Genotype-by-sequencing of Arctocephalus pusillus: flipper tissue from pups
Flipper clippings were collected from 52 CFS and 42 AFS pups between 19982019 using non-destructive methods. Permission to collect specimens was granted by the Department of Natural Resources and Environment, Australia (Wildlife Permits 10000623, 10001493 and 10000706), and by the Ministry of Fisheries and Marine Resources, Namibia, to Sea Search Research and Conservation. Ethical approval for the collection and use of sampled specimens was obtained from the Stellenbosch University Institutional Research Ethics Committee (Animal Care and Use), South Africa (Reference No. ACU-2021-21659), the Phillip Island Nature Parks Animal Experimentation Ethics Committee, Australia (Permits 2.99 and 4.01) and the Macquarie University Animal Care and Ethics Committee, Australia (Approval No. 97001). The CFS colonies sampled were the Cape Cross (n = 16), Pelican Point (n = 17), Sinclairs Island (n = 2), Kleinzee (n = 15) and Mossel Bay (n = 2) colonies. The Australian fur seal colonies sampled were the Lady Julia Percy (n = 18), Seal Rocks (n = 13) and Skerries (n = 11) colonies (Fig. 1a). Samples were collected from pups younger than 6 months of age to allow colonies of birth to be identified (Oosthuisen, 1991) and stored in 96% ethanol until DNA extraction. DNA was extracted using a NucleoSpinTM Tissue Kit (Macherey-Nagel) and DNA quantity and quality assessed with Qubit fluorometer analysis and gel electrophoresis. DNA extractions with concentrations > 20 nanograms per millilitre (ng/ml) were sent to CD Genomics in New York, USA for genotype-by-sequencing (GBS) library preparation using the PstI and MspI enzymes on an Illumina platform. Two laboratory blanks (negative controls) were included during DNA extraction and sequencing. Bioinformatic analyses were performed using Stellenbosch Universitys high-performance computer (University of Stellenbosch, 2025). Illumina sequences were demultiplexed, filtered for phred scores < 10 and cleaned of barcodes and residual GBS restriction sites using the Process_radtags program in Stacks v2.59 (Rochette et al., 2019).