Molecular phylogenetics of the genus trichosporon inferred from mitochondrial cytochrome B gene sequences

Mitochondrial cytochrome b (cyt b) genes of 42 strains representing 23 species of the genus Trichosporon were partially sequenced to determine their molecular phylogenetic relationships. Almost half of the 22 strains investigated (from 11 different species) contained introns in their sequences. Analysis of a 396-bp coding sequence from each strain of Trichosporon under investigation showed a total of 141 (35.6%) variable nucleotide sites. A phylogenetic tree based on the cyt b gene sequences revealed that all species of Trichosporon except Trichosporon domesticum and Trichosporon montevideense had species-specific cyt b genes. Trichosporon sp. strain CBS 5581 was identified as Trichosporon pullulans, and one clinical isolate, IFM 48794, was identified as Trichosporon faecale. Analysis of 132-bp deduced amino acid sequences showed a total of 34 (25.75%) variable amino acid sites. T. domesticum and T. montevideense, Trichosporon asahii and Trichosporon asteroides, and Trichosporon gracile and Trichosporon guehoae had identical amino acid sequences. A phylogenetic tree constructed with the ascomycetes Saccharomyces douglasii and Candida glabrata taken as outgroup species and including representative species from closely related genera species of Trichosporon clustered with other basidiomycetous yeasts that contain xylose in their cell wall compositions. These results indicate the effectiveness of mitochondrial cyt b gene sequences for both species identification and the phylogenetic analysis of Trichosporon species.     

Update on the genus Malassezia.

Malassezia yeasts are commensals of normal human skin, but also cause pityriasis versicolor, seborrhoeic dermatitis and evidence is accumulating that they play a significant role in atopic eczema/dermatitis syndrome (AEDS; formerly atopic dermatitis). The taxonomy of the genus has changed considerably and is likely to change more in the future. Our understanding of the interaction between Malassezia and the host demonstrates that it has the paradoxical ability to both stimulate and suppress the immune response directed against it and there is a fine balance in its existence at the interface between commensalism and pathogenicity.     

Taxonomy of genus Malassezia: progress and problems]

The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M. pachydermatis and six lipid-dependent species, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae. Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.     

Phylogenetic analysis of members of the metabolically diverse genus Gordonia based on proteins encoding the gyrB gene

Members of the metabolically diverse genus Gordonia were isolated from various biotopes including pristine and polluted sites around Taiwan. Identification, comparison and diversity assessment based on the gyrB gene were carried out using a newly developed primer pair for gyrB. The 16S rRNA gene was also sequenced for comparison. A 1.2-kb fragment of the gyrB gene of 17 Gordonia strains including type strains was determined by direct sequencing of PCR amplified fragments. A total of 25 strains (8 of which were retrieved from a public database) of the genus Gordonia form a distinct phyletic line in the GyrB-based tree and are separated from other closely related species of genera of the suborder Corynebacterineae. Sequence similarity of the gyrB sequence from twelve Gordonia type strains ranged from 79.3 to 97.2%, corresponding to between 270 and 41 nucleotide differences, while there was only a 0.3-3.8% difference in 16S rRNA gene sequence similarity at the interspecies level. Phylogenetic analysis based on the GyrB sequence deduced from the gyrB gene is consistent with that of DNA-DNA hybridization results and provides a better discrimination within the species of Gordonia compared to the 16S rRNA gene. The present study demonstrates that gyrB gene analysis will aid in describing novel species belonging to the genus Gordonia.     

Identification of Candida dubliniensis based on the specific amplification of mitochondrial cytochrome b gene.

Candida dubliniensis, a recently described Candida species, is frequently isolated from oral candidiasis in human immunodeficiency virus infected individuals. To detect the organism rapidly, we have developed specific oligonucleotide primers based on the sequences of mitochondrial cytochrome b gene. These primers selectively amplified DNA only from C. dubliniensis; the DNAs of all pathogenic Candida species tested, as well as those of medically relevant yeasts such as Cryptococcus neoformans, and Trichosporon cutaneum, were not amplified. This is the first report describing the effectiveness of cytochrome b gene in PCR based detection of an organism, and we hope the system will be useful as a microbiological tool for rapid detection of C. dubliniensis.     

Nucleotide sequence of the cytochrome B gene and adjacent regions from rat liver mitochondrial DNA

Summary The nucleotide sequence of a 1.76 Kbp segment from rat liver mitochondrial DNA has been determined. It contains the gene for the cytochrome b apoprotein and the tRNA genes for threonine, glutamic acid, and proline. The arrangement of these genes in rat liver mitochondria is the same as in human (Anderson et al. 1981) or mouse (Bibb et al. 1981) mitochondria. The comparison of the cytochrome b sequences so far determined, reveals that several regions in these proteins are highly conserved. Abbreviations: Cyt b, cytochrome b apoprotein; U.R.F., unidentified reading frame (Anderson et al. 1981). Other abbreviations follow IUB-IUPAC conventions     

Characterization of the mitochondrial cytochrome b gene from Venturia inaequalis

A new class of agricultural fungicides derived from the group of antifungal strobilurins acts as specific respiration inhibitors by binding to mitochondrial cytochrome b. The cytochrome b gene was cloned and sequenced from the mitochondrial genome of Venturia inaequalis, the causal agent of apple scab. The gene was 10.65 kbp in size and contained seven exons and six introns. The exons encoded a protein of 393 amino acids. Comparison of the deduced amino-acid sequence with cytochrome b proteins from other fungi revealed highest homologies to the respective proteins of Aspergillus nidulans, Podospora anserina and Neurospora crassa. All amino acids of the V. inaequalis cytochrome b at positions altered in mutants of Saccharomyces cerevisiae resistant to strobilurins, and other fungi with reduced sensitivities to strobilurins, were identical to wild-type isolates of several fungi. The cloning and characterization of the V. inaequalis cytochrome b gene is the initial step in the assessment of resistance risks inherent to the strobilurin fungicides. Received: 2 May / Accepted: 7 August 1997     

Identification and phylogenetic relationship of the most common pathogenic Candida species inferred from mitochondrial cytochrome b gene sequences

We sequenced a 396-bp region of the mitochondrial cytochrome b gene of the most common clinically important Candida species: Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae. The recently described species of Candida, C. dubliniensis, associated with mucosal candidiasis in human immunodeficiency virus-infected individuals, was also included. Two to five strains of each species were examined. Some species represented intraspecies variation, which was not more than 1.8% (DNA). However, interspecies variations were more than 10 and 7%, respectively, for DNA and amino acid sequences. Multiple alignments of nucleotide and deduced amino acid sequences revealed species-specific nucleotides and amino acids. Nucleotide- and amino acid-based phylogenetic trees were constructed and are discussed. Using the database, it is possible to identify presumptive Candida species within a working day.     

Polymorphic variants in the human mitochondrial cytochrome b gene.

We report the polymorphic variants of the human cytochrome b gene based on sequence analysis in 32 Caucasian individuals. We found 27 variants (12 synonymous changes and 15 amino acid replacements). Of these, 15 (8 silent changes and 7 amino acid replacements) have not been previously reported. Based on restriction length polymorphism analysis of patients and their maternal relatives, we conclude that these new amino acid replacements represent maternally inherited polymorphisms. Comparative analysis of the data suggests that four different genotypes can be defined for the human cytochrome b gene.     

Uniparental inheritance of the mitochondrial gene cytochrome b in Plasmodium falciparum

The inheritance of an extrachromosomal 6-kb element has been examined in the human malaria parasite Plasmodium falciparum. A single base pair difference in the cytochrome b gene from the 6-kb element of two different cloned lines of the parasite was identified, and used as a marker in a cross in the mosquito stage of the life cycle. Analysis of 59 individual hybrid oocysts resulting from this cross clearly demonstrated that inheritance of the cytochrome b gene was uniparental. This observation makes it possible to investigate the inheritance and evolution of cytoplasmic traits, including certain forms of drug resistance, in natural populations of this parasite.     

Phylogenetic analysis of Demodex caprae based on mitochondrial 16S rDNA sequence

Demodex caprae infests the hair follicles and sebaceous glands of goats worldwide, which not only seriously impairs goat farming, but also causes a big economic loss. However, there are few reports on the DNA level of D. caprae. To reveal the taxonomic position of D. caprae within the genus Demodex, the present study conducted phylogenetic analysis of D. caprae based on mt16S rDNA sequence data. D. caprae adults and eggs were obtained from a skin nodule of the goat suffering demodicidosis. The mt16S rDNA sequences of individual mite were amplified using specific primers, and then cloned, sequenced, and aligned. The sequence divergence, genetic distance, and transition/transversion rate were computed, and the phylogenetic trees in Demodex were reconstructed. Results revealed the 339-bp partial sequences of six D. caprae isolates were obtained, and the sequence identity was 100 % among isolates. The pairwise divergences between D. caprae and Demodex canis or Demodex folliculorum or Demodex brevis were 22.2–24.0 %, 24.0–24.9 %, and 22.9–23.2 %, respectively. The corresponding average genetic distances were 2.840, 2.926, and 2.665, and the average transition/transversion rates were 0.70, 0.55, and 0.54, respectively. The divergences, genetic distances, and transition/transversion rates of D. caprae versus the other three species all reached interspecies level. The five phylogenetic trees all presented that D. caprae clustered with D. brevis first, and then with D. canis, D. folliculorum, and Demodex injai in sequence. In conclusion, D. caprae is an independent species, and it is closer to D. brevis than to D. canis, D. folliculorum, or D. injai.     

Chitin Synthase 2 (CHS2) gene of Malassezia species]

Malassezia species have been recognized as members of the microbiological flora of human and animal skin; they are also considered to play an important role in the pathogenesis of folliculitis, atopic dermatitis and otitis externa. Therefore, the molecular characteristics were investigated to clarify the epidemiology and the pathogenesis of diseases associated with Malassezia species in human and animals. Molecular investigation was made of 105 clinical isolates of M. pachydermatis from dogs and cats by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD analysis and CHS2 gene analysis indicated that clinical isolates of M. pachydermatis were divided into four distinct genetic types (A, B, C and D). Type A was isolated from lesions of atopic dermatitis, flea allergic dermatitis, otitis externa, pyoderma and seborrheic (dermatitidis) in dogs and cats, and might be predominant on this. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of standard strains of 11 Malassezia species showed 11 distinct clusters of this species.     

Molecular analysis of Cryptococcus neoformans mitochondrial cytochrome b gene sequences

Mitochondrial cytochrome b genes (cyt b) of 40 strains of Cryptococcus neoformans were partially sequenced to determine the genetic relations. With the exception of the type strain of C. neoformans var. neoformans, all strains contained introns in their sequences. Analysis of 386 bp of coding sequence from each strain under investigation revealed a total of 27 (6.99%) variable nucleotide sites and categorized isolates of C. neoformans into nine cyt b types. C. neoformans var. gattii included cyt b types I to V, and C. neoformans var. neoformans comprised types VI to IX. cyt b types were correlated with serotypes. All strains with cyt b types I, IV, and V were serotype B. All other strains except IFM 5878 (serotype B) with cyt b types II and III were serotype C. Serotype D strains had cyt b types VI and IX, and serotype A strains were cyt b type VIII. Of four serotype AD strains, one was cyt b type VII and the remaining three were type VIII. The phylogenetic tree based on deduced amino acid sequences divided the strains only into C. neoformans var. neoformans and C. neoformans var. gattii. These results indicate that cyt b sequences are effective for DNA typing as well as phylogenetic analysis of C. neoformans.     

Phylogenetic relationships of bluetongue viruses based on gene S7

Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.     

Phylogenetic position of Linguatula arctica and Linguatula serrata (Pentastomida) as inferred from the nuclear 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit I gene

Genomic DNA was isolated from a Linguatula serrata female expelled from a dog imported to Norway from Romania and from four Linguatula arctica females collected from semi-domesticated reindeer from northern Norway and subjected to PCR amplification of the complete nuclear 18S rRNA gene and a 1,045-bp portion of the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species differed at two of 1,830 nucleotide positions (99.9 % identity) of the complete 18S rRNA gene sequences and at 102 of 1,045 nucleotide positions (90.2 % identity) of the partial cox1 sequences. The four isolates of L. arctica showed no genetic variation in either gene. The new cox1 primers may facilitate the diagnosis of various developmental stages of L. arctica and L. serrata in their hosts. In separate phylogenetic analyses using the maximum likelihood method on sequence data from either gene, L. arctica and L. serrata clustered with members of the order Cephalobaenida rather than with members of the order Porocephalida, in which the genus Linguatula is currently placed based on morphological characters. The phylogenetic relationship of L. arctica, L. serrata and other pentastomids to other metazoan groups could not be clearly resolved, but the pentastomids did not seem to have a sister relationship to crustaceans of the subclass Branchiura as found in other studies. A more extensive taxon sampling, including molecular characterisation of more pentastomid taxa across different genera, seems to be necessary in order to estimate the true relationship of the Pentastomida to other metazoan groups.     

Phylogenetic relationships in the Lymphocryptovirus genus of the Gammaherpesvirinae

Complete DNA sequences were determined for the glycoprotein B (gB) genes of four viruses from the genus Lymphocryptovirus, whose hosts had been assigned as baboon, orangutan, chimpanzee and gorilla. Together with published sequences for the gB genes of three lymphocryptoviruses, namely the human pathogen Epstein-Barr virus (EBV), a rhesus monkey virus and a marmoset virus, the sequences were used to investigate evolutionary relationships in the genus. The chimpanzee and orangutan viruses' sequences were found to be so close that it is unlikely both represent natural infections in these hosts. Phylogenetic analyses showed that the New World marmoset virus lineage formed a sister clade to that of the Old World viruses, consistent with a cospeciational separation. Within the Old World virus group, resolution of branching pattern was incomplete, and suggestive of a complex history. In particular, it was inferred that separation of the EBV lineage from that of the gorilla virus plus the chimpanzee/orangutan virus may have predated separation of the present day host species.     

Phylogenetic relationships of the genus Eurytrema from domestic and wild animal based on 18S rRNA sequences

Since Loss (1907) established the genus Eurytrema, there were more than eleven species found worldwide from America, Europe to Asia. Adult worms are generally found in pancreatic and bile ducts of wild and domestic ruminants. Some species from wild animal and domestic animal have already differentiated. In this study, we amplified and sequenced the partial 18S rRNA sequences of some Eurytrema species found in wild and domestic animals. The phylogenetic analysis was conducted to show the genetic relationship of these Eurytrema species. The results demonstrated that same species of Eurytrema from domestic animal and wild animal or from separated geological region have a considerable degree of genetic differentiation. Analysis of the 18S rRNA sequences indicated that Eurytrema fukienensis is an independent species and suggested that it may represent the intermediate species between wild and domestic animal.     

Phylogenetic analysis of the genus Cylicocyclus (Nematoda: Strongylidae) based on nuclear ribosomal sequence data

Seven species of Cylicocyclus Ihle, 1922 (Nematoda: Strongylidae) were collected from donkeys from Henan Province, China. Five samples of each species were selected for sequencing. Sixteen different internal transcribed spacer (ITS) sequences representing the seven species of Cylicocyclus were obtained. Sequence differences in the first internal transcribed spacer (ITS-1) among species was lower than that of the second internal transcribed spacer (ITS-2). Phylogenetic analyses were conducted using the combined ITS-1 and ITS-2 data sets from the present study and using reference sequences from the GenBank database. The MP and ML trees were similar in topology. The phylogenetic trees were divided into two clades. Clade I included 8 species of Cylicocyclus; within this group, Cylicocyclus leptostomus (Kotlan, 1920) is nested between different samples of Cylicocyclus ashworthi (LeRoux, 1924), suggesting C. ashworthi may represent a species complex. Clade II included Cylicocyclus elongatus (Looss, 1900) and Cylicocyclus ultrajectinus (Ihle, 1920); however, these two species always clustered with the comparative species (Petrovinema poculatum (Looss, 1900) and Poteriostomum imparidentatum Quiel, 1919), suggesting that C. elongatus and C. ultrajectinus represent members of other genera.     

Taxonomic and phylogenetic relationships between species of the genus Culex (Diptera: culicidae) from Brazil inferred from the cytochrome c oxidase I mitochondrial gene

Species of the genus Culex Linnaeus have been incriminated as the main vectors of lymphatic filariases and are important vectors of arboviruses, including West Nile virus. Sequences corresponding to a fragment of 478 bp of the cytochrome c oxidase subunit I gene, which includes part of the barcode region, of 37 individuals of 17 species of genus Culex were generated to establish relationships among five subgenera, Culex, Phenacomyia, Melanoconion, Microculex, and Carrollia, and one species of the genus Lutzia that occurs in Brazil. Bayesian methods were employed for the phylogenetic analyses. Results of sequence comparisons showed that individuals identified as Culex dolosus, Culex mollis, and Culex imitator possess high intraspecific divergence (3.1, 2.3, and 3.5%, respectively) when using the Kimura two parameters model. These differences were associated either with distinct morphological characteristics of the male genitalia or larval and pupal stages, suggesting that these may represent species complexes. The Bayesian topology suggested that the genus and subgenus Culex are paraphyletic relative to Lutzia and Phenacomyia, respectively. The cytochrome c oxidase subunit I sequences may be a useful tool to both estimate phylogenetic relationships and identify morphologically similar species of the genus Culex.