Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

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Journal of Experimental Marine Biology and Ecology

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The physiological response to increased temperature in over-wintering sea ice algaeand phytoplankton in McMurdo Sound, Antarctica and Tromsø Sound, Norway

Andrew Martin a,1, Andrew McMinn a,⁎, Mark Heath b, Else N. Hegseth c, Ken G. Ryan b

a Institute for Marine and Antarctic Studies, University of Tasmania, Hobart 7001, Australiab School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealandc Department of Arctic and Marine Biology, University of Tromsø, Tromsø N-9037, Norway

⁎ Corresponding author. Tel.: +61 3 6226 2980; fax:E-mail address: Andrew.McMinn@utas.edu.au (A. M

1 Current address: School of Biological Sciences, VictWellington 6140, New Zealand.

0022-0981/$ – see front matter © 2012 Published by Eldoi:10.1016/j.jembe.2012.06.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 March 2012Received in revised form 7 June 2012Accepted 8 June 2012Available online 1 July 2012

Keywords:PhytoplanktonSea iceSea ice algaeWinter Dark survival

The physiological response to increased temperature during dark exposure was examined in phytoplanktonand sea ice algae that had overwintered in McMurdo Sound, Antarctica and Tromsø Sound, Norway. Under icephytoplankton and sea ice algae from McMurdo Sound were incubated in the dark for 22 days and 23 daysrespectively at −2, 4 and 10 °C, while phytoplankton from Tromsø Sound were incubated for 35 days at 4,10 and 20 °C. A fluorescence approach was used to examine algal photophysiology (Fv/Fm, rETRmax and α)and changes in the concentration of chlorophyll a, while the spectrophotometric 2,4,6-tripyridyl-s-triazine(TPTZ) assay was used to quantify water-extractable carbohydrates. Prior to incubation, the photosynthetic pa-rameters documented relatively healthy overwintering communities for both polar regions. Elevated tempera-ture had a considerable impact on the dark survival of Arctic phytoplankton, and, to a lesser extent, Antarcticsea ice algae: photosynthetic health and stored monosaccharides declined during the incubation period, particu-larly at the warmest temperature regimes. In contrast, the concentration of chlorophyll a and polysaccharidesremained relatively constant. When Antarctic sea ice algae were subsequently exposed to low light (~20 μmolphotons m−2 s−1), significant photosynthetic recovery was only observed in cultures maintained at −2 °C. Amore robust response to increased temperature was observed in Antarctic phytoplankton and in general,variability between the −2 °C and 4 °C (Antarctic) and 4 °C and 10 °C (Arctic) temperature regimes wasminimal, which suggests that increasing temperature will not limit the ability of phytoplankton to survive thepolar winter and provide the inocula for bloom events.

© 2012 Published by Elsevier B.V.

1. Introduction

Productivity in polar oceans is characterised by large-scale spatialand temporal variability. This is due not only to the annual expansionand contraction of sea ice in the Arctic and Antarctic, but also to thedramatic seasonal change in light at high latitudes (Lizotte, 2001;Thomas and Dieckmann, 2002). Variation in incident solar radiationhas a profound effect on all aquatic photoautotrophs, but for up to fourmonths of the year polar marine plants need to survive almost totaldarkness (McMinn et al., 2010; Peck, 2005). While most microalgaehave some ability to acclimate to changes in light in the absence ofphotosynthesis, remarkably little is known about the physiologicaland biochemical mechanisms required for dark survival (Zhang et al.,1998). Some taxa produce cysts or other resting forms, some are facul-tatively heterotrophic, while others are able to adjust their metabolicrates or rely on energy storage products (Smayda and Mitchell-Innes,

+61 3 6226 2973.cMinn).oria University of Wellington,

sevier B.V.

1974; Zhang et al., 2003). Laybourn-Parry et al. (2005) documented ac-tive photosynthesis by phytoplankton during winter in Antarctic lakesand concluded that the phytoplankton generally functioned throughoutthe year by employing nutritional versatility. Facultative heterotrophyitself is often only turned on by long periods of darkness (Legrand etal., 1998) but does not appear to be a dark survival strategy adoptedby sea ice algae (Bunt and Lee, 1972;Horner andAlexander, 1972). Rest-ing diatom spores are relatively common in Arctic waters (68–80°N)including the fjords of northern Norway (Eilertsen et al., 1995; Hegsethet al., 1995), Svalbard (Wiktor and Wojciechowska, 2005), the BarentsSea (Rat'kova and Wassmann, 2002), and the Laptev Sea (Tuschling etal., 2000) but in the Antarctic are limited to a few species of diatoms(Chaetoceros spp.) and a number of dinoflagellates (Taylor and McMinn,2002).

Temperature also plays a major role in dark survival (Popels andHutchins, 2002; Smayda and Mitchell-Innes, 1974). Substantialwarming and changes in precipitation are projected by almost all cli-matemodels during the 21st century and average global seawater tem-peratures are expected to rise 2 °C by 2100 (King andHarangozo, 1998).Potential changes to the volume and extent of annual sea ice compli-cates accurate projections of temperature increases in polar oceans

58 A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

(Peck, 2005), but warmer waters are likely to increase metabolic rates.Two different physiological responses occur in different phytoplanktontaxa in response to exposure to darkness; those that are able to reducetheir metabolic rate (Jochem, 1999) and those that are able to utiliselarge quantities of energy storage products, e.g. lipids, starch etc. Respi-ration rates of phytoplankton are poorly known in any environment(Falkowski and Raven, 1997) and in the low chlorophyll SouthernOcean waters, respiration (as measured by O2 uptake) has been seento exceed O2 production,making thesewaters a net source of CO2 ratherthan a sink (Agusti et al., 2004). Elevated temperatures will increase therequirement for light energy, largely through higher respiration rates,and this will probably exacerbate the effect of light limitation. A com-mon and immediate response to a decline in irradiance is an increasein photosynthetic pigments (Falkowski and Raven, 1997). HoweverLüder et al. (2002), working with Antarctic seaweeds, also noticed astrong decrease in the photosynthetic parameters ETRmax and Fv/Fmafter 3 months of darkness, degradation of light harvesting antennaeafter 4 months and degradation of light harvesting complex 1 and/orreaction centres of PSII and/or PSI after 5 months. Pigment contentand photosynthetic performance were at a minimum at the end of6 months, but the plants were able to resume photosynthesis within24 h of the light returning. This photosynthetic response to temperatureappears to be essentially light dependent,with the response at saturatinglight levels being markedly different to that at sub-saturating levels(Ralph et al., 2005). Importantly, protein synthesis in the dark occursat the expense of the consumption of lowmolecularweight compoundsand carbohydrates (Hawes, 1990b; Smith et al., 1990), andmay result ina more rapid draw-down of energy reserves, which will consequentlyshorten the dark survival period of many overwintering taxa.

The overwintering biomass is typically quite low in polar regions(i.e. b0.1 mg chl am−2), but Antarctic sea ice microalgae, for example,contribute between 10-28% of the total primary production in ice-covered regions of the Southern Ocean (Arrigo et al., 1997; Legendreet al., 1992) and over 90% of this biogenic carbon is produced withinfirst-year ice and approximately 60% in the austral spring; November–December (Arrigo and Thomas, 2004). This biomass represents aseasonally-concentrated food source for krill and other under-icegrazers and may provide the inocula for phytoplankton bloom eventsat the receding ice edge in the austral summer (Giesenhagen et al.,1999; Kottmeier and Sullivan, 1988; Lizotte, 2001). In the Arctic, seaice microalgae contribute approximately 26% of the total primary pro-duction (Legendre et al., 1992), although fewer studies have been con-ducted with respect to pelagic and sea ice algal communities comparedto the Antarctic (McMinn andHegseth, 2007). In this study,we examinethe short-term (21–35 days) temperature response of overwinteringphytoplankton and sea ice algae from theAntarctic and Arctic incubatedin the dark. A fluorescence approach was used to examine the photo-physiological state of the algal cells during incubation along withchanges in the concentration of the photosynthetic pigment chlorophylla and the draw-down of stored carbohydrates.

2. Materials and methods

2.1. Antarctic phytoplankton experiment

Under ice phytoplankton were collected from McMurdo Sound inSeptember 2008 via the intake line at Scott Base (77° 51′ S, 166° 45′ E)seawater. Ambient irradiance at the time of collectionwas undetectable(i.e. b0.1 μmol photons m−2 s−1) due to attenuation of the overlyingsea ice and associated snow cover. A minimal flow rate was used to col-lect phytoplankton in the dark over a period of approximately 12 h andthe cells were concentrated using a plankton net with a nominal meshsize of 20 μm. Tominimise physiological stress during the collection pe-riod, cells were frequently transferred to a carboy containing 1 L of sea-water (0.22 μm-filtered seawater, 35‰, –1.8 °C). To remove anyzooplankton such as flagellates, ciliates or copepods, the 1 L stock

solution was passed through a100 μm plankton net. Twenty millitrealiquots of this stock solution were subsequently transferred into18 sterile scintillation vials and incubated in the dark for 22 days atone of three temperatures: −1.8, 4 or 10 °C. Incubation tempera-tures were controlled using the Scott Base flow-through aquarium(−1.8 °C), a standard fridge (4 °C), and a water bath equipped witha heater stirrer (10 °C). The photosynthetic health of the culturesand relative concentration of chlorophyll a was determined prior toincubation from replicate vials and then periodically during thecourse of the experiment. A 10 ml sample of the initial stock solutionwas fixed in gluteraldehyde (final concentration 4%) and relativespecies abundance was subsequently determined using an invertedmicroscope.

2.2. Antarctic sea ice algae experiment

Sea ice algae were collected in September 2009 from 1.9 m thickannual fast-ice at Cape Evans, Antarctica (77° 38′S, 166° 24′E). Thesea ice algae were extracted from the ice matrix approximately threeweeks after the first official sunrise. Based on under ice light data col-lected from this region of McMurdo Sound in 2007 and 2008 byMcMinn et al. (2010), it is possible that the cells used in this incubationexperiment may have been exposed to ~2 μmol photons m−2 s−1 forshort, but undefined, periods of time. While these cells are still consid-ered representative of the overwintering condition, there may never-theless have been a photosynthetic response prior to incubation.Approximately 70 holes (~1.6 m) were drilled in close proximity witha powered ice auger (Jiffy, USA) and cores (~0.3 m, 130 mm diameter)were extracted from the bottom of each hole using a Kovacs ice corer(Kovacs, USA). To minimise light shock, all operations were performedunder a black sheet and each core was transported in a black plastictube. Algal cells were obtained in the field by carefully scraping the bot-tom 10–20 mm of each core (under low light: b1 μmol photonsm−2 s−1) into one of three 20 L carboys (each containing 10 L of0.22 μm-filtered seawater, 35‰, –1.8 °C). The carboys were subse-quently stored in the −1.8 °C flow-through aquarium at Scott Base toallow the cells to settle. After approximately 12 h, the seawater wasdecanted and the sea ice algae were concentrated in approximately1 L of seawater. All storage andmanipulations at Scott Basewere carriedout in the dark.

Twenty millitre aliquots of the stock solution were transferredinto 108 sterile scintillation vials and incubated in the dark for23 days at one of three temperatures: −1.8, 4 or 10 °C. Temperaturewas controlled as described earlier. Triplicate culture vials were period-ically removed from each temperature regime to determine algal pho-tosynthetic health as well as the concentration of chlorophyll a andwater soluble carbohydrates. A 10 ml sample of the initial stock solutionwas fixed in gluteraldehyde (final concentration 4%) and relative spe-cies abundance was subsequently determined using an invertedmicro-scope. The ability of sea ice algae to resume photosynthesis after asustained period in the dark was examined on day 23 by exposing trip-licate vials fromeach treatment to 10 μmol photons m−2 s−1 over a pe-riod of 24 h while maintaining the respective incubation temperature.

2.3. Arctic phytoplankton experiment

Phytoplankton were collected from Tromsø Sound, Norway (69°40′ N, 18° 56′ E) via the intake line at the University of Tromsø, onJanuary 21st 2011, eight days before the first official sunrise of theyear. The temperature of the seawater was 4 °C. Cells were collectedat a minimal flow rate, in the dark, over a period of approximately72 h using a plankton net with a nominal mesh size of 10 μm. To re-move any zooplankton such as flagellates and ciliates, the final stocksolution (approximately 1 L) was passed through a 90 μm planktonnet. Twenty millitre aliquots were subsequently transferred into 108sterile scintillation vials and incubated in the dark for 35 days at one

Table 1Percentage species composition: Antarctic phytoplankton (McMurdo Sound Antarctica,2008), Antarctic sea-ice algae (McMurdo Sound Antarctica, 2009), and Arctic phyto-plankton (Tromsø Sound Norway, 2011).

Antarctic seawater Antarctic sea iceDiatoms DiatomsAchnanthes sp. 0.9 Fragilariopsis curta 58.2Actinoptychus sp. 0.2 Fragilariopsis sublinearis 4.4Asteromphalus sp. 0.1 Gyrosigma sp. 2.4Chaetoceros sp. 1 Nitzchia stellata 26.1Membraneis challengeri 0.6 Navicula director 2.6Corethron sp. 0.2 Navicula glaciei 3.4Coscinidiscus sp. 0.4 Pinnularia quadreata 2.8Entomeneis sp. 0.3Eucampia sp. 0.2 Arctic seawaterFragilariopsis curta 6.2Fragilariopsis cylindrus 0.7 DiatomsFragilariopsis sublinearis 42.1 Chaetoceros sp. spore 6.7Gyrosigma sp. 0.1 Cylindrotheca closterium 2Hantzschia sp. 1.2 Pleurosigma sp. 4.4Melosira sp. 3.4 Thalassiosira sp. 0.8Navicula glaciei 0.4 Unidentified pennate diatom 32.1Navicula sublineata 4.3Nitzschia stellata 0.5 OtherOdontella aurita 11.1 Ciliate 3.2Phaeocystis antarctica 3.1 Dinoflagellate cyst 3.6Phaeocystis sp. 18.2 Dinoflagellate sp. 13.5Staurosira sp. 0.1 Pterosperma sp. 0.4Thalassiosira antarctica 1.5 Unidentified cells 5.2Thalassiosira sp. 1.9 Unidentified spores 28.2OtherDinophysis rotundata 0.3Protoperidinium sp. 1.2Phaeocystis antarctica 3.1Phaeocystis sp. 18.2

59A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

of the following temperatures: 4, 10 or 20 °C. Incubation temperaturesweremaintained using a temperature controlled room at the Universityof Tromsø (4 °C), and standard laboratory culture cabinets (10 °C,20 °C). The photosynthetic health of the phytoplankton, along withchl a and carbohydrate concentration, was determined from triplicatevials at regular intervals (days 7, 13, 21, 28 and 35). At the end ofthe experiment cells were counted in 2 ml samples using an invertedmicroscope.

2.4. Chlorophyll fluorescence measurements

Photosynthetic activity was assessed using rapid-light curves(RLC) on a WaterPAM (Walz, Effeltrich, Germany). PAM fluorometresmeasure quantum yield (ΔF/F′m), photosynthetic efficiency (Fv/Fm)and electron transfer rate (ETR) of photosystem II (PSII) in photosyn-thesis and are a well-accepted method of investigating light adaptationin marine plants (McMinn et al., 2005; Ralph and Gademann, 2005;Schreiber, 2004). Themethod is based on the supply ofweak,modulatedlight pulses (the measuring light) that allows chlorophyll fluorescenceto be monitored without inducing photosynthesis. In the dark-adaptedstate, a minimum fluorescence (Fo) is determined with the measuringlight turned on. When the sample is exposed to actinic light, i.e. lightthat induces photosynthesis, a much higher fluorescence results. Thischaracteristic behaviour is referred to as the Kautsky curve (Schreiber,2004). Maximum fluorescence (Fm) is achieved by exposing the dark-adapted sample to a pulse of very intense light. Themaximumquantumyield of PSII is defined as:

φPSII ¼ Fm−F0ð Þ=Fm ¼ Fv=Fm:

The relative electron transport rate (rETR) can be calculated from

rETR ¼ φpII � E

where E is the irradiance measured in μmol photons m−2 s−1 (Gentyet al., 1989).

Rapid light curves consist of a series of 8 consecutive 10 second in-tervals of actinic light of increasing intensity, followed by a quantumyield measurement at the end of each light interval. Red light emittingdiodes provided the actinic light used in the RLC. Actinic light levelsused were 0, 22, 33, 46, 67, 101, 230, and 342 μmol photons m−2 s−1.rETR vs PAR data were fitted to the exponential function of Jasby andPlatt (1976) using a multiple non-linear regression;

P ¼ Pmax � e −αE=Pmaxð Þ:

Where P = photosynthesis (here equivalent to rETR), Pmax is themaximum photosynthetic rate, αPSII = photosynthetic efficiency ofthe light limiting region of the RLC, E—irradiance (Schreiber, 2004).

2.5. Chlorophyll a analysis

To determine chl a concentrations, the cells from triplicate vialswere filtered onto 47 mm GF/F filters and extracted in 10 ml of metha-nol for 12 h in the dark at 4 °C. The extracted chl a was subsequentlymeasured on a digital fluorometre (10 AU Turner Designs, USA) usingthe acidification protocol of Evans et al. (1987).

2.6. Carbohydrate analysis

Triplicate 20 ml samples were filtered under gentle vacuum pres-sure (b200 mmHg) onto pre-combusted (450 °C for 5 h) GF/F filters(Whatman) and stored at −20 °C until analysis. To extract the watersoluble carbohydrates, each filter was subsequently sealed in a glasstest tube with 10 ml of Milli-Q water and placed in a water bath for1 h at 80 °C (van Oijen et al., 2003). Following extraction, the tubes

were cooled and centrifuged for 5 min at 715 g. The concentration ofcarbohydrates was determined using the 2,4,6-tri pyridyl-s-triazine(TPTZ) spectroscopic method developed by Myklestad et al. (1997).Briefly, monosaccharides and polysaccharides were reduced by hydro-lysis of the glycosidic bonds (0.1 N HCL, 1 h, 150 °C) and subjected toan oxidation reaction at alkaline pH, during which Fe3+ is reduced toFe 2+. The Fe2+was thendetermined colorimetrically after condensationwith the cromogen TPTZ to produce a violet colour of the Fe(TPTZ)22+.Monosaccharides are defined by the pre-hydrolysis concentration,while polysaccharides are obtained by subtracting the pre- from thepost-hydrolysis concentration. D(+)-glucose was used as the standardand reactions were carried out either in the dark or with red light dueto light sensitivity of the analytical reagents (van Oijen, 2004).

2.7. Statistical analyses

One-way ANOVA and post-hoc Tukey tests (SPSS, Version 17.0)were used to determine whether the photosynthetic parameters, chla concentration, or carbohydrate pool varied during the course ofthe experiment at each temperature. A comparison among the tem-perature regimes was performed using the end point data for eachvariable and one-way ANOVA with post-hoc Tukey tests. For therecovery phase of the sea ice algal experiment, mixed-model repeatedmeasures ANOVA was used to compare the effects of incubation treat-ment and tim1e on the photosynthetic parameter Fv/Fm (SPSS, Version17.0).

3. Results

3.1. Antarctic phytoplankton

The overwintering phytoplankton community in McMurdo Soundwas relatively diverse, but essentially dominated by the species

60 A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

Fragilariopsis sublinearis, Phaeocystis sp. and Odontella aurita (Table 1).The maximum quantum yield (Fv/Fm) in the −2 °C treatment at thestart of the experiment was relatively high, 0.53±0.03; it increased sig-nificantly to 0.605±0.008 on day 16 (p=0.004) and then declined to0.576±0.012 on day 22 (Fig. 1A). In the +4 °C treatment, Fv/Fm haddecreased to 0.428±0.083 by day 22 which was significantly differentfrom the start value (p=0.038) and the measurement of 0.614±0.015 obtained on day 16 (p=0.003). Fv/Fm at 10 °C did not vary signif-icantly during the course of the experiment (p>0.05). Fv/Fm in the +4 °C treatment was significantly lower than the −2 °C treatment atthe end of the incubation period (p=0.028). Mean rETRmax increasedsignificantly from 5.238 to >10 across all three temperature regimeson day 16, but declined dramatically on day 22 (Fig. 1B). There wereno significant differences among treatments at the end of the experi-ment (p>0.05). No significant trends were observed with respect tothe photosynthetic parameter α, and there was no variation amongtreatments (p>0.05, Fig. 1C).

Following an initial chl a concentration of 3.45 mg chl a l−1at thestart of the experiment, a significant decline was observed within thefirst seven days at each temperature regime (pb0.05, Fig. 1D). Chl a de-creased in the+4 and+10 °C treatments to b2.0 mg chl a l−1 but only2.86 mg chl a l-1 in the −2 °C treatment. However, subsequent mea-surements on days 16 and 22 were not significantly different for anytemperature and there was no difference among the three temperatureregimes at the end of the experiment (p>0.05, Fig. 1D).

Fig. 1. Photosynthetic parameters and chlorophyll a concentration of overwintering culture(A) Fv/Fm, (B) rETRmax, (C) α, (D) Chl a. Data are means±1 SE. Figure legend: T0 ●; -2 °C

3.2. Antarctic sea ice algae

A relatively depauperate assemblage of microalgal taxa was presentwithin the annual sea ice inMcMurdo Sound at the end of winter, large-ly dominated by the species Fragilariopsis curta and Nitszchia stellata(Table 1) Rapid Light Curves (RLCs) were successfully generated foreach time point during the course of the experiment (T0, days 12, 18and 23). For Fv/Fm, there was a significant decline following the initialmeasurement of 0.55±0.011 across all three treatments (pb0.05)and further differences between time points for the −2 (day 12 vsday 18, p=0.004; day 18 vs day 23, p=0.038) and +10 (day 12 vsday 23, p=0.005; day 18 vs day 23, p=0.037) temperature regimes(Fig. 2A). Fv/Fm was significantly lower at the end of the experimentat +10 °C (0.197±0.048) compared to both the −2 °C (0.393±0.049, p=0.02) and +4 °C (0.380±0.084, p=0.027) treatments. Asimilar trendwas observed with respect to rETRmax, which initially de-clined from 13.99 to b6.0 across all temperatures by day 12 (pb0.01,Fig. 2B). Differences between subsequent time points were only signifi-cant for the +10 °C treatment where rETRmax was lower on day 23(2.45) compared to day 12 (4.8) (p=0.021). The comparison amongtreatments at the conclusion of the experiment was significant(p=0.022) with rETRmax at +10 °C (2.45) being lower than the +4 °C treatment (6.5) (p=0.022, Fig. 2B). There was no variation overtime for any temperature (p>0.05) or significant difference among treat-ments on day 23 for the photosynthetic parameter α (p>0.05, Fig. 2C).

s of Antarctic phytoplankton incubated in the dark for 22 days at −2 °C, 4 °C and 10 °C.○; 4 °C ▼; 10 °C △.

Fig. 2. Photosynthetic parameters, chlorophyll a concentration and water-extractable carbohydrate content of mixed overwintering Antarctic sea-ice algal cultures incubated in thedark for 23 days at−2 °C, 4 °C and 10 °C. (A) Fv/Fm, (B) rETRmax, (C) α, (D) Chl a (E) Monosaccharides, (F) Polysaccharides. Data are means±1 SE. Figure legend: T0 ●; −2 °C ○;4 °C ▼; 10 °C △.

61A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

The chl a concentration was determined for each temperature re-gime on days 6, 12 and 18. There was no significant variation duringthe course of the experiment for any temperature treatment (approx.2 mg chl a l−1) (p>0.05) and no significant difference among the in-cubation temperatures on day 18 (p>0.05, Fig. 2D).

The concentration of water extractable, algal-derived sugars wasexamined on days 6, 12, 18 and 23 (Fig. 2E, F). For the monosaccharidefraction of the carbohydrate pool, there was no significant variation foreither the −2 °C (~3.5 μmol Cl−1) or 4 °C (~3.9 μmol Cl−1) tempera-ture treatments during the course of the experiment (p>0.05). For

Fig. 3. Photosynthetic recovery (Fv/Fm) of mixed Antarctic sea-ice algal cultures exposedto 10 μmol photons m−2 s−1 for 24 h following a 23 day dark incubation at −2 °C, 4 °Cand 10 °C. Respective incubation temperatures were maintained during light exposure.Data are means±1 SE. Figure legend: −2 °C ○; 4 °C ▼; 10 °C△.

62 A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

the sea ice algae incubated at 10 °C, therewas some evidence to suggestthat the concentration of monosaccharides was beginning to decline byday 23 (0.47±0.17, p=0.066), however the concentration of mono-saccharides at the last time point did not differ significantly amongthe three temperature regimes (p>0.05). Polysaccharides weredetected throughout the experimental period and at each incubationtemperature. For the cultures maintained at 4 °C and 10 °C, there wasno difference with respect to time (p>0.05), however the concentra-tion of polysaccharides was more variable at −2 °C (p=0.003) andthere was a significantly higher concentration of polysaccharides onday 18 compared to the three other time points (day 1 vs day 18,p=0.012; day 6 vs day 18, p=0.002; day 18 vs day 23, p=0.008).

After the dark incubation, the sea ice algae were exposed to a shortrecovery phase. Here, the response to low light (~20 μmol pho-tons m−2 s−1) was examined at regular intervals over a period of24 h, during which time treatment replicates were maintained at theirrespective temperatures. The mixed-model repeated measures ANOVArevealed a significant effect of time (pb0.01) and temperature(pb0.01) on recovery rates of Fv/Fm (Fig. 3). For the−2 °C degree treat-ment, Fv/Fm increased from ~0.4 to ~0.5 within an hour of the sea icealgae being exposed to light, and maximum quantum yield had in-creased to ~0.6 by 24 h. The response of algae maintained at a tempera-ture of 4 °Cwas somewhat limited, and therewas no significant increasein Fv/Fm over 24 h for the 10 °C treatment. At the end of the recoveryperiod, Fv/Fm was significantly lower at 10 °C compared to both the−2 °C (pb0.01) and 4 °C (p=0.032) temperature regimes (Fig. 3).

3.3. Arctic phytoplankton

A significant fraction of the phytoplankton biomass in TromsøSound was comprised of unidentified spores, but these typically lackdistinct morphological characteristics that allow for unambiguousidentification under the microscope and germination studies areneeded to confirm the true identify of any single spore/cyst. Most ofthe diatom spores were probably Chaetoceros spp., but without fur-ther definitive germination experiments we are reluctant to specu-late. There was an approximately equal abundance of overwinteringvegetative cells, dominated by several pennate diatoms. BothPleurosigma spp. and Navicula sp. were present, but the most frequenttaxonwas a small unidentified raphoid diatom(Table 1). Prior to the startof the experiment, the photosynthetic parameter Fv/Fmwas 0.43±0.03,indicating a relatively healthy community. For the cultures incubated at4 °C, the maximum quantum yield increased slightly on day 7 (0.46±

0.03), and then began to decline. By day 35, Fv/Fm had decreased to0.18±0.02, which was significantly lower than the maximum quan-tum yield at the start of the experiment (p=b0.001, Fig. 4A). A sim-ilar trend was observed in the 10 °C treatment, with Fv/Fm decliningto 0.1±0.04 by day 35 (p=0.001, Fig. 4A). There was no significantdifference between the Fv/Fm values obtained for the cultures incu-bated at 4 °C and 10 °C at the end of the experiment (p=0.138). Inthe 20 °C temperature regime, Fv/Fm had declined to b0.1 by day13 of the incubation period and the cells appeared to be photosyn-thetically inactive by day 35 (Fig. 4A). Mean rETRmax remained rel-atively stable (~35) during the first 13 days for the culturesmaintained at 4 °C and 10 °C (Fig. 4B), but subsequently declined.Photosynthetic capacity at day 35 for the 4 °C and 10 °C treatmentswas 9.98±1.41 and 10.0±2.98 respectively. Although there wasno difference between the two temperature treatments(p=0.997), these values were both significantly lower than thepre-incubation value of 34.08±7.69 (p=0.004, p=0.002 respec-tively). For the phytoplankton maintained at 20 °C, mean rETRmaxdeclined rapidly to b10 on day 7 and b5 on day 28; both valueswere significantly lower than the pre-incubation value (p=0.001,Fig. 4B). By the end of the experiment, electron transport wasundetectable for phytoplankton incubated at this temperature. A de-cline in the photosynthetic parameter α was observed for each tem-perature regime, although the trend was somewhat variable during thecourse of the experiment (Fig. 4C). On day 35, α had declined from thepre-incubation value of 0.41±0.05 to 0.19±0.03 (p=0.015) in the4 °C treatment and 0.14±0.02 (p=b0.001) in the 10 °C treatment.There was no significant difference between the two treatments(p=0.22). For the phytoplankton incubated at 20 °C, α was 0.10±0.02 on day 28 prior to the crash of the cultures on day 35.

The concentration of chl awas examined on days 7, 21 and 35, butthere was no significant variation in chl a for either the 4 °C (p=0.193)or 10 °C (p=0.18) temperature regimes (Fig. 4D). However, for phyto-plankton cultured at 20 °C, the concentration of chl a declined signifi-cantly during the incubation period (p=0.033) and there was also asignificant difference between the 10 °C (1.29±0.13 μg l−1) and 20 °C(0.79±0.03 μg l−1) treatments at the final time point (p=0.017,Fig. 4D). The monosaccharide fraction of the carbohydrate pool was ex-amined on days 7, 21 and 35 with corresponding polysaccharide dataavailable for days 21 and 35. There was a significant decline in the con-centration of water-extractable monosaccharides for each temperatureregime between days 7 and 35 (Fig. 4E). For the cultures maintained at4 °C, the monosaccharide fraction declined from 10.36±2.14 μmolCl−1 on day 7 to 5.71±1.04 μmol Cl−1 on day 35 (p=0.044). A similartrendwas observed at 10 °Cwith a decline from 10.39±1.28 μmol Cl−1

on day 7 to 5.47±0.65 μmol Cl−1 on day 35 (p=0.035). The lowestmonosaccharide concentration was recorded on day 35 in the 20 °Ctreatment (4.61±0.47 μmol Cl−1). Although the decline in monosac-charides was more apparent for the cultures maintained at 20 °C(p=0.007, Fig 4E), the variation among treatments on day 35 was notsignificant (p=0.594). The concentration of polysaccharides varied be-tween 4.47±0.04 μmol Cl−1 and10.53±2.55 μmol Cl−1, but therewasno evidence for a decline in polysaccharides between days 21 and 35 atany temperature or significant differences among treatments at the endof the experiment (p=0.944, Fig. 4F).

4. Discussion

Research over the last three decades has provided a valuable insightinto the response of sea ice algae and phytoplankton to a range of physi-cochemical variables including light (Griffith et al., 2009; Martin et al.,2011; McMinn et al., 2003), nutrients (Lizotte and Sullivan, 1992;Robinson et al., 1998) temperature and salinity (Arrigo and Sullivan,1992; Palmisano et al., 1987; Ralph et al., 2007), but an understandingof dark survival strategies and the physiological condition of the over-wintering biomass remains fragmentary. This largely reflects the

Fig. 4. Photosynthetic parameters, chlorophyll a concentration and water-extractable carbohydrate content of mixed overwintering Arctic phytoplankton cultures incubated in thedark for 35 days at 4 °C, 10 °C and20 °C. (A) Fv/Fm, (B) rETRmax, (C)α, (D) Chl a (E)Monosaccharides, (F) Polysaccharides. Data aremeans±1 SE. Figure legend: 4 °C○; 10 °C▼; 20 °C△.

63A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

logistical constraints of gaining access to Antarctica, and to a certain ex-tent Arctic regions, at the end of winter. This is the first study to exam-ine the impact of elevated temperature on the physiology of threewintering communities during the biologically significant winter-spring transition. The incubation regimeswere chosen to reflect current

winter-spring temperatures in Antarctic (−2 °C) and Arctic coastal wa-ters (4 °C), a likely increase in temperature associated with globalwarming (4 °C and 10 °C respectively) and an additional temperaturetreatment that can be considered to be outside the optimum for polarcommunities (10 °C and 20 °C respectively).

64 A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

The term dark-survival was first coined by Antia (1976) and wasused to describe the retention of viability in photoautotrophs withoutgrowth during exposure to darkness. The exactmechanisms that enablealgae to survive long periods in the dark remain unknown. Importantly,McMinn et al. (2010) have recently documented that even the very lowirradiances associatedwith the return of daylight in the polar spring canlead to exponential microalgal growth, so every possible effort wasmade in the current study to obtain phytoplankton and sea ice algaethat had not been exposed to light.

4.1. Antarctic phytoplankton

The photosynthetic parameters Fv/Fm, rETRmax, and α, documenta relatively healthy phytoplankton community throughout the periodof incubation. In contrast to Reeves et al. (2011), who dark-incubatedthree Antarctic diatoms at the same temperature range and observeda rapid decline in maximum quantum yield (to b0.1) within 30 days,Fv/Fm values in the current study remained relatively high, even at10 °C. McMinn and Hegseth (2004) have suggested that Fv/Fm valuesbelow 0.125 infer limited photosynthetic activity and a senescentstate and this was also the indicator of cell viability adopted byReeves et al. (2011). In the current study, Fv/Fm increased to ~0.6by day 16 in the cultures maintained at−2 °C and 4 °C, then declinedto 0.42 in the +4 °C treatment by day 22. Maximum quantum yieldwas slightly lower and also less variable in the 10 °C treatment, butviability was still remarkably high considering that the cells had es-sentially been in the dark for four months. Photosynthetic capacity(rETRmax), which can be used to infer the onset of degradation ofthe photosynthetic apparatus, was found to be higher than the valuesreported by Reeves et al. (2011) and also by Lüder et al. (2002), whodocumented values close to 0.0 for rETRmax in the macroalga Palmariadecipiens after a six month period of darkness. Importantly, while pho-tosynthetic capacity is generally considered to be temperature depen-dent in marine microalgae (Meiners et al., 2009; Ralph et al., 2005),we did not observe significant differences among temperature regimesduring a 22 day incubation. The increase in rETRmax observed on day16, while difficult to interpret, illustrates themaintenance of the photo-synthetic apparatus during thewinter. Although rETRmax had declinedin all treatments by day 22, the exposure to elevated temperature doesnot appear to have exacerbated photosynthetic degradation. Light-limited photosynthetic efficiency (α) essentially remained constant ateach incubation temperature, and although there were indications ofa decline in all treatments on day 22, this was not statistically signifi-cant. Had it been possible to extend the incubation period, it wouldhave been particularly interesting to observe whether α continued todecline at the warmer temperatures. While a number of studies havereported a significant response to temperature in marine algae(e.g. Palmisano et al., 1987; Ralph et al., 2005; Verity, 1981), α isnot thought to be linked to photochemical reactions that are tempera-ture dependent (Falkowski and Raven, 1997). It can only be speculatedthat the decline in bothα and Fv/Fm observed on day 22 in thewarmercultures illustrates the onset of a significant temperature threshold.However, it is important to note that maximum rates of primary pro-duction in some Antarctic phytoplankton cultures have been docu-mented at close to 12 °C, with a significant decline only above 17.5 °C(Jacques, 1983). Interestingly, Neori and Holm-Hansen (1982) foundthat 7 °C was optimal for photosynthesis in their Antarctic cultures,which is comparable to the temperatures phytoplankton may experi-ence if carried by surface currents towards the Antarctic Convergence(Palmisano et al., 1987) and considerably higher than the −1.9 °Cthat characterises Antarctic coastal waters formuch of the year. Howev-er, in the absence of light-based metabolism, we suggest that over-wintering or ‘no-growth’ phytoplankton communities are onlymarginally thermally sensitive with respect to photophysiology. Thisfinding must be considered within the context of a relatively short

incubation period, but nevertheless significant change was only reallyevident at the environmentally unrealistic temperature of 10 °C.

4.2. Antarctic sea ice algae

Sea ice algae, by definition, live in or on sea ice and so can nevernaturally experience temperatures significantly above the melting pointof the ice matrix. However, sea ice microalgal communities are over-whelmingly dominated by diatoms and the same species frequentlycomprise phytoplankton blooms. Their use here was not so much tocharacterise the response of sea ice algae per se, but to characterisethe response of diatoms and control for the potential grazing impactof microzooplankon such as dinoflagellates. The photophysiologicalresponse to increased temperature was more apparent in the experi-ment with sea ice algae. Maximum quantum yield was relatively highprior to incubation but declined significantly across all temperature re-gimes by day 6 along with the other photosynthetic parameters rET-Rmax and α. This reduction in photosynthetic performance during thefirst week of the incubation may indicate that the light level inMcMurdo Sound was sufficient for the cells to have resumed photosyn-thesis prior to the extraction of the algae from the ice.When returned tothe dark, the cells may have subsequently reverted to a condition of‘physiological stasis’. With respect to temperature, there was no evi-dence to suggest thatmaintenance of either chl a or of the carbohydratepool was influenced by an increase in temperature from−2 to 4 °C. Al-though the rate of recovery in Fv/Fm was clearly greater for cultures at−2 °C, the cells at 4 °C were capable of resuming photosynthesis andFv/Fm increased slightly within 24 h. Even at the environmentallyunrealistic temperature of 10 °C, the cells remained photosyntheti-cally active for three weeks. This finding is in contrast to Reeves etal. (2011), who documented dark survival periods of just 7–14 daysfor a number of axenic Antarctic cultures incubated at 10 °C. However,the declines observed in Fv/Fm and α and the concentration of mono-saccharides observed at 10 °C is clearly indicative of physiological stressand this temperature may represent a thermal threshold. While a lon-ger period of incubation is required to validate this trend, there wasno evidence of a recovery in Fv/Fm during 24 h of subsequent light ex-posure. This is particularly interesting as Reeves et al. (2011) were ableto document significant recovery (both Fv/Fm and NPQ) following a30 day dark incubation at −2, 4 and 10 °C, but not after a period of60 days. Monosaccharides generally remain stable in phytoplanktonthat are photosynthetically active, but are used by diatoms to produceβ-1,3-glucan, a storage polysaccharide that is mobilised during dark-ness to fuel continued protein synthesis (Alderkamp et al., 2007;Hawes, 1990a,b; Lindqvist and Lignell, 1997). Elevated temperature isthought to increase both the rate of protein synthesis and the photonflux at which saturation occurs (Hawes, 1990b); how these processesare affected by longer periods of darkness remains largely unknown.For example, Reeves et al. (2011) traced the concentration of algal-derivedmonosaccharides and chl a in three species of Antarctic diatom,but observed no significant changes during a two month dark incuba-tion. Palmisano and Sullivan (1982) suggested that the storage andutilisation of intracellular carbon might be more important at theonset of winter, rather than the period of dark survival. The data pres-ented here from an in situ sea ice community essentially supports thishypothesis and, like Reeves et al. (2011), we are able to show thatmain-tenance metabolism does not require a significant drawdown of storedenergy products under non-photosynthetic conditions.

The potential for phytoplankton or sea ice algae to utilise alterna-tive metabolic pathways during the winter months is an importantconsideration, however it has yet to be established whether hetero-trophic metabolism is a significant dark survival strategy. For exam-ple, Horner and Alexander (1972) found the in situ metabolism ofradio-labelled organic substrates by Arctic sea ice algae to be negligi-ble while the dark survival of four algal species was unaffected by theaddition of organic supplements (Bunt and Lee, 1972). Conversely,

65A. Martin et al. / Journal of Experimental Marine Biology and Ecology 428 (2012) 57–66

Palmisano and Sullivan (1982) demonstrated that Antarctic diatomswere able to metabolise an exogenous source of glucose in a 30 daywinter-summer simulation experiment, while Rivkin and Putt (1987)quantified the incorporation of both glucose and amino acids into pro-teins and other complex organic polymers. The microalgal uptake ofboth carbon- and nitrogen-containing compounds in the dark clearlywarrants further exploration, however limited experimental work hasshown that the incorporation and the concentration of dissolved organicmatter (DOM)within the ice appears to be highly variable (Thomas andDieckmann, 2004).

4.3. Arctic phytoplankton

While there are many primary production and biomass measure-ments from spring and summer in the Arctic (e.g. Hegseth, 1998; Hilland Cota, 2005), winter data are still scarce (but see Eilertsen andDegerlund, 2010; Eilertsen et al., 1981) making it particularly difficultto model ecosystem responses to climate change in high-latitudes ofthe northern hemisphere. Of particular importance to the currentstudy is the fact that the annual cycle of light and dark will remainunchanged, at either pole, regardless of potential increases in tempera-ture. With respect to dark survival, it can be considered timely to con-trast a ‘cold and dark’ (Antarctic) with a ‘warm and dark’ (Arctic)ecosystem to provide an insight into likely changes in southern polar re-gions. Unlike the Antarctic, which is surrounded by deep and narrowcontinental shelves, more than 60% of Arctic seas are above continentalshelves that are relatively shallow. Thus, even during the wintermonths, convective processes are thought to support a moderate rateof primary production south of the Arctic Circle (Backhaus et al.,2003). However, there is little evidence to suggest that pelagic vegeta-tive cells survive the winter and provide the inocula for bloom eventsfurther north, for instance in the northern fjords of Norway (Eilertsenand Degerlund, 2010). In fact the only taxon that Eilertsen andDegerlund (2010) consider capable of dark survival in a vegetativeform is the centric diatom Skeletonema costatum sensu lato, a specieswhich comprises a significant fraction of the phytoplankton biomassduring summer months but appeared to be absent from TromsøSound in late January 2011. The process whereby resuspended restingspores act as seed populations for bloom events has been shown experi-mentally (Eilertsen et al., 1995), and a model developed by Backhaus etal. (1999) suggests that this processmay be representative of Arctic wa-ters. As expected, spores made a sizeable contribution to the observedbiomass in the current study. The relative abundance of the five vegeta-tive autotrophic diatom species is a particularly interesting finding.While the observed taxa may not make a significant contribution tothe spring biomass, we provide the first evidence that a number of Arc-tic phytoplankton species are capable of surviving during long dark pe-riods in a vegetative form. Thiswarrants further investigation as it couldexplain the presence of many spring bloom diatom species that ap-parently do not produce spores.

The ability of photoautotrophs to survive long periods of darknessin polar regions has important ecological implications for primaryproduction and marine ecosystem dynamics. Our results show thatover-wintering phytoplankton and sea ice algal communities can sur-vive in the absence of light-based metabolism for extended periods.Maintenance metabolism did not deplete stored energy reserves orthe photosynthetic pigment chlorophyll a under temperature increasesthat were ecologically relevant with respect to climate change projec-tions (−2 to 4 °C and 4 to 10 °C). It is not likely that warming willlimit the dark survival of marinemicroalgae or their subsequent contri-bution to bloom events in polar oceans.

Acknowledgements

We acknowledge the logistical support of Antarctica New Zealandand in particular thank the staff at Scott Base in 2008 and 2009. A

McMinn received financial assistance from the Australian ResearchCouncil and K.G. Ryan acknowledges the support of the Foundationof Research, Science & Technology (VICX0706). [SS]

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