DOI: 10.5061/dryad.bzkh189cw
title: Pronounced differentiation on the Z chromosome and parts of the autosomes in crowned sparrows contrasts with mitochondrial paraphyly: implications for speciation

description: When a single species evolves into multiple descendent species, some parts
 of the genome can play a key role in the evolution of reproductive
 isolation while other parts flow between the evolving species via
 interbreeding. Genomic evolution during the speciation process is
 particularly interesting when major components of the genome—for instance,
 sex chromosomes vs. autosomes vs. mitochondrial DNA—show widely differing
 patterns of relationships between three diverging populations. The
 golden-crowned sparrow (Zonotrichia atricapilla) and the white-crowned
 sparrow (Zonotrichia leucophrys) are phenotypically differentiated sister
 species that are largely reproductively isolated despite possessing
 similar mitochondrial genomes, likely due to recent introgression. We
 assessed variation in more than 45,000 single nucleotide polymorphisms
 (SNPs) to determine the structure of nuclear genomic differentiation
 between these species and between two hybridizing subspecies of Z.
 leucophrys. The two Z. leucophrys subspecies showed moderate levels of
 relative differentiation and patterns consistent with a history of
 recurrent selection in both ancestral and daughter populations, with much
 of the sex chromosome Z and a large region on the autosome 1A showing
 increased differentiation compared to the rest of the genome. The two
 species Z. leucophrys and Z. atricapilla show high relative
 differentiation and strong heterogeneity in the level of differentiation
 among various chromosomal regions, with a large portion of the sex
 chromosome (Z) showing highly divergent haplotypes between these species.
 Studies of speciation often emphasize mitochondrial DNA differentiation,
 but speciation between Z. atricapilla and Z. leucophrys appears primarily
 associated with Z chromosome divergence and more moderately associated
 with autosomal differentiation, whereas mitochondria appear highly similar
 due apparently to recent introgression. These results add to the growing
 body of evidence for highly heterogeneous patterns of genomic
 differentiation during speciation, with some genomic regions showing lack
 of gene flow between populations many hundreds of thousands of years
 before other genomic regions.
description: <strong>Sample Collection</strong>
 For this project, we collected blood samples for genomic analysis
 from individuals of wild <em>Z. leucophrys</em> and
 <em>Z. atricapilla </em>in south-western British Columbia. The
 dataset includes a metadata file in .csv format, with locality, date and
 time of collection, measurements, barcodes, etc. associated with all
 samples. The first round of fieldwork was conducted at
 the Iona Island Bird Observatory (IIBO) in Richmond, British Columbia, in
 Spring 2019. Birds were captured passively in mist nets and banded as part
 of the station’s normal migration monitoring operations. We then took
 10–40 μL of blood from the brachial vein of each bird of the target
 species and stored these samples in 500 μL of Queen’s Lysis Buffer (Seutin
 et al., 1991). We measured wing chord, tail length, tarsus length, mass,
 culmen length, beak depth, and beak width. A total of 19 <em>Z.
 atricapilla</em> and 14 <em>Z. leucophrys</em> of
 indeterminate subspecies were sampled during this initial
 period. The second round of field sampling focused on
 increasing the sample size of <em>Z. leucophrys</em> and was
 conducted between June 16 and 26, 2020 at the Vancouver Campus of the
 University of British Columbia (UBC). Singing birds were located, and a
 mist net was set up nearby. Song recordings of local <em>Z.
 leucophrys</em> were used to attract birds to the net. Once birds
 were captured, they were immediately removed, banded, sampled, and
 measured using the methods outlined above. We sampled an additional 16
 breeding <em>Z. leucophrys</em> individuals during this
 collection period, all of the <em>pugetensis</em> subspecies
 based on their bill colour and location. Additionally, we took samples of
 pectoral muscle tissue from seven unprepared specimens from the freezer of
 the Cowan Tetrapod Collection at the Beaty Biodiversity Museum of UBC that
 had been salvaged from throughout BC. Bioinformatics
 Pipeline The dataset includes all the scripts used to
 process the raw GBS reads acquired from the Genome Quebec Innovation
 Centre. Reads were demultiplexed using custom scripts
 from Baute et al. (2016), and sequences were trimmed for quality using
 Trimmomatic (version 0.36; Bolger et al., 2014). The reads were then
 aligned to the <em>Taeniopygia guttata</em> reference genome
 version bTaeGut2.pat.W.v2 (Warren et al., 2010; Rhie et al., 2021; GenBank
 accession number GCA_008822105.2) using BWA-MEM (version 0.7.15; Li
 & Durbin, 2009). The resulting SAM files were converted to BAM
 files using Picard (version 2.23.8; Broad Institute
 https://broadinstitute.github.io/picard/), and single-end and paired-end
 BAM files were combined using SAMtools (version 1.3.2; Li et al., 2009).
 Single Nucleotide Polymorphisms (SNPs) were identified using the
 HaplotypeCaller tool in GATK (version 3.8; McKenna et al., 2010), which
 produced a gvcf file for each individual. Individual g.vcf files were then
 combined into a single vcf file using the GenotypeGVCFs tool in GATK. We
 filtered genomic sites using VCFtools (version 1.16; Danecek et al.,
 2011), first by removing indels and SNPs with more than 2 alleles. Using a
 custom Perl script (Owens et al., 2016), we removed sites with a mapping
 quality lower than 20 and a heterozygosity above 0.6. We then filtered for
 individuals with more than 60% missing data using VCFtools, removing three
 <em>Z. atricapilla </em>and<em> three Z.
 leucophrys</em> from the dataset. Finally, we used VCFtools to
 filter out SNPs missing from more than 20% of individuals and SNPs with a
 minimum allele frequency of less than 0.05. A
 comparison of mean coverage for each individual across the sex chromosomes
 (W and Z) and a representative autosome (chromosome 3), revealed that
 coverage across the W chromosome was approximately three times higher than
 across the autosomes (Fig. S2). This was true for known female birds, as
 well as known males. Since females are heterogametic for the W chromosome
 and males are homogametic for the Z (Irwin, 2018), we expect coverage
 across the W to be roughly half that of autosomal coverage in females, and
 zero in males. These results indicate that many of the sequences from our
 short-read dataset that mapped to the Zebra Finch W chromosome cannot be
 from the <em>Zonotrichia</em> W chromosome. These likely
 represent sequences found on autosomes in
 <em>Zonotrichia</em>, but on the W chromosome in the Zebra
 finch, or repetitive elements found at high frequency in Zonotrichia, but
 infrequently in the Zebra Finch. After filtering and removal of the W
 sequences, a total of 45,986 SNPs were retained for analysis.
 Analysis Finally, this dataset contained all
 scripts used to analyze the SNP dataset. For detailed methods of these
 analyses, please refer to the associated article.
description: The metadata file is in .csv format, which can be opened in any
 text editor. The files containing scripts used to
 process samples are in .txt format and can be opened in any text editor.
 These scripts were written in bash for use in a UNIX environment. They
 require they require additional scripts from scripts from Baute et al.
 (2016), and the programs Trimmomatic (version 0.36; Bolger et al., 2014),
 BWA-MEM (version 0.7.15; Li & Durbin, 2009), Picard (version
 2.23.8; Broad Institute https://broadinstitute.github.io/picard/), SAMtools (version 1.3.2; Li et al., 2009), GATK (version 3.8; McKenna et al., 2010), VCFtools (version 1.16; Danecek et al., 2011), and Plink (version 1.9; Chang et al., 2015). The files containing scripts used for analyses were written in and require <em>R</em> (version 3.6.3; R Core Team), and the <em>R </em>package pcaMethods (version 1.78.0; Stacklies et al., 2007).
description: # Extreme sex chromosome differentiation, likely driven by inversion,
 contrasts with mitochondrial paraphyly between species of crowned sparrows
 --- This dataset contains all the metadata, barcodes, and photos
 associated with the samples included in the study "Extreme sex
 chromosome differentiation, likely driven by inversion, contrasts with
 mitochondrial paraphyly between species of crowned sparrows" by
 Mcallum, Askelson, Fogarty, Natola, Nikelski, Huang and Irwin, as well as
 all scripts used in the bioinformatics pipline (Bash and perl) and data
 analyses (R). Associated sequences can be found on the SRA as part of the
 BioProject "Genotyping-by-sequencing reads from the bird genera
 Sphyrapicus, Zonotrichia, and Leiothlypis" ## Description of the Data
 and file structure barcodes_plate1.txt - Contains sample names barcodes
 for samples included in this study on plate 1 ("Plate 1 -
 Zonotrichia, and Leiothlypis GBS Library" on SRA). This is used by
 the demultiplex and trim perl script included in
 Zonotrichia-all-command-line-scripts-tidy.txt. barcodes_plate2.txt -
 Contains sample names barcodes for samples included in this study on plate
 2 ("Plate 2 - Sphyrapicus, Zonotrichia, and Leiothlypis GBS
 Library" on SRA). This is used by the demultiplex and trim perl
 script included in Zonotrichia-all-command-line-scripts-tidy.txt.
 photo-archive.zip - Contains high-resolution photographs in .jpg format of
 all birds sampled in this study. Zonotrichia-metadata.csv - Contains
 metadata for all samples included in this study. Samples are organized by
 by sample name. Zonotrichia-metadata-README.txt - Contains detailed
 descriptions of the contents of each column of Zonotrichia-metadata.csv.
 Zonotrichia-all-command-line-scripts-tidy.txt - Contains all Bash and Perl
 scripts used to demulitplex, trim, and align our sequences, call and
 filter snps, and calculate Fst and Linkage disequilibrium.
 ld_heatmap_plotting__sparrows.R - Contains all R scripts used to produce
 figures 7B and S7B. genomics_R_functions_V2.R - Contains a variety of R
 functions from Irwin et al. 2016 that are used by several other R scripts
 used in this project. genotype-by-individual-plot.R - Contains R scipts
 used to produce figures 7A, S6, and S7A. GCSP_vs_WCSP_fst_nuclear_genome.R
 - Contains R scripts used to calculate pairwise genome-wide Fst between
 groups (table 1) and produce prelimiary per-snp Fst manhattan plots
 (Figure S5), and PCAs (figures 1B and S3)
 genotype_by_individual_function.R - Contains R function used by
 genotype-by-individual-plot.R Sparrow_slidingWindow.R - Contains R scripts
 that calculate Fst, Pi_between, and Pi_within accross sliding windows,
 perform analyses using these sliding windows, and prodce summary plots of
 these analyses (figures 2, 3, 4, 6, S4). Additionally, contains scripts
 used to compare the Z chromosome to the autosomes (table 2) and produce
 figure 5. ## Sharing/access Information Links to other publicly accessible
 locations of the data: Sequences associated with this project are uploaded
 to the SRA under BioProject "Genotyping-by-sequencing reads from the
 bird genera Sphyrapicus, Zonotrichia, and Leiothlypis" as BioSamples
 "Plate 1 - Zonotrichia, and Leiothlypis GBS Library" and
 "Plate 2 - Sphyrapicus, Zonotrichia, and Leiothlypis GBS
 Library". URL of BioProject: Was data derived from another source? No
 data was derived from another source. Some of the scripts used are derived
 from other sources. Please refer to the comments in the scripts and the
 manuscript for sources.