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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.
The collection aims to showcase the range of Southern Ocean diatom species found in the major hydrological provinces of the Australian Sector of the Southern Ocean along the 140 degrees E. The collection includes specimens collected in the Sub-Antarctic Zone (SAZ), Polar Frontal Zone (PFZ) and Antarctic Zone (AZ). Samples were collected with McLane Parflux time series sediment traps placed at several depths in the SAZ (47 degrees S site), PFZ (54 degrees S site) and AZ and (61 degrees S site) during the decade 1997-2007. The shortest sampling intervals were eight days and corresponded with the austral summer and autumn, whereas the longest interval was 60 days and corresponded with austral winter. Split aliquots were obtained for taxonomic analysis via scanning electron microscopy (SEM). For improved taxonomic imaging, samples were treated with hydrochloric acid and hydrogen peroxide to remove carbonates and organic matter, respectively. A micropipette was used to transfer the suspension of selected samples to a round-glass cover slip following standard decantation method outlined by Barcena and Abrantes (1998). Samples were air-dried and coated with gold for SEM analysis. SEM analysis was carried out using a JEOL 6480LV scanning electron microscope. Taxonomy Diatoms include all algae from the Class Bacillariophyceae and follow the standardised taxonomy of World Register of Marine Species (WoRMS). Order Asterolamprales Family Asterolampraceae Asteromphalus hookeri Ehrenberg Asteromphalus hyalinus Karsten Order Achnanthales Family Cocconeidaceae Cocconeis sp. Order Bacillariales Family Bacillariaceae Fragilariopsis curta (Van Heurck) Hustedt Fragilariopsis cylindrus (Grunow) Krieger Fragilariopsis kerguelensis (O'Meara) Hustedt Fragilariopsis pseudonana (Hasle) Hasle Fragilariopsis rhombica (O'Meara) Hustedt Fragilariopsis separanda Hustedt Nitzschia bicapitata Cleve Nitzschia kolaczeckii Grunow Nitzschia sicula (Castracane) Husted var. bicuneata (Grunow) Hasle Nitzschia sicula (Castracane) Husted var. rostrata Hustedt Pseudo-nitzschia heimii Manguin Pseudo-nitzschia lineola (Cleve) Hasle Pseudo-nitzschia turgiduloides Hasle Order Chaetocerotanae incertae sedis Family Chaetoceraceae Chaetoceros aequatorialis var. antarcticus Cleve Chaetoceros atlanticus Cleve Chaetoceros dichaeta Ehrenberg Chaetoceros peruvianus Brightwell Chaetoceros sp. Order Corethrales Family Corethraceae Corethron spp. Order Coscinodiscales Family Coscinodiscaceae Stellarima stellaris (Roper) Hasle et Sims Family Hemidiscaceae Actinocyclus sp. Azpeitia tabularis (Grunow) Fryxell et Sims Hemidiscus cuneiformis Wallich Roperia tesselata (Roper) Grunow Order Hemiaulales Family Hemiaulaceae Eucampia antarctica (Castracane) Mangin Order Naviculales Family Plagiotropidaceae Tropidoneis group Family Naviculaceae Navicula directa (Smith) Ralfs Family Pleurosigmataceae Pleurosigma sp. Order Rhizosoleniales Family Rhizosoleniaceae Dactyliosolen antarcticus Castracane Rhizosolenia antennata f. semispina Sundstrom Rhizosolenia antennata (Ehrenberg) Brown f. antennata Rhizosolenia cf. costata Gersonde Rhizosolenia polydactyla Castracane f. polydactyla Rhizosolenia simplex Karsten Proboscia alata (Brightwell) Sundstrom Proboscia inermis (Castracane) Jordan Ligowski Order Thalassiosirales Family Thalassiosiraceae Porosira pseudodenticulata (Hustedt) Jouse Thalassiosira ferelineata Hasle et Fryxell Thalassiosira gracilis (Karsten) Hustedt Thalassiosira lentiginosa (Janisch) Fryxell Thalassiosira oestrupii (Ostenfeld) Hasle var. oestrupii Fryxell et Hasle Thalassiosira oliveriana (O'Meara) Makarova et Nikolaev Thalassiosira tumida (Janisch) Hasle Order Thalassionematales Family Thalassionemataceae Thalassionema nitzschioides var. lanceolatum Grunow Thalassiothrix antarctica Schimper ex Karsten Data available: 73 SEM images of the most abundant diatom species found at the three sampling sites. Samples were collected by several sediment traps placed at different depths in the Subantarctic Zone (47 degrees S site), Polar Frontal Zone (54 degrees S site) and Antarctic Zone (61 degrees S site) during the decade 1997-2007. The collection site and date for each species image can be found in Table 1 (see the word document in the download file).
Diatom and biogenic particle fluxes were investigated over a one-year period (2001-02) at the southern Antarctic Zone in the Australian Sector of the Southern Ocean. Two vertically moored sediment traps were deployed at 60 degrees 44.43'S 139 degrees 53.97' E at 2000 and 3800 m below sea-level. In these data sets we present the results on the temporal and vertical variability of total diatom flux, species composition and biogenic particle fluxes during a year. A detailed description of the field experiment, sample processing and counting methods can be found in Rigual-Hernandez et al. (2015). Total fluxes of particulates at both traps were highly seasonal, with maxima registered during the austral summer (up to 1151 mg m-2 d-1 at 2000 m and 1157 mg m-2 d-1 at 3700 m) and almost negligible fluxes during winter (up to 42 mg m-2 d-1 at 2000 m and below detection limits at 3700 m). Particulate fluxes were slightly higher at 2000 m than at 3700 m (deployment average = 261 and 216 mg m-2 d-1, respectively). Biogenic silica (SiO2) was the dominant bulk component, regardless of the sampling period or depth (deployment average = 76% at 2000 and 78% at 3700 m). Highest relative contribution of opal was registered from the end of summer through early-autumn at both depths. Secondary contributors were carbonate (CaCO3) (7% at 2000 m and 9% at 3700 m) and particulate organic carbon (POC) (1.4% at 2000 m and 1.2% at 3700 m). The relative concentration of carbonate and POC was at its highest in austral spring and summer. Diatom frustules from 61 taxa were identified over the entire experiment. The dominant species of the diatom assemblage was Fragilariopsis kerguelensis with a mean flux between 53 x 106 and 60 x 106 valves m-2 day-1 at 2000 m (annualized mean and deployment average, respectively). Secondary contributors to the diatom assemblage at 2000 and 3700 m were Thalassiosira lentiginosa, Thalassiosira gracilis var. gracilis, Fragilariopsis separanda, Fragilariopsis pseudonana, Fragilariopsis rhombica, Fragilariopsis curta and Azpeitia tabularis. Data available: two excel files containing sampling dates and depths, raw counts, relative abundance and fluxes (valves m-2 d-1) of the diatom species, and biogenic particle fluxes found at 2000 m and 3700 m depth. Each file contains four spreadsheets: raw diatom valve counts, relative abundance of diatom species and valve flux of diatom species and biogenic particle composition and fluxes. Detailed information of the column headings is provided below. Cup - Cup (=sample) number Depth - vertical location of the sediment trap in meters below the surface Mid-point date - Mid date of the sampling interval Length (days) - number of days the cup was open Girdle bands instead of valves were counted for Dactyliosolen antarcticus Castracane. Therefore, D. antarcticus girdles counts were not included in relative abundance calculations
Diatom and biogenic particle fluxes were investigated over a two-year and six-year periods at the Subantarctic and Polar Frontal Zones, respectively, in the Australian Sector of the Southern Ocean. Both sites were located along ~ 140 degrees E: station 47 degrees S was set on the abyssal plain of the central SAZ whereas station 54 degrees S was placed on a bathymetric high of the Southeast Indian Ridge in the PFZ. The data sets contain diatom species and biogeochemical flux data measured at 1000 m at the 47 degrees S site between 1999-2001 and at 800 m at the 54 degrees S site during six selected years between 1997-2007. All traps were MacLane Parflux sediment traps: conical in shape with a 0.5 m2 opening area and equipped with a carousel of 13 or 21 sampling cups. Shortest intervals corresponded with the austral summer and autumn ranging typically between 4.25 and 10 days, whereas the longest intervals were 60 days and corresponded with winter. Total fluxes of particulates at both traps were highly seasonal, with maxima registered during the austral spring and summer and very low fluxes during winter. Seasonality was more pronounced in the 54 degrees S site. Biogenic silica (SiO2) was the dominant bulk component in the PFZ while carbonate (CaCO3) dominated the particle fluxes at the SAZ. POC export was relatively similar between sites despite significant differences in the total diatom flux. Diatom frustules from 94 taxa were identified over the entire experiment. The dominant species of the diatom assemblage was Fragilariopsis kerguelensis at both sites, representing 43% and 59% of the integrated diatom assemblage at the 47 degrees S and 54 degrees S sites, respectively. Secondary contributors to the diatom assemblage at the 47 degrees S were Azpeitia tabularis, Thalassiosira sp. 1, Nitzschia bicapitata, resting spores of Chaetoceros spp., Thalassiosira oestrupii var. oestrupii, Hemidiscus cuneiformis and Roperia tesselata. Subordinate contributions to the diatom assemblage correspond to Pseudo-nitzschia lineola cf. lineola, Pseudo-nitzschia heimii, Thalassiosira gracilis group and Fragilariopsis pseudonana, Fragilariopsis rhombica and Thalassiosira lentiginosa. Data available: two excel files containing sampling dates and depths, raw counts, relative abundance and fluxes (valves m-2 d-1) of the diatom species, and biogenic particle fluxes measured at 1000 m and 800 m depth at the 47 degrees S and 54 degrees S sites, respectively. Each file contains four spreadsheets: raw diatom valve counts, relative abundance of diatom species and valve flux of diatom species and biogenic particle composition and fluxes. Detailed information of the column headings is provided below. Cup - Cup (=sample) number Depth - vertical location of the sediment trap in meters below the surface Mid-point date - Mid date of the sampling interval Length (days) - number of days the cup was open Girdle bands instead of valves were counted for Dactyliosolen antarcticus Castracane. Therefore, D. antarcticus girdles counts were not included in relative abundance calculations. Dates of data collection: 47 degrees S site: July 1999 - October 2001 (two-year record) 54 degrees S site: September 1997 - February 1998, July 1999 - August 2000, November 2002 - October 2004 and December 2005 - October 2007 (six-year record).