Antonie van Leeuwenhoek 71: 325±328, 1997. 325
c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Lipomyces mesembrius sp. nov., a member of the L. starkeyi species-complex
J.P. van der Walt1, A. Botha1 & M.Th. Smith2 1 Department of Microbiology & Biochemistry (UNESCO-MIRCEN), University of the Orange Free State, PO Box 339, Bloemfontein 9300, South Africa; 2 Centraalbureau voor Schimmelcultures Yeast Division, Julianalaan 67, 2628 BC Delft, The Netherlands
Accepted 19 August 1996
Key words: Lipomyces, species delimitation, genetic isolation
Abstract
Lipomyces starkeyi is known to be associated with three strains-clusters showing high mutual nDNA reassociation within each cluster, but which reassociate ambiguously with the type of L. starkeyi. Representative strains of L.
starkeyi and Cluster were examined for possible genetic exchange by the prototrophic selection technique. Since
no genetic recombination was detected, the strains are presumed to be genetically isolated. Cluster is consequently assigned to the rank of species as Lipomyces mesembrius. A description of the new species is given. Lipomyces kononenkoae ssp. spencermartinsiae has been raised to the rank of species as L. spencermartinsiae.
Introduction type of L. starkeyi and the representative strain of Clus-
ter was examined by the prototrophic selection tech- Smith et al. (1995a), in their revision of the genus nique (Pomper & Burkholder 1949) as developed by Lipomyces Lodder and Kreger-Van Rij (1952) by Johannsen (1978, 1980) and Van der Walt et al. (1982). nuclear genome comparison, established that the type species, L. starkeyi, was associated with three relat- ed strain clusters of uncertain taxonomic status. While Materials and methods mutual nDNA reassociation values within each clus- ter were predictive of conspeci®city (86±97%), the Strains studied reassociation between representatives of clusters and the type of L. starkeyi, nevertheless, fell within the Lipomyces starkeyii indeterminate range of 60±63%. Given the ambiguity of these lower values and the lack of crisp phenotyp- CBS 1807T CBS 1807leu
ic differentiation of the strains, Smith et al. (1995a) CBS 1807cys CBS 1807ura deferred the classi®cation of the three Clusters , and The notations cys, leu and ura denote requirements
. Because homology values below 65±70% are con- for L-cysteine L-leucine and uracil respectively. sidered to be taxonomically indeterminate (Kurtzman Representative strains of L. starkeyi Cluster CBS 1987), and fail to distinguish between infra-speci®c 7661.
taxa and genetically isolated species, the taxonomic
status of Clusters , and has to be assessed by hybridization in terms of the biological species con- cept. Induction and characterization of auxotrophic Since the observations by Roberts (1957) on the mutants life-cycle of L. lipofer established that spore-to-spore matings are not feasible for hybridization purposes in Auxotrophic mutants CBS 1807cys CBS 1807leu and Lipomyces, the possible genetic exchange between the CBS 1807ura were obtained by UV irradiation as 326
Table 1. Intraspeci®c crosses of auxotrophic mutants Table 2. Crosses of CBS 7661 and auxotrophic mutants of CBS of CBS 1807 enumerated on minimal recovery medium 1807 in which recombinants were not detected on minimal recovery with D-glucose as carbon source medium with D-galactose as carbon source
Parental strains Recombinant/108 Parental strains Viable count ml 1 propagules of the in which recombinants mated populated were not detected
4 7
CBS 1807ura CBS 1807cys 2.7 10 CBS 7661URAgal CBS 1807uraGAL 4.9 10
4 9
CBS 1807ura CBS 1807leu 1.5 10 CBS 7661CYSgal CBS 1807cysGAL 4.5 10
4 7
CBS 1807cys CBS 1807leu 4.5 10 CBS 7661LEUgal CBS 1807leuGAL 1.7 10
GAL and gal denote the ability and inability to utilize D-galactose respectively. described by Meyer et al. (1993) and characterized according to Johannsen (1978). mutants for hybridization purposes. Consequently the failure to detect recombinants between CBS 7661 and Crossing of mutant strains the auxotrophic mutants of CBS 1807 establishes that the two type strains are virtually incapable of exchang- The adopted procedures are fully described by ing genetic material and as such, genetically isolated. Johannsen (1978).Intraspeci®c and interspeci®c cross- Irrespective of this isolation, the two populations rep- es made, are listed in Tables 1 and 2. Parental strains resented by CBS 1807 and CBS 7661 are genetically were mass-mated on 1 : 10 diluted YM agar and kept close, and best interpreted as sibling taxa. The extent at 20 C until maximum sporulation had occurred (4± of their genetic divergence could possibly be assessed 6 weeks). Two further serial transfers on the same from rRNA and mtDNA base sequence analyses. medium and temperature were made at intervals of 4± 6 weeks. Possible back mutation by the mutants was Taxonomic aspects monitored in all crosses. Recombinant formation in intraspeci®c and interspeci®c crosses was enumerated Given (i) genetic separation of CBS 1807T and CBS on minimal recovery medium with D-glucose and D- 7661 and (ii) the high mutual nDNA reassociation galactose respectively. The results are given in Tables 1 values (86±97%) indicative of conspeci®city that cir-
and 2. The restoration of prototrophy in the recovered cumscribe Cluster , it is proposed that this taxon, in recombinants was con®rmed by subculturing in Difco terms of the biological species concept, be assigned to Yeast Nitrogen Base without amino acids. Ascospore Lipomyces mesembrius Botha, Van der Walt et M.Th. formation by the intraspeci®c recombinants was con- Smith. sp. nov. (Lipomycetaceae).
®rmed on 1 : 10 diluted YM agar. In agaro malti post triduum in 25 C cellulae vege-
tativae gemmantae incapsulatae globosae ellipsoideae vel ovoideae, 3.5±9.0 3.5±8.0 m, vacuolatae glob- Results ulos lipidi continente, singulae binaeque. Cultura post
hebdomades 4 in 18 C cremeibruneola vel dilute bru- All three mutants were found to be stable and showed neola opacca glabra viscosa effusior nitida, margine no back-mutation throughout the study. integro. In agaro farinae Zeae maydis confecto post
decemduum in 25 C hyphae et pseudohyphae nul-
lae. Asci plerumque af®xi, saccati vel tortioter tubi- Discussion lares, uni vel multispori, 9.0±27.0 3.0±11.0 m,
pariete tarde deliquescente. Ascosporae maturae glo- In the absence of detectable back-mutation, the recov- bosae vel ellipsoideae, 2.0±4.0 2.0±3.5 m, succi- ery of sporulating prototrophic recombinants from the nae glabrae vel verruculosae globulos lipidi continente, intraspeci®c crosses of CBS 1807ura, CBS 1807cys conglutinantes ubi liberatae. Fermentatio gaseosa nul- 4 and CBS 1807leu at frequencies of 1.5±4.5 10 la. Glucose melibioso succinato citrato pro fonte carbo- per 108 propagules of the mated populations (Table nis utens, neque D-glucosamino D-riboso D-arabinoso 1), con®rms the competence and suitability of these L-rhamnoso m-erythritolo m-inositolo D-glucuronato 327
D-galacturonato DL-lactato methanolo D-galactonato Fermentation: Absent. nec 1,2 propanodiolo. Usio D-galactosi L-sorbosi D-
Utilization of carbon sources1)
xylosi L-arabinosi sucrosi maltosi , -trehalosi - methyl-D-glucosidi cellobiosi salicini arbutini lactosi D-glucose + Salicin V D-mannitol V raf®nosi melezitosi inulini amyli solubile glyceroli D-galactose V Arbutin V Galactitol V ribitoli xylitoli L-arabinitoli D-glucitoli D-mannitoli L-sorbose V Melibiose + m-Inositol -
galactitoli D-glucono- -lactoni D-gluconati ethanoli D-glucosamine - Lactose V D-glucono-
2,3 butanodioli variabile. Ethylamino hydrochlorido -lactone V cadaverino dihydrochlorido L-lysino et imidazolo pro D-ribose - Raf®nose V D-gluconate V fonte nitrogeni utens, neque nitrate nec nitrite. Sine D-xylose V Melezitose V D-glucuronate - vitaminis externis supplementis crescens. In 35 C non L-arabinose V Inulin V D-galacturonate - increscens. In agaro extracto fermenti confecto quin- D-arabinose - Soluble starch V DL-lactate - quaginta partes glucosi per centum pondere continente L-rhamnose - Glycerol V Succinate + non increscens. Materia amyloidea formans. Ureum Sucrose V m-Erythritol - Citrate +
non ®ndens. Ubiquinonum majus Q9. G + C acidi Maltose V Ribitol V Ethanol V
, Trehalose V Xylitol V Methanol - deoxyribonucleati 47.0 0.3 ± 48.0 0.6 per centum.
-Me-D-glucoside V L-arabinitol V Propane 1,2 diol - Typus cultura CBS 7661 in collectione Centraalbureau Cellobiose V D-glucitol V Butane 2,3 diol V
voor Schimmelcultures (Baarn et Delphis Batavorum D-galactonate - in Hollandia) in sicco et vivo praeservatus. ) 1 Data from Smith et al. (1995a). Growth on malt agar Utilization of nitrogen sources.
After 3 days at 25 C the budding, vegetative cells Potassium nitrate - Cadaverine dihydrochloride +
are encapsulated, globose, ellipsoid to ovoid, 3.5± Sodium nitrite - L-lysine + 9.0 3.5±8.0 m, vacuolated with a lipid globule, and Ethylamine hydrochloride + Imidazole + occur singly or in pairs. The culture is greyish-cream, Growth at 35 C: Absent. mucoid, smooth and shiny, with an entire margin. After Growth in vitamin-free medium: Positive. 4 weeks at 18 C the streak culture is brownish-cream Growth in 0.1% cycloheximide: Positive. to light brown, opaque, smooth, viscous, spreading Growth on 50% (m/m) glucose-yeast extract agar: and shiny; the margin is entire. Absent. Dalmau plate cultures on corn meal agar: After 10 Formation of amyloid: Positive, colouring blue-green days at 25 C neither hyphae nor pseudohyphae are with iodine. formed. Splitting of urea: Absent.
Major ubiquinone system: Q9. Ascus formation Mol. % G + C : 47.0 0.3 ± 48.0 0.6 (Smith et al., 1995a). The asci are as a rule attached, saccate to irregularly
tubular or contorted, one- to multispored, 9.0±27.0 Etymology: mesembrius from the Gr. adj. 3.0±11.0 m, with slowly deliquescent walls. The " o& southern or meridional, referring to the
mature ascospores are globose to ellipsoid, amber- species' occurrence in southern Africa. coloured, 2.0±4.0 2.0±3.5 m glabrous to verru- culose, containing a lipid globule, and conglutinative when liberated. Actively sporulating cultures have a brown tinge. 328
Strains currently assigned to L. mesembrius needs to be reassessed in terms of genetic and geo- graphical isolation. It should, morever, be noted that The species is known from the following ®ve South yeast systematicists have made no effort at formulating African isolates: CBS 7661, CBS 7600, CBS 7601, de®nitive guidelines that permit the recognition of non- CBS 7605 and CBS 7737. Strain CBS 7737 differs hierarchial, infra-speci®c taxa as either subspecies, from the other four strains in 22 physiological charac- varieties or forms. In the absence of such guidelines, ters. For further details of the strains the publication of the conventional use of the unde®ned variety remains Smith et al. (1995a) should be consulted. taxonomically meaningless, and genetically inexplicit.
Type: culture CBS 7661 maintained in the dried and living state in the Collection of the Centraalbureau voor Acknowledgement Schimmelcultures, Baarn and Delft (The Netherlands), respectively. The authors thank E.L. Janse-van Rensburg for deter- Given the fact that reassociation values as high mining the Coenzyme Q system of strains of the new as 60% preclude genetic exchange between the type species. strains of L. starkeyi and L. mesembrius,itmay be inferred that reassociation values as low as 47% observed for the types of the two subspecies in L. References kononenkoae (Smith et al. 1995a), likewise implies the genetic isolation of the two putative, infraspeci®c Johannsen E (1978) Hybridization studies within the genus taxa. It is consequently proposed that the subspecies Kluyveromyces van der Walt emend van der Walt. Ph.D. The- sis Vol I and II, Rhodes University, Grahamstown, South Africa spencermartinsiae be raised to the rank of species: Ð (1980) Hybridization studies within the genus Kluyveromyces. Lipomyces spencermartinsiae (Van der Walt & Antonie van Leeuwenhoek 46: 177±189 M.Th. Smith) Van der Walt & M.Th. Smith stat. nov. Kurtzman CP (1987) Prediction of biological relatedness among yeasts from comparison of nuclear DNA complementarity. In: et sp. nov. Hoog GS de, Smith MTh & Weijman ACM (Eds) The Expand- ing Realm of Yeast-like Fungi. (pp 459) Elseviers Science Publ. Basionym: Lipomyces kononenkoae Nieuwdorp et Amsterdam al. ssp. spencermartinsiae Van der Walt & M.Th. Smith Lodder J & Kreger-van Rij NJW (1952) The yeasts, a taxonomic study. North-Holland Publ. Co. Amsterdam in Antonie van Leeuwenhoek 68, 84, 1995. Meyer PS, Preez JC du, Wing®eld BD & Kilian SG (1993) Eval- Relevant to the foregoing is the ®nding that no uation of Candida blankii hybrids for biomass production. J. recombinants were detected in crosses of strains of L. Biotechnol. 29: 267±275 starkeyi and L. tetrasporus that reassociate for some Naumov GI, Naumova ES, Lantto R, Louis EJ & Korhola M (1992a) Genetic homology between Saccharomyces and its sib- 20% (Smith et al. 1995a). ling species, S. paradoxus and S. bayanus: electrophoretic kary- otypes. Yeast 8: 599±612 Naumov GI, Naumova ES & Korhola M (1992b) Genetic identi®ca- Conclusions tion of natural Saccharomyces sensu stricto yeasts from Finland, Holland and Slovakia. Antonie van Leeuwenhoek 61: 237±243 Pomper S & Burkholder PR (1949) Studies on the biochemical genet- The recognition of L. starkeyi and L. mesembrius as ics of yeast. Proc. Natl. Acad. Sci. USA 35: 456±464 sibling species, introduces the concept of species- Roberts C (1957) Observations on the yeast Lipomyces. Nature (Lon- complexes in this genus. Such species-complexes are don) 179: 1198±1199 Smith MTh, Poot GA, Batenburg-van der Vegte W & Walt JP van der probably more prevalent among the ascogenous yeasts (1995a) Species delimitation in the genus Lipomyces by nuclear than previously anticipated. Their existence in Saccha- genome comparison. Antonie van Leeuwenhoek 68: 75±87 romyces has been described by Naumov et al. (1992a, Smith MTh, Cock AWAN de, Poot GA & Steensma HY (1995a) Genome comparison in the yeastlike fungal genus Galactomyces b) and in Galactomyces and Geotrichum by Smith Redhead et Malloch. Int. J. Syst. Bacteriol. 45: 826±831 et al. (1995b). Similar complexes appear evident in Walt JP van der, Yarrow D, Opperman A & Holland L (1982) Pichia Williopsis (Kurtzman 1987), although the constituent kodama sp. nov., a new homothallic yeast species. J. Gen. Appl. taxa have hitherto not been described. Microbiol. 28: 155±160 Since no taxonomically sound conclusions can be drawn from ambiguous nDNA reassociation values, the rank of the infra-speci®c yeast taxa which have been delimited solely on the basis of such values,