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Can bottom ice algae tolerate irradiance and temperature changes?
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Authors: Meghana A Rajanahally a*, Dalice Simb, Ken G Ryana, Peter Conveyc,d
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a
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Wellington 6140, New Zealand
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b
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Wellington, PO Box 600, Wellington 6140, New Zealand
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c
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0ET, United Kingdom.
School of Biological Sciences, Victoria University of Wellington, PO Box 600,
School of Mathematics, Statistics and Operations Research, Victoria University of
British Antarctic Survey, NERC, High Cross, Madingley Road, Cambridge CB3
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d
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Malaya, 50603 Kuala Lumpur, Malaysia
National Antarctic Research Center, Institute of Graduate Studies, University of
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Corresponding author at: Victoria University of Wellington, PO Box 600,
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Wellington 6140, New Zealand Tel: +91 80 26561095
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Email address: meghana.rajanahally@gmail.com
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Keywords
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Sea ice, ice melt, photophysiology, quantum efficiency of PSII, electron transport
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rates
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Abbreviations
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PAR, photosynthetically-active radiation, UV-B, Ultraviolet B, MAAs, mycosporine -
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like amino acids, PSII, photosystem II, Ek, saturation irradiance, HPLC, high
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performance liquid chromatography, PAM, pulse amplitude modulation, RLC, rapid
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light curves, F’m, maximum fluorescence in the light, Fom, maximum fluorescence
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level , Fv/Fm, maximum quantum yield of PS II, F’m – Ft, ratio of variable
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fluorescence, ETR, electron transport rate, F’, fluorescence yield, Tc, critical
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temperature where permanent damage occurs, Tp, temperature of peak fluoroscence,
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ϕPSII, effective quantum yield of PS II
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Abstract
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Sea ice algae are significant primary producers of the ice-covered marine
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environment, growing under typically cold, dim conditions. During ice break-up they
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are released to the water column, where temperatures can be several degrees higher
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and irradiance can increase by orders of magnitude. To determine how sea ice algae
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respond to such rapid changes, we carried out incubations to examine their tolerance
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to environmentally realistic levels of change in temperature and PAR, as expressed by
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photosynthetic response and production of mycosporine-like amino acids (MAAs).
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The algae were also exposed to a broader range of temperatures, to evaluate their
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potential to function in warmer seas in the event, for instance, of anthropogenic
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transfer to locations further north. When subjected to PAR (0 - 100 mol m-2 s-1) at
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ecologically relevant temperatures (-1°C, 2°C, 5°C), the algae showed tolerance,
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indicated by a lack of decline in the quantum efficiency of photosystem II (PSII). The
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data show that bottom ice algae can tolerate increasing temperature and PAR
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comparable to the changes experienced during and after sea ice melt. MAA
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production increased at higher PAR and temperature. At ambient PAR levels,
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increased temperatures resulted in lower ϕPSII. However, as PAR levels were
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increased, higher temperature reduced the level of stress as indicated by higher ϕPSII
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values. This result suggests, for the first time in sea ice algal studies, that higher
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temperatures can ameliorate the negative effects of increased PAR. Exposure to much
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higher temperatures suggested that the algae were capable of retaining some
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photosynthetic function at water temperatures well above those currently experienced
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in some of their Antarctic habitats. However, when temperature was gradually
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increased past 14°C, the photosystems started to become inactivated as indicated by a
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decrease in quantum yield, suggesting that the algae would not be viable if transferred
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to lower latitude cold temperate areas.
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1. Introduction
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The seasonal formation and melting of sea ice is one of the most striking features of
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the Antarctic marine environment (Convey, et al., 2014). As sea ice algae play an
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important role in primary production in the Southern Ocean and are the only source of
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fixed carbon for all other life in ice-covered habitats of this region (Arrigo, et al.,
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1997), it is important to understand their responses to environmental variability and
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change. During sea ice melt algae are exposed to higher temperatures and levels of
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UV-B and PAR than they experience in the ice matrix (Arrigo and Sullivan, 1992,
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Ralph, et al., 2007, Ryan, et al., 2011). Indeed, temperatures above zero and even up
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to 5°C can be experienced by algae derived from sea ice in shallow waters as well as
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in the open sea during summer in the Antarctic Peninsula region and northern zones
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of the Southern Ocean close to the Polar Front (Morley, et al., 2010). Likewise, PAR
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levels exceeding 100 mol m-2 s-1 can be experienced during thinning and melting of
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sea ice (Ryan, et al., 2011). Therefore, quantifying the ability of algae to withstand
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these conditions is ecologically relevant.
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Productivity in Antarctic sea ice and Southern Ocean algae is mainly restricted to a
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three to four month period in summer (Arrigo, 2014, Peck, et al., 2006, Smetacek and
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Nicol, 2005). Antarctic bottom ice algal communities are thought to be extremely
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shade-adapted, with low Ek values (saturation irradiance, a measure of the intensity of
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PAR required to permit maximum photosynthesis), and are able to adjust
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photosynthetic rates to suit changing diurnal irradiance levels (Arrigo, et al., 1997,
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Ryan, et al., 2009). Low PAR conditions are typical of the under-ice environment
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(Lizotte, 2001). Measurements of daily average PAR under 2.5 m thick spring pack
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ice in the Ross Sea region have shown a range of 5.6 to 10.6 μmol m-2 s-1 in the
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under-ice habitat (Lazzara, et al., 2007). Under 1.8 m thick fast ice in McMurdo
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Sound, PAR levels can be even lower, with as little as 1 μmol m-2 s-1 measured at
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noon on a clear sunny day in spring (Ryan, et al., 2011). Low photosynthetic rates,
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high photosynthetic pigment concentrations and the abundance of chloroplasts in algal
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cells are strong indications of the shade-adapted features of these microalgal
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communities (Lazzara, et al., 2007). In an Arctic study (Manes and Gradinger, 2009),
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ϕPSII increased as PAR levels increased from 0 to 32 mol m-2 s-1 and temperatures
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increased from -3.9°C to -1.6°C within the sea ice in the transition from winter to
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summer.
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The photosynthetic response of all marine algae to temperature is dependent on the
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level of PAR (Davison, 1991). However, although the photochemical reactions of
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photosynthesis are light-dependent, carbon fixation is an enzyme-driven process and
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therefore highly temperature-dependent (Öquist, 1983). In Arctic sea ice diatoms,
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photosynthetic efficiency decreases with increasing temperature (Palmisano, et al.,
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1987). Claquin et al. (2008) showed that several temperate marine algae were able to
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survive a large temperature range, with all the species studied being viable at least
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between 7 and 24°C. In contrast, polar diatoms can only tolerate temperature variation
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over a range of as little as 7°C (Suzuki, et al., 1995). Some species of diatoms found
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in sea ice have also been observed in areas close to the Polar Front where water
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temperatures are known to reach upto 5°C (Atkinson, 1994).
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To protect themselves from environmental stresses, many microorganisms produce
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mycosporine-like amino acids (MAAs) (Vincent, 1988). Other than their production
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being a response to exposure to biologically damaging short wavelength UV-B
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radiation (Klisch and Hader, 2008), MAAs may also perform osmoprotective
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functions (Arrigo and Thomas, 2004, Oren, 1997) and provide antioxidative
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protection (Dunlap and Yamamoto, 1995, Oren and Gunde-Cimerman, 2007).
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Unprecedented global climate change trends have become apparent over the past
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century. In Antarctica, there are also strong regional trends, and region-specific
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effects such as the consequences of the annual formation of the anthropogenic
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Antarctic ozone hole (Convey, et al., 2009, Dixon, et al., 2012, Turner, et al., 2009,
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2012). There is currently an overall positive trend in the annual mean ice extent in the
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Antarctic of 1.2 - 1.8% per decade (Turner, et al., 2013), although this is forecast to
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reverse over the next century with the latest IPCC AR5 models predicting that there
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will be a nearly ice-free state around Antarctica in February within the next century
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(http://www.ipcc.ch/report/ar5/wg1/#.UuhKY2SBoYI, Web 1 May 2014). However,
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regional sea ice extent and duration have also significantly reduced in the
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Bellinghausen Sector and west of the Antarctic Peninsula, and maximum ice loss is
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expected in the central Weddell Sea and the Bellingshausen-Amundsen Seas
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(Lefebvre and Goosse, 2008), at the same time as increases of about 4.5% in the Ross
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Sea (Turner, et al., 2009). Changes in sea ice extent and duration can have significant
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impacts on the phytoplankton community, affecting community composition and
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species abundance (Montes-Hugo, et al., 2009, Schloss, et al., 2012) and having
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consequential impacts elsewhere in the food chain (Forcada, et al., 2006). These
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changes have also been linked with reductions in krill stocks and their replacement by
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salps, effects that in turn impact the entire Southern Ocean food web (Atkinson, et al.,
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2004, Trivelpiece, et al., 2011).
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Separately, there is also a risk of transfer by human agency of Antarctic marine algae
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into lower latitude regions north of the Antarctic Polar Front such as near the sub-
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Antarctic islands, and cold temperate regions around Australia (especially Tasmania),
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New Zealand and southern South America (Lewis, et al., 2003). The Polar Front has
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long formed a barrier to movement of species to and from Antarctic waters (Barnes, et
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al., 2006). The marine environment of Antarctica itself is to some extent protected
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from this danger, as ships generally do not take on sufficient cargo at Antarctic
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research stations to require discharge of externally derived ballast water. However,
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there is a largely unappreciated risk of ballast water, taken on after cargo offloading in
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Antarctica, being discharged north of the Polar Front. The survival of Antarctic
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continental shelf species of microalgae in ballast water provides evidence that such
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algal transport is possible (Lewis, et al., 2003).
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This field-based study focused on measurement of photosynthetic responses using
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chlorophyll fluorescence techniques and MAA production of fresh-collected mixed
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species ice algal communities obtained in the southern Ross Sea. First, these
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communities were exposed to a relatively narrow range of experimentally-imposed
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temperatures shortly after collection, mimicking the scale of temperature changes that
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might influence the timing of sea ice melt, or be experienced after the melt is
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completed in parts of the Southern Ocean. Second, an initial study was made of their
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response to exposure to a wider range of temperatures, to determine the bounds of
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their flexibility and potential of functioning at temperatures more typical of cold
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temperate regions north of the Polar Front. Finally, the critical temperatures affecting
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thylakoid integrity, which determine irreversible changes in photosynthetic function,
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were examined. Such damage has been observed in coral algal symbionts (Buxton, et
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al., 2012, Fitt, et al., 2001, Tchernov, et al., 2004). Determining critical temperatures
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could help in identifying tipping points where small changes in the environment cause
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rapid and extensive changes in an ecosystem (Clark, et al., 2013, Scheffer, et al.,
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2001).
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2. Methods
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2.1 Study Location
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Fresh bottom ice algae were collected from sea ice, ranging from 1.8 to 3.2 m
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thickness, at Gondwana Station, Terra Nova Bay (74° 38’S 164° 13’W) during
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November 2007 and at Cape Evans (77° 38’S 166° 24’E) during November 2010
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(Fig. 1). These algae were worked on immediately following collection and
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extraction, as described below. Experiments were also conducted with algae collected
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and transported to Wellington, New Zealand, from Granite Harbour (77° 0’S 162°
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54’E) during the 2009/10 season. Experiments and measurements on the latter
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commenced within 6 d of collection in the field.
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2.2 Field parameters and algal community composition
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PAR levels were recorded at solar noon on a cloudless day in each season at Granite
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Harbour and Cape Evans using a SpectroSense 2 meter with a SKR 1850 radiometer
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(bandwith 400 – 700 nm) (Skye Instruments, UK).
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Ice thickness and dominant algal taxa were monitored over the 3-5 week durations of
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the study seasons. Samples for taxonomic examination were taken from every core
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used in the studies. However, no taxonomic data were subsequently derived for Terra
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Nova Bay due to loss of samples during transit to New Zealand. In previous years,
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diatoms including Nitzschia stellata, Entomoneis kjellmannii, Berkelya adeliensis,
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Navicula sp. and Fragillariopsis curta have dominated the community at this location
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(Ryan, et al., 2011).
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2.3 Collection methods
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Core samples were obtained from the bottom 20 cm of the sea ice using a Kovaks
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(Lebanon, NH, USA) 13 cm diameter coring drill. Three to five replicate cores were
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collected at each location. During collection, the cores were protected from PAR
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exposure by performing all operations underneath a black sheet, then immediately
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transferring them into opaque black plastic bags and placing into insulated boxes
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(Ryan, et al., 2009, 2012). The bottom of each core was then cut into a standard size
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(width × height × length of 4 × 4 × 10 cm) in a dimly lit room (1-10 mol m-2 s-1), and
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melted into three times the quantity of filtered seawater (35‰, 0.22 μm, -1.8°C) in the
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dark over a period of 12 h (Mundy, et al., 2011, Ryan, et al., 2006). If slowly melted
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in the dark in this way, sea ice algae do not undergo cell lysis due to osmotic shock
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(Mikkelsen and Witkowski, 2010). Once melted, the suspensions obtained were
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homogenized using a small plastic paddle mounted on a battery drill (Ryobi,
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Australia) and filtered through a 20 m plankton mesh to remove large chains of cells.
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These homogenized samples were then used in the experiments described below,
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normally within 1-2 h of preparation.
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Aliquots taken from the melted ice core samples were used to count algal cell
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numbers and to assess taxonomic composition. Cell counts were performed on
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samples from all cores using custom-made counting slides under an inverted
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microscope. Cells in a volume of 0.5 mL were allowed to settle for 2-4 h before
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counts were made. Identification to at least genus level was made for at least 300 cells
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per sample, following Scott & Marchant (2005).
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2.4 PAR Exposure Methodology
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At Cape Evans, 50 mL sub-samples of melted bottom ice samples (n=5 cores) were
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incubated under each of three PAR levels using 5W LED lights (Greenlights, Taiwan)
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(100 mol m-2 s-1, 45 mol m-2 s-1, 1 mol m-2 s-1) at either –1°C, 2°C or 5°C for 48 h
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in a temperature controlled water bath (Haake, Cleveland, OH, USA) filled with a
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15% ethylene glycol mixture in water. The different experimental light levels were
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chosen as they represent those typical in the sea ice profile from the bottom to the
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middle, where these algal communities are found (Table 1). The 50 mL samples were
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incubated in 70 mL opaque specimen containers, each covered with appropriate
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neutral density filters to allow exposure to specific PAR levels.
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Samples obtained from Granite Harbour were used to examine responses to
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conditions that might be experienced following transfer to waters beyond the
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Southern Ocean. Here, bottom ice samples (n=3 cores) were again melted into filtered
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seawater after collection and then transported refrigerated at 4°C to Wellington (New
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Zealand) within a period of 5 d, where they were used in experiments within 24 h.
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Fifty mL sub-samples were again incubated under each of three PAR levels (100
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mol m-2 s-1, 45 mol m-2 s-1, 1 mol m-2 s-1) at 4°C, 14°C or 24°C for 48 h as
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described above. The incubation at 4°C was carried out for 72 h to observe responses
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over a longer period of time.
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Samples of 1 mL for cell counts were taken at the start and end of each incubation in
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all treatments, preserved using 1% glutaraldehyde in seawater and stored at 4°C. The
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remaining 48 mL of suspension from each incubation was used for measurement of
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MAA content. This was filtered onto GF/F filters, which were then placed in
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containers with 10 mL 100% HPLC grade methanol and extracted at 4C for 24 h.
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The methanol extract was then stored at -20C, and analysed using High Performance
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Liquid Chromatography (HPLC) at Victoria University of Wellington following Ryan
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et al. (2002).
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2.5 PAM fluorometry of bottom ice algae
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To study the photosynthetic responses of sea ice algae to (a) a range of temperatures
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mimicking sea ice melt and Southern Ocean conditions, and (b) a wider range of
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temperatures to determine their limits of photosynthetic activity, Pulse Amplitude
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Modulation (PAM) fluorometry was used. PAM is a widely used methodology that
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provides a rapid, non-invasive assessment of the photosynthetic apparatus of algal
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cells (Bilger and Schreiber, 1990) making it a useful tool for non-invasive study and
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for smaller volumes of cultures. It has been used widely in studies of the
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photosynthetic activity of sea ice algae (eg:Hawes, et al., 2012, Kühl, et al., 2001,
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McMinn, et al., 2003, Mou, et al., 2013, Ralph and Gademann, 2005, Ralph, et al.,
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2005, Runcie and Riddle, 2006, Ryan, et al., 2006), and is a useful tool for examining
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the ability of a photosynthetic organism to tolerate environmental stressors and the
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extent of damage caused by these stresses (Maxwell and Johnson, 2000).
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Immediately after collection and thawing of ice core samples as described above,
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photosynthetic properties of the algal community at Cape Evans were examined using
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a Water PAM (Walz, Effeltrich, Germany) fluorometer to measure effective quantum
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yield for photosystem II (ϕPSII). Before commencing observations, the cells were dark
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acclimated. Thereafter, all PAM measurements were made on cells exposed to light as
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the cells were subjected to actinic light used to make the PAM measurements.
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The Water PAM fluorometer was also used to generate rapid light curves (RLC) at
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Cape Evans. To reduce light and temperature stress, the transfer was carried out in a
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cooled laboratory in near dark conditions and the Water PAM was contained within
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an insulated box maintained at low temperature by ice blocks. An RLC describes the
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effective quantum yield as a function of irradiance (Ralph and Gademann, 2005).
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Each sample was treated with a series of eight increasing actinic light treatments (0, 8,
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20, 35, 55, 75, 96, 114, 150 mol m-2 s-1), after which a strong saturating pulse was
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applied and ϕPSII was recorded. The RLC takes 90 sec to generate. The electron
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transport rate (ETR) values were calculated by multiplying the ϕPSII value by the
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irradiance just applied. As the ϕPSII value is a ratio and ETR is derived from this
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parameter, it is termed relative ETR (rETR). An RLC permits derivation of different
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parameters that can be used to describe the photosynthetic properties of an algal
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sample, including rETRmax (the maximum value for rETR), photosynthetic
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efficiency (α) and saturation irradiance (Ek) (Ryan, et al., 2009). To determine these
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parameters, the rETR data were imported into Microsoft Excel v 10.0 (Microsoft,
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USA) and the curve was fitted with a “waiting-in-line” function (Ritchie, 2008). An
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RLC was generated for each sample at time 0, 2, 4, 6, 12, 24 and 48 h.
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2.6 Measurement of MAAs
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A 10 mL aliquot from each sub-sample was evaporated at 30°C on a heating block
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and resuspended in a mixture of 40 μL HPLC grade methanol and 160 μL 0.13%
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acetic acid. The resulting solutions were analysed by reverse-phase HPLC (Dunlap
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and Chalker, 1986). Chromatography was carried out on a Phenomenex C18 column
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(Gemini 5 micron; 110 Å; 250 x 4.6 mm) using a Phenomenex RP.8 security guard
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cartridge (4 x 2 mm ID), an Agilent 1100 HPLC system (Agilent, Loveland, CO,
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USA) and Chemstation software (Agilent, Loveland, CO, USA). Injection volumes
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were 5 L and elution was performed using a flow rate of 0.6 mL min-1 at 30C. An
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isocratic mobile phase consisting of 0.1% acetic acid and 20% methanol in water was
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used. Spectral data were accumulated in a fixed range and chromatograms plotted at
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310, 330 and 360 nm. MAA peaks were identified using comparison with publicly
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available online absorption spectra and retention times. Relative concentrations were
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estimated based on the area under the peaks at 360 nm, which was standardized per
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cell using the cell count data.
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2.7 Thylakoid integrity
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As temperature increases, a threshold temperature is reached where irreversible
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damage occurs in the thylakoid membranes. This damage can be demonstrated
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through measurement of fluorescence yield (F’) as temperatures are increased (Hill, et
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al., 2009). At the critical temperature (Tc), permanent damage is evident as an
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irreversible reduction in maximum quantum yield. At the temperature of maximum
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fluorescence (Tp) there is complete loss of photosynthesis and maximum quantum
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yield becomes zero (Hill, et al., 2009).
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To estimate Tc and Tp, at Terra Nova Bay in 2007, a small incubation chamber system
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was set up in an Imaging PAM (Walz, Effeltrich, Germany) to measure
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photosynthetic yield (ϕPSII). This consisted of a 600 mL tub containing nine short
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opaque tubes fixed in place with silicon sealant. The tub was connected to a water
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bath and coolant was continuously circulated through the chamber using a 65 W
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aquarium pump to maintain a constant temperature. This system allowed temperatures
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inside the sample tubes to be controlled. Five mL of sea ice algal cell suspension was
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poured into six of the nine tubes (two replicates from each of the three ice core
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samples). The other three tubes contained filtered seawater to monitor background
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readings and temperature. The samples were stabilized at -1.8°C over a period of 5
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min and then subjected to a temperature increase of 2°C every 5 min. After every
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increase, the samples were held in the dark at that temperature for 5 min and then a
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saturating pulse was applied and ϕPSII was recorded. In a second set of experiments, F’
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readings only were recorded after each temperature increase. The temperature was
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increased until F’ or ϕPSII reached zero.
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2.8 Statistical Analyses
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First, a repeated measures ANOVA was applied to test specifically for changes over
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time within each treatment. The assumptions of normality and equal variance were
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satisfied. The data from successive time points are likely not to be independent.
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Therefore the covariance matrix - the matrix of all covariances between time points -
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was examined. If that matrix was spherical (as confirmed by Mauchly’s test of
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sphericity), then a standard repeated measures ANOVA calculation was carried out.
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If, however, the sphericity assumption did not hold, then the Greenhouse Geisser
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adjustment was applied (Greenhouse and Geisser, 1959).
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Repeated measures ANOVA was used to analyse effective quantum yield and
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rETRmax, with ‘treatment’ being the irradiance treatments and time points of 0, 2, 4, 6,
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12, 24 and 48h. The significance of differences between pairs of time points was
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adjusted using Bonferroni’s correction. As the separate experiments conducted at the
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different exposure temperatures were completed at different times, each dataset
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obtained was analysed separately.
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Finally, one-way ANOVA was used to compare the different treatment groups at
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specific time points. Post hoc tests using Bonferroni’s correction were used for
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subsequent pair-wise comparisons, e.g. between different pairs of treatment groups at
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a time point, or between different time points within a treatment group. All
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differences referred to are statistically significant at p < 0.05.
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3. Results
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3.1 Environmental parameters and taxonomic composition
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Environmental parameters at the time of collection and community taxonomic
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composition are summarised in Table 1. The sea ice thickness at Granite Harbour was
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greater than at Terra Nova Bay and Cape Evans and, correspondingly, the light level
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at the bottom of the sea ice at Granite Harbour was half of that at the other two sites.
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The communities at Granite Harbour and Cape Evans were clearly different. Granite
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Harbour was dominated by Navicula spp., Entomoneis spp. and Fragilariopsis curta
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while the Cape Evans community included Nitzschia stellata, Fragilariopsis spp.,
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Navicula spp. and chains of Berkeleya spp.
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3.2 Response of algae to temperature and light changes
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3.2.1 Quantum Yield
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Initially a repeated measures ANOVA was conducted with time as the ‘within’ factor
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and temperature and treatment as ‘between’ factors. This showed significant effects of
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time (p<0.001), time by temperature (p<0.001) and time by treatment (p=0.046), but
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the three-way interaction between time, temperature and treatment was not significant
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(p=0.487) (data not shown). Consequently repeated measures analyses were carried
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out for each exposure temperature separately to test for the effect of all treatments and
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time, and for each treatment separately to test for the effect of temperature and time.
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The general response of algae to the treatments at all PAR levels at -1°C and 2°C was
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an initial rapid change in ϕPSII followed by settling over time to a more or less
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constant value by 48 h (Fig. 2). Algae incubated at higher PAR levels showed rapid
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initial decreases before recovering to the same ϕPSII values as those incubated at lower
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PAR levels. At 5°C, ϕPSII for algae at all three PAR levels had an initial decrease
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followed by an increase that continued until the end of the 48 h incubation period.
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At -1°C (Fig. 2a), there were varying significant contributions to differences in ϕPSII
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by time, the time by treatment interaction for algae incubated at different PAR
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treatments, and between treatments (averaged over time) (Table 2a). For algae
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incubated at 100 mol m-2 s-1, there was a decrease in ϕPSII after 2 h, followed by
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recovery by 6 h to reach levels lower than but not significantly different to those
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achieved by algae incubated at 1 mol m-2 s-1 and 45 mol m-2 s-1. All these treatments
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then showed no further change. ϕPSII for algae incubated at 0 mol m-2 s-1 showed an
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increase over 48 h, relative to the start of the incubation, but final values were not
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significantly different from those of the other treatments.
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At 2°C (Fig. 2b), both 45 and 100 mol m-2 s-1 incubations showed an initial decrease
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before recovery. There were significant differences in ϕPSII by time and between
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treatments averaged over time for algae incubated at different PAR treatments (Table
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2a). For algae incubated at 0 mol m-2 s-1, there was a significant increase over 48 h
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relative to the start of the incubation. However, despite similar magnitudes of change
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being observed, with the relatively large variability in the data obtained, no significant
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changes were identified for algae incubated at other exposures.
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In the higher light intensity incubations at 5°C (Fig. 2c) ϕPSII had an initial decrease
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and then recovery. However, the only significant differences identified over time were
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for algae incubated under different PAR treatments (Table 2a). ϕPSII for algae
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incubated at 0 and 1 mol m-2 s-1 showed a significant increase over 48 h relative to
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the start of the incubation but did not differ from each other. No significant changes in
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ϕPSII were identified for algae incubated at 45 or 100 mol m-2 s-1.
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3.2.2 Relative ETRmax
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The initial repeated measures ANOVA showed significant effects of time (p<0.001),
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time by temperature (p<0.001), time by treatment (p<0.001), and the interaction
381
between time, temperature and treatment (p<0.001) (data not shown). As above,
382
repeated measures analyses were then carried out for each temperature and treatment
383
separately.
384
rETRmax for algae incubated at -1°C and 2°C showed similar general responses (Fig.
385
3a and b). At both these temperatures, the rETR for algae incubated at the higher PAR
386
levels increased rapidly before stabilizing at the same rETRmax as the other exposures
387
by 48 h (Fig. 3). The only exception to this generalisation was the algae exposed to 45
388
mol m-2 s-1, where the initial increase in rETRmax was maintained until 48 h. At 5°C,
389
there were initial changes in rETRmax that remained roughly constant until 24 h, after
390
which rETRmax for algae incubated at the higher PAR levels showed a continuing
391
increase.
392
At -1°C (Fig. 3a), rETRmax was significantly greater at the higher PAR exposures
393
(Table 3a), although the variability in the data meant that no significant differences
394
were detected between 0 and 48 h. At 2°C (Fig. 3b), there were significant increases
395
in rETRmax over the time points between 0 and 48 h for algae incubated at all PAR
396
exposures (Table 3a). However, only algae incubated at 45 mol m-2 s-1 showed an
397
increase in rETRmax over 48 h, relative to the start of the incubation. At 5°C (Fig. 3c),
398
there were significant increases in rETRmax over the time points between 0 and 48 h
17
399
for algae incubated at the high PAR exposures. Algae incubated at 45 mol m-2 s-1
400
showed significantly greater rETRmax compared with those incubated at 0 and 1 mol
401
m-2 s-1 at 48 h.
402
403
3.2.3 Higher temperature exposures
404
At 4°C (Fig. 4a), ϕPSII in algae incubated at 100 mol m-2 s-1 reached zero after 24 h.
405
At 14°C (Fig. 4b), ϕPSII in algae incubated at 1 mol m-2 s-1 declined over 48 h,
406
whereas algae incubated at 45 mol m-2 s-1 and 100 mol m-2 s-1 reached zero after 4
407
h. At 24°C (Fig. 4c), ϕPSII for algae at all PAR treatments reached zero after 6 h. There
408
were no significant changes in MAA production in all PAR treatments at all
409
temperatures over the 48 h (data not shown).
410
411
3.2.4 MAAs
412
The initial repeated measures ANOVA showed significant effects of time (p<0.05),
413
time by temperature (p<0.05), time by treatment (p<0.05), but the interaction between
414
time, temperature and treatment was not significant (p>0.05). Again, repeated
415
measures analyses were then carried out for each temperature separately (Table 2.4).
416
The algae showed a general increase in MAA production at all temperatures when
417
incubated at the higher PAR treatments. However, there was no significant increase
418
(Table 4) in MAA production for algae incubated at -1 and 2°C (Figure 5a,b). At 5°C,
419
algae incubated at the higher PAR treatments (Figure 5c) showed a significant
420
increase (p<0.05) in MAA production over the 48 h with algae incubated at 100 mol
421
m-2 s-1 having a significantly higher (p<0.05) MAA production than those incubated at
422
45 mol m-2 s-1.
423
18
424
3.2.5 Thlyakoid integrity
425
As incubation temperature was slowly increased, ϕPSII declined with increasing
426
temperature, with photosynthesis ceasing (Tp) by 27°C (Fig. 6a). This temperature
427
also corresponds to that at which the maximum value of F’ was obtained (Fig 6b). The
428
response of F’ to increasing temperature (Fig. 6b) was constant until ~12°C, then
429
increased markedly at ~14°C, reaching a peak at 24°C. Recording ceased at 48°C as
430
there was no change in F’ for five consecutive readings. These data indicate that the
431
temperature at which permanent damage to the thylakoid membrane was initiated (Tc)
432
was ~14°C.
433
434
4. DISCUSSION
435
Studies of sea ice algae in their natural environment suggest that they are generally
436
photo-inhibited by PAR of 20 mol m-2 s-1, indicating potentially serious impacts on
437
photosynthetic parameters over the short term during melting of sea ice (Ryan, et al.,
438
2011). At a PAR level of 1 mol m-2 s-1 (a typical under-ice level) in the present study,
439
algae were able to maintain their ϕPSII throughout the duration of the incubation. The
440
higher PAR exposures also did not cause a decrease in ϕPSII over the 48 h incubation
441
periods, suggesting that these algae, if they do survive being released from the ice into
442
the water column, may continue to function under the new radiation regime and go on
443
to become part of the spring pelagic algal bloom. These results extend the
444
observations of Ryan et al. (2011), where quantum yield for bottom ice algae
445
decreased over a 4 h incubation period during ice melt. Similarly here, quantum yield
446
decreased over the first 4 h of the manipulation after which it recovered to reach
447
levels similar to those at 0 h.
19
448
The initial quantum yield for algae used in these experiments was relatively low
449
although previously used protocols were implemented (Martin, et al., 2012, McMinn,
450
et al., 2010, Ryan, et al., 2009, Ryan, et al., 2012). The explanation for this is unclear,
451
and yield values recovered to more normal levels in low temperature incubations. The
452
anomalous initial values may be an artefact relating to the strong strand-forming
453
characteristic of Berkeleya sp. that was an important component of the samples
454
obtained from the Cape Evans site. At an ambient temperature of -1°C, the algae used
455
in this study showed no evidence of being stressed by exposure to PAR over a period
456
of 48 h, even at irradiances well above those experienced in their natural environment.
457
At ambient PAR levels, increased temperatures resulted in lower ϕPSII. However, as
458
PAR levels were increased, higher temperature reduced the level of stress as indicated
459
by higher ϕPSII values. This result suggests, for the first time in sea ice algal studies,
460
that higher temperatures can ameliorate the negative effects of increased PAR, which
461
has previously been noted to cause decreased quantum yield and increased
462
photoinhibition in sea ice algae in eastern Antarctica (Ralph, et al., 2005).
463
Compounding factors such as higher PAR and temperature causing damage to PSII
464
complexes have been proposed to enhance photo-induced viability loss and reduce
465
ϕPSII (Van de Poll, et al., 2006). Similar compounding stressors were present in the
466
current study, although here temperature played a greater role in the decrease of ϕPSII
467
than PAR. Analogous amelioration of the effects of increased UV radiation by higher
468
temperatures has also been observed in temperate diatoms, possibly indicating that an
469
increase in temperature could result in higher metabolic activity, leading to a faster
470
turnover in the D1 protein of the electron transport chain in PS II (Halac, et al., 2010).
471
Such a mechanism would result in an increased rate of recovery of PS II.
20
472
Relative ETRmax maintained the same rates at all PAR levels over 48 h exposure at
473
ambient temperatures, despite initial increases, meaning that the algae did not undergo
474
significant photo-inactivation. As temperature was increased, however, exposure to
475
higher PAR levels resulted in higher rates of rETRmax, which could again be due to
476
faster turnover in the D1 protein. Even at the higher temperature of 5°C and PAR of
477
100 mol m-2 s-1, initial decreases in ϕPSII were followed by recovery over a period of
478
48 h. Previous studies of Antarctic bottom ice algal communities have also shown a
479
high resilience to increased irradiance, as decreases in photosynthetic efficiency were
480
rapidly reversed when the algae were returned to low PAR conditions (Petrou, et al.,
481
2011). The main mechanism involved in acclimation was hypothesized to be heat
482
dissipation through activation of the xanthophyll cycle (Petrou, et al., 2011). This
483
finding, combined with the data obtained here, indicate that bottom ice algae may not
484
be obligately shade-adapted as some studies have previously concluded (McMinn, et
485
al., 2003, 2007, Thomas and Dieckmann, 2002), but have plasticity in tolerating
486
changing irradiance.
487
Even with increasing temperatures, the bottom ice algae acclimated to higher
488
irradiances and showed recovery in ϕPSII after initial declines when exposed for a
489
period of 48 h. The current study indicates that bottom ice algae can acclimate to rates
490
of change in irradiance such as would be experienced, for instance, during melting of
491
sea ice. Previous studies have shown quantum yield declined in a dose-dependent
492
manner when PAR was gradually increased (Ryan, et al., 2009). Furthermore, when
493
algae were suddenly exposed to the same final irradiances, ϕPSII dropped to the same
494
values as seen in those exposed to a gradual increase in irradiance, indicating that the
495
previous exposure history did not influence their response to high irradiance (Ryan, et
496
al., 2009).
21
497
Manipulations involving a relatively small and ecologically realistic temperature
498
range (-1 to 5°C) led to significant changes in MAA production in algae incubated at
499
5°C under 100 mol m-2 s-1. The concentration of MAAs per cell generally increased
500
in higher PAR treatments, and was further enhanced at higher than ambient
501
temperatures. This is the first demonstration in the marine environment that MAA
502
production by algae can be stimulated by PAR and temperature stress even in the
503
absence of UV radiation.
504
As demonstrated by the ϕPSII response to larger-scale temperature manipulation,
505
temperature ultimately imposed a far greater level of stress than PAR. At lower
506
temperatures, algae were capable of tolerating PAR of up to 45 mol m-2 s-1, but this
507
tolerance rapidly decreased at higher temperatures. At 4°C, algae sourced from
508
Granite Harbour exposed to 100 mol m-2 s-1 showed a rapid decline in quantum
509
yield, contrasting with the response observed at 5°C in algae from Terra Nova Bay.
510
This could again be due to the differences in species composition of the algal
511
communities present at both sites, and also the inevitable disturbance in conditions
512
experienced during transit from the field to the laboratory. The thylakoid integrity
513
data complement these findings, indicating that the photosynthetic apparatus of sea
514
ice algae is able to tolerate increasing temperatures, at least over a relatively limited
515
temperature range. The initial increase in F’ observed with temperature is consistent
516
with damage occurring at the reaction centre itself rather than within the electron
517
transport chain (Ralph and Gademann, 2005).
518
During sea ice melt, algae face increases in PAR and temperature, and reduced
519
salinity. This study has demonstrated that the mixed algal communities of the Ross
520
Sea can tolerate higher PAR levels if temperature does not increase. Sea ice algae are
521
known to acclimate to higher irradiances over periods of at least a week (Sobrino and
22
522
Neale, 2007). However, this has been confirmed in only a few species and cannot yet
523
be generalized. As exposure temperature was increased in the current study there was
524
an increase in MAA production, which likely acts as a photoprotective compound.
525
Initial decreases in quantum yield at a temperature as high as 5°C were followed by
526
recovery, even at higher PAR levels, which suggests that bottom ice algae are not
527
shade-obligate species as previously thought (McMinn, et al., 2003, 2007, Thomas
528
and Dieckmann, 2002) and are capable of acclimation (Petrou, et al., 2011). Further
529
work with longer incubations is required to clarify the time required for acclimation
530
and to document recovery rates when the algae return to favourable conditions.
531
532
Acknowledgements
533
We acknowledge support from VUW grant 100241, and FRST Grant VICX0706.
534
MAF permits 2009037596 and 2010040450 were used to transport cultures and
535
samples from Antarctica to New Zealand. We thank Antarctica New Zealand for
536
logistic support in the field. Phil Lester, Kevin Newsham and an anonymous referee
537
are thanked for advice and critical comments in the development of this manuscript.
538
PC is supported by NERC funding to the BAS core programme ‘Ecosystems’. This
539
paper also contributes to the international SCAR research programme ‘AnT-ERA’.
540
Fig.1 was prepared by Peter Fretwell, BAS.
541
542
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