. & Yarrow, D. (1990). rcas!.\· D-mannitol. D-glucitol. glu­ Charac/cris/ics and Me/lliticalion. 2nd edn. Cambridge: cono-£5-lactone. D-g:luconate. 2-keto-g:luconate. 5­ Cambridge University Press. keto-gluconate. citric acid. meso-inositol. methanol. Fowell, R. R. (1969). Sporulation and hybridization of yeasts. In D-glucosamine. N-acetyl-D-glucosamine and hexa­ The rellS/S. 1'01.1. Biola!!!, of' reas/s. pp. 303-383. Edited hv A. KNO~. decane. NaN02 • ethylamine and L-lysine are H. Rose & J. S. Harriso'n. New York: Academic Press. utilized as sole sources of nitrogen but cadaverine Kurtzman, C. P. (1984). Pichia Hansen. In Thc rcas/s. a is not utilized.