Computational protocol: The Genetic Diversity of Mesodinium and Associated Cryptophytes

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[…] Nucleic acids of environmental samples were extracted from frozen cells collected by centrifugation (4000 RPM, 10 min) or on SterivexTM filters (EMD Millipore) using either the PowerWater Sterivex DNA Isolation Kit (MO BIO Laboratories, Inc.) or a hot detergent lysis method as described by , modified to exclude zirconia-silica bead disruption. All cultures as well as the North Carolina Bloom sample were extracted using the Qiagen DNeasy Plant Mini Kit. PCR was conducted using GoTaq (Promega) or GoTaq G2 Hot Start mix in 50 mL reactions, with a final concentration of 2.5 mM MgCl2, 200 μM dNTPs, 2.5 U GoTaq Flexi polymerase, and 0.1 μM primers for normal and 0.2 μM for hot start. Primers for Mesodinium spp. (Table ) were designed to amplify the majority of the SSU and LSU rRNA genes, and the entire ITS region, resulting in a ∼1880 bp amplicon. Gene fragments of the large subunit of the ribulose-1,5-bisphosphate carboxylase oxygenase gene (rbcL) were PCR amplified from DNA extracts using primers targeting cryptophyte plastids. A new primer, crypt_rbcLR2 (5′-CAGTGAATACCACCTGAAGCTA-3′), designed using an alignment of cryptophyte rbcL sequences (See Supplementary Data Sheet for accession numbers) was used in combination with L2F (). PCR conditions were: 95°C for 5 min followed by 40 cycles of 95°C for 60 s, 55°C for 60 s, and 72°C for 90 s followed by 72°C for 7 min. The genus-specific primers MESO_245F and MESO_28S_R were used to amplify a combined fragment of the Mesodinium spp. 18S-ITS-28S genes. PCR conditions were: 95°C for 5 min followed by 40 cycles of 95°C for 60 s, 57°C for 60 s, and 72°C for 90 s followed by 72°C for 7 min. PCR products were visualized by agarose gel electrophoresis and later excised and purified from the gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research). Clone libraries were constructed from gel purified fragments using the pGEM-T Easy Vector in the pGEM-T Easy Vector System II cloning kit (Promega Corporation) according to the manufacturer’s protocol. Selected clones were submitted for Sanger sequencing with a single primer to either Beckman Coulter Genomics (Single Pass Sequencing) or the W. M. Keck Ecological and Evolutionary Genetics Facility at the Marine Biological Laboratory (Woods Hole). Full-length Sanger sequencing of select Mesodinium clones were run at Genewiz (Boston) (see Supplementary Data Sheet for accession numbers). Sequences were edited and assembled into contigs using Sequencher (Gene Codes Corporation). We sequenced and analyzed 687 cryptophyte rbcL clones (accession numbers in progress) from environmental samples. For Mesodinium spp., a total of 903 clones were sequenced (accession numbers in progress) from all stations. Using a sequence similarity criterion of 98 and 99% for cryptophytes and Mesodinium, respectively, we constructed independent contigs and generated consensus sequences for use in our global alignment for each sample. This process helped to reduce the number of sequences used in our phylogenetic analyses while maintaining meaningful diversity data. [...] Consensus sequences from assembled contigs were used for separate alignments of Mesodinium spp. and cryptophytes, in combination with available sequences in Genbank and sequences generated from cultures in our laboratory (see above). Alignments were constructed using the Clustal X algorithm (), and refined by eye using MacClade 4.08a (). All maximum likelihood (ML) phylogenetic trees were executed with PhyML 3.0 () using 100 bootstrap replicates and the general time reversible (GTR) substitution model, while estimating the gamma distribution parameter, proportion of invariable sites, and the transition/transversion ratio. Phylogenetic trees were constructed using TreeDyn 198.3 (). Assignment of Mesodinium and cryptophyte species was determined by their nearest neighbor match of a known species within ML phylogenetic trees. […]

Pipeline specifications

Software tools Sequencher, Clustal W, MacClade, PhyML, TreeDyn
Application Phylogenetics
Organisms Teleaulax amphioxeia