Reconstruction of molecular phylogeny of closely related <Emphasis Type="Italic">Amorphophallus</Emphasis> species of India using plastid DNA marker and fingerprinting approaches

Plastid DNA markers sequencing and DNA fingerprinting approaches were used and compared for resolving molecular phylogeny of closely related, previously unexplored Amorphophallus species of India. The utility of individual plastid markers namely rbcL, matK, trnH–psbA, trnLC–trnLD, their combined dataset and two fingerprinting techniques viz. RAPD and ISSR were tested for their efficacy to resolves Amorphophallus species into three sections specific clades namely Rhaphiophallus, Conophallus and Amorphophallus. In the present study, sequences of these four plastid DNA regions as well as RAPD and ISSR profiles of 16 Amorphophallus species together with six varieties of two species were generated and analyzed. Maximum likelihood and Bayesian Inference based construction of phylogenetic trees indicated that among the four plastid DNA regions tested individually and their combined dataset, rbcL was found best suited for resolving closely related Amorphophallus species into section specific clades. When analyzed individually, rbcL exhibited better discrimination ability than matK, trnH–psbA, trnLC–trnLD and combination of all four tested plastid markers. Among two fingerprinting techniques used, the resolution of Amorphophallus species using RAPD was better than ISSR and combination of RAPD +ISSR and in congruence with resolution based on rbcL.     

Comparative evaluation of automated ribotyping and RAPD-PCR for typing of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. occurring in dental caries

A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (?vec et al., Folia Microbiol 54:53–58, 2009) was characterized by automated ribotyping performed by the RiboPrinter^® microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.     

Yeast species diversity in apple juice for cider production evidenced by culture-based method

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.     

Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.     

Yeasts from peat in a tropical peat swamp forest in Thailand and their ability to produce ethanol,indole-3-acetic acid and extracellular enzymes

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens, Debaryomyces fabryi, Meyerozyma guilliermondii, Saturnispora diversa and Schwanniomyces polymorphus var. africanus, and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides, Papiliotrema flavescens, Papiliotrema laurentii and Rhodotorula mucilaginosa. Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus, Cy. subsufficiens and D. fabryi. None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.     

Taxonomic hierarchy of the phylum <Emphasis Type="Italic">Firmicutes</Emphasis> and novel <Emphasis Type="Italic">Firmicutes</Emphasis> species originated from various environments in Korea

This study assessed the taxonomic hierarchy of the phylum Firmicutes as well as elucidated the isolation and classification states of novel Firmicutes species isolated from Korean territory. The hierarchical classification system of the phylum Firmicutes has been developed since 1872 when the genus Bacillus was first reported and has been generally adopted since 2001. However, this taxonomic hierarchy is still being modified. Until Feb. 2017, the phylum Firmicutes consisted of seven classes (Bacilli, Clostridia, Erysipelotrichia, Limnochordia, Negativicutes, Thermolithobacteria, and Tissierellia), 13 orders, 45 families, and 421 genera. Firmicutes species isolated from various environments in Korea have been reported from 2000, and 187 species have been approved as of Feb. 2017. All Firmicutes species were affiliated with three classes (Bacilli, Clostridia, and Erysipelotrichia), four orders (Bacillales, Lactobacillales, Clostridiales, and Erysipelotrichales), 17 families, and 54 genera. A total of 173 species belong to the class Bacilli, of which 151 species were affiliated with the order Bacillales and the remaining 22 species with the order Lactobacillales. Twelve species belonging to the class Clostridia were affiliated within only one order, Clostridiales. The most abundant family was Bacillaceae (67 species), followed by the family Paenibacillaceae (56 species). Thirteen novel genera were created using isolates from the Korean environment. A number of Firmicutes species were isolated from natural environments in Korean territory. In addition, a considerable number of species were isolated from artificial resources such as fermented foods. Most Firmicutes species, belonging to the families Bacillaceae, Planococcaceae, and Staphylococcaceae, isolated from Korean fermented foods and solar salterns were halophilic or halotolerant. Firmicutes species were isolated from the whole territory of Korea, especially large numbers from Provinces Gyeonggi, Chungnam, and Daejeon.     

Genetic Diversity of <Emphasis Type="Italic">Arcobacter</Emphasis> Species of Animal and Human Origin in Andhra Pradesh,India

Arcobacter is an emerging foodborne pathogen having zoonotic significance. Enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive sequence-based PCR (rep-PCR) analysis of a total of 41 Arcobacter isolates revealed a greater degree of genetic diversity. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power for ERIC and rep-PCR was found to be 0.997 and 0.996, respectively. Close clustering between isolates of animal and human origin are indicative of probable zoonotic significance.     

Identification of yeasts during alcoholic fermentation of <Emphasis Type="Italic">tchapalo</Emphasis>, a traditional sorghum beer from Côte d’Ivoire

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d’Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d’Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.     

Taxonomic revisions in the Microstromatales: two new yeast species,two new genera,and validation of <Emphasis Type="Italic">Jaminaea</Emphasis> and two <Emphasis Type="Italic">Sympodiomycopsis</Emphasis> species

Environmental sampling yielded two yeast species belonging to Microstromatales (Exobasidiomycetes, Ustilaginomycotina). The first species was collected from a leaf phylloplane infected by the rust fungus Coleosporium plumeriae, and represents a new species in the genus Jaminaea, for which the name Jaminaea rosea sp. nov. is proposed. The second species was isolated from air on 50% glucose media and is most similar to Microstroma phylloplanum. However, our phylogenetic analyses reveal that species currently placed in Microstroma are not monophyletic, and M. phylloplanum, M. juglandis and M. albiziae are not related to the type species of this genus, M. album. Thus, Pseudomicrostroma gen. nov. is proposed to accommodate the following species: P. glucosiphilum sp. nov., P. phylloplanum comb. nov. and P. juglandis comb. nov. We also propose Parajaminaea gen. nov. to accommodate P. albizii comb. nov. and P. phylloscopi sp. nov. based on phylogenetic analyses that show these are not congeneric with Jaminaea or Microstroma. In addition, we validate the genus Jaminaea, its respective species and two species of Sympodiomycopsis and provide a new combination, Microstroma bacarum comb. nov., for the anamorphic yeast Rhodotorula bacarum. Our results illustrate non-monophyly of Quambalariaceae and Microstromataceae as currently circumscribed. Taxonomy of Microstroma and the Microstromataceae is reviewed and discussed. Finally, analyses of all available small subunit rDNA sequences for Jaminaea species show that J. angkorensis is the only known species that possess a group I intron in this locus, once considered a potential feature indicating the basal placement of this genus in Microstromatales.     

Identification of yeasts isolated from raffia wine (<Emphasis Type="Italic">Raphia hookeri</Emphasis>) produced in Côte d’Ivoire and genotyping of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains by PCR inter-delta

Raffia wine is a traditional alcoholic beverage produced in several African countries where it plays a significant role in traditional customs and population diet. Alcoholic fermentation of this beverage is ensured by a complex natural yeast flora which plays a decisive role in the quality of the final product. This present study aims to evaluate the distribution and the diversity of the yeast strains isolated in raffia wine from four sampling areas (Abengourou, Alépé, Grand-Lahou and Adzopé) in Côte d’Ivoire. Based on the D1/D2 domain of the LSU rDNA sequence analysis, nine species belonging to six genera were distinguished. With a percentage of 69.5 % out of 171 yeast isolates, Saccharomyces cerevisiae was the predominant species in the raffia wine, followed by Kodamaea ohmeri (20.4 %). The other species isolated were Candida haemulonii (4.1 %), Candida phangngensis (1.8 %), Pichia kudriavzevii (1.2 %), Hanseniaspora jakobsenii (1.2 %), Candida silvae (0.6 %), Hanseniaspora guilliermondii (0.6 %) and Meyerozyma caribbica (0.6 %). The molecular characterization of S. cerevisiae isolates at the strain level using the PCR-interdelta method revealed the presence of 21 profiles (named I to XXI) within 115 isolates. Only four profiles (I, III, V and XI) were shared by the four areas under study. Phenotypic characterization of K. ohmeri strains showed two subgroups for sugar fermentation and no diversity for the nitrogen compound assimilations and the growth at different temperatures.     

Characterization of the fungal flora of dolo,a traditional fermented beverage of Burkina Faso,using MALDI-TOF mass spectrometry

A cross-sectional study was conducted in Bobo-Dioulasso, Burkina Faso, to identify the yeast diversity associated with the manufacture of dolo, a traditional fermented beverage of Burkina Faso. From sixty specimens spread onto chromogenic medium plates, sixty-two strains were isolated then identified using MALDI-TOF analysis. Seven yeast species were identified, Saccharomyces cerevisiae (39%) followed by Pichia manshurica (18%) being the most frequent. Forty-three percent of the samples contained Candida species, notably Candida albicans. In conclusion, the combined use of a chromogenic medium and MALDI-TOF analysis reveals a higher diversity in yeast species present in the dolo than previously thought.     

New family of pectinase genes <Emphasis Type="Italic">PGU1</Emphasis>b–<Emphasis Type="Italic">PGU3b</Emphasis> of the pectinolytic yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.     

Phylogenetic analysis of <Emphasis Type="Italic">Plectosphaerella</Emphasis> species based on multi-locus DNA sequences and description of <Emphasis Type="Italic">P. sinensis</Emphasis> sp. nov.

Species in Plectosphaerella are well known as pathogens of several plant species causing fruit, root and collar rot and collapse. In an investigation of endophytic fungi associated with cucurbit plants in China, we isolated 77 strains belonging to the genus Plectosphaerella. To identify the isolated strains, we collected the type or reference strains of all currently accepted species in Plectosphaerella except P. oratosquillae and conducted a phylogenetic analysis. Phylogenetic analysis of the partial 28S rDNA sequences showed that all species in Plectosphaerella were located in one clade of Plectosphaerellaceae. Based on multi-locus phylogenetic analysis of the ITS, CaM, EF1, TUB and morphological characteristics, all species in Plectosphaerella were well separated. Three endophytic strains from stems of Cucurbita moschata, Citrullus lanatus and Cucumis melo from North China were assigned to a new species described as P. sinensis in this paper. The new species differs morphologically from other Plectosphaerella species by irregular chlamydospores, and the dimensions of phialides and conidia. The other endophytic strains from several cucurbit plants were identified as P. cucumerina.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Seasonal and altitudinal changes of culturable bacterial and yeast diversity in Alpine forest soils

The effect of altitude and season on abundance and diversity of the culturable heterotrophic bacterial and yeast community was examined at four forest sites in the Italian Alps along an altitude gradient (545–2000 m). Independently of altitude, bacteria isolated at 0 °C (psychrophiles) were less numerous than those recovered at 20 °C. In autumn, psychrophilic bacterial population increased with altitude. The 1194 bacterial strains were primarily affiliated with the classes Alpha-, Beta-, Gammaproteobacteria, Spingobacteriia and Flavobacteriia. Fifty-seven of 112 operational taxonomic units represented potential novel species. Strains isolated at 20 °C had a higher diversity and showed similarities in taxa composition and abundance, regardless of altitude or season, while strains isolated at 0 °C showed differences in community composition at lower and higher altitudes. In contrast to bacteria, yeast diversity was season-dependent: site- and altitude-specific effects on yeast diversity were only detected in spring. Isolation temperature affected the relative proportions of yeast genera. Isolations recovered 719 strains, belonging to the classes Dothideomycetes, Saccharomycetes, Tremellomycetes and Mycrobotryomycetes. The presence of few dominant bacterial OTUs and yeast species indicated a resilient microbial population that is not affected by season or altitude. Soil nutrient contents influenced significantly abundance and diversity of culturable bacteria, but not of culturable yeasts.     

Yeast population of the Kindo Peninsula lichens

Yeast abundance and species diversity in the lichens collected at the Kindo Peninsula (Karelia) were studied. A total of 14 lichen species analyzed belonged to the genera Bryoria, Cladonia, Hypogymnia, Icmadophila, Nephroma, Peltigera, and Ramalina. Abundance of cultured yeasts in lichens was intermediate between soil and phyllosphere. The average yeast number on lichens was ~2.5 × 10³ CFU/g, while it exceeded 8 × 10³ CFU/g on plants and reached only 1 × 10³ CFU/g in soil. Yeast population of different parts of Cladonia lichens was found to vary significantly in abundance, species diversity, and community structure. The highest yeast abundance and diversity were revealed in the growth zone. Fifteen yeast species were isolated from lichens, including 6 basidiomycetous and 9 ascomycetous ones. Unlike soils and plants, yeast population of lichens consisted mainly of ascomycetous species, with predominance of Candida sphagnicola and anamorphous yeasts of the genus Dothiora. These results show that yeasts from different taxonomic and ecological groups are a necessary component of lichens; conditions favoring the preservation and development of specific yeast communities differing from the typical soil and phyllosphere yeast complexes are formed in the lichens of northern taiga forests.     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Malcofaunal Diversity of Chilika Lake,Odisha, India

Investigation during the period of 3 years from 2007 to 2010 on the malacofauna of Chilika lake revealed the occurrence of 126 molluscan taxa belonging to 56 families, 18 orders of three classes in the bottom sediment. Of these 61 species belonged to Bivalvia, 64 species belonged to Gastropoda and one species belonged to Polyplacophora. Maximum Bivalvia and Gastropoda taxa were found in the outer channel region of the lake. The dominating species were Crassostrea cuttackensis, Saccostrea cucullata, Brachidontes undulatus, Meretrix meretrix among bivalves and Cerethideopsilla cingulata, Bullia vittata, Nassarious stolatus, Indothias lacera, Natica tigrina, Turritella attenuata were from the gastropods. Occurrence of a large number of marine taxa is most probably associated with the opening of new lagoon during 1st August 2008.     

A RAPD fingerprinting of sibling species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group

A comparative analysis of the sibling species of Drosophila virilis was performed by RAPD-PCR technique using a set of random primers. The degree of relatedness was studied by cluster analysis (UPGMA) and multidimensional scaling. The resulting pattern of species relationships contradicts the classical taxonomy. The main result of the cluster analysis is that D. virilis does not cluster with the remaining three species of its phylad, while according to multidimensional scaling, D. virilis is equidistant from all the species of its group, from both the species of its phylad and the species of the montana phylad. The montana phylad is extremely heterogeneous; moreover, the species D. littoralis, D. ezoana, and D. kanekoi appear to be closer to the virilis phylad than to the other species of the montana phylad, wherein these species are traditionally included. The phylogenetic relationships between the studied species discovered using RAPD fingerprinting comply with the results obtained using protein markers and quantitative traits.