Fungal Diversity (2012) 52:123–139 DOI 10.1007/s13225-011-0115-z A novel ascosporogenous yeast species, Zygosaccharomyces siamensis, and the sugar tolerant yeasts associated with raw honey collected in Thailand Sujinan Saksinchai & Motofumi Suzuki & Panuwan Chantawannakul & Moriya Ohkuma & Saisamorn Lumyong Received: 17 February 2011 / Accepted: 4 June 2011 / Published online: 24 July 2011 # Kevin D. Hyde 2011 Abstract Diversity of yeasts in association with bees and their food sources has been explored during the last decade. In Thailand, there has been no study of yeast identification in honey and bees. Hence, a total of 186 yeast strains were isolated from 37 honey samples of 12 different bee species. On the basis of morphological and physiological characteristics, 55 representative strains were chosen and identified by sequence analysis of the 26S rDNA D1/D2 domain and the ITS region. The data were compared with the published sequences and the results showed the occurrence of 19 ascomycetous and 1 basidiomycetous yeast species. Six strains of the new species were isolated. Phylogenetic analysis of the 26S rDNA D1/D2 sequence revealed that they were conspecific and most closely related to Zygosaccharomyces mellis. Based on the ITS sequence, the new species was clustered with the type " and clearly distinguished from the type !. Sequence analysis of combined ITS-26S rDNA D1/D2 showed similar results. The occurrence of these two types, with a divergence of more than 1% in their sequences, and low DNA relatedness among them suggested that members of the type β can be regarded as separate species. An analysis of the morphological and physiological characteristics was performed. Ascospore formation was observed on acetate agar and Gorodkowa agar. The new Zygosaccharomyces species differed physiologically from Z. mellis in 4 assimilation tests. This data supports the hypothesis that the new species, Zygosaccharomyces siamensis, is a novel ascosporogenous yeast. The type strain is JCM 16825T (=CBS 12273T) and a description is given here. Keywords Ascosporogenous yeast . Zygosaccharomyces siamensis . Sugar tolerant yeasts . Raw honey . Thailand Introduction S. Saksinchai The Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand S. Saksinchai Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand M. Suzuki : M. Ohkuma Microbe Division/Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Wako, Saitama 351-0198, Japan P. Chantawannakul : S. Lumyong (*) Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand e-mail: scboi009@chiangmai.ac.th The honeybees (Apidae: Apini) are the most famous of all insects owing to their importance for the pollination of crops, their social organization, and the honey they produce. The single genus Apis has been separated into 11 distinct species historically based on morphological, behavioral, and physiological characteristics, and also on the geographic distribution (Engel 1999). At present the genus Apis contains 10 generally recognized species (Arias and Sheppard 2005; Raffiudin and Crozier 2007), however, the validity of Apis laboriosa has been questioned based on the lack of morphological differentiation from Apis dorsata (Engel 1999; Koeniger et al. 1991), despite differences in behavior and ecology (Kirchner et al. 1996; Underwood 1990). Among Apis species distributed in Southeast and East Asia, five are found in Thailand. Apis andreniformis, Apis 124 cerana, Apis dorsata and Apis florea are native species, whereas Apis mellifera is introduced (Chaiyawong et al. 2004; Hepburn et al. 2005; Insuan et al. 2007; Oldroyd and Wongsiri 2006; Rinderer et al. 1995; Smith et al. 2000; Wongsiri et al. 1990, 1996). The stingless bees (Apidae: Meliponini) are a large monophyletic group of highly eusocial bees, with vestigial stings, found in abundance in warm humid forests around the globe. They are the major visitor of many flowering plants and play an important role in the pollination process of plant life, particularly for native plants and wild flowers in tropical and subtropical regions (Heard 1999; Michener 1974; Roubik 1989). Stingless bees have more species than Apis, and are both distinctive and diverse. They are classified into 50–60 genera, with about 400 described species (Michener 2007; Roubik 2006). A large numbers of Asian Meliponini species have been recorded, of them, 32 species are currently recognized in Thailand (Boontop et al. 2008; Jongjitvimol et al. 2005; Jongjitvimol and Wattanachaiyingcharoen 2006, 2007; Klakasikorn et al. 2005; Schwarz 1939). Attempts to characterize yeast communities associated with bees and their food sources have previously been reported. Most have shown that certain yeast species are isolated repeatedly from some bees. Torulopsis magnoliae (=Candida magnoliae) was the most common isolate found in pollen stored in comb cells of the hive (bee bread) of Apis mellifera (Gilliam 1979, 1997; Gilliam et al. 1974; Péter et al. 2009). It was accompanied by minor ones, one of them, identified as Trichomonascus apis, represented a novel heterothallic yeast species in the Trichomonascus clade (Péter et al. 2009). Inglis et al. (1993) have reported that Candida bombicola (=Starmerella bombicola) was prevalent taxon recovered from nectar, pollen and provisions, but rarely in guts or frass of the alfalfa leafcutter bee (Magachile rotundata). The predominant yeast species found in larval provisions, larvae, and pupae of the solitary bees, Diadasina distincta and Ptilotrix plumata, was Candida batistae (Rosa et al. 1999). Two new ascomycetous yeasts belonging to the Starmerella clade were discovered in nests of two solitary bees. Candida riodocensis was isolated from pollen-nectar provisions, larvae and fecal pellets in nests of Megachile sp., and Candida cellae was found in pollen-nectar provisions of Centris tarsata (Pimentel et al. 2005). A few individuals of Trigona spp. were found to harbor several novel yeast species in the same clade, three of which have been described as Candida floris, Candida powellii and Candida tilneyi (Lachance et al. 2001a; Rosa et al. 2007). Starmerella melliponinorum has been isolated from various substrates associated with five stingless bees. Rosa et al. (2003) and Teixeira et al. (2003) have shown that S. melliponinorum was found in garbage pellets, pollen provisions, adult bees, honey and propolis of Tetragonisca angustula. It was also found in Fungal Diversity (2012) 52:123–139 adult bees and honey of Melipona quadrifasciata and Frieseomelitta varia, and in adult bees of Melipona rufiventris and Trigona fulviventris. The stingless bee T. angustula showed a strong association with S. melliponinorum, but also vectored Zygosaccharomyces machadoi (Rosa and Lachance 2005). Strains identified as members of the Candida apicola complex have been shown to be abundant in nests of M. quadrifasciata (Rosa et al. 2003). Zygosaccharomyces is a yeast genus often associated with food spoilage (James and Stratford 2003; Steels et al. 1999). Currently the genus is comprised of 14 species (Kurtzman 2003; Rosa and Lachance 2005; Torriani et al. 2011), four of which, Z. bailii, Z. bisporus, Z. lentus and Z. rouxii, are well recognized as the causes of significant amounts of food and beverage spoilage. Another related species, Z. mellis is remarkable for extreme osmotolerance and is a spoiling agent of high sugar foods and honey. Physiological characteristics that make Zygosaccharomyces yeasts particularly problematic include their ability to ferment sugar, osmotolerance, resistance to preservatives, formation of heat-resistant ascospores and fructophily (James and Stratford 2003; Steels et al. 1999, 2002). A previous study (Suezawa et al. 2008), using 26S rDNA D1/D2 and ITS sequencing, of the yeasts isolated from miso and soy sauce in Japan revealed two types of Z. mellis. In this study, we conducted a survey on yeast communities associated with raw honey collected from both honeybees and stingless bees in Thailand to demonstrate the distribution of sugar tolerant yeasts as well as to see whether there is a specific relationship between the yeasts and their insect hosts. In addition, we carried out 26S rDNA D1/D2 and ITS sequence analyses of isolated strains from raw honey and authentic strains of Z. mellis in order to type for differentiation. The morphological and physiological characteristics of all six strains of Z. siamensis were examined, and compared with Z. mellis Fabian & Quinet. Materials and methods Yeast isolation and grouping Collections were made in 2006, 2007 and 2009 in six provinces of Thailand. Four nests of Apis cerana, 1 of A. dorsata, 6 of A. florea, 8 of A. mellifera, 1 of Homotrigona fimbriata, 1 of Lepidotrigona doipaensis, 1 of L. terminata, 1 of Tetragonilla collina, 1 of Tetragonula fuscobalteata, 4 of T. laeviceps, 7 of T. pagdeni, and 2 of Trigona spp. were sampled. Honey was aseptically squeezed, diluted in approximately 10 volumes of sterile water, and vortexed for 1 min. One hundred microliters of successive decimal dilutions were spread on Yeast extract-Malt extract (YM) agar (1% glucose, 0.5% peptone, 0.3% malt extract, 0.3% Fungal Diversity (2012) 52:123–139 125 yeast extract, 2% agar) supplemented with 100 mg l-1 chloramphenicol. The plates were incubated at 25°C and examined periodically. Representative yeast colonies were purified and maintained in YM slants or 30% (v/v) glycerol. On the basis of morphological and physiological characteristics (Guo et al. 2006; Yarrow 1998), representative yeast strains were chosen. method (Kimura 1980) and the phylogenetic trees were constructed for selected yeast species with the neighborjoining method (Saitou and Nei 1987) on a CLUSTAL_X package (Thompson et al. 1997). The topology of the phylogenetic tree was tested by performing bootstrap resampling from 1,000 replicates (Felsenstein 1985). Organism, fermentation and assimilation tests Results and discussion The summary of the new Zygosaccharomyces species isolated in this study are described in Table 1, and are maintained in the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Wako, Saitama, Japan. Duplicate of the strain JCM 16825T has also been deposited at the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands (CBS 12273T). The composition of all culture media used in this study, as well as the protocols for fermentation and assimilation tests were in accordance with the methods of Yarrow (1998). Sugar tolerant yeasts associated with raw honey Ribosomal DNA (rDNA) sequencing and sequence analysis Genomic DNA was prepared using YeaStar Genomic DNA Kit™ (Zymo Research, California, USA) according to the manufacturer’s protocol. A polymerase chain reaction (PCR) was performed according to methods described for the amplification of 26S rDNA D1/D2 domain (Kurtzman and Robnett 1997) or the ITS region (White et al. 1990). The amplicons were sequenced using the ABI BigDye® Terminator V3.1 Cycle Sequencing RR-100 kit and an ABI Model 3130xl Genetic Analyzer (Applied Biosystems, Foster City, California, USA) following the manufacturer’s instructions. Sequence data were aligned by the BioEdit program (Isis Pharmaceuticals, California, USA). The sequences determined in this study were deposited in the DDBJ database. The accession numbers of 26S rDNA D1/D2 and ITS sequences are shown in Figs. 1, 2, 3, 4, 5, 6 and 7, and compared with those available in the GenBank database at the DDBJ using the BLASTN program (Altschul et al. 1997). The nucleotide substitution rate was determined by Kimura’s two-parameter Table 1 Summary of new Zygosaccharomyces species isolated in this study JCM: Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama, Japan CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands T type strain Isolation No. HB61T HB67 HB69 HB73 HB137 SB140 Six yeast isolates were obtained from Apis cerana, 2 from A. dorata, 10 from A. florea, 12 from A. mellifera, 2 from Homotrigona fimbriata, 1 from Lepidotrigona doipaensis, 1 from L. terminata, 1 from Tetragonilla collina, 2 from Tetragonula fuscobalteata, 5 from T. laviceps, 10 from T. pagdeni and 3 from Trigona spp. Among the ascomycetous yeasts, the genus Candida was the most commonly isolated, with a high frequency of Candida apicola in raw honey of six of the twelve bee species investigated (Table 2). The 26S rDNA D1/D2 domains showed sequence variations among isolates (Fig. 1). Three strains from the honeybees (A. florea and A. mellifera) differed from the type strain by two bases (sequences identical to C. apicola UFMG Jat 515.1 isolated from Melipona rufiventris pollen in Brazil; Lachance et al. 2010) and thus fit Kurtzman and Robnett’s (1998) criterion for conspecificity. Another two strains from the stingless bees (L. doipaensis and T. laeviceps) differed from the type strain by four bases (sequences identical to C. apicola UFMG Jat 128.2 isolated from M. rufiventris in Brazil; Lachance et al. 2010, Rosa et al. 2003) and fell outside the inclusion limits proposed by Kurtzman and Robnett (1998). D1/D2 sequencing was unsuccessful for the strain HB108 and SB50, indicating that they might have heterogeneous D1/D2 copies. Based on the approach used in many recently published descriptions of yeast species, where sampling is sometime poor and even occasionally limited to single strain, it is conceivable that strains identified as members of the C. apicola complex (Starmerella clade) included in the present study could be Collection numbers JCM No. CBS No. 16825T 16826 16827 16828 16829 16830 12273T – – – – – Source Honey, Honey, Honey, Honey, Honey, Honey, Apis mellifera Apis mellifera Apis mellifera Apis mellifera Apis dorsata Tetragonula pagdeni Locality Year of isolation Chantaburi Chumphon Chumphon Chantaburi Chiang Mai Chiang Mai Feb. 2006 May 2006 May 2006 Feb. 2007 Feb. 2009 Jun. 2007 126 Fungal Diversity (2012) 52:123–139 Fig. 1 Phylogenetic tree drawn from neighbor-joining analysis based on the 26S rDNA D1/D2 region sequence data, showing the placement of strains identified as members of Candida apicola complex (Kurtzman and Robnett 1997; Lachance et al. 2010; Rosa et al. 2003; Tofalo et al. 2009). Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.01 substitutions per site. Saccharomyces cerevisiae CBS 1171T was used as an outgroup assigned to as many as separate species (Kurtzman and Robnett 1997; Lachance et al. 2010; Rosa et al. 2003; Tofalo et al. 2009). The parsimony network generated from D1/D2 sequences suggests on the contrary that all strains but CBS 4353 could be viewed as members of a cohesive evolutionary unit, although the inclusion of strain MUCL 45721/CRGF 96 might appear dubious. Inclusion of ITS sequences in the parsimony network analysis reinforced the notion that most strains should be treated as members of a single evolutionary population, but caused strain MUCL 45721/CRGF 96 to be excluded at the 95% limit. The dependence of these observations on adequate sampling further suggests that the description of new species to accommodate strains CBS 4353 and MUCL 45721/CRGF 96 should await the isolation of a sufficient number of additional strains (Lachance et al. 2010). Candida metapsilosis was the prevalent species found associated with T. pagdeni, and was also isolated at a lower frequency from A. florea (Table 2, Fig. 2). Two nests of A. mellifera yielded Candida parapsilosis and Candida orthopsilosis, which have been previously reported on bees, beetles and other insects (Gilliam 1979, 1997; Gilliam et al. 1974; Suh et al. 2005, 2008). Candida parapsilosis, the second most common yeast species isolated from blood cultures (Bassetti et al. 2006; Messer et al. 2006; Safdar et al. 2002), is a complex of three genetic groups (I, II and III) according to the results from genotypic methods (Enger et al. 2001; Lehmann et al. 1992; Lin et al. 1995; Lott et al. 1993; Messer et al. 2006; Nosek et al. 2002; Roy and Meyer 1998). Tavanti et al. (2005) reported a multilocus sequence typing scheme and suggested that C. parapsilosis groups II and III should be reclassified as C. orthopsilosis and C. metapsilosis, while C. parapsilosis was retained for group I. In addition, C. parapsilosis group IV has also been recognized among Brazilian clinical isolates based on their ITS sequences (Iida et al. 2005). Candida metapsilosis was recovered relatively rarely in clinical samples, evidenced by its paucity in culture collections and comments in previous reports (Asadzadeh et al. 2009; Gomez-Lopez et al. 2008; Gonçalves et al. 2010; Hensgens et al. 2009; Kocsubé et al. 2007; Lockhart et al. 2008; Mirhendi et al. 2010; Pryce et al. 2006; Silva et al. 2009; Tavanti et al. 2005, 2007; Tay et al. 2009). A high frequency of C. metapsilosis observed in raw honey raises the possibility that C. metapsilosis might be the progenitor, associated with a non-mammalian environment, of both the related species C. parapsilosis and C. orthopsilosis, which evolved by adaptation to commensalism in human or other mammalian niches (Tavanti et al. 2005). Therefore, it is cautioned that insects may play an important role in dispersing opportunistic yeasts in nature. Moreover, Fungal Diversity (2012) 52:123–139 127 Fig. 2 Phylogenetic tree of the relationship between Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis based on analysis of the 26S rDNA D1/D2 region sequence. The tree was constructed for selected yeast species, with reference to Asadzadeh et al. (2009), Correia et al. (2004), Daniel and Meyer (2003), Daniel et al. (2009), Iida et al. (2005), Kosa et al. (2006), Kurtzman and Robnett (1997), Mirhendi et al. (2010), Suh et al. (2005) and Tapia-Tussell et al. (2006) with the neighbor-joining method. Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.01 substitutions per site. Saccharomyces cerevisiae NRRL Y12632NT was used as an outgroup insect associations may have been important in the evolution of these yeasts (Suh et al. 2008). Strains identified as Starmerella meliponinorum were isolated from raw honey of A. florea, T. collina, T. laeviceps, T. pagdeni and Trigona sp. (Table 2). The population sizes of S. meliponinorum in raw honey varied between 2.0(±1.0)×102 and 9.8(±3.1)×104 colony forming units (CFU) ml-1. Similar results were observed by Teixeira et al. (2003) with three stingless bees in Brazil. These results suggest that the yeast is metabolically active and able to grow at the expense of the sugars present in these resources, improving their nutritional quality. Yeasts are known to be a source of proteins and vitamins for many insects (Lachance et al. 2001b; Morais et al. 1994; Zacchi and Vaughan-Martini 2002). Suh et al. (2005) have indicated that the presence of yeast allows insects to survive on nutrient-limiting substrates. The high counts further suggest that S. meliponinorum is not an agent of food spoilage for these bees, but rather that a close mutualistic association exists between the yeast and the insects (Rosa et al. 2003; Teixeira et al. 2003). Another strain, classified as Starmerella bombicola, was isolated from A. florea (Fig. 3). It has been described as a species occurring in grapes and fermenting must (Csoma and Sipiczki 2008). Starmerella bombicola is also a yeast associated with flowering plants and their insects (Brysch-Herzberg 2004; Golonka 2002; Rosa and Lachance 1998; Rosa et al. 2003; Sipiczki et al. 2005), and this could be its principle ecological habitat. Pichia guilliermondii was the most common species found in raw honey (Table 2). It is ubiquitous (Spencer and Spencer 1997) and is not likely to be associated specifically with a host insect (Zacchi and Vaughan-Martini 2002). Strain originally designed as HB45 differs from P. guilliermondii (type strain, NRRL Y-2075) by less than two to three nucleotide substitutions (Fig. 4). The result indicates that it can be either conspecific or a sister species according to Kurtzman and Robnett (1998). The other strain, SB146 isolated from T. pagdeni, has a sequence which is identical to Pichia caribbica (type strain, NRRL Y-27274) and Candida fermentati (type strain, NRRL Y-27041). On the basis of phenotype, C. fermentati is believed to be a synonym of P. guilliermondii. However, they differ from each other by three bases, and it appears that C. fermentati and P. guilliermondii may represent two separate but closely related species (Vega et al. 2003). Furthermore, according to the new classification 128 Fungal Diversity (2012) 52:123–139 Fig. 3 Phylogenetic tree of the relationship between Starmerella bombicola and Starmerella meliponinorum based on analysis of the 26S rDNA D1/D2 region sequence. The tree was constructed for selected yeast species, with reference to Brysch-Herzberg (2004), Csoma and Sipiczki (2008), Golonka (2002), Kurtzman and Robnett (1997), Rosa and Lachance (1998), Rosa et al. (2003), Sipiczki et al. (2005) and Teixeira et al. (2003) with the neighbor-joining method. Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.02 substitutions per site. Saccharomyces cerevisiae CBS 1171T was used as an outgroup of P. guilliermondii complex proposed by Vaughan-Martini et al. (2005), they are divided into three species viz. (i) P. guilliermondii (teleomorph of Candida guilliermondii), (ii) Candida carpophila, and (iii) P. caribbica (teleomorph of C. fermentati). Recently, P. guilliermondii and P. caribbica were assigned to the new genus Meyerozyma (Kurtzman and Suzuki 2010). Other yeast species were occasionally isolated from raw honey (Table 2). Blastobotrys terrestris (identity confirmed by sequencing), member of the Trichomonascus clade, was found here in A. cerana. It has been discovered in soil, whereas the related species Blastobotrys chiropterorum and Blastobotrys serpentis have been described as yeasts associated with bat liver and snake intestine (Bhadra et al. 2008b; Meyer et al. 1998). Candida etchellsii has been isolated from fermenting cucumber brine, sputum, concentrate lemon juice, sugar, sausages, soy sauce mashes and miso pastes (Meyer et al. 1998; Suezawa et al. 2006). This yeast was also found to be associated with bees and other beetles (Lachance et al. 2001b; Rosa et al. 2003). The population sizes of C. etchellsii obtained from A. cerana and T. fuscobalteata were small: 1.5 (±0.7)×102 and 2.8(±1.8)×102 CFU ml-1, respectively. It is likely that C. etchellsii is not used by the bees as a food source, but rather that it may be associated with a transient insect species. Candida sorbosivorans was recovered from two samples of A. mellifera in Chantabuti and Nan provinces. It has been described as a species occurring in contaminated industrial material involved in a cascade continuous process for oxidizing sorbitol (D-glucitol) to L-sorbose (James et al. 2001). Trindade et al. (2002) showed that C. sorbosivorans is also a yeast associated with Brazilian tropical fruits, and this could be its principal ecological habitat. Candida fermenticarens, Candida versatilis and Pichia kudriavzevii (synonym of Issatchenkia orientalis) have been isolated from sugar, fruit juice, berries or other visiting substrates (Meyer et al. 1998; Kurtzman 1998). Bees are attracted by sugar-rich foods and it is likely that they are the principal source of infection of these substrates (Rosa et al. 2003; Stratford et al. 2002). This study found Debaryomyces hansenii in raw honey of H. fimbriata. It has previously been isolated from adult bees, beetle gut and tree bark (Bhadra et al. 2008a; Rosa et al. 2003; Suh et al. 2005). This halotolerant and osmotolerant yeast is a frequent contaminant of human food. It was reported to cause spoilage of fruit jam, dairy products, meat Fungal Diversity (2012) 52:123–139 Fig. 4 Phylogenetic tree of the relationship between Meyerozyma caribbica and Meyerozyma guilliermondii based on analysis of the 26S rDNA D1/D2 region sequence. The tree was constructed for selected yeast species, with reference to Burgaud et al. (2010), Čadež et al. (2010), Cocolin et al. (2002), Daniel and Meyer (2003), Daniel et al. (2009), Duarte et al. (2004), El-Latif Hesham et al. (2006), Hellström et al. (2010), Kurtzman and Robnett (1997), Laitila et al. (2006), Nguyen et al. (2007), Nisiotou et al. (2010), Rao et al. (2007a, b), 129 Sette et al. (2010), Suezawa and Suzuki (2007), Suh and Blackwell (2004), Tapia-Tussell et al. (2006), Urubschurov et al. (2008) and Vega et al. (2003) with the neighbor-joining method. Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.01 substitutions per site. Saccharomyces cerevisiae NRRL Y-12632T was used as an outgroup 130 Fungal Diversity (2012) 52:123–139 Fig. 5 Phylogenetic tree drawn from neighbor-joining analysis based on 26S rDNA D1/D2 region sequence data, showing the placement of yeast species closely related to Zygosaccharomyces siamensis (Kurtzman and Robnett 1998; Kurtzman et al. 2001; Rosa and Lachance 2005; Steels et al. 1999). Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.01 substitutions per site. Saccharomyces cerevisiae CBS 1171T was used as an outgroup and sausages (Fröhlich-Wyder 2003; Prista and LoureiroDias 2007; Samelis and Sofos 2003; Tokuoka 1993; Westall and Filtenborg 1998). The basidiomycetous pigmented species of Rhodotorula, along with Erythrobasidium, Occultifur, Sakaguchia and Sporobolomyces, were grouped in the Erythrobasidium clade (Fell et al. 2000). The occurrence of Rhodotorula nymphaea, recovered here from A. florea, is less well understood. It has been isolated from China and Thailand (Abdurehim unpublished; Sjamsuridzal et al. unpublished). Given that other species of Rhodotorula have been isolated from deep-sea environments such as benthic animals and sediments (Nagahama et al. 2001a, b, 2003). Schizosaccharomyces japonicus was accidentally discovered in H. fimbriata. With respect to the source of isolation, however, this fission yeast was originally described as a species isolated from strawberry fields of Kyusyu University in Japan (Yukawa and Maki 1931), and may represent a transient species vectored by bees. Yamadazyma ohmeri (Etchells et Bell) Billon-Grand was first described as Endomycopsis ohmeri Etchells et Bell (Billon-Grand 1989; Etchells and Bell 1950), and was then transferred to the genus Pichia Hansen (Kreger-van Rij 1964). Billon-Grand (1989) introduced the new genus Yamadazyma Billon-Grand to accommodate 16 species from the genus Pichia. On the basis of rRNA sequences, Yamada et al. (1995a) concluded that Y. ohmeri was not closely related to any other species of either Pichia or Yamadazyma. Accordingly, the genus Kodamaea Yamada, Suzuki, Matsuda et Mikata was proposed for that species (Yamada et al. 1995b). In this study, Kodamaea ohmeri also found in A. florea, showed a strong Fungal Diversity (2012) 52:123–139 131 Fig. 6 Phylogenetic tree of relationship between Zygosaccharomyces mellis and Zygosaccharomyces siamensis based on analysis of the ITS sequence (Fujii unpublished; Suezawa et al. 2008). The tree was constructed by the neighbor-joining method. Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.02 substitutions per site association with the small hive beetle Aethina tumida, a pest of the European honeybee A. mellifera (Benda et al. 2008; Torto et al. 2007). It is unknown whether a small hive beetle or a honeybee was responsible for the introduction of this yeast into the beehive. Because yeasts are commonly found in flowers and their associated insects, and because either can become contaminated during the common behavioral interaction between honeybees and small hive beetles, either or both routes of entry are feasible (Benda et al. 2008; Lachance et al. 2001b). The distribution of yeasts associated with raw honey collected in Thailand is complex, as it involves twelve introduced bee species, most of which appear to use different resources. The relatively small number of yeast species found in honey communities may indicate either a limited biodiversity in bee isolates from natural environments, or could be due to the difficulty of cultivating many species outside of a beehive. Thanh et al. (2002) has shown that a yeast associated with the woodlouse has an absolute requirement for exogenous siderophores, which are produced by some common soil fungi. Analogously, the presence of growth factors produced either by the host insect itself or by other microbial endosymbionts may be necessary in order to elucidate the entire yeast microflora of this habitat (Zacchi and Vaughan-Martini 2002). Yeast communities associated with raw honey may be affected by low water activity and Fig. 7 Phylogenetic tree of relationship between Zygosaccharomyces mellis and Zygosaccharomyces siamensis based on sequence analysis of combined ITS-26S rDNA D1/D2 (Fujii unpublished; Suezawa et al. 2008). The tree was constructed by the neighbor-joining method. Bootstrap values were calculated from 1,000 replicates, and values below 50% were omitted. The sequence accession numbers are shown in parentheses. The bar indicates the sequence dissimilarity value of 0.01 substitutions per site 132 Table 2 Frequency of occurrence of yeast species associated with the honeybees and the stingless bees Yeast/bee species A. cerana Blastobotrys terrestris Candida apicola Candida etchellsii Candida fermenticarens Candida metapsilosis Candida orthopsilosis Candida parapsilosis Candida sorbosivorans Candida versatilis Debaryomyces hansenii Debaryomyces mellissophilus Kodamaea ohmeri Meyerozyma caribbica Meyerozyma guilliermondii Pichia kudriavzevii Rhodotorula nymphaea Schizosaccharomyces japonicus Starmerella bombicola Starmerella meliponinorum Zygosaccharomyces siamensis sp. nov. 1 A. dorsata A. florea A. mellifera 1 1 2 H. fimbriata L. doipaensis L. terminata T. collina T. fuscobalteata 1 1 T. laeviceps T. pagdeni 1 1 Trigona spp. 1 1 1 3 1 1 2 1 1 2 1 2 4 1 1 1 3 1 1 1 1 1 1 1 4 3 1 1 1 Fungal Diversity (2012) 52:123–139 1 1 Fungal Diversity (2012) 52:123–139 133 high sugar concentration. Therefore, sugar tolerant yeast species could be found commonly in honey habitats. To our knowledge, this is the first report of sugar tolerant yeast isolation from raw honey. At the next stage of our study, we will attempt to focus on yeasts associated with bee food sources (e.g. pollen provisions, propolis or gabage pellets), as well as with bee life stages (i.e. eggs, larvae, pupae, adults). Furthermore, the contribution of yeasts to the ecology of the alimentary canal of bees requires further study. Zygosaccharomyces siamensis sp. nov., an ascosporogenous yeast species The 26S rDNA D1/D2 sequences of the strains JCM 16825TJCM 16830 were identical. The closest species to the six strains in terms of pairwise sequence similarity was Zygosaccharomyces mellis (Fig. 5), but with 0.6% nucleotide substitutions (3 out of 531 nucleotide positions). Zygosaccharomyces mellis could be divided into two types (type α and type β; Suezawa et al. 2008) based on the ITS sequence (Fig. 6). The ITS sequences of all eight strains of the type β (S70, F70, G70, JCM 16825T, JCM 16826, JCM 16827, Z. mellis NBRC 0485 and Z. mellis NBRC 1732) were not identical to that of Z. mellis NBRC 1615T (type α). Zygosaccharomyces siamensis was clearly distinguished from the type α (29% substitutions). Sequence analysis of combined ITS-26S rDNA D1/D2 showed similar results (Fig. 7). The extent of sequence divergence between these two types was 13.2% in their sequences and low DNA relatedness among them (72% complementarity; Kurtzman 1990), indicating that members of the type β could be regarded as separate species. ITS sequencing was successful for the strains JCM 16825T-JCM 16827, but failed for the strains JCM 16828-JCM 16830, indicating that the latter three might have heterogeneous ITS copies. Egli and Henick-Kling (2001) suggested that cloning of the PCR amplicons prior to sequencing is necessary to investigate this further. One to four ascospores were formed on acetate agar and Gorodkowa agar, usually two which were round to oval (Fig. 8). In terms of physiological characteristics, the strains JCM 16825T-JCM 16830 could assimilate galactose and erythritol, and grow at 37°C as well as in the presence of salt (16% NaCl/5% glucose) in contrast to Z. mellis Fabian & Quinet (Tables 3 and 4). Accordingly, the six strains are considered to represent a novel ascosporogenous yeast species. Hence, it is described as Zygosaccharomyces siamensis sp. nov. in this study. Latin diagnosis of Zygosaccharomyces siamensis Saksinchai, M. Suzuki, Chantawannakul, Ohkuma et Lumyong sp. nov. In medio liquido cum extracto malti post dies 3 ad 25°C, cellulae vegetativae globosae aut ellipsoideae (4.3-5.3×5.5- Fig. 8 Ascospore formation observed in Zygosaccharomyces siamensis when grown on Gorodkowa agar at 25°C for 2 weeks. Bar = 5 μm 6.4 μm), singulae, binae et fasciculatae. In agaro cum extracto malti post dies 5 ad 25°C, coloniae parva, convexa, glabra et candida. Cultura in striis in agaro cum extracto malti post dies 30 ad 17°C incrementum fuscum pallidum, butyrosum, centrum coloniae sublatum, margo glabro vel undulato. In agaro Gorodkowa post dies 14 ad 25°C, asci ovoidei per conjugationem cellularum vegetativarum oriuntur 1–4 ascosporas globosae aut ovales continentes non liberi. Glucosum (lente) et maltosum (lente, variablie) fermentantur at non galactosum, sucrosum, lactosum, raffinosum et trehalosum. Glucosum, galactosum (lente), maltosum (lente), trehalosum (exigue, variabile), glycerolum (lente), erythritolum, D-mannitolum (lente), D-glucitolum (lente), Dgluconicum (exigue, variabile) et glucono-δ-lactonum (lente) assimilantur. L-Sorbosum, sucrosum, cellobiosum, lactosum, melibiosum, raffinosum, melezitosum, inulinum, amylum solubile, D-xylosum, L-arabinosum, D-arabinosum, Dribosum, L-rhamnosum, D-glucosaminum, N-acetyl-D-glucosaminum, methanolum, ethanolum, ribitolum, galactitolum, α-methyl-D-glucosidum, salicinum, DL-lactatum, succinatum, citratum, inositolum, hexadecanum, decanum, 2-ketoD-gluconicum, 5-keto-D-gluconicum, saccharatum, Dglucuronicum, xylitolum, L-arabinitolum et butane-2,3-diolum non assimilantur. Vitamina externa crescentiae sunt neccessaria. Ethylaminum hydrochloricum, L-lysinum et cadaverinum dihydrochloricum (exigue) assimilantur, at non kalium nitricum et natrium nitrosum. Crescit in agaro extracto fermenti confecto 60 partes glucosi per centum. In liquido 16 partes natrium hydrochloricum 5 partes glucosi per centum crescit (exigue). Maxima temperatura crescentiae: 37°C. Typus. JCM 16825T (=CBS 12273T). Isolata ex mellis, Chantaburi Provincia, Thailandia. Typus lyophilus conservatur in Collectione Culturarum JCM, RIKEN, Wako, Saitama, Japan et in collectione zymotica CBS, Trajectum ad Rhenum, Hollandia. 134 Fungal Diversity (2012) 52:123–139 Table 3 Responses of Z. siamensis on fermentation, assimilation and other growth testsa,b Fermentation: Glucose Galactose Sucrose Assimilation: Glucose +(s) − − Maltose Lactose Raffinose l&w/− − − Trehalose − + D-Xylose − D-Mannitol +(l) Galactose +(l) L-Arabinose L-Sorbose − D-Arabinose Sucrose − D-Ribose Maltose +(l) L-Rhamnose Cellobiose − D-Glucosamine Trehalose w/− N-Acetyl-D-glucosamine Lactose − Methanol Melibiose − Ethanol Raffinose − Glycerol Melezitose − Erythritol Inulin − Ribitol Soluble starch − Galactitol Additional assimilation tests and other growth characteristics: 2-Keto-D-gluconate − Ammonium 5-Keto-D-gluconate − Nitrite Saccharate − Ethylamine Glucono-δ-lactone +(l) L-Lysine D-Glucuronate − Cadaverine − − − − − − − − +(s) + − − D-Glucitol α-Methyl-D-glucoside Salicin D-Gluconate DL-Lactate Succinate Citrate Inositol Hexadecane Nitrate Vitamin-free +(l) − − w/− − − − − − − − + − + + +/w 15% NaCl/5% glucose 16% NaCl/5% glucose Growth at 19°C Growth at 25°C Growth at 30°C +/w +/w + + + Xylitol L-Arabinitol − − 50% Glucose yeast extract + Growth at 34°C + Butane 2,3 diol Decane − − 60% Glucose yeast extract 10% NaCl/5% glucose 12.5% NaCl/5% glucose + + +/w Growth at 35°C Growth at 37°C Growth at 40°C + + − a Combined test results from JCM 16825T -JCM 16830 b −, negative; +, positive; l, latent (longer than 7 days); s, slow; w, weak Description of Zygosaccharomyces siamensis Saksinchai, M. Suzuki, Chantawannakul, Ohkuma, Lumyong sp. nov. Growth in YM broth After 3 days at 25°C, the cells are spherical to ellipsoidal, (4.3-5.3×5.5-6.4 μm), and occur singly, in pairs, and in small clusters. Growth on YM agar After 5 days at 25°C, colonies are white, convex, smooth and opalescent. Streak culture on YM agar after 1 month at 17°C is butyrous, white to tannish-white, and raised with a central depression. Colony margins are entire with irregular lobes. Growth on the surface of assimilation media Pellicles are not formed. Table 4 Differential phenotypic characteristics between Z. siamensis and Z. mellis Characteristic Z. siamensis Z. mellisa Assimilation: Galactose Erythritol 16% NaCl/5% glucose Growth at 37°C +(l) + +/w + − − − − a Data from Kurtzman and Fell (1998); Meyer et al. (1998) Formation of ascospores After 2 weeks at 25°C on acetate agar and Gorodkowa agar, conjugated pairs of cells give rise to oval asci containing one to four round-to-oval ascospores, usually two ascospores which are not liberated (Fig. 8). Species showing the type of conjugation described are ordinarily homothallic (Kurtzman et al. 2001). Physiological tests Reactions on the fermentation and assimilation tests commonly used in yeast taxonomy are given in Table 3. As differences in phenotypic characteristics are Fungal Diversity (2012) 52:123–139 galactose and erythritol assimilation, as well as growth at 37°C and in the presence of salt (16% NaCl/5% glucose, Table 4), Z. siamensis can be distinguished from Z. mellis on the basis of sequence comparison and ascospore formation. Type JCM 16825T (=CBS 12273T) is preserved as a lyophilized preparation in the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Wako, Saitama, Japan, as well as in the Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. The strain was isolated in February 2006 from raw honey of the European honeybee (Apis mellifera) collected in Chantaburi province, Thailand (Table 1). Etymology The species epithet siamensis (si.am.en′sis N.L. fem. adj. siamensis) refers to Siam, the historic name of Thailand, the country where the yeast strains were isolated. During a survey of yeasts associated with raw honey collected in Thailand, Z. siamensis has been recovered from 5 samples of Apis mellifera, Apis dorsata and Tetragonula pagdeni. Of these, only one sample of A. dorsata contained other yeast species in the genus Candida, all in small numbers. The yeast counts ranging from 1.0 (±0) × 102 to 8.2 (±0.8) × 103 cfu ml-1, with the higher counts observed in two samples of A. mellifera. These results indicate that the yeast is metabolically active and able to grow in honey (Snowdon and Cliver 1996). Isolation of Z. siamensis, Z. mellis types α and β (Suezawa et al. 2008), and Zygosacharomyces species (Fujii unpublished) from honey may indicate the normal environment of that strains and further suggests that the yeasts may be an agent of spoilage, due mostly to its tolerance to high osmotic pressure (James and Stratford 2003). Therefore, good manufacturing practice should be concerned with avoiding the proliferation of such yeast in honey. Acknowledgements This work was funded by the Thai Government Science and Technology Scholarship for Ph.D. Study, awarded to S. Saksinchai, grant RSA5280010 from the Thailand Research Fund, the National Research University, and Office of the Higher Education Commission. 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