Always ready? Primary production of Arctic phytoplankton at the end of the polar night

The end of the polar night with the concurrent onset of photosynthetic biomass production ultimately leads to the spring bloom, which represents the most important event of primary production for the Arctic marine ecosystem. This dataset shows, for the first time, significant in situ biomass accumulation during the dark–light transition in the high Arctic, as well as the earliest recorded positive net primary production rates together with constant chlorophyll a‐normalized potential for primary production through winter and spring. The results indicate a high physiological capacity to perform photosynthesis upon re‐illumination, which is in the same range as that observed during the spring bloom. Put in context with other data, the results of this study indicate that also active cells originating from the low winter standing stock in the water column, rather than solely resting stages from the sediment, can seed early spring bloom assemblages.

In the polar oceans, several months of darkness prevent phototrophic biomass accumulation during winter. While traditionally, phototrophic organisms have been assumed to overwinter mainly in the form of resting stages, the presence of active cells in the water column in the middle of the polar night as well as the importance of mixotrophic lifestyle during low-light periods have been increasingly acknowledged in the recent years ). Yet, knowledge on the ecophysiological characteristics of these assemblages is still rare. Measurements and experiments during the polar night in Svalbard indicate that phytoplankton are capable to reinitiate photosynthesis almost immediately upon reillumination in the laboratory, even if in situ primary production is not measurable (Berge et al. 2015;Kvernvik et al. 2018). From experiments with single strains of polar phytoplankton, there is increasing evidence that species differ in the extent to which they manage to decrease metabolic energy needs in the dark as well as in their capacity to reestablish photosynthesis and growth upon re-illumination, yet overall capacities for recovery are remarkable (Kennedy et al. 2019;Lacour et al. 2019;Morin et al. 2019;van de Poll et al. 2020a). In view of this, it seems to be the return of sufficient sunlight (Cohen et al. 2020) for net growth rather than germination cues or molecular clocks that determines the start of phototrophic production.
The theoretical lower light limit of photosynthesis is with 0.01 μm photons m À2 s À1 quite low (Raven et al. 2000), but net pelagic phytoplankton growth has mostly been assumed to start at 1-5 μmol photons m À2 s À1 (Siegel et al. 2002;Boss and Behrenfeld 2010). More recently, extremely efficient usage of low light by Arctic phytoplankton and ice-algae with positive growth rates in extremely low-light environments of 0.17-0.25 μmol photons m À2 s À1 in and under sea ice have been observed (Hancke et al. 2018;Randelhoff et al. 2020). This is consistent with satellite-based observations that indicate that rates of biomass accumulation are actually highest in the early phase of Arctic spring, when biomass is still very low (Behrenfeld et al. 2017).
Due to logistic challenges, however, almost no discrete measurements of phytoplankton biomass development and productivity during the Arctic winter-spring transition exist (but see van de Poll et al. 2020b). To this end, surface chlorophyll a (Chl a) and particulate organic carbon (POC) concentrations as proxies for biomass as well as net primary production were measured at a coastal Arctic site (79 N, Svalbard, Norway) during the transition from polar night to daylight in February 2018.

Seawater sampling was conducted in February 2018 in
Kongsfjorden, an open fjord on the west-coast of the Svalbard (Hop & Wiencke 2019). Due to logistical constrains so early in the year, sampling was conducted nearshore at 10 m water depth from the outer jetty of the harbor of Ny-Ålesund (78 55.72 0 N, 11 56.22 0 E) during a phase where no boat traffic occurred.
Average wind speed and incoming incident photosynthetically active radiation (PAR) were measured close to the nearby AWIPEV Atmospheric Observatory (Maturilli 2020). In short, a ThiesClima combined wind sensor was used to derive classic hourly averaged wind speed (m s À1 ) were recorded at 10 m height. Hourly averaged incoming PAR in the range of 370-695 nm was calculated by subtracting UV and IR from the global incoming radiation, which was measured using pyranometers with different shading domes (Maturilli et al. 2019). Data were converted from planar measurements in (W m À2 ) to scalar data in (μmol photons m À2 s À1 ).
Manual conductivity-temperature-depth (CTD) profiles were collected with a XR-620 CTD (RBR Ltd), equipped with a fluorescence sensor. Discrete samples from 0.5 m water depth were collected by single 5 L Niskin hauls. Samples for determination of total Chl a were gently vacuum-filtered (max. À200 kPa relative to atmosphere) onto precombusted (15 h, 500 C) GF/F filters (Whatman) and immediately frozen and stored at À20 C. For extraction, samples were placed in 6 mL 90% cooled acetone, homogenized using a cell mill (Precellys) and left overnight at À20 C. Chl a concentrations were determined on a fluorometer (Trilogy, Turner Designs), using an acidification step (1 M HCl) to determine phaeopigments Knap et al. (1996). Similar to Chl a, samples for POC and particulate organic nitrogen (PON) were gently vacuum-filtered onto precombusted GF/F filters and stored at À20 C. Samples were acidified with HCl to remove inorganic carbon and dried for at least 12 h at 60 C prior to sample preparation. Analysis was performed using a CHNS-O elemental analyzer (Euro EA 3000, HEKAtech).
Species presence was determined from samples collected with a small Apstein hand-net with 20 μm mesh size. The concentrated sample was transferred into a 50 mL falcon tube, fixed with Lugols solution (1% final concentration) and stored in the dark at 4 C. At the home laboratory, samples were settled for at least 24 h in an Uthermoehl chamber, and analyzed on an inverted light microscope (Axiovert Observer, Zeiss).
Potential net primary production rates were determined in duplicate 500 mL samples by incubation with NaH 14 CO 3 spike (53.1 mCi mmol À1 ; Perkin Elmer; applied specific activity of 0.5 μCi mL À1 ) in unscratched 500 mL polycarbonate bottles (Nalgene) for 24 h under reference conditions (1.0 AE 0.6 C and 10 AE 3 μmol photons m À2 s À1 ) together with a dark control. For in situ net primary production (NPP) (specific activity of 0.5 μCi mL À1 ), two 500 mL samples were incubated at 0.2 m water depth in the harbor of Ny-Ålesund. Alongside with the polycarbonate incubation bottles, a min-iPAR logger (PME) with a cosine-corrected underwater quantum sensor LICOR) was mounted to log the irradiances during the incubations. Please note that polycarbonate blocks UV, so that UV stress and inhibition were excluded. For both types of incubations, two 100 μL aliquots were mixed with 100 μL ethanolamine immediately after spiking to determine the total amount of added NaH 14 CO 3 . After 24 h, incubated samples were filtered onto GF/F filters, acidified with 200 μL of 1 M HCl and left to degas overnight. After addition of 10 mL of scintillation cocktail (Ultima Gold AB, PerkinElmer), samples were vortexed and left to stand in the dark for approximately 12 h before counting on the liquid scintillation counter, using automatic quench correction and a counting time of 5 min. For blank determination, one replicate was immediately acidified with 0.5 mL 6 M HCl and treated as samples. Subtracted blank values were 25% AE 25% (n = 23) of the incubated sample counts. The disintegrations per minute of the dark control were subtracted from those of the light-incubated bottles. Potential and in situ NPP was calculated following Nielsen (1955).

Results and discussion
Surface phytoplankton Chl a concentrations and primary productivity were measured in February 2018 spanning the period when the sun reappears and raises over horizon on the 17 th of February, that is, covering the official end of the polar night on Svalbard. Day length increased from less than 2 h on the 18 th to more than 5 h on the 24 th of February. During the sampling period, daily average incoming irradiance levels increased from 2 μmol photons m À2 s À1 on the 15 th to 10 μmol photons m À2 s À1 on the 21 st , with the irradiances between 22 nd and 24 th of February being lower due to cloud cover (5-7 μmol photons m À2 s À1 , Fig. 1A). At the same time, peak irradiances increased from 16 to 60 μmol photons m À2 s À1 .
During the first sampling on 15 February, surface Chl a concentrations were with 0.05 AE 0.001 μg L À1 (Fig. 1C) well above previously reported winter values 0.01-0.02 μg L À1 from Kongsfjorden (Berge et al. 2015;Hegseth et al. 2019), but below prebloom concentrations of 0.1-0.2 μg L À1 measured in the first half of April (Hegseth et al. 2019;van de Poll et al. 2020b;Hoppe et al. 2021). Manual CTD casts indicated no detectable stratification down to 9 m water depth (data not shown), but low daily average wind speeds of 2-5 m s À1 during the first week of the study (Fig. 1B) suggest the potential of a stabilization of the upper water column. In the following days, surface Chl a concentrations increased more than threefold to 0.18 AE 0.012 μg L À1 on 21 February. Exponential fitting indicated a Chl a-based specific growth rate constant of 0.46 d À1 (R 2 = 0.98), corresponding to a doubling of Chl a roughly every 36 h. As measured POC:Chl a ratios showed no clear trend (Table 1) Chl a may be used as a proxy for phytoplankton biomass for the short time span of this study. Such high POC:Chl a ratios, indicative a high nonphototrophic component of the particulate organic carbon pool, are commonly observed in high Arctic environments outside of bloom situations (Riedel et al. 2008;Campbell et al. 2016;Leu et al. 2020;Matthes et al. 2021). In combination with the observed increase in volume-based NPP, in situ as well as under reference conditions (Fig. 2), the development in Chl a (Fig. 1C) thus indicates significant biomass buildup during the polar night-day transition period.
While concurrent Chl a or biomass standing stocks cannot be estimated due to the lack of deeper samples further inside the fjord, the available data indicate nonetheless that phytoplankton can grow fast during this period (Fig. 1C). The resulting growth rate constant of 0.46 d À1 in the surface argues for an astonishingly high rate of biomass accumulation under such low light levels, exemplifying how efficiently Arctic phytoplankton can use light and exceed light compensation already at very low intensities. This is consistent with Randelhoff et al. (2020) who showed based on Argo float data that phytoplankton biomass accumulation in Baffin Bay (69-75 N) started already in February, so only days to weeks after the end of the polar night at this range of latitudes, despite snow and ice cover strongly reducing light availability in the surface ocean. Despite the similarity with regard to the early onset of biomass accumulation, the Chl a-derived growth rates in the latter study were more than an order of magnitude lower than those observed here, which likely stem from stronger light attenuation due to ice and snow cover in the study site of Randelhoff et al. (2020). Together, both studies emphasize that Arctic phytoplankton possess the abilities to perform photosynthesis under very low irradiances. The extent to which this actually occurs will depend on the specific environmental setting and the present phytoplankton assemblage.
After the period of low winds and increasing Chl a concentrations, daily wind speeds increased to values between 9 and 15 m s À1 during the 22 nd to 24 th of February (Fig. 1B). The peak of hourly wind speeds of 18 m s À1 (i.e., gale force) was measured in the night between 22 nd and 23 rd . After the storm, surface Chl a concentrations had decreased to values of 0.08 AE 0.002 μg L À1 on February 24 th (Fig. 1C). This indicates that biomass accumulation was constrained to the surface waters before, and wind-induced mixing diluted the biomass. One should note that while water depth was shallow at the sampling location, the fjord is already deeper than 300 m less than 2 km from the shore (Tverberg et al. 2019). Given that Chl a concentrations after this strong wind event were still more than four times higher than those measured in the middle of the polar night, significant accumulation must have taken place even prior to the sampling, potentially from the beginning of the Civil Twilight (solar elevation above À6 ; Cohen et al. 2020) on the first of February or even before. Given that wind speeds were low (Fig. 1) and wind directions came from rather southwesterly to southerly directions (Maturilli 2018), significant advection from the outside of the fjord seems rather unlikely (Cottier et al. 2007;Tverberg et al. 2019). Also, even if the biomass was laterally advected, it would still have been formed under very similar conditions as observed in this study, despite potentially even lower irradiances if originating from more deeply mixed waters outside the sheltered fjord.
To investigate primary production in surface waters during the study period, two incubations were conducted in situ at 0.2 m water depth. In the first incubation, that took place on the 15 th of February under in situ daily average irradiances of 1.4 μmol photons m À2 s À1 and peak irradiances at 7.6 μmol photons m À2 s À1 (Fig. 2E), in situ NPP was not detectable (Fig. 2B,D). Given the above-described Chl a increase from the 17 th to the 19 th as well as the as the fact that the machinery for light harvest and carbon fixation was present and activated (see below), the apparent lack in situ NPP is likely due to detection limits and uncertainties of the applied method  (AE 0.003 μmol C L À1 d À1 for the current dataset) rather than being truly zero. More sensitive methods would have to be used to prove either of these options. Moreover, it should be noted that, according to the manufacturer, the incubation bottles have an 80-90% light transmission capacity may have blocked some of the incoming light. Six days later, that is, on the 21 st , rates had increased above the measurement uncertainty, now being 0.009 AE 0.001 μmol C L À1 d À1 and 0.90 AE 0.09 μg C (μg Chl a) À1 d À1 for volume-and Chl aspecific estimates, respectively (Fig. 2B,2D). By then, the polar night had ended and daily average in situ irradiances were 3.0 μmol photons m À2 s À1 , with peak irradiances of 16.7 μmol photons m À2 s À1 (Fig. 2E). Please note that carbon fixation data estimated from dark incubations (Table 1), run in parallel to all incubations, was subtracted from the carbon fixation measurements in the light to derive NPP (Fig. 2). Dark fixation was with 0.004 μmol C L À1 d À1 in the same range as NPP on 15 th of February, but only half as much as NPP on the 21 st . Also, irradiances in the incubation bottes may have been 10-20% lower than in situ (see above), potentially making the measured rates even more astonishing. To my knowledge, this is the earliest positive in situ NPP ever measured in the Arctic spring (i.e., only 4 d after the end of the polar night).
To provide a better understanding of the observed in situ data, NPP was also measured under reference conditions of 1 C and 10 μmol photons m À2 s À1 over the study period. Volumebased reference-NPP increased from 0.004 AE 0.001 μmol C L À1 d À1 on the 15 th to 0.016 AE 0.001 μmol C L À1 d À1 on the 21 st of February ( Fig. 2A), mirroring the development in Chl a. Similar to the in situ measurements, C-fixation in the dark was in the  (Table 1), and PAR (μmol photons m À2 s À1 ) in the different incubations (E). Gray shaded areas indicates polar night (i.e., solar elevation below À6 ).
same range as reference-NPP at the first sampling time point (i.e., 77%), but did not increase over time so that at the last time point, it accounted only for an additional 19% of C-fixation measured in the light (Table 1). When normalized to Chl a, however, C-fixation values ranged between 0.786 and 0.979 μg C (μg Chl a) À1 d À1 without a temporal trend (Fig. 2C). This indicates that no substantial changes in photoacclimation and photosynthetic efficiency occurred over the study period. In fact, these values are in the same range to those derived from the subsequent spring bloom assemblage observed in April and May (0.88 AE 0.26 μg C (μg Chl a) À1 d À1 at 10 μmol photons m À2 s À1 ; Hoppe et al. 2021). Such surprisingly high rates of Chl a-specific carbon fixation indicate that Arctic phytoplankton either sustain fully functional and rather efficient photosystems throughout the polar night, or that they recover really quickly. Applying the same dark-control and normalization, the here measured values are even slightly lower than Chl a-specific NPP measured by Kvernvik et al. (2018) in the same study area in January, that is, in the middle of the polar night.
These findings indicate an extraordinary ability of phytoplankton to sustain similar photosynthetic efficiencies throughout the most extreme changes in their environment, being a change from completely darkness to 24 h sunshine in spring. This is in line with laboratory experiments with the Arctic diatom Chaetoceros neogracilis, where growth rate measured after 1 month of darkness were only slightly influenced by the irradiances applied upon re-illumination (Lacour et al. 2019). It also fits into the increasing perception that Arctic coastal primary producers possess high capacities to compensate for environmental variability keeping species composition, ecophysiological parameters, and productivity comparably stable (Panči c et al. 2015;Hoppe et al. 2018a,b;Wolf et al. 2021Wolf et al. , 2019. Whether or not some metabolic functions other than photosynthetic capacities still shift to some sort of resting mode is yet to be investigated. A major drawback for understanding the conditions under which phytoplankton growth is initiated is that we currently do not have satisfactory estimates of phytoplankton respiration under different conditions (Behrenfeld and Boss 2018), despite knowing that respiration declines with decreasing irradiance (Geider et al. 1986;Halsey et al. 2010). Even though it seems plausible to assume relatively low costs of cell maintenance, especially of the photosynthetic apparatus, due to the cold and dark conditions, cells likely need alternative energy sources to survive the extended darkness of the polar night. Only some of the observed species and functional groups seem to be capable of phagotrophy, with the important group of diatoms not being able to use this energy source (McKie-Krisberg and Sanders 2014;Stoecker and Lavrentyev 2018;Jimenez et al. 2021). Osmotrophy, that is, the uptake of organic compounds from the surrounding seawater, on the other hand, seems to be a mechanism that all protists can employ to some degree (Worden et al. 2015;Mitra et al. 2016). Especially in a coastal system such as Kongsfjorden, where riverine and glacial runoff supply organic material, this seems a likely effective energy source.
Ratios of POC:N and POC:Chl a were determined on three occasions (Table 1) and showed rather high and variable values of 8.13 AE 0.86 mol C (mol N) À1 and 873 AE 363 g C (g Chl a) À1 , respectively, indicating a substantial contribution of nonphototrophic particulate organic matter that is supported by mixo-and heterotrophic trophic modes. In line with high POC:Chl a ratios, light microscopic analysis indicated a mixed protist community composition of picoplankton (probably including single-celled Micromonas pusilla), unidentified flagellates (probably including singlecelled Phaeocystis pouchetii) and diatoms (belonging to the genera of Pseudo-Nitzschia, Pleurosigma, Entomoneis, Nitzschia, Licmophora, and Thalassiosira) occurring together with ciliates and larger dinoflagellates. Thus, already at the end of the polar night, the same assemblages that usually also dominate the early spring bloom biomass in the study system were present (Hegseth et al. 2019); C. Hoppe unpubl. results). Furthermore, there is a significant overlap in the pelagic species composition with samples from December and January as well as in mid-March (Vader et al. 2015;Kvernvik et al. 2018). In April and May 2018, the Kongsfjorden spring bloom was in fact dominated by species that also occurred in winter, that is, diatoms such as Nitzschia and Thalassiosira, together with Fragilariopsis and Navicula during the exponential phase and flagellates such as P. pouchetii during the post-bloom phase (C. Hoppe unpubl. results). Thus, the present dataset indicates that, rather than having to be seeded from resting spores that overwinter in the surface sediment (Hegseth and Tverberg 2013), the spring bloom can also originate from this early assemblage that overwinters as active cells in the water column. This finding can fundamentally change the way we look at Arctic coastal overwintering and seeding mechanisms, and calls for a better understanding of their relative importance under different conditions. It also has implication for future projections, as active cells require more energy reserves than resting stages (McQuoid and Hobson 1996). Thus, winter energy demands of the former will increase more strongly with global warming and the increasing inflow of warmer Atlantic waters into the Arctic, putting this overwintering strategy at risk.
In conclusion, this dataset provides unprecedented insight into the overwintering strategies of Arctic phytoplankton and the initiation of its spring blooms. It also urges us to develop a better understanding of the physiological adaptations to prolonged darkness, for example, with respect to alternative energy sources of active cells, germination cues of resting stages and reinitiation mechanisms of photosynthesis. The ability to acclimate to and thrive in such low light environments, as studied here, indicates a large potential of primary production to occur also in the extensive twilight areas of the world's oceans. This new knowledge will also be very valuable for improving parameterizations of primary productivity in biogeochemical and ecological models. The surprisingly early and high rates of biomass production together with the persistence of the phytoplankton assemblage in terms of photosynthetic efficiency and species composition throughout winter and spring showcases the high capacities of Arctic phytoplankton to compensate for large ranges of environmental variability. In the high North, there seem to always be phytoplankton that is ready to capture and efficiently use the scarce and only periodically available key resource of light and thus most likely did not specifically adapt to the polar night.