Computational protocol: Unraveling the diversification history of grasshoppers belonging to the “Trimerotropis pallidipennis” (Oedipodinae: Acrididae) species group: a hotspot of biodiversity in the Central Andes

Similar protocols

Protocol publication

[…] Total genomic DNA was extracted from tissue of the hind leg that had been preserved in ethanol using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA). We used a polymerase chain reaction (PCR) to amplify two mitochondrial (mtDNA) and two nuclear (nDNA) DNA sequences: (1) the mitochondrial dehydrogenase subunit 5 gene fragment (NADH5), (2) the mitochondrial cytochrome c oxidase subunit I gene fragment (COI), (3) the internal transcribed spacer 2 (ITS2); and (4) the histone 3 gene (HIS3) fragment. Detailed information on primers can be found in . DNA fragments were amplified in 50-uL reactions consisting of 1× reaction buffer, 3 mM MgCl2, 1 unit of Taq- DNA-Polymerase (Invitrogen, Argentina), 0.1 mM of each dNTP, 100 ng of each primer (Invitrogen, Argentina) and 50–100 ng of DNA template. Polymerase chain reactions were run under the following conditions: 94 °C for 3 min, followed by 30 cycles of 94 °C for 1 min (denaturation), 48–60 °C (NAHD5 50 °C, COI 46 °C, HIS3 57 °C, ITS2 55 °C) for 1 min (annealing) and 72 °C for 2 min (elongation), with a final elongation step at 72 °C for 10 min. PCR Products were purified with a Purification Kit (AccuPrep; Bioneer Corporation, Daejeon, South Korea) to eliminate unused reagents. Finally, these were sequenced at the “Unidad de Secuenciación y Genotipificado” (FCEyN; UBA, Buenos Aires, Argentina). Sequences were inspected, trimmed and aligned using Geneious v.7.0.6.Information on levels of sequence variation was obtained through MEGA v.7.0.21 program (). [...] A phylogeny was constructed in a Bayesian framework for all four gene fragments of 67 individuals (52 specimens belonging to the ingroup and 15 to the different outgroups (), which were simultaneously analyzed using the program BEAST v.1.8.2 (); a Markov chain Monte Carlo (MCMC) simulation was run for 10 million generations, sampling trees every 1,000 generations. Each genetic region was treated as unlinked for substitution models but as linked for trees. The optimal evolutionary model for each dataset was inferred using JModelTest (), on the basis of the Akaike Information Criterion (AIC) (Akaike, 1973, 1974), as suggested by . Tracer v.1.6 was used to verify proper “mixing” of chains. TreeAnnotator v.1.7.5 () was used to choose the maximum clade credibility tree with the “mean node heights” option from the output trees. Output parameters were examined in Tracer v.1.5 to determine whether the effective sample size of the parameters was >200. The ages of nodes were determined assuming a COI substitution rate of 3.54% and 3.2% pairwise divergence per million years (Myr) (; , respectively). The tree was also estimated using the “Metropolis-coupled Markov chain Monte Carlo” (MCMCMC) algorithm implemented in MrBayes v.3.2 () with the concatenate dataset. Default prior distributions were used. The analysis was run over 1,000,000 generations with sampling every 100 generations. The tree space was explored by four chains (one cold chain and three incrementally heated chains). Convergence was checked with Tracer v.1.6 () and the average standard deviations of the split frequencies of the two runs were below 0.01.A haplotype network was built with a concatenated data set that included both mitochondrial genes, using TCS program v.1.21 (), based on the statistical parsimony method described by .Pairwise genetic distances between clades identified in the phylogenetic analysis were estimated using MEGA v.7.0.21 program (). [...] A biogeographic analysis was carried out to reconstruct the distribution history of the Trimerotropis pallidipennis complex. We used three reconstruction approaches, which differ slightly in the allowed cladogenetic events (; ): a statistical dispersal-vicariance analysis (S-DIVA; ) based on the original formulation of ; a dispersal-extinction-cladogenesis analysis (Lagrange DEC model; ); and a statistical dispersal-extinction-cladogenesis analysis (Lagrange S-DEC model; ) using the program RASP v. 3.2 (). The analysis was based on 1,000 post-burn-in trees from the MrBayes analysis (see item “Phylogenetics analyses” in Material and Methods). Trimerotropis populations were clustered into four regions based on their geographic distribution: (A) East of the Andes, (B) North and Central America, (C) Chilean Pacific slope and (D) Peruvian Pacific slope and Central Andean Highlands of Peru. […]

Pipeline specifications

Software tools Geneious, MEGA-V, BEAST, jModelTest, MrBayes, RASP
Applications Phylogenetics, GWAS