Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Diatom growth rates from samples obtained on LMG1411.

Diatom isolates were obtained from the Western Antarctic Peninsula surface waters.

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). 

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 to p<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 –FeLL and +FeSL for u, Fv:Fm, and  oPSII. All tests passed the Shapiro-Wilks Normality tests unless otherwise stated, in which case p-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 < 0.05).

Cultures for high throughput sequencing of mRNA were grown in acid-washed 2L polycarbonate bottles in iron-replete conditions under growth-saturating light (90 umol photons m-2 s-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’s 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 ug of 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.​

BCO-DMO Data Processing Notes:

-reformatted column names to comply with BCO-DMO standards
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The Southern Ocean surrounding Antarctica is changing rapidly in response to Earth's warming climate. These changes will undoubtedly influence communities of primary producers (the organisms at the base of the food chain, particularly plant-like organisms using sunlight for energy) by altering conditions that influence their growth and composition. Because primary producers such as phytoplankton play an important role in global biogeochemical cycling, it is essential to understand how they will respond to changes in their environment. The growth of phytoplankton in certain regions of the Southern Ocean is constrained by steep gradients in chemical and physical properties that vary in both space and time. Light and iron have been identified as key variables influencing phytoplankton abundance and distribution within Antarctic waters. Microscopic algae known as diatoms are dominant members of the phytoplankton and sea ice communities, accounting for significant proportions of primary production. The overall objective of this project is to identify the molecular bases for the physiological responses of polar diatoms to varying light and iron conditions. The project should provide a means of evaluating the extent these factors regulate diatom growth and influence net community productivity in Antarctic waters. The project will also further the NSF goals of making scientific discoveries available to the general public and of training new generations of scientists. It will facilitate the teaching and learning of polar-related topics by translating the research objectives into readily accessible educational materials for middle-school students. This project will also provide funding to enable a graduate student and several undergraduate students to be trained in the techniques and perspectives of modern biology.

Although numerous studies have investigated how polar diatoms are affected by varying light and iron, the cellular mechanisms leading to their distinct physiological responses remain unknown. Using comparative transcriptomics, the expression patterns of key genes and metabolic pathways in several ecologically important polar diatoms recently isolated from Antarctic waters and grown under varying iron and irradiance conditions will be examined. In addition, molecular indicators for iron and light limitation will be developed within these polar diatoms through the identification of iron- and light-responsive genes -- the expression patterns of which can be used to determine their physiological status. Upon verification in laboratory cultures, these indicators will be utilized by way of metatranscriptomic sequencing to examine iron and light limitation in natural diatom assemblages collected along environmental gradients in Western Antarctic Peninsula waters. In order to fully understand the role phytoplankton play in Southern Ocean biogeochemical cycles, dependable methods that provide a means of elucidating the physiological status of phytoplankton at any given time and location are essential.