Computational protocol: “Darwin’s butterflies”? DNA barcoding and the radiation of the endemic Caribbean butterfly genus Calisto (Lepidoptera, Nymphalidae, Satyrinae)

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Protocol publication

[…] A total of 110 Calisto specimens representing 31 putative taxa were sampled (). All specimens were collected in 1994–1999 by the first author. None of the specimens were subjected to any chemical treatment before desiccation. The climate of the regions ensured quick drying of specimens, which were stored at a room temperature (18–25°C) for over 10 years. DNA was extracted from a single leg removed from each specimen. Specimens were mostly unprepared (papered), with the exception of several individuals.We amplified a 655-bp segment of mitochondrial cytochrome oxidase subunit I, from the COI barcode region. All polymerase chain reactions and DNA sequencing were carried out following standard DNA barcoding procedures for Lepidoptera as described previously (, ). Photographs of all specimens used in the analysis as well as specimen collection data and sequences are available in the Barcode of Life Data System (BOLD) at as well as in GenBank (accession numbers JN197297--JN197406). All voucher specimens are deposited at the McGuire Center for Lepidoptera and Biodiversity (Florida Museum of Natural History, University of Florida).We chose two genera as outgroups: Eretris, which thought to be Calisto’s closest relative on the mainland, based on wing shape and relative proximity to the Caribbean, and the southern Andean genus Auca Hayward, 1953 (Satyrinae: Pronophilina), which we have observed to be morphologically and behaviorally similar to Calisto (e. g., Auca’s association with bunch grass in arid lowland habitats is very similar to species in the Calisto lyceius complex) (Sourakov pers. obs.). Though geographically distant from Calisto, the inclusion of such a Pronophilina member from the southern Andes could provide insight into the origin of Calisto should the genus prove to be non-monophyletic and also provides an additional point of comparison for the pairwise divergence analysis. Hence, we obtained five additional sequences from GenBank (), including two species of Auca, Auca coctei (GenBank number DQ338833) and Auca barrosi (GenBank number DQ338832) (), and two species of Eretris, Eretris sp. (GenBank number GQ357229) and Eretris sp.2 (GenBank number GQ864764) (). We also obtained one additional sequence of Calisto pulchella (GenBank number GQ357225) ().Sequences were aligned using BioEdit software () and manually edited. Sequence information was entered into the Barcode of Life Data System ( along with an image and collateral information for each voucher specimen. Detailed specimen records and sequence information, including trace files, are available in the LOWA project file in the BOLD website. All sequences are also available through GenBank.Sequence data were analyzed using Bayesian inference (BI), as implemented in Mr Bayes 3.1.2 (; ). A GTR substitution model with gamma-distributed rate variation across sites and a proportion of invariable sites was specified before running the program for 5,000,000 generations with default settings. The first 2500 trees (out of 10000) were discarded prior to computing a consensus phylogeny and posterior probabilities.Maximum parsimony (MP) analysis was performed using a heuristic search as implemented in MEGA4 (). We used the close-neighbor-interchange algorithm with search level 3 () in which the initial trees were obtained by random addition of sequences (100 replicates). We used nonparametric bootstrap values () to estimate branch support on the recovered tree, with the bootstrap consensus tree inferred from 1000 replicates (MP tree is provided as Supplementary file). The Kimura 2-parameter model of base substitution was used to calculate genetic distances in MEGA4 software (). Dendroscope () was used to edit trees for publication. […]

Pipeline specifications

Software tools BioEdit, MEGA, Dendroscope
Application Phylogenetics