DOI: 10.5061/dryad.4mw6m907m
title: Temporal and spatial variation in population structure among brooding sea stars in the genus Leptasterias

description: 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. 
description: <i>Sample Collection and DNA Extraction</i>
 Three hundred forty-five adult <i>Leptasterias
 </i>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 <i>Leptasterias
 </i>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. <i>Control
 Region Amplification</i> Forward primer E16Sa
 (Smith<i>, </i>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. <i>COI
 Amplification</i> Primers were designed for this
 study from published mitochondrial cytochrome oxidase subunit I (COI)
 sequences of <i>L. aequalis </i>and <i>L.
 hexactis</i> (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. <i>Intron Amplification
 </i> Five Exon Primed Intron Crossing (EPIC) loci
 (Chenhuil et al., 2010; Gérard et al., 2013) were screened for
 amplification in <i>Leptasterias</i> 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. <i>Sequencing
 Reactions</i> 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).