Computational protocol: Range extension for the common dolphin (Delphinus sp.) to the Colombian Caribbean, with taxonomic implications from genetic barcoding and phylogenetic analyses

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Protocol publication

[…] Sequences obtained from the D-loop and Cytb were edited and aligned manually using the software Geneious v4.8.5. []. In order to identify the species, we conducted a barcoding and cluster analysis advance with 1000 replicates on the “DNA Surveillance”, a validated and curated database of cetacean sequences at the University of Auckland [, ]. This analysis provides a species identification tree indicating what species is most closely related to the problem sequence. The software uses phylogenetic algorithms, providing evolutionary distances between problem (query) and reference sequences []. To compare data shown by “DNA Surveillance”, we conducted a nucleotide blast (blastn) on the National Center for Biotechnology Information (NCBI) website [] using the megablast program in order to find highly similar sequences for our query. For NCBI results we took into account the affinity between the match of sequences and the Expected value (E), which describes the random background noise, being lower or closer to zero if the match between sequences is significant []. [...] A total of 347 bp consensus sequence for Cytb was compiled, analyzed and compared with 190 published sequences in GenBank from around the world (). As well, a 447 bp consensus sequence for D-loop was compiled, analyzed and compared with 190 published sequences from various locations worldwide (). Haplotypes were defined using the R script RemoveRedundantTaxa [], which is a substitute for a MacClade utility.jModelTest v2.1.7 [, ] was used to find the best-fitting model of nucleotide substitution among the sequences of both mitochondrial markers haplotypes. In the case of Cytb the best model was HKY+I, and for the control region the best model was K80+G. Each gene was analyzed independently since there were no sequences for both genes from the same individuals in all locations. Two different trees were obtained: one for Cytb and one for D-loop. The software package BEAST [] was used to generate the phylogenetic relationship tree, establishing Globicephala macrorhynchus as outgroup. The program was ran with 10 million MCMC generations sampling every 1,000 generations, Yule process was specified as the species tree prior, and a Normal distribution for the tmrca. The HKY+I and K80+G models were used for Cytb and D-loop respectively. An uncorrelated lognormal distribution relaxed molecular clock was chosen. The program TRACER v1.6 [] was used to check mixing and convergence of parameters and posterior distribution against generations. Finally, TreeAnnotator v1.8.2 [] was run to summarize the trees obtained in one consensus tree. […]

Pipeline specifications

Software tools Geneious, BLASTN, MacClade, jModelTest, BEAST
Application Phylogenetics