Metschnikowia vanudenii sp. nov. and Metschnikowia lachancei sp. nov., from flowers and associated insects in North America

Metschnikowia species often thrive in insectflower ecosystems where they proliferate owing to the high sugar concentration of the nectar. The sugar composition of floral nectar varies among plant species, but shows a predominance of hexoses (glucose and fructose) and the disaccharide sucrose. Several recently described Metschnikowia species (Hong et al., 2001; Kurtzman & Droby, 2001; Lachance et al., 2001a, b; Lachance & Bowles, 2002) add to the abundance of flower-associated species over those of aquatic origin, suggesting that the proliferation of terrestrial flowering plants and associated insects has markedly impacted genetic diversification in Metschnikowia. The large number of nucleotide substitutions among Metschnikowia species in the D1/D2 domain of large subunit rDNA, especially among the large-spored species, further supports the idea that the genus is highly diverged and that there are numerous undetected species.

In the present study, two novel species of Metschnikowia are proposed. These species, to be named Metschnikowia vanudenii and Metschnikowia lachancei, were isolated in North America from the common milkweed Asclepias syriaca and associated insects. M. vanudenii shares with Metschnikowia gruessii the formation of aeroplane cells, i.e. superimposed pairs of cells that form a cross-like configuration. The second species, M. lachancei, is characterized by clavate ascospores, which differentiates it from all other recognized species of Metschnikowia.

Yeast strains.
The five known strains of M. vanudenii were obtained from North America. The type strain of M. vanudenii (PYCC 4650^T) was isolated by M. A. Lachance, in 1986, from a walk plate by allowing a muscoid fly collected from the common milkweed Asclepias syriaca in Guelph, Ontario, Canada, to walk over the solidified medium (Lachance et al., 2001a). The origin of the remaining four strains (PYCC 4603, PYCC 4606, PYCC 4648 and PYCC 4649) was nectar from flowers of A. syriaca sampled in the province of Québec, Canada. The type strain of M. lachancei (PYCC 4605^T) was isolated in 1987 from nectar of the flower A. syriaca in South Bombay, NY, USA. W. T. Starmer recovered a second strain of this species (PYCC 5767) from a flower of Calystegia sepium (hedge bindweed), in Newfound Gap, Smoky Mountains National Park, TN, USA. All strains examined in this work have been deposited (living and dried) in the Portuguese Yeast Culture Collection (PYCC), Monte de Caparica, Portugal, as well as in other collections, and accession numbers are given in Table 1.

Table 1. Origin and DNA base composition of the Metschnikowia strains examined

Phenotypic characterization.
The morphological and physiological characteristics of the strains were determined by conventional techniques described by Yarrow (1998). Sporulation was induced using the conditions recommended by Pitt & Miller (1968).

DNA base composition and DNADNA reassociations.
For thermal denaturation and reassociation experiments, DNA was purified by chromatography on hydroxylapatite columns following the protocol of Britten et al. (1970). The mean nuclear DNA (nDNA) G+C content from Metschnikowia strains was determined by thermal denaturation (T_m) (Marmur & Doty, 1962) using a Gilford Response II Spectrophotometer and its thermal programming software. The G+C content was determined at least twice for each strain in 0·1x SSC (Owen et al., 1969). The extent of DNA relatedness was determined spectrophotometrically as described by Seidler & Mandel (1971) and modified by Kurtzman et al. (1980), using a Gilford Response II Spectrophotometer and its thermal kinetics program.

rDNA sequencing and sequence analysis.
Methods for nDNA isolation, amplification by PCR of the 26S rDNA domain D1/D2 and sequencing with the ABI TaqDyeDeoxy Terminator Cycle Sequencing kit and the ABI model 377 automated DNA sequencer (PE Biosystems) were previously described (Kurtzman & Robnett, 1998). Sequence data were visually aligned with QEDIT 2.15 (SemWare, Marietta, GA, USA). Phylogenetic relationships were calculated with the maximum-parsimony program of PAUP*4.0b3a (Swofford, 1998), as well as with the neighbour-joining program using the Kimura two-parameter distance measure. Schizosaccharomyces pombe was the designated outgroup species in all analyses. Confidence limits for phylogenetic trees were estimated from bootstrap analysis (1000 replications). The nucleotide sequences for the new species reported here, as well as for reference species, have been deposited with GenBank under the accession numbers given in Fig. 3.

(65K): Fig. 3. Neighbour-joining tree with the Kimura two-parameter distance correction showing phylogenetic placement of M. vanudenii and M. lachancei among species of the genus Metschnikowia, related Candida species and reference species Saccharomyces cerevisiae and Schizosaccharomyces pombe (outgroup species in the analysis). Bootstrap values (1000 replications) are given for nodes with >50 % support. T, Type strain; NT, neotype strain. Culture collection prefixes: Y and YB, ARS Culture Collection (NRRL); CBS, Centraalbureau voor Schimmelcultures; KCTC, Korean Collection for Type Cultures.

Latin diagnosis of Metschnikowia vanudenii Giménez-Jurado, Kurtzman & Spencer-Martins sp. nov.
In medio liquido cum dextroso et peptono et extracto levedinis post dies 3 ad 25 °C cellulae sunt ovoideae aut cylindrateae (35x79 µm), binae vel in catenis brevis. Chlamydosporae praesentes plerumque, globosus (68x810 µm) refractivae. Reproductio vegetativae per gemmatae multipolarem et cellulae cruciformis formatur. Post dies 7 ad 25 °C neque pseudohyphae nec hyphae formatur, sedimentum abundus. Cultura in medio agaro cum dextroso et peptono et extracto levedinis post dies 7 ad 25 °C albida vel cremea butyrosa cum margine glabro. In agaro malti post dies 7 ad temperatura ambeunte, chlamydosporae globosus refractivae abundus et cellulae conjugationem tubi junctioniz formatur. In agaro farinae Zea mays post dies 7 ad 25 °C pseudohyphae rudimentari formatur. Asci in agaro V8 dilutus post dies 12 ad 17 °C ellipsoidopedunculati (68x2545 µm), pedunculi cylindraci, 2-spori. Ascosporae aciculares vel filiformes. Fermentatio glucosum. Assimilat glucosum, sucrosum, maltosum, L-sorbosum, cellobiosum (variabile), α,α-trehalosum (variabile), melezitosum, amylum solubile (variabile), D-xylosum (variabile), ethanolum (variabile), glycerolum (variabile), ribitolum (variabile), D-mannitolum (variabile), D-sorbitolum, salicinum (variabile), arbutinum, D-glucosaminum, glucono-δ-lactonum (variabile), D-glutamatum (variabile), N-acetyl-glucosaminum (variabile) et 2-ketogluconatum ad non D-galactosum, lactosum, melibiosum, raffinosum, inulinum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, erythritolum, D-glucitolum, methyl-α-D-glucosidum, acidum lacticum, acidum succinicum, acidum citricum, myo-inositolum, methanolum, acidum glucoronicum, acidum gluconicum, acidum tartaricum et acidum malicum. Assimilat L-lysini, ethylamini, cadaverini, D-glucosaminum (variabile), betanini, D-glutaminum ad non kallii nitratis, sodii nitrosi, creatino nec creatinino. Vitaminae externae ad crescentiam necessariae sunt. Crescit in 30 °C sed non in 35 °C. Materia amyloidea non formatur. Ureum non finditur. Ubiquinonum majus Q-9. Proportio molaris guanini plus cytosini in acidi deoxyribonucleati 44·4 mol%.

Typus: PYCC 4650^T, ex flore, exsicatus et vivus in collectione zymotica Centraalbureau voor Schimmelcultures (CBS 9134^T), Trajectum ad Rhenum, praeservatus.

Description of Metschnikowia vanudenii Giménez-Jurado, Kurtzman & Spencer-Martins sp. nov.
Growth in glucose (2 %) yeast extract (0·5 %) peptone (1 %) broth.
After 3 days at 25 °C, vegetative cells are ovoid to cylindrical (35x79 µm) and occur mostly in pairs, tetrads or in short chains. Some cells develop into characteristic aeroplane or cross formations. Chlamydospores, when present, are globose to subglobose in shape (68x810 µm), highly refractile and contain several spherical lipid globules. Some strains produce few or no chlamydospores. No pseudohyphae or hyphae were observed in this medium. Cell division occurs by multilateral budding. After 1 week at 25 °C, abundant sediment is formed.

Growth on glucose (2 %) yeast extract (0·5 %) peptone (1 %) agar.
After 1 week at room temperature, strains form white to cream, smooth to papillate, glossy, butyrous colonies with entire margins.

Growth on malt extract agar.
After 1 week at room temperature, strains form copious amounts of subglobose chlamydospores with large, easily seen oil droplets. Cross or aeroplane configurations are present in strains PYCC 4606, PYCC 4648 and PYCC 4650^T.

Dalmau plate cultures on corn-meal agar.
After 1 week at 25 °C, growth under semi-anaerobic conditions showed development of primitive pseudohyphae.

Formation of ascospores.
Asci with ascospores were formed by PYCC 4650^T after incubation for 12 days at 17 °C on dilute (1 : 15) V8 agar (Fig. 1). Asci initially developed from ellipsoid chlamydospores, which further elongated resulting in a cylindrical peduncle shape. Mature asci (68x2545 µm) contain two acicular to filiform ascospores, which are noticeably pointed at one end. Most asci are persistent, although a few become deliquescent.

(147K): Fig. 1. Phase-contrast photomicrographs of M. vanudenii PYCC 4650T grown on dilute V8 agar at 17 °C. Mature ascus containing two filiform ascospores. Scale bar, 10 µm.

Ascosporulation was not observed for strains PYCC 4603, PYCC 4606, PYCC 4648 and PYCC 4649. These four strains were mixed pairwise in all possible combinations with just one pair, PYCC 4648xPYCC 4649, showing evidence of mating. Conjugated cells and apparent zygotes were observed, but ascospores were not seen. Additionally, when this pair of strains was mixed, the frequency of cross-like cells markedly increased over that seen for the individual strains. The observation of conjugants suggests that M. vanudenii might be heterothallic with haploid strains showing varying degrees of mating competence.

Physiological and biochemical characteristics
See Table 2.

Table 2. Physiological and biochemical characteristics that differentiate M. lachancei sp. nov. from M. vanudenii sp. nov. Species: M. lachancei strains PYCC 4605T and PYCC 5767; M. vanudenii strains PYCC 4650T, PYCC 4603, PYCC 4606, PYCC 4648 and PYCC 4649. All strains tested positive for glucose fermentation. Glucose, sucrose, maltose, arbutin, melezitose and xylitol were assimilated by all strains, but D-ribose, L-arabinose, D-arabinose, L-rhamnose, methyl-α-D-glucoside, melibiose, lactose, raffinose, inulin, starch, m-erythritol, galactitol, m-inositol, D-glucuronic, DL-lactic acid, citric acid, malic acid, L-tartaric acid and methanol were not. Ethylamine, cadaverine and betaine, as sole nitrogen sources, were assimilated by all strains, but nitrate, nitrite, creatine and creatinine were not. All strains were able to grow in vitamin-free medium, 50 % glucose, 10 % NaCl/5 % glucose and at 30 °C, but were not able to grow in YNB with 0·01 % cycloheximide and at 35 °C. None formed starch-like compounds. Abbreviations: W, weak; V, variable; +, positive; -, negative.

Etymology.
The specific epithet vanudenii, Latin gen. of van Uden, was chosen in honour of the late Professor Nicolau van Uden for his important contributions to the study of the yeast genus Metschnikowia as well as for his work on the physiology and biochemistry of yeasts.

Origin and deposits.
The type strain PYCC 4650^T [UWO(PS) 86AW4.1] was isolated from a walk plate sampling of a muscoid fly collected from the common milkweed Asclepias syriaca in Guelph, Ontario, Canada. This strain has been deposited (living and dried) in the Portuguese Yeast Culture Collection (PYCC), Centro de Recursos Microbiológicos (CREM), Universidade Nova de Lisboa, Caparica, Portugal, and under number CBS 9134 in the Centraalbureau voor Schimmelcultures in Utrecht, The Netherlands.

Latin diagnosis of Metschnikowia lachancei Giménez-Jurado, Kurtzman, Starmer & Spencer-Martins sp. nov.
In medio liquido cum dextroso et peptono et extracto levedinis post dies 3 ad 25 °C cellulae sunt ellipsoideae aut cylindrateae (37x83 µm), singulae, binae vel aggregatae, gemmationem multipolarem reproducentes. Breves pseudohyphae formatur. Chlamydosporae non formatur. Post dies 7 ad 25 °C sedimentum parcum formatur. Cultura in medio agaro cum dextroso et peptono et extracto levedinis post dies 7, albida vel cremea cum margo crenatus. In agaro farinae Zea mays post dies 7 ad 25 °C pseudohyphae vera cum conidia globosa formatur. In agaro V8 dilutus post dies 34 ad 17 °C asci clavatispedunculati, pedunculi cylindrateae (36x1423 µm), 2-spori in quoque asco. Ascosporae clavatus vel pyriformis. Fermentatio glucosum. Assimilat glucosum, D-galactosum, D-glucosaminum, D-xylosum, sucrosum, maltosum, α,α-trehalosum, cellobiosum, salicinum, arbutinum, melezitosum, glycerolum, xylitolum, D-sorbitolum, D-mannitolum, glucono-δ-lactonum, 2-ketogluconatum, acidum gluconicum, D-glutamatum et N-acetyl-glucosaminum, et ad non L-sorbosum, D-ribosum, L-arabinosum, D-arabinosum, L-rhamnosum, ethanolum, methanolum, methyl-α-D-glucosidum, melibiosum, lactosum, raffinosum, inulinum, amylum solubile, erythritolum, ribitolum, D-glucitolum, myo-inositolum, acidum glucoronicum, acidum lacticum, acidum succinicum, acidum citricum, acidum malicum et acidum tartaricum. Assimilat L-lysini, ethylamini, cadaverini, betanini, D-glutaminum ad non kallii nitratis, sodii nitrosi, D-glucosaminum, creatino nec creatinino. Vitaminae externae ad crescentiam necessariae sunt. Crescit in 30 °C sed non in 35 °C. Materiae amyloidea non formatur. Ureum finditur. Ubiquinonum majus Q-9. Proportio molaris guanini plus cytosini in acidi deoxyribonucleati 46·9 mol%.

Typus: PYCC 4605^T, ex flore, in collectione zymotica Centraalbureau voor Schimmelcultures (CBS 9131^T), Trajectum ad Rhenum, praeservatus.

Description of Metschnikowia lachancei Giménez-Jurado, Kurtzman, Starmer & Spencer-Martins sp. nov.
Growth in glucose (2 %) yeast extract (0·5 %) peptone (1 %) broth.
After 3 days at 25 °C, vegetative cells are ellipsoidal to cylindrical (37x813 µm) and occur in pairs, tetrads or small chains. Cell division occurs by multilateral budding. Sparse slender pseudohyphae are formed. Chlamydospores are not formed. Some cells contain refractile globules. After 1 week at 25 °C sediment is formed.

Growth on glucose (2 %) yeast extract (0·5 %) peptone (1 %) agar.
After 1 week at room temperature, the streak culture is cream to off-white coloured, smooth, glossy and with a lobate margin.

Dalmau plate cultures on corn-meal agar.
After 1 week at 25 °C, growth under semi-anaerobic conditions shows the presence of slender pseudohyphae. Cells become elongated and eventually form branching pseudohyphae with terminal spherical conidia. Branching occurs from the quadrants of the parent cell with secondary branching occurring quite often. No crosswalls form between hyphal cells.

Formation of ascospores.
Asci with ascospores were observed in cultures of PYCC 4605^T after 34 days incubation at 17 °C in dilute (1 : 15) V8 agar medium. Asci are clavate in shape and develop directly from vegetative cells. Some asci develop directly after conjugation of two cells followed by differentiation at one end of the conjugant giving rise to a long cylindrical peduncle. Mature asci (36x143 µm) usually contain two spindle-shaped ascospores, side by side, with their pointed ends oriented towards the peduncle or elongated portion of the ascus (Fig. 2), but occasionally asci with one protruding ascospore are observed. Asci occur either in pairs or groups of short chains, show different stages of development and are usually persistent. Older asci show ascospores with swollen ends containing visible vacuolar and lipidic material. No ascospores were observed in strain PYCC 5767.

(123K): Fig. 2. Phase-contrast photomicrograph of M. lachancei PYCC 4605T. Formation of asci containing clavate ascospores, after 1 week at 17 °C in dilute (1 : 14) V8 agar. Scale bar, 10 µm.

Physiological and biochemical characteristics
See Table 2.

Etymology.
The specific epithet lachancei, Latin gen. of Lachance, was chosen in honour of Professor Marc-André Lachance, in recognition of his important contribution to yeast taxonomy and the discovery of exotic species of the yeast genus Metschnikowia.

Origin and deposits.
The type strain PYCC 4605^T [=UWO(PS) 7ASB2.3^T] was isolated from nectar of the flower Asclepias syriaca in South Bombay, NY, USA. This strain has been deposited (living and dried) in the PYCC CREM, Universidade Nova de Lisboa, Caparica, Portugal, and under number CBS 9131 in the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

G+C content and nDNA relatedness
Initial characterization of the two new species was from mol% G+C content, which revealed that they separated into two groups, M. lachancei with 46·9 mol% and M. vanudenii with a strain mean of 43·5 mol%. This latter value is similar to the G+C content of M. reukaufii (Table 1). nDNA reassociation experiments demonstrated M. vanudenii, M. lachancei and M. gruessii to be separate species (Table 3), which is consistent with relationships determined from rDNA sequence analysis (Fig. 3).

Table 3. DNA relatedness (%) among isolates of M. vanudenii and M. lachancei and the type strains of M. gruessii and M. reukaufii Strains: 1, M. vanudenii PYCC 4650T; 2, M. vanudenii PYCC 4649; 3, M. vanudenii PYCC 4648; 4, M. vanudenii PYCC 4606; 5, M. vanudenii PYCC 4603; 6, M. lachancei PYCC 4605T; 7, M. gruessii PYCC 4382T; 8, M. reukaufii PYCC 4446T. Data are means of two or more determinations. ND, Not determined.

Phylogeny
Phylogenetic analysis of the D1/D2 domain of the large subunit rDNA revealed that M. vanudenii and M. lachancei fit within the Metschnikowia clade (Fig. 3). The two new species form a distinct lineage with M. gruessii and cluster with those members of Metschnikowia that produce small ascospores. We thank Christie J. Robnett for determining the D1/D2 26S rDNA sequences for the two new species.