Nine species of diatoms were isolated from the Western Antarctic Peninsula along the PalmerLTER sampling grid in 2013 and 2014. Isolations were performed using an Olympus CKX41 inverted microscope by single cell isolation with a micropipette (Anderson 2005). Diatom species were identified by morphological characterization and 18S rRNA gene (rDNA) sequencing. DNA was extracted with the DNeasy Plant Mini Kit according to the manufacturer’s protocols (Qiagen). Amplification of the nuclear 18S rDNA region was achieved with standard PCR protocols using eukaryotic-specific, universal 18S forward and reverse primers. Primer sequences were obtained from Medlin et al. (1982). The length of the region amplified is approximately 1800 base pairs (bp). Pseudo-nitzschia species are often difficult to identify by their 18S rDNA sequence, therefore, additional support of the taxonomic identification of P. subcurvata was provided through sequencing of the 18S-ITS1-5.8S regions. Amplification of this region was performed with the 18SF-euk and 5.8SR_euk primers of Hubbard et al. (2008). PCR products were purified using either QIAquick PCR Purification Kit (Qiagen) or ExoSAP-IT (Affymetrix) and sequenced by Sanger DNA sequencing (Genewiz). Sequences were edited using Geneious Pro software (http://www.geneious.com, Kearse et al., 2012) and BLASTn sequence homology searches were performed against the NCBI nucleotide non-redundant (nr) database to determine species with a cutoff identity of 98%.
Diatom phylogenetic analysis was performed with Geneious Pro and included 71 additional diatom 18S rDNA sequences from publically available genomes and transcriptomes, including those in the MMETSP database. Diatom sequences were trimmed to the same length and aligned with MUSCLE (Edgar 2004). A phylogenetic tree was created in Mega with the Maximum-likelihood method of tree reconstruction, the Jukes-Cantor genetic distance model (Jukes and Cantor 1969), and 100 bootstrap replicates.
Isolates were maintained at 4 deg C in constant irradiance at intensities of either 10 umol photons m-2 s-1 (low light) or 90 umol photons m-2 s-1 (growth saturating light) and with media containing high and low iron concentrations. Cultures were grown in the synthetic seawater medium, AQUIL, enriched with filter sterilized vitamin and trace metal ion buffer containing 100 umol L-1 EDTA. The growth media also contained 300 μmol L-1 nitrate, 200 umol L-1 silicic acid and 20 umol L-1 phosphate. Premixed Fe-EDTA (1:1) was added separately for total iron concentrations of either 1370 nmol L-1 or 3.1 nmol L-1. Cultures were grown in acid-washed 28 mL polycarbonate centrifuge tubes (Nalgene) and maintained in exponential phase by dilution. Specific growth rates of successive transfers were calculated from the linear regression of the natural log of in vivo chlorophyll a fluorescence using a Turner 10-AU fluorometer (Brand et al. 1981).
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