DOI: 10.5061/dryad.866t1g1xj
title: Data from: Divergent population structure in five common rockfish species of puget sound, WA suggests the need for species-specific management

description: Quantifying connectivity between endangered or threatened marine
 populations is critical information for scientifically sound management.
 Of the 86 species managed by the Pacific Fishery Management Council (PFMC)
 on the West Coast of the United States, over 75% are rockfishes, and of
 those species, 27 were once deemed ‘at risk’. Although most stocks have
 been successfully rebuilt along the Washington Coast, Puget Sound stocks
 have yet to recover despite fisheries closures. The connectivity of Puget
 Sound stocks to coastal populations is relatively unknown, despite the
 potential of recruitment subsidies from the coast and considerable
 interest in reopening many fisheries for recreational use. The importance
 of accurate connectivity estimates was demonstrated by recent research on
 two of three Endangered Species Act (ESA) listed species in Puget Sound,
 which showed that one species was not sufficiently distinct to warrant
 listing as a separate Puget Sound distinct population segment (DPS). The
 common assumption of isolation of Puget Sound populations, which stems
 primarily from research on three hybridizing species, may therefore be
 erroneous. This study aimed to provide an analysis of the connectivity of
 five Puget Sound rockfish populations and identify distinct population
 segments where appropriate. Samples from five species (Black (Sebastes
 melanops), Yellowtail (S. flavidus), Redstripe (S. proriger), Greenstriped
 (S. elongatus), and Puget Sound (S. emphaeus)) were collected in three
 areas within and one area outside of Puget Sound and analyzed at over
 12,000 restriction-site associated DNA sequencing (RADseq) loci. We found
 unique species-specific patterns of genetic diversity, attributable to
 multiple extrinsic and intrinsic factors. In particular, Black and Puget
 Sound Rockfish showed no genetic differentiation; Yellowtail and
 Greenstriped Rockfish were structured according to known geographic
 barriers; and Redstripe Rockfish revealed evidence for temporal genetic
 differentiation, suggesting that irregular recruitment influences
 population structure. None of the species followed the DPS boundaries
 generally assumed for rockfish, further emphasizing the importance of
 species-specific management for the effective recovery of these rockfish
 populations.
description: <strong>Sampling Procedure </strong>
 We used 279 samples from five species of rockfish (Black
 <em>(S. melanops</em>), Yellowtail (<em>S.
 flavidus</em>), Redstripe (<em>S. proriger</em>),
 Greenstriped (<em>S. elongatus</em>), and Puget Sound (S.
 emphaeus), Figure 1) that were collected in 1999-2021 in previous surveys
 of WDFW, NOAA-NMFS, and the Department of Fisheries and Oceans (DFO
 Canada). Individual fin clips were preserved in 95% ethanol or dried on
 filter paper. Samples were collected from multiple locations (see Figure
 3), grouped into four different regions: 1) southern Puget Sound (Puget
 Sound proper, south of Admiralty Inlet, SPS), 2) British Columbia
 (Canadian Salish Sea north of the US/Canada border, BC), 3) northern Puget
 Sound (US Salish Sea north of Admiralty Inlet, NPS and east of the
 Victoria Sill) and 4) the US west coast (US Pacific Coast west of Victoria
 Sill, WC). Due to differences in the abundance and distribution of species
 across this geographic range, we have no Puget Sound Rockfish from WC and
 one Greenstriped Rockfish from NPS.  <strong>DNA
 Extraction, Library Preparation, and Sequencing </strong>
 Genomic DNA was extracted using the Nexttec DNA isolation kit
 (Nexttec Incorporated, Middlebury, VT, USA) following the manufacturer’s
 protocol and quantified using a Qubit Fluorometer (ThermoFisher
 Scientific, Waltham, MA, USA). DNA concentration was normalized to 125ng
 in 10 µL of molecular-grade water. Restriction site-associated DNA
 sequencing (RADseq) libraries were prepared using a version of the Ali et
 al. (2016) protocol without the targeted bait capture step, referred to in
 the literature as BestRAD
 (https://github.com/merlab-uw/Protocols/blob/main/bestRAD). Briefly,
 genomic DNA was digested using the SbfI enzyme. An adapter (P1) containing
 a forward amplification primer site, an Illumina sequencing primer site,
 and an individual 6 bp barcode was ligated to each fragment at the
 restriction site end. Fragments were then randomly sheared using
 sonication and size-selected to 300-500 bp in length. Subsequently, P2
 adapters were ligated to the reverse end and libraries were amplified by
 PCR. Each library was assessed for quality on a 1% agarose gel and a
 Bioanalyzer DNA 1000 kit (Agilent Technologies, Santa Clara, CA).
 Libraries were pooled in equimolar amounts and sequenced on a NovaSeq
 (paired-end, 116 bp or 150 bp) at the University of Oregon, either an S4
 or SP run type. Ninety-six individuals were randomly included in one of
 six RADseq libraries to avoid any lane effect (Leigh et al.,
 2018). <strong>Initial
 Filtering </strong> Raw sequence data were
 quality-checked using multiQC (Ewels et al., 2016). Prior to SNP calling
 and genome alignment, raw sequences were demultiplexed using
 process_radtags in the STACKS v2.60 pipeline (Catchen et al., 2011;
 Rochette et al., 2019). Sequences were trimmed to 104 bases and filtered
 for quality. Individuals with fewer than 250,000 total reads were excluded
 from downstream analysis (Krohn et al., 2018). Our paired-end sequences
 were then aligned to the Honeycomb Rockfish (Sebastes umbrosus) genome
 from GenBank (NCBI Accession Number: PRJNA562243) with Bowtie2 v2.4. using
 the ‘very-sensitive’ option (Langmead & Salzberg, 2012). The
 Honeycomb Rockfish genome is one of only two annotated full genomes and
 was chosen due to its closer phylogenetic relationship to our species
 (Hyde & Vetter, 2007a). Following genome alignment, SNP calling
 and basic population genetics statistics were calculated using the gstacks
 and populations modules from the STACKS pipeline. SNPs were called if they
 had a minimum mapping quality of 40. SNPs were filtered following
 published recommendations (O’Leary et al., 2018) requiring that loci meet
 the following criteria: minimum genotype depth ≥ 5, mean minimum read
 depth ≥ 15, genotype call rate ≥ 80%. We subsampled SNPs by choosing one
 SNP on each RADtag with the highest minor allele frequency. SNPs with
 genotype frequencies that were significantly different than expectations
 under Hardy-Weinberg Equilibrium (HWE) were removed using the following
 procedure: p-values were calculated across samples for each population
 using the r package pegas v1.1 (Paradis, 2010). P-values were then
 combined across samples for each locus using Fisher’s combination of
 probabilities, and adjusted to q-values for the false discovery rate
 (Benjamini & Hochberg, 1995). Loci with q-values below 0.05 were
 considered significantly out of HWE and removed from downstream
 analysis.  <strong>Misidentification
 Analysis</strong> To identify any cases of 1)
 misidentification of species during field sampling or 2) interspecific
 hybridization in our dataset, raw sequences for all individuals were
 analyzed together for eight species (five from this study, and Brown,
 Quillback, and Copper Rockfish from Wray et al (in prep)) immediately
 after genome alignment. SNP calling, and basic population genetics
 statistics were calculated using the gstacks and populations modules from
 the STACKS pipeline. SNP filtering followed the same protocol as in the
 species-specific analyses (O’Leary et al., 2018). Unlike Different from
 the species-specific analysis, however, we intended to we avoided SNPs
 with fixed differences between species, because this would likely find
 differences between only two species. Therefore, we chose the first SNP on
 each RADtag using the –write-single-snp option in populations.
 Additionally, we did not filter for HWE because a reduction of
 heterozygosity due to species-specific (subpopulation) structure would
 likely influence HWE p-values due to the Wahlund effect (REF). We plotted
 all eight species together in a principal components analysis (PCA). Any
 individuals that visually grouped with a species different than its field
 identification were removed from downstream analysis. 
description: # Divergent Population Structure In Five Common Rockfish Species Of Puget
 Sound, WA Suggests The Need For Species-Specific Management
 [https://doi.org/10.5061/dryad.866t1g1xj](https://doi.org/10.5061/dryad.866t1g1xj) **Description of the data and file structure** This repository contains the raw fastq files (gzipped) and end product files (in VCF format) for a study on the population structure of five Rockfish species common in Puget Sound, WA using RADseq. We found species-specific patterns of genetic differentiation, attributable to both extrinsic and intrinsic factors. Specifically, Black and Puget Sound Rockfishes showed no genetic differentiation; Yellowtail and Greenstriped Rockfish were structured according to known geographic barriers; and Redstripe Rockfish revealed evidence for temporal genetic differentiation, suggesting irregular recruitment influences population structure. ## Description of the data and file structure Here are a few notes about the zipping structure: * *Species*_R1.zip contains the raw forward and reverse reads for each sample run sequenced on a SP run type. *Species*_R2.zip contains the raw forward and reverse reads for each sample run sequenced on a S4 run type. Each file is in gzipped fastq format. To get the full forward and reverse reads for all individuals within a species, you must download the *Species*_R1.zip and *Species*_R2.zip file. * Final_VCF_Files.zip contains all the final VCF files for the intraspecific analyses. There should be one file for each species. All VCF files were filtered for high-quality SNPs and samples (see methods for filtering details). * Sample_Metadata contains all relevant information on each sample (location, weight, length, etc.). Use this file to identify which sample you need from the *Species*_R1.zip, *Species*_R2.zip, or Final_VCF_Files.zip. ## Code/Software All relevant code is provided in this [github repository(opens in new window)](https://github.com/anita-wray/rockfish_RADseq)