S1: Supplementary Materials and Methods

Samples and DNA Extraction

Epipodial tentacle or foot clippings were preserved in ethanol or water and stored at -80ºC. DNA was extracted from approximately 25-mg of tissue using a DNeasy Tissue Kit (Qiagen Inc., Valencia, California) and eluted in 200-µl of TE buffer (10-mM Tris pH 8.0, 1-mM EDTA). A 5-ml aliquot of the stock DNA was diluted with 45-ml of DNA-grade water, placed in 96-well trays, and stored at 4ºC for use in the polymerase chain reaction (PCR).

Cytochrome Oxidase Subunit One (COI)

Reactions were carried out in 25-ml volumes, including 5-ml of dilute template DNA, 0.5-U REDTaq DNA polymerase (Sigma-Aldrich Corporation, St. Louis, Missouri), 2.5-ml 10X PCR buffer, 2.5-mM MgCl2 final concentration, 2-mM each dNTP final concentration, and 5-pmoles each primer. The amplified fragments were sequenced on a MegaBACE™ 500 DNA sequencer (Amersham Biosciences Corp., Piscataway, New Jersey) using the forward primer AB-COIF and a DYEnamic™ ET dye terminator sequencing kit (Amersham Pharmacia Inc., Piscataway, New Jersey). Sequences were aligned and edited to 483-bp using Sequencher 4.2 (Gene Codes Corporation, Ann Arbor, Michigan).

Microsatellites

Reactions were carried out in 25-ml volumes, including 5-ml of dilute template DNA, 0.5-U REDTaq DNA polymerase, 2.5-ml 10X PCR buffer, 2.5-mM MgCl2 final concentration, 2-mM each dNTP final concentration, and 5-pmoles each primer. Forward primers were 5’ end-labeled with one of three dyes HEX, TET, or 6-FAM for fluorescent visualization. Cycling conditions were modified slightly from Miller et al. (2001) for optimization.

Following amplification, 2-ml of a denaturing solution containing xylene cyanol and bromophenol blue dyes in 99% formamide were added to each 5-ml of PCR product. Samples were loaded onto vertical 0.4-mm thick large format (33 x 39 cm) 6% polyacrylamide denaturing gels containing 33% formamide and 7-M urea. ROX-labeled MegaBACE™ ET900-R Size Standard (Amersham Pharmacia Inc., Piscataway, New Jersey) or Genemark 350 Fluorescent DNA Ladder (Northern Biotechnology, Schofield, Wisconsin) was run concurrently as a size standard. Gels were electrophoresed at 60-Watts for two to three hours and scanned on a Typhoon 9410 Variable Mode Imager (Molecular Dynamics Inc., Sunnyvale, Califonia). Hka3 allele sizes were scored using FragmeNT Analysis v1.2 software (Molecular Dynamics Inc., Sunnyvale, California). Hka28, Hka40, Hka56, and Hka80 allele sizes and AFLP banding patterns were viewed with ImageQuaNT software (Molecular Dynamics Inc., Sunnyvale, California) and scored by hand.

Amplified Fragment Length Polymorphisms (AFLPs)

Multiple pairs of restriction enzymes were tested on two individuals per population; EcoRI and MseI were found to give bands in the appropriate size range (≤ 2000 bp). After ligation of adapters complementary to the restriction sites, all sixteen possible pre-amplification primer combinations were tested. Pre-amplification products were diluted 10-fold with 10-mM Tris-EDTA pH 8.0 for use in the final amplification step. EcoRI amplification primers were 5’ end-labeled with the fluorescent dye HEX for visualization.

Both pre-amplification and amplification reactions were carried out in 20 mL volumes, including 5-ml of template (concentrated stock DNA or pre-amplification product), 1-U MasterTaq DNA polymerase (Eppendorf AG, Hamburg, Germany), 1X Taq Buffer, 1X TaqMaster PCR Enhancer, 2-mM each dNTP final concentration, 6-pmoles of MseI primer, and 6-pmoles EcoRI pre-amplification primer or 1-pmole of EcoRI amplification primer. Touchdown PCR cycling conditions for the pre-amplification included an initial two minute 95°C denaturation followed by 10 cycles of 95°C for 30-s, 50°C for 30-s (-1ºC per cycle), and 72°C for 30-s. The annealing temperature for the remaining 25 cycles was 40ºC. A touchdown protocol was also used for the final amplification, but initial annealing temperatures were raised to 56°C or 58°C depending on the primer combination.

Following amplification, 1-ml of a denaturing solution containing xylene cyanol and bromophenol blue dyes in 99% formamide were added to each 5-ml of PCR product. Samples were loaded onto vertical 0.4-mm thick large format (33 x 39 cm) 7.5% polyacrylamide denaturing gels containing 33% formamide and 7-M urea, electrophoressed (3-4 hours, 60-Watts), and scanned as with the microsatellite gels.

AFLP Reproducibility Test

A set of the first 12 individuals from the northern California and southern California populations (N = 24) were subjected to a complete replication of the AFLP protocol for 11 markers from the first four primer combinations listed in Table 2. The presence or absence of product bands was independently rescored by hand, and the genotypes were compared between the original scoring and the rescoring. Of 264 genotypes, five (< 2%) were scored differently between runs. Gels were visually identical in side-by-side comparisons, so variability was due to scoring error and not irreproducibility in the AFLP assay.

Validation of Genetic Markers

No breeding experiments were carried out to assess the genetic bases of the markers employed in this study. However, we have previously confirmed the maternal inheritance of mtDNA and the Mendelian inheritance of microsatellite polymorphisms in the closely related white abalone (H. sorenseni), using is same genetic markers in laboratory crosses (Gruenthal and Burton 2005). Although we have no family data on AFLPs, Liu et al. (2006) found that 95% of 387 AFLP markers exhibited normal Mendelian segregation in the Pacific abalone (H. discus hannai).