DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region

Abstract

Correct identification of forensically important insects, such as flies in the family Calliphoridae, is a crucial step for them to be used as evidence in legal investigations. Traditional identification based on morphology has been effective, but has some limitations when it comes to identifying immature stages of certain species. DNA-barcoding, using COI, has demonstrated potential for rapid and accurate identification of Calliphoridae, however, this gene does not reliably distinguish among some recently diverged species, raising questions about its use for delimitation of species of forensic importance. To facilitate DNA based identification of Calliphoridae in the Caribbean we developed a vouchered reference collection from across the region, and a DNA sequence database, and further added the nuclear ITS2 as a second marker to increase accuracy of identification through barcoding. We morphologically identified freshly collected specimens, did phylogenetic analyses and employed several species delimitation methods for a total of 468 individuals representing 19 described species. Our results show that combination of COI + ITS2 genes yields more accurate identification and diagnoses, and better agreement with morphological data, than the mitochondrial barcodes alone. All of our results from independent and concatenated trees and most of the species delimitation methods yield considerably higher diversity estimates than the distance based approach and morphology. Molecular data support at least 24 distinct clades within Calliphoridae in this study, recovering substantial geographic variation for Lucilia eximia, Lucilia retroversa, Lucilia rica and Chloroprocta idioidea, probably indicating several cryptic species. In sum, our study demonstrates the importance of employing a second nuclear marker for barcoding analyses and species delimitation of calliphorids, and the power of molecular data in combination with a complete reference database to enable identification of taxonomically and geographically diverse insects of forensic importance.

Keywords: Calliphoridae; Caribbean; DNA-barcoding; Diptera; Forensic entomology.

Figure 1. Map of collecting localities of all specimens used for the molecular analysis.

(Image credit: https://commons.wikimedia.org/wiki/File:Caribbean_map_blank.png#filelinks).

Figure 2. Summary of the Bayesian tree based on the COI dataset including 442 individuals, with the results of four different species delimitation approaches in addition to morphology, genetic distances of >2% mtDNA, ITS2 and the concatenated matrix.

See Fig. S1 for bootstrap support values.

Figure 3. Bayesian tree based on ITS2 dataset including 158 specimens.

Individual terminal taxa have been replaced with species names, while full taxon clade structure is retained. Colors represent different species based on morphology. See Fig. S2 for bootstrap support values.

Figure 4. Bayesian tree based on the concatenate dataset including 137 specimens.

Individual terminal taxa have been replaced with species names, while full taxon clade structure is retained. Colors represent different species based on morphology. See Fig. S3 for bootstrap support values.