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

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[…] A region of the mitochondrial genome encoding COI was amplified in a single fragment using the primers LCO1490 (), and C1-N-2776 (). Those primers amplified successfully in all Calliphoridae except Lucilia Robineau-Desvoidy. From the eight Caribbean species of Lucilia, only Lucilia retroversa amplified successfully using these primers. For the remaining Lucilia species two different primer-pairs were used. The Primer 1 () with C1-N-2191 () and the C1-J-1751 () with C2-N-3014. For the second internal transcribed spacer ITS2 we used the primers ITS4 and ITS5.8 (). The primer sequences and protocols are listed in . Amplified fragments were sequenced in both directions by University of Arizona Genetics Core. Sequences were interpreted from chromatograms using Phred and Phrap (; ) using the Chromaseq module () in Mesquite 3.03 () with default parameters. The sequences were then proofread by examining chromatograms by eye. Alignments were done using MAFFT () through the online portal EMBL-EBI with default settings. The matrices were exported to Mesquite 3.03 () and the translation of coding sequences to proteins for COI were checked for potential errors. [...] The COI gene was partitioned by codon positions, each partition and ITS2 gene were exported from Mesquite for model choice. The appropriate models were chosen using jModeltest v2.1.4 (), and the AIC criterion (). The corresponding model of evolution was used for the Bayesian analysis: GTR + Γ + I for COI1st, F81+ I for COI2nd, GTR + Γ for COI3rd and HKY + Γ + I for ITS2. We ran the MC3(Metropolis Coupled Markov Chain Monte Carlo) chain in MrBayes v3.2.3 () through the online portal Cipres Science Gateway v3.3 (). The analysis was run for 20,000,000 generations, sampling every 1,000 generations, and the sample points of the first 5,000,000 generations were discarded as ‘burnin’, after which the chains had reached stationarity as determined by analysis in Tracer (). Maximum likelihood (ML) analysis of the concatenated matrix was done in Garli () using the same partitioning scheme and models. Sequences were submitted to GenBank and BOLD. [...] We used MEGA6 to calculate genetic distances within and among species level clades suggested by the barcoding analysis of the COI data and by morphology. We used the species delimitation plugin in Geneious 8.1.5 (; ) to estimate species limits under Rosenberg’s reciprocal monophyly P(AB) () and Rodrigo’s P(RD) method (). For this analysis we used a 317 taxa subset of our data, produced by reducing the most densely sampled species like Co. minima, Co. macellaria, Ch. rufifacies and L. retroversa to 38 exemplars since P(RD) probability cannot be computed when there are more than 40 exemplars per clade. We also estimated the probability of population identification of a hypothetical sample based on the groups being tested P ID (Strict) and P ID (Liberal). The genealogical sorting index (gsi) statistic () was calculated using the gsi webserver (http://genealogicalsorting.org) on the estimated tree. As genetic distances in MEGA6, gsi and species delimitation metrics from Geneious require a priory species designation, 26 putative species were assigned to the data based on combined analysis of phylogenetic topology from COI and morphological and geographic information. Finally, we used a single locus Bayesian implementation (bPTP) of the Poisson tree processes model () to infer putative species boundaries on a given single locus phylogenetic input tree available on the webserver: http://species.h-its.org/ptp/. The analysis was run as a rooted tree from the MrBayes analysis, for 500,000 generations with 10% burnin removed. For gsi and bPTP analysis we reduced the data to 103 taxa representing the 26 putative species because of limitations of the server. […]

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