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A total of 296 isolates of Saccharomyces cerevisiae sampled from naturally fermenting grape musts from various locations in Lebanon were typed by interdelta fingerprinting. Of these, 88 isolates were compared with oenological strains originating from various countries, using microsatellite characterization at six polymorphic loci. These approaches evidenced a large diversity of the natural oenological Lebanese flora over the territory as well as in individual spontaneous fermentations. Several cases of dominance and perenniality of isolates were observed in the same wineries, where fermentations appeared to involve lineages of sibling isolates. Our work thus evidenced a “winery effect” on strains’ relatedness. Similarly, related or identical strains were also detected in vicinal wineries, suggesting strain circulation within small geographical areas and a further “vicinity effect”. Moreover, and despite its diversity, the Lebanese flora seemed interrelated, on the basis of microsatellite loci analysis, in comparison to worldwide communities. We finally tested the ability of 21 indigenous strains to act as potential starters for winemaking. Seven of them passed our pre-selection scheme and two of them at least may be good candidates for use provided pilot-scale assays confirm their suitability.

The most common fermented beverage, lager beer, is produced by interspecies hybrids of the brewing yeast Saccharomyces cerevisiae and its wild relative S. eubayanus. Lager-brewing yeasts are not the only example of hybrid vigour or heterosis in yeasts, but the full breadth of interspecies hybrids associated with human fermentations has received less attention. Here we present a comprehensive genomic analysis of 122 Saccharomyces hybrids and introgressed strains. These strains arose from hybridization events between two to four species. Hybrids with S. cerevisiae contributions originated from three lineages of domesticated S. cerevisiae, including the major wine-making lineage and two distinct brewing lineages. In contrast, the undomesticated parents of these interspecies hybrids were all from wild Holarctic or European lineages. Most hybrids have inherited a mitochondrial genome from a parent other than S. cerevisiae, which recent functional studies suggest could confer adaptation to colder temperatures. A subset of hybrids associated with crisp flavour profiles, including both lineages of lager-brewing yeasts, have inherited inactivated S. cerevisiae alleles of critical phenolic off-flavour genes and/or lost functional copies from the wild parent through multiple genetic mechanisms. These complex hybrids shed light on the convergent and divergent evolutionary trajectories of interspecies hybrids and their impact on innovation in lager brewing and other diverse fermentation industries., Work supported by the National Science Foundation (grant nos. DEB-1253634 to C.T.H. and DGE-1256259 (Graduate Research Fellowship to Q.K.L.), the USDA National Institute of Food and Agriculture Hatch Project (nos. 1003258 and 1020204 to C.T.H.) and in part by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science nos. DE-SC0018409 and DE-FC02-07ER64494). Q.K.L. was also supported by the Predoctoral Training Program in Genetics, funded by the National Institutes of Health (grant no. 5T32GM007133). D.P. is a Marie Sklodowska-Curie fellow of the European Union’s Horizon 2020 research and innovation programme (grant no. 747775).

Additional file 2: Table S1. Relative composition (%) of the FA fraction of LS3 cells after growth in various substrates. Table S2. Final yields after 72 h of cultivation of LS3 and CBS8244T in YNB-based glucose media (C/N = 60) at different temperatures. Table S3. Time course analysis of the FA profiles of strains LS3 and CBS 8244T cultivated in YNB-based glucose media (C/N = 60) at various temperatures.

Additional file 1: Figure S1. Polymorphisms in the ITS-D1D2 nucleotide sequence. Figure S2. Growth of B. adeninivorans and B. raffinosifermentans in three different substrates. Figure S3. Organic acids excreted in lipogenic culture conditions. Figure S4. FA composition of strains of B. raffinosifermentans and B. adeninivorans after growth in lipogenic medium. Figure S5. Growth curves and microscopic images of strains LS3 and CBS 8244T cultured in glucose lipogenic medium at various temperatures. Figure S6. Relative FA composition of strains LS3 and CBS 8244T at various times and temperatures of cultivation in glucose lipogenic medium.

The use of the nuclear DNA reassociation technique has led taxonomists to consider Saccharomyces uvarum a synonym of S. bayanus. The latter, however, is not a species but a hybrid harbouring S. eubayanus (Seu) and S. uvarum (Su) subgenomes with a minor DNA contribution from S. cerevisiae (Sc). To recognize genetically pure lines of S. uvarum and putative interspecies hybrids among so-called S. bayanus strains present in public culture collections, we propose the use of four markers that were defined from the S. bayanus CBS 380T composite genome, namely SeuNTS2 (rDNA), ScMAL31, MTY1 and SuMEL1. Saccharomyces carlsbergensis CBS 1513 was found to be similar to S. bayanus except that it carries the SeuMEL1 allele. Different marker combinations revealed that among 33 strains examined only a few were similar to CBS 380T, but many pure S. uvarum lines and putative Su/Seu-related hybrids occurred. Our results demonstrated that these hybrids were erroneously considered authentic S. bayanus and therefore the varietal state ‘Saccharomyces bayanus var. uvarum comb. nov. Naumov’ is not valid. Our markers constitute a tool to get insights into the genomic makeup of Saccharomyces interspecies hybrids. We also make a proposal to name those hybrids that may also be applicable to other fungal hybrids.

Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae · S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto. � 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.

Summary A PCR-RFLP method has been improved for the rapid identification of the four species of Saccharomyces sensu stricto . We used the NTS2-ETS sequence of the rDNA as target and the amplification reaction was realized by chosing a pair of primers that could anneal to the end and the beginning of the 5S and 18S conserved sequences, respectively. PCR products obtained from S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus (synonym S. carlsbergensis ) type strains displayed a clear polymorphism when digested with several restriction enzymes. Using only three enzymes: Ban I, Alu I and Taq I we were able to differentiate S. cerevisiae, S. paradoxus and the S. bayanus/S. pastorianus complex which shared the same pattern. The S. bayanus taxon now regroups the former species S. abuliensis, S. globosus, S. heterogenicus, S. intermedius, S. inusitatus, S. uvarum . PCR-RFLP of these species showed that, except for S. abuliensis and S. uvarum , they shared the same patterns as S. bayanus type strain. That lead us to conclude that there are two subgroups in S. bayanus and that S. abuliensis and S. uvarum belong to a distinct subgroup. Remarkably, wine strains that were previously identified as S. bayanus by genetic hybridization techniques, as well as strains isolated from cider fermentation, belong to the S. uvarum subgroup.

International audience; Recent changes in the epidemiology of candidiasis highlighted an increase in non- Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently mis-identified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. These two PCR/restriction fragment length polymorphism methods may be used as reference tools [either alternatively or adjunctively to the existing ribosomal DNA (26S or ITS) sequence comparisons] for unambiguous determination of the Candida species for which phenotypic characterization remains problematic.

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380(T) and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380(T) harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380(T) and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380(T) or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.

Significant differences in electrophoretic karyotyping patterns were found among 27 strains of Y. lipolytica. Twenty-one of these strains were classified into four groups of similar karyotypes while six strains showed unique karyotypes. Chromosomal DNAs of different strains were hybridized with cloned genes of Y. lipolytica (URA3, LEU2, ARS18 and ARS68), which revealed four different bands in most strains. We conclude that the haploid chromosome number of Y. lipolytica is at least four, and possibly five or six. Electrophoretic karyotyping and hybridization with cloned genes of Y. lipolytica provided evidence of a large divergence between Y. lipolytica and related species of Saccharomycopsis, Endomycopsella and Endomyces.

The intergenic spacer rDNA amplification and AluI fingerprinting (IGSAF) method detected four distinct groups among 170 Debaryomyces hansenii strains: D. hansenii var. hansenii; Candida famata var. famata; D. hansenii var. fabryi and C. famata var. flareri. IGS sequence comparison of representative strains showed that D. hansenii var. hansenii and C. famata var. famata belonged to one species, whereas D. hansenii var. fabryi and C. famata var. flareri belonged to two different ones. This confirmed the following three species recently reinstated: D. hansenii (=C. famata), Debaryomyces fabryi and Debaryomyces subglobosus (=Candida flareri). Accordingly, growth at 37 degrees C may no longer be used to differentiate D. hansenii from D. fabryi. Riboflavin production is more specific for D. fabryi and D. subglobosus strains. IGSAF identified all the other 17 species of the genus Debaryomyces, six of them sharing with D. hansenii an rRNA gene unit harbouring two 5S rRNA genes. The phylogenetic tree established with IGS sequences was congruent with the one based on ACT1, GPD1 and COX2 sequences depicting a distinct D. hansenii clade close to the D. subglobosus, Debaryomyces prosopidis and D. fabryi clade. Description of Debaryomyces vietnamensis sp. nov. (type strain CBS 10535(T), MUCL 51648(T)), closely related to Debaryomyces nepalensis is given.

Seventy six yeast strains isolated form bili bili and others sample were identified and typed in purpose of selecting appropriate starter culture. Identification techniques included conventional phenetic method, PCR/RFLP of NTS2 rDNA region, partial sequencing of the D1/D2 region of 26S rDNA and karyotyping using contour clamped homogenous electric field (CHEF) technique. The Saccharomyces cereviseiae strains were also compared to industrial strains according to their fermentation profiles on maltose in the presence of 2- eoxy-D-glucose and to their karyotypes. We observed that the fermentation of bili bili was carried out by an indigenous natural flora predominantly represented by highly polymorphic S. cerevisiae strains whereas early steps in the process were carried out mainly by Kluyveromyces maxianus strains. All of the S. cerevisiae strains which were used in trial fermentation gave a good rate of fermentation suggesting that they may be used as starter cultures. African Journal of Biotechnology Vol. 4 (7), pp. 646-656, 005

Considerable amounts of molecular and genetic data indicate that interspecific hybridisation may not be rare among natural strains of Saccharomyces sensu stricto. Although a post-zygotic barrier operating during meiosis usually prevents the production of viable spores, stable hybrids can arise which can even evolve into distinct species. This study was aimed to analyse the genome of a fertile Saccharomyces cerevisiae x S. uvarum hybrid and monitor its changes over four filial generations of viable spores. The molecular genetic analysis demonstrated that the two species did not contribute equally to the formation and stabilisation of the hybrid genome. S. cerevisiae provided the mitochondrial DNA and the more stable part of the nuclear genome. The S. uvarum part of the hybrid nuclear genome became progressively smaller by loosing complete chromosomes and genetic markers in the course of successive meiotic divisions. Certain S. uvarum chromosomes were eliminated and/or underwent rearrangements in interactions with S. cerevisiae chromosomes. Numerous S. uvarum chromosomes acquired S. cerevisiae telomere sequences. The gradual elimination of large parts of the S. uvarum genome was associated with a progressive increase of sporulation efficiency. We hypothesise that this sort of genomic alterations may contribute to speciation in Saccharomyces sensu stricto.

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.

94 ref.; International audience

PCR/RFLP of the NTS2 (IGS2) of rDNA was applied to differentiate two closely related yeast species, Kluyveromyces lactis var. lactis (referred to as K. lactis) and K. marxianus. Using specific primers, the NTS2 region was amplified from DNA of both K. lactis and K. marxianus type and collection strains. AluI restriction of amplified fragments generated patterns characteristic for each species. The NTS2 region from K. lactis var. drosophilarum and related species K. aestuarii, K. africanus, K. dobzhanskii, and K. wickerhamii could also be amplified with the same primers, but AluI patterns generated were clearly different. PCR/RFLP of the NTS2 appears thus to be a convenient method for rapid identification of K. lactis and K. marxianus, frequently found in dairy products. This test was validated therefore on K. lactis and K. marxianus from natural habitats. We showed that all yeast strains collected from whey samples and scoring blue on X-gal glucose plates were either K. lactis or K. marxianus. For application purposes, we propose here an approach for quickly screening for K. lactis/marxianus and Saccharomyces cerevisiae in dairy products using X-gal coloured and lysine growth media.Key words: yeast, Kluyveromyces, ribosomal DNA, karyotype, taxonomy.

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.

Saccharomyces uvarum is proposed as a proper species within the complex Saccharomyces sensu stricto. Molecular characteristics including the similarity of the restriction profile of the non-transcribed spacer 2 (NTS2) and of the D1/D2 sequences of the rDNA, as well as other genotypic and phenotypic characteristics confirm that this group of strains is highly homogeneous and distinguishable from other species of the Saccharomyces sensu stricto group.

The genome of Saccharomyces servazzii was analyzed with 2570 random sequence tags totalling 2.3 Mb. BLASTX comparisons revealed a minimum of 1420 putative open reading frames with significant homology to Saccharomyces cerevisiae (58% aa identity on average), two with Schizosaccharomyces pombe and one with a human protein, confirming that S. servazzii is closely related to S. cerevisiae. About 25% of the S. servazzii genes were identified, assuming that the gene complement is identical in both yeasts. S. servazzii carries very few transposable elements related to Ty elements in S. cerevisiae. Most of the mitochondrial genes were identified in eight contigs altogether spanning 25 kb for a predicted size of 29 kb. A significant match with the Kluyveromyces lactis linear DNA plasmid pGKL-1 encoded RF4 killer protein suggests that a related plasmid exists in S. servazzii. The sequences have been deposited with EMBL under the accession numbers AL402279–AL404848.

By analyzing 2830 random sequence tags (RSTs), totalling 2.7 Mb, we explored the genome of the marine, osmo- and halotolerant yeast, Debaryomyces hansenii. A contig 29 kb in length harbors the entire mitochondrial genome. The genes encoding Cox1, Cox2, Cox3, Cob, Atp6, Atp8, Atp9, several subunits of the NADH dehydrogenase complex 1 and 11 tRNAs were unambiguously identified. An equivalent number of putative transposable elements compared to Saccharomyces cerevisiae were detected, the majority of which are more related to higher eukaryote copia elements. BLASTX comparisons of RSTs with databases revealed at least 1119 putative open reading frames with homology to S. cerevisiae and 49 to other genomes. Specific functions, including transport of metabolites, are clearly over-represented in D. hansenii compared to S. cerevisiae, consistent with the observed difference in physiology of the two species. The sequences have been deposited with EMBL under the accession numbers AL436045–AL438874.

PCR/RFLP of the NTS2 sequence of rDNA was shown to be suitable for differentiating Saccharomyces sensu stricto species. We previously showed that, within the presently accepted S. bayanus taxon, strains formerly classified as S. uvarum represented a distinct subgroup (Nguyen and Gaillardin, 1997). In this study, we reidentified 43 more strains isolated recently from wine, cider and various fermentation habitats, and confirmed by karyotyping, hybridization and mtDNA analysis the homogeneity of strains from the S. uvarum subspecies. Molecular typing of nuclear and mitochondrial genomes of strains preserved in collections, and often originating from beer like S. pastorianusNT, revealed the existence of hybrids between S. uvarum and S. cerevisiae. Surprisingly, S. bayanusT CBS380 appeared itself to be a hybrid between S. uvarum and S. cerevisiae. This strain has a mitochondrial genome identical to that of S. uvarum, and a very similar karyotype with 13 isomorphic chromosomes, six of which at least hybridize strongly with S. uvarum chromosomes or with a S. uvarum specific sequence. However, four of the chromosome bands of S. bayanusT bear Y' sequences indistinguishable from those of S. cerevisiae, a feature that is not observed among presently isolated S. uvarum strains. Because of the hybrid nature of S. bayanus(T) and of the scarcity of similar hybrids among present days isolates, we propose to reinstate S. uvarum as a proper species among the Saccharomyces sensu stricto complex.

In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series.

s. The following are averages for the citations in the two databases for the period 1990-2000: K. marxianus (and synonym K. fragilis) 649, K. lactis 736, K. polysporus 13. Fabospora and Zygofabospora averaged one citation each. Article 51.1 of the International Code of Botanical Nomenclature is clear that a legitimate name must not be rejected merely because it is inappropriate or disagreeable, or because another is preferable or better known. However, Article 14.2 states that conservation aims at retention of those names which best serve stability of nomenclature. Placing K. lactis and K. marxianus in a genus other than Kluyveromyces would cause considerable confusion, and such a classification would face almost certain rejection by the industrial/biotechnological and genetics communities. Thus, it is proposed to conserve the generic name Kluyveromyces with a new type: K. marxianus (E. C. Hansen) van der Walt (Antonie van Leeuwenhoek Ned. Tijdschr. Hyg. 31: 347, 1965), based on: Saccharomyces marxianus E. C. Hansen (Medd. Carlsberg Lab. 2: 220, 1888), the first described species of this clade. With acceptance of this proposal, the two species strongly supported phylogenetically in the K. polysporus clade (K. polysporus, K. yarrowii van der Walt & al.) will have to be placed in a new genus. Lyophilized (freeze-dried) neotype (designated by Lodder & Kreger-van Rij, The Yeasts, A Taxonomic Study, 1952) cultures of K. marxianus (CBS 712, NRRL Y8281) are available, respectively, from the Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands) and the ARS Culture Collection (Peoria, Illinois, U.S.A.).