Multilocus phylogeographical analysis of Trypanosoma (Megatrypanum) genotypes from sympatric cattle and water buffalo populations supports evolutionary host constraint and close phylogenetic relationships with genotypes found in other ruminants q Herakles A. Garcia a,b , Adriana C. Rodrigues a , Franjo Martinkovic c , Antonio H.H. Minervino d , Marta Campaner a , Vânia L.B. Nunes e , Fernando Paiva f , Patrick B. Hamilton g , Marta M.G. Teixeira a,⇑ a Departamento de Parasitologia, Universidade de São Paulo, Instituto de Ciências Biomédicas II, Av. Prof. Lineu Prestes 1374, 05508-900 São Paulo, SP, Brazil b Departamento de Patología Veterinaria, Faculdad de Ciencias Veterinarias, Universidad Central de Venezuela, Maracay, Venezuela c Department for Parasitology and Parasitic Diseases, Veterinary Faculty, University of Zagreb, Zagreb, Croatia d Instituto de Biodiversidade e Floresta, Universidade Federal do Oeste do Pará, PA, Brazil e Centro de Ciências Biológicas, Agrárias e da Saúde, Universidade para o Desenvolvimento do Estado e da Região do Pantanal, Campo Grande, MS, Brazil f Departamento de Parasitologia Veterinária, Universidade Federal do Mato Grosso do Sul, MS, Brazil g Biosciences, College of Life and Environmental Sciences, University of Exeter, United Kingdom article info Article history: Received 27 June 2011 Received in revised form 19 August 2011 Accepted 26 September 2011 Available online 29 October 2011 Keywords: Trypanosoma (Megatrypanum) genotypes Multilocus phylogeny Phylogeography Host restriction Host switching Evolution Ruminant parasites abstract Species of the subgenus Trypanosoma (Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle (Bos taurus), but only one in water buffalo (Bubalus bubalis). However, the small number of isolates exam- ined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associa- tions and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate loca- tions in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochon- drial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all iso- lates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymor- phisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, ante- lope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corre- sponds to the subgenus T. (Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host– parasite co-evolution, indicating that host switches have occurred across ruminants followed by diver- gences, giving rise to new trypanosome genotypes adapted exclusively to one host species. Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Trypanosoma (Megatrypanum) is a complex of phylogenetically related species and genotypes nested within a monophyletic assemblage formed by trypanosomes that apparently only infect species of Ruminantia (former Artiodactyla). Trypanosoma (Mega- trypanum) theileri is a prevalent and cosmopolitan parasite of cattle from the tropics to near the Arctic Circle (Hoare, 1972; Wells, 1976). Species of the subgenus T. (Megatrypanum) are also highly prevalent in other livestock species such as water buffalo, sheep and goats, but not pigs or horses, and there are numerous reports in wild bovids, such as African buffalo and antelopes (e.g., duikers, sitatunga, nyala, kudu) and European and North American bovids (e.g., elk, bison, moose, caribou) and cervids (mules and fallow, red, rein and roe deer), South American brocket deer and sika deer 0020-7519/$36.00 Ó 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2011.09.001 q Note: The nucleotide sequences reported in this paper are available in GenBank under Accession Numbers listed in Table 1. ⇑ Corresponding author. Tel.: +55 11 3091 7268; fax: +55 11 3091 7417. E-mail address: mmgteix@icb.usp.br (M.M.G. Teixeira). International Journal for Parasitology 41 (2011) 1385–1396 Contents lists available at SciVerse ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara