Phylogenetic analysis of isolated biofuel yeasts based on 5.8S-ITS rDNA and D1/D2 26S rDNA sequences

The utilization of agro-industrial wastes such as whey as raw materials for the production of bio-ethanol is gaining importance as a result of the attractiveness of renewable fuel alternatives due to exhaustion of fossil fuel sources coupled with the positive impact to the environment. Here, we report the isolation of two Kluyveromyces spp. designated as BM4 and P41, able to produce ethanol as main fermentation product from fermenting whey. Three different molecular biological approaches including, the RFLP analysis of the 5.8S-ITS rDNA, the sequence of the 5.8S-ITS rDNA region and the sequence of the D1/D2 domain of the 26S rRNA gene were applied for accurate identification. While RFLP analysis of 5.8S-ITS region failed to accurate the differentiation between the two species, sequencing of this region and D1/D2 region of the 26S rRNA gene verified the identification. PCR amplification and sequence analysis of 5.8S-ITS rRNA and D1/D2 domain of the 26S rRNA genes revealed that the isolates BM4 and P41 were highly related to Kluyveromyces marxianus and Kluyveromyces lactis with homology of 99% for both. In addition, phylogenetic analysis indicated that both BM4 and P41 shared a cluster with K. marxianus and K. lactis, respectively. The fermentative performance of both strains on cheese whey to produce ethanol was evaluated at different parameters such as incubation temperature, initial pH, whey sugar concentrations, and yeast concentrations. Results show that the maximum ethanol productions achieved at pH 4.5 and 35 °C were 5.52% and 5.05% for K. marxianus and K. lactis, respectively. Our results demonstrated that K. marxianus and K. Lactis could be recommended for cheese whey bioremediation in the environment and produce renewable biofuel.     

The use of karyotyping in the systematics of yeasts

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungiHortaea werneckii, Filobasidiella (=Cryptococcus)neoformans, andMalassezia species.Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes ofFilobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varietiesneoformans andbacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains ofMalassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas inM. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.     

Molecular characterisation of Hanseniaspora species

The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined. From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups. This subdivision was supported by electrophoretic chromosome patterns. Hanseniaspora guilliermondii, H. uvarum and H. valbyensis show 6–7 bands (8 to 9 chromosomes), while the second group comprises the species H. occidentalis, H. osmophila and H. vineae which have only 5 chromosomes.     

Molecular characterisation of the species of the genus Zygosaccharomyces

The restriction fragments polymorphisms of the mitochondrial DNA and the PCR fragment that comprised the internal transcribes spacers and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 40 strains from the 10 species of the genus Zygosaccharomyces, including the new species Z. lentus were examined. The RFLP's of the ITS-5.8S region showed a specific restriction pattern for each species, including the new species Z. lentus. The only exception were the species Z. cidri and Z. fermentati that produced identical restriction profiles. The electrophoretic chromosome patterns confirmed the differences between the species of this genus, including the phylogenetic closest species Z. cidri and Z. fermentati. They present few chromosomes ranging from 3 bands (4 or 5 chromosomes) for Z. florentinus to 7 bands (8 to 10 chromosomes) for Z. cidri and Z. fermentati. The strain level resolution power of RFLP's of mtDNA of this genus enabled the characterisation of strains from the same species, even where they are isolated from the same substrate. However, in the cases of Z. bailii and Z. lentus, electrophoretic karyotyping there was considerable variation.     

Sequence-based identification of species belonging to the genus Debaryomyces

In the present study we assessed the identification by sequence analysis of the 15 species belonging to the genus Debaryomyces. We found that the following species can be identified both quickly and correctly by direct sequence comparison of the ribosomal 5.8S-ITS region: D. carsonii, D. etchelsii, D. maramus, D. melissophilus, D. occidentalis and D. yamadae. In contrast, the species D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. polymorphus, D. pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae showed high sequence similarity in ribosomal regions with one or several species. In these cases, sequence comparison of the ACT1 gene is proposed to ensure unequivocal strain designation.     

Phylogenetic analysis of basidiomycetous yeasts by means of 18S ribosomal RNA sequences: relationship ofErythrobasidium hasegawianum and other basidiomycetous yeast taxa

The basidiomycetous yeast genusErythrobasidium Hamamoto, Sugiyama & Komagata, based on the type speciesE. hasegawianum Hamamoto et al., is characterized by filobasidiaceous basidia and the Q-10 (H₂) system as its major ubiquinone. It is tentatively placed in the Filobasidiaceae. The molecular characterization is based on 18S ribosomal RNA sequence comparisons among the basidiomycetous yeasts, and the ultrastructural characterization on the cell wall and hyphal septal pores inE. hasegawianum clearly indicate a close relationship with the teliospore-forming yeastsRhodosporidium toruloides andLeucosporidium scottii. Our molecular phylogeny with statistical analysis suggests that the existing taxonomic system of basidiomycetous yeasts, based primarily on the morphology of basidia including the teliospores (probasidia), should be revised.     

The expanding realm of ballistosporous yeasts

The recent progress in the systematics of ballistosporous yeasts is discussed. The extensive isolation studies carried out in the last decade resulted in a marked increase in the number of ballistosporous yeast species which now number nearly fifty. The increased number of species has expanded the complexity of taxonomic properties in the following ways: expansion of the range of mol% G+C of nuclear DNA to 39–68.5, thus overlapping that of all basidiomycetous yeasts; increased complexity of conidiogenesis with the finding of the generaBallistosporomyces andKockovaella. Based on partial sequencing of 18S ribosomal RNA (positions 1451–1618 inSaccharomyces cerevisiae), it is suggested that mode of conidiogenesis has little value for defining genera. Consequently, ballistosporous yeasts merely represent the ballistosporous stage of various taxa which cover the whole evolutionary spectrum of basidiomycetous yeasts. The importance of continuing isolation of ballistosporous yeasts is stressed, which together with molecular studies, will aid further progress in the systematics of basidiomycetous yeasts.     

Characterization of the genome of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from red berry wines

Using restriction analysis of noncoding rDNA regions, multiplex PCR, and molecular karyotyping, we have examined Saccharomyces strains isolated from red berry wine materials in Russia, Belarus, and Ukraine. According to the molecular analysis, all strains belong to the species S. cerevisiae. A correlation was revealed between microsatellite fingerprints of the strains and the source of their isolation. The strains isolated from juices and from the surface of different berries showed distinct PCR profiles. The genome compositions of interspecific Saccharomyces hybrids of natural and laboratory origin were studied.     

Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l⁻¹. A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)₅ and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

Molecular systematics of the genus Allodontichthys (Cyprinodontiformes: Goodeidae)

Phylogenetic analyses of cytochrome c oxidasesubunit I (627bp) and mitochondrial controlregion (338bp aligned) sequences for all knownspecies of Allodontichthys wereperformed. Allodontichthys was recoveredas monophyletic, and A. hubbsi wassupported as the most primitive member of thegenus in all analyses. Combined-data analysessuggest that A. polylepis is sister to aclade comprising A. tamazulae and A. zonistius; however, a strict consensus of COIand control region cladograms results in acollapse of this node. The two species thatare broadly sympatric, A. hubbsi and A. tamazulae, are not sister taxa.Allodontichthys was found the sistergroup of a clade including Ilyodon andXenotaenia. These three genera are theonly goodeids to occupy high-gradient riversystems. Ilyodon and Allodontichthys share similar distributions;however, Allodontichthys exhibits greaterthan an order of magnitude more geneticvariation in COI than Ilyodon incomparisons of individuals from the samelocalities in the Ameca and Armeria drainagesystems. This is interpreted to mean Allodontichthys arrived in some westerndrainages significantly before Ilyodon.The Rio Coahuayana appears to be the center oforigin of the genus Allodontichthys. This river basin contains the most primitivemember of the genus, and appears to haveconnected with the Mesa Central, the center of endemism of the Goodeidae. A calibrated rateof molecular change in COI reveals Allodontichthys began diverging approximately6.2 million years ago (mya). Remainingspeciation in the group appears to haveoccurred about 2.7 to 3.3 mya. The hypothesisthat a hard polytomy comprising A. zonistius, A. polylepis, and A. tamazulae could be explained by speciation ofA. polylepis and A. tamazulae fromdifferent A. zonistius ancestors ispresented. Stream capture is evident but notfrequent in the history of Allodontichthys, and speciation does notclearly correlate with documented geologicactivity of the Mexican Transvolcanic Belt.     

Analysis of Pteridium ribosomal RNA sequences by rapid direct sequencing

A total of 864 bases from 5 regions interspersed in the 18S and 26S rRNA molecules from various clones of Pteridium covering the general geographical distribution of the genus was analysed using a rapid rRNA sequencing technique. No base difference has been detected amongst the three major lineages, two of which apparently separated before the breakup of the ancient supercontinent, Pangaea. These regions of the rRNA sequences have thus been conserved for at least 160 million years and are here compared with other eukaryotic, especially plant rRNAs.     

Molecular systematics of the Atlanto-Mediterranean Solea species

Within the genus Solea , four sister lineages have been evolved that actually correspond to the species S. vulgaris, S. senegalensis, S. kleini and S. lascaris . The existence of S. aegyptiaca and S. impar is not supported and these species can be synonymized under S. vulgaris and S. lascaris , respectively. This pattern of taxonomy agrees fully with that proposed by Ben-Tuvia (1990; Journal of Fish Biology 36, 947–960) based on morphological features. In addition, mtDNA data contradict the widely accepted relatedness between S. kleini and S. lascaris based on the shared enlarged forenostril on the blind side. A sample of soles clearly related to S. senegalensis was caught in the Gulf of Taranto (Ionian Sea), representing the most western finding of this species in the Mediterranean Sea.     

Molecular systematics of Acarus siro s. lat., a complex of stored food pests

The astigmatid mite Acarus siro (Linnaeus 1758) is an important agricultural pest and environmental allergen. However, it is likely that many mites described in the literature as A. siro, collected from both outdoor and stored product habitats, may belong to one of its sibling species, A. farris [Ent. Ber. Amst. 2 (26) (1905) 20] or A. immobilis [Bull. Br. Mus. Nat. Hist. 11 (1964a) 413; Acarologia. 6 (Suppl) (1964) 101]. The three species are difficult to separate morphologically, gene exchange between some of them is possible and, although each species displays environmental preferences, they occur together in some environments. This raises a question about their separate species status. In a pilot study, we investigated whether genetic data supported the separate species status of these forms. Both nuclear (the second internal transcribed spacer region [ITS-2] of the ribosomal cistron) and mitochondrial (cytochrome oxidase subunit I, mtcoxI hereafter) loci were employed for this purpose. Mtcox1 data does not conflict the differentiation into three separate species and while the ITS2 data were problematic for this group of mites it suggested that a congener, Acarus gracilis [Ann. Mag. Nat. Hist. 10 (1957) 753], is basal to the A. siro species complex.     

5S and 5.8S ribosomal RNA evolution in the suborder tetrahymenina (Ciliophora: Hymenostomatida)

Summary The nucleotide sequences of the 5S and 5.8S rRNAs of eight strains of tetrahymenine ciliates have been determined. The sequences indicate a clear distinction betweenTetrahymena paravorax and its suggested conspecificT. vorax, but leave the taxonomic distinction betweenT. vorax andT. leucophrys in doubt. The rRNA sequences of sixTetrahymena species and of three other species of the suborder Tetrahymenina have been used to deduce evolutionary schemes in which ancestral rRNA sequences and changes are proposed. These schemes suggest the predominant acceptance of GA and CT transitions in the 5S rDNA during the evolution of the suborder.     

Phylogenetic relationships ofCryptococcus neoformans and some related basidiomycetous yeasts determined from partial large subunit rRNA sequences

The genusCryptococcus was found to be heterogeneous on the basis of partial rRNA sequences. The human-pathogenic speciesC. neoformans, comprising 4 serotypes and havingFilobasidiella neoformans andF. bacillispora as teleomorphs, was found at a relatively large distance fromFilobasidium. Serotypes B and C had identical sequences, while in A and D they were different, with D closer to B and C than to A.Filobasidiella depauperata, which lacks a yeast-like anamorph, clustered withF. neoformans.The genusFilobasidium was clearly separated fromFilobasidiella and clustered withC. albidus, C. kuetzingii, C. gastricus, C. lupi, C. vishniaciae, C. bhutanensis, C. aerius, C. terreus andC. ater. The latter may represent the anamorph ofFilobasidium elegans. The organe to red species ofCryptococcus, as well asC. aquaticus andC. yarrowii, were found completely unrelated with these taxa,C. macerans being affiliated toCystofilobasidium capitatum.The genusTrichosporon was found relatively homogeneous; it includesC. humicola, C. curvatus and the filamentous speciesHyalodendron lignicola. Cryptococcus flavus andC. dimennae probably belong to the Tremellales, though distances between these species are large. The positions ofC. laurentii andC. luteolus remains to be determined.     

Molecular biology and systematics of an extinct Lemur:Pachylemur insignis

The systematic position of the extinctPachylemur insignis has been controversial: some authors consideredPachylemur as a lemur, whereas others viewed it closer toVarecia. Its classification in the genusLemur orVarecia thus remained an open question. DNA extraction from subfossil bones, using a non-destructive method, allowed us to obtain enough material to make a Southern blot. The hybridization ofPachylemur withEulemur fulvus, Lemur catta, andVarecia variegata highly repeated DNA probes showed that only theVarecia probe gave a positive signal on hybridization on thePachylemur blot. These results indicate thatPachylemur must be considered closer to the genusVarecia than toEulemur andLemur.     

DNA relatedness among saturn-spored yeasts assigned to the generaWilliopsis andPichia

Saturn-spored species assigned to the generaWilliopsis andPichia were compared from extent of nuclear DNA complementarity. Of thePichia spp., four were recognized as distinct taxa:P. dispora, P. saitoi, P. zaruensis andPichia sp. nov. AmongWilliopsis spp., the following were accepted:W. californica, W. mucosa comb. nov.,W. pratensis, W. saturnus var.saturnus, W. saturnus var.mrakii comb. nov.,W. saturnus var.sargentensis comb. nov.,W. saturnus var.subsufficiens comb. nov. andWilliopsis sp. nov. The newPichia andWilliopsis species are described elsewhere. Moderate (36–68%) DNA relatedness was detected between the formerPichia sargentensis and varieties ofW. saturnus again demonstrating that nitrate assimilation is not a reliable criterion for separating yeast species.