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Sporobolomyces odoratus sp. nov., a new species in the Sporidiobolus ruineniae clade
FEMS Yeast Research 2 (2002) 9^16
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Sporobolomyces odoratus sp. nov., a new species in the Sporidiobolus ruineniae clade Elisabete Vale¤rio, Ma¤rio Gadanho, Jose¤ Paulo Sampaio * Centro de Recursos Microbiolo¤gicos, Secc a‹o Auto¤noma de Biotecnologia, Faculdade de Cie“ncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal Received 6 June 2001; received in revised form 4 October 2001; accepted 8 November 2001 First published online 7 December 2001
Abstract This report presents the description of a new Sporobolomyces species, Sp. odoratus sp. nov. The new species was characterized by DNA fingerprinting using the micro/minisatellite-primed PCR approach (MSP-PCR). A phylogenetic analysis using the D1/D2 region of the 26S rDNA revealed that Sp. odoratus belongs to a clade that includes Rhodosporidium fluviale and Sporidiobolus ruineniae. An integrated comparison with related species is presented. A number of presumptive strains of S. ruineniae were also investigated using the MSP-PCR method and 26S rDNA sequence analysis. Mating experiments revealed, for the first time, that S. ruineniae includes heterothallic strains, besides those already known to be self-fertile. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Basidiomycetous yeast; Taxonomy ; Sporobolomyces odoratus; Sporidiobolus ruineniae
1. Introduction The anamorphic basidiomycetous genus Sporobolomyces Kluyver and van Niel accommodates species that produce bilaterally symmetrical ballistoconidia and have CoQ 10 or CoQ 10(H2) as their major ubiquinones. The typical attributes of Sporobolomyces include incapacity to utilize inositol and to produce amyloid compounds. In addition, xylose is not present in whole-cell hydrolysates. The asexual genus Rhodotorula Harrison shares with Sporobolomyces all the characteristics mentioned above, except the capacity to produce ballistoconidia. The polyphyletic nature of Sporobolomyces was revealed by sequence analysis of 18S rDNA [1^3] and of the D1/D2 region of the 26S rDNA [4,5]. Within the Urediniomycetes, species like Sporobolomyces roseus Kluyver p van Niel are related to the teleomorphic genus Sporidiobolus Nyland, whereas species like Sp. oryzicola Nakase p Suzuki and S. elongatus Shivas and Rodrigues de Miranda are a⁄liated to the Naohidea^Sakaguchia clade, and species such as Sp. xanthus (Nakase, Okada p Sugiyama)
* Corresponding author. Tel. : +351 (21) 2948300; Fax: +351 (21) 2948530. E-mail address: [email protected] (J.P. Sampaio).
Boekhout and Sp. lactophilus Nakase, Itoh, Suzuki p Bandoni belong to the Agaricostilbum^Kondoa clade. The type species of Sporobolomyces is Sporidiobolus salmonicolor (Fischer p Brebeck) Kluyver p van Niel and represents the anamorphic stage of S. salmonicolor Fell p Tallman. It is possible that, in the future, Sporobolomyces will be restricted to taxa related to the type species, in order to decrease its present heterogeneity. However, any taxonomic change has to take into account the proximity between Sporidiobolus (and related Sporobolomyces species) and Rhodosporidium (and related Rhodotorula species). In fact, sequence analysis of 26S rDNA has indicated a close relationship between Sporidiobolus ruineniae Holzschu, Tredick p Pha¡, Sporidiobolus microsporus Higham ex Fell, Blatt p Statzell-Tallman, Rhodosporidium azoricum Sampaio p Gadanho, Rhodosporidium £uviale Fell, Kurtzman, Tallman p Buck and R. lusitaniae Fonseca and Sampaio [6,7]. These ¢ve species belong to the £uviale^ruineniae group, one of the three clades in which the species of the genera Sporidiobolus and Rhodosporidium, together with their close asexual relatives, are divided [7]. Recently, Takashima and Nakase [8] described Sporobolomyces nylandii Takashima p Nakase and Sporobolomyces poonsookiae Takashima p Nakase, two anamorphic species that belong to the £uviale^ruineniae clade. This report presents the description of an additional Sporobo-
1567-1356 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 1 ) 0 0 0 5 4 - X
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Table 1 List of strains used in MSP-PCR ¢ngerprinting Strain
Rhodosporidium azoricum Sampaio p Gadanho R. azoricum R. £uviale Fell, Kurtzman, Tallman p Buck R. lusitaniae Fonseca p Sampaio R. lusitaniae R. lusitaniae S. microsporus Higham ex Fell, Blatt p Statzell-Tallman S. ruineniae Holzschu, Tredick p Pha¡ var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. coprophilus Kurtzman p Fell S. ruineniae var. coprophilus Sp. nylandii Takashima p Nakase Sp. odoratus Sampaio, Fonseca p Vale¤rio Sp. odoratus
IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC
Sp. odoratus
IGC 5696
PDA contamination, Monte de Caparica, Portugal
Sp. odoratus Sp. odoratus Sp. poonsookiae Takashima p Nakase
IGC 5697 IGC 5698 IGC 5699T
Leaf of PlatanusUacerifolia, Monte de Caparica, Portugal Leaf of lemon tree, Algueira‹o, Portugal Dead leaf of Mangifera indica, Thailand
5062T 5084 4701T 4641T 4599 4642 5617T 4201T 4211 5676 5677 5692 5660T 5596 5693T 5694T 5695
Strain origin
CBS 6568T
CBS CBS CBS CBS CBS
7041T 5001T 5000 5004 5078
CBS 5811T CBS 5624 JCM 10213T ZP 351
JCM 10207T
Isolation source
Observations
Soil, Azores, Portugal Soil, Azores, Portugal Brackish water, USA Woodland soil, Lisbon, Portugal Dry leaf, Arra¤bida, Portugal Plant litter, Arra¤bida, Portugal Herbaceous culm, Jamaica Leaf of Malpighia coccigera, Indonesia Leaf of M. coccigera, Indonesia Unknown substrate, Indonesiaa Unknown substrate, Indonesiaa Garden soil, Taiwan Dung of goat, Pakistan Air, Japanb Dead leaf of Oryza sativa, Thailand Basidiocarp of Myxarium nucleatum, Sesimbra, Portugal Leaf of Acer monspessulanum, Arra¤bida, Portugal
Mating type Mating type Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Mating type Anamorphic Mating type
A1 A2
A1 A2
Absence of ballistoconidia Isolated by J.P. Sampaio, December 1996 Isolated by A. Fonseca and J. Ina¤cio, November 1998 Isolated by J.P. Sampaio and E. Vale¤rio, November 1999 Isolated by A. Fonseca, May 2000 Isolated by A. Fonseca, October 1986
IGC, Portuguese Yeast Culture Collection, FCT-UNL, Portugal; CBS, Centraalbureau voor Schimmelcultures, Yeast Division, Utrecht, The Netherlands ; JCM, Japan Collection of Microorganisms, RIKEN, Japan; ZP, personal collection of J.P. Sampaio. a Isolated by J. Ruinen and deposited at CBS as S. roseus. b Type strain of Pichia rosea; maintained at CBS as Rhodotorula graminis.
E. Vale¤rio et al. / FEMS Yeast Research 2 (2002) 9^16
FEMSYR 1443 3-5-02
Species
E. Vale¤rio et al. / FEMS Yeast Research 2 (2002) 9^16
lomyces species in this clade, Sp. odoratus sp. nov. A comparison with closely related taxa is presented.
2. Materials and methods 2.1. Isolations, morphological characterization and studies of sexual compatibility For the isolations, the ballistoconidium-fall method was employed. For microscopy, cultures were grown on MYP agar (malt extract 0.7% w/v, yeast extract 0.05% w/v, soytone 0.25% w/v and agar 1.5% w/v) at room temperature (20^23‡C) and studied with an Olympus BX50 microscope, using phase contrast optics. For determination of sexual compatibility, pairs of 2^4-day-old cultures were crossed on SG agar (soytone 0.2% w/v, glucose 0.2% w/v and agar 1.5% w/v), incubated at room temperature and examined for production of mycelium and teliospores after 1 week. 2.2. Physiological characterization Physiological and biochemical characterization was performed according to the techniques described by Yarrow [9]. Additional assimilation tests were performed using aldaric acids and aromatic compounds, as described by Fonseca [10] and Sampaio [11], respectively.
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3. Results and discussion Between 1986 and 2000, ¢ve strains of an anamorphic pink-colored ballistoconidial yeast were isolated from several localities in Portugal (Table 1). All the isolates were light pink, dull, exhaled a peculiar odor and produced large quantities of forcibly discharged propagules. The latter characteristic resulted in the formation of numerous small colonies that covered the entire surface of the culture medium. In addition, ballistoconidia were frequently produced at the end of long stalks. All strains were unable to utilize melezitose and to grow at 35‡C. This combination of physiological traits occurs in some species of the £uviale^ruineniae clade, such as S. microsporus, S. ruineniae and R. lusitaniae. Therefore, the isolates were initially compared with the species of this group, regardless of their ability to produce ballistoconidia. The indication that the ¢ve strains were conspeci¢c and distinct from the remaining species of the £uviale^ruineniae clade was obtained in DNA ¢ngerprinting experiments using MSP-PCR. This investigation was conducted using the primers M13 and (GTG)5 and the banding patterns obtained are depicted in Fig. 1. It can be observed that the pro¢les of the various strains of Sp. odoratus are similar, but clearly di¡erent from those of the remaining species in the £uviale^ruineniae clade. In order to determine the phy-
2.3. PCR ¢ngerprinting and rDNA sequence analysis For PCR ¢ngerprinting, the DNA extraction protocol, primers, PCR and electrophoresis conditions and gel image analysis procedures were those reported by Sampaio et al. [12]. A total of 21 strains representing the various species of the £uviale^ruineniae clade were investigated using the micro/minisatellite-primed PCR approach (MSPPCR). The list of cultures used in this study, and information related to them, is presented in Table 1. For rDNA sequence analysis, total DNA was extracted using the protocol of Sampaio et al. [12] and ampli¢ed using primers ITS5 and LR6. Cycle sequencing of the 600^650-bp region at the 5P end of the 26S rDNA D1/D2 domain employed forward primer 5P-GCA TAT CAA TAA GCG GAG GAA AAG and reverse primer 5P-TCC TCC GCT TAT TGA TAT GC. Sequences were obtained with an Amersham Pharmacia ALF Express II automated sequencer using standard protocols. Alignments were made with MegAlign (Dnastar Inc., Madison, WI, USA) and visually corrected. Phylogenetic trees were computed with PAUP version 4.0 using the neighbor-joining method [13]. Distances between sequences were calculated using Kimura’s two-parameter model [14]. Bootstrap analysis [15] was based on 1000 random resamplings.
Fig. 1. MSP-PCR ¢ngerprints of Sp. odoratus and remaining species of the £uviale^ruineniae clade generated with primers M13 (a) and (GTG)5 (b). M, molecular size marker (V DNA cleaved with HindIII and xX174 DNA cleaved with HaeIII).
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logenetic position of the new species, the nucleotide sequence of the D1/D2 region of the 26S rDNA was determined (Fig. 2). Sequence analysis con¢rmed that Sp. odoratus represents a distinct species and that it belongs to the £uviale^ruineniae clade. Currently, the £uviale^ruineniae clade comprises three species of Sporobolomyces and presents two particularities.
It includes species that are able to produce ballistoconidia together with species that do not present this trait. This issue has been addressed by Fell et al. [6] and, more recently, by Gadanho et al. [7]. Secondly, the basidia of some sexual species (R. lusitaniae, S. ruineniae and S. microsporus) are formed at the end of a stalk [7], which does not occur in any of the species belonging to the other two
Fig. 2. Phylogenetic tree of S. odoratus and related taxa of the genera Sporobolomyces, Rhodotorula, Sporidiobolus and Rhodosporidium, obtained by neighbor-joining analysis of the D1/D2 region of the 26S rDNA. Rhodotorula minuta and Saccharomyces cerevisiae were included to root the tree. Percentage bootstrap values of 1000 replicates are given at each node. GenBank accession numbers are indicated after species designation (names in boldface correspond to sequences determined in this study; T, type strain).
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Table 2 Physiological characteristics of the strains of Sp. odoratus Carbon compounds
IGC 5694T
IGC 5695
IGC 5696
IGC 5697
IGC 5698
D-Glucose
+ 3 + 3 3 + D D 3 + 3 + 3 3 + + 3 3 + 3 3 3 + 3 + D + + 3 3 + DW 3 3 + 3 3 3 3 3 3 3 3 + + 3 3 + 3 + 3 3 3 3 3
+ + + 3 3 + D D 3 + 3 + 3 3 + + 3 3 + 3 3 3 D 3 + D + + 3 3 + + 3 3 + 3 + 3 3 3 3 3 3 + + 3 3 + 3 + 3 3 3 3 3
+ + + 3 3 + 3 3 3 + 3 + 3 3 3 + 3 3 + 3 3 3 D 3 + 3 + + 3 3 DW D 3 3 + 3 D 3 3 3 3 3 3 + D 3 3 + 3 + 3 3 3 3 3
+ + + 3 3 + D 3 3 + 3 + 3 3 + + 3 3 + 3 3 3 D 3 + D + + 3 3 + + 3 3 + 3 + 3 3 3 3 3 3 + + 3 3 + 3 + 3 3 3 3 3
+ 3 + 3 3 + D 3 3 + 3 + 3 3 3 + 3 3 + 3 3 3 + 3 + 3 + W 3 3 + W 3 3 DW 3 3 3 3 3 3 3 3 + 3 3 3 + 3 D 3 3 3 3 3
+ + 3 3 3
+ + 3 3 3
+ + 3 3 3
+ + 3 3 3
+ + 3 3 3
D-Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose K,K-Trehalose Methyl-K-D-glucoside Cellobiose Salicin Arbutin Melibiose Lactose Ra⁄nose Melezitose Inulin Soluble starch Glycerol Erythritol Ribitol Xylitol D-Glucitol D-Mannitol Galactitol Inositol Glucono-N-lactone D-Gluconic acid D-Glucuronic acid D,L-Lactic acid Succinic acid Citric acid L-Malic acid L-Tartaric acid D-Tartaric acid m-Tartaric acid Saccharic acid Mucic acid Methanol Ethanol Vanillic acid Veratric acid Ferulic acid p-Hydroxybenzoic acid m-Hydroxybenzoic acid Protocatechuic acid Catechol Gallic acid Salicylic acid Gentisic acid Phenol Nitrogen compounds Nitrate Nitrite Ethylamine L-Lysine Cadaverin
D, delayed; W, weak results.
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Table 2 (continued). Carbon compounds
IGC 5694T
IGC 5695
IGC 5696
IGC 5697
IGC 5698
Creatine Creatinine Other tests Growth in vitamin-free medium Growth with 0.01% cycloheximide Growth with 0.1% cycloheximide Growth at 30‡C Growth at 35‡C Formation of starch-like compounds Splitting of arbutin Hydrolysis of urea Colour reaction with diazonium blue B
3 3
3 3
3 3
3 3
3 3
+ 3 3 3 3 3 + + +
+ 3 3 3 3 3 + + +
+ 3 3 + 3 3 + + +
+ 3 3 3 3 3 + + +
+ 3 3 3 3 3 + + +
D, delayed; W, weak results.
are discrepant between Sp. odoratus and R. lusitaniae. In contrast to R. lusitaniae, the new species utilizes sucrose, ra⁄nose and does not grow with galactitol, ferulic acid, p-hydroxybenzoic acid, gallic acid and catechol. Since the MSP-PCR approach is well suited to characterize large sets of isolates, we also investigated a group of strains suspected to belong to S. ruineniae (IGC 5596, IGC 5676, IGC 5677 and IGC 5692). Three strains, IGC 5676, IGC 5677 and IGC 5692, showed DNA banding pro¢les similar to IGC 4201T and IGC 4211, the two con¢rmed representatives of S. ruineniae var. ruineniae, whereas the patterns of IGC 5596 were comparable to those of IGC 5660T , the single known strain of S. ruineniae var. coprophilus Kurtzman p Fell (Fig. 1). Among the variety rui-
clades (toruloides^glutinis and johnsonii^pararoseus). The phylogenetic tree in Fig. 2 indicates that the closest relative of Sp. odoratus is Sp. nylandii. The two species have 11 di¡erent bases in the D1/D2 region of the 26S rDNA. S. poonsookiae, the third anamorphic species in this clade, has 25 di¡erent bases towards Sp. odoratus. The physiological and biochemical properties of Sp. odoratus are depicted in Table 2 and a comparison with other ballistoconidia-producing taxa is presented in Table 3. The closest relatives of Sp. odoratus are Sp. nylandii, Sp. poonsookiae and R. lusitaniae (Fig. 2). The new species does not utilize D-ribose, cellobiose and catechol and is not able to grow in the presence of 0.1% cycloheximide, in contrast to Sp. nylandii and Sp. poonsookiae. Several physiological tests
Table 3 Salient physiological di¡erences between Sp. odoratus and related ballistoconidia-producing taxa
Cluster 1 toruloides^glutinis Sp. alborubescens Derx (CBS 482T ) Cluster 2 £uviale^ruineniae S. ruineniae Holzschu, Tredick p Pha¡ (CBS 5001T , CBS 5000) S. microsporus Higham ex Fell, Blatt p Statzell-Tallman (CBS 7041T ) Sp. nylandii Takashima p Nakase (IGC 5693T ) Sp. odoratus Sampaio, Fonseca p Vale¤rio (IGC 5694T , IGC 5695, IGC 5696, IGC 5697, IGC 5698) Sp. poonsookiae Takashima p Nakase (IGC 5699T ) Cluster 3 johnsonii^pararoseus Sp. roseus Kluyver p van Niel (JCM 5353LT ) Sp. marcillae Santa Mar|¤a (CBS 4217T ) Sp. ruberrimus Yamazaki p Fujii (CBS 7500AUT , CBS 7501) S. pararoseus Fell p Tallman (CBS 491T , CBS 484, CBS 4216, IGC 4622) S. salmonicolor Fell p Tallman (CBS 490T , CBS 2630, CBS 2643, IGC 4351, IGC 4558, IGC 4623) S. johnsonii Nyland (CBS 5470T , IGC 4353) Sp. holsaticus Windish ex Yarrow p Fell (CBS 1522T )
Malt.
Ribo.
Melz.
Cellob.
Starch
30‡C
35‡C
0.1% Cx.
AmH. Cat.
+
+
+
3
3
+
+
+
+
3
+
+
3
+
3
+
3
+
+
+
3
+
3
+
3
+
3
+
+
+
+ 3
+ 3
+ 3
+ 3
3* 3
+ V
+ 3
+ 3
3 3
+ 3
3
+
3
+
3*
+
3
+
+
+
+ + +
+ + 3
+ + +
+ + V
+ + +
3 3 3
3 3 3
3 3 3
3 + 3
3 3 3
+
3
+
+
+
+
3
3
+
3
V
V
V
V
V
+
+
+
V
+
+ +
V +
V +
+ +
+ 3
+ +
+ +
+ +
3 3
+ +
Results: (+) positive (including delayed and weak results), (3) negative and (V) variable. Data are from the present study and from the Portuguese Yeast Culture Collection database and correspond to the strains indicated between brackets ; * positive results in the original description [8]. Abbreviations: Malt., Maltose ; Ribo., D-Ribose; Melz., Melezitose ; Cellob., Cellobiose ; 0.1% Cx., growth in the presence of 0.1% cycloheximide; AmH., m-hydroxybenzoic acid; Cat., catechol.
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neniae, two strains (IGC 5676 and IGC 5677), previously identi¢ed as Sp. roseus [16], produced numerous ballistoconidia and strain IGC 5692 produced ballistoconidia very rarely. With respect to the variety coprophilus, IGC 5596 did not form ballistoconidia similarly to IGC 5660T , the type strain of this variety. The strain IGC 5596 was originally described as the type of Pichia rosea Nishiwaki and was received from the CBS (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) culture collection as Rhodotorula graminis di Menna. Interestingly, all the seven isolates of S. ruineniae were collected in Asia (Indonesia, Taiwan, Japan and Pakistan) and, although we have recently studied large sets of ballistoconidial yeasts from Portugal using the MSP-PCR approach, none of them belonged to S. ruineniae. Since these four new members of S. ruineniae were not self-fertile, as is the case for the previously characterized strains of this species [17], we investigated their mating compatibility in crossing experiments. From the six pairings inoculated on SG agar, mycelium and teliospores were observed in the crossing of IGC 5676 and IGC 5692. Besides incomplete clamp connections, true clamps were also observed. The teliospores originated from transversally septate basidia similar to those already observed in the homothallic strains. These results indicate that S. ruineniae includes both homothallic and heterothallic strains, a situation that was not reported previously for this species. Description of a sexual yeast species based solely on homothallic isolates followed, later, by the unravelling of heterothallic strains based on molecular typing methods occurred previously for Rhodosporidium kratochvilovae Hamamoto, Sugiyama p Komagata [12]. The examples of S. ruineniae and R. kratochvilovae show the importance of molecular typing in the discovery of new anamorph^teleomorph connections. Moreover, our ¢ndings also demonstrate the relevance of population-focussed studies rather than type strain-oriented investigations. We designate IGC 5676 as the mating type A1 and IGC 5692 as the mating type A2 of S. ruineniae var. ruineniae. The other two strains (IGC 5677 and IGC 5596) did not exhibit any sign of sexual behavior and are presently considered as anamorphic strains of this species. To con¢rm our identi¢cations, we determined the D1/D2 sequences of the two mating strains and also that of IGC 5596. No di¡erences were detected in comparison with the published sequences of S. ruineniae (Fig. 2). With respect to conventional phenotypic criteria, the L-rhamnose test and the mode of teliospore germination can be used to distinguish the two varieties of S. ruineniae [18]. We were able to con¢rm growth with this carbon compound for the strains of the variety ruineniae (weak results for IGC 5677, delayed for IGC 5692 and vigorous for IGC 5676, 4201 and 4211) and absence of growth for the two strains of the variety coprophilus. The reported heterogeneity in teliospore germination could not be investigated because none of the strains of the variety coprophilus produced teliospores.
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3.1. Latin diagnosis of Sporobolomyces odoratus Sampaio, Fonseca p Vale¤rio sp. nov. Post 2 dies in MYP agar cellulae ovoideae ad cylindraceae (2) 3^5U5^8 (9) Wm. Ballistoconidia ovoidea ad reniformia, 3^4U6^10 Wm, ex sterigmatibus longis (2^ 3U10^70 Wm) abundanter oriunda. Mycelium verum non formatur. Post unum mensem ad 20^22‡C cultura in striis subrosea, hebes, super¢cie laevi vel ex parte rugosa, textura butyracea. Assimilatio maltosi, D-ribosi, melezitosi, cellobiosi deest, nullum incrementum ad 35‡C. Cultura odore peculiari. 3.2. Description of Sporobolomyces odoratus Sampaio, Fonseca p Vale¤rio sp. nov. Yeast cells after 2 days on MYP agar ovoid to cylindrical measuring (2) 3^5U5^8 (9) Wm (Figs. 3 and 4). Ballistoconidia abundantly produced, ovoid to reniform, measuring 3^4U6^10 Wm and originating at the end of usually long (2^3U10^70 Wm) sterigmata (Figs. 3 and 4). True mycelium is not formed. After 1 month at 20^22‡C, the streak culture is pale-pink, dull, the surface is smooth or slightly wrinkled and the texture is butyrous. A salient feature is the production of a peculiar odor. Unable to ferment glucose. Additional physiological and biochemical characteristics are depicted in Table 2.
Fig. 3. Line drawings of yeast cells, sterigmata and ballistoconidia of Sp. odoratus grown on MYP agar at room temperature for 2^3 days. a: Cell with long sterigma and immature ballistoconidium (IGC 5697) ; b: budding yeast cells and formation of ballistoconidia (IGC 5694T ); c: released ballistoconidia of IGC 5694T and IGC 5697. Bar = 10 Wm.
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References
Fig. 4. Micrographs of ballistoconidia-forming cells and ballistoconidia of Sp. odoratus grown on MYP agar at room temperature for 2^3 days. a: IGC 5697; b: IGC 5694T . Bar = 10 Wm.
3.2.1. Etymology The speci¢c epithet odoratus ( = fragrant) refers to the peculiar odor of the cultures of this species. 3.2.2. Origin, type and deposits IGC 5694 was isolated by J.P. Sampaio from a basidiocarp of Myxarium nucleatum Wallroth collected at Quinta dos Pinheiros, Sesimbra, 30 km south of Lisbon, Portugal, in December 1996. This strain was deposited lyophilized (holotype) and living (ex-type) in the Portuguese Yeast Culture Collection, Caparica, Portugal. Information on the origin of other strains of this species is presented in Table 1.
Acknowledgements M.G. was supported by a Grant SFRH/BD/1170/2000. The authors are indebted to Dr. Chee-Jen Chen (Southern Taiwan University of Technology, Taiwan) for sending a strain (IGC 5692) of S. ruineniae, to Dr. M. Hamamoto (Japan Collection of Microorganisms, Japan) for sending the type strains of Sp. nylandii and Sp. poonsookiae, to Prof. Isabel Spencer-Martins for critical reading of the manuscript and to Dr. Michael WeiM (University of Tu«bingen, Germany) for preparing the Latin diagnosis.
[1] Nakase, T., Takematsu, A. and Yamada, Y. (1993) Molecular approaches to the taxonomy of ballistosporous yeasts based on the analysis of the partial nucleotide sequences of 18S ribosomal ribonucleic acids. J. Gen. Appl. Microbiol. 39, 107^134. [2] Nakase, T., Suh, O. and Hamamoto, M. (1995) Molecular systematics of ballistoconidium-forming yeasts. Stud. Mycol. 38, 163^173. [3] Hamamoto, M. and Nakase, T. (2000) Phylogenetic analysis of the ballistoconidium-forming yeast genus Sporobolomyces based on 18S rDNA sequences. Int. J. Syst. Evol. Microbiol. 50, 1373^1380. [4] Fell, J.W., Boekhout, T. and Freshwater, D.W. (1995) The role of nucleotide sequence analysis in the systematics of the yeast genera Cryptococcus and Rhodotorula. Stud. Mycol. 38, 129^146. [5] Fell, J.W., Boekhout, T., Fonseca, A., Scorzetti, G. and Statzell-Tallman, A. (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int. J. Syst. Evol. Microbiol. 50, 1351^1371. [6] Fell, J.W., Blatt, G.M. and Statzell-Tallman, A. (1998) Validation of the basidiomycetous yeast, Sporidiobolus microsporus sp nov., based on phenotypic and molecular analyses. Antonie van Leeuwenhoek 74, 265^270. [7] Gadanho, M., Sampaio, J.P. and Spencer-Martins, I. (2001) Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium : R. azoricum sp. nov.. Can. J. Microbiol. 47, 213^221. [8] Takashima, M. and Nakase, T. (2000) Four new species of the genus Sporobolomyces isolated from leaves in Thailand. Mycoscience 41, 357^369. [9] Yarrow, D. (1998) Methods for the isolation, maintenance and identi¢cation of yeasts. In: The Yeasts, a Taxonomic Study, 4th edn. (Kurtzman, C.P. and Fell, J.W., Eds.), pp. 77^100. Elsevier Science, Amsterdam. [10] Fonseca, A. (1992) Utilization of tartaric acid and related compounds by yeasts: taxonomic implications. Can. J. Microbiol. 38, 1242^1251. [11] Sampaio, J.P. (1999) Utilization of low molecular weight aromatic compounds by heterobasidiomycetous yeasts: taxonomic implications. Can. J. Microbiol. 45, 491^512. [12] Sampaio, J.P., Gadanho, M., Santos, S., Duarte, F., Pais, C., Fonseca, A. and Fell, J.W. (2001) Polyphasic taxonomy of the genus Rhodosporidium : R. kratochvilovae and related anamorphic species. Int. J. Syst. Evol. Microbiol. 51, 687^697. [13] Saitou, N. and Nei, M. (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406^425. [14] Kimura, M. (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111^120. [15] Felsenstein, J. (1985) Con¢dence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783^791. [16] Boekhout, T. (1991) A revision of ballistoconidia-forming yeasts and fungi. Stud. Mycol. 33, 1^194. [17] Statzell-Tallman, A. and Fell, J.W. (1998) Sporidiobolus Nyland. In: The Yeasts, a Taxonomic Study, 4th edn. (Kurtzman, C.P. and Fell, J.W., Eds.), pp. 693^699. Elsevier Science, Amsterdam. [18] Kurtzman, C.P. and Fell, J.W. (1991) Molecular relatedness between the basidiomycetous yeasts Sporidiobolus ruinenii and Sporobolomyces coprophilus. Mycologia 83, 107^110.
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Sporobolomyces odoratus sp. nov., a new species in the Sporidiobolus ruineniae clade Elisabete Vale¤rio, Ma¤rio Gadanho, Jose¤ Paulo Sampaio * Centro de Recursos Microbiolo¤gicos, Secc a‹o Auto¤noma de Biotecnologia, Faculdade de Cie“ncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal Received 6 June 2001; received in revised form 4 October 2001; accepted 8 November 2001 First published online 7 December 2001
Abstract This report presents the description of a new Sporobolomyces species, Sp. odoratus sp. nov. The new species was characterized by DNA fingerprinting using the micro/minisatellite-primed PCR approach (MSP-PCR). A phylogenetic analysis using the D1/D2 region of the 26S rDNA revealed that Sp. odoratus belongs to a clade that includes Rhodosporidium fluviale and Sporidiobolus ruineniae. An integrated comparison with related species is presented. A number of presumptive strains of S. ruineniae were also investigated using the MSP-PCR method and 26S rDNA sequence analysis. Mating experiments revealed, for the first time, that S. ruineniae includes heterothallic strains, besides those already known to be self-fertile. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Basidiomycetous yeast; Taxonomy ; Sporobolomyces odoratus; Sporidiobolus ruineniae
1. Introduction The anamorphic basidiomycetous genus Sporobolomyces Kluyver and van Niel accommodates species that produce bilaterally symmetrical ballistoconidia and have CoQ 10 or CoQ 10(H2) as their major ubiquinones. The typical attributes of Sporobolomyces include incapacity to utilize inositol and to produce amyloid compounds. In addition, xylose is not present in whole-cell hydrolysates. The asexual genus Rhodotorula Harrison shares with Sporobolomyces all the characteristics mentioned above, except the capacity to produce ballistoconidia. The polyphyletic nature of Sporobolomyces was revealed by sequence analysis of 18S rDNA [1^3] and of the D1/D2 region of the 26S rDNA [4,5]. Within the Urediniomycetes, species like Sporobolomyces roseus Kluyver p van Niel are related to the teleomorphic genus Sporidiobolus Nyland, whereas species like Sp. oryzicola Nakase p Suzuki and S. elongatus Shivas and Rodrigues de Miranda are a⁄liated to the Naohidea^Sakaguchia clade, and species such as Sp. xanthus (Nakase, Okada p Sugiyama)
* Corresponding author. Tel. : +351 (21) 2948300; Fax: +351 (21) 2948530. E-mail address: [email protected] (J.P. Sampaio).
Boekhout and Sp. lactophilus Nakase, Itoh, Suzuki p Bandoni belong to the Agaricostilbum^Kondoa clade. The type species of Sporobolomyces is Sporidiobolus salmonicolor (Fischer p Brebeck) Kluyver p van Niel and represents the anamorphic stage of S. salmonicolor Fell p Tallman. It is possible that, in the future, Sporobolomyces will be restricted to taxa related to the type species, in order to decrease its present heterogeneity. However, any taxonomic change has to take into account the proximity between Sporidiobolus (and related Sporobolomyces species) and Rhodosporidium (and related Rhodotorula species). In fact, sequence analysis of 26S rDNA has indicated a close relationship between Sporidiobolus ruineniae Holzschu, Tredick p Pha¡, Sporidiobolus microsporus Higham ex Fell, Blatt p Statzell-Tallman, Rhodosporidium azoricum Sampaio p Gadanho, Rhodosporidium £uviale Fell, Kurtzman, Tallman p Buck and R. lusitaniae Fonseca and Sampaio [6,7]. These ¢ve species belong to the £uviale^ruineniae group, one of the three clades in which the species of the genera Sporidiobolus and Rhodosporidium, together with their close asexual relatives, are divided [7]. Recently, Takashima and Nakase [8] described Sporobolomyces nylandii Takashima p Nakase and Sporobolomyces poonsookiae Takashima p Nakase, two anamorphic species that belong to the £uviale^ruineniae clade. This report presents the description of an additional Sporobo-
1567-1356 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 1 ) 0 0 0 5 4 - X
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Table 1 List of strains used in MSP-PCR ¢ngerprinting Strain
Rhodosporidium azoricum Sampaio p Gadanho R. azoricum R. £uviale Fell, Kurtzman, Tallman p Buck R. lusitaniae Fonseca p Sampaio R. lusitaniae R. lusitaniae S. microsporus Higham ex Fell, Blatt p Statzell-Tallman S. ruineniae Holzschu, Tredick p Pha¡ var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. ruineniae S. ruineniae var. coprophilus Kurtzman p Fell S. ruineniae var. coprophilus Sp. nylandii Takashima p Nakase Sp. odoratus Sampaio, Fonseca p Vale¤rio Sp. odoratus
IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC IGC
Sp. odoratus
IGC 5696
PDA contamination, Monte de Caparica, Portugal
Sp. odoratus Sp. odoratus Sp. poonsookiae Takashima p Nakase
IGC 5697 IGC 5698 IGC 5699T
Leaf of PlatanusUacerifolia, Monte de Caparica, Portugal Leaf of lemon tree, Algueira‹o, Portugal Dead leaf of Mangifera indica, Thailand
5062T 5084 4701T 4641T 4599 4642 5617T 4201T 4211 5676 5677 5692 5660T 5596 5693T 5694T 5695
Strain origin
CBS 6568T
CBS CBS CBS CBS CBS
7041T 5001T 5000 5004 5078
CBS 5811T CBS 5624 JCM 10213T ZP 351
JCM 10207T
Isolation source
Observations
Soil, Azores, Portugal Soil, Azores, Portugal Brackish water, USA Woodland soil, Lisbon, Portugal Dry leaf, Arra¤bida, Portugal Plant litter, Arra¤bida, Portugal Herbaceous culm, Jamaica Leaf of Malpighia coccigera, Indonesia Leaf of M. coccigera, Indonesia Unknown substrate, Indonesiaa Unknown substrate, Indonesiaa Garden soil, Taiwan Dung of goat, Pakistan Air, Japanb Dead leaf of Oryza sativa, Thailand Basidiocarp of Myxarium nucleatum, Sesimbra, Portugal Leaf of Acer monspessulanum, Arra¤bida, Portugal
Mating type Mating type Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Self-fertile Mating type Anamorphic Mating type
A1 A2
A1 A2
Absence of ballistoconidia Isolated by J.P. Sampaio, December 1996 Isolated by A. Fonseca and J. Ina¤cio, November 1998 Isolated by J.P. Sampaio and E. Vale¤rio, November 1999 Isolated by A. Fonseca, May 2000 Isolated by A. Fonseca, October 1986
IGC, Portuguese Yeast Culture Collection, FCT-UNL, Portugal; CBS, Centraalbureau voor Schimmelcultures, Yeast Division, Utrecht, The Netherlands ; JCM, Japan Collection of Microorganisms, RIKEN, Japan; ZP, personal collection of J.P. Sampaio. a Isolated by J. Ruinen and deposited at CBS as S. roseus. b Type strain of Pichia rosea; maintained at CBS as Rhodotorula graminis.
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Species
E. Vale¤rio et al. / FEMS Yeast Research 2 (2002) 9^16
lomyces species in this clade, Sp. odoratus sp. nov. A comparison with closely related taxa is presented.
2. Materials and methods 2.1. Isolations, morphological characterization and studies of sexual compatibility For the isolations, the ballistoconidium-fall method was employed. For microscopy, cultures were grown on MYP agar (malt extract 0.7% w/v, yeast extract 0.05% w/v, soytone 0.25% w/v and agar 1.5% w/v) at room temperature (20^23‡C) and studied with an Olympus BX50 microscope, using phase contrast optics. For determination of sexual compatibility, pairs of 2^4-day-old cultures were crossed on SG agar (soytone 0.2% w/v, glucose 0.2% w/v and agar 1.5% w/v), incubated at room temperature and examined for production of mycelium and teliospores after 1 week. 2.2. Physiological characterization Physiological and biochemical characterization was performed according to the techniques described by Yarrow [9]. Additional assimilation tests were performed using aldaric acids and aromatic compounds, as described by Fonseca [10] and Sampaio [11], respectively.
11
3. Results and discussion Between 1986 and 2000, ¢ve strains of an anamorphic pink-colored ballistoconidial yeast were isolated from several localities in Portugal (Table 1). All the isolates were light pink, dull, exhaled a peculiar odor and produced large quantities of forcibly discharged propagules. The latter characteristic resulted in the formation of numerous small colonies that covered the entire surface of the culture medium. In addition, ballistoconidia were frequently produced at the end of long stalks. All strains were unable to utilize melezitose and to grow at 35‡C. This combination of physiological traits occurs in some species of the £uviale^ruineniae clade, such as S. microsporus, S. ruineniae and R. lusitaniae. Therefore, the isolates were initially compared with the species of this group, regardless of their ability to produce ballistoconidia. The indication that the ¢ve strains were conspeci¢c and distinct from the remaining species of the £uviale^ruineniae clade was obtained in DNA ¢ngerprinting experiments using MSP-PCR. This investigation was conducted using the primers M13 and (GTG)5 and the banding patterns obtained are depicted in Fig. 1. It can be observed that the pro¢les of the various strains of Sp. odoratus are similar, but clearly di¡erent from those of the remaining species in the £uviale^ruineniae clade. In order to determine the phy-
2.3. PCR ¢ngerprinting and rDNA sequence analysis For PCR ¢ngerprinting, the DNA extraction protocol, primers, PCR and electrophoresis conditions and gel image analysis procedures were those reported by Sampaio et al. [12]. A total of 21 strains representing the various species of the £uviale^ruineniae clade were investigated using the micro/minisatellite-primed PCR approach (MSPPCR). The list of cultures used in this study, and information related to them, is presented in Table 1. For rDNA sequence analysis, total DNA was extracted using the protocol of Sampaio et al. [12] and ampli¢ed using primers ITS5 and LR6. Cycle sequencing of the 600^650-bp region at the 5P end of the 26S rDNA D1/D2 domain employed forward primer 5P-GCA TAT CAA TAA GCG GAG GAA AAG and reverse primer 5P-TCC TCC GCT TAT TGA TAT GC. Sequences were obtained with an Amersham Pharmacia ALF Express II automated sequencer using standard protocols. Alignments were made with MegAlign (Dnastar Inc., Madison, WI, USA) and visually corrected. Phylogenetic trees were computed with PAUP version 4.0 using the neighbor-joining method [13]. Distances between sequences were calculated using Kimura’s two-parameter model [14]. Bootstrap analysis [15] was based on 1000 random resamplings.
Fig. 1. MSP-PCR ¢ngerprints of Sp. odoratus and remaining species of the £uviale^ruineniae clade generated with primers M13 (a) and (GTG)5 (b). M, molecular size marker (V DNA cleaved with HindIII and xX174 DNA cleaved with HaeIII).
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E. Vale¤rio et al. / FEMS Yeast Research 2 (2002) 9^16
logenetic position of the new species, the nucleotide sequence of the D1/D2 region of the 26S rDNA was determined (Fig. 2). Sequence analysis con¢rmed that Sp. odoratus represents a distinct species and that it belongs to the £uviale^ruineniae clade. Currently, the £uviale^ruineniae clade comprises three species of Sporobolomyces and presents two particularities.
It includes species that are able to produce ballistoconidia together with species that do not present this trait. This issue has been addressed by Fell et al. [6] and, more recently, by Gadanho et al. [7]. Secondly, the basidia of some sexual species (R. lusitaniae, S. ruineniae and S. microsporus) are formed at the end of a stalk [7], which does not occur in any of the species belonging to the other two
Fig. 2. Phylogenetic tree of S. odoratus and related taxa of the genera Sporobolomyces, Rhodotorula, Sporidiobolus and Rhodosporidium, obtained by neighbor-joining analysis of the D1/D2 region of the 26S rDNA. Rhodotorula minuta and Saccharomyces cerevisiae were included to root the tree. Percentage bootstrap values of 1000 replicates are given at each node. GenBank accession numbers are indicated after species designation (names in boldface correspond to sequences determined in this study; T, type strain).
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13
Table 2 Physiological characteristics of the strains of Sp. odoratus Carbon compounds
IGC 5694T
IGC 5695
IGC 5696
IGC 5697
IGC 5698
D-Glucose
+ 3 + 3 3 + D D 3 + 3 + 3 3 + + 3 3 + 3 3 3 + 3 + D + + 3 3 + DW 3 3 + 3 3 3 3 3 3 3 3 + + 3 3 + 3 + 3 3 3 3 3
+ + + 3 3 + D D 3 + 3 + 3 3 + + 3 3 + 3 3 3 D 3 + D + + 3 3 + + 3 3 + 3 + 3 3 3 3 3 3 + + 3 3 + 3 + 3 3 3 3 3
+ + + 3 3 + 3 3 3 + 3 + 3 3 3 + 3 3 + 3 3 3 D 3 + 3 + + 3 3 DW D 3 3 + 3 D 3 3 3 3 3 3 + D 3 3 + 3 + 3 3 3 3 3
+ + + 3 3 + D 3 3 + 3 + 3 3 + + 3 3 + 3 3 3 D 3 + D + + 3 3 + + 3 3 + 3 + 3 3 3 3 3 3 + + 3 3 + 3 + 3 3 3 3 3
+ 3 + 3 3 + D 3 3 + 3 + 3 3 3 + 3 3 + 3 3 3 + 3 + 3 + W 3 3 + W 3 3 DW 3 3 3 3 3 3 3 3 + 3 3 3 + 3 D 3 3 3 3 3
+ + 3 3 3
+ + 3 3 3
+ + 3 3 3
+ + 3 3 3
+ + 3 3 3
D-Galactose L-Sorbose D-Glucosamine D-Ribose D-Xylose L-Arabinose D-Arabinose L-Rhamnose Sucrose Maltose K,K-Trehalose Methyl-K-D-glucoside Cellobiose Salicin Arbutin Melibiose Lactose Ra⁄nose Melezitose Inulin Soluble starch Glycerol Erythritol Ribitol Xylitol D-Glucitol D-Mannitol Galactitol Inositol Glucono-N-lactone D-Gluconic acid D-Glucuronic acid D,L-Lactic acid Succinic acid Citric acid L-Malic acid L-Tartaric acid D-Tartaric acid m-Tartaric acid Saccharic acid Mucic acid Methanol Ethanol Vanillic acid Veratric acid Ferulic acid p-Hydroxybenzoic acid m-Hydroxybenzoic acid Protocatechuic acid Catechol Gallic acid Salicylic acid Gentisic acid Phenol Nitrogen compounds Nitrate Nitrite Ethylamine L-Lysine Cadaverin
D, delayed; W, weak results.
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Table 2 (continued). Carbon compounds
IGC 5694T
IGC 5695
IGC 5696
IGC 5697
IGC 5698
Creatine Creatinine Other tests Growth in vitamin-free medium Growth with 0.01% cycloheximide Growth with 0.1% cycloheximide Growth at 30‡C Growth at 35‡C Formation of starch-like compounds Splitting of arbutin Hydrolysis of urea Colour reaction with diazonium blue B
3 3
3 3
3 3
3 3
3 3
+ 3 3 3 3 3 + + +
+ 3 3 3 3 3 + + +
+ 3 3 + 3 3 + + +
+ 3 3 3 3 3 + + +
+ 3 3 3 3 3 + + +
D, delayed; W, weak results.
are discrepant between Sp. odoratus and R. lusitaniae. In contrast to R. lusitaniae, the new species utilizes sucrose, ra⁄nose and does not grow with galactitol, ferulic acid, p-hydroxybenzoic acid, gallic acid and catechol. Since the MSP-PCR approach is well suited to characterize large sets of isolates, we also investigated a group of strains suspected to belong to S. ruineniae (IGC 5596, IGC 5676, IGC 5677 and IGC 5692). Three strains, IGC 5676, IGC 5677 and IGC 5692, showed DNA banding pro¢les similar to IGC 4201T and IGC 4211, the two con¢rmed representatives of S. ruineniae var. ruineniae, whereas the patterns of IGC 5596 were comparable to those of IGC 5660T , the single known strain of S. ruineniae var. coprophilus Kurtzman p Fell (Fig. 1). Among the variety rui-
clades (toruloides^glutinis and johnsonii^pararoseus). The phylogenetic tree in Fig. 2 indicates that the closest relative of Sp. odoratus is Sp. nylandii. The two species have 11 di¡erent bases in the D1/D2 region of the 26S rDNA. S. poonsookiae, the third anamorphic species in this clade, has 25 di¡erent bases towards Sp. odoratus. The physiological and biochemical properties of Sp. odoratus are depicted in Table 2 and a comparison with other ballistoconidia-producing taxa is presented in Table 3. The closest relatives of Sp. odoratus are Sp. nylandii, Sp. poonsookiae and R. lusitaniae (Fig. 2). The new species does not utilize D-ribose, cellobiose and catechol and is not able to grow in the presence of 0.1% cycloheximide, in contrast to Sp. nylandii and Sp. poonsookiae. Several physiological tests
Table 3 Salient physiological di¡erences between Sp. odoratus and related ballistoconidia-producing taxa
Cluster 1 toruloides^glutinis Sp. alborubescens Derx (CBS 482T ) Cluster 2 £uviale^ruineniae S. ruineniae Holzschu, Tredick p Pha¡ (CBS 5001T , CBS 5000) S. microsporus Higham ex Fell, Blatt p Statzell-Tallman (CBS 7041T ) Sp. nylandii Takashima p Nakase (IGC 5693T ) Sp. odoratus Sampaio, Fonseca p Vale¤rio (IGC 5694T , IGC 5695, IGC 5696, IGC 5697, IGC 5698) Sp. poonsookiae Takashima p Nakase (IGC 5699T ) Cluster 3 johnsonii^pararoseus Sp. roseus Kluyver p van Niel (JCM 5353LT ) Sp. marcillae Santa Mar|¤a (CBS 4217T ) Sp. ruberrimus Yamazaki p Fujii (CBS 7500AUT , CBS 7501) S. pararoseus Fell p Tallman (CBS 491T , CBS 484, CBS 4216, IGC 4622) S. salmonicolor Fell p Tallman (CBS 490T , CBS 2630, CBS 2643, IGC 4351, IGC 4558, IGC 4623) S. johnsonii Nyland (CBS 5470T , IGC 4353) Sp. holsaticus Windish ex Yarrow p Fell (CBS 1522T )
Malt.
Ribo.
Melz.
Cellob.
Starch
30‡C
35‡C
0.1% Cx.
AmH. Cat.
+
+
+
3
3
+
+
+
+
3
+
+
3
+
3
+
3
+
+
+
3
+
3
+
3
+
3
+
+
+
+ 3
+ 3
+ 3
+ 3
3* 3
+ V
+ 3
+ 3
3 3
+ 3
3
+
3
+
3*
+
3
+
+
+
+ + +
+ + 3
+ + +
+ + V
+ + +
3 3 3
3 3 3
3 3 3
3 + 3
3 3 3
+
3
+
+
+
+
3
3
+
3
V
V
V
V
V
+
+
+
V
+
+ +
V +
V +
+ +
+ 3
+ +
+ +
+ +
3 3
+ +
Results: (+) positive (including delayed and weak results), (3) negative and (V) variable. Data are from the present study and from the Portuguese Yeast Culture Collection database and correspond to the strains indicated between brackets ; * positive results in the original description [8]. Abbreviations: Malt., Maltose ; Ribo., D-Ribose; Melz., Melezitose ; Cellob., Cellobiose ; 0.1% Cx., growth in the presence of 0.1% cycloheximide; AmH., m-hydroxybenzoic acid; Cat., catechol.
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neniae, two strains (IGC 5676 and IGC 5677), previously identi¢ed as Sp. roseus [16], produced numerous ballistoconidia and strain IGC 5692 produced ballistoconidia very rarely. With respect to the variety coprophilus, IGC 5596 did not form ballistoconidia similarly to IGC 5660T , the type strain of this variety. The strain IGC 5596 was originally described as the type of Pichia rosea Nishiwaki and was received from the CBS (Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands) culture collection as Rhodotorula graminis di Menna. Interestingly, all the seven isolates of S. ruineniae were collected in Asia (Indonesia, Taiwan, Japan and Pakistan) and, although we have recently studied large sets of ballistoconidial yeasts from Portugal using the MSP-PCR approach, none of them belonged to S. ruineniae. Since these four new members of S. ruineniae were not self-fertile, as is the case for the previously characterized strains of this species [17], we investigated their mating compatibility in crossing experiments. From the six pairings inoculated on SG agar, mycelium and teliospores were observed in the crossing of IGC 5676 and IGC 5692. Besides incomplete clamp connections, true clamps were also observed. The teliospores originated from transversally septate basidia similar to those already observed in the homothallic strains. These results indicate that S. ruineniae includes both homothallic and heterothallic strains, a situation that was not reported previously for this species. Description of a sexual yeast species based solely on homothallic isolates followed, later, by the unravelling of heterothallic strains based on molecular typing methods occurred previously for Rhodosporidium kratochvilovae Hamamoto, Sugiyama p Komagata [12]. The examples of S. ruineniae and R. kratochvilovae show the importance of molecular typing in the discovery of new anamorph^teleomorph connections. Moreover, our ¢ndings also demonstrate the relevance of population-focussed studies rather than type strain-oriented investigations. We designate IGC 5676 as the mating type A1 and IGC 5692 as the mating type A2 of S. ruineniae var. ruineniae. The other two strains (IGC 5677 and IGC 5596) did not exhibit any sign of sexual behavior and are presently considered as anamorphic strains of this species. To con¢rm our identi¢cations, we determined the D1/D2 sequences of the two mating strains and also that of IGC 5596. No di¡erences were detected in comparison with the published sequences of S. ruineniae (Fig. 2). With respect to conventional phenotypic criteria, the L-rhamnose test and the mode of teliospore germination can be used to distinguish the two varieties of S. ruineniae [18]. We were able to con¢rm growth with this carbon compound for the strains of the variety ruineniae (weak results for IGC 5677, delayed for IGC 5692 and vigorous for IGC 5676, 4201 and 4211) and absence of growth for the two strains of the variety coprophilus. The reported heterogeneity in teliospore germination could not be investigated because none of the strains of the variety coprophilus produced teliospores.
15
3.1. Latin diagnosis of Sporobolomyces odoratus Sampaio, Fonseca p Vale¤rio sp. nov. Post 2 dies in MYP agar cellulae ovoideae ad cylindraceae (2) 3^5U5^8 (9) Wm. Ballistoconidia ovoidea ad reniformia, 3^4U6^10 Wm, ex sterigmatibus longis (2^ 3U10^70 Wm) abundanter oriunda. Mycelium verum non formatur. Post unum mensem ad 20^22‡C cultura in striis subrosea, hebes, super¢cie laevi vel ex parte rugosa, textura butyracea. Assimilatio maltosi, D-ribosi, melezitosi, cellobiosi deest, nullum incrementum ad 35‡C. Cultura odore peculiari. 3.2. Description of Sporobolomyces odoratus Sampaio, Fonseca p Vale¤rio sp. nov. Yeast cells after 2 days on MYP agar ovoid to cylindrical measuring (2) 3^5U5^8 (9) Wm (Figs. 3 and 4). Ballistoconidia abundantly produced, ovoid to reniform, measuring 3^4U6^10 Wm and originating at the end of usually long (2^3U10^70 Wm) sterigmata (Figs. 3 and 4). True mycelium is not formed. After 1 month at 20^22‡C, the streak culture is pale-pink, dull, the surface is smooth or slightly wrinkled and the texture is butyrous. A salient feature is the production of a peculiar odor. Unable to ferment glucose. Additional physiological and biochemical characteristics are depicted in Table 2.
Fig. 3. Line drawings of yeast cells, sterigmata and ballistoconidia of Sp. odoratus grown on MYP agar at room temperature for 2^3 days. a: Cell with long sterigma and immature ballistoconidium (IGC 5697) ; b: budding yeast cells and formation of ballistoconidia (IGC 5694T ); c: released ballistoconidia of IGC 5694T and IGC 5697. Bar = 10 Wm.
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References
Fig. 4. Micrographs of ballistoconidia-forming cells and ballistoconidia of Sp. odoratus grown on MYP agar at room temperature for 2^3 days. a: IGC 5697; b: IGC 5694T . Bar = 10 Wm.
3.2.1. Etymology The speci¢c epithet odoratus ( = fragrant) refers to the peculiar odor of the cultures of this species. 3.2.2. Origin, type and deposits IGC 5694 was isolated by J.P. Sampaio from a basidiocarp of Myxarium nucleatum Wallroth collected at Quinta dos Pinheiros, Sesimbra, 30 km south of Lisbon, Portugal, in December 1996. This strain was deposited lyophilized (holotype) and living (ex-type) in the Portuguese Yeast Culture Collection, Caparica, Portugal. Information on the origin of other strains of this species is presented in Table 1.
Acknowledgements M.G. was supported by a Grant SFRH/BD/1170/2000. The authors are indebted to Dr. Chee-Jen Chen (Southern Taiwan University of Technology, Taiwan) for sending a strain (IGC 5692) of S. ruineniae, to Dr. M. Hamamoto (Japan Collection of Microorganisms, Japan) for sending the type strains of Sp. nylandii and Sp. poonsookiae, to Prof. Isabel Spencer-Martins for critical reading of the manuscript and to Dr. Michael WeiM (University of Tu«bingen, Germany) for preparing the Latin diagnosis.
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