RESUMEN
Annual surveys of Irish soil samples identified three isolates, CBS 16921 (UCD88), CBS 18246 (UCD443), and CBS 18247 (UCD483), of an apiculate yeast species within the Hanseniaspora genus. The internal transcribed spacer (ITS) and D1/D2 region of the large subunit (LSU) rRNA sequences showed that these are isolates of the recently described species Hanseniaspora menglaensis, first isolated from Southwest China. No genome sequence for H. menglaensis is currently available. The genome sequences of the three Irish isolates were determined using short-read (Illumina) sequencing, and the sequence of one isolate (CBS 16921) was assembled to chromosome level using long-read sequencing (Oxford Nanopore Technologies). Phylogenomic analysis shows that H. menglaensis belongs to the fast-evolving lineage (FEL) of Hanseniaspora. Only one MAT idiomorph (encoding MATα1) was identified in all three sequenced H. menglaensis isolates, consistent with one mating type of a heterothallic species. Genome comparisons showed that there has been a rearrangement near MATα of FEL species compared to isolates from the slowly evolving lineage (SEL).
RESUMEN
Two protein-coding genes, actin and translation elongation factor-1alpha (EF-1alpha), as well as two ribosomal gene regions, D1/D2 domains of the large subunit and both internal transcribed spacers including the 5.8S gene region, were evaluated regarding their usefulness for reconstruction of phylogenetic relationships in the Hanseniaspora-Kloeckera species group. This included analyses of sequence divergence values, heterogeneity of evolutionary rates and the reliability of the inferred trees. Both protein-coding genes showed greater capacities to resolve at the strain level and between the closely related species of Hanseniaspora-Kloeckera, compared with the ribosomal gene regions. However, to obtain a fully resolved and reliable phylogenetic tree that reflected the biological relationships it was necessary to combine three congruent sequence datasets. The novel species Hanseniaspora pseudoguilliermondii sp. nov. (type strain CBS 8772T) is described as a result of the application of various molecular approaches to delimit species. Furthermore, incongruent gene genealogies of genetically divergent strains of Hanseniaspora occidentalis, as determined by amplified fragment length polymorphism analysis and DNA-DNA reassociation measurements, indicated the presence of two novel varieties, H. occidentalis var. occidentalis (type strain CBS 2592T) and H. occidentalis var. citrica var. nov. (type strain CBS 6783T), which could be distinguished by habitat preference.
Asunto(s)
Saccharomycetales/clasificación , Actinas/genética , Dermatoglifia del ADN , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , ADN Ribosómico/química , ADN Ribosómico/aislamiento & purificación , ADN Espaciador Ribosómico/genética , Proteínas Fúngicas/genética , Genes de ARNr , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Factor 1 de Elongación Peptídica/genética , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , ARN de Hongos/genética , ARN Ribosómico/genética , ARN Ribosómico 5.8S/genética , Saccharomycetales/genética , Análisis de Secuencia de ADNRESUMEN
Fourteen apiculate yeast strains isolated from various sources in South Africa, North America and the Hawaiian islands were found to be genetically divergent from other Hanseniaspora-Kloeckera species by using randomly amplified polymorphic DNA (RAPD)-PCR. After cluster analysis of the RAPD-PCR fingerprints, five groups were recognized. DNA reassociation values among representatives of these groups and strains of Hanseniaspora-Kloeckera species revealed that the strains represent five novel species. Four are described here as novel species of HANSENIASPORA: Hanseniaspora meyeri sp. nov. (type CBS 8734(T)), Hanseniaspora clermontiae sp. nov. (type CBS 8821(T)), Hanseniaspora lachancei sp. nov. (type CBS 8818(T)) and Hanseniaspora opuntiae sp. nov. (type CBS 8733(T)). The fifth novel species, which is represented by only a single strain, CBS 8772, is not introduced as a new taxon. Phylogenetic analyses of the D1/D2 region of the 26S rDNA and internal transcribed spacer (ITS) regions with 5.8S rDNA sequences placed H. meyeri, H. clermontiae, H. lachancei, H. opuntiae and strain CBS 8772 close to Hanseniaspora uvarum and Hanseniaspora guilliermondii. The key characteristics for standard physiological identification of H. clermontiae and H. lachancei were respectively maximal growth temperature and assimilation of 2-keto-D-gluconate. However, physiological characteristics did not allow the distinction of H. opuntiae and strain CBS 8772 from H. guilliermondii or H. meyeri from H. uvarum. These three novel taxa can be identified by either ITS sequencing or PCR-RFLP of ITS regions using restriction enzymes MboII and HinfI.
Asunto(s)
Saccharomycetales/clasificación , Composición de Base , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/genética , Variación Genética , Hawaii , Datos de Secuencia Molecular , América del Norte , Fenotipo , Filogenia , Saccharomycetales/genética , Saccharomycetales/aislamiento & purificación , Saccharomycetales/metabolismo , SudáfricaRESUMEN
Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.