DOI: 10.5061/dryad.w9ghx3fmn
title: Pooled whole genome sequencing from year 2004 and Early-Late SNP data from year 2014

description: Speciation underlies the generation of novel biodiversity. Yet, there is
 much to learn about how natural selection shapes genomes during
 speciation. Selection is assumed to act against gene flow at barrier loci,
 promoting reproductive isolation. However, evidence for gene flow and
 selection is often indirect and we know very little about the temporal
 stability of barrier loci. Here we utilize haplodiploidy to identify
 candidate male barrier loci in hybrids between two wood ant species. As
 ant males are haploid they are expected to reveal recessive barrier loci,
 which can be masked in diploid females if heterozygous. We then test for
 barrier stability in a sample collected ten years later and use survival
 analysis to provide a direct measure of natural selection acting on
 candidate male barrier loci. We find multiple candidate male barrier loci
 scattered throughout the genome. Surprisingly, a proportion of them are
 not stable after ten years, natural selection apparently switching from
 acting against to favoring introgression in the later sample. Instability
 of barrier effect and natural selection for introgressed alleles could be
 due to environment-dependent selection, emphasizing the need to consider
 temporal variation in the strength of natural selection and the stability
 of barrier effect at putative barrier loci in future speciation work.
description: Pooled whole genome sequencing We collected
 the samples used for pooled sequencing from the Långholmen hybrid
 population in the year 2004. The samples were freshly frozen and kept in
 -20°C until genomic DNA was extracted in the year 2010 from half a body
 using a Qiagen kit. We sequenced four samples, each consisting of 24
 individuals: R males, R females (unmated queens), W males and W females
 (unmated queens). These individuals were classified into the two lineages
 based on six to seven diagnostic microsatellite alleles. The sample
 concentrations were checked with Qubit and pooled into the four pools in
 equal amounts. Due to haplodiploidy, this resulted in 24 and 48
 chromosomes sampled for male and female pools, respectively.
 Each pool was sequenced with 100bp paired-end sequencing on its
 own lane in Illumina HiSeq2000 in the Institute for Molecular Medicine
 Finland (FIMM). This resulted in 46,106,000 to 108,204,481 total number of
 reads per pool. We quality trimmed reads by removing up to 20 bp that had
 phred score &lt; 20 using FASTX-Toolkit. Next, we made de novo
 assemblies of each of the four samples with Soapdenovo (Li et al. 2010)
 trying out different kmer sizes (31, 41, 51, 61, 71) for each assembly.
 The R male assembly with kmer size of 41 was best in terms of completeness
 and quality (genome size: 222.6 Mb, 327480 contigs, average contig length:
 679 bp, N50 = 1748 bp) and chosen as our reference assembly. We then
 mapped each sample back to the R male reference assembly after removing
 contigs of the assembly shorter than 500bp using Bowtie2 v2.0.2 (Langmead
 & Salzberg 2012). Reads mapped in proper pairs and with a mapping
 quality superior to 20 were filtered and combined in a single mpileup file
 using samtools 1.4 (Li et al. 2009). Since coverage of the W male pool was
 low (mean = 16, s.d. = 38), overlaps between read pairs were
 kept. SNP genotyping Individuals
 for genotyping were collected from field colonies in year 2014. DNA was
 extracted using Qiagen kit and protocol for insects. Genotyping was done
 at individual level for a total of 196 individuals. Samples were randomly
 assigned to 96-well plates for genotyping. Primer design and genotyping
 were done at LGC genomics using KASP genotyping chemistry. After removal
 of SNPs or individuals with more than 10% missing data, diploid males and
 three ambiguous individuals, we were left with a total of 185 genotyped
 individuals genotyped at 300 SNPs.
description: Please see ReadMe.