Infection, Genetics and Evolution 1 (2002) 225–235 Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi  A. Marcilla a , M.D. Bargues a,∗ , F. Abad-Franch b,c , F. Panzera d , R.U. Carcavallo e , F. Noireau e,f , C. Galvão e , J. Jurberg e , M.A. Miles b , J.P. Dujardin g , S. Mas-Coma a a Departamento de Parasitolog´ ıa, Facultad de Farmacia, Universidad de Valencia, Av. Vicent Andrés Estellés s/n, 46100 Burjassot-Valencia, Valencia, Spain b Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1 E7HT, UK c Instituto “Juan César Garc´ ıa”, Fundación Internacional de Ciencias Sociales y Salud, Casilla Postal 17-1106292 Quito, Ecuador d Sección de Genética Evolutiva, Instituto de Biolog´ ıa, Facultad de Ciencias, Universidad de la República, Calle Igua 4225, 11400 Montevideo, Uruguay e Laboratório Nacional e Internacional de Referˆ encia em Taxonomia de Triatom´ ıneos, Departamento de Entomologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil f URO16, Institut de Recherche pour le Développement (IRD), Av. Agropolis 911, 34032 Montpellier Cedex 1, France g UMR IRD/ORSTOM-CNRS 9926, URO62, Institut de Recherche pour le Développement (IRD), Av. Agropolis 911, 34032 Montpellier Cedex 1, France Received 24 July 2001; received in revised form 12 January 2002; accepted 25 January 2002 Abstract 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. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Panstrongylus species; Triatominae; Chagas disease vectors; rDNA ITS-2 sequences; Taxonomy; Phylogeny 1. Introduction The Triatominae (Hemiptera: Reduviidae) are notorious as the vectors of Trypanosoma cruzi, which infects a great  New nucleotide sequence data reported in this paper are available in the GenBank TM , EMBL and DDBJ databases under the accession numbers listed in Table 1. ∗ Corresponding author. Tel.: +34-96-386-4298; fax: +34-96-386-4769. E-mail address: M.D.Bargues@uv.es (M.D. Bargues). variety of sylvatic and domestic mammals and causes Ameri- can trypanosomiasis (Chagas disease) in humans throughout Latin America. Over 12 million people are infected by this parasite, with about 90 million considered at risk in endemic areas. No vaccine is available and except in the very early stage of the infection, there is no effective chemotherapy (WHO, 1991). A total of 133 species of Triatominae are cur- rently recognised, grouped into 18 genera forming five tribes (Dujardin et al., 2000; Carcavallo et al., 2000). Over half of these species have been naturally or experimentally infected 1567-1348/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S1567-1348(02)00029-1