DOI: 10.5061/dryad.fqz612jt8
title: Genomic diversity and differentiation between island and mainland populations of White‐tailed Eagles (Haliaeetus albicilla)

description: Using whole genome shotgun sequences from 92 white-tailed eagles
 (Haliaeetus albicilla) sampled from Greenland, Iceland, Norway, Denmark,
 Estonia, and Turkey between 1885–1950 and after 1990, we investigate the
 genomic variation within countries over time, and between countries. Clear
 signatures of ancient biogeographic substructure across Europe and the
 North‐East Atlantic are observed. The greatest genomic differentiation was
 observed between island (Greenland and Iceland) and mainland (Denmark,
 Norway and Estonia) populations. The two island populations share a common
 ancestry from a single mainland population, distinct from the other
 sampled mainland populations, and despite the potential for high
 connectivity between Iceland and Greenland they are well separated from
 each other and are characterized by inbreeding and little variation.
 Temporal differences also highlight a pattern of regional populations
 persisting despite the potential for admixture. All sampled populations
 generally showed a decline in effective population size over time, which
 may have been shaped by four historical events: I) isolation of refugia
 during the last glacial period 110‐115,000 years ago, II) population
 divergence following the colonization of the deglaciated areas ~10,000
 years ago, III) human population expansion, which led to the settlement in
 Iceland ~1,100 years ago, and IV) human persecution and exposure to toxic
 pollutants during the last two centuries.
description: Tissue
 was obtained from 92 specimens: 63 contemporary and 29 historic, from six
 different countries. These included 12 contemporary and eight historic
 individuals from Greenland, 25 contemporary and two historic individuals
 from Iceland, 12 contemporary and 13 historic individuals from Norway, 11
 contemporary and five historic individuals from Denmark, three
 contemporary individuals from Estonia, and one historic individual from
 Turkey (Figure 1). The historic specimens were sampled between 1885 and
 1950 (all but the two Icelandic individuals were sampled prior to 1937),
 while all contemporary individuals were sampled post-1990 (full individual
 information is presented in Table 1). Muscle tissue
 and whole blood from contemporary samples from Estonia, Denmark, and
 Greenland (Table 1) were stored at -20 °C until DNA extraction and were
 provided by the Department of Ecoscience, Arctic Research Centre, AU,
 Roskilde, Denmark<strong> </strong>(Estonian, Danish, and
 Greenland samples), Natural History Museum of Denmark, University of
 Copenhagen, Copenhagen, Denmark (Danish samples) and the Greenland
 Institute of Natural Resources, Nuuk, Greenland (Greenland samples). Whole
 blood samples from contemporary samples from Iceland were collected in an
 ongoing monitoring project of the white-tailed eagle in Iceland (led by
 the Icelandic Institute of Natural History) and stored in EDTA at -20 °C
 until DNA extraction. Whole genome shotgun DNA sequences from twelve
 Norwegian individuals were provided by the Department of Natural History,
 University Museum, Norwegian University of Science and Technology (NTNU),
 Trondheim, Norway. DNA extraction, library building, and sequencing of all
 contemporary samples are described in Hansen et al (2021, in
 review). Historic
 samples consisting of toepad clippings, taken with disposable sterile
 scalpel blades, from museum samples provided by The Natural History Museum
 of Denmark, University of Copenhagen, Denmark; Icelandic Institute of
 Natural History, Reykjavik, Iceland, and Department of Natural History,
 NTNU University Museum, Norwegian University of Science and Technology
 (NTNU), Trondheim, Norway. Historic
 samples from Greenland, Iceland, Denmark, Turkey, and five of the thirteen
 Norwegian specimens were processed at the clean laboratory facilities at
 the Globe Institute at the University of Copenhagen. Firstly, to prevent
 cross-contamination from other museum specimens, the samples were cleaned
 with a dilute bleach solution (ca. 5% commercial strength), then rinsed
 with 70% ethanol followed by molecular biology grade water performed using
 a proteinase-based lysis-buffer according to Gilbert et al. (2008). Each
 sample was added 300 µL lysing buffer including 20 µL proteinase K and
 incubated for 3 hours. The supernatant was purified by combining 720 µL
 binding buffer modified as in Allentoft et al. (2015), with 80 µL sample
 lysate, vortexed and centrifuged through a Monarch® DNA Cleanup Column
 (New England Biolabs Inc., Beverly, Massachusetts, USA). The binding step
 was repeated 3 times after which the column was washed with 800 µL PE
 buffer, from where the DNA eluded into 21.5 µL EBT buffer. Throughout the
 entire process, only LoBind Eppendorf tubes were used. The remaining
 eight Norwegian historic specimens were processed at the Norwegian
 University of Science and Technology (NTNU) University Museum’s dedicated
 palaeo-genomics laboratory. For these, the genomic DNA extractions were
 performed with a Qiagen DNeasy Blood & Tissue kit. The
 manufacturer’s protocol was used except that the amount of proteinase K
 was doubled, and the lysis step incubation at 56°C was extended to 15
 hours. The DNA solutions were incubated at 37°C for 10 minutes prior to
 elution. For all
 historic samples, blunt-end Illumina shotgun sequencing libraries were
 prepared using the BEST protocol (Carøe et al. 2018). In both of the aDNA
 laboratories, extraction and library blanks were also included to monitor
 for contamination. Indexed
 libraries from historic samples from Greenland, Iceland, Denmark, Turkey
 and five Norwegian specimens processed at the University of Copenhagen
 were paired-end sequenced on four flow cells with 2x150 bp read length at
 deCODE Genetics in Iceland using an Illumina NovaSeq 6000. The purified
 and indexed libraries for the eight Norwegian specimens processed at NTNU
 were pooled and paired-end sequenced over two runs on the Illumina HiSeq
 4000 platform at the NTNU Genomics Core Facility, and over one run on an
 Illumina NovaSeq 6000 at the University of Oslo Norwegian National
 Sequencing Centre. Fastq file quality of all samples
 was checked using FastQC (Babraham Bioinformatics 2010), then run through
 AdapterRemoval v2 using standard-setting, but providing adapter sequences
 for samples, and using the arguments --collapse and –trimns (Schubert et
 al. 2016). The fastq files were mapped to the golden eagle
 (<em>Aquila chrysaetos</em>)<em> </em>genome
 (GCA_900496995.3) using bwa aln, samse, and sampe, with the flags -q 15
 and -k 1 (Li and Durbin 2009). Although a white-tailed eagle genome is
 available, the golden eagle was deliberately chosen as the reference to
 minimize the potential of mapping biases derived from the fact that the
 available white-tailed eagle genome is not equally related to all
 populations studied here (the published white-tailed eagle genomes come
 from Greenland, UK, and Germany), thus might introduce errors in the
 analyses (Gopalakrishnan et al. 2017). A further benefit of aligning to
 the golden eagle genome is that it has been assembled to chromosome level
 completeness and annotated, thus enabling us to both identify and exclude
 sex chromosomes as needed in some of the downstream analyses, and identify
 the genes present in regions under selection. Picard (Broad Institute
 2020) was used to remove duplicate reads. To identify likely damaged bases
 the base quality score was rescaled with mapDamage 2.0 (Jónsson et al.
 2013). Genotypes were called using GraphTyper2 (Eggertsson et al. 2019)
 with standard settings. The VCF file for the 92 individuals was filtered
 using VCFtools, BCFtools, and VCF-annotate; SNPs had to have a minor
 allele count of one.