Adaptation versus plastic responses to temperature, light, and nitrate availability in cultured snow algal strains

Abstract Snow algal blooms are widespread, dominating low temperature, high light, and oligotrophic melting snowpacks. Here, we assessed the photophysiological and cellular stoichiometric responses of snow algal genera Chloromonas spp. and Microglena spp. in their vegetative life stage isolated from the Arctic and Antarctic to gradients in temperature (5 – 15°C), nitrate availability (1 – 10 µmol L−1), and light (50 and 500 µmol photons m−2 s−1). When grown under gradients in temperature, measured snow algal strains displayed Fv/Fm values increased by ∼115% and electron transport rates decreased by ∼50% at 5°C compared to 10 and 15°C, demonstrating how low temperatures can mimic high light impacts to photophysiology. When using carrying capacity as opposed to growth rate as a metric for determining the temperature optima, these snow algal strains can be defined as psychrophilic, with carrying capacities ∼90% higher at 5°C than warmer temperatures. All strains approached Redfield C:N stoichiometry when cultured under nutrient replete conditions regardless of temperature (5.7 ± 0.4 across all strains), whereas significant increases in C:N were apparent when strains were cultured under nitrate concentrations that reflected in situ conditions (17.8 ± 5.9). Intra-specific responses in photophysiology were apparent under high light with Chloromonas spp. more capable of acclimating to higher light intensities. These findings suggest that in situ conditions are not optimal for the studied snow algal strains, but they are able to dynamically adjust both their photochemistry and stoichiometry to acclimate to these conditions.

Snow algae bloom in an ephemeral snowpack environment, experiencing low temper atur es, v ariable liquid water and nutri-ent a vailabilities , and high light intensities (Gorton et al. 2001, Techel and Pielmeier 2011, Rivas et al. 2016, Ren et al. 2019, Hoham and Remias 2020 ).Temper atur es c har acteristic of ablating snowpac ks ar e stable at or just abov e fr eezing during the ablation period, with the insulating properties of snow resulting in temperatur es r ar el y shifting mor e than 1 • C (Stibal et al. 2007, Burns et al. 2014, Maccario et al. 2015, Rivas et al. 2016 ).Such consistently low temper atur es mean liquid water is not always av ailable, v arying from 0.5% to 15% of the snowpack (Techel and Pielmeier 2011 ), and thus microbial communities must be able to tolerate frequent desiccation.Nutrient availability of the snowpack is also variable across the ablation season, with 80% of the solutes present being released within the first 30% of meltwater (Kuhn 2001 ) during an initial 'ionic pulse' (Costa et al. 2017 ).Nutrient concentrations ar e gener all y oligotr ophic acr oss seasons and r egions , with a vera ge nitr ate and ammonia ∼5 μmol L −1 and 10 μmol L −1 , respectiv el y in Arctic (Larose et al. 2013 ) and Antarctic (Hodson 2006 ) snowpacks during the ablation season.Phosphate availability is also low, with snowpacks sampled in the Antarctic (Dubnick et al. 2017 ) and Rockies (Hamilton and Havig 2017 ) r ar el y containing le v els abov e detection limits (0.24 μmol L −1 and 0.52 μmol L −1 , r espectiv el y).On the snow surface, light intensities are dynamic, with photosynthetically active radiation (PAR) reaching 2000 -3000 μmol photons m −2 s −1 and UV 30% greater than sea level in alpine regions (Gorton et al. 2001, Morgan-Kiss et al. 2006 ).Within snowpacks, light attenuation with depth results in a notable drop in PAR, with intensity at 1 cm depth ∼25% -50% of that measured at the surface depending on pr e v alent weather conditions (Gorton et al. 2001, Stibal et al. 2007 ).
To-date, studies examining the impact of temper atur e on snow algae have focused on growth dynamics, with Chloromonas spp.trending to w ar ds faster gro wth rates but lo w er ov er all carrying capacities ( K ) with warmer temper atur es (Hoham 1975, Hoham et al. 2008 ), demonstrating a balance between thermodynamic and biological processes within their cells.Research into responses to nutrient availability has focused primarily on metabolite production, with consideration to biotechnological applications.For example, nitrate and phosphate limitation drive upregulation of carbohydrate and fatty acid production in the snow alga Chlamydomonas nivalis to balance corresponding declines in amino and organic acid production under nutrient limitation (Lu et al. 2016 ).In other Chlorophyte snow algal strains, nitrate limitation has been shown to stimulate the production of nitrogen-free secondary carotenoid pigments, resulting in a change in bulk cellular colouration (Leya et al. 2009 ).In situ r esearc h into snow algal stoichiometry has found C:N ratios ranging from 16 to 33 (Spijkerman et al. 2012 ), m uc h higher than the 'optimal' Redfield ratio of 6.6 (Redfield 1958 ).
As the quantity and quality of PAR available to snow algae is dynamic in both the short and long term, photosystems need to be flexible to utilize light efficiently, alongside preventing excess light from causing cellular damage (Remias et al. 2005, Pr oc házk ová et al. 2023 ).When exposed to a continuous high light, c hlor ophytes have been shown to downsize the size of their antenna complex after se v er al hours, suc h that cells still absorb adequate light while protecting PSII from photodamage, with the reverse true for long-term, low light acclimation (Melis 1991 ).Evidence of these photoacclimatory mechanisms have been recorded in a number of snow algal species through pulse amplitude modulated (PAM) fluor ometry, with c hanges in both the efficiency of light use and the light intensity at which the PSII becomes saturated when the cells are exposed to varying light intensities (Procházková et al. 2019, Soto et al. 2020 ).
Despite many studies on snow algal responses to k e y abiotic stressors (i.e.Hoham et al. 2009, Leya et al. 2009 ), few have assessed responses to more realistic, multistressor conditions, nor potential inter-or intr aspecific r esponses.Researc h into other cryospheric terr estrial micr oor ganisms, including r ed snow algae (Segawa et al. 2018 ), has identified endemism (Vincent 2000, Conv ey 2010, Vyv erman et al. 2010, Segawa et al. 2017 ), but it is not y et kno wn if similar endemism exists across other snow algal species, nor the role of endemism in modulating responses to abiotic str essors.Her e, we assessed the growth, cellular stoichiometric, and photophysiological capabilities of two snow algal genera ( Microglena spp.and Chloromonas spp.) isolated from both the Arctic and Antarctic in their v egetativ e life sta ge.To identify temper atur e optima, gr owth, cellular stoic hiometry, and photophysiology wer e monitor ed acr oss 5, 10, and 15 • C under n utrient re plete conditions.Subsequentl y, m ultistr essor r esponses to nitr ate av ailability (1, 5, and 10 μmol L −1 ; Hodson 2006 ) and light intensity (50 and 500 μmol photons m −2 s −1 ) were assessed.

Materials and methods
In total, two series of incubations were conducted to determine the growth, photophysiological, and stoichiometric responses of multiple snow algal strains to temperature gradients and subse-quentl y v ariations in nitr ate av ailability and light intensity.Incubations were performed with strains of snow algae from two genera, and on replicates of each strain isolated from both Arctic and Antarctic en vironments .

Snow algal strains
A total of two genera of snow algae both isolated from King George Island, Antarctica and Spitsber gen, Sv albar d w er e inv estigated during the present study (Table 1 ).Clonal algal cultur es wer e acquir ed fr om the Cultur e Collection of Cryophilic Algae (CCCryo) at the Fraunhofer-IBMT in Potsdam (see Table 1 for strain numbers and origin).Cultures were transported to the Low Temperature Experimental Facility (LOWTEX) at the University of Bristol and stor ed in v ented cultur e flasks (Corning, Ne w York, USA) with nutrient replete 3N-BBM prior to experimentation (Bischoff andBold 1963 , Andersen 2005 ).

Temperature incubations
Though snow algal species are generally regarded as psyc hr ophilic, with optimal gr owth temper atur es below 15 • C, some ar e consider ed psyc hr otoler ant, exhibiting pr efer ential gr owth at higher temper atur es (Hoham and Remias 2020 ).To assess the selected strains, an initial incubation experiment was conducted with all strains to monitor responses across a range of temperatures (5 -15 • C) under nutrient replete conditions (3N-BBM) and at a consistent light intensity of 50 μmol photons m −2 s −1 .This data provided important context for our subsequent incubations wher e temper atur e was held constant ar ound optimal gr owth conditions (4 • C), while nitrate availability and light intensity were varied.
Temper atur e incubations were conducted at 5, 10, and 15 • C using model 305a LMS incubators (LMS, Se v enoaks, UK) housed within the LOWTEX facility at the University of Bristol.This temper atur e r ange was selected to ascertain the temper atur e optima for these strains, determining whether they can be better defined as psyc hr ophilic or psyc hr otoler ant.To establish incubations, 5 mL of stock culture of each strain previously maintained under CCCryo r ecommended gr owth conditions (3N-BBM, 4 • C, 50 μmol photons m −2 s −1 ) was inoculated into 45 mL 3N-BBM within a 50 mL Corning culture flask with a vented cap (Corning).A total of N = 8 replicates were established per algal strain and temperature treatment.Irradiance during incubations was provided by OSRAM L 8 W/535 fluorescent tubes (OS-RAM, Munic h, German y) with a mean light intensity of 50 μmol photons m −2 s −1 provided on a 16:8 h L:D cycle.Throughout incubations, sampling for determination of algal abundance (cells mL −1 ) and biovolume ( μm 3 per cell) was conducted three times per week on 50 μL of each culture sampled using a sterile plastic tip on a manual displacement pipette after thorough homogenization.Following subsampling, sample flasks were repositioned r andoml y within incubators to ensur e e v en light distribution.Cell abundance (see below) was used to identify exponential and stationary growth phases of each strain/temperature treatment, during whic h destructiv e sampling of N = 4 replicates was undertaken for photophysiological and stoichiometric analyses (see below).

Nutrient and light incubations
Following temper atur e incubations, the gr owth, photophysiological and stoichiometric responses of all algal strains were tested in response to gradients of nitrate availability and light, while

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temper atur e was held constant at 4 • C. For these incubations, 4 • C was c hosen giv en that all str ains demonstr ated gr owth optima at lo w er temper atur es during this study.Although snowpack conditions are often just above freezing (Burns et al. 2014 ), temperatures belo w 4 • C w ere av oided here to prevent freezing of the cultures during incubations.For each algal species/strain, incubations were conducted in three 1000-OD-MIX algal multicultivator incubators (Photon Systems Instruments, Czech Republic), which allo w ed for specific wavelengths and light intensities to be applied and for the constant oxygenation of cultures.All incubators were maintained in temperature-controlled rooms within LOW-TEX thr oughout the incubations.Eac h of the thr ee m ulticultiv ators housed N = 8 replicate incubations of one nitrate treatment (Low, Medium, or High; Table 2 ), with all other nutrients maintained at replete concentrations as per standard BBM (Bischoff andBold 1963 , Andersen 2005 ).Nitr ate tr eatments (r anging 1 -10 μmol L −1 ; Table 2 ) were selected to replicate the changes in nitr ate av ailability a ppar ent acr oss the ablation season within melting snowpacks on top of supr a glacial ice surfaces, shown to range 1 -10 μmol L −1 in situ , with higher concentrations experienced at the onset of melt (Hodson 2006, Telling et al. 2014, Holland et al. 2022 ).Nitrate assimilation has also been linked to light intensity in green algae (Aparicio and Quiñones 1991 ), as well as increased nitr ate assimilation r ates in snow algae compar ed to ammonia (J ones 1999 ).T hough nitr ate concentr ations wer e not av ailable for the specific sites where the cultures were isolated from, estimates fr om compar able snowpac k envir onments formed the basis for our treatment conditions (Table 2 ).Snow algal responses to nitrate availability were assessed under both low (50 μmol photons m 2 s −1 ) and higher (500 μmol photons m 2 s −1 ) light intensities .T hese wer e ac hie v ed using the 'Warm White' setting of the m ulticultiv ators , with wa velengths peaking at 450 and 600 nm with no UV provision, on a 16:8 h L:D cycle; replicating illumination timescales apparent during the spring ablation seasons in the polar regions where our algal str ains wer e isolated fr om.Though r educed as compar ed to expected in situ light conditions ( ∼ 2000 μmol photons m −2 s −1 ), the light treatments applied here were selected to allow quantification of responses across an order-of-magnitude difference in light intensity, while maintaining viable cultures in the lab.A total of N = 8 replicates were established per algal strain, nitr ate concentr ation and light tr eatment.Prior to the inoculation of cultures, cells that had been growing in 3N-BBM were rinsed in triplicate with treatment media (Low, Medium, or High NO 3 concentr ation) befor e final inoculation into m ulticultiv ators.For this, cultur es wer e centrifuged in an Eppendorf 5804R centrifuge at 300 RCF (Eppendorf, Hamburg, Germany) at 4 • C for 7 min, the supernatant r emov ed, and the pelleted algae resuspended in the selected treatment media.This process was repeated three times, before inoculating 2 mL of the rinsed algae into 70 mL media.
T hroughout incubations , sampling for determination of algal abundance (cells mL −1 ) and biovolume ( μm 3 per cell) was conducted a minimum of three times per week on 50 μL of each culture sampled using a sterile plastic tip on a manual displacement pipette after thorough homogenization.Homogenization was ac hie v ed using 20 mm stirr er bars (VWR International, Lutterworth, UK), sterilized before addition, and a Fisherbrand mini ma gnetic stirr er (Thermo Fisher Scientific , Massachusetts , USA).Cell abundance and biovolume (see belo w) w ere used to identify exponential and stationary growth phases of each strain/nitrate tr eatment, during whic h destructiv e sampling of N = 4 replicates was undertaken for photophysiological and stoic hiometric anal yses (see below).

Abundance , biovolume , and gr owth determination
Throughout both sets of incubations, cell abundance (cells mL −1 ) and biovolume ( μm 3 ) were monitored a minimum of three times per week on 50 μL subsamples fixed with 1% glutaraldehyde final concentration.Cell abundance was measured by counting cells on a modified Fuchs Rosenthal Haemocytometer (0.2 mm by 1/16 mm 2 ; Hawksley, Lancing, UK) using a bright field Olympus BX41 microscope (Germany).Additional to cell counts, ima ges of eac h sample wer e taken at 10x and 40x magnification with a MicroPublisher 6 CCD camera attachment (Teledyne Photometrics, USA) and the width and radius of 15 cells measured per r eplicate using Ima geJ softwar e and calculated to biovolume per cell ( μm 3 cell −1 ) assuming each strain to be a prolate spheroid (Hillebrand et al. 1999 ).To provide an estimate of the total algal biovolume of each culture per time step ( μm 3 per mL −1 ), the mean biovolume per cell was calculated across all 15 cells measured and multiplied by the cellular abundance.To model and summarize the growth of each culture throughout incubations, the 'SummarizeGrowth' function of the 'Gr owthcurv er' R pac ka ge v.0.3.1 (Spr ouffske and Wa gner 2016 ) was used to fit a logistic r egr ession to either algal abundance (cells mL −1 ) or total biovolume ( μm 3 mL −1 ) datasets (A) in relation to incubation time (h) as: (Equation (1); Sprouffske and Wagner 2016 ) The function identifies the optimal values for the maximum possible abundance (carrying capacity; K ) given the cellular abundance (cells mL −1 ) or total biovolume ( μm 3 mL −1 ) measur ed thr oughout the incubation.In addition, the specific Culture collection growth media Bischoff and Bold ( 1963 ), Andersen ( 2005 ) gr owth r ate ( μ) of cultur es during their exponential phase of incubations was calculated from logistic regression trajectories abo ve .

Photophysiology determination
Ra pid light r esponse curv es (RLCs; Perkins et al. 2006 ) were performed using PAM fluorometry to characterize the photophysiological responses of all species/strains during exponential and stationary growth phases of both sets of incubations.Measurements were conducted on 3 mL subsamples of eac h cultur e using a Walz Water -P AM fluorometer with attached red-light emitter/detector cuvette system and stirrer (Walz GmBH).Each sample was dark adapted for a minimum of 5 min under incubation temper atur e prior to RLC measurement.RLCs consisted of nine sequential light steps of 20 s duration ranging in irradiance from 0 to 2000 μmol photons m −2 s −1 .The maximum quantum efficienc y ( F v /F m ) w as calculated fr om minim um ( F 0 ) and maxim um ( F m ) fluorescence yields measured in the dark-adapted state during the initial RLC step of 20 s darkness (Consalvey et al. 2005 ).Electr on tr ansport thr ough photosystem II (PSII) was calculated across subsequent light steps in relative units (rETR) assuming an equal division of light between PSI and PSII (Consalvey et al. 2005 ).Analysis of all RLC data (rETR versus PAR) follo w ed Eilers and Peeters ( 1988 ) with calculation of the r elativ e maxim um electr on tr ansport r ate (rETRmax), the maxim um light utilization coefficient ( α), and the light saturation coefficient (Ek).Nonphotoc hemical quenc hing (NPQ) was calculated for each light step after Consalvey et al. ( 2005 ) and reported as NPQ(Ek), i.e. the level of NPQ a ppar ent at Ek.

Stoichiometry determination
The cellular carbon (C) and nitrogen (N) contents of all algal str ains wer e measur ed during exponential and stationary gr owth phases of both sets of incubations to determine stoichiometric responses to growth conditions.In all cases, a subsample of known v olume, as w ell as blanks of each media type, wer e filter ed onto pr e weighed, pr ecombusted (450 • C for 5 h) 13 mm diameter GF/A filters (1.6 μm r etention; Cytiv a Whatman, Maidstone, UK), whic h wer e subsequentl y fr ozen at −20 • C until anal ysis.Filters wer e freeze-dried for 24 h to remove all w ater, rew eighed and wrapped in individual 16 mm tin disks prior to elemental analysis using a Vario PYRO cube ® (Elementar, Stockport, UK).The detection limits of elemental concentrations were 0.001% for both elements measured, and the coefficient of variation (CV) for C and N according to 12 replicates of an or ganic anal ytical standard (NC Soil Standard 338 40025, cert.341506, C = 2.31%, N = 0.23%; ThermoFisher Scientific, Br emen, German y) wer e 5.32% and 2.94%, r espectiv el y.The molar content of carbon and nitrogen per cell was derived from the total recorded carbon and nitrogen area as: where Ar x is the relative atomic mass of nutrient x (e.g.carbon), x [%] is the derived percentage of nutrient x present in a processed sample, w is the total weight of the processed sample, and A tot is the total number of cells filtered onto the processed sample (e.g. cells mL −1 × mL filtered).This was then used to calculate the molar C:N ratio.

Data analysis
The analysis and plotting of data were completed using R v.4.2.1 (R Core Team 2022 ).Data were first checked for homogeneity of variance and normal distribution.Two-and three-way analysis of variance (ANOVA) tests were used to compare measured par ameters for gr owth, photophysiology, and stoichiometry between treatments, with post hoc Tuk e y HSD analysis applied to all significant ANOVA results.

Temper a ture incuba tions
All snow algal strains demonstrated higher carrying capacities ( K ) but lo w er specific gro wth rates ( μ) when incubated at 5 • C as compared to 10 or 15 • C (Fig 1 ).This trend was most pronounced for total biovolume datasets ( μm 3 mL −1 ), whereby a ∼90% increase was observed in K at 5 • C as compared to higher temperatures (Fig 1 A and B); with exponential phase μ ∼1500% smaller for all strains at 5 • C (Fig 1 C and D).This indicated a metabolism that maximized total biomass production ( K ) through slo w er specific gro wth rates ( μ) over longer periods at lo w er temperatures for all snow algal str ains incubated her e; consistent with Hoham ( 1975 ), who observed 20x higher K at 5 To be considered a true psychrophile, microbes should possess the ability to grow at 0 • C, with optimal growth at or below 15 • C (Morita 1975, Finster 2008, Cv etk ovska et al. 2017 ).Psyc hr otoler ant species are generally defined as showing growth between 7 and 35 • C, with optimal growth below 20 • C (Finster 2008 ); though strict definitions are still debated (Hoham andRemias 2020 , Hüner et al. 2022 ).For oligotrophic eukaryotic communities, carrying capacity ( K ) has been proposed as the most a ppr opriate measur e of optimal gr owth giv en that micr oor ganisms ar e gov erned by both thermodynamics and biology (Feller and Gerday 2003 ).Her e, incr easing temper atur e incr eases individual r eaction r ates, but metabolic processes that are heat labile are compromised (Loppes et al. 1996 ,  (Remias et al. 2005, Lukeš et al. 2014 ).Taken together, these datasets demonstrated the impact of decreased temperatures on downstream metabolic carbon sinks as compared to the ov er all ca pacity for photochemistry of our incubated snow algal strains (Ensminger et al. 2006 ).Photoautotr ophs gener all y striv e to maintain an equilibrium between energy supply (electron transport) and energy utilization (carbon fixation) as environmental forcings c hange, suc h that with reduced temperatures, the efficiency of the light-independent reactions of photosynthesis can be suppressed (Maxwell et al. 1994 ).This results in cold temperatures mimicking the effect of high light on algal photophysiology, inducing a corr esponding suppr ession of electr on tr ansport (Mor gan- Kiss et al. 2006 ).Inter estingl y, thr ee of our four strains sho w ed a greater induction of NPQ at higher temper atur es during the present study (excepting Antarctic Chloromonas sp.; Fig 2 E), likely reflecting the higher le v els of electr on tr ansport a ppar ent at these temper atur es (Ser ôdio and Lav aud 2011 , Blommaert et al. 2021 ).In contrast, the Antarctic Chloromonas sp.showed decreased Fv/Fm, rETRmax and ele v ated NPQ at lo w er temper atur es, indicating a gr eater degr ee of stress for this species at 5 • C.
The C:N ratio of all snow algal str ains av er a ged 5.7 ± 0.3 during n utrient re plete temper atur e incubations (Fig 2 F), a ppr oac hing the Redfield C:N stoichiometry of 6.6 (Redfield 1958 ), with no significant c hange r elativ e to temper atur e despite the v ariable gr owth r ates ( μ) and carrying ca pacities ( K ) a ppar ent acr oss incubations.In the marine envir onment, numer ous studies hav e demonstrated how marine phytoplankton preferentially grow where the nutrient availability matches their optimal requirements (Arteaga et al. 2014 ), such that variability in marine POM stoichiometry does not reflect species plasticity, but differences in assemblage composition (Quigg et al. 2003, Daines et al. 2014, Garcia et al. 2018 ).When we compar e the gr owth and stoic hiometric responses of snow algae gr own her e under nutrient replete conditions (temper atur e incubations) to those a ppr oac hing in situ nitr ate concentr ations (see below) our data highlights how similar dynamics would not be expected for micr oalgal comm unities in snowpac k envir onments.

Nitr a te and light incubations
When grown at 4 • C across a range of nitrate concentrations designed to a ppr oximate in situ nitr ate av ailability (1-10 μmol L −1 ), snow algal growth (both μ and K ) was significantly decreased as compared to growth under nutrient replete (3N-BBM) conditions, with the decrease in K exacerbated by higher-light (500 μmol photons m −2 s −1 ) conditions.Under low light (50 μmol photons m −2 s −1 ), biovolume-based carrying capacities ( K ; μm 3 mL −1 ) were 17.4% ± 9.9% of those a ppar ent under 3N-BBM for the same light and temper atur e conditions (contr ast Fig 3 A with Fig 1 A and B), despite a r elativ e nitr ate av ailability of just 0.01% -0.11% 3N-BBM (Table 2 ).Assuming a linear increase in K with nitrate availability until maximal K is ac hie v ed (estimated as K measured under 3N-BBM), 100% K would be a ppar ent at just 0.64% of 3N-BBM nitrate a vailability, i.e .56 μmol L −1 .This is ∼5 -10x higher than concentr ations pr e viousl y measur ed in snowpac ks (Hodson 2006, Lar ose et al. 2013 ), though 10x lo w er than optimal nitrate requirements of nonsnowpack inhabiting Chlorophytes (Corredor et al. 2021 ).Under high light, biovolume-based K was ∼20% lower than that ac hie v ed under low light across incubations (Fig 3 A), suggesting that higher light served to further restrict total biomass production under conditions of low nitrate availability.
Under low light, low nitrate conditions, C:N ratios were ∼200% ± 90% higher for all strains as compared to C:N observed under Cultured snow algal C:N stoichiometry here was thus consistent with in situ values recorded during snow algal blooms under compar able nitr ate concentr ations (0.5 -7 μmol L −1 ), whereby C:N ranged 16 -33 (Spijkerman et al. 2012 ).Taken together, these data intonate that snow algae blooming within snowpacks are likely not growing at their optimum C:N a vailabilities , but rather showing stoichiometric plasticity relative to ambient nitrate availability.This contrasts with expectations for marine microalgal comm unities, wher eby taxonomic differences in community composition are believed to be responsible for varying POM stoichiometry as opposed to plastic responses of individual species themselves (Quigg et al. 2003, Arteaga et al. 2014, Daines et al. 2014, Garcia et al. 2018 ).Despite the clear growth preferences for lo w er temper atur es shown her e, C:N r esponses indicated that snow algal species may not be adapted to the oligotrophic nature of their snowpack environments in relation to nitrate availability.Recently, Williamson et al., ( 2021 ) demonstrated low N and P cellular quotients of Streptophyte glacier algae sampled from surface ice of the Greenland Ice Sheet, concluding that lo w er N and P cellular r equir ements likel y r eflected ada ptation of glacier to their oligotrophic icy environment.Ho w ever, given that glacier algae have onl y r ecentl y been br ought into cultur e (Remias and Pr oc házk ová 2023 ), Williamson et al. ( 2021 ) were not able to confirm this by culturing glacier algae under nutrient replete conditions and observing cellular responses.Our data show clear stoichiometric plasticity of multiple snow algal strains when grown under snowpack nitr ate concentr ations as opposed to str ains ada pted to ov er all lo w er nutrient concentrations .T hough it should be noted that all  str ains wer e ca pable of gr owth at these nitr ate concentr ations, with no differences apparent in photophysiology or stoichiometric r esponses acr oss the 1 -10 μmol L −1 r ange of nitr ate tr eatments emplo y ed.
A total of three of the four strains (excluding Arctic Microglena sp.) demonstrated higher specific growth rates ( μ) under the higher light treatment (500 μmol photons m −2 s −1 ); though K r emained depr essed at higher-light.For these thr ee str ains, abundance-based specific growth rates ( μ) were higher but more variable (0.053 ± 0.077) than under low light (0.032 ± 0.034) (Fig 3 C).This is supported by both gener all y consistent or increased Fv/Fm and rETRmax (Fig 4 A and B) across these three strains between the low (Fv/Fm: 0.50 ± 0.14; rETRmax: 39.04 ± 21.07) and high-light conditions (Fv/Fm: 0.58 ± 0.21; rETRmax: 50.21 ± 35.65).These data suggest that any photoinhibition induced by the higher light le v el was not sufficient to exceed photopr otectiv e ca pabilities of these str ains (Mojzeš et al. 2020, Maltsev et al. 2021 ), allowing for a reduction in stress (Fv/Fm) and higher specific growth rates, indicated by rETRmax and μ.
Intr a-specific differ ences wer e observ ed in photophysiological responses betw een lo w and high-light conditions .For example , the rETRmax of the Arctic Microglena sp. was ov er all ∼50% that of the Antarctic strain across all light treatments (20.2 ± 6.0 and 47.3 ± 21.0, r espectiv el y; Fig 4 B).The Arctic Microglena sp. also sho w ed a lo w er efficienc y of light use ( α) under high-light, suggesting photoacclimation through reduction of antenna size to protect PSII fr om photodama ge (Melis 1991 ), with a compar able rETRmax ac hie v ed acr oss the two light treatments for this Arctic strain,.For Chloromonas sp.there were also intra-specific differences .T he Arctic strain maintained comparable Fv/Fm between the two light intensities, while the Antarctic strain's Fv/Fm increased by ∼90%-150% under high light (Fig. 4 A).This suggested that our Antarctic Chloromonas sp.pr efer entiall y gr e w under higher light, whic h was not a ppar ent for either Microglena sp.. NPQ did not vary between light treatments for Antarctic Chloromonas sp., but was ∼600% ± 200% higher ov er all as compar ed to the other thr ee str ains (Fig 4 E), suggesting a greater dependence on NPQ processes in this strain.The capacity of another closely related snow alga, Chloromonas kaweckae , to cope with higher light conditions has also been noted in situ, where it has been found in the upper 3 cm of the snowpack (Pr oc házk ová et al. 2023 ).
This study has highlighted diversity in studied snow algal strain responses to environmental stressors (temperature, light, and nitr ate av ailability), and how they behav e differ entl y to other micr oalgal comm unities.Nutrient r eplete temper atur e incubations demonstrated the psychrophilic nature of these strains, with carrying capacity ( K ) taken as an a ppr opriate metric of growth.Incubations also demonstrated how colder temper atur es mimic the effects of high light on algal photophysiology.Under nutrient replete conditions, the C:N ratio of studied strains approached Redfield stoichiometry (6.6), whereas significantly elevated C:N (17.8 ± 5.9) was evident when strains were grown in nitrate concentr ations that r eflected snowpac k conditions .T hese data highlight the plasticity in snow algal cellular stoichiometry and suggest comm unities gr owing in situ ar e not r eceiving their optimal nutrient r equir ements.Pr eliminary estimates indicate optimal nitr ate concentr ations 5 -10x that found in snowpacks, but 10x lo w er than that of other nonsnowpack inhabiting chlorophytes.Higher-light conditions under in situ nitrate concentrations elucidated intra-specific differences in algal photophysiology, with suppressed parameters for the Arctic Microglena sp. and pr efer ential r esponses fr om the Antarctic Chloromonas sp.strain.

F igure 1 .
Gro wth parameters for the temperature incubations.All panels show mean ± SE, N = 4. Lo w er case letters indicate homogenous subsets determined through a tw o-w ay ANOVA analysis of respective parameters in relation to temperature and strain.Upper case letters indicate homogenous subsets determined through one-way ANOVA analysis in relation to strain only.(A) Biomass carrying capacity -K (lc: F6,35 = 1.77,UC: F3,35 = 13.45,P < .05).(B) Cell count carrying capacity -K (lc: F6,36 = 3.87, UC: F3,36 = 13.52, both P < .05).(C) Biomass specific growth rate during exponential phaseμ (lc: F6,36 = 0.90, UC: F3,36 = 0.16).(D) Cell count specific growth rate during exponential phaseμ (lc: F6,36 = 0.69, UC: F3,36 = 0.79).F eller and Gerda y 2003 , Ca vicc hioli 2016 ).Accordingl y, maximal K at 5 • C during the present study supports the psyc hr ophilic natur e of our incubated snow algal strains, despite the higher specific gr owth r ates ( μ) observ ed at 15 • C. In contr ast, other studies hav e defined related snow algal species as psyc hr otoler ant (Seabur g et al. 1981 , Lukeš et al. 2014 , Bar c yt ė et al. 2018 ) but these used different metrics to define optimal growth.Photophysiology datasets further confirmed the low temperatur e pr efer ence of incubated snow algal str ains and indicated the mechanism of reduced μ under lo w er gro wth temperatures.Maximum quantum yields in the dark-adapted state (Fv/Fm; an inv erse pr oxy of str ess in micr oalgae; Consalv ey et al. 2005 ) wer e significantl y incr eased ( ∼115% ± 7%) at lo w er incubation temperatur es r elativ e to 15 • C during the exponential gr owth phase for all strains except Antarctic Chloromonas sp.(Fig 2 A).In contrast, maxim um electr on tr ansport r ates (rETRmax) wer e decr eased by ∼50% at 5 • C as compared to 10 and 15 • C (Fig 2 B) for all strains; consistent with patterns pr e viousl y observ ed for C. cf.niv alis

Table 1 .
Algal species, sample origin and location details, and associated CCCryo culture strain number.All cultures were isolated from snow fields in the given locations.Microglena cf .sp.-002bNorth of Artigas Base freshwater lake, Fildes Peninsula, Maxwell Bay, King George Island, South Shetland Islands, Antarctica.

Table 2 .
Nitr ate concentr ations used to r eflect c hanging snowpac k envir onments for the incubations as well as the nutrient replete 3N-BBM media used for the nutrient replete temper atur e incubations.
• C compared to 15 • C for Chloromonas pichinc hae during compar able labor atory incubations.Hoham et al. ( 2008 ) also recorded similar comparisons in K between 5 and 15 • C for Chloromonas tughillensis , another Chloromonas species with a low temper atur e optim um.