Online Resource 3

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Online Resource 3
To obtain sequences of msh1, total genomic DNA was extracted from ethanol (EtOH)-preserved material using a CTAB (2% hexadecyltrimethylammonium bromide) protocol (with proteinase K in final concentration of 167 μg/mL) and a single chloroform-only extraction (modified from France et al. 1996). Using the polymerase chain reaction (PCR), we amplified approximately 460-990 bp of the 5’-region of msh1 for 14 specimens; variability in amplicon length depended on which combination of forward primers (i.e. ND4L2475F or ND42625F, Brugler and France 2008; ND42599F, France and Hoover 2002; MSH3010F, Thoma et al. 2009) and reverse primers (i.e. MSH3101R, McFadden et al. 2011; MUT3458R, Sánchez et al. 2003) that were used. Each PCR used 0.5 U of either TaKaRa Ex Taq™ polymerase or DyNAzyme II™ DNA polymerase with the following reaction components: 1X TaKaRa Ex Taq buffer [Mg2+ free, with added MgCl2 of 1.5 mM] or 1X Optimized DyNAzyme buffer [includes 1.5 mM MgCl2], 0.4 mM dNTPs, 0.24-0.32 µM of each primer, 0-2.5 µg acetylated bovine serum albumin [Promega], 7-90 ng of genomic DNA, and brought to a final volume of 25 µL with H20. PCRs were carried out using the following cycle profile: initial denaturation at 94°C for 2 to 3 min followed by 30 to 40 cycles of denaturation at 94°C for 20 to 30 s, annealing at 45 to 50°C for 30 to 45 s, extension at 72°C for 50 s to 1 min, and a final extension at 72°C for 6 min. PCR products were purified by enzymatic digestion (1.52 U of ExoI and 0.15 U FastAP™ thermosensitive alkaline phosphatase [Fermentas] per 1 µL PCR product; modification of Werle et al. 1994) or from low melting point (LMP) agarose by digestion with agarase (5 U per 100 μl melted 1% LMP agarose; Sigma-Aldrich). Purified PCRs were cycle-sequenced at the University of Louisiana at Lafayette using ABI BigDye® Terminator v1.1 Cycle Sequencing Kit (1/4 reactions) and purified with Sephadex G-50 columns (GE Healthcare). Purified products were electrophoresed on an ABI PRISM® 3130xl Genetic Analyzer and sequence traces were edited using Sequencher™ v4.7 (Gene Codes). DNA sequences were submitted to GenBank. A multiple alignment, comprised of msh1 nucleotide sequences, was created using MUSCLE (Edgar 2004). The trimmed alignment of ~700 bp was submitted to DnaSP v5.00.07 (Librado and Rozas 2009) to generate a sequence file designating unique genetic types (i.e. haplotypes); sites with missing data were excluded.


Brugler MR, France SC (2008) The mitochondrial genome of a deep-sea bamboo coral (Cnidaria, Anthozoa, Octocorallia, Isididae): genome structure and putative origins of replication are not conserved among octocorals. J Mol Evol 67:125-136
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792-1797
France SC, Hoover LL (2002) DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471:149–155
France SC, Rosel PE, Agenbroad JE, Mullineaux LS, Kocher TD (1996) DNA sequence variation of mitochondrial large subunit rRNA provides support for a two subclass organization of the Anthozoa (Cnidaria). Mol Mar Biol Biotech 5:15–28
Librado P, Rozas J (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452
McFadden CS, Benayahu Y, Pante E, Thoma JN, Nevarez PA, France SC (2011) Limitations of mitochondrial gene barcoding in Octocorallia. Molecular Ecology 11:19-31
Sánchez JA, McFadden CS, France SC, Lasker HR (2003) Molecular phylogenetic analyses of shallow-water Caribbean octocorals. Mar Biol 142:975–987
Thoma JN, Pante E, Brugler MR, France SC (2009) Deep-sea octocorals and antipatharians show no evidence of seamount-scale endemism in the NW Atlantic. Mar Ecol Prog Ser 397:25-35
Werle E, Schneider C, Renner M, Völker M, Fiehn W (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res 22:4354-4355

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