Phylogenetic multi-locus codon models and molecular clocks reveal the monophyly of haematophagous reduviid bugs and their evolution at the formation of South America James S. Patterson a,b,1 , Michael W. Gaunt a, * a Infectious and Tropical Diseases Department, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK b South African National Bioinformatics Institute, Western Cape University, Cape Town, Western Cape, South Africa article info Article history: Received 15 October 2009 Revised 21 April 2010 Accepted 24 April 2010 Available online 7 May 2010 Keywords: Triatominae monophyly Hemiptera Reduviidae Multi-locus codon models Molecular clock for the insects abstract We previously constructed a single molecular clock to date insect evolution that remains a cornerstone within entomological dating. The insect clock predicts that triatomine bugs, the vectors of South Amer- ican trypanosomiasis, originated with the formation of South America. We addressed this hypothesis using the insectivorous reduviid bugs and their phylogenetic relationship with the haematophagous reduviid bugs, as well as their biogeographic distribution. Putative paraphyly or monophyly of Triatominae, by non-haematophagous reduviids, have both previously been hypothesized and identified. We sampled a broad range of predatory reduviids, viz. Ectrichodiinae, Emesinae, Hammacerinae, Harpac- torinae, Reduviinae, Salyavatinae, Steniopodainae and Vesciinae, including both New World and Old World representatives and sequenced the nuclear 28S ribosomal gene locus and the mitochondrial loci 5 0 cytochrome oxidase 1 (cox1 [COI]), cox1 3 0 , cytochrome oxidase 2 (cox2 [COII]) and cytochrome oxidase b(cob [cytb]). Robust evidence for the monophyly of Triatominae was observed in 5/5 loci using codon/ nucleotide (28S) based maximum likelihood phylogenies, 3/5 loci using codon-based Bayesian phyloge- nies and in cox2 using amino acid Bayesian phylogenies. Several South American members of the Redu- viinae, that are morphologically and phylogenetically a sister group to triatomine bugs, have a modal divergence date with the Triatominae of 109–107 million years ago (MYA). This creates a scenario where the closest (non-haematophagous) ancestor to triatomine bugs evolved immediately prior to the breakup of Gondwanaland whilst the triatomine bugs evolved 95 MYA, putatively linking the origin of haemato- phagous behaviour to the origin of South America and in particular infers a delayed onset to the evolution of haematophagy. The placement of the enigmatic tribe Bolboderini as an ingroup to the Triatominae monophyly, confirms the 95 MYA node as the most ancient in the subfamily. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Triatomine bugs are the vectors of South American trypanoso- miasis the most important parasitic disease in South America (Schofield and Galvão, 2009). A molecular clock study of the insects predicted that the origin of triatomine bugs coincided with the for- mation of the South American continent (Gaunt and Miles, 2002a). The ancient date for the origin of triatomine bugs is also suggested by the recovery of a Triatoma fossil from Dominican amber (Poinar, 2005). The age of Dominican amber is controversial and stands at either 20–15 MYA based on foraminifera or 40–35 MYA based on coccoliths (Poinar, 2005). The immediate ancestors to the Triatominae, the predatory assassin bugs (Hemiptera: Heteroptera: Cimicomorpha: Reduvii- dae) are one of the largest and most morphologically diverse families of the true bugs, composed of 23–32 subfamilies with some 6000 species (Maldonado-Capriles, 1990). With the exception of a few aberrant plant feeding members (Berenger and Pluot-Sigwalt, 1997) and the haematophagous triatomines, all reduviids are predators on other invertebrates. Reduviids apart from triatomine bugs represent a global distribution with a bewil- dering array of morphology and insect host specificity (see Schuh and Slater (1995)). The reduviids bugs have become an important evolutionary model of haematophagous behaviour following a comprehensive hypothesis that blood-feeding arose from independently, i.e. on numerous occasions, from arthropod predatory ancestors resulting in the subfamily Triatominae (Fig. 1)(Bargues et al., 2000; Scho- field and Dujardin, 1999). This would be a drastic departure from 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.04.038 * Corresponding author. E-mail address: michael.gaunt@lshtm.ac.uk (M.W. Gaunt). 1 Present address: CIMPAT, Departamento de Ciencias Biológicas, Universidad de Los Andes, Cra. 1 No. 18 A-10, Bogotá, Colombia. Molecular Phylogenetics and Evolution 56 (2010) 608–621 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev