Computational protocol: Uncovered Diversity of a Predominantly Andean Butterfly Clade in the Brazilian Atlantic Forest: a Revision of the Genus Praepedaliodes Forster (Lepidoptera: Nymphalidae, Satyrinae, Satyrini)

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Protocol publication

[…] Total genomic DNA was isolated from 58 individuals of Praepedaliodes species using Invisorb® Spin Tissue Mini Kit (STRATEC Molecular, Germany). The mitochondrial DNA (mtDNA) gene cytochrome oxidase subunit I (COI, 1499 bp) was sequenced, including the barcode region proposed by Hebert et al (), which is the 5′ portion of the COI (658 bp), according to published protocols (Wahlberg & Wheat ). Sequences were aligned with all sequences available for Praepedaliodes genus in Genbank, with 14 individuals as outgroups, 12 of which belonging to eight genera of the Pedaliodes complex, one belonging to the genus Pseudomaniola Röber, 1889 (a representative of another clade of the subtribe Pronophilina), and one belonging to the genus Atlanteuptychia Freitas, Barbosa & Mielke, 2013, a representative of the subtribe Euptychiina, considered as sister group of Pronophilina (Table ). The final matrix comprised 73 individuals of the ten species of Praepedaliodes, and the mentioned outgroups. Bayesian analyses (BI) were carried out using the program MrBayes 3.2 (Ronquist et al ) on the CIPRES portal (Miller et al ). The model-jumping feature of the program was utilized, thereby sampling all possible GTR submodels according to their posterior probability (Ronquist et al ). The gamma parameter was also included to allow site rate variation. Four simultaneous chains were run for 10 × 106 generations for two runs, sampling trees every 1000 cycles. The first 2500 trees were discarded as “burn in” based on when the runs had converged and reached equilibrium. The convergence of the likelihood traces of the independent runs was assessed with TRACER v1.5, and the ESS (effective sample size) values were verified to be above 300 for all parameters, which indicates that they were sufficiently sampled to estimate their posterior distributions (Drummond et al ).Pairwise genetic distances between individuals (based on barcodes only) (Table ) were calculated using MEGA v. 6.0 (Tamura et al ), under Kimura two-parameter (K2P) model of nucleotide substitution (Kimura ), and the frequency distribution of genetic divergence was plotted using pairwise values. […]

Pipeline specifications

Software tools MrBayes, CIPRES Science Gateway, MEGA-V
Applications Phylogenetics, GWAS