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.

Study population and data collection. 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. 

Microsatellite genotyping. 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 (> 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 (>98%) were scored at all loci (mean proportion of loci typed >0.99). Mean allelic diversity at these loci was 11.25 ± 1.17 alleles/locus (range: 3–31 alleles/locus). 

Double Digest Restriction Associated DNA (ddRAD) sequencing. 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.
     
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^-6) 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.