The ITS-2 of the nuclear rDNA as a molecular marker for populations, species, and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease.

:The nucleotide sequences of the rDNA second internal transcribed spacer (ITS-2) of 31 populations of 12 and 3 species of the two main Triatominae tribes Triatomini and Rhodniini, including the most important Chagas disease vectors, were obtained. Sequence comparisons and parsimony, distance, and maximum-likelihood analyses indicate that ITS-2 is a useful marker for resolving supraspecific, specific, subspecific, and even sometimes population-level relationships in Triatominae. Results were markedly different between species of Triatomini and Rhodniini, suggesting polyphyly. Phylogenetic trees support an old divergence between South American and North-Central American Triatomini and query the validity of some genera (Dipetalogaster, Psammolestes). The very low sequence variation between species of the phyllosoma complex suggests that subspecific ranking would be more appropriate. Triatoma dimidiata proves to be a clearly differentiated species, with several populations evidencing a clinal variation along a north-south axis and a population from Yucatan showing differences consistent with specific status.

Marcilla A ，Bargues MD ，Ramsey JM ，Magallon-Gastelum E ，Salazar-Schettino PM ，Abad-Franch F ，Dujardin JP ，Schofield CJ ，Mas-Coma S ... -  《MOLECULAR PHYLOGENETICS AND EVOLUTION》

Ribosomal DNA ITS-1 intergenic spacer polymorphism in triatomines (Triatominae, Heteroptera).

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.

Tartarotti E ，Ceron CR 《BIOCHEMICAL GENETICS》

Genetic variation, population structure, and phylogenetic relationships of Triatoma rubida and T. recurva (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert, insect vectors of the Chagas' disease parasite Trypanosoma cruzi.

Nucleotide and amino acid sequence data from the mitochondrial cytochrome b (Cytb) and cytochrome c oxidase subunit I (COI) gene segments were used to gain insights into the population biology and phylogenetic relationships of two species of hematophagous kissing bugs (Hemiptera: Reduviidae: Triatominae) from the Sonoran Desert of northwestern Mexico and southern Arizona, USA, Triatoma rubida (Uhler, 1894) and T. recurva (Stål, 1868), both of which are vectors of the protozoan parasite Trypanosoma cruzi responsible for Chagas' disease. Analysis of molecular variance of gene sequences indicated significant structure among populations of both species from widely separated geographic localities. Phylogenetic analyses of gene and amino acid sequences employing both Bayesian and parsimony methods showed that T. recurva clustered within the phyllosoma complex of Triatoma species from central and southern Mexico with high statistical support, and that it was closely related to T. longipennis. Triatoma dimidiata also was shown to be closely related to the phyllosoma complex, as was T. sanguisuga which has historically been assigned to the lecticularia complex. Analyses of gene sequences were unable to confidently resolve relationships of T. rubida, although weak support for a T. nitida+T. rubida clade was seen under certain conditions. A provisional calibration of a mitochondrial DNA molecular clock for T. rubida, based on geological dates for the vicariant separation of the Baja California peninsula from mainland Mexico, suggested that pairwise sequence divergences for the Cytb and COI genes were 1.1-1.8% and 0.6-1.0% per million years, respectively. Two highly supported sympatric lineages of T. rubida uhleri from southern Arizona, which are hypothesized to have diverged approximately 550,000-900,000 years ago, were detected in the Cytb gene trees.

Pfeiler E ，Bitler BG ，Ramsey JM ，Palacios-Cardiel C ，Markow TA ... -  《MOLECULAR PHYLOGENETICS AND EVOLUTION》

Populations, hybrids and the systematic concepts of species and subspecies in Chagas disease triatomine vectors inferred from nuclear ribosomal and mitochondrial DNA.

In Chagas disease, triatomine vectors are the main target for control measures because of the absence of effective drugs. The broad usefulness of nuclear rDNA and mtDNA sequences explains why triatomine studies using these markers have increased so pronouncedly in recent years. This indicates the appropriateness of an updated review about these molecular markers, concentrating on aspects useful for research on Chagas disease vectors. A comparative analysis is presented on the efficiency, weight of their different characteristics, limitations and problems of each of the different DNA markers in the light of the results obtained in studies on populations, hybrids, subspecies and species of the subfamily Triatominae. The use of a standardized composite haplotype code nomenclature for both nuclear rDNA and mtDNA markers is strongly encouraged to avoid difficulties in comparative studies. Triatomine aspects related to concerted evolution, microsatellites, minisatellites and insertions/deletions in nuclear rDNA and silent/non-silent mutations, pseudogenes and weaknesses of partial sequences in mtDNA are analysed. Introgression and hybrids, nuclear and mitochondrial DNA strengths, and compared evolutionary rates of nuclear rDNA and mtDNA in triatomines are discussed. Many conclusions are obtained thanks to the availability, for the first time in triatomines, of a complete sequence of a protein-coding mtDNA gene as ND1 from very numerous triatomine species covering from different populations of a species up to members belonging to different tribes. The evolutionary rates of each nuclear rDNA marker and mtDNA marker are analysed by comparison at subspecies level (intrapopulational, interpopulational, between morphs, and between subspecies) and species level (close and distant species of the same genus, species of different genera, and species of different tribes). Weaknesses of mtDNA for systematic-taxonomic purposes detected recently and newly in insects and triatomines, respectively, are discussed in detail. Emphasis is given to taxonomic units and biological entities presenting well-known problematics, both from the systematic-taxonomic and/or epidemiological-control points of view, as well as to molecular situations which can give rise to erroneous conclusions. All these aspects constitute the background on which the key question about the systematic concepts of species and subspecies in triatomines is focused. The global purpose is to facilitate future work on triatomines by highlighting present gaps, how better choice the appropriate markers, and marker aspects which should be taken into account. Key characteristics as alpha, CI and transformation rate matrices ought to be obtained and noted to get appropriate results and allow correct interpretations. The main aim is to offer a baseline for future fundamental research on triatomines and applied research on transmission, epidemiology and control measures related to Chagas disease vectors.

Mas-Coma S ，Bargues MD 《-》

Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi.

Panstrongylus species are widely distributed throughout the Americas, where they act as vectors of Trypanosoma cruzi, agent of Chagas disease. Their intraspecific relationships, taxonomic position and phylogeny in relation to other Triatomini were explored using ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS-2) sequence polymorphisms and maximum parsimony, distance and maximum likelihood analyses of 10 populations representing six species of the genus (P. megistus, P. geniculatus, P. rufotuberculatus, P. lignarius, P. herreri and P. chinai). At the subspecific level, P. megistus appeared more homogeneous than P. rufotuberculatus and P. geniculatus (both with broader distribution). Several dinucleotide microsatellites were detected in the sequences of given species. Many of these microsatellites (GC, TA, GT and AT) showed different number of repeats in different populations and thus, may be very useful for population differentiation and dynamics analyses in future studies. The sequences of P. lignarius (considered sylvatic) and P. herreri (a major disease vector in Peru) were identical, suggesting that these species should be synonymised. Intrageneric analysis showed a clear separation of P. rufotuberculatus, with closest relationships between P. geniculatus and P. chinai, and P. megistus occupying a separate branch. Genetic distances between Panstrongylus species (0.11585-0.22131) were higher than those between Panstrongylus and other Triatomini (16 species from central and North America and South America) (0.08617-0.11039). The distance between P. megistus and P. lignarius/herreri (0.22131) was the largest so far recorded in the tribe. The pronounced differences in length and nucleotide composition suggest a relatively old divergence of Panstrongylus species. P. rufotuberculatus was closer to Mesoamerican Triatoma, Meccus and Dipetalogaster species than to other Panstrongylus. All Panstrongylus clustered with the Mesoamerican clade; P. rufotuberculatus clustered with the phyllosoma complex and T. dimidiata, with D. maxima and T. barberi in a basal position. The rest of Panstrongylus appeared paraphyletically in the tree. This is evidence suggesting polyphyly within the genus Panstrongylus, whose species may be related to the ancestors giving rise to central and North American Triatomini.

Marcilla A ，Bargues MD ，Abad-Franch F ，Panzera F ，Carcavallo RU ，Noireau F ，Galvão C ，Jurberg J ，Miles MA ，Dujardin JP ，Mas-Coma S ... -  《INFECTION GENETICS AND EVOLUTION》