We present evidence for and investigate potential mechanisms driving habitat-linked genetic divergence within a bird species endemic to a single 250 km² island. The island scrub-jay (Aphelocoma insularis) exhibits microgeographic divergence in bill morphology across pine-oak ecotones on Santa Cruz Island, California (USA) similar to adaptive differences described in mainland congeners over much larger geographic scales. To test whether individuals exhibit genetic differentiation related to habitat type and divergence in bill length, we genotyped over 3,000 single nucleotide polymorphisms (SNPs) in 123 adult island scrub-jay males from across Santa Cruz Island using restriction site-associated DNA sequencing (RADseq). Neutral landscape genomic analyses revealed that genome-wide genetic differentiation was primarily related to geographic distance and differences in habitat composition. We also found 168 putatively adaptive loci associated with habitat type using multivariate redundancy analysis (RDA) while controlling for spatial effects. Finally, two genome-wide association analyses revealed a polygenic basis to variation in bill length with multiple loci detected in or near genes known to affect bill morphology in other birds. Our findings support the hypothesis that divergent selection at microgeographic scales can cause adaptive divergence in the presence of ongoing gene flow.

Aphelocoma insularis RAD-sequencing and genotyping

We generated RADseq libraries for adult male Island Scrub-jays using the restriction enzyme SbfI. We then sequenced 100 bp single-end reads on two Illumina HiSeq 2500 lanes at the Genomics Core Facility at the University of Oregon. Sequenced reads were quality‐filtered and demultiplexed, and individual barcodes were removed using STACKS version 2.3b pipeline. We aligned sequences to the Florida scrub-jay (Aphelocoma coerulescens) draft reference genome using BWA-MEM. We used the default settings of ref_map.pl in STACKS to identify SNPs and exported the raw SNP matrix using the “populations” module of STACKS. 

• Bioinformatic pipeline code available in: 001-bioinformatics_scripts_final.Rmd, radiator_script_150_ISSJ.R

Filtered VCF file for neutral popualtion genetics  

Additional filtering was performed using the RADIATOR package. Filters are summarized in supplemental Table S1.  We mapped scaffolds to individual chromosomes of the Zebra Finch genome and imputed missing data using BEAGLE and putatively adaptive loci identified by PCADAPT were removed. VCF containes 3,342 SNPs from 123 individuals. ISSJ.ZF.ordered_imputed_BEAGLE_neutral.vcf

• Circuitscape resistance file: ISSJ_resistance_data.csv

Filtered VCF file for genotype-by-environment association and genome-wide association  

• VCF without putatively adaptive loci removed and containes 3,345 SNPs. ISSJ.ZF.ordered_imputed_BEAGLE.vcf
• Individual sampling locality data and habitat composition is available in: issj_filtered.csv, ISSJ_environmental_data_final.csv
• Associated files for GWA analyses: tguttata_gene_ensembl.csv, ISSJ.ZFcorr.csv, Beagle_imputed_GWAS.assoc.txt
• GEA code: 200–Identification_of_Population_Structure_Associated_with_Habitat_final.Rmd
• GWA code: 300-Identification_of_loci_Underlying_Variation_in_Morphology_final_bedops.Rmd