DOI: 10.5061/dryad.np5hqbzvd
title: Neutral and adaptive drivers of genomic change in introduced brook trout (Salvelinus fontinalis) populations revealed by pooled sequencing

description: Understanding the drivers of successful species invasions is important for
 conserving native biodiversity and for mitigating the economic impacts of
 introduced species. However, whole-genome resolution investigations of the
 underlying contributions of neutral and adaptive genetic variation in
 successful introductions are rare. Increased propagule pressure should
 result in greater neutral genetic variation, while environmental
 differences should elicit selective pressures on introduced populations,
 leading to adaptive differentiation. We investigated neutral and adaptive
 variation among nine introduced brook trout (Salvelinus fontinalis)
 populations using whole-genome pooled sequencing. The populations inhabit
 isolated alpine lakes in western Canada and descend from a common source,
 with an average of ~19 (range of 7-41) generations since introduction. We
 found some evidence of bottlenecks without recovery, no strong evidence of
 purifying selection, and little support that varying propagule pressure or
 differences in local environments shaped observed neutral genetic
 variation differences. Putative adaptive loci analysis revealed
 non-convergent patterns of adaptive differentiation among lakes with
 minimal putatively adaptive loci (0.001%-0.15%) that did not correspond
 with tested environmental variables. Our results suggest that (i)
 introduction success is not always strongly influenced by genetic load,
 (ii) observed differentiation among introduced populations can be
 idiosyncratic, population-specific, or stochastic, and (iii)
 conservatively, in some introduced species, colonization barriers may be
 overcome by support through one aspect of propagule pressure or benign
 environmental conditions.
description: This dataset was collected by
 sampling <em>Salvelinus fontinalis</em> from lakes in the
 Rocky Mountains of Alberta and BC. DNA extractions from fin tissue were
 conducted using <em>Qiagen</em> blood and tissue kits (Qiagen,
 Germany) and the manufacturer protocol. To ensure equal quantities of DNA
 in the pooled samples, DNA quality and quantity were initially assessed by
 1% Agarose gel electrophoresis using HindIII digested Lambda DNA run at
 100V to assess possible DNA degradation. Multiple quality tests per
 individual were conducted on a <em>Qubit</em> Fluorometer 2.0
 (Invitrogen, USA) selecting for quantity &gt;20 ng/µL and confirmed in
 a <em>NanoDrop</em> spectrophotometer (Thermo Scientific, USA)
 as well as estimates of 260/230 and 260/280 ratios greater than
 &gt;1.8 quality. Individual DNA was then pooled by
 sex, and population (18 total pools) with 20 individuals in each pool;
 exceptions were Cobb females (n=17) and both McNair sexes (n=8) due to low
 population and sample sizes. Twenty individuals per pool were chosen to
 ensure a balance between available population samples and to have equal
 representation between sexes (two pools of 20 per population) while
 maintaining accurate unbiased allele frequency estimates (Anand et al.,
 2016; Boitard et al., 2012). Albeit from different sexes, the adoption of
 two pools per population also provided a degree of sampling replication
 for some population genomic analyses, such as genomic-wide diversity and
 genetic differentiation. Fifty µL of each individual sample was selected
 for each pool at a dilution of 10 ng/µL, with DNA concentrations confirmed
 both prior to and post using a <em>Qubit</em> Fluorometer. DNA
 was pooled together at a final concentration of 3 ng/µL, confirmed using a
 <em>Qubit</em> Fluorometer.  Genomic libraries of these pooled
 DNA samples were prepared by Génome Québec Innovation Centre, Montréal,
 Québec, Canada via a shotgun approach with PCR with <em>Illumina
 TrueSeq</em> LT adaptors (Illumina, USA).  All pools passed quality
 and quantity requirements and were sequenced each on two lanes of
 <em>NovaSeq 6000 S4</em> flowcell (Illumina) and paired-end
 reads of 100 base pairs (bp). Coverage was estimated based on the
 assumption that the brook trout genome is approximately 3Gb, based on the
 Animal Genome Size Database (http://www.genomesize.com/). A reference genome of charr (<em>Salvelinus </em>sp<em>.</em>) available from NCBI (ASM291031v2, Christensen et al., 2018; Genome size = ~2.4GB, scaffold N50 = 1.02Mbp, Contig N50 = 55.6Kbp, masked mapping) was used due to its close phylogenetic and karyotypic relationship with brook trout (Timusk et al., 2011). The reference genome was prepared using Burrows-Wheeler Aligner (BWA) v 0.7.12 (Li & Durbin, 2009), indexed with SAMtools v 1.5 (Li et al., 2009), and a dictionary was created using Picard tools v 2.17.11 (http://broadinstitute.github.io/picard/, accessed 20-11-2019) to permit sequence alignment. SNP discovery was performed using the PPalign module of the PoolParty pipeline v 0.8 (Micheletti & Narum, 2018); the methods and packages in this module are detailed below. Mapping, alignment trimming, and filtering to the charr reference genome were performed with BWA-MEM v 0.7.12, SAMtools v 1.5 using a mapping quality threshold of 10 and SAMblaster v 0.1.24 (Faust & Hall, 2014), while filtering for a quality score threshold of 20 was performed by BBMap v 37.93 (Bushnell, sourceforge.net/projects/bbmap/, accessed 20-11-2019), and summarized with Fastqc v 0.11.7 (Andrews, 2010). SNP filtering was carried out using common parameters for salmonid species, found below (Horn et al., 2020). Duplicate sequences and unpaired reads were filtered using SAMtools, BBMap, and Picard tools with a minimum fastq trimming length of 25bp. An indel window of 15bp was used to mask SNPs around indel regions. SNP calling was facilitated conservatively by BCFtools v 1.5 (Li et al., 2009) with a quality score of 20, a minimum global allele frequency of 0.05, and a minimum global coverage of 10. Raw reads were checked for quality using FastQC and MultiQC v1.7 (Ewels et al., 2016). After SNP calling, multiallelic SNPs that could be paralogs were removed following Létourneau et al., 2018; Narum et al., 2017; and Terekhanova et al., 2019. Finally, for all analyses, the PPanalyze module of PoolParty was used to filter out duplicated loci and filter for loci common between all populations with minimum global coverage of 20, maximum global coverage of 100, and minimum allele frequency of 0.05. Of all tested SNPs, the proportion of putatively adaptive loci - i.e. loci with greater deviation from average - was negligible (0.15%), and therefore putatively adaptive loci were not removed. Following alignment, <em>mpileup</em> files were run through the PPstats module of PoolParty to estimate depth of coverage, alignment statistics, and genome coverage. Collectively, a total of 362,493 SNPs remained that were common among all populations in the dataset (and biallelic, with scaffold removed); these were used for all subsequent genomic analyses, except the Cochran Mantel Haenszel (CMH) tests described below.
description: These .sync files are labeled for the entire dataset and for the
 common loci dataset. The latter is used for the majority of our published
 work. Information on the format of the files can be found in the README
 file.