A peer-reviewed open-access journal ZooKeys 609: 107–120Molecular (2016) phylogeny of the forensically important genus Cochliomyia 107 doi: 10.3897/zookeys.609.8638 RESEARCH ARTICLE http://zookeys.pensoft.net Launched to accelerate biodiversity research Molecular phylogeny of the forensically important genus Cochliomyia (Diptera: Calliphoridae) Sohath Yusseff-Vanegas1, Ingi Agnarsson1 1 Department of Biology, University of Vermont, 109 Carrigan Drive, Burlington, VT 05405, USA Corresponding author: Sohath Yusseff-Vanegas ([email protected]) Academic editor: Pierfilippo Cerretti | Received 3 April 2016 | Accepted 25 July 2016 | Published 8 August 2016 http://zoobank.org/37FC24A0-F546-44F5-95F7-15B8121D86AB Citation: Yusseff-Vanegas S, Agnarsson I (2016) Molecular phylogeny of the forensically important genusCochliomyia (Diptera: Calliphoridae). ZooKeys 609: 107–120. doi: 10.3897/zookeys.609.8638 Abstract Cochliomyia Townsend includes several abundant and one of the most broadly distributed, blow flies in the Americas, and is of significant economic and forensic importance. For decades,Cochliomyia hominivorax (Coquerel) and C. macellaria (Fabricius) have received attention as livestock parasites and primary indica- tor species in forensic entomology. However, C. minima Shannon and C. aldrichi Del Ponte have only been subject to basic taxonomy and faunistic studies. Here we present the first complete phylogeny of Cochlio- myia including numerous specimens per species, collected from 13 localities in the Caribbean. Four genes, the mitochondrial COI and the nuclear EF-1α, 28S rRNA, and ITS2, were analyzed. While we found some differences among gene trees, a concatenated gene matrix recovered a robustly supported mono- phyletic Cochliomyia with Compsomyiops Townsend as its sister group and recovered the monophyly of C. hominivorax, C. macellaria and C. minima. Our results support a close relationship between C. minima and C. aldrichi. However, we found C. aldrichi containing C. minima, indicating recent speciation, or issues with the taxonomy of the group. We provide basic information on habitat preference, distribution and feeding habits of C. minima and C. aldrichi that will be useful for future forensic studies in the Caribbean. Keywords Forensic entomology, Caribbean region, habitat preferences, Cochliomyia minima, Cochliomyia aldrichi, Cochliomyia macellaria, Cochliomyia hominivorax Copyright S. Yusseff-Vanegas and I. Agnarsson. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 108 Sohath Yusseff-Vanegas & Ingi Agnarsson / ZooKeys 609: 107–120 (2016) Introduction Cochliomyia Townsend is endemic to the Americas and includes only four species: Cochliomyia minima Shannon, C. aldrichi Del Ponte, C. macellaria (Fabricius) and C. hominivorax (Coquerel). All of them are flesh eaters during their larval stage and are locally abundant. In particular, Cochliomyia macellaria is one of the most broadly distributed blow flies in the New World (Whitworth 2010). These species vary in habitat preference, feeding habits, dispersal abilities, and morphology among the species (Hall 1948, Whitworth 2010). For instance, C. aldrichi, C. minima and C. macellaria are primarily carrion feeders, while, C. hominivorax is an obligate parasite of mammals (Hall 1948, Stevens and Wallman 2006, McDonagh et al. 2009, Mc- Donagh and Stevens 2011). Cochliomyia hominivorax and C. macellaria have been intensely studied due to their commercial and forensic importance. Cochliomyia macellaria is one of the most forensically important species commonly found on decomposing remains. This species is considered important for post mortem interval estimations (Smith 1986, Byrd and Castner 2010) being among the first species to colonize corpses. In contrast, Cochlio- myia hominivorax is an obligate parasite with its larvae producing myiasis and feeding on living tissue (Hall 1948, Guimaraes et al. 1983). This species is one of the most im- portant insect pests of livestock in the Neotropics causing economic losses of billions of dollars every year (Vargas-Terán et al. 2005). Both species are common throughout the year in tropical, warm and humid areas (Hall 1948). Cochliomyia macellaria can be found in temperate climates from Canada to Argentina during the summer months (Whitworth 2010). Cochliomyia hominivorax initially ranged from southern United States to northern Argentina (Guimaraes et al. 1983) but has been eradicated from North America, Central America, Puerto Rico and the Virgin Islands (Vargas-Terán et al. 2005). It is worth noting that in 1988 this species was introduced in Libya and it was successfully eradicated in 1992 based on the sterile insect technique (SIT). This was the major international effort and avoid a major disaster for the livestock indus- try of Africa and Southern Europe (Lindquist et al. 1992). Despite those successfully eradications C. hominivorax continues to be an economically important pest in South America and parts of the Caribbean (Vargas-Terán et al. 2005). The other two congeners,C. minima and C. aldrichi, are poorly known and re- search has been limited to descriptive morphology and faunistics (Hall 1948, Dear 1985, Whitworth 2010). These two species are restricted to the West Indies andC. al- drichi has been reported in the Florida Keys (Whitworth 2010). Dear (1985) listed C. minima for the Florida Keys, however Whitworth (2010) concluded that Dear mistak- enly identified one C. aldrichi specimen as C. minima. Forensically important insects in the Caribbean are generally understudied and these two species have not played an important role in forensic entomology. Yet, due to their abundance and broad distribu- tion in this region, including Cuba, Dominican Republic, Jamaica, Puerto Rico, Vir- gin Islands, Bahamas and Cayman Islands (Hall 1948, Dear 1985, Whitworth 2010) they have an enormous forensic potential. For example, recent studies conducted in Molecular phylogeny of the forensically important genus Cochliomyia 109 Puerto Rico showed that C. minima is abundant and widely distributed on the island, and that adults are attracted to, and feed on, carrion (Yusseff-Vanegas 2014). Although the adult morphology of the four species is well known (Hall 1948, Dear 1985, Whitworth 2010), studies on the relationship among Cochliomyia spe- cies have not been conducted yet. Morphological studies have provided synapomor- phies of Cochliomyia that clearly diagnose it from all other Calliphoridae (Hall 1948; Dear 1985; Whitworth 2010). These include short and filiform palpus and phallus with extremely elongated paraphallus and a complex distiphallus (Dear 1985, Figs 37–44). Prior studies on the relationships among Calliphoridae (Stevens 2003, Harvey et al. 2008, McDonagh and Stevens 2011) and the subfamily Chrysomyinae (Singh and Wells 2011), including C. macellaria and C. hominivorax, supported Cochliomyia monophyly, and placed it as sister to Compsomyiops Townsend. However, the mono- phyly of the genus has not been formally tested with thorough sampling of all species, and the relationships among its species remain unknown. Furthermore, DNA-based methods can provide reliable identification of specimens by non-experts and will be particularly important for the identification of larval stages ofC. minina and C. al- drichi that remain poorly known. For example, only the third instar of C. minima has been described (Yusseff-Vanegas 2014). Here we provide a robust phylogenetic hypothesis of Cochliomyia based on four genes sequenced from 38 individuals collected throughout the Caribbean, includ- ing for the first time molecular data about C. minima and C. aldrichi. Our main goals are to test the monophyly of this genus and the validity of, and relationships among, its species. Methods Specimens and DNA extraction A total of 44 specimens were included in this study, 38 representing the ingroup plus six outgroup species [Chrysomya megacephala (Fabricius), C. rufifacies(Macquart), Hemilucilia sp., Lucilia cuprina (Wiedemann), Compsomyiops fulvicrura (Robineau- Desvoidy) and Compsomyiops callipes (Bigot)]. All sequences used here are new except for Compsomyiops fulviclura and C. callipes (Table 1). The specimens were collected in the Caribbean (Jamaica, Cuba, Dominican Republic, Puerto Rico, Saint Barts Mar- tinique and Dominica) from 2011 to 2013 and in the following countries, Colombia (2014), Florida (2013) and Mexico (2010 and 2012) (Table 1). All specimens were killed and preserved in 95% ethanol and stored at -20 °C. The adults were examined with a Leica MZ16 stereomicroscope and identified using the Whitworth (2010) keys. The DNA was isolated from thoracic muscle or two legs of each individual with the QIAGEN DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). Voucher specimens were deposited at the UVM Natural History Museum (in the Zadock Thompson Zoological Collections) and sequences were submitted to GenBank. 110 Sohath Yusseff-Vanegas & Ingi Agnarsson / ZooKeys 609: 107–120 (2016) Table 1. Specimen details, collection information and GenBank accession numbers. Species name –Voucher Number Location CO1 EF-1α ITS2 28S rRNA Cochliomyia macellaria CO002 Colombia, El refugio Dry Forest KX529522 KX529616 KX529574 KX529487 Cochliomyia macellaria CO010 Colombia, Choco, Jardín botánico del Pacífico KX529545 KX529617 KX529575 KX529488 Cochliomyia