Please cite this article in press as: Lima, L., et al., Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylo- genetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit). Acta Trop. (2015), http://dx.doi.org/10.1016/j.actatropica.2015.07.015 ARTICLE IN PRESS G Model ACTROP-3683; No. of Pages 12 Acta Tropica xxx (2015) xxx–xxx Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit) Luciana Lima a , Oneida Espinosa-Álvarez a , Paola A. Ortiz a , Javier A. Trejo-Varón b , Julio C. Carranza b , C. Miguel Pinto c,d , Myrna G. Serrano e , Gregory A. Buck e , Erney P. Camargo a , Marta M.G. Teixeira a,∗ a Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil b Laboratorio de Investigaciones en Parasitología Tropical, Facultad de Ciencias, Universidad del Tolima, Ibagué, Colombia c Division of Mammals, National Museum of Natural History, Smithsonian Institution, Washington, DC, USA d Centro de Investigación en Enfermedades Infecciosas y Crónicas, Escuela de Biología, Pontificia Universidad Católica del Ecuador, Quito, Ecuador e Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA a r t i c l e i n f o Article history: Received 3 June 2015 Accepted 15 July 2015 Available online xxx Keywords: Trypanosoma cruzi Chiroptera Multilocus analysis Phylogeny Genotyping Evolution a b s t r a c t Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six dis- crete typing units (DTUs) designated as TcI–TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydro- genase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, mul- tilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI–Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured popu- lations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU. © 2015 Published by Elsevier B.V. ∗ Corresponding author. Fax: +55 11 30917417. E-mail addresses: lulima@usp.br (L. Lima), oneidae07@usp.br (O. Espinosa-Álvarez), paolaortiz27@gmail.com (P.A. Ortiz), jatrejovaron@gmail.com (J.A. Trejo-Varón), jcarranza@ut.edu.co (J.C. Carranza), mpinto@amnh.org (C.M. Pinto), gabuck@vcu.edu (G.A. Buck), erney@usp.br (E.P. Camargo), mmgteix@icb.usp.br (M.M.G. Teixeira). 1. Introduction Trypanosoma cruzi is a genetically and ecologically hetero- geneous zoonotic taxon formally recognised as formed by six Discrete Typing Units (DTUs) designated as TcI–TcVI. The ratio- nale for the recent DTU classification proposed to accommodate the known genetic diversity within T. cruzi, as well as biological, eco-epidemiological and pathological features of each DTU have been recently reviewed (Zingales et al., 2012). Isolates of distinct DTUs circulate in a complex network of trans- mission cycles involving humans and other mammals of virtually all http://dx.doi.org/10.1016/j.actatropica.2015.07.015 0001-706X/© 2015 Published by Elsevier B.V.