Antonie van Leeuwenhoek (2013) 103:229–238 DOI 10.1007/s10482-012-9804-1 ORIGINAL PAPER Two new anamorphic yeasts species, Cyberlindnera samutprakarnensis sp. nov. and Candida thasaenensis sp. nov., isolated from industrial wastes in Thailand Jamroonsri Poomtien • Sasitorn Jindamorakot Savitree Limtong • Pairoh Pinphanichakarn • Jiraporn Thaniyavarn • Received: 16 April 2012 / Accepted: 25 August 2012 / Published online: 26 September 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Three yeast strains were isolated from industrial wastes in Thailand. Based on the phylogenetic sequence analysis of the D1/D2 region of the large subunit rRNA gene, the internal transcribed spacer (ITS1-5.8S rRNA gene-ITS2; ITS1-2) region, and their physiological characteristics, the three strains were found to represent two novel species of the ascomycetous anamorphic yeast. Strain JP52T represent a novel species which was named Cyberlindnera samutprakarnensis sp. nov. (type strain JP52T; = BCC 46825T = JCM 17816T = CBS 12528T, MycoBank no. MB800879), which was differentiated from the closely related species Cyberlindnera mengyuniae CBS 10845T by 2.9 % sequence divergence in the D1/D2 region and 4.4 % sequence divergence in the ITS1-2. Strain JP59T and JP60 were identical in their D1/D2 and ITS1-2 regions, which were closely related to those of Scheffersomyces spartinae CBS 6059T by 0.9 and 1.0 % sequence divergence, respectively. In addition, supportive evidence of actin gene and translational elongation factor gene by sequence divergence of 6.5 % each confirmed their distinct status. Furthermore, JP59T and JP60 differentiated from the closely related species in some biochemical and physiological characteristics. These two strains were assigned as a single novel species which was named Candida thasaenensis sp. nov. (type JP59T = BCC 46828T = JCM 17817T = CBS 12529T, MycoBank no. MB800880). J. Poomtien
P. Pinphanichakarn
J. Thaniyavarn (&) Faculty of Science, Department of Microbiology, Chulalongkorn University, 254 Phayathai Road, Bangkok 10300, Thailand e-mail: Jiraporn.Th@chula.ac.th Introduction S. Jindamorakot Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand S. Limtong Faculty of Science, Department of Microbiology, Kasetsart University, 50 Phaholyothin Road, Bangkok 10900, Thailand Keywords New anamorphic yeast species
Cyberlindnera samutprakarnensis sp. nov.
Candida thasaenensis sp. nov The assignment of yeast and fungi strains to the family, and especially to the genera and species level has previously been primarily based upon morphology and then physiological/biochemical characters. However, it has become apparent that this is somewhat problematic. Comparative molecular analyses of sequence variation in regions that show relatively similar mutation rates across species, such as the D1/D2 variable regions of the large subunit (LSU) RNA gene, the complete small subunit (SSU) RNA gene or 123 230 the non-coding internal transcribed spacers (ITS1-5.8S rRNA-ITS2; ITS1-2) region are increasingly used for the rapid and development stage independent approach for identification of species. As an example of the use of molecular approaches in ascomycetous yeasts, the yeast genera Williopsis and Pichia were originally classified as individual species in several diverse clades of ascomycetous yeasts. The classification of Williopsis saturnus species complex was designated as five varieties of var. saturnus, var. mrakii, var. sargentensis, var. suaveolens, and var. subsufficiens by nuclear DNA reassociation (Kurtzman 1991). Therefore, to clarify the taxonomic status of Williopsis saturnus, pairwise nucleotide differences among type strains of five varieties of W. saturnus for D1/D2 region of LSU rRNA gene, ITS1-2 rRNA, and EF-1a genes was performed, which showed that the five varieties of W. saturnus represent closely related, but genetically distinct taxa that can be regard as species of Lindnera (Kurtzman et al. 2008). The genus Cyberlindnera was introduced as a replacement name for Lindnera, with 21 new combinations (Minter 2009). Cyberlindnera mengyuniae and Cyberlindnera rhizosphaerae were later described to be related to this clade (Chen et al. 2009; Mestre et al. 2010). Five novel species of genus Candida in the Cyberlindnera clade were proposed, hence the Cyberlindnera clade contains 23 recognized teleomorphic species and 12 Candida species are also related to this clade (Chang et al. 2012). For resolving the clade containing the coenzyme Q-9 producing species of Pichia, P. spartinae, P. acaciae, and P. guilliermondii were assigned to Yamadazyma and clustered among several clades (Billon-Grand 1989; Kurtzman and Robnett 1998). Kurtzman and Suzuki (2010) further classified the species of these genera into 12 well-supported clades, based on the sequence analysis of the D1/D2 region of the LSU and the SSU rRNA genes. In this analysis, P. spartinae was proposed to belong to the genus Scheffersomyces. S. spartinae, which does not ferment D-xylose is the basal species in the genus Scheffersomyces, and its placement is weakly supported. The addition of new species to the clade may strengthen placement of S. spartinae in the genus or provide evidence that it is a member of an undescribed sister genus. The new taxon Candida gosingica was proposed by Chang et al. (2011) as a novel species that was quite closely related to S. spartinae, based on sequence analysis of the SSU, the D1/D2 domain of the LSU, and the ITS1-2 region of the rDNAs. 123 Antonie van Leeuwenhoek (2013) 103:229–238 In the course of a survey on the biosurfactantsproducing yeasts, we isolated and screened for the effective biosurfactant-producing yeasts which could reduce the surface tension value to below 40 mN m-1, three anamorphic yeast strains were found to represent two novel species which closely related to Cyber. mengyuniae and S. spartinae are described as Cyberlindnera samutprakarnensis sp. nov. for strain JP52T and Candida thasaenensis sp. nov. for strains JP59T and JP60. Materials and methods Yeast strains Strain JP52T was collected from the wastewater of a cosmetic factory (Milott laboratory Ltd.) in Bangplee, Samutprakarn province, Thailand, using an enrichment technique, ten grams of each sample were inoculated in 90 ml of YM broth (pH 4.5) supplemented with 100 lg ml-1 chloramphenicol and 1 % (v/v) palm oil for acquiring biosurfactant-producing yeast and then incubated at room temperature on a rotary shaker at 200 rpm. The enriched cultures were purified on YM agar plate. Strains JP59T and JP60 were isolated from a sediment pond in a palm biodiesel production plant in Thasae, Chumporn province, Thailand, by standard plate culture technique; 10 g of each sample were suspended in 90 ml of sterile saline solution (0.85 % (w/ v) NaCl). The serials dilution were spread onto acidified YM agar (pH4.5) supplemented with 1 % (v/v) palm oil, 100 lg ml-1 chloramphenicol, and 0.2 % (w/v) sodium propionate incubated at 30 °C for 3 days. The purified culture was maintained in YM broth supplemented with 10 % (w/v) glycerol and stored at -80 °C. Examination of taxonomic characteristics The three strains, JP52T, JP59T and JP60, were characterized morphologically, physiologically, and biochemically following standard yeast identification methods (Yarrow 1998). The nitrogen assimilation was examined on solid media with starved inoculum (Nakase and Suzuki 1986a), while the ability to grow at various temperatures was determined in YM broth using metal block baths. Vitamin requirements were determined by the method of Komogata and Nakase (1967). Antonie van Leeuwenhoek (2013) 103:229–238 Ubiquinone system Ubiquinones were analyzed from freeze-dried cells derived from 2-days-old cultures grown in YM broth on a rotary shaker at 150 rpm at 25 °C. Ubiquinones were extracted, purified, and identified according to the method of Nakase and Suzuki (1986b). Ubiquinone isoprenologues were identified by HPLC using a Cosmosil (Waters C18) column and eluted with a 2:1 (v/v) ratio of methanol: isopropanol as the mobile phase at a flow rate of 1 ml min-1 and the elutant was detected with UV spectrophotometry at 275 nm. DNA sequencing and phylogenetic analysis The nucleotide sequence of the D1/D2 region of the LSU rRNA gene and ITS1-2 region, including the DNA extraction, PCR primers, and reaction conditions, and the sequencing of amplicons were determined according to the methods outlined in Kurtzman and Robnett (1998) and White et al. (1990). The actin gene ACT1 and the elongation factor 2 gene EF2 were amplified using the primers ACT1 (59-TACCCA ATTGAACACGGTAT-39) and ACT2 (59-TCTGAA TCTTTCGTTACCAAT-39) for ACT1 and EFIIF1 (59-AAGTCTCCAAA CAAAGCATAAC-39) and EFIIR2 (59-GGGAAAGCTTGACCACCAGTAGC -39) for EF2. PCR was performed according to the method described by Diezmann et al. (2004). Sequence divergence comparison of D1/D2 LSU rRNA gene and the ITS1-2 regions of the yeast strains with those similar sequences in the GenBank database, obtained by the nucleotide BLAST analysis tool (Altschul et al. 1997), were performed as detailed below. The sequences data of JP52T, JP59T, and JP60 have been deposited in the GenBank database under the following accession numbers: D1/D2, AB598079, AB598080, and AB686643; ITS1-2, AB695388, AB686644, and AB686645; ACT1 and EF2 of JP59T, JP60 AB742430, AB742431, AB742433, and AB742434, respectively. The phylogenetic placement of the proposed novel species along with neighboring taxa was analyzed using the concatenated ITS1-2 and D1/D2 region of the LSU rRNA gene. The sequences of novel strains and related species which were retrieved from GenBank (accession number indicated in Figs. 1, 2) were aligned using the CLUSTAL X ver. 2.0 software (Larkin et al. 2007) and phylogenetic trees were constructed from the evolutionary distance data with Kimura’s two-parameter 231 correction (Kimura 1980) using the Neighbor–Joining (NJ) distance based (Saitou and Nei 1987). Sites where gaps existed in any sequences were excluded. Bootstrap analysis (Felsenstein 1995) was performed with 1,000 random resamplings. Results and discussion In the course of a survey of biosurfactants-producing yeast, we collected ninety-four strains which could reduce the surface tension value to below 40 mN m-1. Among them, three strains were differentiated from any known species and represent two novel species. Strain JP52T was obtained from the wastewater of a cosmetics factory and another two strains, JP59T and JP60, from the sediment ponds of a palm biodiesel production plant. All three strains demonstrated multilateral budding proliferation; ascospore was not found after 28 days on YM agar, 5 % Malt extract agar or Acetate agar at 25 °C, revealing negative diazonium blue B (DBB) and urease reactions. Yeast strain JP52T had Q-7 as the major ubiquinone, while JP59T and JP60 had the Q-9 ubiquinone system. The phylogenetic position of the three strains was determined from the rRNA gene sequences from the D1/D2 region of the LSU rRNA gene and the ITS1-2 region. Strain JP52T was closely related to Cyber. mengyuniae CBS 10845T by 2.9 % (16 substitutions, 7 indels) and 4.4 % (28 substitutions, 49 indels) in the D1/D2 region and ITS1-2 region; then close to Cyberlindnera saturnus varieties, Cyber. saturnus, Cyber. suaveolens, Cyber. subsufficiens, Cyber. mrakii, and Cyber. sargentensis by 4.6–5.3 % sequence divergence (Table 1, upper triangle). According to (Kurtzman and Robnett 1998), strain with [1 % nucleotide substitution in the D1/D2 region usually separate species. In the phylogenetic tree based on the concatenated ITS1-2 and D1/D2 region of LSU rRNA gene sequences, strain JP52T was placed in the Cyberlindnera clade, distinct from the described species of Cyberlindnera (Fig. 1). Strain JP52T differed from Cyber. mengyuniae in their ability to assimilate maltose, cellobiose, melezitose, soluble starch, rhamnose, salicin, and succinic acid, and from Cyber. mrakii as shown in Table 2. On the basis of morphological, biochemical, physiological, and chemotaxonomic characteristics, the sequence analysis of the D1/D2 region of the LSU rRNA gene and ITS 123 232 Antonie van Leeuwenhoek (2013) 103:229–238 94 Candida easanensis ST-225T (HM461688 / AY634571) 56 Candida maesa GJ8L01T (HM461661 / FJ527053) Candida pattaniensis BCC 11799T (HM461657 / AY634568) Candida stauntonica GY13L05T (HM461658 / FJ527095) Candida taoyuanica GY15S07T (FJ873419 / FJ527145) Cyberlindnera japonica NRRL YB-2750T (CBS7209* / EF550323) 76 100 Cyberlindnera amylophila NRRL YB-1287T (CBS7020* / EF550319) Cyberlindnera mississippiensis NRRL YB-1294T (CBS7023* / U74597) 66 66 Cyberlindnera fabianii NRRL Y-1871T (AF335967 / EF550321) Cyberlindnera veronae NRRL Y-7818T (AF335966 / EF550322) Cyberlindnera americana NRRL Y-2156T (CBS5644* / EF550328) 68 60 67 Candida mycetangii NRRL Y-6843T (CBS8675* / EF550330) Candida maritima NRRL Y-17775T (CBS8176* / EF550332) 76 Candida hungchunana NC3W71T (HQ623543 / HM461702) Cyberlindnera meyerae NRRL Y-17236T (CBS7076* / EF550327) Cyberlindnera euphorbiae NRRL Y-17232T (CBS8033* / U73580) 92 Cyberlindnera samutprakarnensis JP52T (AB695388 / AB598079) Cyberlindnera mengyuniae CBS10845T (EU043159 / EU043158) T 54 Cyberlindnera mrakii NRRL Y-1364 (EU307973 / EF550317) 81 Cyberlindnera saturnus NRRL Y-17396T (EU307970 / EU550316) 81 73 93 91 73 Cyberlindnera suaveolens NRRL Y-17391T (EU307977 / EU307993) 84 Cyberlindnera sargentensis NRRL YB-4139 T (EU307980 / U94936) Cyberlindnera subsufficiens NRRL YB-1657 T (EU307975 / EF550318) Candida vartiovaarae NRRL Y-6701 T (CBS4289* / EF550315) Candida takata EN25S01T (FJ873431 / FJ527238) 61 Cyberlindnera misumaiensis NRRL Y-17389T (CBS8062* / EF550306) Cyberlindnera lachancei NRRL Y-27008T (CBS8557* / EF550313) Cyberlindnera petersonii NRRL Y-3808T (CBS5555* / EF550311) 73 Cyberlindnera jadinii NRRL Y-1542T (DQ249199 / EF550309) Cyberlindnera maclurae NRRL Y-5377T (CBS8671* / EF550310) Wickerhanomyces pijperi NRRL YB-4309 (HM156502 / EF550335) Knuc 0.01 Fig. 1 NJ-based phylogenetic tree for Cyberlindnera samutprakarnensis nov. (JP52T) based on the concatenated ITS and D1/D2 region of the LSU rRNA gene sequence. Wickerhanomyces pijperi NRRL YB-4309T is used as the outgroup. The numerals at each node represent the percentages from 1,000 replicate bootstrap resamplings (excluded when \50 %). Sequences were retrieved from the NCBI Genbank databases and CBS database (*). Bar 0.01 substitutions per nucleotide position region, we concluded that the strain JP52T represent a novel species of the Cyberlindnera clade, but they did not exhibit ascospore formation in sporulation media for 4 weeks; therefore, JP52T is proposed to be a new species which was named Cyberlindnera samutprakarnensis sp. nov. (MycoBank No. MB800879). Strains JP59T and JP60 had identical sequences of D1/D2 region of the LSU rRNA gene and ITS1-2 region. The sequence analysis of D1/D2 region and ITS1-2 region of these two strains showed that Scheffersomyces spartinae is the most closely related species by differed with 0.9 % sequence divergence 123 Antonie van Leeuwenhoek (2013) 103:229–238 51 233 Debaryomyces nepalensis NRRl Y-7108T (AB053099 / U45839) Debaryomyces robertsiae NRRl Y-6670T (AB054019 / U45805) Debaryomyces renaii ATCC MYA-4749 (HQ999977 / HQ999953) 97 Schwanniomyces etchellsii NRRl Y-7121T (CBS2011* / U45809) Debaryomyces coudertii NRRl Y-7425T(AM992914 / U45846) 61 64 Schwanniomyces capriottii NRRl Y-7423T (AB054102 / U45841) T 87 Schwanniomyces pseudopolymorphus NRRl Y-4229 (AB054101 / U45845) Schwanniomyces polymorphus JCM7443T (AB054103 / AB054994) 51 Schwanniomyces yamadae NRRl Y-11714T(AB054022 / U45837) Schwanniomyces occidentalis var.persoonii NRRl Y-7400T (AB054020 / U45840) Candida multigemmis IFO10247T (CBS6524 */ U45782 100 Candida saitoana NRRL Y-17316T (HQ652067 / U45762) Candida glaebosa NRRL Y-6949T (FJ153208 / U45757) 100 75 Candida pseudoglaebosa NRRL Y-17911T (HQ652066 / U71072) 94 Candida fluviatilis NRRL Y-7711T (HQ652068 / U45717) Candida palmioleophila NRRL Y-17323T (CBS7418 / U45758) 100 Scheffersomyces spartinae NRRl Y-7322T (EU343815 / U45764) Candida gosingica SJ7S11T (FJ153193 / EF460550) 81 55 Candida thasaenensis JP59T (AB686644 / AB598080) 87 Candida thasaenensis JP60 (AB686645 / AB686643) 95 Candida ergastensis NRRL Y-17652T (HG999966 / U45746) Candida coipomoensis NRRl Y-17651T (AJ606466 / U45747) 80 76 Candida lignicola ST33T (EF627975 / AY845350) 100 Scheffersomyces stipitis NRRL Y-7124T (CBS5773* / U45741) 96 Scheffersomyces segobiensis NRRL Y-11571T (AB054118 / U45742) 100 Candida shehatae var. shehatae NRRl Y-12858T (AJ606464 / U45761) Candida shehatae var. insectosa NRRL Y-12854T (HQ652064 / U45773) 51 Candida quercitrusa NRRL Y-5392T (AM158924 / U45831) Candida fragi NRRL Y-17910T (AY344066 / U71071) 71 83 Candida oleophila NRRL Y-2317T (AY528671 / U45793) 55 98 Candida boleticola NRRL Y-569006T (AY569005 / U45777) Candida schatavii NRRL Y-569006T (AY528671 / U45795) Candida zeylanoides JCM 9454T (AB278160 / U45853) 63 Candida santamariae var. membranifaciens NRRl Y-17647T (AY542870 / U45785) Candida sophiae-reginae NRRL Y-17688T (HQ623553 / U45817) Wickerhamia fluorescens NRRL YB-4819T (GU246255 / U45719) Knuc 0.01 Fig. 2 NJ-based phylogenetic tree for C. thasaenensis nov. (JP59T, JP60) based on the concatenated ITS and D1/D2 region of the LSU rRNA gene sequence. Wickerhamia fluorescens NRRL YB-4819T is used as the outgroup. The numerals at each node represent the percentages from 1,000 replicate bootstrap resamplings (excluded when\50 %). Sequences were retrieved from the NCBI Genbank databases and CBS database (*). Bar 0.01 substitutions per nucleotide position (5 substitutions, 2 indels) and 1.02 % sequence divergence (6 substitutions, 0 indels), respectively. ITS1-2 region sequence analysis is supportive information for yeast identification with a similar amount of intraspecific variation (James et al. 1996; Sugita et al. 1999; Bai et al. 2001, 2002; Scorzetti et al. 123 234 Antonie van Leeuwenhoek (2013) 103:229–238 Table 1 The sequence divergence of the D1/D2 LSU rDNA (upper triangle) and ITS1-2 region (lower triangle) of strain JP52T and their related species, Cyber. mengyuniae, Cyber. JP52 Cyber. men Cyber. mra mrakii, Cyber. saturnus, Cyber. sargentensis, Cyber. suaveolens, and Cyber. subsufficiens Cyber. sat Cyber. sar Cyber. sua Cyber. sub JP52 0 16 (7) 25 (7) 27 (12) 25 (7) 27 (12) 27 (7) Cyber. mengyuniae 28 (49) 0 36 (74) 36 (69) 38 (69) 36 (69) 32 (74) Cyber. mrakii 36 (127) 14 (5) 0 1 (0) 0 1 (0) 4 (0) Cyber. saturnus 40 (114) 13 (5) 2 (1) 0 0 0 4 (0) Cyber. sargentensis 37 (122) 13 (5) 7 (1) 5 (2) 0 0 4 (0) Cyber. suaveolens 40 (114) 13 (5) 3 (1) 1 (0) 4 (2) 0 4 (0) Cyber. subsufficiens 38 (117) 16 (5) 6 (4) 4 (3) 5 (5) 5 (3) 0 The numbers in front of the parenthesis are indicating substitutions and numbers in parenthesis are the number of indels Table 2 Differential phenotypic characteristics of Cyberlindnera samutprakarnensis sp. nov., C. thasaenensis sp. nov., and their closely related species Characteristic 1 2 3 4 5 6 - - - - ? w/- Galactose - ? - - - - L-sorbose - ND - - ? ? Sucrose ? ? - ? ? ? Maltose ? - V ? ? ? Cellobiose ? - ? ? ? ? Trehalose ? ? V ? ? ? Raffinose ? ? ? - - - Melezitose ? - V ? ? ? Inulin ? ? V - - - Soluble starch ? - - D - - ? - V ? - - Glycerol Salicin ? ? ? - ? V ? ? ? S ? Succinic acid ? - ? ? ? ? Citric acid ? ? V ? - ? 50 % glucose - - - ? ? - 0.1 % cycloheximide - ND - - ? - 37 °C ? ? V ? - ? 42 °C ? ? - - - - Fermentation of: Maltose Assimilation of: L-rhamnose Growth at/with: ? Positive, D delayed, S slow, V variable, - negative, ND not determined Species 1 Cyber. samutprakarnensis, 2 Cyber. mengyuniae, 3 Cyber. mrakii, 4 C. thasaenensis, 5 C. gosingica, 6 S. spartinae. Data for Cyber. mengyuniae were taken from Chen et al. (2009). C. gosingica were taken from Chang et al. (2011). Cyber. mrakii and S. spartinae were taken from Kurtzman et al. (2011) 123 Antonie van Leeuwenhoek (2013) 103:229–238 2002; Kurtzman and Robnett 2003). However, multigene analysis provides a surer appraisal of kinship, hence the pairwise sequence analysis of actin gene ACT1 and transitional elongation factor gene EF2 of both S. spartinae and C. gosingica with strains JP59T provided better supportive information to resolve the novel species and confirm distinct status. Strains JP59T and JP60 were differed from both S. spartinae and C. gosingica with 6.5 % sequence divergence (35 substitutions, 0 indels) and 4.8 % sequence divergence (26 substitutions, 0 indels), respectively, in the actin gene and translational elongation factor gene. Phylogenetic analysis based on the concatenated sequences of ITS and D1/D2 region showed that strains JP59T and JP60 were single species which phylogenetically related to S. spartinae and C. gosingica with a high bootstrap confidence (Fig. 2). Strain JP59T and JP60 can be distinguished from S. spartinae, the most closely related species by its ability to assimilate L-rhamnose and that it can grow in 50 % (w/v) glucose; and from C. gosingica, by its ability to assimilate L-rhamnose, glycerol, and citrate, its ability to grow at 37 °C and its inability to assimilate L-sorbose, 0.1 % cycloheximide and maltose is not fermented (Table 2). The molecular and physiological characteristics described above demonstrated that strain JP59T and JP60 represents a novel species, Candida thasaenensis sp. nov. (MycoBank No. MB800880) which is proposed for the two strains with the type strain JP59T. 235 medium. Pseudomycelium and true mycelium are not formed on Dalmau plates on corn meal agar (CMA) after 14 days at 25 °C. Ascospores are not produced on 5 % malt extract agar, Fowell’s acetate agar, and YM agar after 28 days at 25 °C. The major ubiquinone is Q-7. Glucose, sucrose, fructose, and raffinose (may be weak) fermentation are positive. But galactose, maltose, lactose, and melibiose fermentation are negative. Assimilation of carbon compounds: glucose, sucrose, maltose, cellobiose, trehalose, lactose (slow), melibiose (slow), raffinose, melezitose, inulin, soluble starch, D-xylose, D-ribose(slow), L-rhamnose, ethanol, glycerol, D-mannitol, D-glucitol, methyl-a-D-glucoside(slow), salicin, glucono-d-lactone, 2-ketogluconic acid (slow), succinic acid, citric acid, propane-1,2diol, and butane-2,3-diol are assimilated. No growth occurs on galactose, L-sorbose, L-arabinose, D-arabinose, D-glucosamine, N-acetyl-D-glucosamine, methanol, erythritol, ribitol, galacitol, xylitol, L-arabinitol, inositol, D-gluconic acid, 5-ketogluconic acid, DL-lactic acid, D-glucuronic acid, and D-galacturonic acid. Assimilation of nitrogen compounds: potassium nitrate, sodium nitrite, ethylamine, L-lysine, and cadaverine are positive. Growth in vitamin-free medium is negative. Starch-like compounds are not produced. Growth on 50 % glucose yeast extract agar is negative. Growth in the presence of 0.1 % cycloheximide is negative. Grows at 40–42 °C but not at 45 °C. Liquefaction of gelatin is negative. Acid formation on chalk agar is positive. Urease activity and DBB reaction are negative. The MycoBank number is MB800879. Description of Cyberlindnera samutprakarnensis Poomtien, Jindamorakot, Pinphanichakarn, Limtong, and Thaniyavarn, sp. nov Type strain Cyberlindnera samutprakarnensis (sa.mut.pra.karn. en0 sis. N.L. fem.adj. samutprakarnensis referring to Samutprakarn, Thailand, where this strain was isolated). After growth in YM broth for 3 days at 25 °C, cells in the sediment are ovoid to ellipsoidal, 2–4 9 4–8 lm, and occur singly, in pairs and sometimes arranged in chain, budding, or in pairs (Fig. 3A). Flocculent and sediment are formed after 3 days. After the growth on YM agar for one month at 25 °C, the streak culture is whitish to cream-colored, butyrous, circular, smooth, glistening, and convex with entire margins. A pellicle is not present during growth on the surface of assimilation Holotype is strain JP52T, isolated from the wastewater of a cosmetic factory (Milott laboratory Company Ltd.), collected in Bangplee, Samutprakarn Prov., Thailand, in Feb. 2009, by Jamroonsri Poomtien. It was deposited at (i) the BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, Thailand, as BCC 46825T; (ii) the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Hirosawa, Wako, Saitama, Japan, as JCM 17816T, and (iii) Centraalbureau voor Schimmelcutures (CBS), Utrecht, The Netherlands, as CBS 12528T. The strain is maintained by freezing and/or lyophilization. 123 236 Antonie van Leeuwenhoek (2013) 103:229–238 A C B Fig. 3 Morphology of two new yeast species. (A) Morphology of vegetative cells of Cyberlindnera samutprakarnensis sp. nov. JP52T in sediment grown in YM broth for 3 days at 25 °C. (B) Vegetative cells of Candida thasaenensis sp. nov. JP59T grown in YM agar for 3 days at 25 °C. Scale bars = 10 lm. (C) Pseudomycelia of C. thasaenensis sp. nov. JP59T developed on slide culture with CMA, after 7 days at 25 °C. Scale bars = 30 lm Etymology After growth in YM broth for three days at 25 °C, cells in sediment are spheroidal, ovoidal shape (2.7–4.7 9 3.3–6.0 lm), or ellipsoidal and elongate (2.7–3.3 9 6.7–21.3 lm) in shape, occur singly, in pair, or form in chains (Fig. 3B). Pseudomycelium is well developed. After the growth on YM agar for one month at 25 °C, the streak culture is pale yellowish. Membranous growth generally results from profuse formation of filaments. Yeast colonies are raised, moderately rough, dull, and fringed with pseudomycelium. A pellicle is not present during growth on the surface of assimilation medium. In Dalmau plates after seven days of slide culture on CMA, potato dextrose agar (PDA), and YM agar at 25 °C, pseudomycelium The specific epithet ‘‘samutprakarnensis’’ was derived from ‘‘Samutprakarn Province,’’ where a sample of wastewater containing Cyber. samutprakarnensis was collected. Description of Candida thasaenensis Poomtien, Jindamorakot, Pinphanichakarn, Limtong, and Thaniyavarn, sp. nov Candida thasaenensis (tha.sae.nen.sis. N.L. fem. adj. thasaenensis referring to Thasae, Thailand, where two strain were isolated). 123 Antonie van Leeuwenhoek (2013) 103:229–238 is abundant on these three media but true hypha do not develop. The extensive pseudomycelium as Candidatype consisting of spheriodal blastospores singly and in short chains are formed on media CMA (Fig. 3C) and YM, and form clusters like mycotoruloides-type on PDA. Ascospores are not produced on 5 % malt extract agar, Fowell’s acetate agar, and YM agar after 28 days at 25 °C. The major ubiquinone is Q-9. Glucose, fructose, and sucrose (may be weak) fermentation are positive. But galactose, maltose, lactose, melibiose, and raffinose fermentation are negative. Assimilation of carbon compounds: glucose, sucrose, maltose, cellobiose, trehalose, melezitose, soluble starch (latent), L-rhamnose (slow), ethanol, glycerol, ribitol, D-mannitol, D-glucitol (slow), xylitol, methyl-a-D-glucoside, salicin, glucono-d-lactone (slow), 2-ketogluconic acid, DL-lactic acid (slow), succinic acid, citric acid, and propane-1,2-diol are assimilated. No growth occurs on galactose, L-sorbose, lactose, melibiose, raffinose, inulin, D-xylose, L- arabinose, D- arabinose, D-ribose, D-glucosamine, N-acetyl-D-glucosamine, methanol, erythritol, galacitol, L-arabinitol, inositol, D-gluconic acid, 5-ketogluconic acid, D-glucuronic acid, D-galacturonic acid, and butane-2, 3-diol. Assimilation of nitrogen compounds: ethylamine, L-lysine, and cadaverine are positive. But potassium nitrate and sodium nitrite assimilation are negative. Growth in vitamin-free medium is negative. Starch-like compounds are not produced. Growth on 50 % glucose yeast extract agar is positive. Growth in the presence of 0.1 % cycloheximide is negative. Grows at 37 °C but not at 40 °C. Liquefaction of gelatin is negative. Acid formation on chalk agar is positive. Urease activity and DBB reaction are negative. The MycoBank number is MB800880. Type strain Holotype is strain JP59T, isolated from a soil sediment pond of a palm oil biodiesel production plant (Chumporn Palm Oil industry), collected in Thasae, Chumporn Prov., Thailand, in May 2009, by Jamroonsri Poomtien. It was deposited at (i) the BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, Thailand, as BCC 46828T, (ii) Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Hirosawa, Wako, Saitama, Japan, as JCM 17817T, and (iii) Centraalbureau voor Schimmelcutures (CBS), 237 Utrecht, The Netherlands, as CBS 12529T. The strain is maintained by freezing and/or lyophilization. Etymology The specific epithet ‘‘thasaenensis’’ was chosen because this species was isolated from Thasae Chumporn Province. Acknowledgments We thank Mrs. Somjit Am-In for helping in several experiments performed at the BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology. This work was partially supported financially by the Chulalongkorn. 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