Mitochondrial DNA sequences of triatomines (Hemiptera: Reduviidae): phylogenetic relationships

The phylogenetic relationships among 18 species of Triatominae were inferred based on mitochondrial DNA (mtDNA) sequences. The species of Triatoma included 11 belonging to the infestans complex [T. infestans (Klug), T. guasayana Wygodzinsky & Abalos, T. sordida (St?l), T. platensis Neiva, T. brasiliensis Neiva, T. rubrovaria (Blanchard), T. vitticeps (St?l), T. delpontei Roma?a & Abalos, T. maculata (Erichson), T. patagonica Del Ponte, and T. matogrossensis Leite & Barbosa] and four others of the same genus but of different complexes [T. circummaculata (St?l), T. protracta (Uhler), T. dimidiata (Latreille), and T. mazzottii Usinger]. As possible outgroups we used Mepraia spinolai Mazza, Panstrongylus megistus (Burmeister), and Rhodnius prolixus St?l. We analyzed mtDNA fragments of the 12S and 16S ribosomal RNA genes from each of the 18 species, as well as of the cytochrome oxidase I (COI) gene from nine. The 12S, 16S, and COI gene sequences were analyzed individually and combined. All of the phylogenetic analyses unambiguously supported two clusters: one including T. infestans, T. platensis, and T. delpontei, and the other T. sordida and T. mutagrossensis. Inclusion of T. circummaculata into the infestans complex was confirmed, although this is in disagreement with the morphological classification. On the other hand, our analyses showed that T. dimidiata is closely related to a phylosoma complex species, T. mazzottii. This is consistent with the tentative classification previously made based on morphological characters. The issue of the monophyly of the genus Triatoma remains unresolved.     

Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences

Bat lyssaviruses Aravan and Khujand were isolated in southern Kyrgyzstan in 1991 and in northern Tajikistan in 2001, respectively. Preliminary studies with anti-nucleocapsid monoclonal antibodies suggested that the viruses were distinct from other lyssavirus serotypes. These data were supported by sequencing of the N gene of Aravan virus. In the present study, we sequenced the entire N, P and G genes of both Aravan and Khujand viruses and compared them with respective sequences of other lyssaviruses available from GenBank. The results suggested that each virus should be considered as a newly recognized genotype according to the current approaches for genotype definition (amount of nucleotide identity of the N gene and bootstrap support of joining to certain phylogenetic groups). Use of different phylogenetic methods and comparison of different parts of the genomes generally suggested that Khujand virus was mainly related to genotype 6, while Aravan virus, on the one hand, was related to Khujand virus, and, on the other hand, demonstrated moderate similarity to genotypes 4, 5 and 6. The potential significance of these new lyssaviruses for veterinary and public health should not be underestimated.     

Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles

Identification and characterization of functional elements in the noncoding regions of genomes is an elusive and time-consuming activity whose output does not keep up with the pace of genome sequencing. Hundreds of bacterial genomes lay unexploited in terms of noncoding sequence analysis, although they may conceal a wide diversity of novel RNA genes, riboswitches, or other regulatory elements. We describe a strategy that exploits the entirety of available bacterial genomes to classify all noncoding elements of a selected reference species in a single pass. This method clusters noncoding elements based on their profile of presence among species. Most noncoding RNAs (ncRNAs) display specific signatures that enable their grouping in distinct clusters, away from sequence conservation noise and other elements such as promoters. We submitted 24 ncRNA candidates from Staphylococcus aureus to experimental validation and confirmed the presence of seven novel small RNAs or riboswitches. Besides offering a powerful method for de novo ncRNA identification, the analysis of phylogenetic profiles opens a new path toward the identification of functional relationships between co-evolving coding and noncoding elements.In all living organisms, the genome regions located between protein-coding sequences are home to a wide diversity of functional elements that include noncoding RNA (ncRNA) genes, DNA regulatory elements, untranslated regions (UTRs) of genes, transposable and self-replicating elements, and a variety of other transcribed or nontranscribed functional sequences. As these elements are often key players in gene regulation and thus in the global cell interaction network, their systematic identification and characterization has become a major challenge in biology.Computational protocols developed to collect and characterize noncoding elements in genomic sequences rely, to a large extent, on comparative genomics. The most common strategies involve, first, collecting sequences under selective pressure and, second, analyzing the aligned sequences using various classifiers that exploit criteria such as nucleotide composition, folding potential, fold conservation, or covariation between distant positions (Rivas and Eddy 2001; Washietl et al. 2005; Pedersen et al. 2006; Torarinsson et al. 2006). In general, such classifiers are designed to detect structured RNAs among noncoding elements with no further distinction between regulatory elements, repeats, or artifacts produced by sequence comparison algorithms.Comparative genomics entails a significant amount of expert intervention, especially in obtaining the right genome set to optimize the specificity and sensitivity of RNA detection. Although a number of studies have successfully identified ncRNAs in several animal (Missal et al. 2005; Washietl et al. 2007) and microbial genomes (Altuvia 2007), the pace at which such studies are performed and published lags far behind the rate of genome sequence output. Most of the complete genomes sequenced to date have escaped the scrutiny of RNA experts, and their potential for novel RNA functions lies unexplored. Recently, Livny et al. (2008) introduced an automated procedure, SIPHT, which combines conserved sequence detection and the presence of adjacent Rho-independent terminators. The procedure is sufficiently automated to be applied to all available bacterial genomes; however, the requirement for a terminator motif introduces a bias against the significant fraction of RNA elements that are not followed by a detectable terminator.Nucleic acid phylogenetic profiling (NAPP) addresses both the issue of high-throughput noncoding sequence identification and that of their functional characterization. Phylogenetic profiling (Ragan and Gaasterland 1998; Pellegrini et al. 1999) posits that genes belonging to the same metabolic or regulatory pathway tend to occur concomitantly in a given set of organisms. By clustering proteins based on their occurrence profile in many species, one therefore obtains clusters of functionally related proteins, which enables functional assignments to uncharacterized sequences. While this principle has been successfully applied to protein annotation (Pellegrini et al. 1999; Enault et al. 2003; Srinivasan et al. 2005), to date, no application to ncRNA annotation has emerged. An obstacle to building nucleic acid profiles over a large evolutionary time frame is certainly the lower sensitivity of DNA sequence similarity searches compared to searches at the amino acid sequence level. However, in spite of this limitation, Janky and van Helden (2008) were able to obtain clear phylogenetic profiles for promoter DNA elements, by focusing their analysis on upstream sequences of orthologous genes. In this study, we apply phylogenetic profiling for the first time to the complete noncoding DNA in bacterial genomes, with the objective of classifying all conserved noncoding elements in a single computing process.     

Environmental kinetoplastid-like 18S rRNA sequences and phylogenetic relationships among Trypanosomatidae: paraphyly of the genus Trypanosoma

Using kinetoplastid-like sequences from deep-sea environmental samples as an outgroup, we applied phylogenetic analysis to 18S rRNA sequences of the families Trypanosomatidae and Bodonidae (Eugelenozoa: Kinetoplastida). The monophyly of the genus Trypanosoma was not supported by a number of different methods. Rather, the results indicate that the American and African trypanosomes constitute distinct clades, therefore, implying that the major human disease agents T. cruzi (cause of Chagas' disease) and T. brucei (cause of African sleeping sickness) are not as closely related to each other as they were previously thought to be. Likewise, the results did not support monophyly of the genera Leishmania, Leptomonas, Bodo and Cryptobia.     

18S rRNA gene sequences and phylogenetic relationships of European hard-tick species (Acari: Ixodidae)

The complete 18S rRNA gene sequences of the following six European hard-tick species were obtained by direct PCR cycle sequencing and silver-staining methods: Rhipicephalus pusillus, Boophilus annulatus, Dermacentor marginatus, Hyalomma lusitanicum, Haemaphysalis punctata, and Ixodes ricinus. Differences observed in the sequence alignment of these six species together with the 18S rRNA gene sequences of 13 other hard-tick species demonstrate that this gene is a good marker for supraspecific differentiation as well as genus grouping among hard ticks. Phylogenetic analyses strongly support that Hyalomma species share a common ancestor with Rhipicephalinae and, consequently, Hyalomminae should no longer be considered an independent subfamily. However, no definitive conclusion could be reached to support or oppose the separation of the subfamilies Haemaphysalinae and Amblyomminae. Accepted: 15 July 1997     

Molecular identification and phylogenetic relationships of trichomonad isolates of galliform birds inferred from nuclear small subunit rRNA gene sequences

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.     

The radiation of Thaparocleidus (Monogenoidea: Dactylogyridae: Ancylodiscoidinae): phylogenetic analyses and taxonomic implications inferred from ribosomal DNA sequences

The monophyly of Thaparocleidus Jain 1952 and its phylogenetic relationship with Pseudancylodiscoides were assessed using the D1–D2 domains of the large-subunit ribosomal deoxyribonucleic acid sequences, and taxonomic implications were discussed concerning the relative taxonomic importance of the characters of the reproductive complex and those of the haptoral scleties for future genus erection and combination within the Ancylodiscoidinae. Thaparocleidus contained divergent genetic lineages and was not resolved as a monophyletic group. The first lineage was represented by two species found on Pangasium sutchi (Pangasidae), and the second one contained 12 species all from Silurus astus (Siluridae). Three clades were observed for species from S. astus, consistent with results of morphological analyses, indicating that 12 Thaparocleidus species could be divided into three groups by morphological examinations of the male copulatory organ (MCO). Pseudancylodiscoides spp. was more closely related to Thaparocleidus spp. from S. astus, and morphologically, it was found that shapes of MCOs of these species were the same type but different from that of Thaparocleidus spp. from P. sutchi. Therefore, based on results of molecular phylogenetic analyses and morphological examinations, we propose to abolish Pseudancylodiscoides and to erect a new genus to accommodate Thaparocleidus species from S. astus and Pseudancylodiscoides spp. It is also suggested to erect another new genus to accommodate two Thaparocleidus species from the P. sutchi, whose MCO shapes are different from other Thaparocleidus species.     

Comparative chromosome and mitochondrial DNA analyses and phylogenetic relationships within common voles (Microtus, Arvicolidae)

The four species of common voles within the genus Microtus – M. kirgisorum, M. transcaspicus, M. arvalis, and M. rossiaemeridionalis – are so closely related that neither morphological features nor paleontological evidence allow clarification of their phylogeny. Analysis of vole karyotypes and mitochondrial DNA sequences, therefore, is essential for determining their phylogenetic relationships. A comparison of high resolution GTG-banding patterns allows us to ascertain the similarity between the karyotypes of these species, revealing that they are composed of rearrangements of the same chromosomal elements. Based on this analysis, we propose possible routes of chromosomal divergence involved in speciation within this group of voles and construct a phylogenetic tree of their karyotypes. We suggest that two different karyotypic variants existed during the course of vole evolution – one resulting in M. rossiaemeridionalis and M. transcaspicus, the other, M. kirgisorum and M. arvalis. As an alternative approach FITCH and KITSCH computer programs were used to construct a phylogenetic tree of vole molecular evolution based on a pairwise comparison of mitochondrial cytochrome b sequences and the divergence time of the species was determined. The correlation between the trees constructed using karyologic and molecular approaches is discussed in the context of other available data.     

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy.     

Study of the KIR gene profiles and analysis of the phylogenetic relationships of Rajbanshi population of West Bengal,India

The natural killer (NK) cells have distinct receptors called killer cell immunoglobulin-like receptors (KIR) which are responsible for regulating NK cell responses to infections and malignancy. The extensive variations in the number and type of KIR genes can be used as a tool to understand the differentiation of populations and also for tracing genetic background. In this study, we have aimed to analyze the KIR gene polymorphism in the Rajbanshi population of West Bengal, India. To our knowledge this is the first report on the KIR gene polymorphism in the Rajbanshis, a population widely distributed in the Terai and Dooars region of West Bengal, India. Herein, we have studied the gene distribution of 14 KIR genes (KIR3DL1–3DL3, KIR 2DL1–2DL5, 2DS1–2DS5 and 3DS1) and two pseudogenes (KIR3DP1 and 2DP1). The gene frequencies and genotypic frequencies were calculated, based on which statistical analyses were performed. The presence of a considerable number of genotypic profiles suggests substantial diversity in the KIR gene pool of the Rajbanshis in the region studied. Apart from the framework genes (KIR2DL4, 3DL2, 3DL3 and 3DP1) present in all the individuals, the gene frequencies of other KIR genes varied between 0.84 and 0.15. Moreover the KIR polymorphisms of the Rajbanshis were also compared with that of available published data of the populations of other ethnicities. Though the Rajbanshi population showed a tendency to cluster with other Indian population based on KIR gene frequencies, the influence of Tibeto–Burman Lineage on their KIR genotypic profiles cannot be overlooked. Furthermore, evidences from previously published data on Y chromosome haplogroup diversity study on Rajbanshis support the view. Our results will not only help to understand the genetic background of the Rajbanshi population, but also in tracing the population migration events in the North–Eastern part of India and in illustrating the extensive genetic admixture amongst the different linguistic groups of the country and also in KIR-related disease researches.     

The Hyrcanus group of Anopheles (Anopheles) in China (Diptera: Culicidae): species discrimination and phylogenetic relationships inferred by ribosomal DNA internal transcribed spacer 2 sequences

The Hyrcanus group comprises many closely related species with wide distributions in the Oriental and Palaearctic regions. The sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA were determined for 12 species in China--An. crawfordi, An. hyrcanus, An. junlianensis, An. kunmingensis, An. kweiyangensis, An. lesteri, An. liangshanensis, An. peditaeniatus, An. pullus, An. sinensis, and two unknown species within the group. The length of the ITS2 ranged from 436 bp in An. hyrcanus to 469 bp in An. crawfordi, with GC contents of 44.9-46.8%. Intraspecific variation was found in three species (An. junlianensis, An. liangshanensis, and An. pullus) at the level of 0.0-0.4%, whereas interspecific differences ranged from 1.6% between An. liangshanensis and An. kunmingensis to 50.8% between An. peditaeniatus and sp. 1. The ITS2 comparisons revealed two unknown species, verified the valid species status for An. kunmingensis, and found An. pullus in China. We agree that An. anthropophagus is a junior synonym of An. lesteri. The validation of An. junlianensis awaits recognition of the molecular identity of the entity identified as An. yatsushiroensis. The ITS2 divergences were used for inferring phylogenetic relationships among 12 species in China. The estimation revealed close relationships among An. liangshanesis, An. kunmingensis, An. kweiyangensis, An. lesteri, and An. sinensis. Our study emphasizes the need for the molecular identity of the species members in integrated studies in systematics, bionomics, and population genetics for the Hyrcanus group.     

Species delimitation and phylogenetic relationships of Chinese Leishmania isolates reexamined using kinetoplast cytochrome oxidase II gene sequences

Leishmaniasis is a geographically widespread disease caused by protozoan parasites belonging to the genus Leishmania and transmitted by certain species of sand fly. This disease still remains endemic in China, especially in the west and northwest frontier regions. A recent ITS1 phylogeny of Chinese Leishmania isolates has challenged some aspects for their traditional taxonomy and cladistic hypotheses of their phylogeny. However, disagreement with respect to relationships within Chinese Leishmania isolates highlights the need for additional data and analyses. Here, we test the phylogenetic relationships among Chinese isolates and their relatives by analyzing kinetoplast cytochrome oxidase II (COII) gene sequences, including 14 Chinese isolates and three isolates from other countries plus 17 sequences retrieved from GenBank. The COII gene might have experienced little substitution saturation, and its evolutionary process was likely to have been stationary, reversible, and homogeneous. Both neighbor-joining and Bayesian analyses reveal a moderately supported group comprising ten newly determined isolates, which is closely related to Leishmania tarentolae and Endotrypanum monterogeii. In combination with genetic distance analysis as well as Bayesian hypothesis testing, this further corroborates the occurrence of an undescribed species of Leishmania. Our results also suggest that (1) isolate MHOM/CN/93/GS7 and isolate IPHL/CN/77/XJ771 are Leishmania donovani; (2) isolate MHOM/CN/84/JS1 is Leishmania tropica; (3) the status referring to an isolate MRHO/CN/62/GS-GER20 from a great gerbil in Gansu, China, as Leishmania gerbilli, formerly based on multilocus enzyme electrophoresis, is recognized; and (4) E. monterogeii is nested within the genus Leishmania, resulting in a paraphyletic Leishmania. In addition, the results of this study enrich our understanding of the heterogeneity and relationships of Chinese Leishmania isolates.     

A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species

Gene trees of Plasmodium species have been reported for the nuclear encoded genes (e.g. the Small Subunit rRNA) and a mitochondrial encoded gene, cytochrome b. Here, we have analyzed a plastid gene coding for caseinolytic protease ClpC, whose structure, function and evolutionary history have been studied in various organisms. This protein possesses a 220-250 amino acid long AAA domain (ATPases associated with a variety of cellular activities) that belongs to the Walker super family of ATPases and GTPases. We have sequenced the AAA motif of this gene, encoding the protein from nine different species of Plasmodium infecting rodents, birds, monkeys, and humans. The codon usage and GC content of each gene were nearly identical in contrast to the widely varying nucleotide composition of genomic DNAs. Phylogenetic trees derived from both DNA and inferred protein sequences have consistent topologies. We have used the ClpC sequence to analyze the phylogenetic relationship among Plasmodium species and compared it with those derived from mitochondrial and genomic sequences. The results corroborate well with the trees constructed using the mitochondrially encoded cytochrome b. However, an important element distinguishes the trees: the placement of Plasmodium elongatum near the base of the plastid tree, indicating an ancient lineage of parasites in birds that branches from the tree prior to other lineages of avian malaria and the human parasite, P. falciparum.     

Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes

To gain a deeper insight into the evolution of mating-type genes from filamentous ascomycetes, a comprehensive sequence analysis of PCR-amplified sequences corresponding to A- and a-specific mating-type sequences was undertaken. The study included nine homothallic (compatible) and eight heterothallic (incompatible) members of the genera Neurospora and Sordaria. Distance and parsimony trees based on gene fragments from the mat a-1 and mat A-1 genes were compared with trees derived from partial DNA sequences of the gpd glyceraldehyde-3-phosphate dehydrogenase gene. In contrast to the sequences from the gpd gene, mating-type genes show striking sequence differences, suggesting that these genes evolve very rapidly. Strong inter-relationships were found among homothallic, as well as among heterothallic, members of both genera, indicating that in each genus a change from one reproductive strategy to another might result from one single event. Received: 6 April / 2 June 1999     

Orthopoxvirus DNA: A comparison of restriction profiles and maps

Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus-cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAS of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus.     

Mitochondrial DNA sequences reveal close relationships between social parasitic ants and their host species

In the tribe Leptothoracini, the phylogenetic relationship of socially parasitic ants (Doronomyrmex kutteri, D. goesswaldi and Harpagoxenus sublaevis) and their host species Leptothorax acervorum has been controversial. Even more controversial is the relationship between the socially parasitic ant Chalepoxenus muellerianus and its host species Leptothorax unifasciatus, L. nigriceps, L. interruptus and L. recedens. On the basis of morphological, ecological and ethological criteria it has been argued that socially parasitic ants and their respective hosts always evolved from common ancestors, and hence it has been postulated that these species should be included in common taxonomical groups. This would require the division of the tribe Leptothoracini into two subgroups, one comprising the subgenus Leptothorax (s. str.) and the other the subgenus Myrafant, together with their respective parasitic genera. We have used the polymerase chain reaction (PCR) to compare a 360-bp sequence of the mitochondrial cytochrome b gene of 14 species belonging to the tribe Leptothoracini and an outgroup species Tetranorium impurum (Tetramoriini). The results generally agree with the morphological studies which suggest that a common ancestral species differentiated into host and parasite species. This relationship is very obvious within the Leptothorax (s. str.) group but less pronounced in the species belonging to the Myrafant group. Leptothorax (Temnothorax) recedens shows a greater sequence divergence than the outgroup species T. impurum.