DOI: 10.5061/dryad.5dv41nsbj
title: American Crow SNPs and microsatellite data

description: Infectious diseases can cause steep declines in wildlife populations,
 leading to changes in genetic diversity that may affect the susceptibility
 of individuals to infection and the overall resilience of populations to
 pathogen outbreaks. Here, we examine evidence for a genetic bottleneck in
 a population of American crows (Corvus brachyrhynchos) before and after
 the emergence of West Nile virus (WNV). More than 50% of marked birds in
 this population were lost over the two-year period of the epizootic,
 representing a 10-fold increase in adult mortality. Using analyses of SNPs
 and microsatellite markers, we tested for evidence of a genetic bottleneck
 and compared levels of inbreeding and immigration in the pre- and post-WNV
 populations. Counter to expectations, genetic diversity (allelic diversity
 and the number of new alleles) increased after WNV emergence. This was
 likely due to increases in immigration, as the estimated membership
 coefficients were lower in the post-WNV population. Simultaneously,
 however, the frequency of inbreeding appeared to increase: mean inbreeding
 coefficients were higher among SNP markers, and
 heterozygosity-heterozygosity correlations were stronger among
 microsatellite markers, in the post-WNV population. These results indicate
 that loss of genetic diversity at the population level is not an
 inevitable consequence of a population decline, particularly in the
 presence of gene flow. The changes observed in post-WNV crows could have
 very different implications for their response to future pathogen risks,
 potentially making the population as a whole more resilient to a changing
 pathogen community, while increasing the frequency of inbred individuals
 with elevated susceptibility to disease.
description: <strong>Study population and data collection.
 </strong>Crows in the Ithaca, New York, population are cooperative
 breeders. They live in groups of up to 14 birds, including a socially
 bonded pair of adults as well as 0-12 auxiliary birds, which are usually
 offspring from previous broods). Although auxiliaries usually do not
 contribute offspring to the brood, molecular work in the post-WNV
 population indicates that auxiliary males occasionally do sire extra-pair
 offspring with the female breeder, arising both through incest (mothers
 mating with their adult auxiliary sons) and through matings between
 non-relatives (e.g., unrelated step-mothers and adult auxiliary
 males).  Genetic samples were collected from crow
 nestlings from 1990–2011. We collected blood (~150 ul) from the brachial
 vein of nestlings and banded them with unique combinations of metal bands,
 color bands, and patagial tags on days 24–30 after hatching. DNA was
 extracted from samples using DNeasy tissue kits (Qiagen, Valencia, CA)
 following the manufacturer’s protocol. All fieldwork with American crows
 was carried out under protocols approved by the Institutional Animal Care
 and Use Committees of Binghamton University (no. 537-03 and 607-07) and
 Cornell University (no. 1988–0210).  The pre-WNV
 dataset included samples collected between 1990 and 2002. The 2002
 nestlings were sampled prior to WNV emergence, as nestlings fledge the
 nest between May and July, whereas WNV mortality typically occurs between
 August and October in this crow population. The post-WNV samples were
 collected between 2005 and 2011. Samples collected immediately after WNV
 emergence (2003 and 2004) were not included in the analysis to allow time
 for the birds to respond to the population loss. We maximized independence
 of the birds selected for analysis by including only one randomly chosen
 offspring per brood and no more than two broods per family group in the
 pre-and post-WNV samples, with each brood per family group separated by
 the maximum number of years possible within the pre- or post-WNV sampling
 periods (1990–2002 pre-WNV; 2005–2011 post-WNV; Figure S1). Birds were
 randomly and independently selected (with replacement) for the SNP and
 microsatellite analyses; therefore, there was little overlap among
 individual birds included in these marker sets. Of the 286 individual
 birds included in this analysis, 22 were common to both marker sets (15
 pre-WNV; 7 post-WNV).  The 20-year time period of this
 study may have encompassed 2–4 breeding cohorts (approximately 1–2 pre-
 and 1–2 post-WNV, with a sharp turn-over immediately after WNV emergence).
 Crows can produce offspring as early as two years after hatching, but most
 do not begin breeding independently until at least 3–4 years after
 hatching. Breeding initiation is limited at least in part by breeding
 vacancies, which are created by the death of one or both members of an
 established breeding pair. Such breeding vacancies likely increased in
 availability after the emergence of WNV. 
 <strong>Microsatellite genotyping. </strong>A total
 of 222 crows (n = 113 and 109 crows pre- and post-WNV, respectively) were
 genotyped at 34 polymorphic microsatellite loci that were optimized for
 American crows. Alleles were scored using the microsatellite plugin for
 Geneious 9.1.8. We used GenePop version 4.7 to test for linkage
 disequilibrium between all pairs of loci, departures from Hardy–Weinberg
 equilibrium (HWE), and null allele frequency. Locus characteristics (e.g.,
 alleles/ locus, tests of Hardy–Weinberg equilibrium and null allele
 frequencies) are given in the supplementary materials (Table
 S1).  Departures from HWE expectations were observed at
 two loci (PnuA3w from the pre-WNV sample and Cb06 from the post-WNV
 sample) after Bonferroni correction (Table S1); these loci were removed
 from subsequent analysis. In 561 pairwise comparisons, four pairs of loci
 appeared to be in linkage disequilibrium (Cb20 and Cb21; Cb14 and CoBr36;
 CoBr22 and Cb17, and CoBr12 and Cb10), but this linkage was only apparent
 at both time points (the pre-WNV and post-WNV populations) for Cb20 and
 Cb21. We removed both Cb20 and Cb21 from the analysis but retained the
 other loci because apparent linkage at only a single time point was
 unlikely to be a result of physical linkage. Two additional loci (Cb17 and
 Cb10) had a high frequency of null alleles (&gt; 0.1) and were removed
 from the dataset. All subsequent analyses are therefore based on 28 loci.
 We scored all birds at a minimum of 26 of these 28 loci, and most
 (&gt;98%) were scored at all loci (mean proportion of loci typed
 &gt;0.99). Mean allelic diversity at these loci was 11.25 ± 1.17
 alleles/locus (range: 3–31 alleles/locus). 
 <strong>Double Digest Restriction Associated DNA (ddRAD)
 sequencing.</strong> We performed ddRAD sequencing on 86 randomly
 selected crows (43 pre-WNV and 43 post-WNV).  100-500 ng of DNA were
 digested with SbfI-HF (NEB, R3642L) and MspI-HF (NEB, R016S) restriction
 enzymes. Samples were ligated with a P2-MspI adapter and pooled in groups
 of 18-20, each with a unique P1 adapter. Pooled index groups were purified
 using 1.5X volumes of homemade MagNA made with Sera-Mag Magnetic
 Speed-beads (FisherSci). Fragments 450-600 bp long were selected using
 BluePippin (Sage Science) by the Cornell University Biotechnology Resource
 Center (BRC). After size selection, unique index barcodes were added to
 each index group by performing 11 cycles of PCR with Phusion® DNA
 polymerase (NEB). Reactions were purified using 0.7X volumes of MagNA
 beads and pooled in equimolar ratios for sequencing on the Illumina HiSeq
 2500 at the BRC, with single end reads (100 bp). The sequencing was
 performed with an added Illumina PhiX control (15%) due to low 5’
 complexity. Pre- and post-WNV samples were library prepared together and
 sequenced on a single lane to avoid the introduction of a library or lane
 effect.<br>     <br>We used FASTQC v0.11.9 (Babraham
 Bioinformatics; http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to assess read quality. We trimmed reads to 147 bp using fastX_trimmer (FASTX-Toolkit) to exclude low-quality data at the 3’ end of reads. Next, we eliminated reads with Phred scores below 10, then eliminated reads in which 5% or more bases had Phred scores below 20 (fastq_quality_filter). The fastq files were demultiplexed using the process_radtags module in STACKS v2.52 pipeline to create a file with sequences specific to each individual.  We first scaffolded the American Crow reference genome (NCBI assembly: ASM69197v1, Accession no: GCA_000691975.1) into putative pseudochromosomes using the synteny-based Chromosemble tool in Satsuma2 (Grabherr et al. 2010) and the Hooded Crow genome (NCBI assembly: ASM73873v5, Accession no: GCA_000738735.5). We aligned sequence reads to the American Crow pseudochromosome assembly using BWA-MEM (Li & Durbin 2009). We called SNPs in ANGSD (Korneliussen et al. 2014) using the GATK model, requiring SNPs to be present in 80% of the individuals (0.95 postcutoff, SNP p-value 1e<sup>-6</sup>) with a minimum allele frequency of 0.015. We removed bases with quality scores below 20 (-minQ 20), bad reads (-remove_bads), mapping quality below 20 (-minMapQ20), base alignment quality below 1 (-baq), more than two alleles (-skipTriallelic), and heterozygote bias (-hetbias_pval 1e-5), requiring the minimum depth per individual to be at least two and read depth higher than 1,800. These filters resulted in 16,200 SNPs. To reduce differences in missingness between the pre- and post-WNV populations, we excluded loci that had less than 80% called genotypes per population, resulting in 5,151 SNPs.