DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region
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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 identify 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 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.
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2017. DNA-barcoding of forensically important blow flies (Diptera: Calliphoridae) in the Caribbean Region. PeerJ Preprints 5:e3014v1 https://doi.org/10.7287/peerj.preprints.3014v1note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
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Supplemental Information
Phylogenetic relationship within Calliphoridae based on a Bayesian analysis of nucleotide data from COI
Numbers indicate posterior probability support values. Specimen voucher codes referred to in Table 1 are shown following species names. For specimens from Lesser Antilles (LA), the three capital letters before the voucher code refers to the name of the islands abbreviated a follows: SBA, St. Barthelemy; SAB, Saba; BAR, Barbuda; NEV, Nevis; KIT, St. Kitts; MTQ, Martinique; ANT, Antigua; GUA, Guadeloupe; MON, Montserrat; EUS, St. Eustatius; SMA, St. Martin, SLU, St. Lucia; BBD Barbados.
Phylogenetic relationship within Calliphoridae based on a Bayesian analysis of nucleotide data from ITS
Numbers indicate posterior probability support values. Specimen voucher codes referred to in Table 1 are shown following species names. For specimens from Lesser Antilles (LA), the three capital letters before the voucher code refers to the name of the islands abbreviated a follows: SBA, St. Barthelemy; SAB, Saba; BAR, Barbuda; NEV, Nevis; KIT, St. Kitts; MTQ, Martinique; ANT, Antigua; GUA, Guadeloupe; MON, Montserrat; EUS, St. Eustatius; SMA, St. Martin, SLU, St. Lucia; BBD Barbados.
Phylogenetic relationship within Calliphoridae based on based on partitioned Bayesian analysis of the combined gene (COI and ITS2) data set
Numbers indicate posterior probability support values. Specimen voucher codes referred to in Table 1 are shown following species names. For specimens from Lesser Antilles (LA), the three capital letters before the voucher code refers to the name of the islands abbreviated a follows: SBA, St. Barthelemy; SAB, Saba; BAR, Barbuda; NEV, Nevis; KIT, St. Kitts; MTQ, Martinique; ANT, Antigua; GUA, Guadeloupe; MON, Montserrat; EUS, St. Eustatius; SMA, St. Martin, SLU, St. Lucia; BBD Barbados.
Additional Information
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Sohath Z Yusseff-Vanegas conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Ingi Agnarsson conceived and designed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):
All specimens were collected under appropriate permits: USA, Florida, Everglades, United States Department of the Interior National Park Service EVER-2013-SCI-0028; Puerto Rico, DRNA: 2011-IC-035 (O-VS-PVS15-SJ-00474-08042011); Jamaica, NEPA, reference number #18/27; USA, USDI National Park Service,EVER-2013-SCI-0028; Costa Rica, SINAC, pasaporte científico no. 05933, resolución no. 019-2013-SINAC; Cuba, Departamento de Recursos Naturales, PE 2012/05, 2012003 and 2012001; Dominican Republic, Ministerio de Medio Ambiente y Recursos Naturales, no 0577, Mexico, SEMARNAT scientific collector permit FAUT-0175 issued to Dr. Oscar Federico Francke Ballve, Oficio no. SGPA/DGVS/10102/13; Colombia, Authoridad Nacional de Licencias Ambientales, 18.497.666 issued to Alexander Gómez Mejía; Saba, The Executive Council of the Public Entity Saba, no 112/2013; Martinique, Ministère de L’Écologie, du Développement Durable, et de L‘Énergie; Nevis, Nevis Historical & Conservation Society, no F001; Barbados, Ministry of Environment and Drainage, no 8434/56/1 Vol. II.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The GenBank accession numbers are in Table 1.
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as a supplementary file.
Funding
Funding for this work comes from National Science Foundation (DEB-1314749 and DEB-1050253) to I. Agnarsson and G. Binford. Development of this project was further supported by a UVM APLE grant to Omar Neyra and Cole Rachman. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
