Sample collection, DNA extraction, and pooling

Small craft and SCUBA were used to sample Southern and Central California, USA subtidal coastal waters. Foot tissue from 46 K. kelletii (urn:lsid:marinespecies.org:taxname:491054) adults (>60 mm shell length; Rosenthal 1970) – 23 individuals in 2015 and 23 individuals in 2016 – were sampled non-lethally from each of 13 subtidal locations (~15 m depth) across the species’ historical and expanded range. By only including adults, we aimed to represent the local, established population and avoid the bias in population structure that could arise from recent recruitment if juveniles were included. Collected tissue samples were then frozen on dry ice or liquid nitrogen for transport to the California Polytechnic State University (San Luis Obispo, CA) and stored at -80 ℃ until processed for DNA extraction.

DNA extraction was performed using an optimized version of the ‘salting-out’ protocol developed by Li et al. (2011) and modified by Daniels et al. (2023a), where the full extraction protocol is detailed. Briefly, 30 mg of tissue was lysed with proteinase K and RNase A in a warm water bath. Subsequently, DNA was separated from proteins, which precipitated in the presence of ammonium acetate by centrifugation, and was then purified from the supernatant via ethanol washes. Finally, precipitated DNA was resuspended in Tris-EDTA (1xTE) buffer and stored at -20 ºC until further analyses.

DNA quality was assessed visually using a 1% agarose gel in Tris-Acetic Acid-EDTA (TAE) buffer, GelRed (Biotium, Inc) gel stain, and referenced to the 200–10,000 bp Hyperladder I (Bioline, Meridian Bioscience). Because 91.3% of all extractions produced high molecular weight bands (>10 kb) with faint smears from degraded DNA, all specimens were included in the study. Extractions were quantified using the AccuClear Ultra High Sensitivity dsDNA quantification kit (Biotium, Inc) with 3 standards and measured using a SpectraMax M2 microplate reader (Molecular Devices, LLC). Finally, an equimolar amount of DNA from each of the 46 individuals collected at each location was pooled by the collection site (population), ensuring that each library had the same number of individuals. In total, 598 individuals belonging to 13 populations spanning approximately 800 km were included in the analysis.

Library preparation and sequencing

Equimolar pooled ezRAD (Toonen et al. 2013) libraries were generated following the detailed protocol of Knapp et al. (2016) for all 13 sites. Briefly, genomic DNA was digested using the isoschizomer restriction enzymes MboI and Sau3AI (New England Biolabs, Ipswich, MA). Digestions were performed in a total volume of 50 µl, containing 25 µl of dsDNA (~1 µg), 5 µl of NEB Cutsmart Buffer (provided with restriction enzymes), 18 µl of HPLC grade water, 1 µl MboI (10 units), and 1 µl Sau3AI (10 units) under the following thermocycler profile: 37 ºC for 18 h, then deactivation at 65 ºC for 20 min. After digestion, samples were cleaned using Mag-Bind TotalPure NGS (Omega Bio-Tek) beads at a 1:1.18 (DNA:beads) ratio to remove fragments < 200 bp (Norcross, GA). Libraries were prepared for sequencing using the KAPA Hyper Prep DNA kit (Roche Sequencing and Life Science) following a modified version of the manufacturer protocol (see Knapp et al. 2016). Quality control by a Bioanalyzer and sequencing of the libraries on one lane of an Illumina HiSeq2500 were performed in the DNA Technologies and Expression Analysis Core Laboratory at the University of California (Davis, CA).

958359_v1_kelletia_kelletii_pooled_rad.csv