domain: sde sdu.ocean sdv

text:
Virginie Galindo
email: virginie.galindo@gmail.com

Michel Gosselin

Johann Lavaud

C J Mundy

Brent Else

Jens Ehn

Marcel Babin

Søren Rysgaard

Pigment composition and photoprotection of Arctic sea ice algae during spring

Keywords: Arctic, Snow melt, Ice algae, Pigments, Photoacclimation, Light memory

From the beginning of spring to the melt period, ice algae in the bottom of Arctic sea ice experience a large irradiance range, varying from <0.1% up to 25 or 30% of the incoming visible radiation. The increase in spring is usually rapid, with a varying photoacclimative response by bottom ice algae to protect themselves against excess light, such as changes in cellular pigment composition. This study focused on the temporal variation in pigment composition of bottom ice algae under 2 contrasting snow depths (thin and thick) during spring.

Controlled experiments were also carried out to investigate the photoprotective capacity of ice algae to relatively high irradiances during a short-term period (<6 h). Bottom ice algae were able to photoacclimate rapidly and effectively to irradiance ranging from 10 to 100 mol photons m 2 s 1 . However, we observed contrasting responses in photoacclimation depending on the ice algal community composition and their light history. Our experimental results suggest that the xanthophyll cycle (diadinoxanthin to diatoxanthin conversion) and D1-protein recycling play an important role in stabilizing photoprotection in ice algae. In addition, bottom ice algae likely employed a 'cellular light-exposure memory' strategy in order to improve their photoacclimative response to changing light exposure. According to our data, this process could be maintained over at least 2 wk. Hence, ice algae may be more resilient to varying light conditions than previously thought, and may be well-adapted for the expected future light regime changes associated with variability in snow and sea ice cover.

INTRODUCTION

The ice algal community resides mostly in the bottom 10 cm of sea ice (Smith et al. 1990[START_REF] <jrn>riedel | Winter-spring dynamics in sea-ice carbon cycling in the coastal Arctic Ocean[END_REF][START_REF] <jrn>juhl | Seasonal development and differential retention of ice algae and other organic fractions in first-year Arctic sea ice[END_REF]) and is largely dominated by diatoms [START_REF] <jrn>poulin | The pan-Arctic biodiversity of marine pelagic and sea-ice unicellular eukaryotes: a first-attempt assessment[END_REF]), a very abundant and productive group of unicellular algae [START_REF] <jrn>malviya | Insights into global diatom distribution and diversity in the world's ocean[END_REF]. Diatoms are successfully adapted to polar conditions due to their unique low-light acclimation [START_REF] <jrn>petrou | Southern Ocean phytoplankton physiology in a changing climate[END_REF][START_REF] <jrn>lacour | Growth, chl a content, photosynthesis, and elemental composition in polar and temperate microalgae[END_REF]. Ice algae represent the initial and most significant source of primary production during the winter-spring transition in Arctic waters. They contribute 5-20% of total marine primary production in Arctic seasonally ice-covered waters (e.g. [START_REF] <jrn>michel | Variability in oceanographic and ecological processes in the Canadian Arctic Archipelago[END_REF][START_REF] Arrigo | Primary producers and sea ice[END_REF][START_REF] Miller | Removal of snow cover inhibits spring growth of Arctic ice algae through physiological and behavioral effects[END_REF]) and >50% in perennially ice-covered waters [START_REF] <jrn>legendre | Ecology of sea ice biota[END_REF][START_REF] <jrn>gosselin | New measurements of phytoplankton and ice algal production in the Arctic Ocean[END_REF]. Primary production of ice algae is influenced by many environmental variables such as temperature [START_REF] Arrigo | The influence of salinity and temperature covariation on the photophysiological characteristics of Antarctic sea ice microalgae[END_REF], salinity [START_REF] <jrn>ryan | Acclimation of Antarctic bottom-ice algal communities to lowered salinities during melting[END_REF], and nutrient availability [START_REF] Mp | Photosynthetic capacity in microalgae associated with Antarctic pack ice[END_REF][START_REF] Denman | Modeling ice algal growth and decline in a seasonally ice-covered region of the Arctic (Resolute Passage, Canadian Archipelago)[END_REF], but mainly by irradiance in the range of photosynthetically active radiation (EPAR; 400 to 700 nm), which is principally regulated by the thickness of snow and ice cover [START_REF] <jrn>mundy | Variability of snow and ice thermal, physical and optical properties pertinent to sea ice algae biomass during spring[END_REF][START_REF] Aumack | Light and nutrient effects on the settling characteristics of the sea ice diatom Nitzschia frigida[END_REF] in addition to the annual cycle and cloudiness. EPAR reaching the bottom of first-year sea ice is often <1% of incident irradiance [START_REF] Arrigo | A simulated Antarctic fast-ice ecosystem[END_REF][START_REF] <jrn>lazzara | Light environment and seasonal dynamics of microalgae in the annual sea ice at Terra Nova Bay (Ross Sea, Antarctica)[END_REF]) at the beginning of spring, and thus the ice algal community is adapted to perform photosynthesis under very low irradiance (e.g. Cota 1985[START_REF] <jrn>johnsen | Photoadaptation of sea-ice microalgae in the Barents Sea[END_REF], Kühl et al. 2001). However, the rapid increase in light intensity and day length from winter to the spring melt period [START_REF] <jrn>sakshaug | Light and productivity of phytoplankton in polar marine ecosystems: a physiological view[END_REF] results in a rapid increase in EPAR of up to 25 or 30% of incident irradiance at the bottom ice [START_REF]On the aggregate-scale partitioning of solar radiation in Arctic sea ice during the Surface Heat Budget of the Arctic Ocean (SHEBA) field experiment[END_REF][START_REF] <jrn>campbell | Remote estimates of ice algae biomass and their response to environmental conditions during spring melt[END_REF], exposing the ice algae to a large range of EPAR. Light conditions can also change rapidly on a daily basis, mainly due to opposite and/or cumulative effects of melting and storms (rain or snow) that modify the snow cover thickness and optical properties. Furthermore, the acceleration of global warming in the Arctic affects snow and sea ice cover. Snow cover declined by 40% between 1989and 2009[START_REF] <jrn>screen | Declining summer snowfall in the Arctic: causes, impacts and feedbacks[END_REF][START_REF] Je | Future Arctic climate changes: adaptation and mitigation time scales[END_REF]) and during some periods, precipitation has switched from snow to rain (Comiso & Hall 2014), while sea-ice extent has retreated by 45% in the last 3 decades [START_REF] <jrn>screen | Dramatic interannual changes of perennial Arctic sea ice linked to abnormal summer storm activity[END_REF][START_REF] <jrn>stroeve | The Arctic's rapidly shrinking sea ice cover: a research synthesis[END_REF]. All of these changes, accompanied by an earlier melt onset [START_REF] <jrn>markus | Recent changes in Arctic sea ice melt onset, freeze up, and melt season length[END_REF], are expected to increase the EPAR levels transmitted through the sea-ice cover. This increase in transmitted EPAR is foreseen to enhance ice algal biomass [START_REF] <jrn>poulin | The pan-Arctic biodiversity of marine pelagic and sea-ice unicellular eukaryotes: a first-attempt assessment[END_REF]) and production [START_REF] <jrn>wassmann | Footprints of climate change in the Arctic marine ecosystem[END_REF]), but ice algae may also face light stress [START_REF] <jrn>leu | A (too) bright future? Arctic diatoms under radiation stress[END_REF][START_REF] <jrn>petrou | Southern Ocean phytoplankton physiology in a changing climate[END_REF] which may be of importance since they are adapted to very low light levels.

In order to sustain photosynthesis under changing light conditions, ice algae use some photobehavioral (i.e. vertical migration in pennate diatoms; [START_REF] Aumack | Light and nutrient effects on the settling characteristics of the sea ice diatom Nitzschia frigida[END_REF]) and photophysiological features described as photoprotection and photoacclimation. These photoprotective mechanisms allow algae to minimize oxidative photodamage generated by excess light exposure, and specifically to maintain photosystem II (PSII) photochemistry. They include changes in cellular photosynthetic and photoprotective pigment composition [START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF], with an increase in antioxidant carotenes and xanthophylls depending on incident irradiance. On shorter time scales (i.e. <1 h) of light fluctuations, the most important photoprotective processes are the repair of damaged PSII [START_REF] <jrn>petrou | Rapid photoprotection in sea-ice diatoms from the East Antarctic pack ice[END_REF]) and thermal dissipation of excess energy [START_REF] <jrn>goss | Biodiversity of NPQ[END_REF]. This process comprises a fast (seconds to minutes) enzymatic light-driven conversion of xanthophyll cycle (XC) pigments [START_REF] <jrn>goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. In diatoms, the XC consists of the de-epoxidation of diadinoxanthin (DD) to diatoxanthin (DT) [START_REF] <edb>brunet | Pigments and photoacclimation processes[END_REF][START_REF] <jrn>kuczynska | Photosynthetic pigments in diatoms[END_REF]. In polar conditions, the XC is very important for optimizing algal photosynthetic activity during the spring-summer transition when transmitted irradiance at the ice-water interface increases [START_REF] <jrn>kashino | Strategies of phytoplankton to perform effective photosynthesis in the North Water[END_REF][START_REF] <jrn>katayama | Photoprotective responses of an ice algal community in Saroma-Ko Lagoon, Hokkaido, Japan[END_REF][START_REF] Sy | Strategy of photo-protection in phytoplankton assemblages in the Kongsfjorden, Svalbard, Arctic[END_REF][START_REF] <jrn>katayama | Photoprotection and recovery of photosystem II in the Southern Ocean phytoplankton[END_REF].

As bottom ice algae strongly contribute to Arctic marine primary production during spring, several studies have described their photophysiological responses to a change in EPAR levels from winter to spring (e.g. [START_REF] <jrn>gosselin | Responses of sea-ice microalgae to climatic and fortnightly tidal energy inputs (Manitounuk Sound, Hudson Bay)[END_REF][START_REF] Barlow | Photoadaptive strategies in sea-ice microalgae[END_REF][START_REF] <jrn>kudoh | Photosynthetic acclimation and the estimation of temperate ice algal primary production in Saroma-ko Lagoon, Japan[END_REF][START_REF] Ss | Small scale vertical gradients of Arctic ice algal photophysiological properties[END_REF], through the spring season (e.g. [START_REF] <jrn>michel | Photo-adaptation of sea-ice microalgae in springtime: photosynthesis and carboxylating enzymes[END_REF][START_REF] <jrn>cota | Physical control of Arctic ice algal production[END_REF][START_REF] Ban | Comparative analysis of photosynthetic properties in ice algae and phytoplankton inhabiting Franklin Bay, the Canadian Arctic, with those in mesophilic diatoms during CASES 03-04[END_REF][START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF]) and from spring to summer [START_REF] <jrn>kühl | Photosynthetic performance of surface-associated algae below sea ice as measured with pulse amplitude modulated (PAM) fluorometer and O2 microsensors[END_REF], with a special focus on the potential deleterious effects of ultraviolet radiation (UVR) [START_REF] <jrn>enberg | Solar PAR and UVR modify the community composition and photosynthetic activity of sea ice algae[END_REF][START_REF] <jrn>leu | A (too) bright future? Arctic diatoms under radiation stress[END_REF]. A few works have also focused on the impact of change in snow cover on the photoacclimation of ice algae [START_REF] <jrn>juhl | Effects of snow removal and algal photoacclimation on growth and export of ice algae[END_REF], Lund-Hansen et al. 2014), but little is known about their capacity to acclimate to rapid light changes through pigment photoprotection [START_REF] <jrn>kudoh | Xanthophyll-cycle of ice algae on the sea ice bottom in Saroma Ko lagoon, Hokkaido, Japan[END_REF][START_REF] <jrn>katayama | Photoprotective responses of an ice algal community in Saroma-Ko Lagoon, Hokkaido, Japan[END_REF][START_REF] <jrn>petrou | Southern Ocean phytoplankton physiology in a changing climate[END_REF]. Therefore, the main objectives of this study were to (1) determine the change in pigment composition including XC pigments related to the photoprotective response of the bottom ice algal community under 2 dominant snow depths (thin and thick) from the beginning of spring to early summer, and (2) investigate the short-term photoacclimation response to an experimental light gradient of bottom ice algae acclimated to 2 snow cover sites.

MATERIALS AND METHODS

Study site, sample collection and experiments

The study was conducted on landfast first-year sea ice, located north of Davis Strait near Qikiqtarjuaq, Nunavut, Canada (67° 28' N, 63° 47' W;

), as part of the Green-Edge project.

Sea ice samples were collected for measurements of pigments every 2 d from 27 April to 6 July 2015.

For the time series, ice sampling was performed at thick (>25 cm) and thin (15 to 20 cm less than thick snow) snow depths. Snow depth and ice thickness were measured on each ice sampling day with a ruler and an ice thickness gauge (Kovacs Enterprises), respectively.

Transmittance of EPAR at the ice-water interface was also measured on each sampling day (see details below). The bottom 3 cm sections were extracted from sea-ice cores using a 14 cm internal diameter ice corer (Mark V Coring System; Kovacs Enterprises). For pigment analysis, at least 2 ice cores were extracted per snow site and pooled immediately in a dark isothermal container to avoid brine drainage losses. These ice core samples were melted in 0.2 m filtered seawater (FSW) (3 parts FSW to 1 part melted ice) to minimize osmotic stress on the microbial community during melting [START_REF] Bates | Ultraviolet attenuation by dissolved and particulate constituents of first-year ice during late spring in an Arctic polynya[END_REF][START_REF] <jrn>garrison | Organism losses during ice melting: a serious bias in sea ice community studies[END_REF]. Ice-water interface samples for salinity and nutrient determination were collected through an auger hole with a battery-operated plastic submersible pump (Cyclone®) secured to the end of an articulated under-ice arm.

Three distinct types of experiments with ice algae were carried out on 11 occasions between 6 and 31 May 2015 ( ). For these experiments, the bottommost 1 cm of 3 sea-ice cores were quickly scraped [START_REF] Hirawake | Contribution of benthic microalgae to ice covered coastal ecosystems in northern Hokkaido, Japan[END_REF][START_REF] Martin | Phytoplankton and sea ice algal biomass and physiology during the transition between winter and spring (McMurdo Sound, Antarctica)[END_REF] and pooled in a dark isothermal container with 0.2 m FSW (ca. 38 parts FSW to 1 part melted ice) in order to reduce the time of melting, which can impact physiological processes. Indeed, too long of a dark period during the ice melting could result in DT to DD de-epoxidation [START_REF] <jrn>goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. In our study, ice samples were melted in less than 30 min. The melted ice was gently mixed, and nutrients were sampled before the water was distributed into clear polycarbonate bottles under dim light conditions (i.e. diffusive light in a Polarhaven shelter with opaque walls and 2 end windows covered by 4 layers of black plastic sheeting).

During the first experiment (Table 1), three 500 ml polycarbonate bottles (EPAR transmittance of ca. 80%) containing ice melt water were placed in each experimental chamber.

Each chamber was then exposed to one light intensity (10, 50, 100 or 200 mol photons m 2 s 1 ) using cool white dimmable LEDS (9 W) and neutral density light filters (LEE Filters). During the experimental period, irradiance at the ice-water interface ranged from 0.3 to 8 mol photons m 2 s 1 , and therefore ice algae were acclimated to different light levels in their natural environment. The experimental chambers were continuously cooled with running seawater pumped from the ice-water interface using a small electric submersible pump (Lifegard Aquatics QuietOne Model 1200, 317 GPH). The ice algae were exposed to their respective treatments for 3 h and then placed at the lowest light levels achievable in the field (<5 mol photons m 2 s 1 , incubators covered by 4 layers of black plastic sheeting) for a 2 h dark-recovery period. The same experiment was repeated on 7 occasions and the data from the same ice algae (from thin or thick snow) and the same period (before or during snowfall) were averaged. Subsampling for pulse-amplitude modulation (PAM) fluorometry measurements (i.e. chlorophyll a [chl a]

fluorescence) occurred at 0, 0.5, 1, 2, 3 and 5 h, while pigment composition was assessed at 0, 3 and 5 h.

In the second experiment (Table 1), the relative importance of photoprotection and photorepair mechanisms were assessed using 2 inhibitors: dithiothreitol (DTT) and lincomycin.

The xanthophyll inhibitor DTT prevents the de-epoxidation of DD into DT (Olaizola et al. 1994[START_REF] Rousseau | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF] In the third experiment (Table 1), duplicate 2 l polycarbonate bottles containing melted ice water from both snow covers were incubated in an opaque-walled incubator located on the snow, exposed to incident irradiance (EPAR + UVR) and continuously cooled with running seawater pumped from the ice-water interface using a small electric submersible pump (Pondmaster magnetic drive utility pump, Model 500 GPH). Subsamples were collected for PAM fluorescence and pigment analysis after 30 min and every hour thereafter for 3 h.

Irradiance measurement

During the study period, transmitted irradiance was measured every sampling day between 10:00 and 11:00 h (local time; UTC -05:00) using a compact-optical profiling system (C-OPS; Biospherical Instruments). Incident and under-ice downward irradiances were recorded at 19 individual wavelengths (from 320 to 780 nm) by a cosine light sensor. The vertical profile of irradiance under the sea ice was measured by the IcePRO version of the instrument, which is specifically design to fit and sink through a 25 cm diameter ice auger hole. EPAR was computed by constructing a piecewise cubic hermit interpolating polynomial (PCHIP) using downward irradiance measured at all C-OPS wavelengths and then numerically integrating from 400 to 700 nm to obtain final units of mol photons m 2 s 1 (Mobley 1994). The transmitted irradiance at the ice-water interface was calculated as detailed in Belzile et al. (2000). The water column diffuse attenuation coefficient was determined from the linear portion of triplicate measurements of the natural logarithm of the transmitted irradiance versus depth. Due to the alteration of light profiles underneath the ice because of the auger hole and the snow added on it, the EPAR plots were linear, on average, from 10 to 50 m. The r 2 of the relationship between the natural logarithm of the transmitted irradiance versus depth was always >0.99.

To estimate incident irradiance and EPAR at the ice-water interface over a day, we used incident downward shortwave (305 to 2800 nm) radiation, which was measured continuously (as 1 min averages). The shortwave radiation measurements were made using the upward-looking cosine response pyranometer on a 4-sensor net radiometer (Kipp & Zonen; model CNR4) placed at an undisturbed site approximately 20 m west of the meteorological station at a height of 1 m above the snow. The instrument included a heater/ventilator unit (Kipp & Zonen; model CNF4) that cycled on for 5 min at the beginning of every hour to keep the instrument lenses free of snow and frost. We noticed that the heater/ventilator caused a transient increase in measured shortwave radiation under certain atmospheric conditions (light winds, clear skies). In these cases, we used a smoothing algorithm to remove minor data spikes. Incident downward shortwave radiation was converted to EPAR (multiplied by 0.47) as described by [START_REF] <jrn>papaioannou | Relationships of photosynthetically active radiation and shortwave irradiance[END_REF]. Then the EPAR value in W m 2 was converted into mol photons m 2 s 1 using a factor of 4.15 as described in [START_REF] <jrn>halverson | High-resolution observations of chlorophyll-a biomass from an instrumented ferry: influence of the Fraser River plume from 2003 to 2006[END_REF]. As algae act as scalar collectors with a maximum interception of light incident from all directions due to the arrangement of their photosynthetic tissues, scalar irradiance is the preferred measurement (Kirk 2011). To calculate scalar irradiance at the ice-water interface, we first estimated downward irradiance at the icewater interface by multiplying the incident downwelling EPAR on the surface by the calculated EPAR transmittance (as detailed above). This subsequent downward irradiance was converted to downward scalar irradiance by dividing by an average cosine factor of 0.7 (Ehn & Mundy 2013).

To simplify, we assumed that the upward scalar irradiance was negligible at the ice-water interface. Due to this assumption, we note that our scalar irradiance estimates are likely conservative. Although no direct measurements have been reported, the modeling study of [START_REF] <jrn>pavlov | Altered inherent optical properties and estimates of the underwater light field during an Arctic under-ice bloom of Phaeocystis pouchetii[END_REF] estimated scalar irradiance at the ice bottom to be ~1.8 times greater than EPAR, in comparison to our estimate that was scaled by a factor of 1.4.

Salinity and nutrients

Salinity of melted ice samples was measured with a hand-held conductivity meter (330i/SET; WTW) calibrated against a 15 N KCl solution at 20°C. Samples for nutrient determination at the ice-water interface and in the experiments were filtered through a precombusted (5 h at 450°C) Whatman GF/F glass-fiber filter (nominal porosity of 0.7 m) inserted in a filter holder. The filtrate was collected into 20 ml polyethylene vials after thorough rinsing.

Samples were poisoned with mercuric chloride (final concentration 10 g ml 1 ) according to [START_REF]Stability of solutions of nutrient salts during storage[END_REF], and stored in the refrigerator until analysis. Nitrate plus nitrite (hereinafter NOX), nitrite, phosphate and silicic acid were analyzed using a Bran Luebbe Seal autoanalyzer according to the method of [START_REF] Aminot | Dosage automatique des nutriments dans les eaux marines: méthodes en flux continu[END_REF]. The analytical detection limits for NOX, phosphate and silicic acid were 0.05, 0.02 and 0.2 mol l 1 , respectively.

Chl a variable fluorescence

To study the photosynthetic responses of algae, PAM fluorometry was used. It is a widely used methodology that provides a rapid, non-invasive assessment of the photosynthetic apparatus of algal cells [START_REF] <jrn>parkhill | Fluorescence-based maximal quantum yield for PSII as a diagnostic of nutrient stress[END_REF]. It is also a useful tool to examine the ability of photosynthetic organisms to tolerate environmental stressors and the extent of damage caused by these stresses [START_REF] <jrn>maxwell | Chlorophyll fluorescence-a practical guide[END_REF]. Fluorescence measurements were made with a water-PAM cuvette version with blue LEDs (Walz) inside the unheated field laboratory (Polarhaven shelter), which was set up at the sampling station. All samples were placed in a small cooler containing 2 ice packs during their dark-acclimation for at least 30 min before the measurement of fluorescence.

The samples were stirred during measurements.

Minimum (F0) and maximum fluorescence (Fm) levels were assessed, and the maximum quantum yield of PSII photochemistry (Fv/Fm) was calculated as follows:

Fv/Fm = (Fm -F0) / Fm (1)
Fm was obtained using a saturating pulse of ca. 3000 mol photons m 2 s 1 for 0.8 s. When measured on a community, Fv/Fm changes must be interpreted with care (see, for example, [START_REF] <jrn>parésys | Quantitative and qualitative evaluation of phytoplankton communities by trichromatic chlorophyll fluorescence excitation with special focus on cyanobacteria[END_REF]. Nevertheless, in our study, sea-ice algae communities were largely dominated by diatoms (see 'Results'). Hence, Fv/Fm variations can be largely attributed to changes in the photosynthetic efficiency of diatoms.

Rapid light curves (RLCs) were also generated to determine the photosynthetic parameters under both snow covers as a function of snow conditions (before or during snow events). Dark-acclimated samples were exposed to successive increasing actinic light ranging from 0 to 139 mol photons m 2 s 1 for 30 s each. The actinic light was measured with a spherical micro quantum sensor US-SQS (Walz). The relative electron transport rate (rETR; dimensionless) values were calculated as follows:

rETR = PSII × E (2)
where PSII = (Fm' -F') / Fm', known as the operational PSII quantum yield and E is the irradiance applied.

Data from RLCs were fitted using the following equation (Eilers & Peeters 1988, Zonneveld 1998) using a Levenberg-Marquardt regression algorithm:

rETR= E / (aE 2 + bE + c) (3)
where E is irradiance, and a, b and c are regression coefficients to fit the rETR versus E curve.

RLC measurements allow us to describe the main photosynthetic properties of an algal sample, including the maximum (rETRmax), the light use efficiency () represented by the initial slope of the curve (mol photons m 2 s 1 ) 1 and the light saturation coefficient (Ek; mol photons m 2 s 1 ) [START_REF] Kg | The shortterm effect of irradiance on the photosynthetic properties of Antarctic fast-ice microalgal communities[END_REF]) using the following expressions:

 = 1 / c (4) rETRmax = [b + 2(ac) 0.5 ] 1 (5) Ek = rETRmax /  (6)
As for typical 14 C P-E curves, these photosynthetic parameters were used to compare the photosynthetic performances and photoacclimation properties of algal communities.

Pigments and CHEMTAX analysis

The identification and concentration of algal pigments were determined by reverse-phase HPLC. Samples (50 to 200 ml, depending on the biomass) were filtered onto 25 mm Whatman GF/F filters using a vacuum pump (0.8 mm Hg), wrapped in aluminum foil and stored immediately at -80°C until analysis. For the time series, the algal pigments of the bottom 3 cm of the ice were extracted at -20°C in 3 ml 100% methanol, disrupted by 10 s of sonication (Bandelin Sonopuls HD2200) and filtered 1 h later through 25 mm Whatman GF/F filters. The pigments were separated and quantified as described in Ras et al. ( 2008), a method adapted from [START_REF] <jrn>van Heukelem | Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments[END_REF]. Pigments were analyzed using an Agilent Technologies 1200

Series system with a narrow reversed-phase C8 Zorbax Eclipse XDB column (150 × 3 mm, 3.5 m particle size) which was maintained at 60°C. A diode-array detector allowed measuring the absorption of most pigments at 450 nm, while chl a and its derivatives were detected at 667 nm.

For the light experiments, the algal pigments were extracted as described in Alou-Font et al.

( 2013,2016) and disrupted by 10 s of sonication (Heat Systems; model XL2010). The remaining pigment samples were analyzed using an Agilent Technologies 1200 Series but with a Symmetry C8 column (150 × 4.6 mm, 3.5 m particle size; Waters Corporation). Pigments were detected with a G1315P diode-array absorbance detector (400 to 700 nm) and chlorophylls were detected by fluorescence (excitation at 400 nm and emission at 650 nm; G1321A fluorescence detector).

We used the HPLC separation method described in Zapata et al. (2000). For both methods, pigments were identified based on retention time and spectral properties of external pigment standards, even for degradation pigments (chlorophyllide a, pheophytin a, pheophorbide a) (DHI Lab Products) [START_REF] Es | Data sheets aiding identification of phytoplankton carotenoids and chlorophylls[END_REF]. Limits of detection and quantification were estimated as in [START_REF] Bidigare | Analysis of algal pigments by highperformance liquid chromatography[END_REF] and pigments with concentrations below the limit of detection were not reported. In this study, total chl a (Tchl a) mentioned thereafter corresponds to the sum of chl a, chlorophyllide a, chl a allomer and epimer measured by HPLC. The ratios of photoprotective carotenoids (PPC; including DD, DT, violaxanthin, zeaxanthin and ,-carotene) to photosynthetic carotenoids (PSC; including fucoxanthin, peridinin, 9'-cis-neoxanthin and alloxanthin) were also calculated. The de-epoxidation state (DES) index, an indicator of photoprotection [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF], was expressed as DES = DT / (DD + DT) and calculated for the different experiments.

The contribution of major algal groups to chl a was determined from marker pigment ratios using the CHEMical TAXonomy (CHEMTAX) program (Mackey et al. 1996, version

Statistical analysis

Normality of the data was determined using the Shapiro-Wilk test with a 0.05 significance level. When the data were normally distributed, we used parametric tests. For the time series and the third experiment, paired t-tests were used to compare paired variables from the thin and thick snow cover sites. For non-parametric data, a Wilcoxon signed rank test was used instead. To determine differences among the 3 sampling periods, a 1-way ANOVA by ranks (Kruskal-Wallis test) was performed and completed by a multiple mean comparison test using rank sums (Dunn's test) adjusted with the Bonferroni method. In addition, Pearson's linear correlations (r) on parametric data were used to infer the relationship between 2 variables. For the first experiment, 1-way repeated ANOVA was performed followed by a post hoc Tukey's test to identify averages that were significantly different between treatments. A t-test was also conducted to determine the difference in algal responses (e.g. decline) depending on snow cover.

RESULTS

Change in environmental conditions

Because of temporal changes in snow cover depth resulting from major snow precipitation events on 16 and 20 May, we distinguish 3 sampling periods (i.e. before and during snow events, and snowmelt;

). Snow depth remained relatively stable at both sites until 12

May, and increased from ca. 10 to 30 cm and from ca. 25 to 51 cm on 20 May at thin and thick snow cover sites, respectively (Fig. 2a). Thereafter, snow depth slowly decreased at both sites until 9 June, when it decreased faster until reaching 0 cm on 20 June at the thin snow site and later at the thick snow site. Mean sea-ice thickness ranged from 132 to 96 cm during the sampling season (Fig. 2b) and was significantly thinner during the melting period (mean = 116 cm) than during the 2 previous periods (mean = 124 cm) (Dunn's test, p < 0.05).

Nutrient concentrations at the ice-water interface varied from 5.38 to 1.38 mol l 1 for NOX (Fig. 2c), from 0.22 to 0.04 mol l 1 for NO2  , from 7.4 to 5.5 mol l 1 for Si(OH)4 and from 0.92 to 0.60 mol l 1 for PO4 3 . NOX concentrations oscillated between 4.21 and 5.38 mol l 1 from the beginning of the sampling period until 10 June and then started to decrease progressively down to 1.38 mol l 1 at the end of sampling (Fig. 2c). Mean concentrations of NOX, Si(OH)4 and PO4 3 were significantly lower during the melting period (after 10 June) than during the 2 previous periods (Dunn's test, p < 0.05).

EPAR transmittance through the ice and snow remained relatively constant at around 0.06% of incident irradiance until 31 May and then increased progressively up to 4.6% on 6 July due to the complete snowmelt (Fig. 2d). Mean EPAR transmittance was significantly higher after 9 June than before this date (Dunn's test, p < 0.05).

Effect of snow events and snow cover depth on ice algal biomass, pigment composition and photosynthetic properties

During the first sampling period (i.e. before snow events), bottom ice Tchl a concentration, used as an index of ice algal biomass, was significantly higher under the thin snow (9.28 mg m 2 ) than under the thick snow (5.14 mg m 2 ) cover (paired t-test, t = 3.632, df = 11, p < 0.01) ( a). During the following 2 periods (i.e. snow events and snowmelt), no significant difference in algal biomass was observed between the 2 snow cover conditions (paired t-test, t = 1.556, df = 9, p = 0.15 during snow events and t = 0.566, df = 4, p = 0.60 during snowmelt). During the snow events period, bottom ice Tchl a concentration reached a maximum value of 22 mg m 2 on 27 May and 32 mg m 2 on 2 June at the thick and thin snow sites, respectively. Thereafter, Tchl a concentrations decreased progressively to reach a minimum value of 0.18 mg m 2 at the thick snow site during the snowmelt period. No samples were collected at the thin snow site after 24 June.

The concentrations of PSC and PPC followed the same trend as Tchl a. PSC variations were mainly influenced by fucoxanthin, while PPC were mostly governed by DD and ,carotene. The PPC:PSC ratio was relatively constant before and during snow events, with values around 0.1 wt:wt, followed by an increase up to 0.81 wt:wt during the snowmelt period (Fig. 3b).

Before snow events, the PPC:PSC ratio was significantly higher under thin snow (0.12 wt:wt) than under thick snow (0.09 wt:wt) (paired t-test, t = 2.393, df = 11, p < 0.05). This ratio was positively correlated with EPAR transmittance at the ice-water interface under both thin (r = 0.816, p < 0.001) and thick snow covers (r = 0.828, p < 0.001) and during the snowmelt period (r = 0.812, p < 0.001). Over the entire sampling period, the ratio of (DD+DT) to Tchl a followed the same trend as PPC:PSC (Fig. 3b,c). (DD+DT):Tchl a was stable around 0.03 wt:wt before and during snow events and increased to 0.28 wt:wt during the snowmelt period under both snow cover conditions. It was significantly higher under thin snow (0.04 wt:wt) than under thick snow (0.02 wt:wt) cover before snow events (paired t-test, t = 3.460, df = 11, p < 0.01) and was positively correlated with ice bottom EPAR transmittance during the snowmelt period (r = 0.667, p < 0.05).

The contributions of major algal groups to chl a were determined via CHEMTAX analysis, which showed that diatoms were always dominant under both snow covers during the entire sampling period (>82%;

). Diatoms Type 2, containing fucoxanthin and chl c2 and c3, were likely associated with pennate diatoms in Alou-Font et al. ( 2013), while diatoms Type 1 were associated with centric diatoms. Some differences occurred depending on the snow cover and snow periods. Before snow events, diatoms Type 2 (i.e. pennate diatoms) totally dominated under the thin snow cover, while the algal community was more heterogeneous under thick snow with the presence of diatoms Type 1 (16%), cryptophytes (14%) and dinoflagellates (3%).

Thereafter, during snow events, the algal community was more heterogeneous under both snow covers, but dinoflagellates (3%) and cryptophytes (2%) were relatively more abundant under thin snow cover. Finally, diatoms entirely dominated the algal community during the snowmelt period (ca. 99%) with a dominance of diatoms Type 1 (i.e. centric diatoms), but diatoms Type 2 (i.e. pennate diatoms) were 4 times more abundant under thick snow cover (20 vs. 5%). Hence, despite the dominance of diatoms in all samples, some differences in algal community (heterogeneous or diatom types) occurred depending on the environmental conditions.

Before the period of snow events, there was a clear difference in photosynthetic properties and photoacclimation in ice algae living under thin or thick snow cover ( ). As expected,  was lower and rETRmax was higher under thin snow. As a result, Ek was twice as high (ca. 45 mol photons m 2 s 1 ) under thin snow. During the snow event period, ice algae under the thin snow cover maintained rETRmax and increased  resulting in a Ek decrease of half, reaching a value similar to that under the thick snow cover before the snow event period.

Interestingly, ice algae under thick snow cover maintained  (probably because it was already at its maximum), yet increased rETRmax to a similar level as ice algae under thin snow cover during snow events.

Effect of a natural range of light exposure on the photophysiology of bottom ice algae as a function of snow conditions

Two types of experiments were performed to determine the short-term effect of enhanced irradiance on the photophysiological response of bottom ice algae (see Table 1). The aim of the first experiment was to assess the short-term effect of enhanced irradiance over a range typically observed at the ice-water interface (10 to 200 mol photons m 2 s 1 ; see [START_REF] <jrn>ryan | Chlorophyll fluorescence imaging analysis of the responses of Antarctic bottom-ice algae to light and salinity during melting[END_REF][START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF]) on the photophysiological response of bottom ice algae acclimated to different light environments depending on the snow conditions (i.e. snow depth and snow events). Before snow events, the Fv/Fm ratio, a proxy for the maximum quantum yield for PSII photochemistry, was significantly lower after a 3 h light-exposure to the highest light treatment (i.e. 200 mol photons m 2 s 1 ) (1-way repeated ANOVAs completed by post hoc Tukey's tests, q = 4.644 and p < 0.05 for thin snow, and q = 3.464 and p < 0.05 for thick snow cover) ( a,b). The decrease in Fv/Fm was greater for thick snow (t-test, t = 24, p < 0.05). After 2 h of darkness, Fv/Fm did not change. For the thin snow cover (Fig. 5a), the Fv/Fm response was different with an increase throughout the light exposure and recovery period, especially for the 10 and 50 mol photons m 2 s 1 treatments, and it reached final values higher than those at T0 (ca. 0.55). For thick snow cover (Fig. 5b), Fv/Fm strongly decreased during the first 30 min of illumination in light treatments of 10, 50 and 100 mol photons m 2 s 1 , followed by stabilization and a recovery to T0 values during dark recovery. During snow events (Fig. 5c,d ) to a maximum level of 0.14 to 0.20 wt:wt. While it was strongly positively correlated with the increase of EPAR at the ice-water interface (r = 0.85, p < 0.001), no significant difference was observed before or during snow events or between snow covers (thin or thick).

After 2 h of dark-recovery, DES decreased back to its T0 values, independent of snow events and snow depth conditions.

In a second, complementary experiment, we aimed to determine the respective importance of DD de-epoxidation and of the synthesis of PSII D1 (psbA) protein to support bottom ice algae in maintaining their photochemical performance when exposed to their natural light environment. For that purpose, bottom ice algae were collected under thin snow cover and incubated at the ice-water interface in the absence (control) and presence of the inhibitor of DD de-epoxidation (i.e. DTT), and of D1 protein synthesis (i.e. lincomycin) ( ). During the experiment, estimated ice bottom EPAR transmittance progressively decreased from 105 to 53 mol photons m 2 s 1 from 11:00 to 17:00 h local time (Fig. 7a), a range of irradiance encompassed by that of the first experiment. In all treatments, Fv/Fm decreased during the first 4 h (Fig. 7b; ca. 25 to 30%) until reaching similar values at mid-afternoon (15:00 h) and increased slightly thereafter concomitantly with the sharpest EPAR decrease (from 75 to 53 mol photons m 2 s 1 ). There was a greater Fv/Fm decline after 2 h light exposure in the presence of DTT and lincomycin (ca. 20 to 25%) in comparison with the control conditions (5%). It is noteworthy that Fv/Fm response in control conditions after 2 h light exposure was similar to the response observed in our controlled experimental light treatment (Expt 1, thin snow cover, 100

mol photons m 2 s 1 ; Fig. 5a). In control conditions, DES increased for the first 2 h from 0.063 to 0.086 wt:wt and stabilized until 15:00 h, after which it decreased back to T0 values, while EPAR at the ice-water interface reached its lowest values (Fig. 7a). In DTT-treated samples, DES was stable around its T0 value. At T0, DES in lincomycin-treated samples was significantly higher than in the control and DTT treatments, and did not change during the day, even when EPAR at the ice-water interface decreased. At 17:00 h, Fv/Fm in DTT-treated samples was at 80% of the initial ratio, as for the control, while Fv/Fm in lincomycin-treated samples was only at 40% of the initial ratio. However, DES in lincomycin-treated samples was the highest by a factor of 2 in comparison with other treatments.

Effect of high light exposure on the photophysiology of ice algae released from bottom sea ice

The aim of the third experiment (Table 1) was to assess the photoprotective capability of bottom ice algae when exposed to a sudden increase in irradiance, similar to what they would experience when carried into adjacent ice-free areas by surface currents after their release in the water column. Our hypothesis was that bottom ice algae from thin snow cover should be less light-sensitive than those from thick snow cover. To test this hypothesis, bottom ice algae were collected under thin and thick snow covers and exposed to incident irradiance ( ). During the experiment, incident EPAR was constant at an average of 1107 ± 95 mol photons m 2 s 1 (Fig. 8a).

Fv/Fm of bottom ice algae from thin and thick snow covers reached the same minimum value (ca. 0.05), illustrating a high level of photoinhibition (i.e. -75% in Fv/Fm) when exposed to these over-saturating light intensities (10 times higher than the maximum average of bottom ice; see Fig. 8a) after a 3 h period. Nevertheless, the pattern of Fv/Fm variations was very different between the 2 algal communities (Fig. 8b). For algae from thin snow cover, Fv/Fm decreased progressively during the first 2 h of light exposure, and sharply during the last hour of exposure.

For thick snow cover, Fv/Fm showed a rapid drop close to 0 after the first 0.5 h, followed by a slight but significant recovery to its final value.

At the same time, ice algae underwent substantial changes in pigment content, with a Tchl a decrease (Fig. 8c) and DES increase (Fig. 8d); mean values were significantly different under thin and thick snow cover conditions (paired t-test, t = 12.394, df = 5, p < 0.001 for Tchl a and Wilcoxon signed rank test, W =21, p < 0.05 for DES). More specifically, under thin snow cover, Tchl a decreased by 20% (from 1228 to 987 g chl a l 1 ) during the first 0.5 h of light exposure, followed by a slower but consistent decrease of 23% (from 987 to 759 g chl a l 1 ) during the rest of the experiment. Simultaneously, DES increased from a value of 0.09 to 0.19 wt:wt during the first 0.5 h of light exposure and then stabilized around a value of 0.18 wt:wt at end of the experiment. Under thick snow cover, Tchl a followed a consistent decrease of 33%, from 146 to 98 g l 1 during the 3 h light exposure, while DES did not show significant changes, varying between 0.12 and 0.15 wt:wt.

DISCUSSION

Effect of snow cover on the taxonomic composition and photosynthetic properties of bottom ice algae

According to previous studies at similar latitudes (ca. 70° N) (Renaud et al. 2007[START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF], the ice algal bloom starts between the end of March and beginning of April. In 2015, snow events around mid-May may have extended the ice algal bloom period by reducing EPAR penetration through the ice and thermally insulating the ice, which consequently delayed the melting process that usually terminates the bloom (e.g. Fortier et al. 2002[START_REF] <jrn>mundy | Variability of snow and ice thermal, physical and optical properties pertinent to sea ice algae biomass during spring[END_REF], Campbell et al. 2015). Thus, a positive relationship was observed between snow depth and Tchl a concentration (r = 0.693, p < 0.001), contrary to observations for early spring when light is limiting [START_REF] <jrn>mundy | Influence of snow cover and algae on the spectral dependence of transmitted irradiance through Arctic landfast first-year sea ice[END_REF][START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF]), but consistent with later bloom conditions (Campbell et al. 2015[START_REF] <jrn>leu | Arctic spring awakeningsteering principles behind the phenology of vernal ice algae blooms[END_REF].

Under both snow covers (thin and thick), the bottom ice algal community was mainly composed of diatoms (Fig. 4), as confirmed by Imaging Flow CytoBot data (P. L. Grondin pers.

comm.) and as also reported previously [START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF]. Whilst taxonomic studies in the Arctic have shown that pennate diatoms contribute to at least 68% of the abundance of total ice algae [START_REF] <jrn>von Quillfeldt | High number of diatom species in first-year ice from the Chukchi Sea[END_REF][START_REF] <jrn>poulin | The pan-Arctic biodiversity of marine pelagic and sea-ice unicellular eukaryotes: a first-attempt assessment[END_REF], we observed a change from a dominance of pennate diatoms (e.g. diatoms Type 2) before snow events to centric diatoms (e.g. diatoms Type 1) during the snowmelt period. This diatom community change from the beginning to the end of spring has recently been observed in Dease Strait (Nunavut) during spring 2014 [START_REF] <unknown>campbell | Seasonal dynamics of algal and bacterial communities in Arctic sea ice under variable snow cover[END_REF]. The transition from pennate to centric diatoms could be associated with the increase in light conditions and changes in physico-chemical properties (e.g. lower brine salinity and limited nutrient availability) of sea ice through the spring season. Furthermore, before snow events (e.g.

at the beginning of spring) the algal community under thick snow cover was more heterogeneous, with the presence of cryptophytes and dinoflagellates. This observation was already documented by Różańska et al. (2009) in Franklin Bay, where diatoms were less abundant in sea ice under thick snow than under thin snow. They suggested that flagellates were more abundant due to their capacity to be mixotrophic instead of purely autotrophic (Sherr et al.

2013, Unrein et al. 2014

). Thus, the algal composition changed markedly depending on snow cover but also over the different sampling periods.

As the snow melting period progresses, ice algae grow and need to acclimate to higher light intensities. Before snow events, PPC:PSC and (DD+DT):Tchl a ratios were higher under thin than thick snow cover conditions (Fig. 3b,c) in accordance with higher light transmittance [START_REF] Brunelle | Variability of phytoplankton light absorption in Canadian Arctic seas[END_REF][START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF]). These findings were further corroborated by a lower , a higher rETRmax and a higher Ek under thin snow cover (details in Table 2), a typical During the snowmelt period, bottom ice algae can be exposed for prolonged periods to relatively high light conditions. Thus, they need to be able to protect themselves through, e.g. the synthesis of carotenoids and other antioxidants. The lower bottom ice PPC:PSC ratio (up to 0.81) than previously reported in the Canadian Beaufort Sea (up to 1-3. plasticity and are able to acclimate well to all environments, but perform best under pelagic conditions. Thus, in a future Arctic where sea ice will be thinner and consequently light intensities higher, the bottom ice algae could shift from pennate to centric diatoms, based on their differential photoacclimative ability.

Ice algal photophysiological response: the 'cellular light memory' hypothesis

When comparing the photophysiological response of ice algae as a function of snow depth before and during snow events, we observed that ice algae under thick snow cover were more light-sensitive, as indicated by the more rapid decrease of Fv/Fm under light exposure from 10 to 200 mol photons m 2 s 1 (Fig. 5). Before snow events, the algal community under thick snow cover was more heterogeneous with the presence of cryptophytes and dinoflagellates, which are more sensitive to greater light intensities than diatoms [START_REF] <jrn>richardson | Adaptation of unicellular algae to irradiance: an analysis of strategies[END_REF][START_REF] <jrn>demers | Rapid light-induced changes in cell fluorescence and in xanthophyll-cycle pigments of Alexandrium excavatum (Dinophyceae) and Thalassiosira pseudonana (Bacillariophyceae): a photo-protection mechanism[END_REF]. Differences in photoacclimative and photoprotective strategies have been reported

between diatoms and other algal clades [START_REF] Lester | Aspects of resilience of polar sea ice algae to changes in their environment[END_REF][START_REF] <jrn>petrou | Southern Ocean phytoplankton physiology in a changing climate[END_REF][START_REF] <jrn>lacour | Growth, chl a content, photosynthesis, and elemental composition in polar and temperate microalgae[END_REF] as well as among diatom strains (Lavaud & Goss 2014, [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF][START_REF] <jrn>petrou | Southern Ocean phytoplankton physiology in a changing climate[END_REF]). In addition, the lower DES before the experiment (T0) in this algal community confirmed that it was low-light acclimated before the experiment. Since our experiments were performed under constant temperature (ca. -1.2°C) and nutrient-sufficient conditions, the differences in Fv/Fm and DES between thin and thick snow cover depths could be attributed to the algal community composition and/or the light history of the cells. However, during snow events, the algal community was similar between snow covers, thus the difference in light sensitivity, such as the quicker response of DES and the smaller decrease of Fv/Fm, must be associated with the different light history. The algal community and the light history are then 2 essential factors which must be taken into account when looking at photoprotection and photoacclimation of bottom ice algae.

The differential photophysiological response as a function of snow depth was even more pronounced when ice algae were exposed to over-saturating light stress (ca. 1000 mol photons m 2 s 1 for 3 h) that mimicked their release from ice and exposure at the surface of open waters.

Our experiment confirmed that bottom ice algae from thin snow cover were less light-sensitive than those from thick snow cover. Their light response was supported by their capacity to reduce excitation pressure on PSII (Fv/Fm relatively stable for 2 h) due to a higher DES and synthesis of DT (1.75 times higher than in ice algae from thin snow cover). A similar response was observed Surprisingly, ice algae under the same snow depth but during different time periods (ca.

25 cm, i.e. thick snow before snow events and thin snow during snow events; Fig. 2a), did not show the same light response (Fig. 5b,c). For all light intensities, the Fv/Fm decrease was stronger for ice algae under thick snow before snow events, indicating a higher light sensitivity.

The ice algae under thin snow during snow events showed a similar response to those under thin snow before snow events (Fig. 5a,c) even if they had spent 13 d under the new light conditions (i.e. less light availability because of thicker snow cover) when the experiment took place. This observation suggests that ice algae from thin snow kept their acclimation status similar to that before the snow events, even if they were exposed to lower irradiance (due to snow events) for several days. A similar process, so-called 'cellular light memory', has been reported in higher Based on similar features for photochemistry and light dissipation between higher plants and diatoms, we surmise that diatoms also possess this process of cellular light memory.

Photoprotective mechanisms in low-light acclimated algae

Our study confirms that ice algae were adapted to very low light intensities, but could maintain photosynthesis over a range of irradiances corresponding to those measured at the icewater interface (up to ca. 100 mol photons m 2 s 1 ; Fig. 7a). Indeed, fast (within 30 min light exposure) and major changes in PSII photochemistry (decrease in Fv/Fm) and DD de-epoxidation (increased DES) were observed for irradiance of 100 mol photons m 2 s 1 according to the literature and this study. It is noteworthy that ice algae could be more light resistant when aggregated (a behavioral feature we could not assess with the design of our experiments). In fact, aggregation of ice algae during spring and generated self-shading are 2 processes often mentioned in Arctic studies [START_REF] Rn | A laboratory study on O2 dynamics and photosynthesis in ice algal communities: quantification by microsensors, O2 exchange rates, 14 C incubations and a PAM fluorometer[END_REF][START_REF] Assmy | Floating ice-algal aggregates below melting Arctic sea ice[END_REF][START_REF] <jrn>fernández-Méndez | Composition, buoyancy regulation and fate of ice algal aggregates in the central Arctic Ocean[END_REF], and it could expand their range of light resistance beyond 100 mol photons m 2 s 1 .

In order to better understand the photophysiological response of ice algae, we compared the effects of presence and absence of an inhibitor of de-epoxidation of DD into DT (i.e. DTT) and of PSII photodamage repair (i.e. lincomycin). This single experiment with duplicate samples

gives us an idea of the relative importance of DT and PSII D1 protein synthesis in photoprotection and in the maintenance of photosynthesis under an irradiance range (ca. 50 to 100 mol photons m 2 s 1 ) that ice algae experience at the ice-water interface (Fig. 7a).

Although this type of experiment has been previously conducted (DTT: Kudoh et al. 2003[START_REF] <jrn>griffith | Diadinoxanthin cycle of the bottom ice algal community during spring in McMurdo Sound, Antarctica[END_REF]lincomycin: Petrou et al. 2010), this was the first time that such a combined protocol was applied on an Arctic ice algal community. As during our controlled experiments, bottom ice algae were able to maintain their photosynthetic capacity (mostly high and stable Fv/Fm) under these light conditions. Differential light-response between control conditions and inhibitor treatments indicate that PSII photochemical functionality was supported by both DT and D1-psbA protein synthesis with an apparently stronger photoprotective capacity by DT synthesized from DD de-epoxidation (i.e. Fv/Fm decrease was significantly higher when DTT was added as compared to lincomycin). This is in agreement with previous reports on Antarctic bottom ice algae, showing that they are well adapted to their changing in situ light environment as indicated by no/low PSII photodamage due to DT and D1-psbA protein synthesis [START_REF] <jrn>petrou | Rapid photoprotection in sea-ice diatoms from the East Antarctic pack ice[END_REF](Petrou et al. , 2011)). However, when ice algae were exposed to an over-saturating light stress (ca. 1000

mol photons m 2 s 1 ), their photochemical capacity was nearly abolished after 3 h, although efficient DT-driven photoprotection occurred during the first 2 h. This response could be associated with high EPAR but also with the presence of UVR in this experiment, while it was absent in the others. Some studies showed that pennate diatoms were relatively tolerant of UVR (Zacher et al. 2007, Wulff et al. 2008), while a recent study on ice algal communities in the Baltic Sea observed that UVR was one of the controlling factors [START_REF] <jrn>enberg | Solar PAR and UVR modify the community composition and photosynthetic activity of sea ice algae[END_REF]. In addition, studies on Antarctic algal cultures exposed to UVR observed an increase in mortality (McMinn et al. 1999), a reduction in photosynthesis (Schoeld et al. 1995, Villafañe et al. 2004) and a decline in productivity (Marcoval et al. 2007). Such light conditions can occur when ice algae released from sea ice are exported towards the surface of open waters along a receding ice edge. Our observation strongly points to the inability of ice algae to manage with a high rate of PSII photodamage generating photoinhibition. However, as mentioned above, they can also protect themselves via self-shading by forming aggregates when released into the water column [START_REF] Rn | A laboratory study on O2 dynamics and photosynthesis in ice algal communities: quantification by microsensors, O2 exchange rates, 14 C incubations and a PAM fluorometer[END_REF][START_REF] Assmy | Floating ice-algal aggregates below melting Arctic sea ice[END_REF][START_REF] <jrn>fernández-Méndez | Composition, buoyancy regulation and fate of ice algal aggregates in the central Arctic Ocean[END_REF]. This process enhances the sinking rate of sea ice algae, especially of diatoms [START_REF] <jrn>raven | Seasonal variation in benthic community oxygen demand: A response to an ice algal bloom in the Beaufort Sea, Canadian Arctic?[END_REF][START_REF] Aumack | Light and nutrient effects on the settling characteristics of the sea ice diatom Nitzschia frigida[END_REF],

and their export to deeper water layers where they can feed both pelagic and benthic food webs [START_REF] <jrn>kohlbach | The importance of ice algae-produced carbon in the central Arctic Ocean ecosystem: food web relationships revealed by lipid and stable isotope analyses[END_REF].

CONCLUSIONS

In this study, bottom ice algae were dominated by diatoms and were well adapted to daily and weekly changes in their light environment during Arctic spring. This is in agreement with previous studies in Antarctica (e.g. McMinn et al. 2007[START_REF] <jrn>mangoni | Photoacclimation in Antarctic bottom ice algae: an experimental approach[END_REF], Petrou et al. 2011[START_REF] Lester | Aspects of resilience of polar sea ice algae to changes in their environment[END_REF]. Their rapid response to excess light exposure is supported by central photoprotective processes such as XC and the repair of photodamaged D1 protein in PSII. We found that the photoprotective ability of bottom ice algae depends on their light history, controlled by the overlying snow depth through its influence on light transmittance to the bottom ice environment. We propose that, in order to acclimate to their permanently changing light environment driven by snow events and winds, bottom ice algae perform 'cellular light memory' similar to higher plants [START_REF] Hebda | Evidence for light wavelength-specific photoelectrophysiological signaling and memory of excess light episodes in Arabidopsis[END_REF]. According to our data, cellular light memory can prolong over at least 2 wk, enabling ice algae to improve their photoacclimative response to changing light conditions over different periods (days vs. weeks).

In a context of global warming in the Arctic, it has been predicted that the snow cover will potentially undergo rapid changes during future Arctic spring due to the combined effects of an increase in snow events [START_REF] <jrn>singarayer | Vertical fine structure of particulate matter and nutrients in sea ice of the high Arctic[END_REF], AMAP 2011) yet a decrease in snow depth (Webster et al. 2014) and duration [START_REF] Tv | Multiple effects of changes in Arctic snow cover[END_REF][START_REF] <jrn>derksen | Spring snow cover extent reductions in the 2008-2012 period exceeding climate model projections[END_REF][START_REF] Je | Future Arctic climate changes: adaptation and mitigation time scales[END_REF]). This will directly and strongly affect the light availability for bottom ice algae. The high plasticity of ice algae to acclimate to relatively large variations in irradiance over very different time scales suggests that ice algae may be more resilient to future changes than previously anticipated. However, ice algae will likely face more frequent light stress due to disturbed sea ice dynamics and especially an earlier melt [START_REF] <jrn>leu | A (too) bright future? Arctic diatoms under radiation stress[END_REF]. As shown in our study, bottom ice algae can only sustain reversible photoinhibition under high-light conditions for a limited period (less than 3 h), greatly reducing their productivity and survival potential once released into the water column at the ice edge [START_REF] <jrn>yamamoto | Photosynthetic characteristics of sinking microalgae under the sea ice[END_REF]. In this context, the timing between snowmelt, the release of ice algae and their stage of development will certainly affect their photoacclimative and photoprotective responses. Thus, it would be of interest to achieve similar light-response experiments on bottom ice algae during the decline of their spring bloom and the beginning of their release from sea ice. calculated from 5 samples for thin snow and 9 samples for thick snow

  high-light acclimation response (McMinn & Hegseth 2004, Manes & Gradinger 2009, Katayama & Taguchi 2013).

  photoprotection. In addition, the dominance of centric diatoms at the end of spring(Campbell et al. 2017, our Fig. 4) seems to represent the ideal algal community to seed an under-ice bloom. In fact, centric diatoms, as observed with Chaetoceros sp. byPetrou & Ralph (2011), possess a high

  in ice algae from Antarctic pack ice(Petrou et al. 2011). Higher DT synthesis illustrates well the response of diatoms to higher average irradiance[START_REF] <jrn>wilhelm | Light acclimation in diatoms: from phenomenology to mechanisms[END_REF] as reported from different algal communities which inhabit substrates and which are dominated by diatoms, i.e. ice algal communities[START_REF] Arrigo | Primary producers and sea ice[END_REF], Petrou et al. 2011[START_REF] <jrn>katayama | Photoprotective responses of an ice algal community in Saroma-Ko Lagoon, Hokkaido, Japan[END_REF] as well as benthic diatoms that inhabit intertidal sediments (Laviale et al. 2015). Higher DT cellular content can originate from DD de-epoxidation and de novo synthesis when light conditions are harsher (Lavaud & Goss 2014). Increased DT (and DD) synthesis was recently shown to be directly dependent on the redox state of the plastoquinone (PQ) pool (Lepetit et al. 2013) and thus on the excitation pressure on the photosynthetic machinery, and it is likely related to the parallel synthesis of specific PSII light-harvesting antenna proteins (LHCx; see Wu et al. 2012, Lepetit et al. 2017). Higher DT content provides a stronger capacity to prevent/limit the harmful effects of excess light exposure on photosynthetic efficiency, namely PSII photodamage (Wu et al. 2012, Lepetit et al. 2013) and lipid peroxidation of thylakoid membranes (Lepetit et al. 2010). DT acts via 2 main processes: the dissipation of excess excitation energy in PSII antenna through NPQ (Lavaud & Goss 2014, Goss & Lepetit 2015) and the direct scavenging of reactive oxygen species (ROS) as proposed by Lepetit et al. (2010).

  plants and it can persist for several days[START_REF] Hebda | Evidence for light wavelength-specific photoelectrophysiological signaling and memory of excess light episodes in Arabidopsis[END_REF][START_REF] <jrn>karpiński | Cellular light memory, photo-electrochemical and redox retrograde signaling in plants[END_REF]. The cellular memory of excess light exposure is based on a complex network in plant tissues which starts in the plastids of leaves, and which orchestrates a physiological response to improve photoacclimation under changing light conditions. It is associated with photoelectrochemical retrograde signaling due in part to changes in PSII redox state, and with photoprotective processes such as the XC-related NPQ. Although diatoms are unicellular organisms, they possess a photoelectrophysiological activity across thylakoid membranes[START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF], a redox-based plastid-to-nucleus retrograde signaling pathway[START_REF] <jrn>lepetit | High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]) and a strong NPQ tightly associated with the XC (Lavaud & Goss 2014).

Fig. 5 .

 5 Fig. 5. Maximum quantum yield of PSII photochemistry (Fv/Fm) of bottom ice algae exposed to 10, 50, 100 and 200 mol photons m 2 s 1 . Bottom ice algae were collected under (a,c) thin and (b,d) thick snow cover sites (a,b) before and (c,d) during snow events. Samples were exposed to the different light conditions for 3 h, followed by 2 h of recovery at low light (<5 mol photons m 2 s 1 ). Values are mean ± SD of experiments performed on (a) 14 May (n = 3), (b) 6 and 12 May (n = 6), (c) 25 and 29 May (n = 6) and (d) 27 and 31 May (n = 6)

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

  Figure 4.

  Figure 5

Figure 7 .

 7 Figure 7.

Figure 8

 8 Figure 8

  whereas lincomycin prevents the transcription of chloroplast-encoded D1 proteins (psbA) and therefore inhibits the repair of photodamaged PSII (Petrou et al. 2010).

	Duplicate 500 ml polycarbonate bottles containing melted ice water from a thin snow cover site
	were prepared (1) without inhibitor (i.e. control), (2) with DTT (final concentration 100 mol l 1 ;
	Olaizola et al. 1994) and (3) with lincomycin (final concentration 600 mol l 1 ; Kropuenske et
	al. 2009). Approximately 5 min after adding the chemical, the bottles were incubated for 6 h at
	the ice-water interface using a custom-built under-ice arm. Subsamples for PAM fluorescence
	and pigment analysis were collected every 2 h between 11:00 and 17:00 h (local time; UTC -
	05:00).

  1.95 as used in[START_REF] Sw | Composition and significance of picophytoplankton in Antarctic waters[END_REF]. According to[START_REF] <jrn>mackey | CHEMTAX-a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton[END_REF], the accuracy of CHEMTAX calculations is optimized when calculations are done on smaller groups of samples, with stable pigment ratios. Thus, samples were separated into 4 data sets with similar environmental (snow depth) and biological (algal bloom phase) conditions. CHEMTAX was used separately on each data set, and the average was calculated for each condition. As the data sets were separated in the same way as those in[START_REF] Alou-Font | Snow cover affects ice algal pigment composition in the coastal Arctic Ocean during spring[END_REF], we used the same initial pigment ratio matrices defined therein. Therefore, to improve biomass estimation, 10 successive runs of CHEMTAX using the output from each run as the input for the next was used, as recommended by Latasa(2007) and used in recent studies (e.g.[START_REF] <jrn>eker-Develi | Phytoplankton taxonomy based on CHEMTAX and microscopy in the northwestern Black Sea[END_REF][START_REF] <jrn>liu | High-resolution phytoplankton diel variations in the summer stratified central Yellow Sea[END_REF][START_REF] <jrn>wang | The modification and optimizing of the CHEMTAX running in the South China Sea[END_REF]. The final ratio matrices are displayed in TableS1in the Supplement at www.int-

res.com/articles/suppl/mXXXpXXX_supp.pdf.

  ), the Fv/Fm decrease for thin snow cover was significantly different from those before snow events (t-test, t = 3.702, df = 7, p < 0.01) at 200 mol photons m 2 s 1 . Fv/Fm reached the same minima after 3 h of light exposure (ca. 0.30 to 0.35). For lower light intensity treatments, Fv/Fm was rather stable throughout the experiment, except for the 100 mol photons m 2 s 1 treatment for thick snow, which showed a stronger decrease and lower recovery than before snow events. Because Fv/Fm is also influenced by nutrient availability in situ[START_REF] <jrn>lin | The fate of photons absorbed by phytoplankton in the global ocean[END_REF] as well as in experimental conditions[START_REF] <jrn>parkhill | Fluorescence-based maximal quantum yield for PSII as a diagnostic of nutrient stress[END_REF], we verified that nutrient concentrations were not limiting in our incubation bottles (see TableS2in the Supplement). Furthermore, due to low primary production (maxima ranged from 0.23 to 0.73 mol C l 1 h

1

) at the beginning of the experiment (data not shown), dissolved inorganic carbon concentrations (ca. 2105 ± 9.12 mol kg 1 ) were sufficient to provide enough carbon for the photosynthesis of ice algae. In addition to Fv/Fm, DES (an index of DD deepoxidation in bottom ice algae) was assessed (

). At T0, DES varied between 0.05 and 0.09 wt:wt and it increased significantly under the 2 highest light treatments (100 and 200 mol photons m 2 s 1

  . Below this light intensity, the non-significant changes in Fv/Fm and DES indicate that bottom ice algae were acclimated to an irradiance ranging between 50 and 100 mol photons m 2 s 1 and probably closer to 50 mol photons m 2 s 1 according to our experiments. This observation agrees with the results of Juhl & Krembs (2010), who showed that Nitzschia frigida (the most abundant bottom ice pennate diatom in the Arctic) could acclimate up to 110 mol photons m 2 s 1 in laboratory conditions. Also similar to our data,[START_REF] <jrn>cota | Physical control of Arctic ice algal production[END_REF] reported that the optimal photosynthetic irradiance range for ice algae was from 16 to 100 mol photons m 2 s 1 . When exposed to 200 mol photons m 2 s 1 , and despite the significant increase of DES index, ice algae underwent an important drop down of their photochemistry as indicated by the very low Fv/Fm (ca. 0.3) and its only partial recovery, which well illustrates PSII photoinactivation and/or photodamage[START_REF] <jrn>petrou | Rapid photoprotection in sea-ice diatoms from the East Antarctic pack ice[END_REF]. This light-response has already been reported for ice algae in Antarctica[START_REF] <jrn>petrou | Rapid photoprotection in sea-ice diatoms from the East Antarctic pack ice[END_REF](Petrou et al. , 2011)). In contrast, studies in Greenland and Antarctica found that highly shade-adapted ice diatoms showed a photoinhibitory response at irradiances as low as 25 to 50 mol photons m 2 s 1 (e.g.[START_REF] <jrn>kühl | Photosynthetic performance of surface-associated algae below sea ice as measured with pulse amplitude modulated (PAM) fluorometer and O2 microsensors[END_REF][START_REF] Ryan | Spring sea-ice photosynthesis, primary productivity and biomass distribution in eastern Antarctica, 2002-2004[END_REF][START_REF] <jrn>mangoni | Photoacclimation in Antarctic bottom ice algae: an experimental approach[END_REF][START_REF] <jrn>ryan | Chlorophyll fluorescence imaging analysis of the responses of Antarctic bottom-ice algae to light and salinity during melting[END_REF]. Nutrient availability (Cota & Horne 1989) could well explain this discrepancy with our data as well as differential photoadaptation abilities among diatom species and communities between our data and others[START_REF] Barnett | Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of southern Europe[END_REF][START_REF] <jrn>petrou | Southern Ocean phytoplankton physiology in a changing climate[END_REF]. Thus,

environmental (e.g. nutrient concentrations and light conditions depending on snow depth) and biological (e.g. algal community composition) conditions influence the photo-response of ice algae, which can acclimate to a range of irradiances from 25 up to 100 mol photons m 2 s 1

Acknowledgements. We acknowledge the contributions of the Canada Excellence Research Chair (CERC) and Canada Research Chair (CRC) programs, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), Canada Economic Development, and Fonds de recherche du Québec -Nature et technologies (FRQNT) through the Québec-Océan research cluster. The GreenEdge project is funded by the following French and Canadian programs and agencies: ANR (Contract #111112), CNES (project

Thin and thick To assess the photoprotective capability of bottom ice algae when they are exposed to a sudden increase of irradiance

Before snow events

In situ irradiance at water surface (before and during snow events and snowmelt). Average was calculated for each snow cover from