Infection, Genetics and Evolution 19 (2013) 280–286 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid High phylogeographic structure in sylvatic vectors of Chagas disease of the genus Mepraia (Hemiptera: Reduviidae) ⇑ Ricardo Campos a,b, Fernando Torres-Pérez b, Carezza Botto-Mahan c, Ximena Coronado a, Aldo Solari a, a Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile b Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso 2373223, Chile c Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile article info abstract Article history: The hematophagous Hemiptera of the subfamily Triatominae are a very diverse group with a variety of Available online 9 May 2013 morphs, behaviors and distributions. They have great epidemiological importance because many of its members are vectors of the protozoan Trypanosoma cruzi, the agent of Chagas disease. Mepraia is a genus Keywords: of Triatominae endemic to Chile responsible for transmitting T. cruzi in the sylvatic cycle. Mepraia Chagas disease includes three species, M. gajardoi (18° 300–26° 300 S) M. spinolai (26° 300–34° 200 S) and the recently Phylogeography described M. parapatrica in intermediate zones (24° 360–26° 510 S). Using mitochondrial DNA sequences, Triatominae vectors we inferred historical processes that led to the current structure of populations. Phylogeographic analy- Mepraia gajardoi ses identified three lineages, congruent with current taxonomy, and populations were highly structured. Mepraia spinolai Chile The times to the most recent common ancestor suggest that M. spinolai is the oldest lineage. We discuss the taxonomic and biogeographic implications of our results. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction et al. (1994). Lent and Wygodzinsky (1979) described the spinolai complex as a taxonomic group composed of Triatoma eratyrusifor- Chagas disease is a serious human parasitic disease in the mis Del Ponte 1929, Triatoma breyeri Del Ponte 1929 and Triatoma Americas caused by the flagellate protozoan Trypanosoma cruzi, (Mepraia) spinolai Porter 1934. T. eratyrusiformis and T. breyeri are and transmitted by blood-sucking insects of the subfamily Tri- geographically separated from T. spinolai (M. spinolai) by the Andes atominae (Hemiptera: Reduviidae) (Lent and Wygodzinsky, 1979; Range (Lent and Wygodzinsky, 1979). The same authors proposed Schofield et al., 2006). Despite the research effort dedicated to that the species of the spinolai complex share a common ancestor the subfamily in recent years, the taxonomic status, evolutionary that was separated by the uplifting of the Andes during the Mio- relationships and phylogeographic structure of several species re- cene about 20 million years ago (Moreno et al., 2006; Campos main controversial, due in part to their high degree of morpholog- et al., 2013). ical plasticity (Dujardin et al., 1999). Two genera of Triatominae Three species currently are included in the genus: M. spinolai, M. occur in Chile: Triatoma and Mepraia. Triatoma infestans is the main gajardoi and Mepraia parapatrica (Frías and Atria, 1998; Frías et al., domestic vector of T. cruzi, now controlled in several countries 1998; Frías, 2010). Until 1998, Mepraia spinolai was the only spe- including Chile through widespread interventions under the cies of the genus, distributed in coastal and interior valleys in Chile Southern Cone Initiative (Dias and Schofield, 1999). Mepraia is en- between 18° and 34° S(Lent and Wygodzinsky, 1979). However, on demic to the semiarid and arid regions distributed in coastal and the basis of karyotypes, morphological characters and experimen- interior valleys of northern and central Chile. While Triatoma is tal crosses, coastal desert populations between 18° and 26° S were mainly associated with domestic settings in Chile, Mepraia is found described as M. gajardoi (Frías et al., 1998; Jurberg et al., 2002). The in sylvatic areas mainly among rock piles, but it occasionally colo- remaining populations from 26° to 34° S maintained the status of nizes domestic and peridomestic habitats (Frías and Atria, 1998; M. spinolai inhabiting in the interior mountains from the Atacama Schofield et al., 1998; Cattan et al., 2002). Mepraia was first de- Region to the Metropolitan Region (Frías et al., 1998). Studies using scribed with the inclusion of Triatoma spinolai Porter 1934 as Me- molecular markers suggested that the geographical criterion ini- praia spinolai by Mazza et al. (1940), and revalidated by Lent tially proposed by Frías et al. (1998) to separate the two Mepraia species should be reviewed (Calleros et al., 2010). A recent study ⇑ Corresponding author at: Programa de Biología Celular y Molecular, ICBM, using geometric morphometrics on wings of Mepraia species Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile. Tel.: identified two distinct groups (congruent with M. gajardoi and +56 2 978 6062; fax: +56 2 735 5580. M. spinolai), but also reported a new wing phenotype; insects with E-mail address: [email protected] (A. Solari). 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.04.036 R. Campos et al. / Infection, Genetics and Evolution 19 (2013) 280–286 281 vestigial wings in the areas that limit the range of the two species rion (Akaike, 1974) on the concatenated matrix implemented in (Campos et al., 2011). In that area (border of the Antofagasta and the program jmodelTest 0.1.1 (Posada, 2008). Nodal supports were Atacama Regions; 24°–26° S) a new species (M. parapatrica) was estimated by the bootstrap method (Felsenstein, 1985) with 1000 recently described (Frías, 2010). The complex taxonomy of Mepraia replicates using PhyML 3.0 (Guindon and Gascuel, 2003). We con- may be resolved using an integrative approach that includes mor- sidered branches receiving >70% bootstrap support to be well-sup- phological and molecular characters. To our knowledge, no study ported (Hillis and Bull, 1993; Wilcox et al., 2002). has evaluated the historical processes that may have occurred Bayesian analyses were performed using MrBAYES v.3.1.2b within Mepraia leading to its current phylogeographic pattern. (Ronquist and Huelsenbeck, 2003), using as prior parameters val- In this study, we used molecular data from natural populations ues obtained from jModeltest. We ran Metropolis-coupled Markov of Mepraia to explore genetic structure across northern Chile and Chain Monte Carlo simulations (MCMCMC) with four incremen- the phylogeographic relationships of M. parapatrica with M. gajar- tally heated chains. From random starting trees, four independent doi and M. spinolai. Our study also aims to estimate the date of the runs (two replicas of two simultaneous, independent runs each) of most recent common ancestor (MRCA) of Mepraia, and discuss the 1 Â 107 generations each were performed, with the resulting trees biogeographic scenario that yielded the current phylogeographic sampled every 1000 generations. We determined when a station- structure of the three species. ary state was reached (to discard the burn-in samples; 1000 trees) by plotting the logarithmic likelihood scores of sample points against generations. The last 9000 trees were used to compute a 2. Materials and methods 50% majority rule consensus tree. The percentage of samples that recover any particular clade in this tree represents that clade’s pos- 2.1. Insect collection terior probability; we considered P P 95% as evidence for signifi- cant support (Alfaro et al., 2003). Trees were visualized using the A total of 164 nymphs and adults of Mepraia from 13 localities FigTree v1.1.2 program, available at http://tree.bio.ed.ac.uk/soft- in Chile ranging from 18° to 33° S were collected between 2007 ware/figtree/. T. infestans, T. breyeri and T. eraturiformis were used and 2010 in the coast and interior valleys (Table 1). Two Triatoma as outgroups based on their phylogenetic proximity to the spinolai eratirusiformis from Salinas de Bustos, and two Triatoma breyeri complex (Hypsa et al., 2002; de Paula et al., 2005). The Median from Patquia Viejo were also collected; both locations belong to Joining method (Bandelt et al., 1999) implemented in Network the Department of Independencia, Province of La Rioja, Argentina 4.2.0.1 software was used alternatively to assess intraspecific rela- (courtesy of S Catalá). Insects were manually collected by trained tionships of all Mepraia samples. The pairwise differences between people. Bugs were dissected in the laboratory and the limbs were populations were calculated using the fixation index (Fst) and the kept in 70% ethanol at À20 °C. significance of the values was obtained by 1000 permutations using Arlequin 3.5.1 (Excoffier and Lischer, 2010). Genetic struc- 2.2. Mitochondrial DNA extraction, amplification, and sequencing ture was estimated using an analysis of molecular variance (AMO- VA; Excoffier et al., 1992) using Arlequin 3.5.1 (Excoffier and Genomic DNA from legs was extracted using the DNA extraction Lischer, 2010); the analysis assesses the proportion of genetic var- Ò kit EZNA Tissue DNA according to manufacturer’s instructions. A iation explained by different population hierarchies. The level hier- 636-bp fragment of the mitochondrial cytochrome oxidase sub- archies were defined on the basis of the phylogenetic tree and unit-I (COI) gene and a 682-bp fragment of the cytochrome b haplotype network. (Cyt b) gene were amplified via polymerase chain reaction (PCR) using the primers LCO1490 (forward) (50-GGTCAACAAATCATAAA- 0 0 GATATTGG-3 ) and HCO2198 (reverse) (5 -TAAACTTCAGGGTGAC- 2.4. Molecular diversity and neutrality tests CAAAAAATCA-30) for the Cyt b gene (Folmer et al., 1994), and the 0 0 primers 7432 (forward) (5 -GGACGWGGWATTTATTATGGATC-3 ) We evaluated genetic variability of concatenated COI and Cyt b 0 0 and 7433 (reverse) (5 -GCWCCAATTCARGTTARTAA-3 ) for the COI sequences (1022-nt) for each population using DnaSp 5.1 (Librado gene (Monteiro et al., 2003).