Computational protocol: Genomic Environment Impacts Color Vision Evolution in a Family with Visually Based Sexual Selection

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Protocol publication

[…] Using DNeasy blood and tissue kits (QIAGEN) DNA was extracted from tissue samples of single specimens of: Heterandria formosa, Xiphophorus helleri, Poecilia caymanensis, P. vittata, P. nigrofasciata, P. latipinna, P. velifera, P. petenensis, P. mexicana, P. minor, P. reticulata, P. bifurca, P. picta, P. parae, and P. wingei. (Note that Heterandria formosa is not to be confused with Poecilia formosa—two distinct members of the family Poeciliidae from different genera). Also, P. wingei was formerly considered a strain of P. reticulata but has been shown to be a distinct species (). Primers specific to 5′ and 3′ UTR regions were designed using genomic data: LWS and SWS2 loci—Poecilia wingei () and Xiphophorous helleri (); SWS1 and RH1 loci—Xiphophorus maculatus (GenBank accessions: AGAJ01036758.1 and AGAJ01019341.1, respectively). UTR primers of RH2-1 were taken from . For primer sequences see , online. We follow and refer to LWS loci by their location relative to one another, with LWS-R being a retrotransposed gene in a separate linkage group compared with LWS-1, LWS-2, and LWS-3. PCR products of LWS-2 were generated in two overlapping segments, each with one UTR and one internal primer. The LWS opsin genes share a duplication history that predates the family Poeciliidae (, ; ). By generating sequencing products with UTR primers, we ensure that phylogenetic clustering of opsins within species are gene conversion events rather than independent duplications (). All sequencing was performed by Molecular Cloning Laboratories (MCLAB; San Francisco, CA, USA). Sequence chromatograms were viewed and analyzed using SeqMan Pro (Lasergene 8.0; DNASTAR) when sites were ambiguous we compared overlapping reads and called a base by the highest peak.A phylogeny of the species used in this study was inferred from ND2 mitochondrial sequences retrieved from GenBank (, online). LWS and SWS2 sequences for P. wingei were taken from (GenBank Accession: HM540108 and HM540107) and X. helleri from (GenBank Accession: GQ999832 and GQ999833). The LWS and RH2-1 sequences of P. mexicana and P. latipinna were taken from (GenBank Accessions: JF823552 – JF823560). PCR products could not be reliably amplified for the RH2-2 locus in the majority of the species so this opsin gene was left out of all analyses. All sequences generated are available under GenBank Accessions KX768552 - KX768664. [...] The divergence of the opsin classes occurred prior to the emergence of the family Poeciliidae (), making alignment of introns and UTRs across classes difficult. Therefore, we inferred a series of trees based on several sets of sequences: 1) mitochondrial ND2, 2) all opsins: exon sequence only, 3) SWS1: full sequence (UTR, introns and exons), 4) SWS2: full sequences, 5) RH1: full sequence, 6) RH2-1: full sequence, 7) LWS: exons and introns, and 8) LWS: UTR only sequence (see in the online for all UTR lengths).For each set, sequences were aligned using a command line implementation of Mafft v7.221 () and edited manually using AliView v1.17.1 () to ensure that intron–exon boundaries were consistent. Best-fit models of molecular evolution were determined using MrModelTest 3.04 (). Maximum likelihood phylogenetic trees were reconstructed using RAxML v8.2.9 (). We performed Bayesian analysis of each aligned data partition using BEAST v 1.8.2 () under a log normal relaxed molecular clock model (). We ran four independent chains for 50 million generations each; we assessed convergence of the runs graphically using Tracer v1.5 () and through evaluation of the effective sample size estimates (ESS) for each parameter (). ESS values above 200 were taken as evidence of parameter stability. We generated maximum clade credibility (MCC) phylogenetic trees from the resulting distributions of trees using TreeAnnotator v1.8.2 (). ML bootstrap values and Bayesian posterior probabilities were employed to assess support for inferred topologies. […]

Pipeline specifications

Software tools DNASTAR Molecular Biology Suite, MAFFT, AliView, MrModelTest, RAxML, BEAST
Application Phylogenetics
Organisms Ilex paraguariensis, Homo sapiens