Computational protocol: A distinct species, Dodonaformosana, detected in the Dodonaeugenes species complex: clarification of the taxonomic status of the Punch butterfly in Taiwan

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[…] DNA was extracted from two legs or thorax muscle using the Puregene DNA Isolation kit (Gentra Systems, Minnesota, USA). Mitochondrial cytochrome c oxidase 1 (COI) and cytochrome c oxidase 2 (COII) genes were amplified using the primers listed in Supplementary file 2. Polymerase Chain Reaction (PCR) was performed in a 30 μL volume, containing 23.5 μL of sterile ddH2O, 1 μL of extracted DNA, 1 μL of 10 μM dNTP, 3 μL of 10X PCR reaction buffer, 0.6 μL of each 10 μM primer, and 0.3 μL of Power Taq (Genomics Biosci & Tech, Taiwan). PCR was carried out using two settings as follows: (1) Standard: initial denaturation of 5 mins at 95 °C, followed by 40 cycles consisting of denaturation of 30 s at 95 °C, annealing of 30 s at 57–47 °C, and extension of 30–60s at 72 °C, and final extension of 7 mins at 72 °C; (2) Touchdown: initial denaturation of 5 mins at 95 °C, followed by 20 cycles consisting of three steps of 30 s at 95 °C, 30 s at 65–55 °C (-0.5 °C per cycle), and 30 s at 72 °C, and then additional 20 cycles consisting of 30 s at 95 °C, 30 s at 55–45 °C, and 30 s at 72 °C, and final extension of 7 mins at 72 °C. The quality of PCR products were visually checked on 1–2% agarose gels. If DNA fragments were correctly amplified, the PCR products were sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). [...] DNA sequences were checked and corrected by eye using Sequencher 4.10 (Gene Codes, Ann Arbor, USA). Sequence matrices were aligned using MUSCLE (), and the aligned datasets were saved in the fasta or nexus format for subsequent analyses. Genetic distances were calculated using Kimura-2-parameter models implied by MEGA 6.0 (), and general sequence information was calculated using web server DIVEIN (). All sequences were submitted to GenBank under the accession numbers KX866690-KX866733 (listed in Suppl. material ). [...] Molecular phylogenies were reconstructed under the Bayesian inference (BI) and Maximum Likelihood (ML) criteria. The BI analysis was performed in MrBayes v. 3.2.6 (). The best-fit data partitioning and substitution models (Table ) were selected using the results produced by PartitionFinder v 1.1.1 (). Two independent runs for three partition schemes were performed with eight chains (seven heated and one cold), and five million generations with sampling every 100 generations were set. The first 25 % of generated trees were discarded as burn in (default setting) and the remaining trees were used for producing a majority rule tree with posterior probability for each nodal support. To check the quality of our Bayesian phylogenies, the effective sample size (ESS) of each parameter was over 200, and the convergence test of Marko Chain Monte Carlo (MCMC) chains was checked by Tracer 1.6 (). The ML analysis was done using RAxML Pthreads-based version 8 (; ), and the optimal substitution model and partitioning schemes were found using PartitionFinder (Table ). ML analysis was done with three partitions under the GTRGAMMA model, with 1000 replications for calculating bootstrap support values. Takashia nana was used as a single outgroup in both BI and ML analyses. […]

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