Journal of Heredity 2011:102(3):260–268 Ó The American Genetic Association. 2011. All rights reserved. doi:10.1093/jhered/esr012 For permissions, please email: [email protected]. Advance Access publication March 25, 2011 Colonization Process of the Brazilian Common Vesper Mouse, Calomys expulsus (Cricetidae, Sigmodontinae): Downloaded from A Biogeographic Hypothesis http://jhered.oxfordjournals.org/ FABRI´CIA FERREIRA DO NASCIMENTO*, LUCIANA G. PEREIRA*, LENA GEISE,ALEXANDRA M. R. BEZERRA, PAULO S. D’ANDREA, AND CIBELE R. BONVICINO From the Genetics Division, Instituto Nacional de Caˆncer, Rua Andre´ Cavalcante, 37, 4 andar, 20231-050, Rio de Janeiro, RJ, Brazil (Nascimento and Bonvicino); the Laborato´rio de Biologia e Parasitologia de Mamı´feros Silvestres Reservato´rios, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil, 4365, 21045-900, Rio de Janeiro, RJ, Brazil (Pereira, D’Andrea, and Bonvicino); the Laborato´rio de Mastozoologia, Departamento de Zoologia, IB, Universidade do Estado do Rio de Janeiro, Rua Sa˜o Francisco Xavier 524, 220559-900, Rio de Janeiro, RJ, Brazil (Geise); and Departamento de Zoologia, Universidade at Fundação Oswaldo Cruz-Biblioteca de Manguinhos/Oswaldo Cruz Foundation-Ma on October 6, 2016 de Brası´lia, 70910-900, Brası´lia, DF, Brazil (Bezerra). *These authors contributed equally to this work. Address correspondence to Cibele R. Bonvicino at the address above, or e-mail: [email protected]. Abstract Riverine barriers have been associated to genetic diversification and speciation of several taxa. The Rio Sa˜o Francisco is one of the largest rivers in South America, representing the third largest river basin in Brazil and operating as a geographic barrier to gene flow of different taxa. To evaluate the influence of the Rio Sa˜o Francisco in the speciation of small rodents, we investigated the genetic structure of Calomys expulsus with phylogenetic and network analyses of cytochrome b DNA. Our results suggested that C. expulsus can be divided into 3 subpopulations, 2 on the left and another one on the right bank of this river. The time of divergence of these subpopulations, using a Bayesian framework, suggested colonization from the south to the north/northeast. Spatial analysis using a clustering method and the Monmonier’s algorithm suggested that the Rio Sa˜o Francisco is a biogeographic barrier to gene flow and indicated that this river may play a role in the incipient speciation process of these subpopulations. Key words: Calomys, cytochrome b, Phyllotini, phylogeography, Rio Sa˜o Francisco, South America Natural geographic barriers have been associated to genetic Distrito Federal). These regions extend from the humid diversification and speciation of different taxa (Miller et al. Atlantic Forest to the drier domains of Caatinga and Cerrado 2006; Rodriguez-Robles et al. 2008). In South America, most (Godinho and Godinho 2003; IBAMA 2007), representing studies have been focused on the Amazonas river basin a geographic barrier for several taxa (Rodrigues 2003; (Patton et al. 2000; Cheviron et al. 2005; Funk et al. 2007), the Coutinho-Abreu et al. 2008; Passoni et al. 2008). largest basin in the world (Meade et al. 1991), although other The genus Calomys, comprising Sigmodontinae species watercourses have also been shown to be effective barriers of the tribe Phyllotini, is widely distributed across South for several vertebrates (Rodrigues 2003; Passoni et al. 2008). America, in Venezuela, Colombia, Peru, Bolivia, Paraguay, The Rio Sa˜o Francisco is one of the longest rivers of South southeastern Brazil, Uruguay, and Argentina (Musser and America, extending for 2830 km, and its basin is the third Carleton 2005). Different species of Calomys inhabit largest in Brazil, accounting for 7.6% of the country’s seasonally dry tropical environments, from Cerrado to territory and encompassing an area of approximately 645.000 Caatinga (Almeida et al. 2007). The complex taxonomic km2 (Godinho and Godinho 2003; Kohler 2003). This river arrangement of this genus is a consequence of the irrigates several regions of different morphoclimatic domains morphological similarities between congeneric species, and climate settings of 6 territories (States of Minas Gerais, a situation that is common in other Sigmodontinae genera. Bahia, Pernambuco, Sergipe, Alagoas, part of Goia´s, and Hershkovitz (1962), based on morphological characters, 260 Nascimento et al. Calomys expulsus Colonization reported the first taxonomic review of Calomys with only 4 Francisco, mainly in regions of the Cerrado and Caatinga species, Calomys lepidus, C. sorellus, C. callosus,andC. laucha. morphoclimatic domains (Figure 1a). DNA was isolated Recently, karyological, taxonomic, and phylogenetic studies from livers preserved in 100% ethanol following Sambrook showed that a considerably higher diversity of this genus has et al. (1989).Cytb DNA (1143 bp) was PCR amplified using been underestimated. Olds (1988) and Salazar-Bravo et al. primers L14724 (Irwin et al. 1991) and CIT-REV (Casado (2001) recognized 10 Calomys species, albeit with different et al. 2010). Amplicons were purified with GFX PCR DNA taxonomic arrangements, whereas other authors recognized and Gel Band Purification Kit (GE Healthcare, Brazil) and Downloaded from 12 or 13 species (Musser and Carleton 2005; Almeida et al. sequenced using the PCR primer L14724 and internal 2007, respectively). primers CitbAot (Menezes et al. 2010) and CB-in1 (Cassens Steppan and Sullivan (2000) and Haag et al. (2007) et al. 2000). Sequencing was carried out using an ABI Prism postulated that Calomys is a monophyletic genus with high 377 and 3730 platforms. Electropherograms were manually levels of genetic differentiation that took place during checked using BIOEDIT (version 7.0.8.0; Hall 1999) and a long period of intrageneric divergence. Molecular CHROMAS (version 1.45; MacCarthy 1998). http://jhered.oxfordjournals.org/ phylogenetic studies divided Calomys in 2 groups, one with lowland species and another with highland species Alignment, Test of Substitution Saturation, and Genetic (Salazar-Bravo et al. 2001). Subsequently, phylogenetic Distance Estimation analysis based on a higher number of species showed that We analyzed 23 cyt b sequences generated in this study and the large-bodied lowland species (C. fecundus, C. expulsus, C. 49 cyt b GenBank sequences, 35 C. expulsus, 9 from other venustus, C. tocantinsi, C. callosus, C. cerqueirai,andan Calomys species, 1 Auliscomys micropus,1Phyllotis magister,1 undescribed species from Beni) were included in a separate Eligmodontia typus,1Akodon montensis, and 1 Sigmodon hispidus, clade (Almeida et al. 2007; Bonvicino et al. 2010). Calomys which were manually aligned using BIOEDIT. These last 5 at Fundação Oswaldo Cruz-Biblioteca de Manguinhos/Oswaldo Cruz Foundation-Ma on October 6, 2016 expulsus has also been placed as the most basal lineage of species were used as outgroups in phylogenetic trees. the large-bodied group (Almeida et al. 2007). GenBank accession numbers are listed in Table 1 and Calomys shows an interesting pattern of geographic Supplementary Table 1. distribution and is one of the few Sigmodontinae genera The DNA substitution saturation index (I ; Xia et al. restricted to dry habitats (Almeida et al. 2007). Furthermore, ss 2003), calculated with DAMBE (version 5.2.5; Xia and Xie small-bodied size species are mainly found in open 2001), was used for detecting phylogenetic signals. Two vegetation (strict or mixed grassland and savanna habitats), analyses were performed, one with C. expulsus with out- whereas large-bodied species, including C. expulsus, can also group data and another with only C. expulsus. Results were be found in forest formations (Almeida et al. 2007). Calomys compared with a critical I value (I ) following Xia and expulsus is endemic to Brazil, occurring from Caatinga to ss ss.c Lemey (2009). Cerrado in the east and central-west regions, respectively. Pairwise genetic distances were estimated with the This species was the first to diverge from the large-bodied modified Log-Det for closely related taxa (Tamura and stock, probably by an initial invasion of a new habitat (forest Kumar 2002) using MEGA (version 4; Tamura et al. 2007). formations) associated to change in body size (Almeida et al. The Hasegawa–Kishino–Yano (HKY) (Hasegawa et al. 1985) 2007; Cordeiro-Estrela P, personal communication). These plus a proportion of invariable sites and gamma-distributed features and the fact that C. expulsus is the most ancient substitution rates (HKY þ I þ C) was selected as the DNA lineage among large-bodied species are indicative of the substitution model with MODELGENERATOR (version presence of structured populations. 0.84; Keane et al. 2006) using the Bayesian information To investigate whether the Rio Sao Francisco has been ˜ criterion for phylogenetic reconstructions (see below). acting as a barrier to C. expulsus dispersal and to identify structured populations, we analyzed mitochondrial cyto- Phylogenetic Reconstruction and Divergence Time chrome b (cyt b) DNA sequences from specimens collected Estimates along a wide range of its geographic distribution, both at the left and right bank of this river. Our results suggested that Phylogenetic reconstructions based on maximum likelihood specimens could be subdivided in 3 subpopulations and that (ML) were carried out with PHYML (version 3.0; Guindon colonization occurred from the south to the north and and Gascuel 2003). The tree topology space was searched northeast