Philippe-Israël Morin
email: philippe-israel.morin.1@ulaval.ca

-Israël

; Lacour

; Grondin

Flavienne ; Bruyant

; Ferland

; Forget

; Massicotte

; Donaher

; Campbell
email: dcampbell@mta.candonaher@mta.ca

; Lavaud

& Babin

Response of the sea-ice diatom Fragilariopsis cylindrus to simulated polar night darkness and return to light

Keywords: Diatoms polar night darkness survival Fragilariopsis cylindrus, sea-ice diatom, diatoms, acclimation, dark survival, polar night, light return, darkness

Arctic photoautotrophic communities must survive through polar night darkness until light returns in spring. We tracked changes in the cellular resource allocations and functional capacities of a polar sea-ice diatom, Fragilariopsis cylindrus, to understand acclimation processes in both darkness and during the subsequent return to light. We measured parameters at specific time-points over 3 months of darkness, and then over 6 days after a return to light. Measured parameters included cell number and size, cellular carbon and nitrogen quotas, lipid and pigment contents, concentration of key proteins of the photosynthetic system, photosynthetic parameters based on both variable fluorescence and carbon assimilation, and the level of non-photochemical quenching.

A stable functional state was reached within a few days after the transition to dark and was then maintained throughout three months of darkness. The dark period resulted in a decrease of lipid droplet cell quota (-6%), chlorophyll a cell quota (-41%) and the maximum carbon fixation rate per cell (-98%). Return to light after 1.5 months of darkness resulted in a strong induction of non-photochemical quenching of excitation and a fast recovery of the maximum carbon fixation rate within 1 day, followed by a rapid increase in the cell number. Return to light after three months of darkness showed an increase of mortality or a profound down-regulation induced over longer periods of darkness.

Introduction

Diatoms experience a wide range of environmental conditions across the oceans, with some imposing extreme stresses upon the cells. Light spans one of the largest ranges of environmental variation as diatoms may transition from high light exposure in the sunlit surface layer to darkness due to ocean mixing or during the night. Beyond diel cycles, diatoms may survive weeks in total darkness during deep ocean mixing events [START_REF] Cullen | The kinetics of algal photoadaptation in the context of vertical mixing[END_REF][START_REF] Marshall | Open-ocean convection: observations, theory, and models[END_REF], and possibly up to centuries during sedimentation events [START_REF] Mcquoid | Viability of phytoplankton resting stages in the sediments of a coastal Swedish fjord[END_REF][START_REF] Godhe | Linking the planktonic and benthic habitat: genetic structure of the marine diatom Skeletonema marinoi[END_REF][START_REF] Härnström | Hundred years of genetic structure in a sediment revived diatom population[END_REF]. At high latitudes, darkness sometimes lasts as long as ca. six months as a consequence of the sea ice covered with snow and low or even negative sun elevation during the polar night. Given the photoautotrophic nature of diatoms, their survival of a lack of sunlight for up to 6 months is remarkable and has motivated many studies in the past decades to understand the related acclimation processes. So far, in experiments studying the response to prolonged darkness, microalgal or diatom cell growth recovered after the imposed dark period (Table 1). Spore production might explain diatom survival during prolonged darkness [START_REF] Doucette | Thalassiosira antarctica: vegetative and resting stage chemical composition of an ice-related marine diatom[END_REF], but spores have only rarely been observed in experiments [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF][START_REF] Zhang | Dark survival of marine microalgae in the high Arctic (Greenland Sea)[END_REF].

A "vegetative" or physiological resting state could be more prevalent for overwintering as resting cells have the ability to rapidly recover to their active state [START_REF] Anderson | The ultrastructure and cytochemistry of resting cell formation in Amphora coffaeformis (Bacillariophyceae)[END_REF][START_REF] Sicko-Goad | Rejuvenation of Melosira granulata (Bacillariophyceae) resting cells from the anoxic sediments of Douglas lake, Michigan. I. Light microscopy and 14 C uptake[END_REF]. Heterotrophic nutrition has also been considered as a means for dark survival [START_REF] Lewin | Heterotrophy in diatoms[END_REF][START_REF] White | Growth of two facultatively heterotrophic marine centric diatoms[END_REF][START_REF] Hellebust | Heterotrophic nutrition[END_REF], but the extent of its contribution remains uncertain, as it is not always detected [START_REF] Horner | Algal population in Arctic sea ice: an investigation of heterotrophy[END_REF][START_REF] Popels | Factors affecting dark survival of the brown tide alga Aureococcus anophagefferens (Pelagophyceae)[END_REF][START_REF] Mcminn | Dark survival in a warming world[END_REF].

In several experiments on microalgae, not all on polar diatoms, a physiological resting state during prolonged darkness has been characterized by a low rate of metabolic activities. The metabolic activity of chlorophytes was greatly lowered after 10 days in the dark [START_REF] Jochem | Dark survival strategies in marine phytoplankton assessed by cytometric measurement of metabolic activity with fluorescein diacetate[END_REF]. The particulate organic carbon and nitrogen cell quotas in three Antarctic diatoms remained stable over 80 days in the dark, also suggesting a lowered metabolism with limited consumption of reserves [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF]. Despite low rates of metabolic activity, consumption of energy reserves likely fuels basal metabolic needs shortly after transition to total darkness [START_REF] Palmisano | Physiology of sea ice diatoms. I. Response of 3 polar diatoms to a simulated summer-winter transition[END_REF]. [START_REF] Mock | Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus[END_REF] found that 60% of all genes were down-regulated in the F. cylindrus transcriptome after 7 days of darkness, but genes involved in starch, sucrose and lipid metabolism were up-regulated. [START_REF] Schaub | Effects of prolonged darkness and temperature on the lipid metabolism in the benthic diatom Navicula perminuta from the Arctic Adventfjorden, Svalbard[END_REF] also found patterns of lipid consumption in a benthic Arctic diatom to be faster in the first two weeks of a two-month long dark experiment. In other experiments with either a temperate diatom [START_REF] Handa | Carbohydrate metabolism in the marine diatom Skeletonema costatum[END_REF], a Chlorophyceae [START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF] or a Pelagophyceae [START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF], similar patterns of rapid consumption early during the dark period occurred with preferential catabolism of proteins and carbohydrate reserves.

Photophysiology also appears to be strongly downregulated in prolonged darkness. Among Chlorophycaeae [START_REF] Hellebust | Effects of environmental conditions on the rate of photosynthesis and some photosynthetic enzymes in Dunaliella tertiolecta Butcher[END_REF][START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF], Pelagophyceae [START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF]) and temperate [START_REF] Griffiths | Factors affecting the photosynthetic capacity of laboratory cultures of the diatom Phaeodactylum tricornutum[END_REF] or polar [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF] diatoms, the maximum rate of carbon fixation (Pmax) per chlorophyll a [START_REF] Hellebust | Effects of environmental conditions on the rate of photosynthesis and some photosynthetic enzymes in Dunaliella tertiolecta Butcher[END_REF][START_REF] Griffiths | Factors affecting the photosynthetic capacity of laboratory cultures of the diatom Phaeodactylum tricornutum[END_REF][START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF][START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF] or per cell [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF] strongly decreased within the first weeks under prolonged darkness. In the experiment of [START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF], a drop in the absolute concentration of carbon fixation enzyme RuBisCO was also detected. [START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF], however, recently measured in a polar diatom a rather stable level of RuBisCO-to-carbon ratio, despite a strong decrease in Pmax per carbon in prolonged darkness. In other studies, the maximum quantum yield of photochemistry (ΦM) and the maximum relative electron transport rate (rETRmax) also decreased within several weeks of darkness whether studying polar algal communities [START_REF] Martin | The physiological response to increased temperature in over-wintering sea ice algae and phytoplankton in McMurdo Sound, Antarctica and Tromso Sound, Norway[END_REF], cultures of polar diatoms [START_REF] Reeves | The effect of prolonged darkness on the growth, recovery and survival of Antarctic sea ice diatoms[END_REF][START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF], benthic diatom communities [START_REF] Wulff | Exposure to sudden light burst after prolonged darkness -a case study on benthic diatoms in Antarctica[END_REF] or even Rhodophyta thalli [START_REF] Luder | Acclimation of photosynthesis and pigments during and after six months of darkness in Palmaria decipiens (Rhodophyta): a study to simulate Antarctic winter sea ice cover[END_REF].

At the structural level of the photosynthetic apparatus, the molecular components of the light-harvesting antennae (pigments, proteins) of a green algae (Baldisserotto et al., 2005a;[START_REF] Ferroni | Acclimation to darkness in the marine chlorophyte Koliella antarctica cultured under low salinity: hypotheses on its origin in the polar environment[END_REF] and a Xanthophyceae (Baldisserotto et al., 2005b) were partially dismantled or degraded after 2-3 months of darkness. However, in these experiments, the light-harvesting antennae appeared to keep a certain level of organization to re-use light as soon as it became available once again. Generally, a decrease in the chlorophyll a cell quota or absolute concentration occurred in previous dark experiments (Table 1), though not always, particularly for dark experiments shorter than 1 month. Chlorophyll a remained stable in these shorter term experiments whether expressed as cell quota [START_REF] Hellebust | Effects of environmental conditions on the rate of photosynthesis and some photosynthetic enzymes in Dunaliella tertiolecta Butcher[END_REF][START_REF] Doucette | Thalassiosira antarctica: vegetative and resting stage chemical composition of an ice-related marine diatom[END_REF], absolute concentration [START_REF] Griffiths | Factors affecting the photosynthetic capacity of laboratory cultures of the diatom Phaeodactylum tricornutum[END_REF][START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF][START_REF] Reeves | The effect of prolonged darkness on the growth, recovery and survival of Antarctic sea ice diatoms[END_REF] or chl-to-carbon ratio [START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF]. In benthic diatoms, [START_REF] Veuger | Long-term pigment dynamics and diatom survival in dark sediment[END_REF] also measured a decrease in the dry weight concentration of other pigments, including the photoprotective diadinoxanthin/diatoxanthin pigments, with the largest decrease attributable to the photosynthetic ones (chloroyphyll a, chloroyphyll c, Fucoxanthin).

In polar regions, when the polar night ends, incident irradiance increases, the snow and seaice covers then melt, and spring blooms of ice algae and phytoplankton take place [START_REF] Wassmann | Future Arctic Ocean seasonal ice zones and implications for pelagic-benthic coupling[END_REF]. Much of the annual production, and most of the new production in the Arctic Ocean, occur at that time of the year [START_REF] Sakshaug | Primary and secondary production in the Arctic Seas[END_REF][START_REF] Perrette | Near-ubiquity of ice-edge blooms in the Arctic[END_REF][START_REF] Ardyna | Parameterization of vertical chlorophyll a in the Arctic Ocean: impact of the subsurface chlorophyll maximum on regional, seasonal, and annual primary production estimates[END_REF]. Sea-ice algae dominated by pennate diatoms are the first to exploit the return of light in spring, before the phytoplankton bloom develops [START_REF] Mundy | Variability of snow and ice thermal, physical and optical properties pertinent to sea ice algae biomass during spring[END_REF][START_REF] Leu | Arctic spring awakening -Steering principles behind the phenology of vernal ice algal blooms[END_REF][START_REF] Wassmann | Arctic marine ecosystems in an era of rapid climate change[END_REF]. At least a few diatom cells from all species present at any time in polar oceans must survive overwintering in the full darkness to inoculate the populations that grow during summer. The stress imposed by the return of light in spring may further compromise the survival of overwintering populations after such a long period of darkness. Thus, their ability to recover is of crucial importance as regard to their fate.

Despite the numerous studies on microalgae dark survival, only a few have measured physiological parameters during the recovery upon light return (Table 1). In general, the low photosynthetic performances observed during the dark period, whether measured at the photochemistry level [START_REF] Luder | Acclimation of photosynthesis and pigments during and after six months of darkness in Palmaria decipiens (Rhodophyta): a study to simulate Antarctic winter sea ice cover[END_REF][START_REF] Wulff | Exposure to sudden light burst after prolonged darkness -a case study on benthic diatoms in Antarctica[END_REF][START_REF] Martin | The physiological response to increased temperature in over-wintering sea ice algae and phytoplankton in McMurdo Sound, Antarctica and Tromso Sound, Norway[END_REF], the carbon fixation level [START_REF] Griffiths | Factors affecting the photosynthetic capacity of laboratory cultures of the diatom Phaeodactylum tricornutum[END_REF][START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF][START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF] or both [START_REF] Kvernvik | Fast reactivation of photosynthesis in arctic phytoplankton during the polar night[END_REF][START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF], recover within the first days of re-illumination. [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF] and [START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF] also measured a recovery in particulate nitrogen and carbon levels which requires energy to be available rapidly after re-illumation. Recent studies on polar phytoplankton communities [START_REF] Kvernvik | Fast reactivation of photosynthesis in arctic phytoplankton during the polar night[END_REF]) and a polar diatom culture [START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF] focused on the ability to restore growth with different irradiance intensities. Regardless of the light intensity, polar microalgae appeared to recover their photophysiological capacity within 48 hours.

Although dark survival of microalgae has received considerable attention in the past decades, our understanding of more specifically polar night darkness survival in diatoms remains limited for several reasons. Some of the former studies have shown dark survival for numerous microalgal species over periods representative of the polar night and even beyond (rows 3,5,9,10,13-15,18-20,23,26-28, Table 1). They however documented a limited suite of physiological and biochemical parameters, which did not allow to fully understand the involved cellular mechanisms. Some other experiments did measure several parameters and provided a complete transcriptional profiling of sequenced genes, but for only a short dark period (< 20 days) (rows 4,6, Table 1); 3) Other experiments studied nonpolar species (or not diatoms) with detailed characterization and are to be interpreted with caution relative to diatoms in the polar environment (rows 12,21, Table 1); 4). A combination of these limitations is not uncommon (rows 7,8,11,16,17,22,24,25,29-31, Table 1).

Our study (row 1, Table 1) aimed at overcoming the limitations described above with an integrative characterization of a sea ice diatom, Fragilariopsis cylindrus, tracking physiological and metabolic acclimation over a darkness period representative of the polar night, and over its resumption of growth upon return to light. Our results are largely consistent with earlier findings on parameters measured in common across the studies, but we significantly expand previous knowledge by parallel monitoring of multiple physiological and metabolic features. The length of each experiment is shown in days, hours-h or years-y when specified for Dark ** indicates only growth potential was verified upon a light return.

… indicates more species were studied.

Materials & Methods

Cell culturing

Axenic cultures of Fragilariopsis cylindrus (Grunow) Krieger (strain NCMA3323) were freshly obtained from the National Center for Marine Algae and Microbiota. F. cylindrus is the only polar diatom with a sequenced and published genome (see [START_REF] Mock | Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus[END_REF]. It was grown in semi-continuous cultures in pre-filtered L1 medium [START_REF] Guillard | Stichochrysis immobilis is a diatom, not a chrysophyte[END_REF]. Cultures in triplicate were started in 50 ml borosilicate tubes and then sequentially transferred to larger vessels several times until a final transfer of 5 l of culture to 20-l polycarbonate round vessels (Fig. S1). Thereafter, further additions of L1 media served to increase culture volume while matching growth rate so that the cell density remained steady [START_REF] Wood | Measuring growth rates in microalgal cultures[END_REF]. Light was provided continuously with DURIS® E3 LED bands (GW JCLMS1.EC, 4000 K) at a scalar irradiance of approximately 30 µmol photons m -2 s -1 as measured with a QSL-100 quantum sensor (Biospherical instruments, San Diego, CA, USA) placed in the centre of the vessel. Scalar irradiance ranged from 29.5 to 33.5 µmol photons m -2 s -1 depending on the culture vessel position in the growth chamber (Fig. S1).

This irradiance was chosen based on the irradiance at which the growth rate saturated (KE): 0.244±0.041 d -1 . Each culture was gently mixed with a 12.5 cm magnetic stirrer and bubbled with air filtered through a 0.3 µm capsule filter (Carbon CAP, Whatman™ 6704-7500). Temperature of the growth chamber (CARON, model 7901-33-2) was kept at 0°C

for the duration of the experiment.

Sampling design

The first two samplings took place once the growth rate, cell diameter and Chlorophyll a (Chla) were steady for a minimum of 10 cell generations (MacIntyre & Cullen 2005a), one day before the transition from light to dark (referred to as the -1-day sampling), and on the day of the transition just before turning off the light (referred to as the 0-day sampling).

Both are collectively referred to as light-acclimation sampling days. In order to avoid light limitation of growth, the cultures were kept optically thin during the acclimation period (between 4×10 4 to 6×10 5 cell mL -1 ). The cell suspension density was ~ 5×10 5 cell ml -1 before the dark transition. The light system was then switched off and each vessel was carefully covered with opaque material. Sampling in the dark began 24 hours following the transition and subsequent dark samplings followed after 5, 14, 28, 63 and 83 days of darkness as shown with the timeline in Fig. 1. Syringes used for sampling culture volume were completely opaque, as well as the tubes connecting to the cultures vessels, and all subsamples were contained in opaque tubes until their respective measurements. Every immediate manipulation (e.g. filtrations, fluorescence determinations and 14 C incubations)

was completed under very low green light levels in order to avoid excitation of the photosystems. The Light return 1 experiment took place after 1.5 months (48 days) of darkness. Culture volume was carefully transferred to gently aerated 3-l vessels cooled to 0°C and illuminated at 30 µmol photons m -2 s -1 with a slightly different light spectrum (Fig. S2). This second light system was provided by a customized LED system comprising 8 colours independently variable in intensity and mounted on 6 LED panels around the 3-l vessels (Fig. S3). The cultures were sampled at 30 minutes, 2 hours, 5 hours, 1 day, 3 days and 6 days following re-illumination (Fig. 1). The Light return 2 experiment took place after 3 months (90 days) in the dark following the same procedure (Fig. 1). All manipulations were completed under very low green light levels for both light return experiments (See background light in Fig. S3)

At sampling time-points culture samples were harvested to measure the parameters described below with a few exceptions. The relative electron transport rate (rETR) and nonphotochemical quenching (NPQ) were measured for the light return experiments and for several time-points during the dark period (14, 28, 47 and 83 days). Carbon fixation rates

were measured at all points except after 3 months of darkness. Lipid droplets were measured from 5 hours to 6 days following both light return experiments and for every time-point during the dark period. The maximum quantum yield of PSII (Photosystem II) photochemistry (ΦM) and the effective absorption cross-section (σPSII) were also measured 2 days after the transition to dark. Table 1 summarizes the measurements made for every sampling time-point.

Cell number and volume and culture axenicity

Cells were counted and sized using a Beckman Multisizer 4 Coulter Counter. Three consecutive countings were recorded for each culture sampling point. Total cell counts did not differentiate cell viability; hence mortality could not be assessed through this method.

However, mortality can be suspected when looking at the flow cytometry data (see Lipid droplets section and Fig. S4). The flow cytometry data indicated that the number of debris, likely the result of dying cells, appeared to increase with the duration of the experiment, especially during the Light return 2 experiment. The cell volume was calculated using the sphere-equivalent diameter. The biovolume was calculated as cell volume x cell number for each culture. Axenicity of each culture was verified with petri dishes prepared as described in MacIntyre & Cullen (2005b). Axenicity was confirmed once before the acclimation period.

Carbon and nitrogen cell content

Three technical replicates were harvested per algal culture; aliquots of 20 ml were filtered onto binder-free glass-fiber filters (GF/F) (0.7 µm, 25 mm) pre-combusted at 500°C for 24 hours. Filters were then dried at 60°C for at least 12 hours and kept desiccated before elemental analysis with a CHN analyzer (2400 Series II CHNS∕ O; Perkin Elmer, Norwalk, CT, USA).

Lipid droplets

Cells were assessed for their lipid droplets content using the molecular probe BODIPY® 505/515 (www.lifetechnologies.com) according to [START_REF] Brennan | Enhancement of BODIPY 505/515 lipid fluorescence method for applications in biofuel-directed microalgae production[END_REF] using a flow cytometer (488 nm excitation, 520 nm emission, Millipore Guava easycyte flow cytometer) in a 96-well plate. BODIPY fluorescence emitted from the lipid droplets was quantified for each cell as relative fluorescence units (RFU). For samples analyzed at each time-point, a total of 9 wells were prepared (three technical replicates per culture) and BODIPY fluorescence was measured on 5000 cells for each well. Before fluorescence measurements, samples were incubated for 1 h on ice in the dark. Each well contained 300 µl of algal culture marked with 4 µl of a BODIPY solution (final concentration 0.33 µM / 1.32% DMSO). For each time-point sampling, technical replicates were pooled together for each culture and averaged for their mean RFU according to a target function (Fig. S4).

Pigment content

For each culture, 10 ml were filtered onto glass-fiber filters (GF/F) (0.7 µm, 25 mm, Millipore). Filters were immediately flash-frozen in liquid nitrogen and stored at -80°C until analysis. Pigment separation was performed with high performance liquid chromatography (HPLC) according to [START_REF] Zapata | Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases[END_REF]. Before HPLC analyses, pigments were extracted in 95% methanol and sonicated for 20 seconds three times.

Samples were then centrifuged (4500 rpm) 15 minutes at 4°C and filtered on polytetrafluoroethylene (PTFE) membranes (0.2 µm). Data were analysed using the ChromQuest 5.0 software.

Photosynthetic proteins

For PsbA (PSII protein D1) and RbcL (RuBisCO large subunit) quantification, 30 ml of each culture was harvested onto GF/F filters, flash-frozen and stored at -80°C. Protein extraction was performed using the FastPrep-24 and bead lysing 'matrix D' (MP Biomedicals), using 3 cycles of 60 seconds at 6.5 m s -1 in 300 µL of 1X extraction buffer (Agrisera AS08_300 with 0.4 M of the protease inhibitor AEBSF added), then spun at 16 000 g for 5 minutes [START_REF] Li | Rising CO2 Interacts with growth light and growth rate to alter photosystem II photoinactivation of the coastal diatom Thalassiosira pseudonana[END_REF]. For each protein extract supernatant we then estimated the content of total nitrogen derived from the original sample using the parallel determinations of total N content per mL of sample. We then loaded each well of gels with a volume of protein extract sufficient to deliver equivalent total nitrogen across wells. We chose this approach for loading because total nitrogen determinations are more reliable than total protein determinations. Separation of proteins was done in a Bolt 4-12%

Bis Tris SDS-PAGE gel (Invitrogen). Proteins were quantified by western-blotting with anti-PsbA (AS05 084) or anti-RbcL (AS01 017) antibodies (www.agrisera.se) [START_REF] Li | A hard day's night: diatoms continue recycling photosystem II in the dark[END_REF]. Chemiluminescent images were obtained using ECL Ultra reagent (Lumigen, TMA-100) and a VersaDoc CCD imager (Bio-Rad). Band densities for samples were determined against the standard curve using the ImageLab software (v 4.0, Bio-Rad).

Variable fluorescence

Variable in vivo Chla fluorescence at 680 nm was measured using a Fluorescence subsequently exposed to a rapid light curve (RLC) protocol using 8 step-wise increasing irradiances from 1 to 111 µmol photons m -2 s -1 and for 10 or 30 seconds each [START_REF] Lefebvre | Duration of rapid light curves for determining the photosynthetic activity of microphytobenthos biofilm in situ[END_REF]). After each irradiance-step, Fm' was probed with an actinic flash (500 ms), while a detecting modulated light source measured Fs. Although the instrument allows excitation of fluorescence at four different wavelengths, actinic light was only provided by the actinic LEDs peaking at 655 nm (Fig. S2). To calculate the PSII-specific rETR, the achieved quantum yield of charge separation in PSII (ΦPSII) at each irradiance step was multiplied by the corresponding irradiance (E):

𝛷 (2a) 𝑟𝐸𝑇𝑅 𝛷 ⋅𝐸 ( 2b
)
where Fm' and Fs are the maximum and steady-state fluorescence of light acclimated cells, respectively. To calculate the maximum relative electron transport rate (rETRmax), a function was fitted to the data by least-square fit according to [START_REF] Eilers | A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton[END_REF]:

𝑟𝐸𝑇𝑅 𝐸 (3)
where a, b and c are expressed as:

𝑎 ・ (4a) 𝑏 ・ (4b) 𝑐 (4c)
and where s is the initial slope, Im is the optimal irradiance and Pm is the maximal production rate of the fit. The dynamic non-photochemical quenching (NPQd, also referred as NPQ in the text) was calculated for each irradiance-step as:

𝑁𝑃𝑄𝑑 (5)
and the sustained and total non-photochemical quenching (NPQs, NPQt) were calculated as:

𝑁𝑃𝑄𝑠 (6a) 𝑁𝑃𝑄𝑡 𝑁𝑃𝑄𝑑 𝑁𝑃𝑄𝑠 (6b)
where Fm and Fm' are the maximum fluorescence of dark and light (incubation-irradiance) acclimated cells, respectively, and Fm24h is the maximum fluorescence of dark-acclimated cells for 24 hours to allow complete relaxation of NPQS. NPQ calculations were computed with the 30 seconds RLC protocol. To calculate the maximum non-photochemical quenching (NPQmax) and developed NPQ at 30 µmol photons m -2 s -1 (NPQ30), a function was fitted to the data by least-square fit according to [START_REF] Serôdio | A model for describing the light response of the nonphotochemical quenching of chlorophyll fluorescence[END_REF]:

𝑁𝑃𝑄 𝐸 𝑁𝑃𝑄 ⋅ 50 ( 7
)
where NPQmax is the maximum NPQ value, E50 is the irradiance at which 50% of NPQmax is reached and n is the Hill coefficient of the fit (the sigmoidicity of the curve).

C incubations

A sample was first collected for each culture, and inoculated with inorganic 14 C (NaH 14 CO3, 2 µCi ml -1 ). Samples were then processed as described in [START_REF] Bruyant | Diel variations in the photosynthetic parameters of Prochlorococcus strain PCC 9511: Combined effects of light and cell cycle[END_REF]. Inoculated culture aliquots of 1 ml were dispensed into 24 glass scintillation vials of 7 ml cooled in separate thermo-regulated alveoli (0°C). The vials were exposed to 24 where P is the rate of carbon fixation [mg C m -3 h -1 ], R is the total activity (DPM), N is the number of hours of incubation, RS is the sample count (DPM) corrected for quenching, RB is the blank (or dark sample) count (DPM) corrected for quenching and W is the total weight of carbon dioxide available. The relationship between the rate of carbon fixation (P)

and irradiance (E) was fitted to the equation determined by [START_REF] Platt | Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton[END_REF] to obtain the photosynthetic coefficients:

𝑃 𝑃 1-𝑒𝑥𝑝 - 𝑒𝑥𝑝 - 𝑃 ( 9
)
where PS is the maximum carbon fixation rate in absence of photoinhibition [µg C m -3 h -1 ],

α is the initial slope of the carbon fixation vs. irradiance curve [µg C m -3 h -1 (μmol photons m -2 s -1 ) -1 ], E is the incubation irradiance (μmol photons m -2 s -1 ), β is the photoinhibition coefficient [µg C m -3 h -1 (μmol photons m -2 s -1 ) -1 ] and P0 is the intercept of the curve [µg C m -3 h -1 ]. The maximum carbon fixation rate at saturating irradiance (Pmax) was calculated as follow:

𝑃 𝑃 (10)

Statistical analysis: mean comparison of parameters between sampling periods

Different time windows were considered to follow the evolution of the measured parameters. The time windows considered are those with large changes in tracked parameter levels. Generally, the full duration of a given phase of the experiment, e.g. 83 days of darkness (or 63 days for carbon fixation parameters), is the period considered for statistical comparison. In some cases, within a given phase, there was an apparent change in the direction of the variation, e.g. see the number of cell per ml that increases from 0 to 28 days of darkness and decreases after from 28 to 83 days of darkness (Fig. 2a). For a given parameter, the time at which this directional change occured splits a given phase of the experiment into two periods for statistical comparison, e.g. D0-28 and D28-83 for the number of cell per ml in the dark. For the dark period (D), there were four main periods considered:

up to the first month (D0-28), up to the second month (D0-63), up to the third month (D0-83)

and between the first and the third months (D28-83). For the light return periods (L1, L2), the periods used for comparisons are specified in the text relatively to time ranges of interest, e.g. from 30 minutes to three days of illumination (L130min-3d). To determine whether there was a significant variation in the measured parameters during the dark experiment, we used linear mixed models that included an error term (or random effect) on the culture to account for the pseudo-replication of the data. For the light return periods, the mixed effect models also included a fixed effect on the light experiments (whether it was the first or the second light return experiment) and an interaction term between the two fixed effects (time and experiment). To test for temporal autocorrelation for each measured parameter, models were compared including a first order autocorrelation structure or not. ΔAIC was computed between each paired model to determine if the two models showed equivalent power or not.

The models were considered equivalent if ΔAIC < 5 and the model without a first autocorrelation structure was then chosen. For ΔAIC > 5, the model with the lowest AIC was chosen (supplementary files, model_significance_dark & model_significance_light).

Temporal autocorrelation was found to be present only rarely and was accounted for when necessary. The linear mixed-effects models were fitted in R using the nlme package v3.1-137 [START_REF] Pinheiro | nlme: Linear and Nonlinear Mixed Effects Models[END_REF]. All statistical analyze were performed in R 3.5.2 (R Core Team S1, S2 and S3.

Photosynthetic apparatus dismantlement

When cells are exposed to a long period of darkness, the photosynthetic machinery is not operating to convert light to chemical energy. A metabolic cost is associated with sustaining the molecular components of the photosynthetic apparatus [START_REF] Geider | Respiration and microalgal growth: a review of the quantitative relationship between dark respiration and growth[END_REF][START_REF] Quigg | Protein turnover in relation to maintenance metabolism at low photon flux in two marine microalgae[END_REF][START_REF] Li | The nitrogen costs of photosynthesis in a diatom under current and future pCO2[END_REF]. Based on the assumption of a lower metabolism and slower protein turnover in darkness [START_REF] Li | A hard day's night: diatoms continue recycling photosystem II in the dark[END_REF], renewal of degraded photosynthetic components should be limited. Hence, Chla and the main photosynthetic accessory pigment Fucoxanthin (Fuco) cell quotas decreased as in past experiments [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF]Baldisserotto et al. 2005a;[START_REF] Veuger | Long-term pigment dynamics and diatom survival in dark sediment[END_REF]. Chla and Fuco decreased significantly after 3 months of darkness by 41% and 48% respectively (D0-83, P-valueChla = 6.88 x 10 -3 , P-valueFuco = 1.27 x 10 -3 ) (Fig. 3a). For the same period, the photoprotective pigments (Diadinoxanthin (DD) + Diatoxanthin (DT)) cell quotas decreased, although not significantly, by 20% (D0-83, P-value = 4.51 x 10 -1 ). The pool decrease was attributable to the DD form since DT fully returned to its epoxidized form (DD) after 1 day of darkness (Fig. 3b). Thus, the photosynthetic (Chla + Chlc +Fuco) to photoprotective pigments ratio decreased significantly by 31% (D0-83, P-value = 4.31 x 10 - 6 ) (Fig. 3c). As a likely consequence of this specific pigment degradation, σPSII decreased significantly by 27% (D0-83, P-value = 1.41 x 10 -4 ) (Fig. 3c).

Photosynthetic proteins cell quotas (PsbA and RbcL) decreased significantly after 3 months of darkness (D0-83) by 85% and 72% respectively (D0-83, P-valuePsbA = 2.03 x 10 -4 , P-valueRbcL = 1.44 x 10 -4 ). They reached a much lower detected level relative to t0 than did the photosynthetic pigments (Fig. 4a). The massive decrease for PsbA is indicative of a PSII core complex degradation as seen in other experiments with a green chlorophyte and a snow xanthophycean algae (Baldisserotto et al. 2005a;Baldisserotto et al. 2005b;[START_REF] Ferroni | Acclimation to darkness in the marine chlorophyte Koliella antarctica cultured under low salinity: hypotheses on its origin in the polar environment[END_REF]) under prolonged darkness. Based on the photosynthetic proteins results, one would expect that carbon fixation capacity was largely suppressed in absence of these key proteins, particularly RbcL. Indeed, the carbon fixation vs. irradiance curves showed a major significant decrease within 2 months in α and in Pmax per cell by 92% and 98% respectively (D0-63, P-valueα = 1.16 x 10 -3 , P-valuePmax = 4.58 x 10 -6 ) (Figs. 4b,c) (Fig. 5a), as reported before [START_REF] Hellebust | Effects of environmental conditions on the rate of photosynthesis and some photosynthetic enzymes in Dunaliella tertiolecta Butcher[END_REF][START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF][START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF][START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF][START_REF] Kvernvik | Fast reactivation of photosynthesis in arctic phytoplankton during the polar night[END_REF][START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF]. Pmax per cell decreased faster than the RbcL cell quota, possibly because of an early inactivation of the carbon fixation enzyme [START_REF] Hellebust | Effects of environmental conditions on the rate of photosynthesis and some photosynthetic enzymes in Dunaliella tertiolecta Butcher[END_REF][START_REF] Macintyre | Activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in three marine microalgae[END_REF][START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF].

Interestingly, in the study of [START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF], the RuBisCO-to-carbon ratio did not change for cultures of Chaetoceros neogracile exposed to 1 month of darkness, despite a strong decrease in Pmax per carbon. Note that the RbcL cell quota in our study also did not decrease significantly during the first month in darkness (D0-28, P-value = 5.41 x 10 -1 ) before it began to decrease, which suggest that an initial decrease in RuBisCO activity, rather than its pool, explains the initial decrease in Pmax. Modification of the carbon fixation enzyme to an inactive state may be triggered by dark transition [START_REF] Parry | RuBisCO regulation: a role for inhibitors[END_REF]. Pmax also decreased faster relative to α, which significantly lowered the light saturation parameter Ek by 72% (D0-63, P-value = 5.38 x 10 -4 ) (Pmax/α, Fig. S10). This decrease of Ek during prolonged darkness increased the risk of photoinhibition upon subsequent re-illumination, because a given re-illumination level would rise farther above Ek, resulting in excess excitation. The photoprotective NPQ (Eqn 5) was indeed induced even for the lowest RLC irradiances, most likely because of impaired electron sink capacities such as carbon fixation [START_REF] Huner | Energy balance and acclimation to light and cold[END_REF][START_REF] Joliot | Inhibition of CO2 fixation by iodoacetamide stimulates cyclic electron flow and non-photochemical quenching upon far-red illumination[END_REF]) (Fig. S11).

While the carbon fixation curve parameters decreased, ΦM (Eqn 1) showed no significant variation after three months of darkness (D0-83, P-value = 9.96 x 10 -1 ) even though a decrease has previously been observed in prolonged darkness in diatoms [START_REF] Reeves | The effect of prolonged darkness on the growth, recovery and survival of Antarctic sea ice diatoms[END_REF][START_REF] Martin | The physiological response to increased temperature in over-wintering sea ice algae and phytoplankton in McMurdo Sound, Antarctica and Tromso Sound, Norway[END_REF][START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF]) (Fig. 4b). The internally normalized fluorescence ratio ΦM reflects the photochemical activity of the remaining PSII capable of at least a single turnover within the remaining pool of viable cells. But the PsbA determinations (Fig. 4a) show that the content of PSII decreased significantly. Analyses of rETRmax (Fig. 4c) also that electron transport away from the remaining PSII was also suppressed. Note that the drop in rETRmax is not available in Fig. 4c because no measurements were made during the first two weeks of the dark period (see Materials & Methods, sampling design). Nevertheless, together with Pmax, Fig. 4c strongly suggests that rETRmax was high before the dark transition. Thus, the combination of the drop in PSII content and the drop in rETRmax can together explain the drop in carbon fixation curve parameters. Furthermore, the 14 C incubations lasted 20 minutes. Viable cells taken out of an extended period of darkness and exposed to 14 C incubations may suffer more from impaired electron sink capacities than during a nearly instantaneous measurement of photochemical activity using a single saturating flash.

Light exposure after darkness

The dark acclimated F. cylindrus cultures were re-exposed to light after 1.5 months and after 3 months of darkness and monitored for the same physiological parameters. Previous experiments that studied the light transition from prolonged darkness in polar diatoms are scarce. However, Kvernik et al. ( 2018) and [START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF] recently studied the ability of dark acclimated polar phytoplankton communities and Chaetoceros neogracile culture, respectively, to resume photophysiolocal activity and cell growth over a wide range of irradiance. We found results consistent with their studies, with the addition of other physiological and metabolic features that complement our understanding of the acclimation processes at stake for polar night survival and return to light.

Photosensitivity and photosynthetic apparatus reassembly F. cylindrus cells acclimated to darkness largely dismantled key catalytic complexes of their photosynthetic apparatus, while retaining much of their pigment bed. To limit photodamage, diatoms mainly rely on NPQ mediated by the xanthophyll cycle [START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF], especially for F. cylindrus growing at low temperatures, which limits other physiological responses [START_REF] Petrou | Rapid photoprotection in sea-ice diatoms from the East Antarctic pack ice[END_REF][START_REF] Petrou | Heterogeneity in the photoprotective capacity of three Antarctic diatoms during short-term changes in salinity and temperature[END_REF]. Given the low photosynthetic capacities reached during the dark period, a rapid NPQ response was expected to occur immediately upon light return to dissipate excessive excitation. For the Light return 1 experiment, the highest NPQ30 was indeed observed immediately upon reillumination (Fig. 6a). This level of NPQ significantly decreased by 69% to 'a steady state' within 3 days of re-illumination (L130min-3d, P-value = 5.43 x 10 -11 ). The de-epoxidation state (DES: DT/(DD+DT)) showed consistent variations (64% significant decrease, L130min-3d, P-value = 2.35 x 10 -3 ) indicating that NPQ was mostly related to DT (Lavaud & Goss 2014) (Fig. 6a). The NPQ30 was close to the NPQmax induced during the RLC (at 111 μmol photons m -2 s -1 , Fig. S11).

In the Light return 2 experiment, the NPQ induction showed a different pattern. Despite the presence of DT (Fig. 3b), NPQ remained low (Fig. 6b). This particular inconsistency between DT synthesis and NPQ has previously been observed with Phaeodactylum tricornutum [START_REF] Lavaud | In diatoms, the transthylakoid proton gradient regulates the photoprotective non-photochemical fluorescence quenching beyond its control on the xanthophyll cycle[END_REF]Lavaud & Lepetit 2013) and F. cylindrus [START_REF] Kropuenske | Photophysiology in two major Southern Ocean phytoplankton taxa: photoprotection in Phaeocystis antarctica and Fragilariopsis cylindrus[END_REF]). According to [START_REF] Kropuenske | Photophysiology in two major Southern Ocean phytoplankton taxa: photoprotection in Phaeocystis antarctica and Fragilariopsis cylindrus[END_REF], 30 minutes of dark-acclimation before the NPQ measurements are not sufficient to achieve complete re-epoxidation of DT for highly light-stressed cells, so that NPQ remains 'locked-in' and relaxes only over several hours [START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF]. To allow this sustained part of NPQ to relax, extra samples were dark acclimated for 24 hours to calculate NPQt (Eqn 6a, 6b). NPQt developed at 30 μmol photons m -2 s -1 was higher after 2 hours, 5 hours and 1 day, but not after 30 minutes of re-illumination (Fig. S11). Possibly, for this short timing, the PSII light-harvesting system was still too dismantled to activate a fully dynamic NPQ response immediately upon re-illumination.

In the Light return 1 experiment, Chla and Fuco cell quotas first decreased over 3 days of re-illumination, although not significantly (L130min-3d, P-valueChla = 9.96 x 10 -1 , P-valueFuco = 7.76 x 10 -2 ) (Fig. 3a). The simultaneous decrease of Chla and Fuco and significant increase of DD cell quota (L130min-3d, P-value = 2.05 x 10 -4 ) (Fig. 3b) led in turn to a significant 43% decrease of the Photosynthetic/Photoprotective pigment ratio until the third day of the Light return 1 experiment (L130min-3d, P-value = 4.01 x 10 -5 ) (Fig. 3c). After 3 days, photosynthetic pigments stopped decreasing and started to build up as previously observed in past experiments on polar and non-polar diatoms [START_REF] Griffiths | Factors affecting the photosynthetic capacity of laboratory cultures of the diatom Phaeodactylum tricornutum[END_REF][START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF][START_REF] Nymark | Molecular and photosynthetic responses to prolonged darkness and subsequent acclimation to re-illumination in the diatom Phaeodactylum tricornutum[END_REF]). The late build-up of photosynthetic pigments, although not achieving statistical significance (L13d-6d, P-valueChla = 8.32 x 10 -2 , P-valueFuco = 5.56 x 10 -2 ), stabilised the Photosynthetic/Photoprotective pigment ratio until the 6 th day of the Light return 1 experiment (L13d-6d, P-value = 5.90 x 10 -1 ).

In the Light return 2 experiment, variations in Chla and Fuco cell quotas were not significantly different to the Light return 1 experiment according to the interaction terms (P-value Chla = 8.21 x 10 -2 , P-valueFuco = 7.63 x 10 -2 ) (Fig. 3a), but DD cell quota did not increase similarly as in the Light return 1 experiment and thus was significantly different, (P-valueDD = 2.96 x 10 -4 ) (Fig. 3b). Nevertheless, in both light experiments, the Photosynthetic/Photoprotective pigment ratio significantly decreased until the 6 th day of light exposure by 30% and 35% respectively (L130min-6d, P-value = 5.34 x 10 -3 / L230min-6d, P-value = 2.19 x 10 -3 ) (Fig. 3c). Thus, the pigment composition of the light harvesting antennae shifted gradually to a higher proportion of xanthophylls (DD+DT), typical of high light acclimated algal cells [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria[END_REF][START_REF] Kropuenske | Photophysiology in two major Southern Ocean phytoplankton taxa: photoprotection in Phaeocystis antarctica and Fragilariopsis cylindrus[END_REF][START_REF] Lepetit | High light acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]). The σPSII variations were similar to Photosynthetic/Photoprotective pigment, although not decreasing significantly (L130min-6d, P-value = 9.92 x 10 -1 / L230min-6d, P-value = 1.39 x 10 -1 ) (Fig. 3c), and are in agreements with the observations by [START_REF] Kvernvik | Fast reactivation of photosynthesis in arctic phytoplankton during the polar night[END_REF] on Arctic microalgae communities.

The abundance of photosynthetic proteins PsbA and RbcL increased during the Light return 1 experiment (Fig. 4a). The PsbA cell quota remained stable within the first 5 hours (L130min-5h, P-value = 1.00 x 10 0 ), consistent with rapidly-induced photoprotection protecting a further degradation of PsbA [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF]. It then significantly increased until the 6 th days (L15h-6d, P-value = 2.86 x 10 -8 ) to end up near t0 values, supporting a reassembly of the PSII reaction center (RCII) back to the pre-acclimation state. The RbcL cell quota was nearly undetectable within the first day, but then increased significantly into a quantifiable range until the 6 th day (L11d-6d, P-value = 2.38 x 10 -14 ) (Fig. 4a). However, Pmax per cell increased significantly within 1 day of light exposure (L1dark-1d, P-value = 1.54

x 10 -8 , much of the increase occurred between the previously measured dark level (D28) and the first 30 minutes of illumination) (Fig. 4c) (Fig. 5b) as also observed by [START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF]. The discrepancies with the apparent delayed recovery of RbcL content results from RbcL in darkness and early re-illumination falling below a quantifiable range. Indeed, the low residual content of RuBisCO would operate at maximal activity within 1 day of light exposure [START_REF] Macintyre | Photosynthesis and regulation of RuBisCO activity in net phytoplankton from Delaware Bay[END_REF][START_REF] Macintyre | Activation and deactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in three marine microalgae[END_REF]. ΦM also increased rapidly within 1 day of light exposure (L130min-1d, P-value = 9.55 x 10 -6 ) (Fig. 4b), supporting a fast recovery of the photophysiology [START_REF] Luder | Acclimation of photosynthesis and pigments during and after six months of darkness in Palmaria decipiens (Rhodophyta): a study to simulate Antarctic winter sea ice cover[END_REF][START_REF] Kvernvik | Fast reactivation of photosynthesis in arctic phytoplankton during the polar night[END_REF][START_REF] Lacour | Decoupling light harvesting, electron transport and carbon fixation during prolonged darkness supports rapid recovery upon re-illumination in the Arctic diatom Chaetoceros neogracilis[END_REF].

Despite the slow synthesis of photosynthetic proteins and the decrease of the Photosynthetic/Photoprotective pigment ratio, the cells achieved efficient coupling from RCII photochemistry to carbon fixation within 1 day in the Light return 1 experiment.

In the Light return 2 experiment, PsbA and RbcL cell quotas were initially nearly undetectable. The recovery slopes of Pmax per cell and ΦM appeared lower and are significantly different from those in the Light return 1 experiment according to the interaction terms (P-valuePmax = 7.32 x 10 -6 , P-valueΦM = 5.79 x 10 -9 ) (Figs. 4b,c). The recovery in the Light return 2 experiment was possibly slowed by a more extensive dismantling of the photosynthetic apparatus and electron sink capacities, and by a likely substantial population of dead cells under longer darkness acclimation (3 months vs. 1.5 months). As in the dark experiment, the variations in ΦM likely reflected the fluorescence ratio recovery within the PSII pool of remaining viable cells, while Pmax reflected the whole cell population (including dead cells and empty frustules). Thus, the speed of recovery was indeed lower in the Light return 2 experiment as shown by the variations in ΦM.

Metabolic recovery

All together the cells rapidly (within 1 day) acclimated to the applied irradiance (30 µmol photons m -2 s -1 ) in the Light return 1 experiment, as illustrated by the rapid significant increase of Ek (L1dark-1d, P-value = 2.95 x 10 -5 , much of the increase occurred between the previously measured dark level (D28) and the first 30 minutes of illumination) (Fig. S10).

The interaction term is also not significant for Ek, meaning that the increases in Ek for the Light return experiments 1 and 2 are statistically equivalent (P-value = 1.44 x 10 -1 ). Lacour [START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF]. The results of the present experiment suggest that F. cylindrus had a lag phase longer than 6 days before reaching detectable exponential growth after 3 months of darkness, yet recovery of exponential growth could not be confirmed within the timescale of our measures.

Conclusions

F. cylindrus achieved a physiological resting state a few days following the transition to dark and maintained it throughout until the return to light (Fig. 7). This rather stable state was characterized by very low consumption of energy reserves, a slow decrease of photosynthetic pigments, a faster decrease in key photosynthetic protein complexes, and very low photosynthetic capacities. Subsequent transition back to light after 1.5 months first triggered fast photoprotection followed by the renewal of photosynthetic components.

Rapid recovery of photophysiology occurred within a few hours of return to light, followed by resumption of cell growth after 1 day of re-illumination. The re-acclimated light state showed similar characteristics to high light grown cells regarding the changes in pigment composition, at least over the initial 6 days. The results from the transition to light after 3 months highlighted an apparent lag phase that increases in length with longer periods of darkness. Mortality in the dark may have delayed the full recovery of the population with an apparent lag phase longer than 6 days.

The results of this study suggest that the low rate of energy consumption for dark survival and high photoprotective capacity upon light return may be two physiological traits that help F. cylindrus to thrive in polar oceans. It remains to be investigated whether mortality or sustained down regulation, or both, are the major factor(s) explaining the stronger physiological drop-down and the delayed recoveries of measured physiological and molecular parameters after prolonged darkness (3 months). Progressive dark and light transitions, rather than sudden shifts as in this experiment, should also be tested and coupled with mortality measurements to determine if a particular light return regime compromises survival more than another one. The role of heterotrophy in dark survival remains to be clarified, because available dissolved organic carbon within and underneath sea-ice [START_REF] Riedel | Winter-spring dynamics in seaice carbon cycling in the coastal Arctic Ocean[END_REF]) could potentially improve diatom survival to the winter polar night. Finally, the expression of genes was not within the scope of this study and should also be addressed in future experiments to uncover the signature of metabolic pathways over a dark period that is significant to the Arctic polar night. Table S2 Mean±SD values of sampling Light return 1 days.

Figure legends

Table S3 Mean±SD values of sampling Light return 2 days.

  (D) and Light (L) return experiments. The measured parameters are separated into four categories (Cell, Metabolism, Molecular components and Photophysiology of the Photosynthetic apparatus). T is the temperature in Celsius degrees. Abbreviations: L = Light return, D =Dark, L-D= Light-Dark transition, POC & PON = Particular Organic Carbon & Nitrogen, Prots = Proteins, Carbs = Carbohydrate, ATP = Adenosine triphosphate, 14 C & 13 C = Radiocarbon, 14 C P. vs E. curves = Photosynthesis versus Irradiance curves of 14 Carbon fixation, Fluo = Fluorescence determinations (PSII variable fluorescence and/or spectrofluorimetry), Chla-b-c = Chlorophyll a-b-c, PSII = Photosystem II, PsbA = PSII protein D1, RbcL = RuBisCO large subunit. * indicates two or more light return experiments.

  L1 = Light return 1 experiment, L2 = Light return 2 experiment, 1: Cell number per mL and cell volume (µm 3 ), 2: Carbon & Nitrogen cell quotas, 3: Lipid droplets cell quota, 4: Pigment (Chlorophyll a, Chlorophyll c, Fucoxantin, Diadinoxanthin, Diatoxanthin) cell quotas, 5: PsbA (PSII protein D1) & RbcL (RuBisCO large subunit) cell quotas, 6: Maximum quantum yield (ΦM) and the effective absorption cross-section for PSII photochemistry (σPSII), 7: Relative electron transport rate (rETR) and nonphotochemical quenching (NPQ), 8: Carbon fixation rate (µg C m -3 h -1 )

  photochemistry (σPSII, Å 2 quanta -1 ), the minimum flux of fluorescence (F0) and the maximum flux of fluorescence of dark acclimated cells (Fm) in relative units[START_REF] Kolber | Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols[END_REF]. σPSII, F0 and Fm were measured on culture sub-samples shortly after harvesting along the dark acclimation and during the Light return 1 and Light return 2 experiments, after 30 minutes of dark acclimation. The maximum quantum yield of PSII (ΦM) was computed as:𝛷(1)A Phyto-PAM fluorometer (Phyto-ML, Heinz Walz GmbH, Germany) was also used to assess complementary fluorescence parameters. Using a different fluorometer did not compromise the interpretation of the data altogether, as the trends in ΦM were similar for both FIRe and Phyto-PAM determinations (FigS5). Phyto-PAM determinations are typically higher than FIRe determinations, but their relative variations are equivalent and comparable[START_REF] Röttgers | Comparison of different variable chlorophyll a fluorescence techniques to determine photosynthetic parameters of natural phytoplankton[END_REF]. For the Phyto-PAM fluorometer, cells were dark-acclimated for 30 minutes when applicable (Light return 1 and Light return 2 experiments) and

  different light levels provided by separate LEDs (LUXEON Rebel, Philips lumileds) from the bottom of each alveolus. The PAR (E, µmol photons m -2 s -1 ) in each alveolus was measured before incubation with a quantum sensor (Heinz Walz GmbH, US-SQL) equipped with a 4π collector. After 20 min of incubation, culture aliquots were fixed with 50 µL of buffered formalin then placed under the fume hood and acidified (250 µL of HCl 50%) for 3 hours to remove the excess inorganic carbon (JGOFS protocol, UNESCO 1994). Finally, 6 mL of scintillation cocktail (Ecolume, MP Biomedicals) were added to each vial prior to counting using a liquid scintillation counter (Perkin Elmer® Tri-Carb 2910TR). This step allowed determining the amount of radiolabeled carbon assimilated by the cells from the number of disintegration per minute (DPM). To determine the total amount (total activity) of bicarbonate added, three 20 µl aliquots of radioactive sample were added to 50 µl of an organic base (ethanolamine) and 6 ml of the scintillation cocktail into glass scintillation vials. The carbon fixation rate was finally computed according to[START_REF] Parsons | Photosynthesis as measured by the uptake of radioactive carbon[END_REF]

  2018). The complete and detailed statistical results are provided in supplementary files. The p-values reported in the text are those for the parameters relative change for the considered periods (supplementary files, posthoc_comparisons_dark & posthoc_comparisons_light) or those for the interaction term between time and experiment to determine if a parameter's variations for both light return periods are similar to each other or not (model_significance_light). The figures presented in the Results and Discussion section show Dark, Light return 1 and Light return 2 data altogether to provide a visual comparison between the three experiments. Light return 1 and Light return 2 data are enlarged in supplementary figures to allow easier comparison between each other. The lines between sampling periods are strictly shown for visual clarity and do not imply interpolated levels of any parameters. The mean values and standard deviations plotted in these figures are available for the main sampling periods in Table

  photons m -2 s -1). In the present study, the time at which Ek matches the actual irradiance the cells were exposed to during light returns testifies to their full ability to perform photochemistry and fix carbon to support anabolism and ultimately cell growth, particularly during the Light return 1 experiment.

Figure 1 .

 1 Figure 1. Timeline of the sampling strategyBefore dark transition, cultures were grown under stable light conditions for 52 days.Cultures were then transferred to complete darkness (grey arrow) at 'day 0' (t0 in the following figures). The vertical lines show the times of sampling, and the dashed lines show transfer of a fraction of the replicate cultures to Light return 1 after 1.5 months (48 days) and Light return 2 after 3 months (90 days) of dark incubation. Upon light return, dark acclimated subsamples were transferred to the same light conditions as before darkness.

Figure 2 .

 2 Figure 2. Cells and reserves a) Biovolume (µm 3 , red) and Cell number per ml (blue); b) Cell volume (µm 3 , red) and lipid droplets cell quota (RFU, blue); c) µg carbon (red) and µg nitrogen per cell (blue) of Fragilariopsis cylindrus cultures kept in the dark at 0°C for up to 3 months and then reexposed to continuous light of 30 µmol photons m -2 s -1 after 1.5 months or 3 months of darkness for the light return experiments 1 and 2, respectively. Values from the light return experiments are shown enlarged in Fig. S6. Each point is the mean of the three cultures with the standard deviation as the error bar, except for the carbon points after the first month of darkness from which a divergent culture replicate was discarded (red dots) from the mean and standard deviation calculations.

Figure 3 .

 3 Figure 3. Photosynthetic and photoprotective pigments a) µg Chlorophyll a (Chla, red) and Fucoxanthin per cell (Fuco, blue); b) µg Diadinoxanthin (DD, red) and Diatoxanthin per cell (DT, blue); c) the effective absorption cross-section for PSII photochemistry (σPSII, Å 2 quanta -1 , red) and photosynthetic / photoprotective pigments (Chla + Chlc + Fuco / DD + DT, blue) of Fragilariopsis cylindrus cultures kept in the dark at 0°C for up to 3 months and then re-exposed to continuous light of 30 µmol photons m -2 s -1 after 1.5 months or 3 months of darkness for the light return experiments 1 and 2, respectively. Values from the light return experiments are

Figure 4 .

 4 Figure 4. Photosynthethic proteins and photosynthesis parameters a) µg PsbA (PSII protein D1, red) and µg RbcL per cell (RuBisCO large subunit, blue); b)α (initial slope of carbon fixation, [µg C cell -1 h -1 (μmol photons m -2 s -1 ) -1 ], red) and ΦM (maximum quantum yield of PSII, dimensionless, blue); c) Pmax (maximum carbon fixation rate, µg C cell -1 h -1 , red) and rETRmax (maximum relative electron transport rate, dimensionless, blue) of Fragilariopsis cylindrus cultures kept in the dark at 0°C for up to 3 months and then re-exposed to continuous light of 30 µmol photons m -2 s -1 after 1.5 months or 3 months of darkness for the light return experiments 1 and 2, respectively. Values from the light return experiments are shown enlarged in Fig.S9. Each point is the mean of the three cultures with the standard deviation as the error bar, except for the PsbA Light return 1 points from which a culture replicate was discarded (red dots) of the mean and standard deviation calculations. Note that other samples for PsbA (after 2 months of darkness and during Light return 2, red dots) and RbcL (after 2 months of darkness, during Light return 1 until day 1 and during Light return 2, blue dots) are to be treated with caution as most of them were detectable but did not fall into a quantifiable range.

Figure

  Figure 5. 14 C incubation curves a) Carbon fixation vs. irradiance curves (µg C cell -1 h -1 ) of Fragilariopsis cylindrus cultures kept in the dark at 0°C for up to 3 months and then exposed to continuous light of 30 µmol photons m -2 s -1 after b) 1.5 months (Light return 1) or c) 3 months of darkness (Light return 2). Each curve is fitted on data points pooled from three cultures for each sampling time.

Figure 6 .

 6 Figure 6. Photoprotective capacity a) NPQ developed at 30 µmol photons m -2 s -1 (NPQ30, dimensionless, red) and Deepoxidation state of Diadinoxanthin (DES, blue) of Fragilariopsis cylindrus cultures kept in the dark at 0°C for up to 3 months and then re-exposed to continuous light of 30 µmol

Figure S1

 S1 Figure S1 Picture of F. cylindrus cultures grown during light acclimation.

Figure S2

 S2 Figure S2 Spectrum of the different light sources used during the experiments.

Figure S3

 S3 Figure S3Picture of F. cylindrus cultures grown during the light return experiments.

Figure S4

 S4 Figure S4 Flow cytometry data for emitted BODIPY fluorescence Figure S5 ΦM determinations from PAM and FIRe fluorometers Figure S6 Figure 2 enlarged for the light return experiments.

Figure S7

 S7 Figure S7Comparison between carbon and nitrogen per cell and per ml.

Figure S8

 S8 Figure S8 Figure 3 enlarged for the light return experiments.

Figure S9 Figure 4

 S94 Figure S9 Figure 4 enlarged for the light return experiments.

Figure S10

 S10 Figure S10Ek 14 C (Pmax/α) and Ek PAM (rETRmax/α).

Figure S11

 S11 Figure S11Curves of dynamic and total non-photochemical quenching.

Table 1

 1 Chronological list of dark survival experiments for microalgal species with the present study highlighted. 174

	Species	Length of experiment (days)	Cell	Parameters Metabolism and Reserves components Photosynthetic apparatus Molecular Photophysiology	T(°C)	References
	Fragilariopsis cylindrus	D: 90 L: 6*	Number Volume	POC & PON Lipids	Pigments RbcL, PsbA	14 C P. vs E. curves Fluo	0	This study
	Chaetoceros neogracile	D: 30 L: 8,14	Number Volume	POC & PON	Pigments RbcL	14 C P. vs E. curves Fluo	0	Lacour et al., 2019
	Arctic phytoplankton community of polar night	D: in situ L: 2	N/A	N/A	Chla	14 C uptake Fluo	1.5, 2	Kvernvik et al., 2018
	Fragilariopsis cylindrus	D: 7	N/A	Gene expression	Gene expression	N/A	-2, 11	Mock et al., 2017
	Navicula cf. perminuta	D: 56	N/A	Lipids, Prots, Carbs	N/A	N/A	0, 7	Schaub et al., 2017
	Phaeodactylum tricornutum	D: 2 L: 1	Number Morphology	N/A	Pigments Gene expression	Fluo	15	Nymark et al., 2013
	Polar algal communities	D: 22-35 L: 1	N/A	Carbs	Chla	Fluo	-2, 4, 10, 20	Martin et al., 2012
	Fragilariopsis cylindrus Thalassiosira antartica…	D: 30-60 L:growth**	N/A	Carbs	Chla	Fluo	-2, 4, 10	Reeves et al., 2011
	Diatom sediment samples	D: 371 L: 1	N/A	N/A	Pigment content (Dark only)	13 C uptake (Light only)	17	Veuger & Van Oevelen 2011
	Sediment sample / isolation of Skeletonema marinoi	D: >100 y L: N/A	Growth (Light only)	N/A	N/A	N/A	10	Harnstrom et al., 2011
	Diatom sediment samples	D: 15-64 L: 1-4 h	N/A	N/A	N/A	Fluo	4-6	Wulff et al., 2008
	Aureococcus anophagefferens	D: 14	Number	POC & PON,	Chlorophyll a	14 C P. vs E. curves		Popels et al.,
		L: 4-5*	Bacteries	Lipids, Prots, Carbs	RbcL	Fluo	6	2007
	Koliella antartica	D:60	Morphology N/A	Chla,b	N/A		Ferroni et al.,
					PSII assembly		5	2007
	Xanthonema sp.	D: 60-90	Number	N/A	Chla,b,carotenoid	N/A		Baldisserotto et
	Koliella antartica		Morphology		PSII assembly		4, 5	al., 2005
	Diatom sediment samples	D: > 55 y L: 30-40	Growth (Light only)	N/A	N/A	N/A	3, 10, 18	McQuoid et al., 2002
	Palmaria decipiens	D: 180	N/A	N/A	N/A	Fluo	0	Luder et al.,
		L: 28						2002

Table 2 :

 2 Sampling time-points of the parameters measured during the dark and light experiments

	Time	1 Cell number & volume	2 Carbon & nitrogen	3 Lipid droplets	4 Pigment	5 Photosynthetic proteins	6 Variable fluorescence (FIRe)	7 Variable fluorescence (PAM)

D : -1 day X X X X X X X D : 0 days X X X X X X X D : 1 day X X X X X X X D : 2 days X D : 5 days X X X X X X X D : 14 days X X X X X X X X D : 28 days X X X X X X X X D : 47 days X D : 63 days X X X X X X D : 83 days X X X X X X X X L1 : 30 min X X X X X X X L1 : 2 hours X X X X X X X L1 : 5 hours X X X X X X X X L1 : 1 day X X X X X X X X L1 : 3 days X X X X X X X X L1 : 6 days X X X X X X X X L2 : 30 min X X X X X X X L2 : 2 hours X X X X X X X L2 : 5 hours X X X X X X X X L2 : 1 day X X X X X X X X L2 : 3 days X X X X X X X X L2 : 6 days X X X X X X X X

Acknowledgements

The authors thank Catherine Lalande and Thibaud Dezutter for their help with the CHN analyzer, Marie-Josée Martineau for her help with pigment analysis, Gabrièle Deslonchamps for her help with the culture media nutrients analysis, Alexandre Dubé and Chris Eberlein for their help with the flow cytometer, Marc-André Lemay for his help with R programming, José Lagunas-Morales and Guislain Bécu for their help with the illumination system of the CARON growth chamber and Nicolas Schiffrine for his help with algal culturing. This work was supported through NSERC Discovery, Fonds de recherche du Québec -Nature et technologies and Canada Excellence Research Chair in Remote Sensing of Canada New Arctic Frontier (M. Babin), and through a Canada Research Chair in Phytoplankton Ecophysiology, the Canada Innovation Foundation and the New Brunswick Innovation Foundation (D. A. Campbell).

Results & Discussion

Prolonged darkness exposure to mimic the polar night Light acclimated cultures of F. cylindrus were exposed to complete darkness over a period of three months to mimic the polar night. Multiple physiological processes were monitored for the first time in a dark experiment to such an extent on the model polar diatom F. cylindrus. The results are discussed with respect to earlier findings on polar and non-polar species to understand diatom dark survival in polar environments.

Cell and reserves

Cell number per ml of algal culture (Fig. 2a) increased slightly but significantly during the first month of the dark period by 27% (D0-28, P-value = 4.26 x 10 -3 ), which is likely due to a final cell division for a subset of the cell population at the beginning of the dark period. For the same period, the culture biovolume (Fig. 2a) did not increase significantly (D0-28, Pvalue = 1.05 x 10 -1 ) because the average cell volume significantly decreased by 8% (D0-28, P-value = 1.08 x 10 -2 ) as a consequence of cell division (Fig. 2b). The cell number and the biovolume then decreased until the end of the third month of darkness, although not significantly (D28-83, P-valuecell number = 1.06 x 10 -1 , P-valuebiovolume = 1.38 x 10 -1 ), to end up at values slightly above the t0 (values just before dark transition). During the first month of darkness, the carbon cell quota significantly decreased by 34% (D0-28, P-value = 3.57 x 10 - 3 ) (Fig. 2c). The Carbon cell quota then significantly increased until the end of the third month (D28-83, P-value = 5.19 x 10 -4 ) to values slightly above the t0. The variations in the nitrogen cell quota were similar to those of cellular carbon but none of the comparisons were significant (D0-28, P-value = 1.11 x 10 -1 ; D28-83, P-value = 9.02 x 10 -2 ). A potential mechanism for long-term dark survival is to lower metabolism and to fine-tune the utilization rate of stored energy products [START_REF] Handa | Carbohydrate metabolism in the marine diatom Skeletonema costatum[END_REF][START_REF] Palmisano | Physiology of sea ice diatoms. I. Response of 3 polar diatoms to a simulated summer-winter transition[END_REF][START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF][START_REF] Jochem | Dark survival strategies in marine phytoplankton assessed by cytometric measurement of metabolic activity with fluorescein diacetate[END_REF][START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF][START_REF] Schaub | Effects of prolonged darkness and temperature on the lipid metabolism in the benthic diatom Navicula perminuta from the Arctic Adventfjorden, Svalbard[END_REF]). In our experiment, the carbon cell quota did decrease for the first month of the dark period.

However, this decrease was likely the consequence of cell division on cell volume. Indeed, the particulate organic carbon concentration per ml of culture did not decrease significantly during the first month (D0-28, P-value = 1.58 x 10 -1 ) (Fig. S7). The lipid droplets cell quota probed with BODIPY (RFU) remained mostly constant within the first month with a slight but significant increase of 5% (D0-28, P-value = 1.18 x 10 -2 ) (Fig. 2b). Lipid droplets then slowly and significantly decreased until the end of the third month in the dark by 11% (D28-83, P-value = 1.14 x 10 -5 ). Hence, this suggests very low dark metabolic rates and low energy reserve consumption as a survival process in F. cylindrus. The study by [START_REF] Mock | Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus[END_REF] on the F. cylindrus transcriptome response to 7 days of darkness showed that metabolic activities were largely suppressed with approximately 60% of all genes downregulated. However, genes involved in starch, sucrose and lipid metabolism were upregulated, which likely fuelled the remaining metabolic needs within the cell for this short period of darkness. It is common that the rate of energy consumption is high in the first weeks of darkness, but decreases as the cells age in darkness [START_REF] Handa | Carbohydrate metabolism in the marine diatom Skeletonema costatum[END_REF][START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF][START_REF] Popels | Physiological responses during dark survival and recovery in Aureococcus anophagefferens (Pelagophyceae)[END_REF][START_REF] Schaub | Effects of prolonged darkness and temperature on the lipid metabolism in the benthic diatom Navicula perminuta from the Arctic Adventfjorden, Svalbard[END_REF]. The results of the present study show a more stable pattern in the carbon, nitrogen and lipid droplets quotas, which is in agreement with global metabolism suppression over a long period of darkness. This global metabolism suppression could also be due, to some extent, to the colder temperature at which polar species grow. The rate of energy reserve depletion is likely to be lower for polar species, despite adaptations that compensate for lower kinetics at low temperature [START_REF] Lyon | Polar microalgae: new approaches towards understanding adaptations to an extreme and changing environment[END_REF], and may contribute to longer darkness survival for polar species than for temperate species as observed by [START_REF] Peters | Prolonged darkness and diatom mortality. II. Marine temperate species[END_REF].

The minor increase of the carbon quota after 1 month was unexpected. Note that if bacterial presence and growth were to account for this increase, a sufficient amount of dissolved organic carbon (DOC) would have had to be initially present in the culture medium to sustain heterotrophic bacterial growth [START_REF] Rivkin | Inorganic nutrient limitation of oceanic bacterioplankton[END_REF]). The cultures were axenically handled and the fresh culture medium was initially free of DOC. It is unlikely that DOC was released from broken cells by the magnetic stirrer because the cell number remained quite steady through the full length of the experiment. Despite the minor increase in cellular carbon toward the end of the dark experiment, the general trend of the cell reserves data supports the interpretation of a suppression of metabolic activity in the dark.

Carbon and nitrogen cell quotas increased significantly by 62% and 66%, respectively, over 6 days for the Light return 1 experiment (L130min-6d, P-valuecarbon = 1.12 x 10 -2 , P-valuenitrogen = 2.10 x 10 -6 ) (Fig. 2c). The lipid droplets cell quota also significantly increased by 14% in 6 days (L15h-6d, P-value = 3.85 x 10 -6 ) (Fig. 2b). However, carbohydrates probably accounted for some of the increase in carbon quotas [START_REF] Myklestad | Production of carbohydrates by marine planktonic diatoms. I. Comparison of nine different species in culture[END_REF][START_REF] Chauton | Gene regulation of carbon fixation, storage, and utilization in the diatom Phaeodactylum tricornutum acclimated to light/dark cycles[END_REF] despite the lack of carbohydrates data to support this statement. The cell volume significantly increased by 31% in 6 days (L130min-6d, P-value = 1.19 x 10 -11 ) (Fig. 2b) which is consistent with a larger content in lipids, carbon and nitrogen. As expected, the cell population entered an exponential growth phase measured between 1 day and 6 days following exposure to light with a mean population growth rate of 0.146±0.010 d -1 (Fig. 2a). This was 40% lower than the mean growth rate measured during the pre-darkness acclimation period (0.244±0.041 d -1 ), possibly as a consequence of the larger size of new cells and of potential mortality before and during light return. For the Light return 2 experiment, the increase in the carbon cell quota was statistically equivalent to the Light return 1 experiment as supported by the interaction term (P-value = 1.28 x 10 -1 ) while nitrogen cell quota, lipid droplets cell quota, cell volume and cell number per ml of algal culture slopes were significantly different from the Light return 1 experiment (P-valuenitrogen = 7.28 x 10 -5 , P-valuelipid = 2.34 x 10 -2 , P-valuecell volume = 3.12 x 10 -11 , P-valuecell number = 1.38 x 10 -7 ) (Figs. 2a,b,c). Cell growth was not observed over the Light return 2 tracked reillumination period. Along with the greater dismantling of the photosynthetic apparatus impacting the speed of photophysiological recovery, mortality may have compromised the population ability to reinitiate detectable growth upon light exposure. Nevertheless, a fraction of the population recovered as the ΦM data suggest that living cells recovered function of the PSII pool after ~ 3 days in Light return 2 (Fig. 4b). For parameters that showed a lag phase, that lag phase may be in fact only apparent, because those parameters were normalized to the total number of cells (including the dead ones). As healthy cells divide and the relative contribution of dead cells thereby decreases, full population recovery may be observed. The lag phase was previously reported to increase with increasing previous dark period [START_REF] Dehning | Survival of Scenedesmus acuminatus (Chlorophyceae) in darkness[END_REF][START_REF] Peters | Prolonged darkness and diatom mortality I: Marine Antarctic species[END_REF]. In antarctic diatoms, the lag phase lasted 4 days following 74 days in the dark (Peters &

Authors Contribution

Morin P. I. is the main author of this work with major contributions to designing and running the experiments, analysing the data and writing the paper. All co-authors helped with running the experiments and/or revising the paper. Campbell D. A., J. Lavaud and M.

Babin helped with the structure of the manuscript and the discussion of the data. photons m -2 s -1 after 1.5 months (Light return 1) or b) 3 months of darkness (Light return 2).

Each point is the mean of the three cultures with the standard deviation as the error bar. cell quotas and with rETRmax (maximum relative electron transport rate), NPQ (nonphotochemical quenching) and Pmax (maximum carbon fixation rate per cell). Note that rETRmax and NPQ levels are hypothesized before dark transition (day 0) as there were no measurements available for this particular sampling time. The number of photosynthetic and photoprotective pigments (green and orange oval shapes, respectively) aims to represent the measured levels per cell and their ratio to each other, rather than an exact view of the photosynthetic apparatus. Legend for levels: +++ (highest), ++ (high), + ( moderately high), -(moderately low), --(low), ---(lowest). Each level is assigned relative to the observed variation for a given parameter within the entire experiment.

Supporting information legend