Rhodotorula benthica sp. nov. and Rhodotorula calyptogenae sp. nov., novel yeast species from animals collected from the deep-sea floor, and Rhodotorula lysiniphila sp. nov., which is related phylogenetically

Rhodotorula minuta is isolated frequently from marine sources such as water, plants and animals. An extensive study of the basidiomycetous yeasts based on the D1/D2 region of 26S rDNA sequences confirmed the heterogeneity of the physiological and biochemical properties of this species and the presence of at least five species among strains related to R. minuta in the Erythrobasidium clade (Fell et al., 2000). Of these five species, only Rhodotorula marina was phylogenetically divergent. The other four species, Rhodotorula laryngis, R. minuta, Rhodotorula pallida and Rhodotorula slooffiae, formed a phylogenetically coherent group. Some strains belonging this group originated from animal sources, such as humans, fish or other terrestrial or marine animals (Phaff et al., 1952; Reiersøl, 1955; Novák & Vörös-Felkai, 1962; Galgóczy & Novák, 1965; Sugiyama & Goto, 1967; Hagler & Ahearn, 1987). We investigated the distribution of yeasts in deep-sea environments in the north-west Pacific Ocean (Nagahama et al., 2001a) and found that some strains isolated from benthic animals such as tubeworms or giant white clams were phylogenetically located within the Erythrobasidium clade. We found that deep-sea isolates within this clade occurred only in animal sources, although R. minuta is frequently found in oceanic regions (Fell, 1976). One of these isolates, related to Sakaguchia dacryoides, was described as Rhodotorula lamellibrachiae (Nagahama et al., 2001b). Others were part of the R. minuta species complex. Phylogenetic analysis of these deep-sea strains and other strains treated as R. minuta showed substantial heterogeneity in their internal transcribed spacer (ITS) sequences (Nagahama et al., 2001a).

We sequenced these and additional strains of R. minuta and its relatives in the ITS5·8S rDNA region and D1/D2 of the 26S rDNA to clarify the phylogenetic relationships and species outlines within the R. minuta species complex. Three novel Rhodotorula species are described in this paper.

Sample collection and isolation.
Samples were collected from Iheya Ridge and Sagami Bay using both a manned and an unmanned submersible (see for example Takami et al., 1997; Nagahama et al., 2001a). Fourteen strains isolated from a clam and tubeworms and 13 strains of R. minuta already in the JCM culture collection were studied (Table 1).

Table 1. Rhodotorula strains examined in the ITS5·8S and D1/D2 regions of the rDNA Tubeworms sampled were of Lamellibrachia sp., and clams sampled were of Calyptogena sp. Sampling site 1 was Sagami Bay (35°00'N, 139°14'E) at a depth of 1156 m (unless indicated otherwise); sampling site 2 was Iheya Ridge (27°47'N, 126°53'E) at 1023 m. Representative strains used in analyses are in bold.

Physiological and biochemical characteristics.
Strains were characterized morphologically and physiologically by standard methods with some modifications (Yarrow, 1998). Assimilation of nitrogen compounds was examined on solid media using a starved inoculum (Nakase & Suzuki, 1986). Vitamin requirements were investigated according to the method of Komagata & Nakase (1967). Ubiquinones were extracted by the method of Yamada & Kondo (1973) with slight modifications and determined by HPLC as described previously (Hamamoto & Nakase, 1995).

Nucleic acid analyses.
DNA extraction and purification for analyses of DNA base composition and DNADNA hybridization were performed following the procedure described by Hamamoto & Nakase (1995) with slight modifications. The DNA base composition was determined by the HPLC method of Tamaoka & Komagata (1984). DNADNA hybridization experiments were performed using fixed-DNA microplates, following the procedures described by Ezaki et al. (1989).

Phylogenetic analysis.
DNA extraction for PCR was performed using the QIAamp DNeasy tissue kit (Qiagen), with some modifications (Nagahama et al., 2001b). The primers used for amplifying and sequencing the 5·8S rDNA and ITS regions were those described by White et al. (1990); the primers for the D1/D2 region of 26S rDNA were those described by Fell et al. (2000). PCR products were purified using Suprec-02 (Takara) and sequenced using a LI-COR model 4000L DNA sequencer. All sequences were aligned using CLUSTAL W 1.81 (Thompson et al., 1994) and adjusted manually. Positions where one or more species contained a length mutation or a region aligned ambiguously were not included in the subsequent phylogenetic analysis. Phylogenetic trees were constructed utilizing the maximum-likelihood (ML) method (Felsenstein, 1981) with the optimal T_s/T_v ratio of the HKY85 model estimated from the neighbour-joining tree in PAUP 4.0b8 (Swofford, 1998). This tree was derived by a heuristic search with random stepwise addition of 100 replicates. The robustness of branches in the tree was evaluated by bootstrap analysis (Felsenstein, 1985) with 100 resamplings.

Phenotypic characteristics of strains isolated from benthic animals
Fourteen strains isolated from benthic animals obviously belonged to the genus Rhodotorula. They were characterized by the presence of carotenoid pigments, no sexual cycle, no fermentation, no ballistospores, no starch-like substance and the absence of inositol utilization. Their assimilation of carbon and nitrogen compounds suggested affinities with R. minuta. The isolates contained Q-9 or -10 as the major ubiquinone isoprenologue.

Groupings based on nucleotide sequence similarity in the ITS1 and ITS2 regions of rDNA
ITS sequences were determined in 14 isolates and 13 additional strains preserved in the JCM culture collection and compared with five published sequences of strains related to the R. minuta species complex. Strains with identical sequences in the ITS regions and 5·8S rDNA were represented by an single sequence. They are represented by 13 single representatives (Table 1, in bold). Similarity values for ITS1 and ITS2 sequences among these single representatives are summarized in Table 2. In this study, we treated strains with greater than 99 % ITS region similarities as conspecific; these were JCM 3777^T with JCM 3781 and SY-90 and JCM 10954^T with JCM 3779 and JCM 8099. Strain SY-84 had more than 99 % similarity to JCM 10954^T, JCM 3779 and JCM 8099 in ITS1, but not in ITS2. Because similarity values between JCM 5951^T and JCM 8105 were borderline, they were treated separately. R. pallida JCM 3780^T showed exceptionally low similarities to others in both ITS regions.

Table 2. Similarity of ITS1 and ITS2 regions and DNA complementarity among strains of R. minuta and related species

DNA complementarity
We performed DNADNA hybridization experiments with representative strains (Table 2). The G+C contents of nuclear DNA in these strains had differences of less than 2 mol%. On the basis of similarities in ITS sequences, the strains were divided into two groups for simplicity and the relative DNA binding values were examined within each group (A and B in Table 2). It should be noted that JCM 10953 was substituted for CBS 2221^T due to a procedural problem in these experiments.

Strains SY-86^T and SY-91^T were most closely related to R. laryngis (approx. 15 % relative binding) and R. pallida (approx. 37 % relative binding), respectively. Values greater than 70 % between JCM 5951^T and JCM 8105 indicate that these strains should be recognized as a single species. The three species represented by SY-86^T, SY-91^T and JCM 5951^T were genetically distinct. Therefore, we describe these as novel species below. Strain SY-84 had considerable relatedness to JCM 10954^T, the type strain of R. slooffiae. These strains may be distinct, but we deferred the resolution of this problem because the relationship of R. slooffiae with SY-84 was less than that of other species.

Phylogenetic relationships among R. minuta and related species
The tree of D1/D2 regions, with an outgroup yeast occurring in the tubeworm, R. lamellibrachiae, reconstructed by means of the ML method, is shown in Fig. 1. Strain SY-91^T, R. laryngis and R. pallida were united with quite high statistical support by the bootstrap method, although the branching order within these three species was uncertain. The relationship between R. minuta and R. slooffiae had somewhat low bootstrap values. This topology contradicted a previously published one (Fell et al., 2000), in which R. minuta was primarily united with R. laryngis. This relatedness based on our tree was also supported by higher value (near 50 %) of DNA complementarity between the two type strains (Table 2) and the phylogenetic relationship based on the ITS5·8S rDNA regions with higher statistical support (data not shown).

(35K): Fig. 1. Phylogenetic tree of R. minuta and related species on the basis of a total of 644 aligned nucleotide sites in the D1/D2 region of the 26S rDNA. The maximum-likelihood tree on HKY85 model (Ts/Tv=2·52558) was constructed as described in the text. Numbers at branches are bootstrap values, derived only for the nodes supported by greater than 50 % (100 replicates). Bar, 0·01 substitutions per site.

Distinctive features other than molecular phylogeny among R. minuta-related species are few. Assimilation of lactose, maltose and sucrose was used previously as criteria to differentiate species within this complex (Novák & Vörös-Felkai, 1962; Galgóczy & Novák, 1965; Phaff & Ahearn, 1970), although these sugar assimilations could not entirely distinguish the three novel species and were occasionally unstable between a species and its synonyms (e.g. R. minuta and Rhodotorula texensis; R. laryngis and Rhodotorula zsoltii). Strain SY-86^T was characterized by having Q-9 as the major ubiquinone isoprenologue, while the other R. minuta-related species contained Q-10. Additional analysis of other molecular species may be required in order to produce a reliable phylogeny among species in the R. minuta complex.

Latin diagnosis of Rhodotorula benthica sp. nov. Nagahama, Hamamoto, Nakase & Horikoshi
In medio liquido YM post 3 dies ad 25 °C, cellulae ovoideae vel ellipsoidae (24x35 µm), singulae aut binae. Post unum mensem pellicula fragilis et incompleta, et sedimentum formantur. Cultura in agaro YM ad 25 °C, subrosea, nitida, mollis vel mucosa et margine glabra. Hyphae et pseudohyphae non formantur. Fermentatio nulla. Glucosum, galactosum (exiguum), L-sorbosum (exiguum), sucrosum, cellobiosum, trehalosum, lactosum, melezitosum, D-xylosum, L-arabinosum, ethanolum, glycerolum, ribitolum (exiguum), D-mannitolum (vel exiguum), D-glucitolum (vel exiguum), salicinum (varium), glucono-δ-lactonum, acidum 2-ketogluconicum, acidum 5-ketogluconicum, acidum DL-lacticum (vel exiguum), acidum succinicum et acidum D-glucuronicum (vel exiguum) assimilantur, at non maltosum, melibiosum, raffinosum, inulinum, amylum solubile, D-arabinosum (aut exiguum), D-ribosum, L-rhamnosum, erythritolum, galactitolum, methyl-α-D-glucosidum, acidum citricum, inositolum nec acidum D-galacturonicum. Non kalium nitricum, natrium nitrosum, ethylaminum, lysinum nec cadaverinum assimilantur. Maxima temperatura crescentiae: 4144 °C. Thiaminum necessaria ad crescentiam. G+C acidi deoxyribonucleati 47·2 mol% (per HPLC). Ubiquinonum majus Q-10.

Typus stirps SY-91^T ex verme tubuloso Lamellibrachia sp., Sagami Bay, Japonia, isolata est. In collectionibus culturarum quas Japan Collection of Microorganisms, Wako, Saitama sustentant, no. JCM 10901^T deposita est.

Description of Rhodotorula benthica sp. nov. Nagahama, Hamamoto, Nakase & Horikoshi
Rhodotorula benthica (ben'thi.ca. N.L. adj. benthica of the benthos, the flora and fauna of the deep sea).

In YM broth (Difco) after 3 days of culture at 25 °C, cells are ovoidal to ellipsoidal (24x35 µm) and occur singly or in parentbud pairs. A sediment and incomplete fragile pellicle are formed after 1 month. After 1 month on YM agar at 25 °C, the streak culture is light-pink in colour, glistening, soft to slimy and has an entire margin. In Dalmau plate cultures on cornmeal agar (Difco), no branching hyphae or pseudohyphae are formed. Fermentation ability is negative. The following carbon compounds are assimilated: D-glucose, galactose (weak), L-sorbose (weak), sucrose, cellobiose, trehalose, lactose, melezitose, D-xylose, L-arabinose, ethanol, glycerol, ribitol (weak), D-mannitol (or weak), D-glucitol (or weak), salicin (variable), glucono-δ-lactone, 2-ketogluconic acid, 5-ketogluconic acid, DL-lactic acid (or weak), succinic acid and D-glucuronic acid (or weak); no growth occurs on maltose, melibiose, raffinose, inulin, soluble starch, D-arabinose (or weak), D-ribose, L-rhamnose, erythritol, galactitol, methyl α-D-glucoside, citric acid, inositol or D-galacturonic acid. Nitrogen compound assimilation tests are negative for potassium nitrate, sodium nitrite, ethylamine, lysine and cadaverine. The maximum temperature for growth is 4144 °C. Thiamin is required for growth. No growth occurs on 50 % glucose/yeast extract agar. Growth occurs in the presence of 100 p.p.m. cycloheximide. Growth in the presence of 10 % sodium chloride is weak or negative. No starch-like substances are produced. The Diazonium blue B reaction is positive. Urease activity is positive. The major ubiquinone is Q-10. The G+C content of the nuclear DNA is 47·2 mol% (by HPLC).

The type strain, strain SY-91^T (=JCM 10901^T=CBS 9124^T), was isolated from a tubeworm, Lamellibrachia sp., collected from the deep-sea floor in Sagami Bay, Japan.

Latin diagnosis of Rhodotorula calyptogenae sp. nov. Nagahama, Hamamoto, Nakase & Horikoshi
In medio liquido YM post 3 dies ad 25 °C, cellulae ovoideae vel ellipsoidae (24x37 µm), singulae aut binae. Post unum mensem annulus tenuis et sedimentum formantur. Cultura in agaro YM ad 25 °C, subaurantiaca, nitida, mollis et margine glabra. Hyphae et pseudohyphae non formantur. Fermentatio nulla. Glucosum, galactosum (exiguum), L-sorbosum (exiguum), sucrosum, cellobiosum, trehalosum, lactosum (exiguum), melezitosum, D-xylosum, L-arabinosum (exiguum vel lente), ethanolum (lente), glycerolum, ribitolum (exiguum vel lente), D-mannitolum (vel exiguum), D-glucitolum (exiguum), salicinum (exiguum vel lente), glucono-δ-lactonum, acidum 2-ketogluconicum, acidum 5-ketogluconicum, acidum succinicum et acidum D-glucuronicum (vel lente) assimilantur, at non maltosum, melibiosum, raffinosum, inulinum, amylum solubile, D-arabinosum, D-ribosum, L-rhamnosum, erythritolum, galactitolum, methyl-α-D-glucosidum, acidum DL-lacticum, acidum citricum, inositolum nec acidum D-galacturonicum. Non kalium nitricum, natrium nitrosum, ethylaminum, lysinum nec cadaverinum assimilantur. Maxima temperatura crescentiae: 4144 °C. Thiaminum necessaria ad crescentiam. G+C acidi deoxyribonucleati 48·0 mol% (per HPLC). Ubiquinonum majus Q-9.

Typus stirps SY-86^T ex Calyptogena sp., Sagami Bay, Japonia, isolata est. In collectionibus culturarum quas Japan Collection of Microorganisms, Wako, Saitama sustentant, no. JCM 10899^T deposita est.

Description of Rhodotorula calyptogenae sp. nov. Nagahama, Hamamoto, Nakase & Horikoshi
Rhodotorula calyptogenae (ca.ly'p.to.ge.na.e. N.L. fem. gen. n. calyptogenae of Calyptogena, a genus of giant white clam, from which the organism was isolated).

In YM broth (Difco) after 3 days of culture at 25 °C, the cells are ovoidal to ellipsoidal (24x37 µm) and occur singly or in parentbud pairs. A sediment and thin ring are formed after 1 month. On YM agar after 1 month at 25 °C, the streak culture is light-orange in colour, glistening, soft and has an entire margin. In Dalmau plate cultures on cornmeal agar (Difco), no branching hyphae or pseudohyphae are formed. Fermentation ability is negative. The following carbon compounds are assimilated: D-glucose, galactose (weak), L-sorbose (weak), sucrose, cellobiose, trehalose, lactose (weak), melezitose, D-xylose, L-arabinose (weak or delayed), ethanol (delayed), glycerol, ribitol (weak or delayed), D-mannitol (or weak), D-glucitol (weak), salicin (weak or delayed), glucono-δ-lactone, 2-ketogluconic acid, 5-ketogluconic acid, succinic acid and D-glucuronic acid (or delayed); no growth occurs on maltose, melibiose, raffinose, inulin, soluble starch, D-arabinose, D-ribose, L-rhamnose, erythritol, galactitol, methyl α-D-glucoside, DL-lactic acid, citric acid, inositol or D-galacturonic acid. No assimilation of the nitrogen compounds potassium nitrate, sodium nitrite, ethylamine, lysine and cadaverine occurs. The maximum temperature for growth is 4144 °C. Thiamin is required for growth. No growth occurs on 50 % glucose/yeast extract agar. Growth occurs in the presence of 100 p.p.m. cycloheximide. Growth in the presence of 10 % sodium chloride is weak. No starch-like substances are produced. The Diazonium blue B reaction is positive. Urease activity is positive. The major ubiquinone is Q-9. The G+C content of the nuclear DNA is 48·0 mol% (by HPLC).

The type strain, strain SY-86^T (=JCM 10899^T=CBS 9125^T), was isolated from a giant white clam, Calyptogena sp., collected from the deep-sea floor in Sagami Bay, Japan.

Latin diagnosis of Rhodotorula lysiniphila sp. nov. Nagahama, Hamamoto, Nakase & Horikoshi
In medio liquido YM post 3 dies ad 25 °C, cellulae ovoideae vel ellipsoidae (24x36 µm), singulae aut binae. Post unum mensem annulus tenuis et sedimentum formantur. Cultura in agaro YM ad 25 °C, subrosea vel aurantiaca, nitida, mollis, et margine glabra. Hyphae et pseudohyphae non formantur. Fermentatio nulla. Glucosum, galactosum (exiguum), L-sorbosum (exiguum), sucrosum, cellobiosum, trehalosum, lactosum, melezitosum, D-xylosum, L-arabinosum, D-arabinosum (vel exiguum), ethanolum, glycerolum, ribitolum (exiguum), D-mannitolum, D-glucitolum (exiguum), salicinum (exiguum), glucono-δ-lactonum, acidum 2-ketogluconicum, acidum 5-ketogluconicum, acidum DL-lacticum (exiguum) et acidum succinicum assimilantur, at non maltosum, melibiosum, raffinosum, inulinum, amylum solubile, D-ribosum, L-rhamnosum, erythritolum, galactitolum, methyl-α-D-glucosidum, acidum citricum, inositolum, acidum D-glucuronicum nec acidum D-galacturonicum. Lysinum assimilantur at non kalium nitricum, natrium nitrosum, ethylaminum, nec cadaverinum. Maxima temperatura crescentiae: 3134 °C. Thiaminum necessaria ad crescentiam. G+C acidi deoxyribonucleati 49·1 mol% (per HPLC). Ubiquinonum majus Q-10.

In collectionibus culturarum quas Japan Collection of Microorganisms, Wako, Saitama sustentant, no. JCM 5951^T deposita est.

Description of Rhodotorula lysiniphila sp. nov. Nagahama, Hamamoto, Nakase & Horikoshi
Rhodotorula lysiniphila (ly'si.ni.phi.la. N.L. n. lysina lysine; Gr. adj. philos liking, preferring; N.L. adj. lysiniphila referring to the utilization of lysine as a nitrogen compound).

In YM broth (Difco) after 3 days at 25 °C, the cells are ovoidal to ellipsoidal (24x36 µm) and occur singly or in parentbud pairs. A sediment and thin ring are formed after 1 month. On YM agar after 1 month at 25 °C, the streak culture is light-pink to orange in colour, glistening, soft and has an entire margin. In Dalmau plate cultures on cornmeal agar (Difco), no branching hyphae or pseudohyphae are formed. Fermentation ability is negative. The following carbon compounds are assimilated: D-glucose, galactose (weak), L-sorbose (weak), sucrose, cellobiose, trehalose, lactose, melezitose, D-xylose, L-arabinose, D-arabinose (or weak), ethanol, glycerol, ribitol (weak), D-mannitol, D-glucitol (weak), salicin (weak), glucono-δ-lactone, 2-ketogluconic acid, 5-ketogluconic acid, DL-lactic acid (weak) and succinic acid; no growth occurs on maltose, melibiose, raffinose, inulin, soluble starch, D-ribose, L-rhamnose, erythritol, galactitol, methyl α-D-glucoside, citric acid, inositol, D-glucuronic acid or D-galacturonic acid. Lysine is assimilated. No growth occurs on potassium nitrate, sodium nitrite, ethylamine or cadaverine. The maximum temperature for growth is 3134 °C. Thiamin is required for growth. No growth occurs on 50 % glucose/yeast extract agar. Growth occurs in the presence of 100 p.p.m. cycloheximide. Growth in the presence of 10 % sodium chloride is negative. No starch-like substances are produced. The Diazonium blue B reaction is positive. Urease activity is positive. The major ubiquinone is Q-10. The G+C content of the nuclear DNA is 49·1 mol% (by HPLC).

The type strain is strain JCM 5951^T (=CBS 9126^T).

We thank Dr Y. Nogi for his useful suggestions on technical problems. We are indebted to Dr D. Honda for his skilful advice on phylogenetic analysis using PAUP* 4.0.

Footnotes

†Present address: Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.