Computational protocol: Genetic Evidence for Hybrid Trait Speciation in Heliconius Butterflies

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Protocol publication

[…] When new polymorphisms are introduced by recent introgression at some time after species divergence, this should increase levels of linkage disequilibrium (LD) above the background within the region involved. We analyzed LD across the HmB locus for combined samples of H. heurippa and H. c. cordula. This calculation was made among 199 SNPs from the 24 genetic markers sampled in the HmB linked region. These SNPs were those where the minor allele frequency was greater than 10% (from 60 alleles) and thus, were considered as informative for the LD analysis. The LD computation was executed using the software MIDAS , which considers the distance between pairwise markers and does not assume that the genotypic phases are known. The resulting LD among the SNPs was visualized with the R package LDheatmap plotting the r2 estimates versus physical distance. [...] For all the loci net divergence, fixed differences, shared polymorphisms and nucleotide diversity (π) were estimated with SITES . Additionally, we calculated the number of substitutions per site for synonymous sites (Ks) and non-synonymous sites (Ka) in pairwise comparisons among the three species. The ratio Ka/Ks was determined in order to detect protein evolution in DNAsp v4.90.1 . McDonald-Kreitman test was also performed in DNAsp v4.90.1. [...] In order to confirm the species relationships, genealogical topologies were reconstructed for three fragments within the HmB region, rooted for the 3′ kinesin (6,493 pb) using H. numata as outgroup and unrooted for 5′ kinesin partial sequence (kin_2; 1,100 pb) and sorting nexin (sdp; 402 pb) (). Maximum Parsimony analysis was carried out in PAUP*v4.0b10 using a heuristic search with TBR branch swapping; bootstrap values were calculated with 5,000 replicates using the same search conditions. Modeltest v3.7 was used to determine the most appropriate model for nucleotide substitution based on corrected Akaike information criterion (AICc). For the 3′ kinesin data set Modeltest identified the HKY+I+G model, for 5′ kinesin the K81uf+I+G and for nexin the K80+I. Likelihood reconstructions were also made in PAUP*v4.0b10 based on selected evolutionary models. Heuristic search and bootstrapping were carried out as for parsimony.The 3′ kinesin genealogy was used to enforce a molecular clock hypothesis. When the likelihoods were compared, constant rate evolution was rejected (x 2 = 96.92, df = 48; P<0.001). Then a Bayesian framework, implemented in BEAST v1.4.8 , was employed to obtain an approximate time for the 3′ kinesin introgression. We applied the HKY+I+G model of evolution with four rate categories and assumed a relaxed lognormal clock. Based on the calibration proposed by Wahlberg et al. for Nymphalidae, with Heliconius and Eueides diverged from their common ancestor 18.4 Mya . This date was used as a prior for a probabilistic calibration to determine the splitting time between H. cydno and H. melpomene alleles and between H. heurippa and H. melpomene alleles. The rest of the parameters were sampled keeping the default prior distributions. Two independent runs were implemented, with 50 million steps and burn-ins of 5,000,000. Tracer v1.4 was used to combine runs and observe parameter convergence . Divergence time standard deviations were calculated from 95% confidence/credibility intervals using a normal approximation. We also computed the time of 3′ kinesin introgression under the assumption of species expansion. To perform this calculation, we first tested the fit of the observed mismatch distribution to the theoretical expectation as implemented in Arlequin v. 3.0 . The calculations were made with a neutral mutation rate of ∼2.99×10−10 per base per generation for this region and 10 generations per year. […]

Pipeline specifications

Software tools LDheatmap, DnaSP, MKT, ModelTest-NG, BEAST, Arlequin
Applications Phylogenetics, Population genetic analysis, GWAS
Organisms Heliconius cydno, Ilex paraguariensis