(2011). Extracted genomic DNA was used as template in subsequent PCR reactions. In addition, psbA was amplified selleck inhibitor and sequenced from the C. ovata stock culture following the same methods to ensure Esoptrodinium sequence identity by direct and phylogenetic sequence comparison (below). Reportedly dinoflagellate-specific primers bAf1 (5′-GGTCAAGGTTCTTTCTCTGAYGGNATGCC-3′) and bAr1 (5′-GTTGTGAGCGTTACGTTCRTGCATNACYTC-3′; Zhang et al. 2000) were used to amplify 500 bp of a highly conserved region of the psbA gene. PCR was carried out in 0.5 mL PCR tubes containing 45 μL of Platinum® PCR Super Mix (Invitrogen Corp., Carlsbad, CA, USA), 100 ng of each primer, 20 ng of template DNA, and 2.5 μL Ku 0059436 DMSO with appropriate

(+) and (−) controls. PCR was conducted using a Mastercycler® gradient thermal

block (Eppendorf AG, Hamburg, Germany) and reaction protocol: initial denaturing at 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1:00, followed by 72°C for 4 min. PCR products were visualized and size checked by gel electrophoresis, and purified using polyethylene glycol (Thermo Fisher Scientific Inc., Waltham, MA, USA) and ethanol following Bachvaroff et al. (2009). Purified products were sequenced in both directions using Applied Biosystems BigDye Terminator version 3.1 (GENEWIZ, Inc., South Plainfield, NJ, USA). Alignments used to create final psbA phylogenies were performed with Muscle (Edgar 2004) in MEGA5 (Tamura

et al. 2011) using default parameters 上海皓元医药股份有限公司 as suggested by Hall (2011). Nucleotide sequences were converted to amino acid sequences, aligned, and then reverted back to nucleotides before phylogenetic analysis was performed (Hall 2011), and an overall mean P-distance was calculated to ensure the alignment was reliable. The initial 1,095 nucleotide alignment contained 44 taxa plus three outgroups (Mesostigma viride, Nephroselmis olivacea, and Cyanophora paradoxa) based on Zhang et al. (2000) (Table S1 in the Supporting Information). MEGA5 was used to conduct maximum likelihood (ML) and maximum parsimony (MP) analyses to infer evolutionary history from the psbA alignment. The alignment was analyzed beforehand with jModelTest v0.1.1 (Posada 2008) and the general time reversible (GTR) model plus invariable sites with a Gamma distributed rate of variation (GTR+Ι+Γ) achieved the lowest log-likelihood score. A ML phylogenetic tree was constructed using this model with 8 discrete gamma categories and a Nearest-Neighbor-Interchange heuristic method applied. All gaps and missing data were removed, and the 3rd position in each codon was excluded (Hoppenrath and Leander 2010), resulting in 285 nucleotide positions in the final alignment. Bootstrap (BS) support was conducted with 100 replicates. The MP phylogenetic tree was constructed using a Close-Neighbor-Interchange search method from 10 initial trees.