Fast repetition rate fluorometer (FRRF) study of sea ice algae in low iron conditions. Algae were grown in an ice tank and the measurements were made at the end with a Chelsea Insrtuments FRRF. Materials and Methods (see the download document for original formatting and formulas) 1. Ice tank incubation The polar pennate diatom Fragilariopsis cylindrus, isolated from Antarctic pack ice in 2015 (Davis station, East Antarctica) was incubated in a purpose designed ice tank (Island Research, Tasmania). The ice tank, which was contructed of titanium to minimise dissolved Fe, was placed into a freezer (–20 degrees C), and the ice thickness and temperature gradient controlled by interaction between a basal heater and the adjustable ambient freezer temperature (see Kennedy et al., 2012). This enabled an ice thickness of approximately 5.5 cm to be maintained during the experiment. The diatom F. cylindrus was incubated in Aquil media (Price et al. 1989) buffered with ethylenediaminetetraacetic acid (EDTA) at 150 micro mol photons m−2 s−1 (PAR), a salinity of 35, and a Fe concentration of 400 nM, where the concentration of total inorganic forms of Fe (Fe') was 1.54 nM, this being continuously supplied to the medium and the exact value calculated using the software Visual MINTEQ, ver. 3.1 (https://vminteq.lwr.kth.se). Before a freezing cycle started, the seawater temperature was maintained at 2.5 degrees C, and a sample was obtained to assess the original physiological state of the algae (Day−5, hereafter). After obtaining the sample, the seawater temperature was set to −1.8 degrees C to initiate ice formation in the ice tank. Once ice had formed at Day−2, the under-ice seawater was partially replaced with ultrapure water to reduce the salinity down to 35, because the salinity had increased (to approximately 38) as a result of brine rejection from the ice. After a 2-day acclimation to the new salinity, ice samples were obtained every 5 days for 20 days (i.e., Days 0, 5, 10, 15, and 20). To minimize the heterogeneity among ice cores, ice samples were randomly collected from the tank chamber with a trace metal-free hand drill (2 cm in diameter) from randomly annotated grids on the ice surface, following normal random sampling numbers generated by the software R (https://www.r-project.org/). To assess the effects of melting and high light exposure, the ice was melted at 2.5 degrees C for 2 days. After the ice had completely melted, the seawater was exposed to a high light level, which was adjusted to represent the likely summer light intensity at the surface in ice-edge regions (800 micro mol photons m−2 s−1; MODIS Aqua), Seawater samples were obtained both after the melting and light exposure events (Melt and Light, respectively, hereafter). Fast repetition rate (FRR) fluorometry To monitor the photophysiology of F. cylindrus during the freezing and melting processes, variable chlorophyll a fluorescence (ChlF) measurements were conducted using a bench-top Fast Repetition Rate fluorometer (FRRf) (FastOcean Act2Run Systems, Chelsea Technologies) with Act2Run software (Chelsea Technologies). Ice samples were directly thawed at 2 degrees C in the dark for 30 min, and the slushily melted ice samples were placed in a quartz tube and their flouresence (ChlF) was measured. A single turnover protocol was applied for the ChlF measurements; 100 flashlets with 1 micro second duration at a wavelength 450 nm and 2 micro second intervals for excitation of reaction centres of photosystem II (PSII, hereafter), and 20 flahlets with 1 μs duration and 100 micro second intervals for relaxation. Eighteen light steps were applied to generate a rapid light curve (RLC) from 0 to 1800 μmol photons m−2 s−1, taking less than 5 min to complete one RLC. At each light step (~15 s), at least five induction and relaxation curves were averaged to obtain ChlF yields, described in Table, after calibrating the ChlF yields with filtered seawater. According to the models proposed

Metadata record for data from ASAC Project 102 See the link below for public details on this project. From the abstracts of some of the referenced papers: Six species of marine microalgae, namely Phaeodactylum tricornutum Bohlin, Dunaliella tertiolecta Butcher, Isochrysis galbana Parke, Porphyridium purpureum (Bory) Ross, Chroomonas sp., and Oscillatoria woronichinii Anis., have been examined with respect to their gas exchange characteristics and the inorganic carbon species taken up by the cells from the bulk medium. All species showed a high affinity, in photosynthesis, for inorganic carbon and low CO2 compensation concentrations. Such data are suggestive of operation of a 'CO2-concentrating mechanism' in these microalgae. Direct measurements of internal organic carbon pools in four of the species studied confirm this (O. woronichinii and Chroomonas were not tested). By comparison of achieved photosynthetic rates with calculated rates of CO2 supply from the dehydration of bicarbonate, it was shown that Phaeodactylum, Porphyridium and Dunaliella could utilise the bicarbonate present in the medium. Data for the other species were inconclusive although the pH dependence of K 1/2CO2 for photosynthesis by Oscillatoria indicated that this species too could utilise bicarbonate. Such observations could, however, not be used as evidence that, at least in the eucaryotic algae examined, bicarbonate was the inorganic carbon species crossing the plasmalemma as Phaeodactylum, Porphyridium and Dunaliella, and Isochrysis all showed the presence of carbonic anhydrase activity in intact cells as well as in crude extracts. 'External' carbonic anhydrase activity represented from 1/4 to 1/2 of the total activity in the cells of these algae. It is concluded that, as a consequence of a CO2-concentrating mechanism, photorespiration was suppressed in the marine microalgae examined although the data obtained did not allow any firm conclusions to be drawn regarding the species of inorganic carbon transported into the cell. Analysis of the age composition of a given species within a community is fundamental to any study of population dynamics and to the subsequent analyses of community interactions such as competition, succession and productivity. A problem exists in that calendar age often provides little information on the role played by any given individual plant within a population. For many populations the most useful definition of population structure is obtained from an analysis of both the functional age and the vitality of the component plants. Data from such studies on populations of marine macroalgae are lacking mainly because of the lack of suitable methods. This paper provides a review of the methods which have ben applied to such analyses in both terrestrial and marine communities, discusses these methods in the context of marine algae and presents the results of a case study on the analysis of population structure in the large brown alga Durvillaea potatorum. Evidence is presented for the occurrence of sexual reproduction including plasmogamy and meiosis, events previously undescribed in the life history of Ascoseira mirabilis. Ascoseira is monoecious. Gametangia are formed in chains within conceptacles. Synaptonemal complexes, structures concerned with chromosome pairing in meiosis, have been observed in the nucleus of gametangial initials. Mature male and female gametes have the same size and appearance, and resemble typical brown algal zoids. Sexual interaction begins after the female gamete settles down, and both zygotes and unfused gametes develop into sporophytes. It is concluded that Ascoseira has the same basic pattern of life history that characterises the order Fucales, and it is argued that this is probably the result of convergent evolution rather than being indicative of close phylogenetic relationship. Life histories are of central importance in understanding evolution and phylogeny of brown algae. Like other hereditary

Chlorophyll data was used to measure growth rates of sea ice algae in CO2 incubations. Sea ice brine microalgae was collected from sackholes. Replicate samples were incubated in ambient air (~0.04% CO2), 0.1% CO2, 1.0% CO2 and 2.0% CO2 concentrations. Three incubation experiments were carried out at SIPEX stations 4 (expt 1) 7 (expt 3) and 8 (expt 4). Growth rate calcualtions followed a standard exponential growth model, i.e Bf = Bi x e(rt) Where Bf equals final biomass, Bi equals initial biomass, r = growth rate and t = time (in days).

From the abstract of one of the papers: Phytoplankton biomass and speciation were monitored at an inshore site near Davis Station, East Antarctica during three consecutive summer seasons (December-February, 1992-5). Four distinct phytoplankton assemblages were identified in which the dominant species were: Phaeocystis sp., an undescribed Cryptomonas species, Thalassiosira dichotomica, and a mixed assemblage containing Fragilariopsis spp. and Nitzschia spp. Little interannual consistency was found in either the timing of the appearance or disappearance of the various assemblages. Similarly, the seasonal trends in biomass varied dramatically from year to year. Variations in the phytoplankton community can be ascribed, to some extent, to the random variation in a number of factors, including the date of fast ice break out, water column stratification, temperature and salinity, zooplankton grazing and strong winds. Periods of strong wind result in the introduction of offshore or deeper water masses into the shallow inshore environment, where the physical and chemical conditions allow blooms to develop. A number of the papers listed in the reference section are available as pdf's in the download section.

The distribution and abundance of ice-associated copepods in the fast ice of the Australian Antarctic Territory were investigated over a distance of approximately 650 km between October and December 1995. The six sites where collections were made were: offshore from Mawson station, Larsemann Hills (including Nella Bay), Rauer Islands (ice edge near Filla Is), O'Gorman Rocks and Bluff Island near Davis Station, and Murphy Rocks in the northern Vestfold Hills. Ice cores were obtained using SIPRE ice augers. Five to ten cores were collected along transects several km in length. Thickness of sea ice and snow cover were measured at each sampling site. Chlorophyll a concentrations were determined for each core. Copepods were isolated from the melted core water and identified and counted. Zooplankton tows were also made at each site where cores were collected. Nine species of copepods were identified from the cores. However, of these, only three were recorded regularly: Paralabidocera antarctica, Drescheriella glacialis and Stephos longipes. The abundance of copepods ranged between 0 and 147/L. The highest densities were recorded at the Larsemann Hills and the lowest at Murphy Rocks. Within the cores, the highest abundances were found in the bottom 10 cm of ice, irrespective of the species. Chlorophyll a concentrations ranged between 0.9 and 373 mg/m3. Data available: excel files containing sampling dates, sampling sites and abundances (number per L) of three dominant sea ice copepods, Paralabidocera antarctica, Drescheriella glacialis, Stephos longipes. Data are presented for developmental stages (nauplii, copepodites and adults) where available. Totals are also provided. Vertical distribution in some cores is also provided. Chlorophyll a concentrations (ug/L) provided for most sites. Detailed information about each of the spreadsheets is provided below: The chlorophyll spreadsheet shows chlorophyll concentrations for 5 sites in the AAT. The column headings are: core - reference number of the core collected subsection - depth in the core in cm volume - vol of melted core water volume added - 1 L of filtered seawater for melting % original - amount of total that core water represents (i.e. minus the 1L added) aliquot - volume subsampled for chlorophyll analysis acetone - amount added (mL) for extraction 750, 664, 647, 630 - wavelengths where absorbance was measured chloro a - amount of chlorophyll a in the sample ug/L - chloro a expressed as a concentration The spatial spreadsheet shows species abundances of three copepods at 4 sites N1 to NVI - nauplius stage 1 to 6 of a species CI to CVI - copepodite stage 1 to 6 of a species F or M - female or male of copepodite stage 5 or 6 1,1 etc - cores 1 and 2 from site 1 within a major location (e.g. 2 cores close together in the Larsemann Hills) The temporal spreadsheet shows abundances over time at 2 sites (O'Gorman Rocks, Bluff Is) near Davis and two species (Paralabidocera antarctica and Drescheriella glacialis) on several sampling dates N1 to N3 - total nauplii in each of three cores (i.e. not separated into stages as above) C1 to C3 - total copepodites A1 to A3 - total adults Then at the bottom are the means of each three cores.

Field-based sampling: As part of Australian Antarctic Science project # 4298, a total number of 44 sea ice sites were sampled for bio-optical measurements along 4 transects on land-fast sea ice off Davis Station (Antarctica) during November – December 2015. Measurements included simultaneous hyperspectral down-welling (ice surface) irradiance (triplicate) and under-ice radiance (triplicate) measurements (320 – 900 nm, 3.3 nm resolution) with a TriOS ACC and Trios ARC radiometer, respectively. The radiance measurements were conducted with the TriOS ARC radiometer mounted onto an L-shaped arm (for deployment details see Melbourne-Thomas et al. 2015). Subsequently, snow thickness was measured with a ruler and an ice core was collected directly above the radiometer location. Sea-ice freeboard (tape measure) and ice thickness (ice core length) were also recorded. Ice cores (9 cm internal diameter) were cut into sections, and these were melted in the dark at +4 degrees C, filtered onto GFF filters and then used to measure ice algal pigment content (using High Performance Liquid Chromatography (HPLC) and spectral ice algal absorption coefficients (ap, ad, aph) for entire vertical profiles or for the lower-most 0.1 m of ice cores. The location of the sampling grid had its origin (x=0, y=0) at GPS position: -68.568904, 77.945439. Transects (128m – 512 m in length) started at x=60, x=70, x=80 and x=90 m and were sampled at y-positions of 0m, 0.5m, 1m, 2m, 4m, 8m, 16m, 32m, 64m, 128m, (256m, and 512m) on 19/11/2015, 23/11/2015, 29/11/2015 and 02/12/2015, respectively. Analysis of ice algal chlorophyll a concentration: For pigment analysis, 0.25 to 1.0 litres of melted ice core subsamples were passed through 25 mm diameter glass-fiber (Whatman GF/F) filters. The filters were then frozen and stored below −80 degrees C prior to analysis using HPLC. Samples were extracted over 15 to 18 hours in acetone before analysis by HPLC using a modified C8 column and binary gradient system with an elevated column temperature [Van Heukelem and Thomas, 2001]. Pigments were identified by retention time and absorption spectra from a photo-diode array (PDA) detector, and concentrations were determined from commercial and international standards (Sigma; DHI, Denmark). Analysis of particulate (algal and non-algal) absorption: The optical density (OD) spectra of the particulate material on these filters (see section above) were measured over the 350 to 750 nm spectral range in 0.9 nm increments, using a Cintra 404 UV/VIS dual-beam spectrophotometer equipped with an integrating sphere. The pigments on the sample filter were then extracted using the method of Kishino et al. [1985]'s method to determine the OD of the non-algal particles in a second scan. The OD due to ice algae was then obtained by calculating the difference between the optical density of the total particulate and non-algal fractions. The OD measurements were converted to absorption spectra using blank filter measurements, and by first normalizing the scans to zero at 750 nm and then correcting for the path length amplification using the coefficients of Mitchell [1990]. A detailed description of the method is given in Clementson et al. [2001], and followed SeaWiFS protocols [Muller et al., 2003]. An exponential function was fitted to all spectra of non-algal particulate material: ad(λ) = ad(350 nm) exp[−S(λ − 350 nm)] + b, (1) where ad(λ) is the residual absorption coefficient over the wavelength (λ) range 350 to 750 nm of the particles after methanol extraction, also referred to as absorption of detritus [m−1] although this may include absorption of non-extractable pigments and heterotrophic protists. A non-linear least-squares technique was used to fit Equation 1 to the untransformed data, where S and b are empirically-determined constants. The inclusion of an offset b allows for any baseline correction. In some samples, pigment extraction was incomplete, leaving small residual peaks in

The productivity of Antarctic waters may be controlled by the amount of iron. Experiments have shown that this is probably the case for phytoplankton but as yet we do not know if iron limits the growth of sea ice algae. This study will assess whether iron limits sea ice algae production and will conduct experiments to work out how these algae use iron. Measurements have been made to determine whether sea ice algae are limited by Fe. Sea ice samples were taken and this spreadsheet refers to those ice cores Columns A-G are self explanatory Column G is the depth in the ice core from the bottom Column H is the chlorophyll concentration in mg Chl m-2 Column I is the phaeophytin concentration in mg m-2 J is the total amount of protein in the sample ng m-2 K is the total amount of the protein flavodoxin ng m-2 L is the total amount of ferrodoxin ng m-2 These last two enable the Fe limitation status to calculated (not completed).

Sea ice covers up to 20 million km2 of the Southern Ocean. When present it supports a vigorous ecosystem that provides energy and food for all other marine organisms. Using the latest micro sensor technology, we are examining the factors that effect the productivity of this vital link in the Antarctic marine food web. New data were added to this metadata record in January 2011. These data included FRRF data collected on the CEAMARC, CASO, SIPEX and SAZ-SENSE voyages. A word document in the download file provides details about these datasets, plus those collected on Voyage 1 2009-2010, and voyage 2 2008-2009. The download file also contains a folder labelled "Older data". This data is described below: An explanation of the excel spreadsheet in the download file is as follows: Worksheet 1 is the chlorophyll data Worksheet 3 is the location data CHLOROPHYLL DATA Column A is sample name, the first letter refers to the location data in worksheet 3, the second to the ice flow number and the third to the replicate number Section refers to depth in ice core, measured from the bottom Ignore C Column D is the total volume of melted ice Column E is the volume of D that was filtered Column G is the Fluorometer reading before the addition dilute HCl Column H is the fluorometer reading after the addition of acid Column I is the calculation of chlorophyl concentration in the sample Column K is areal chlorophyll estimate Column L is the mean for the core Column N is the mean for the site Column O is the standard deviation LOCATION DATA Lat, longs and times of each sampling. The first set (B-G) refers to the time sampling started, the second (H-M) to when it finished Project objectives: - Determine the net photosynthesis and primary productivity of the phytoplankton and major sea ice algal communities of the Eastern Antarctic Sea Ice Zone (SIZ). Estimate seasonal and annual algal production and inter annual variability Obtain data on biomass distribution and variability to establish regional relationships between ice thickness, snow cover, and biomass Determine the effects of a) Light b) Nutrients (principally nitrate and iron) c) Temperature on photosynthesis and primary production Determine whether the biomass and productivity of the phytoplankton and sea ice algae in winter and spring limits the biomass or growth of krill Estimate the effects of climate change on Sea ice Zone primary production Taken from the 2008-2009 Progress Report: Progress against objectives: This project used V2, a spring voyage, to collect underway data to determine surface biomass and primary production. Biomass samples (chlorophyll a) were taken every 3 hours. Productivity estimates by PAM were also made every 3 hours. Productivity measurements by FRRF were made every 1 minute. Nutrient samples were taken at the same time as the biomass samples. Analysis of the biomass samples is complete. Preliminary analysis of the productivity data has commenced. This data is being used for a Masters project (Rob Johnson, IASOS). An iron addition experiment accompanied this monitoring. Iron was added to samples taken every 3 hours and the change in photosynthesis (maximum quantum yield) measured with a PAM. The rate of recovery from iron stress was the principal focus. Most of this data has been submitted as metadata. Using The PAM and FRRF simultaneously also enabled a comparison to be made between these different ways of measuring photosynthesis. Progress was also made on the analysis of FRRF productivity and biomass data collected over several years on the L'Astrolabe transect. Analysis involves quantitative manipulation of FRRF data and correlation with chlorophyll, nutrients, temperature and other biological parameters. A publication arising from this work will be submitted this year. Taken from the 2009-2010 Progress Report: Progress against objectives: We participated in V1 of the Aurora Australis, spring 2009. The objective of this project was to measure surface primary