DOI: 10.5061/dryad.vh151q1c
title: Data from: High-throughput identification of informative nuclear loci for shallow-scale phylogenetics and phylogeography

description: One of the major challenges for researchers studying phylogeography and
 shallow-scale phylogenetics is the identification of highly-variable and
 informative nuclear loci for the question of interest. Previous approaches
 to marker generation have generally required extensive testing of loci of
 unknown utility from other systems or development of markers from the
 nearest model organism with genomic resources. Here, we present a fast and
 economical approach to generating thousands of variable, single-copy
 nuclear loci for any system using next-generation sequencing. We performed
 Illumina paired-end sequencing of three reduced-representation libraries
 (RRLs) in chorus frogs (Pseudacris) to identify orthologous, single-copy
 loci across libraries and to estimate sequence divergence at multiple
 taxonomic levels. We also conducted PCR testing of these loci across the
 genus Pseudacris and outgroups to determine whether loci developed for
 phylogeography can be extended to deeper phylogenetic levels. Prior to
 sequencing, we conducted in silico digestion of the most closely-related
 reference genome (Xenopus tropicalis) to generate expectations for the
 number of loci and degree of coverage for a particular experimental
 design. Using the RRL approach, we: (1) identified &gt;100,000
 single-copy nuclear loci, 6,339 of which were shared across individuals
 within species and 904 of which were shared between species, (2) estimated
 average nuclear sequence divergence at 0.1% between alleles within an
 individual, 1.1% between conspecific individuals, and 1.8% between
 species, and (3) determined from PCR testing that 53% of the loci
 successfully amplify within-species and also many amplify to the
 genus-level and beyond (16%). Our study effectively identified nuclear
 loci present in the genome that have levels of sequence divergence on par
 with mitochondrial markers commonly used in phylogeography. Specifically,
 we estimated that ~7% of loci in the chorus frog genome are &gt;3%
 divergent within species; this translates to ~50,000 single-copy loci in
 the genome with &gt;3% divergence. Moreover, successful amplification
 of many loci at deeper phylogenetic levels indicates that the RRL approach
 represents an efficient method for rapid generation of informative markers
 for both phylogenetics and phylogeography. We conclude by making
 recommendations for minimizing the cost and maximizing the efficiency of
 marker development for future studies in this field.
description: Lemmon_Lemmon_Supplemental_Tables_16Nov2011MtDNALociFinalNuclearLoci_ECM7144FinalNuclearLoci_ECM7210FinalNuclearLoci_ECM7278FinalNuclearLoci_ECM7144_ECM7210FinalNuclearLoci_ECM7144_ECM7278FinalNuclearLoci_ECM7210_ECM7278FinalNuclearLoci_ECM7144_ECM7210_ECM7278ReadMeVerifiedNuclearLociRRL_AnalysisScriptsPython scripts used to produce alleles from raw Illumina data.InSilicoPredictionPrograms used to predict the number loci given a particular restriction site. The first program (GetCutterFragLens.java) is first used to generate an expected fragment length distribution (from an uninterleaved fasta file). The second program PredictNLoci.r is used to determine the number of loci contained within a specified size range.All_Primers_And_PCR_Results_LocusBySpeciesThis file contains the primer sequences for all loci tested, as well as the results we obtained for each locus x species combination tested.PCR_Results_LocusBySpecies.xlsxSupplementalFigures