DOI: 10.5061/dryad.qbzkh18ks
title: Anolis carolinensis character displacement SNP

description: Here are six files that provide details for all 44,120 identified single
 nucleotide polymorphisms (SNPs) or the 215 outlier SNPs associated with
 the evolution of rapid character displacement among replicate islands with
 (2Spp) and without competition (1Spp) between two Anolis species. On 2Spp
 islands, A. carolinensis occurs higher in trees and have evolved larger
 toe pads. Among 1Spp and 2Spp island populations, we identify 44,120 SNPs,
 with 215-outlier SNPs with improbably large FST values, low nucleotide
 variation, greater linkage than expected, and these SNPs are enriched for
 animal walking behavior. Thus, we conclude that these 215-outliers are
 evolving by natural selection in response to the phenotypic convergent
 evolution of character displacement. There are two, non-mutually exclusive
 perspective of these nucleotide variants. One is character displacement is
 convergent: all 215 outlier SNPs are shared among 3 out of 5 2Spp island
 and 24% of outlier SNPS are shared among all five out of five 2Spp island.
 Second, character displacement is genetically redundant because the allele
 frequencies in one or more 2Spp are similar to 1Spp islands: among one or
 more 2Spp islands 33% of outlier SNPS are within the range of 1Spp MiAF
 and 76% of outliers are more similar to 1Spp island than mean MiAF of 2Spp
 islands. Focusing on convergence SNP is scientifically more robust, yet it
 distracts from the perspective of multiple genetic solutions that enhances
 the rate and stability of adaptive change. The six files include: a
 description of eight islands, details of 94 individuals, and four files on
 SNPs.  The four SNP files include the VCF files for 94
 individuals with 44KSNPs and two files (Excel sheet/tab-delimited file)
 with FST, p-values and outlier status for all 44,120 identified single
 nucleotide polymorphisms (SNPs) associated with the evolution of rapid
 character displacement. The sixth file is a detailed file on the 215
 outlier SNPs. Complete sequence data is available at Bioproject
 PRJNA833453, which including samples not included in this study. 
 The 94 individuals used in this study are described
 in “Supplemental_Sample_description.txt”
description: <em><strong>Anoles and genomic
 DNA:</strong></em> Tissue or DNA for 160
 Anolis carolinensis and 20 A. sagrei samples were provided by the Museum
 of Comparative Zoology at Harvard University (Table S2). Samples were
 previously used to examine evolution of character displacement in native
 A. carolinensis following invasion by A. sagrei onto man-made spoil
 islands in Mosquito Lagoon Florida (Stuart et al. 2014). One hundred
 samples were genomic DNAs, and 80 samples were tissues (terminal tail
 clip, Table S2). Genomic DNA was isolated from 80 of
 160 A. carolinensis individuals (MCZ, Table S2) using a custom SPRI
 magnetic bead protocol (Psifidi et al. 2015). Briefly, after removing
 ethanol, tissues were placed in 200 ul of GH buffer (25 mM Tris- HCl pH
 7.5, 25 mM EDTA, , 2M GuHCl Guanidine hydrochloride, G3272 SIGMA, 5 mM
 CaCl2, 0.5% v/v Triton X-100, 1% N-Lauroyl-Sarcosine) with 5% per volume
 of 20 mg/ml proteinase K (10 ul/200 ul GH) and digested at 55º C for at
 least 2 hours. After proteinase K digestion, 100 ul of 0.1%
 carboxyl-modified Sera-Mag Magnetic beads (Fisher Scientific) resuspended
 in 2.5 M NaCl, 20% PEG were added and allowed to bind the DNA. Beads were
 subsequently magnetized and washed twice with 200 ul 70% EtOH, and then
 DNA was eluted in 100 ul 0.1x TE (10 mM Tris, 0.1 mM EDTA). All DNA
 samples were gel electrophoresed to ensure high molecular mass and
 quantified by spectrophotometry and fluorescence using Biotium AccuBlueTM
 High Sensitivity dsDNA Quantitative Solution according to manufacturer’s
 instructions. Genotyping-by-sequencing (GBS) libraries
 were prepared using a modified protocol after Elshire et al. (Elshire et
 al. 2011). Briefly, high-molecular-weight genomic DNA was aliquoted and
 digested using ApeKI restriction enzyme. Digests from each individual
 sample were uniquely barcoded, pooled, and size selected to yield insert
 sizes between 300-700 bp (Borgstrom et al. 2011). Pooled libraries were
 PCR amplified (15 cycles) using custom primers that extend into the
 genomic DNA insert by 3 bases (CTG). Adding 3 extra base pairs
 systematically reduces the number of sequenced GBS tags, ensuring
 sufficient sequencing depth. The final library had a mean size of 424 bp
 ranging from 188 to 700 bp .
 <em><strong>Anolis
 SNPs:</strong></em> Pooled libraries were
 sequenced on one lane on the Illumina HiSeq 4000 in 2x150 bp paired-end
 configuration, yielding approximately 459 million paired-end reads ( ~138
 Gb). The medium Q-Score was 42 with the lower 10% Q-Scores exceeding 32
 for all 150 bp. The initial library contained 180 individuals with
 8,561,493 polymorphic sites. Twenty individuals were <em>Anolis
 sagrei</em>, and two individuals (Yan 1610 & Yin 1411)
 clustered with <em>A. sagrei</em> and were not used to define
 <em>A. carolinesis</em>’ SNPs.
 <em>Anolis carolinesis </em>reads were aligned to the
 <em>Anolis carolinensis</em> genome (NCBI RefSeq accession
 number:/GCF_000090745.1_AnoCar2.0). Single nucleotide polymorphisms (SNPs)
 for <em>A. carolinensis</em> were called using the GBeaSy
 analysis pipeline (Wickland et al. 2017) with the following filter
 settings: minimum read length of 100 bp after barcode and adapter
 trimming, minimum phred-scaled variant quality of 30 and minimum read
 depth of 5. SNPs were further filtered by requiring SNPs to occur in
 &gt; 50% of individuals, and 66 individuals were removed because they
 had less than 70% of called SNPs. These filtering steps resulted in 51,155
 SNPs among 94 individuals. Final filtering among 94 individuals required
 all sites to be polymorphic (with fewer individuals, some sites were no
 longer polymorphic) with a maximum of 2 alleles (all are bi-allelic),
 minimal allele frequency 0.05, and He that does not exceed HWE (FDR
 &lt;0.01). SNPs with large He were removed (2,280 SNPs). These SNPs
 with large significant heterozygosity may result from aligning paralogues
 (different loci), and thus may not represent polymorphisms. No SNPs were
 removed with low He (due to possible demography or other exceptions to
 HWE). After filtering, 94 individual yielded 44,120 SNPs. Thus, the final
 filtered SNP data set was 44K SNPs from 94 indiviuals.
 <em><strong>Statistical
 Analyses:</strong></em> Eight <em>A.
 carolinensis </em>populations were analyzed: three populations from
 islands with native species only (1Spp islands) and 5 populations from
 islands where A. carolinesis co-exist with <em>A. sagrei
 </em>(2Spp islands, Table 1, Table S1). Most analyses pooled the
 three 1Spp islands and contrasted these with the pooled five 2Spp
 islands. Two approaches were used to define SNPs with
 unusually large allele frequency differences between 1Spp and 2Spp
 islands: 1) comparison of FST values to random permutations and 2) a
 modified FDIST approach to identify outlier SNPs with large and
 statistically unlikely FST values. <em>   
  Random Permutations:</em> FST values were
 calculated in VCFTools (version 4.2, (Danecek et al. 2011)) where the
 p-value per SNP were defined by comparing FST values to 1,000 random
 permutations using a custom script (below). Basically, individuals and all
 their SNPs were randomly assigned to one of eight islands or to 1Spp
 versus 2Spp groups. The sample sizes (55 for 2Spp and 39 for 1Spp islands)
 were maintained. FST values were re-calculated for each 1,000
 randomizations using VCFTools. <em>     Modified
 FDIST:</em> To identify outlier SNPs with
 statistically large FST values, a modified FDIST (Beaumont and Nichols
 1996) was implemented in Arlequin (Excoffier et al. 2005). This modified
 approach applies 50,000 coalescent simulations using hierarchical
 population structure, in which demes are arranged into k groups of d demes
 and in which migration rates between demes are different within and
 between groups. Unlike the finite island models, which have led to large
 frequencies of false positive because populations share different
 histories (Lotterhos and Whitlock 2014), the hierarchical island model
 avoids these false positives by avoiding the assumption of similar
 ancestry (Excoffier et al. 2009). References 
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description: Original sample (tissue or DNA) are available at MCZ and specific
 individuals and their catalogue number are described in Table S2 on
 journal site or here in table
 “<em>Supplemental_Sample_description.txt</em>”.
 Original sequence data are available at NCBI Bioproject
 PRJNA833453. Sequences are names as “Spp_Island_Status_MCZ#_sex “ where
 “Spp” is Ac or As (Ac = <em>Anolis </em><em>carolinensis</em>, As = <em>A. </em><em>sagrei), </em>islands are as defined in Figure 1 and Supplemental Table 1 & 2 (or here as “<em>Supplemental_islands_descripiton.txt</em>” and “<em>Supplemental_Sample_description.txt</em>”)