DOI: 10.5061/dryad.mkkwh711m
title: Genomic data support the taxonomic validity of Middle American livebearers Poeciliopsis gracilis and Poeciliopsis pleurospilus (Cyprinodontiformes: Poeciliidae)

description: Poeciliopsis (Cyprinodontiformes: Poeciliidae) is a genus comprised of 25
 species of freshwater fishes. Several well-known taxonomic uncertainties
 exist within the genus, especially in relation to the taxonomic status of
 Poeciliopsis pleurospilus and P. gracilis. However, to date, no studies
 have been conducted to specifically address the taxonomic status of these
 two species. The goal of this study was to examine the taxonomic validity
 of P. pleurospilus and P. gracilis using genomic data (ddRADseq) in
 phylogenetic, population genetic, and species delimitation frameworks.
 Multiple analyses support the recognition of both taxa as distinct species
 and also permits us to revise their respective distributions. A species
 delimitation analysis indicates that P. pleurospilus and P. gracilis are
 distinct species from one another, each of which consists of two distinct
 lineages that are geographically structured. Phylogenetic and population
 genetic analyses demonstrate clear evidence that individuals of P.
 gracilis are distributed north and west of the Isthmus of Tehuantepec in
 both Pacific and Atlantic river systems in Mexico, whereas individuals of
 P. pleurospilus are distributed in both Atlantic and Pacific river systems
 south and east of the Isthmus of Tehuantepec, from southern Mexico to
 Honduras.
description: Three<b>
 </b>double digest restriction enzyme DNA (ddRAD) libraries (batch
 information available in S3 Table) were prepared following a modified
 version (S1 Appendix) of the protocol from Peterson et al. 2012. Each
 sample was digested with MspI and PstI restriction enzymes and ligated to
 common (5’- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
 - 3’) and unique oligos (S3 Table). We then used a BluePippin
 machine to size select for 300 – 500 bp fragments, then libraries were
 sent to the University of Oregon’s Genomic and Cell Characterization Core
 Facility (GC3F) for Illumina Sequencing on the Hiseq 4000 for 100 bp
 single-end reads. The raw data files
 returned from GC3F were run through FastQC
 v0.11.3 to check the overall quality of the reads from the Illumina run.
 The FastQ file output from the previous step was input into the ipyrad pipeline
 for assembly and initial filtering. Reads that contained more than 5 bases
 with a low-quality Phred score (&lt;33) were excluded. Reads were then
 clustered based on an 85% similarity threshold and reads with less than 6x
 coverage were filtered out. A maximum of 5 ambiguous base calls and 5
 heterozygous sites per read were allowed during filtering. Additional
 filtering using VCFtools excluded individuals with more than 95% missing
 data, and single nucleotide polymorphism (SNP) loci with a 60% call rate
 or lower. We retained one dataset that consisted of all specimens that was
 used for our phylogenetic inference (oneout.recode.vcf). A separate
 dataset that consisted only of ingroup individuals was used for the
 population genetic analyses (ingroup.recode.vcf).
description: S1 Appendix. Laboratory Protocol. S1 Fig. a-score
 optimization – spline interpolation. S1 Table.
 Sampling localities of ingroup individuals. S2 Appendix.
 ipyrad summary statistics. S2 Table. Sampling localities of
 outgroup individuals. S3 Appendix. Bioinformatic
 workflow. S3 Table. Barcodes and batch
 information. S4 Appendix. Treefile of phylogenetic
 inference. S4 Table. Parameters used for assembly
 of concatenated dataset. ingroup.recode - VCF file
 containing alignment of ingroup individuals. 
 oneout.recode - VCF file containing alignment of ingroup
 individuals plus one outgroup specimen (<i>Brachyrhaphis
 rhabdophora</i>).