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Kluyveromyces osmophilus , a single-strain species isolated from Mozambique sugar, has been treated a synonym of Zygosaccharomyces mellis . Analyses of D1/D2 LSU rRNA gene sequences confirmed that the species belongs to the genus Zygosaccharomyces but showed it to be distinct from strains of Z. mellis . During studies of yeasts associated with stingless bees in Brazil, nine additional isolates of the species were obtained from unripe and ripe honey and pollen of Scaptotrigona cfr. bipunctata , as well as ripe honey of Tetragonisca angustula . The D1/D2 sequences of the Brazilian isolates were identical to those of the type strain of K. osmophilus CBS 5499 (=ATCC 22027), indicating that they represent the same species. Phylogenomic analyses using 4038 orthologous genes support the reinstatement of K. osmophilus as a member of the genus Zygosaccharomyces . We, therefore, propose the name Zygosaccharomyces osmophilus comb. nov. (lectotype ATCC 22027; MycoBank no. MB 833739).

The yeast Kluyveromyces marxianus possesses advantageous traits like rapid growth, GRAS (generally regarded as safe) status and thermotolerance that make it very suitable for diverse biotechnological applications. Although physiological studies demonstrate wide phenotypic variation within the species, there is only limited information available on the genetic diversity of K. marxianus. The aim of this work was to develop a multilocus sequence typing (MLST) method for K. marxianus to improve strain classification and selection. Analysis of housekeeping genes in a number of sequenced strains led to the selection of five genes, IPP1, TFC1, GPH1, GSY2 and SGA1, with sufficient polymorphic sites to allow MLST analysis. These loci were sequenced in an additional 76 strains and used to develop the MLST. This revealed wide diversity in the species and separation of the culture collection and wild strains into multiple distinct clades. Two subsets of strains that shared sources of origin were subjected to MLST and split decomposition analysis. The latter revealed evidence of recombination, indicating that this yeast undergoes mating in the wild. A public access web-based portal was established to allow expansion of the database and application of MLST to additional K. marxianus strains. This will aid understanding of the genetic diversity of the yeast and facilitate biotechnological exploitation.

A phylogenetic analysis of 17 species belonging to the genus Kluyveromyces and 12 reference and outgroup species was performed using mitochondrial cytochrome-c oxidase II gene sequences. The genus Kluyveromyces appears as a polyphyletic taxon formed by species included within the following four main groups. The Kluyveromyces phaffii group encompasses the species Kluyveromyces blattae, K. phaffii and Kluyveromyces yarrowii. The Kluyveromyces marxianus group is a monophyletic group consisting of the species Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, K. marxianus and Kluyveromyces wickerhamii. The monophyletic Kluyveromyces thermotolerans group is formed by K. thermotolerans, Kluyveromyces waltii and Saccharomyces kluyveri (which appears in the mitochondrial tree as the sister clade of the K. marxianus group). Finally, the Saccharomyces cerevisiae group contains the remaining Kluyveromyces species, as well as the reference Saccharomyces species (sensu lato and sensu stricto) and Candida glabrata (the phylogenetic relationships within this group are unclear according to the bootstrap test). The phylogenetic relationships obtained for this mitochondrial gene are, for the most part, congruent with previous trees based on nuclear rRNA sequences, except for the position of K. yarrowii and the close relationship between the K. marxianus and K. thermotolerans groups. These differences, as well as the existence of these groups, are discussed in the context of previous studies based on phenotypic, genetic and molecular data. Although additional studies are required to decipher the phylogenetic relationships between the genus Kluyveromyces and the closely related genera Saccharomyces, Torulaspora and Zygosaccharomyces, future changes to their taxonomic status should take account of the existence of these four groups of Kluyveromyces species.