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\u2019s 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).\u00a0Pseudo-nitzschia\u00a0species are often difficult to identify by their 18S rDNA sequence, therefore, additional support of the taxonomic identification of\u00a0P.\u00a0subcurvata\u00a0was 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%.
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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.
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Isolates were maintained at 4 deg C in constant irradiance at intensities of either 10\u00a0umol\u00a0photons m-2\u00a0s-1\u00a0(low light) or 90\u00a0umol\u00a0photons m-2\u00a0s-1\u00a0(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\u00a0umol\u00a0L-1\u00a0EDTA. The growth media also contained 300 \u03bcmol L-1\u00a0nitrate, 200\u00a0umol\u00a0L-1\u00a0silicic acid and 20\u00a0umol\u00a0L-1\u00a0phosphate. Premixed Fe-EDTA (1:1) was added separately for total iron concentrations of either 1370 nmol L-1\u00a0or 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\u00a0log of\u00a0in vivo\u00a0chlorophyll\u00a0a\u00a0fluorescence using a Turner 10-AU fluorometer (Brand et al. 1981).\u00a0
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Statistical analyses of growth rates and photophysiological data were performed with SigmaPlot 12.5 (SysStat Software Inc.). To test for significant differences between treatments, Two-Way Analysis of Variance (ANOVA) was performed with a significance level set\u00a0to\u00a0p<0.05. ANOVA also tests for normality using Shapiro-Wilks and Equal Variance tests. Because ANOVA does not test all interactions, an unpaired t-test was performed between \u2013FeLL and +FeSL for u, Fv:Fm, and \u00a0oPSII. All tests passed the Shapiro-Wilks Normality tests unless otherwise stated, in which case\u00a0p-values are representative of the Mann-Whitney Rank Sum test. Post-hoc Tukey tests were also performed in order to determine which treatments differed significantly (p\u00a0< 0.05).
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Cultures for high throughput sequencing of mRNA were grown in acid-washed 2L polycarbonate bottles in iron-replete conditions under growth-saturating light (90\u00a0umol\u00a0photons m-2\u00a0s-1). After reaching late exponential/early stationary phase, cultures were harvested onto polycarbonate filters (3.0 um pore size, 25 mm) and stored at -80 deg C. Total RNA was extracted using the RNAqueous 4PCR Kit (Ambion) according to the manufacturer\u2019s protocols. Residual genomic DNA was eliminated by DNAseI digestion at 37 deg C for 45 min. An Agilent Bioanalyzer 2100 was used to determine RNA integrity. mRNA libraries were generated with ~2\u00a0ug\u00a0of total RNA and prepared with the Illumina TruSeq Stranded mRNA Library Preparation Kit. Samples were individually barcoded and pooled prior to sequencing on the Illumina MiSeq platform at the High Throughput Sequencing Facility (HTSF) at UNC-Chapel Hill. Sequencing resulted in approximately 0.7-2 million paired-end reads of 2x300bp per sample.\u200b
\nDiatom growth rates from samples obtained on LMG1411.\n
Diatom isolates were obtained from the Western Antarctic Peninsula surface waters.
BCO-DMO Data Processing Notes:
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