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Experimental Set-up: An unreplicated, 6-level, dose-response experiment was conducted on a natural microbial community over a range of pCO2 levels (343, 506, 634, 953, 1140 and 1641 micro atm). Seawater was collected on the 19th November 2014 approximately 1 km offshore from Davis Station, Antarctica (68 degrees 35' S, 77 degrees 58' E) from an area of ice-free water amongst broken fast-ice. The seawater was collected using a thoroughly rinsed 720L Bambi bucket slung beneath a helicopter and transferred into a 7000 L polypropalene reservoir tank. Six 650 L polyethene tanks (minicosms), located in a temperature-controlled shipping container, were immediately filled via teflon lined house via gravity with an in-line 200 micron Arkal filter to exclude metazooplankton. The minicosms were simultaneously filled to ensure they contained the same starting community. The ambient water temperature at time of collection was -1.0 degrees C and the minicosms were maintained at a temperature of 0 degrees C plus or minus 0.5 degrees C. At the centre of each minicosm there was an auger shielded for much of its length by a tube of polythene. This auger was rotated at 15 rpm to gently mix the contents of the tanks. Each minicosm tank was covered with an acrylic air-tight lid to prevent pCO2 off-gasing outside of the minicosm headspace. The minicosm experiment was conducted between the 19th November and the 7th December 2014. Initially, the contents of the tanks were given a day to equibrate to the minicosms. This was followed by a five day acclimation period to increasing pCO2 at low light (0.8 plus or minus 0.2 micro mol m-1 s-1), allowing cell physiology to acclimated to the pCO2 increase (days 1-5). During this period the pCO2 was progressively adjusted over five days to the target level for each tank (343 - 1641 micro atm). Thereafter pCO2 was adjusted daily to maintain the pCO2 level in each treatment (see carbonate chemistry section below). Following acclimation to the various pCO2 treatments light was progressively adjusted to 89 plus or minus 16 micro mol m-2 s-1 at a 19 h light:5 h dark cycle. The community was incubated and allowed to grow for a further 10 days (days 8-18) with target pCO2 adjusted back to target each day (see carbonate chemistry section below). For a more detailed description of minicosm set-up, lighting and carbonate chemistry see; Davidson, A. T., McKinlay, J., Westwood, K., Thomson, P. G., van den Enden, R., de Salas, M., Wright, S., Johnson, R., and Berry, K.:Enhanced CO2 concentrations change the structure of Antarctic marine microbial communities, Mar. Ecol. Prog. Ser., 552, 93-113, 2016. Deppeler, S. L., Petrou, K., Westwood, K., Pearce, I., Pascoe, P., Schulz, K. G., and Davidson, A. T.: Ocean acidification effects on productivity in a coastal Antarctic marine microbial community, Biogeosciences, 2017. Light microscopy sampling and analysis: Samples from each minicosm were collected on days 1, 3, 5, 8, 10, 12, 14, 16 and 18 for microscopic analysis to determine protistan identity and abundance. Approximately 960 mL were collected from each tank, on each day. Samples were fixed with 20 40 mL of Lugol's iodine and allowed to sediment out at 4 degrees C for greater than or equal to 4 days. Once cells had settled the supernatant was gently aspirated till approximately 200 mL remained. This was transferred to a 250 mL measuring cylinder, again allowed to settle (as above), and the supernatant gently aspirated. The remaining 20 mL. This final 20 mL was transferred into a 30 mL amber glass bottle. All samples were stored and transported at 4 degrees C to the Australian Antarctic Division, Hobart, Australia for analysis. Lugols-fixed and sedimented samples were analysed by light microscopy between July 2015 and February 2017. Between 2 to 10 mL (depending on cell-density) of lugols-concentrated samples was placed into a 10 mL Utermohl cylinder (Hydro-Bios, Keil) and the cells allowed to settle overnight. Due to the large variation in size and taxa, a stratified counting procedure was employed to ensure both accurate identification of small cells and representative counts of larger cells. All cells greater than 20 microns were identified and counted at 20x magnification; those less than 20 microns at 40x magnification. For larger cells (greater than 20 microns), 20 randomly chosen fields of view (FOV) at 3.66 x 106 microns2 counted to gain an average cells per L. For smaller cells (less than 20 microns), 20 randomly chosen FOVs at 2.51 x 105 microns2 were counted. Counts were conducted on an Olympus IX 81 microscope with Nomarski interference optics. Identifications were determined using (Scott and Marchant, 2005) and FESEM images. Autotrophic protists were distinguished from heterotrophs via the presence of chloroplasts and based on their taxonomic identity. Electron microscopy sampling and analysis: A further 1 L was taken on days 0, 6, 13 and 18 for analysis by Field Emission Scanning Electron Microscope (FESEM). 25 These samples were concentrated to 5 mL by filtration over a 0.8 micron polycarbonate filter. Cells were resuspended, the concentrate transferred to a glass vial and fixed to a final concentration of 1% EM-grade gluteraldehyde (ProSciTech Pty Ltd). All samples were stored and transported at 4 degrees C to the Australian Antarctic Division, Hobart, Australia for analysis. Gluteraldehyde-fixed samples were prepared for FESEM imaging using a modified polylysine technique (Marchant and Thomas, 30 1983). In brief, a few drops of gluteraldehyde-fixed sample were placed on polylysine coated cover slips and post-fixed with OsO4 (4%) vapour for 30 min, allowing cells to settle onto the coverslips. The coverslips were then rinsed in distilled water and dehydrated through a graded ethanol series ending with emersion in 100% dry acetone before being critically point dried in a Tousimis Autosamdri-815 Critical Point Drier. The coverslips were mounted onto 12.5 mm diameter aluminium stubs and sputter-coated with 7 nm of platinum/palladium in a Cressington 208HRD coater. Imaging of stubs was conducted by JEOL JSM6701F FESEM and protists identified using (Scott and Marchant, 2005). All units are in cells per L estimates from individual field of view counts (FOV) Protistan taxa and functional group descriptions and abbreviations: Autotrophic Dinoflagellate (AD) - including Gymnodinium sp., Heterocapsa and other unidentified autotrophic dinoflagellates Bicosta antennigera (Ba) Chaetoceros (Cha) - mainly Chaetoceros castracanei and Chaetoceros tortissimus but also other Chaetoceros present including C. aequatorialis var antarcticus, C. cf. criophilus, C. curvisetus, C. dichaeta, C. flexuosus, C. neogracilis, C. simplex Choanoflagellates (except Bicosta) (Cho) - mainly Diaphanoeca multiannulata but also Parvicorbicula circularis and Parvicorbicula socialis present in low numbers Ciliates (Cil) - mostly cf. Strombidium but other ciliates also present Discoid Centric Diatoms greater than 40 microns (DC.l) - unidentified centrics of the genera Thalassiosira, Landeria, Stellarima or similar Discoid Centric Diatoms 20 to 40 microns (DC.m) - unidentified centrics of the genera Thalassiosira, Landeria, Stellarima or similar Discoid Centric Diatoms less than 20 microns (DC.s) - unidentified centrics of the genera Thalassiosira Euglenoid (Eu) - unidentified Fragilariopsis greater than 20 microns (F.l) - mainly Fragilariopsis cylindrus, some Fragilariopsis kerguelensis and potentially some Fragilariopsis curta present in very low numbers Fragilariopsis less than 20 microns (F.s) - mainly Fragilariopsis cylindrus, and potentially some Fragilariopsis curta present in very low numbers Heterotrophic Dinoflagellates (HD) - including Gyrodinium glaciale, Gyrodinium lachryma, other Gyrodinium sp., Protoperidinium cf. antarcticum and other unidentified heterotrophic dinoflagellates Landeria annulata (La) Other Centric Diatoms (OC) - Corethronb pennatum, Dactyliosolen tenuijuntus, Eucampia antarctica var recta, Rhizosolenia imbricata and other Rhizosolenia sp. Odontella (Od) - Odontella weissflogii and Odontella litigiosa Other Flagellates (OF) - Dictyocha speculum, Chrysochromulina sp., unknown haptophyte, Phaeocystis antarctica (flagellate and gamete forms), Mantoniella sp., Pryaminmonas gelidicola, Triparma columaceae, Triparma laevis subsp ramispina, Geminigera sp., Bodo sp., Leuocryptos sp., Polytoma sp., cf. Protaspis, Telonema antarctica, Thaumatomastix sp. and other unidentified nano- and picoplankton Other Pennate Diatoms (OP) - Entomonei kjellmanii var kjellmanii, Navicula gelida var parvula, Nitzschia longissima, other Nitzschia sp., Plagiotropus gaussi, Pseudonitzschia prolongatoides, Synedropsis sp. Phaeocystis antarctica (Pa) - colonial form only Proboscia truncata (Pro) Pseudonitzschia subcurvata (Ps) Pseudonitzschia turgiduloies (Pt) Stellarima microtrias (Sm) Thalassiosira antarctica (Ta) Thalassiosira ritscheri (Tr) *.se = standard error for mean cell per L estimate ie. Tr.se = standard error for the mean cells per L for Thalassiosira ritscheri based on individual FOV estimates as described in methods above. Davis Station Antarctica Experiment conducted between 19th November and 7th December 2014.
An unreplicated, six-level dose-response experiment was conducted using 650 L incubation tanks (minicosms) adjusted to fugacity of carbon dioxide (fCO2) from 343 to 11641 uatm. The minicosms were filled with near-shore water from Prydz Bay, East Antarctica and the protistan composition and abundance was determined by microscopy analysis of samples collected during the 18 day incubation. Abundant taxa with low variance were examined separately, but rare taxa with high variance were combined into functional groups (descriptions below). Cluster analyses and ordinations were performed on Bray-Curtis resemblance matrixes formed from square-root transformated abundance data. This transformation was assessed as appropriate for reducing the influence of abundance species, as judged from a one-to-one relationship between observed dissimilarities and ordination distances (ie. Shepard diagram, not shown). The Bray-Curtis metric was used as it is recommended for ecological data due to its treatment of joint absences (ie. these do not contribute towards similarity), and giving more weight to abundant taxa rather than rare taxa. The data days 1 to 8 and then days 8 to 18 were analysed separately to distinguish community structure in the acclimation period and in the exponential growth phase during the incubation period of the experiment. Hierarchical agglomerative cluster analyses, based on the Bray-Curtis resemblance matrix, was performed using group-average linkage. Significantly different clusters of samples were determined using SIMPROF (similarity profile permutations method) with an alpha value of 0.05 and based on 1000 permutations. An unconstrained ordination by non-metric multidimensional scaling (nMDS) was performed on the resemblance matrix with a primary (`weak') treatment of ties. This was repeated over 50 random starts to ensure a globally optimal solution according to . Clusters are displayed in the nMDS using colour. Weighted average of sample scores are shown in the nMDS to show the approximate contribution of each species to each sample. The assumption of a linear trend for predictors within the ordination was checked for each covariate, and in all instances was found to be justified. A constrained canonical analysis of principal coordinates (CAP) was conducted according to the Vegan protocol using the Bray-Curtis resemblance matrix. This analysis was used to assess the significance of the environmental covariates, or constraints, in determining the microbial community structure. Unlike the nMDS ordination, the CAP analysis uses the resemblance matrix to partition the total variance in the community composition into unconstrained and constrained components, with the latter comprising only the variation that can be attributed to the constraining variables, fCO2, Si, P and NOx. Random reassignment of sample resemblance was performed over 199 permutations to compute the pseudo-F statistic as a measure of significance of each environmental constraint in the structural change of the microbial community. A forward selection strategy was used to choose a minimum subset of significant constraints that still account for the majority of the variation within the microbial community. All analysis were performed using R v1.0.136 and the add-on package vegan v2.4-2. Protistan taxa and functional group descriptions and abbreviations: Autotrophic Dinoflagellate (AD) - including Gymnodinium sp., Heterocapsa and other unidentified autotrophic dinoflagellates Bicosta antennigera (Ba) Chaetoceros (Cha) - mainly Chaetoceros castracanei and Chaetoceros tortissimus but also other Chaetoceros present including C. aequatorialis var antarcticus, C. cf. criophilus, C. curvisetus, C. dichaeta, C. flexuosus, C. neogracilis, C. simplex Choanoflagellates (except Bicosta) (Cho) - mainly Diaphanoeca multiannulata but also Parvicorbicula circularis and Parvicorbicula socialis present in low numbers Ciliates (Cil) - mostly cf. Strombidium but other ciliates also present Discoid Centric Diatoms greater than 40 microns (DC.l) - unidentified centrics of the genera Thalassiosira, Landeria, Stellarima or similar Discoid Centric Diatoms 20 to 40 microns (DC.m) - unidentified centrics of the genera Thalassiosira, Landeria, Stellarima or similar Discoid Centric Diatoms less than 20 microns (DC.s) - unidentified centrics of the genera Thalassiosira Euglenoid (Eu) - unidentified Fragilariopsis greater than 20 microns (F.l) - mainly Fragilariopsis cylindrus, some Fragilariopsis kerguelensis and potentially some Fragilariopsis curta present in very low numbers Fragilariopsis less than 20 microns (F.s) - mainly Fragilariopsis cylindrus, and potentially some Fragilariopsis curta present in very low numbers Heterotrophic Dinoflagellates (HD) - including Gyrodinium glaciale, Gyrodinium lachryma, other Gyrodinium sp., Protoperidinium cf. antarcticum and other unidentified heterotrophic dinoflagellates Landeria annulata (La) Other Centric Diatoms (OC) - Corethronb pennatum, Dactyliosolen tenuijuntus, Eucampia antarctica var recta, Rhizosolenia imbricata and other Rhizosolenia sp. Odontella (Od) - Odontella weissflogii and Odontella litigiosa Other Flagellates (OF) - Dictyocha speculum, Chrysochromulina sp., unknown haptophyte, Phaeocystis antarctica (flagellate and gamete forms), Mantoniella sp., Pryaminmonas gelidicola, Triparma columaceae, Triparma laevis subsp ramispina, Geminigera sp., Bodo sp., Leuocryptos sp., Polytoma sp., cf. Protaspis, Telonema antarctica, Thaumatomastix sp. and other unidentified nano- and picoplankton Other Pennate Diatoms (OP) - Entomonei kjellmanii var kjellmanii, Navicula gelida var parvula, Nitzschia longissima, other Nitzschia sp., Plagiotropus gaussi, Pseudonitzschia prolongatoides, Synedropsis sp. Phaeocystis antarctica (Pa) - colonial form only Proboscia truncata (Pro) Pseudonitzschia subcurvata (Ps) Pseudonitzschia turgiduloies (Pt) Stellarima microtrias (Sm) Thalassiosira antarctica (Ta) Thalassiosira ritscheri (Tr) *.se = standard error for mean cell per L estimate ie. Tr.se = standard error for the mean cells per L for Thalassiosira ritscheri based on individual FOV estimates as described in methods above.
This data set was collected during an ocean acidification mesocosm experiment performed at Davis Station, Antarctica during the 2014/15 summer season. It includes: - description of methods for all data collection and analyses. - diatom cell volume - bulk silicification - species specific silicification via fluorescence microscopy - bulk community Fv/Fm on day 12 - single-cell PAM fluorometry data (maximum quantum yield of PSII: Fv/Fm) A natural community of Antarctic marine microbes from Prydz Bay, East Antarctica were exposed to a range of CO2 concentrations in 650 L minicosms to simulate possible future ocean conditions up to the year ~2200. Diatom silica precipitation rates were examined at CO2 concentrations between 343 to 1641 micro atm, measuring both the total diatom community response and that of individual species, to determine whether ocean acidification may influence future diatom ballast and therefore alter carbon and silica fluxes in the Southern Ocean. Described and analysed in: Antarctic diatom silicification diminishes under ocean acidification (submitted for review) Methods described in: Antarctic diatom silicification diminishes under ocean acidification (submitted for review) Location: Prydz bay, Davis Station, Antarctica (68 degrees 35'S, 77 degrees 58' E) Date: Summer 2014/2015 Worksheet descriptions: Bulk silicification - raw data Measured total and incorporated biogenic silica using spectrophotometer for all tanks on day 12 after 24 h incubation with PDMPO - raw data Bulk Fv/Fm - dark-adapted maximum quantum efficiency of PSII (Fv/Fm) on whole community - raw data Measured Fv/Fm of individual cells from 3 mesocosm tanks. Single-cell silicificiation, Fluorescence microscopy - raw data Measured autofluorescence and PDMPO fluorescence of individual diatoms from 6 mesocosm tanks Single-cell PAM, dark-adapted maximum quantum efficiency of PSII (Fv/Fm) - raw data Measured Fv/Fm of individual cells from 3 mesocosm tanks. Cell volume Calculated cell volume (um3) of 7 species from minicosm tanks 1 and 6 - raw data Abbreviations: Fv/Fm Maximum quantum yield of PSII PDMPO 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole Tant Thalassiosira antarctica DiscLg Large Discoid centric diatoms Stella Stellarima microtrias Chaeto Chaetoceros spp. Prob Proboscia truncata Pseu Pseudonitzschia turgiduloides FragLg Fragilariopsis cylindrus / curta Centric Large Discoid centric diatoms LargeThalassiosira Large Discoid centric diatoms