Temporal genetic studies of low-dispersing organisms are rare. Marine invertebrates lacking a planktonic larval stage are expected to have lower dispersal, low gene flow, and a higher potential for local adaptation than organisms with planktonic dispersal. Leptasterias is a genus of brooding sea stars containing several cryptic species complexes. Population genetic methods were used to resolve patterns of fine-scale population structure in central California Leptasterias species using three loci from nuclear and mitochondrial genomes. Historic samples (collected between 1897 and 1998) were compared to contemporary samples (collected between 2008 and 2014) to delineate changes in species distributions in space and time. Phylogenetic analysis of contemporary samples confirmed the presence of a bay-localized clade and revealed an additional bay-localized and previously undescribed clade of Leptasterias. Analysis of contemporary and historic samples indicates two clades are experiencing a constriction in their southern range limit and suggests a decrease in clade-specific abundance at sites at which they were once prevalent. Historic sampling revealed a dramatically different distribution of diversity along the California coastline compared to contemporary sampling and illustrates the importance of temporal genetic sampling in phylogeographic studies. These samples were collected prior to significant impacts of Sea Star Wasting Disease (SSWD) and represent an in-depth analysis of genetic structure over 117 years prior to the SSWD-associated mass die-off of Leptasterias. 

Sample Collection and DNA Extraction

Three hundred forty-five adult Leptasterias individuals were collected from 17 intertidal sites on the Pacific Coast between December 2008 and July 2014 (Table 1, DFW Scientific Collecting Permit SC-12882). Ray samples were collected from individuals separated by at least one meter when possible to avoid family groups and tissue was stored in 95% ethanol. Samples are stored at San Francisco State University. Alaskan samples were provided courtesy of Marnie Chapman, Sara Caldwell, and Sherry Tamone from the University of Alaska Southeast.

Historic samples of Leptasterias spp. collected between 1897 and 1998 were obtained from the Invertebrate Zoology collection at the California Academy of Sciences or gifted from Dave W. Foltz (Louisiana State University). Historic samples were collected on the Pacific coast ranging from Lonesome Cove, Washington to Diablo Canyon, California (Table 1). Tube feet were sampled from historic samples and transferred from ethanol into milliQ water and left on a shaker for two days to remove excess ethanol prior to extraction. All DNA extractions were carried out using NucleoSpin Tissue Columns (Macherey-Nagel Inc, Bethlehem, PA, USA), except for samples collected in 2008 from Marshall Gulch, Bodega Bay and Mussel Rock, which were extracted with a phenol-chloroform extraction.

Control Region Amplification

Forward primer E16Sa (Smith, Arndt, Gorski, & Fajber, 1993) and reverse primer Star-L (Flowers & Foltz, 2001) were used for amplification of 286 bp of the putative control region and 8 bp of the conserved 3’ end of the large ribosomal subunit 16S gene (henceforth referred to in entirety as D-Loop for simplicity). PCR reactions for contemporary samples had the following components: 5-500 ng DNA template, 0.2 µM of each primer, 1X PE II Buffer, 1 mM dNTPs, 2.5 mM MgCl₂, 1.25 µg BSA, 1 unit of Taq DNA polymerase (New England Biolabs, NEB, Ipswich, MA, USA) and milliQ water up to 25 uL final volume. Thermal cycling conditions were: initial denaturation at 94°C for 120 seconds, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 45°C for 60 seconds, extension at 72°C for 60 seconds, and a final extension at 72°C for 300 seconds. A separate thermocycling reaction was run for historic samples with a total of 35 cycles if amplification was unsuccessful after 30 cycles.

COI Amplification

Primers were designed for this study from published mitochondrial cytochrome oxidase subunit I (COI) sequences of L. aequalis and L. hexactis (Hrincevich et al., 2000; Foltz et al., 2008). Forward primer COILF, 5’ GCA-GGA-TTT-ACC-CAC-TGA-TTT-C 3’ and reverse primer COILR, 5’CCT-GGC-TTC-ACA-GGC-AGA-T 3’ amplified 378 bp of COI, 68 bp of tRNA-Arg and 90 bp of ND4L genes (henceforth referred to as COI for simplicity). PCR reactions were carried out using the same reaction concentrations and volumes as in D-Loop amplification. Thermal cycling conditions were: initial denaturation at 96°C for 120 seconds, 35 cycles of denaturation at 94°C for 30 seconds, annealing at 46°C for 30 seconds, extension at 72°C for 60 seconds, and a final extension at 72°C for 300 seconds. For all historic samples, thermocycling conditions were run for 40 cycles.

Intron Amplification

Five Exon Primed Intron Crossing (EPIC) loci (Chenhuil et al., 2010; Gérard et al., 2013) were screened for amplification in Leptasterias based on successful amplification in other echinoderm taxa: i1, i9, i39, i43, and i51. One EPIC locus which offered the highest resolution among sites and clades was chosen and optimized for population genetic analyses. The i51 primer pair amplified a region in the gene group UDP-N-acetylglucosaminyl-transferase (Chenhuil et al., 2010). Primers were redesigned for i51 to decrease primer degeneracy. Forward primer i51LF GAT-CGA-CCC-AGC-CAC-ATT and reverse primer i51LR TTG-AAG-CAA-CAG-GGG-AGA-AG were exclusively used to amplify a 277 base pair intronic region. PCR reactions were the same as D-Loop and COI but used 0.1 µM of each primer. Thermal cycling conditions were: initial denaturation at 96°C for 60 seconds, 35 cycles of denaturation at 94°C for 40 seconds, annealing at 45°C for 30 seconds, extension at 72°C for 40 seconds, and a final extension at 72°C for 120 seconds.

Sequencing Reactions

Amplification of PCR templates was assessed with gel electrophoresis using a 1.5% agarose gel stained with ethidium bromide. PCR products were cleaned using a SAP/EXO reaction following manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). Cycle sequencing reactions were carried out in the reverse direction using the 1/8 reaction BigDyeTerminator v3.1 (Applied Biosystems, ABI, Foster City, USA). Products were sequenced using an ABI 3130 genetic analyzer. Cloning was used to resolve and confirm a subset of alleles for i51 in heterozygous individuals using Vector System II, pGEM-T (Promega, Madison, WI).