SHORT COMMUNICATION Recent African derivation of Chrysomya putoria from C. chloropyga and mitochondrial DNA paraphyly of cytochrome oxidase subunit one in blowflies of forensic importance J. D. WELLS 1 ,N.LUNT 2 andM. H. VILLET 2 1 Department of Biology, West Virginia University, Morgantown, West Virginia, U.S.A. and 2 Department of Zoology & Entomology, Rhodes University, Grahamstown, South Africa Abstract. Chrysomya chloropyga (Wiedemann) and C.putoria (Wiedemann) (Diptera: Calliphoridae) are closely related Afrotropical blowflies that breed in carrionandlatrines,reachinghighdensityinassociationwithhumansandspread- ing to other continents. In some cases of human death, Chyrsomya specimens provideforensicclues.Becausetheimmaturestagesofsuchfliesareoftendifficult to identify taxonomically, it is useful to develop DNA-based tests for specimen identification. Therefore we attempted to distinguish between C.chloropyga and C.putoria using mitochondrial DNA (mtDNA) sequence data from a 593-bp region of the gene for cytochrome oxidase subunit one (COI). Twelve specimens from each species yielded a total of five haplotypes, none being unique to C.putoria. Therefore it was not possible to distinguish between the two species using this locus. Maximum parsimony analysis indicated paraphyletic C.chloropyga mtDNA with C.putoria nested therein. Based on these and previously published data, we infer that C.putoria diverged very recently from C.chloropyga. Key words. Calliphoridae, Chrysomya, Diptera, blowflies, cytochrome oxidase, evolution, forensic entomology, molecular systematics, mitochondrial DNA, para- phyletic species, reciprocal mitochondrial DNA monophyly, South Africa, U.S.A. Mitochondrial DNA (mtDNA) sequence data have often been advocated for species identification of forensically important insect specimens (Sperling etal., 1994; Malgorn & Coquoz, 1999; Vincent etal., 2000; Wallman & Donnellan, 2001; Wells & Sperling, 2001; Wells etal., 2001). This approach is easier to accomplish in cases of reciprocal mtDNA monophyly corresponding to morpho- logical species. When reciprocal mtDNA monophyly exists, a phylogenetic analysis of reference (identified) sequences plus the unknown sample will group the unknown with its own species, provided that the relevant species is included among the reference sequences. This method can also pro- duce an objective statement about the strength of this con- clusion, based on measures of branch support such as a bootstrap value (Wells & Sperling, 2001). Apparently some sister species have diverged too recently fordetectableautapomorphiestoresultfrommutationsand lineage sorting (Avise, 2000). For example, Wallman & Donnellan (2001) were unable to distinguish between the cytochrome oxidase haplotypes of certain Australian Calli- phora species. For purposes of forensic science, the molecu- lar systematics of very closely related carrion-insect species deserve close attention because these specimens are difficult to identify using morphological criteria. Two such Diptera are the ‘coppery-tailed blowfly’ Chrysomya chloropyga (Wiedemann) and the ‘tropical latrine blowfly’ Chrysomya putoria (Wiedemann), both originally Afrotropical in Correspondence: Dr J. D. Wells, Department of Biology, West Virginia University, 53 Campus Drive, Morgantown, WV 26506, U.S.A. Fax: þ 13042936363; e-mail: jdwells@mail.wvu.edu Medical and Veterinary Entomology (2004) 18, 445–448 # 2004 The Royal Entomological Society 445