Comparative Thermophysiology of Marine Synechococcus CRD1 Strains Isolated From Different Thermal Niches in Iron-Depleted Areas

Marine Synechococcus cyanobacteria are ubiquitous in the ocean, a feature likely related to their extensive genetic diversity. Amongst the major lineages, clades I and IV preferentially thrive in temperate and cold, nutrient-rich waters, whilst clades II and III prefer warm, nitrogen or phosphorus-depleted waters. The existence of such cold (I/IV) and warm (II/III) thermotypes is corroborated by physiological characterization of representative strains. A fifth clade, CRD1, was recently shown to dominate the Synechococcus community in iron-depleted areas of the world ocean and to encompass three distinct ecologically significant taxonomic units (ESTUs CRD1A-C) occupying different thermal niches, suggesting that distinct thermotypes could also occur within this clade. Here, using comparative thermophysiology of strains representative of these three CRD1 ESTUs we show that the CRD1A strain MITS9220 is a warm thermotype, the CRD1B strain BIOS-U3-1 a cold temperate thermotype, and the CRD1C strain BIOS-E4-1 a warm temperate stenotherm. Curiously, the CRD1B thermotype lacks traits and/or genomic features typical of cold thermotypes. In contrast, we found specific physiological traits of the CRD1 strains compared to their clade I, II, III, and IV counterparts, including a lower growth rate and photosystem II maximal quantum yield at most temperatures and a higher turnover rate of the D1 protein. Together, our data suggests that the CRD1 clade prioritizes adaptation to low-iron conditions over temperature adaptation, even though the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most iron-limited waters.


INTRODUCTION
Marine picocyanobacteria contribute to the biogeochemical cycling of various elements, most notably carbon, contributing ∼25% of ocean net primary productivity, of which the Synechococcus genus alone is responsible for about 16% (Flombaum et al., 2013). The large geographic distribution of these organisms, extending from the equator to subpolar waters, is largely attributable to their extensive genetic and functional diversity (Zwirglmaier et al., 2008;Farrant et al., 2016;Doré et al., 2020). Amongst the nearly 20 clades within subcluster (SC) 5.1, the most abundant and diversified Synechococcus lineage in oceanic ecosystems Scanlan et al., 2009;Ahlgren and Rocap, 2012), only four (clades I, II, III, and IV) were thought to largely dominate in situ. Clades I and IV mainly thrive in temperate and cold, nutrient-rich waters, while clades II and III reside in warm, oligotrophic or mesotrophic areas (Zwirglmaier et al., 2008;Mella-Flores et al., 2011), suggesting the existence of cold (I/IV) and warm (II/III) Synechococcus "thermotypes." This hypothesis was subsequently confirmed by work demonstrating that strains representative of these different clades exhibit distinct thermal preferenda (Mackey et al., 2013;Pittera et al., 2014;Breton et al., 2020;Six et al., 2021), a feature notably linked to differences in the thermostability of light-harvesting complexes (Pittera et al., 2017), lipid desaturase gene content (Pittera et al., 2018) and the ability of some strains to induce photoprotective light dissipation at colder temperatures using the orange carotenoid protein (OCP; Six et al., 2021). Field studies using global ocean datasets have allowed to refine the respective ecological niches of the different thermotypes, with clade I extending further north than clade IV (Paulsen et al., 2016;Doré et al., 2022) and clades II and III predominating in N-and P-depleted waters, respectively, but also to highlight the importance of a fifth clade within SC 5.1, the CRD1 clade (Farrant et al., 2016;Sohm et al., 2016;Kent et al., 2019). Initially thought to be limited to the Costa Rica dome area (Saito et al., 2005;Gutiérrez-Rodríguez et al., 2014), the latter clade was recently found to be a major component of Synechococcus communities in iron (Fe)-depleted areas (Farrant et al., 2016;Sohm et al., 2016;Ahlgren et al., 2020). Furthermore, analysis of the global distribution of these organisms using high-resolution marker genes has highlighted large within-clade microdiversity associated with niche differentiation in marine Synechococcus (Farrant et al., 2016;Larkin and Martiny, 2017;Xia et al., 2019), as also observed in Prochlorococcus (Kashtan et al., 2014;Larkin et al., 2016). Using the petB gene encoding cytochrome b 6 , Farrant et al. (2016) showed that most major clades encompassed several Ecologically Significant Taxonomic Units (ESTUs), i.e., genetically related subgroups within clades occupying distinct oceanic niches. This is notably the case for ESTU IIB that occupies a cold thermal niche in sharp contrast with IIA, the dominant ESTU within clade II that occupies warm, mesotrophic, and oligotrophic Fe-replete waters. Similarly, three distinct ESTUs with distinct thermal niches were identified within the CRD1 clade and the cooccurring clade EnvB (a.k.a. CRD2; Ahlgren et al., 2020): (i) CRD1B/EnvBB are found in cold mixed waters in cooccurrence with ESTUs IA, IVA and IVC, (ii) CRD1C/EnvBC dominate in warm, high-nutrient low-chlorophyll (HNLC) regions such as the central Pacific Ocean, and (iii) CRD1A/EnvBA are present in both environments and thus span a much wider range of temperatures than CRD1B and C (Farrant et al., 2016). This suggests that these three CRD1 ESTUs could correspond to different thermotypes.
In order to test this hypothesis, we used strains representative of each of the three CRD1 ESTUs to determine the fundamental thermal niches of these organisms as compared to typical cold (clades I and IV) and warm (clades II and III) thermotypes. Furthermore, given the strong influence of temperature on optimal functioning of the photosynthetic apparatus in marine Synechococcus (Pittera et al., 2014(Pittera et al., , 2017Guyet et al., 2020), we also examined the effect of temperature acclimation on the photophysiology of CRD1 ESTUs compared to their clade I and IV counterparts and show that CRD1 thermotypes actually differ more strongly in this respect to members of clades I-IV than from each other.

Strains and Growth Conditions
The eight Synechococcus spp. strains used in this study were retrieved from the Roscoff Culture Collection (RCC 1 ), including representative strains of the three known CRD1 ESTUs (CRD1A-C) and one or two of each of the four dominant clades in the field (clades I-IV) used as controls (Table 1 and Supplementary Figure 1). Cells were grown in 50 mL flasks (Sarstedt, Germany) in PCR-S11 culture medium (Rippka et al., 2000) supplemented with 1 mM sodium nitrate. Cultures were acclimated for at least 2 weeks in temperature-controlled chambers across a range of temperatures dependent on the thermal tolerance of each strain and under a continuous light of 75 µmol photons m −2 s −1 (hereafter µE m −2 s −1 ) provided by a white-blue-green LED system (Alpheus, France). For each experiment, cultures were grown in triplicate, inoculated at an initial cell density of ∼3 × 10 6 cells mL −1 , and samples were harvested daily to measure growth rate and fluorescence parameters as described below.
In order to compare the capacity of strains to repair the D1 subunit of photosystem II (PSII; see "Measurement of PSII Repair Rate" section), cultures grown in 250 ml flasks at 75 µE m −2 s −1 were acclimated at 18, 22, and 25 • C, temperatures at which all strains were able to grow, were subjected to high light stress (375 µE m −2 s −1 ). Exponentially growing cultures were sampled at T0 and after 15, 30, 60, and 90 min of stress, before shifting cultures back to the initial light conditions and then sampling again after 15, 30, 60 min, and 24 h of recovery (R). While D1 repair measurements were performed at all-time points, cell concentrations were measured by flow cytometry only at T0, T30 min, T90 min, R30 min, and R24 h and liposoluble pigment content was determined only at T0.  I  II  II  III  IV  CRD1  CRD1  CRD1   ESTU b  IA  IIA  IIA  IIIA  IVA  CRD1C  CRD1B  CRD1A   Pigment type c  3aA  3dB  3a  3c  3dA  3cA  3dA

Flow Cytometry
Culture aliquots (200 µl) sampled for flow cytometry were fixed using 0.25% (v/v) glutaraldehyde (grade II, Sigma Aldrich, United States) and stored at −80 • C until analysis (Marie et al., 1999). Cell concentrations were estimated using a Guava easyCyte flow cytometer (Luminex Corporation, United States) and maximum growth rates (µ max ) were calculated as the slope of the linear regression of ln (cell density) vs. time during the exponential growth phase. Synechococcus cells were identified based on their red (695 nm) and orange (583 nm) fluorescence, proxies for their chlorophyll a and phycoerythrin content, respectively. Fluorescence, forward scatter and side scatter values were normalized to that of standard 0.95 µm beads using Guavasoft software (Luminex Corporation, United States).

Fluorescence Measurements
The maximum PSII quantum yield (F V /F M ) was estimated using a Pulse Amplitude Modulation fluorimeter (Phyto-PAM II, Walz, Germany) during the exponential growth phase after 10 min dark acclimation followed by addition of 100 µM of the PSII blocker 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, Sigma-Aldrich, United States; Campbell et al., 1998). The PSII quantum yield was calculated as: where F 0 is basal fluorescence, F M maximal fluorescence level and F V variable fluorescence (Campbell et al., 1998;Six et al., 2007). Fluorescence excitation (with emission set at 580 nm) and emission (with excitation set at 530 nm) spectra were generated using a LS-50B spectrofluorometer (Perkin-Elmer, United States) as described in Six et al. (2004). The fluorescence excitation ratio (Exc 495:550 nm ) was used as a proxy for the PUB:PEB ratio. Phycobilisome (PBS) rod length and the degree of coupling of the PBS to PSII reaction center chlorophylls was then assessed using fluorescence emission spectra by calculating the phycoerythrin (PE, F max = 565-575 nm) to phycocyanin (PC, F max = 645-655 nm) ratio as well as the PC to PBS terminal acceptor (TA; F max = 680 nm) ratio, respectively (Pittera et al., 2017).

Pigment Analyses
Triplicate cultures were harvested during the exponential phase when F V /F M was maximum for each temperature condition. Cultures (50 mL) were subjected to centrifugation in the presence of 0.01% (v/v) pluronic acid (Sigma-Aldrich, Germany) at 4 • C, 14,000 × g for 7 min, using an Eppendorf 5804R (Eppendorf, France). Pellets were resuspended and transferred to 1.5 ml Eppendorf tubes and centrifuged at 4 • C, 17,000 × g for 2 min using an Eppendorf 5417R centrifuge (Eppendorf, France). Once the supernatant was removed samples were stored at −80 • C until further analysis. Pigment content was subsequently assessed using calibrated high-performance liquid chromatography (HPLC 1100 Series System, Hewlett Packard, St Palo Alto, CA), as previously described (Six et al., 2005).

Measurement of the Photosystem II Repair Rate
Each culture acclimated to 75 µE m −2 s −1 and 18, 22 or 25 • C was split into two new 50 mL flasks (Sarstedt Germany) with one used as a control and the other flask supplemented with lincomycin (0.5 mg mL −1 final concentration, Sigma-Aldrich, United States) in order to inhibit protein synthesis and thus D1 repair . Both sub-cultures were then subjected to light stress by exposing cultures to 375 µE m −2 s −1 continuous light (at the same temperature), and F V /F M measured at different time points as described above. The PSII repair rate for each strain at each temperature was determined from the coefficient differences between the exponential curves fitted over the 90 min time course of F V /F M measurements for control and +lincomycin samples. This light stress experiment was replicated on four independent cultures.

Determination of the Realized Environmental Niches of Major Synechococcus Ecologically Significant Taxonomic Units
The realized niches of CRD1 and clades I-IV ESTUs were determined using petB reads extracted from metagenomic data from the Tara Oceans and Tara Polar circle expeditions, the Ocean Sampling Day (OSD; June 21st 2014) campaign, and petB metabarcodes from (i) various oceanographic cruises (CEFAS, BOUM, Micropolar, RRS Discovery cruise 368 and several in the northwestern Pacific Ocean as detailed in Xia et al., 2017), (ii) two individual sampling sites in the Mediterranean Sea (Boussole, Point B) as well as (iii) a bi-monthly sampling at the longterm observatory site SOMLIT ("Service d'Observation en Milieu Littoral")-Astan located 2.8 miles off Roscoff between July 2009 and December 2011 (Supplementary Table 1).
petB metagenomic recruitment using the Tara and OSD datasets was performed as described previously (Farrant et al., 2016). Synechococcus petB sequences from both metagenomes and metabarcodes were used to define operational taxonomic units (OTUs) at 97% identity using Mothur v1.34.4 (Schloss et al., 2009) that were then taxonomically assigned using a petB reference database (Farrant et al., 2016). OTUs encompassing more than 3% of the total Synechococcus reads for a given sample were grouped into ESTUs and used to determine the whole temperature range occupied by each of the five major Synechococcus ESTUs.

Comparative Genomics
The Cyanorak v2.1 information system (Garczarek et al., 2021) 2 was used to compare the phyletic pattern i.e., the presence/absence pattern of each cluster of likely orthologous genes (CLOG) in each strain, for CRD1 strains and their clades I-IV counterparts for a number of selected genes potentially involved in adaptation to low temperature based on previous literature (see section "Results").

The Fundamental Thermal Niches of CRD1 vs. Clades I-IV Strains
In order to determine the temperature optima and boundary limits of the different CRD1 strains and to compare them to those of typical cold and warm Synechococcus thermotypes, representative strains of each of the three CRD1 ESTUs and strains of clades I, II, III, and IV were grown over a range of temperatures from 6 to 36 • C. The growth responses of all strains to temperature followed a typical asymmetric bellshaped curve over the selected temperature range (Figure 1), with a progressive rise in growth rate (µ) until T opt (the temperature associated with maximum µ: µ max ) was reached, and a sharp decline above T opt . BIOS-U3-1 (CRD1-B) was able to grow between 12 and 29 • C with a T opt at 25 • C (µ max = 0.78 ± 0.02 d −1 ), a growth pattern most similar to that of the clade IV strain BL107, while the clade I strain MVIR-18-1 was able to grow at much lower temperatures, down to 8 • C but could not grow above 25 • C ( Figure 1A). MITS9220 (CRD1-A) and BIOS-E4-1 (CRD1-C) displayed thermal growth characteristics more similar to the clade II (A15-62 and M16.1) and III (WH8102) strains, representatives of warm thermotypes ( Figure 1B). While most strains in this category displayed a minimal growth temperature of 16 • C, large variations between strains were observed at the highest thermal boundary limit 2 http://www.sb-roscoff.fr/cyanorak/ (T max ). Maximum growth temperature was obtained for M16.1 (II; T max : 34 • C), then A15-62 (II) and WH8102 (III; both with T max at 32 • C), MITS9220 (CRD1-A; T max : 31 • C) and finally for BIOS-E4-1 (CRD1-C; T max : 30 • C). The latter strain also displayed the highest minimal growth temperature (T min : 18 • C) and thus possesses the narrowest temperature range for growth of all the strains studied (12 • C vs. 15-18 • C). It is also worth noting that CRD1 strains display a lower maximum growth rate and more generally lower growth rates at most temperatures than their clade I, II, III, and IV counterparts.

The Environmental Realized Niches of CRD1 vs. Clades I-IV Strains
We then compared the fundamental thermal niches of all studied strains, i.e., the whole temperature range over which they can grow in a laboratory setting in the absence of biotic interactions (e.g., competition or predation), with environmental realized niches (sensu Pearman et al., 2008) of the corresponding ESTUs. For this, we determined the distribution limits of each of these ESTUs along the Tara Oceans and Tara Polar circle transects, 203 samples from OSD2014 and additional oceanographic cruises and individual sampling sites, altogether encompassing 413 samples worldwide covering a wide range of temperature conditions (Figure 1, Supplementary Figure 2, and Supplementary Table 1). This made it possible to have much finer estimates of the limits of the thermal niches of the different ESTUs than in the study performed by Farrant et al. (2016), in particular for the cold adapted ESTUs, which were poorly represented in the initial Tara Oceans dataset (Supplementary Figure 2).
This analysis showed that the CRD1B ESTU displayed a reduced thermal tolerance range in the environment (14-24.5 • C) compared to the BIOS-U3-1 strain in culture (12-29 • C), while the typical cold thermotypes colonized larger thermal niches in situ than their representative strains ( Figure 1A). Environmental realized niches indeed ranged from 2.5 to 24 • C for ESTU IA (compared to 8-25 • C for MVIR-18-1) and from 8.5 to 25 • C for ESTU IVA (compared to 12-29 • C for BL107). Interestingly, the median temperature of the CRD1B ESTU is 3 • C higher than that observed for ESTUs IA and IVA.
As concerns warm thermotypes, CRD1C displayed a fairly narrow thermal tolerance range in situ (22-29.5 • C), which, similar to the cold thermotype CRD1B, was even narrower than for its representative strain BIOS-E4-1 (18-30 • C; Figure 1B). Comparatively, the CRD1A ESTU was detected across a wider temperature range (14-30.5 • C) than the other two CRD1 ESTUs and also slightly larger than the corresponding strain in culture (MITS9220, 16-31 • C). Still, the most extended temperature range was observed for ESTU IIA and IIIA (12-32 • C) that reached significantly lower temperature limits than the corresponding clade II (16 to 32-34 • C) and III (16-32 • C) strains. Of note, although both IIA and IIIA ESTUs displayed a similar temperature range, the median temperature of ESTU IIA (25 • C) was about 3 • C higher than that of ESTU IIIA (22 • C) and the maximum median temperature was surprisingly observed for the CRD1C ESTU (26.5 • C). In this context, it is also worth mentioning that although clade II strains are clearly both warm thermotypes, M16.1 displays a significantly higher temperature limit for growth than A15-62 and more generally than all other strains. This suggests that ESTU IIA may encompass two distinct ESTUs, but such a high temperature niche (>32 • C) where they could be discriminated is exceptional and not available in our dataset (Supplementary Table 1).

Comparative Genomics
In order to assess whether the cold, temperate thermotype BIOS-U3-1 (CRD1B) exhibits similar adaptation mechanisms to those previously described for the cold-adapted clades I and/or IV, we examined a number of clusters of likely orthologous genes (CLOGs) from all Synechococcus genomes belonging to clades I-IV and CRD1 present in the Cyanorak v2.1 information system (Garczarek et al., 2021). First, we looked for the occurrence of two amino-acid substitutions in phycocyanin α-(RpcA) and β-subunits (RpcB), which were shown to differ between cold-(Gly in clades I and IV for RpcA43; Ser in RpcB42) and warm-thermotypes (Ala in clades II and III for RpcA43; Asp in RpcB42), these substitutions being potentially responsible for the differential thermotolerance of this phycobiliprotein between thermotypes (Pittera et al., 2017). In all three CRD1 strains, both sites displayed the warmtype residue (Supplementary Figures 3A,B), suggesting that in contrast to typical cold and warm thermotypes, the molecular flexibility of this phycobiliprotein does not differ between CRD1 thermotypes. We then looked at fatty acid desaturases that are essential for regulating membrane fluidity and thus the activity of integral membrane proteins, including photosynthetic complexes (Mikami and Murata, 2003;Pittera et al., 2018;Breton et al., 2020). All three CRD1 strains surprisingly possess in addition to the core 9-desaturase gene desC3, a second 9-desaturase, desC4 (Supplementary Table 2), previously thought to be specific to cold-adapted strains as well as the 12-desaturase desA3 found in both cold-adapted clades I and IV as well as in clade III, a warm thermotype subjected to much stronger seasonal variability than its (sub) tropical clade II counterparts (Pittera et al., 2018). Furthermore, BIOS-U3-1 also possesses desA2, thought to be specific to warm environments, while this gene is in contrast absent from the other two CRD1 warm-adapted strains. Thus, CRD1 strains exhibit a different desaturase gene set and potentially display a larger capacity to regulate membrane fluidity than typical cold-or warm-adapted thermotypes. Finally, while all clades I, III and IV genomes possess the ocp operon, involved in the protection of PSII against photoinactivation through the dissipation of excess light energy (Kirilovsky, 2007) and which was recently shown in marine Synechococcus to play a key role at low temperature (Six et al., 2021), none of the three CRD1 genomes possess this operon.

Photosynthetic Activity and Pigment Content
PSII quantum yield (F V /F M ), used as a proxy of photosynthetic activity, was measured for each strain over their whole temperature growth range. Most strains displayed a decrease in this parameter at both low and high boundary limits of their growth temperature range and this effect was particularly striking for BIOS-U3-1, reaching values down to 0.11 at 14 • C and 0.32 at 28 • C (Figure 2A). Besides MVIR-18-1 that exhibited a quite constant F V /F M over its whole temperature range, the decrease in F V /F M at high temperature was stronger for cold than warm thermotypes that are able to maintain a quite high F V /F M in the warmest growth conditions ( Figure 2B). Finally, as for growth rate, CRD1 strains exhibited lower F V /F M at all temperatures than clade I to IV strains.
The Exc 495:550nm fluorescence excitation ratio, used as a proxy for PUB:PEB ratio, was consistent with the pigment type of each strain (Humily et al., 2013; Table 1 and Supplementary Figure 4A). This ratio remained pretty constant over the whole temperature range for all strains except for the chromatic acclimator BL107 (pigment type 3dA), for which a sharp increase was observed at its maximal growth temperature (28 • C) to reach a value (1.35) intermediate between that typically observed in green light (or white light; 0.6-0.7) and blue light (1.6-1.7). This suggests that the chromatic acclimation process could be affected by growth temperature, at least in this strain. The phycobilisome (PBS) rod lengths and the degree of coupling of PBS to PSII reaction center chlorophylls, as estimated from PE:PC and PE:TA ratios, respectively, showed fairly limited variations over the temperature range, indicating that the phycobiliprotein composition of PBS is quite stable over the growth temperature range of each strain (Supplementary Figures 4B,C). One notable exception was a rise in both ratios for strain A15-62 at its minimal growth temperature, likely attributable to the partial decoupling of individual phycobiliproteins and of the whole PBS from PSII, a phenomenon typically observed under stressful conditions (Six et al., 2007;Guyet et al., 2020). It is also worth noting that MITS9220 and to some extent BIOS-E4-1, exhibited a significantly higher PE:PC ratio than the five other strains, potentially indicating a different phycobiliprotein composition and/or length of PBS rods.
In terms of liposoluble pigments, the β-carotene/chlorophyll a (β-car/Chl a) ratio tended to increase with temperature in BIOS-E4-1 and MITS9220, as observed for the other warm thermotypes, whilst this ratio was more stable in the cold thermotypes BIOS-U3-1 and BL107, and seemed to slightly increase in the lower part of the thermal range for the clade I strain MVIR-18-1 (Figure 3). For all strains, these ratios result from a concomitant increase with temperature of Chl a and β-car content per cell (Supplementary Figure 5), indicating an enhancement of the surface of thylakoids per cell at higher temperatures that was particularly marked for BIOS-E4-1 and A15-62, whilst this variation was fairly limited in the other two CRD1 strains. As these two pigments are present in different proportions in PSI and II (Umena et al., 2011;Xu and Wang, 2017), the higher β-car/Chl a ratio measured in clades I and IV strains also suggests that they may have a higher PSII:PSI ratio than all other strains, including BIOS-U3-1, and that this ratio might be more strongly affected by temperature in warm than cold thermotypes.
As concerns the zeaxanthin/chlorophyll a (Zea/Chl a) ratio, although an increase in this ratio was measured at low temperature for all strains, the amplitude was globally larger for cold than for warm thermotypes, with BIOS-U3-1 behaving very similarly to the clade IV strain BL107 that exhibits the largest variation in this ratio (Figure 3). Changes in this ratio likely originate partially from the decrease in Chl a content in response to cold, a strategy typically used by cells to regulate light utilization under slow growth conditions (Inoue et al., 2001). However, several strains also displayed an increase in their Zea content per cell at low temperature, a response particularly striking in BIOS-U3-1 and A15-62, but that also seems to occur in M16.1 and in the two other CRD1 strains BIOS-E4-1 and MITS9220 (Supplementary Figure 5). Thus, although Zea has been hypothesized to be involved in the photoprotection of coldadapted strains by dissipating excess light energy under low temperature conditions (Kana et al., 1988;Breton et al., 2020), this process seems to be present in both cold and warm-adapted CRD1 strains and in most warm thermotypes as well. In this context, it is also worth noting that the two clade II strains, A15-62 and M16.1, displayed fairly distinct temperature-induced variations in their Zea:Chl a ratios and individual pigment contents, possibly linked to their different isolation temperatures (see section "Discussion" below).

Photosystem II Repair Capacity
The ability of the different strains to repair PSII in response to light stress (375 µE m −2 s −1 ) was determined in cultures acclimated to 18, 22, and 25 • C by measuring changes in F V :F M over time after adding the protein synthesis inhibitor lincomycin, or not (Supplementary Figure 6). While a decrease in F V :F M ratio during the 90 min light stress period was observed in both cultures supplemented with lincomycin and controls, this ratio only re-increased back up to initial F V :F M values, after shifting cultures back to standard light conditions (75 µE m −2 s −1 ), in the control group in most strains and temperature conditions. Thus, all studied strains were able to recover from this light stress, as long as the D1 repair cycle was not inactivated by inhibition of protein synthesis. Yet, a fast decrease in F V :F M was observed for all three CRD1 cultures supplemented with lincomycin, while the +/− lincomycin curves overlapped during the first 15-30 min of light stress in most other strains and conditions. This suggests that the initial decrease in F V :F M in clades I-IV strains was not due to D1 damage but rather to dissipation of light energy as heat through non-photochemical quenching (Campbell et al., 1998), whilst the damage and hence repair of D1 proteins only occurred later on.
The PSII repair rate (R PSII ), as calculated from the time course of F V :F M with and without lincomycin, increased with temperature in most strains, except for BIOS-U3-1 that displayed its highest rate at 22 • C (Figure 4). Strikingly, all three CRD1 strains displayed significantly higher R PSII than clade I-IV strains at all three tested temperatures, a difference ranging from 3-to nearly 40-fold at the lowest common temperature (18 • C). Furthermore, CRD1 strains displayed fairly limited variation in R PSII with temperature (ranging from 1.33 to 1.87-fold) compared to the other strains, the strongest increase in R PSII being observed for the clade I strain MVIR-18-1 (21.5-fold) and the clade III strain WH8102 (5.5-fold). This indicates that CRD1 strains exhibit a constitutively high level of PSII repair compared to the other strains whatever the growth temperature and only trigger a moderate increase in R PSII in response to temperature variations.

DISCUSSION
Temperature constitutes one the strongest driving factors that have shaped genetic diversification and niche partitioning in marine cyanobacteria (Scanlan et al., 2009;Flombaum et al., 2013;Biller et al., 2015) and phytoplankton at large (Sunagawa et al., 2015;Delmont et al., 2020). While temperature has caused one major diversification event in Prochlorococcus, resulting in the divergence of the cold-adapted HLI from the warmadapted HLII clades (Johnson et al., 2006;Kettler et al., 2007), several independent temperature-related diversification events also occurred in the Synechococcus SC 5.1 radiation, leading to the emergence of clades I and IV Zwirglmaier et al., 2008). Here, determination of the temperature optimum and boundary limits (i.e., the fundamental niche) of strains representative of the three CRD1 ESTUs identified in the field (Farrant et al., 2016) showed that different thermotypes can also be delineated within the CRD1 clade, which dominates the Synechococcus populations in low-Fe areas of the world Ocean. Comparison with representative strains of the cold-adapted Synechococcus ecotypes (clades I and IV) on the one hand, and warm-adapted ecotypes (clades II and III) on the other, made it possible to classify (i) the CRD1A strain MITS9220, isolated from the equatorial Pacific Ocean, as a warm thermotype, (ii) the CRD1B strain BIOS-U3-1, isolated from the Chilean upwelling, as a cold temperate thermotype, and finally (iii) the CRD1C strain BIOS-E4-1, isolated from the edge of the South Pacific gyre, a stable, warm, Fe-depleted oceanic region (Claustre et al., 2008), as a warm temperate stenotherm.
As expected from theory (Pearman et al., 2008), the realized environmental thermal niches of CRD1 ESTUs were narrower than their fundamental niches (or similar for MITS9220). In contrast, for ESTUs IA to IVA, the realized environmental niche was significantly more extended toward the low thermal limit than the fundamental niches of their representative strains, this expansion being particularly marked for ESTU IA (Figure 1). This could be due to passive transport of Synechococcus populations by currents into water masses colder than their temperature limits for growth. Alternatively, these ESTUs may exhibit a greater microdiversity than previously assessed (Farrant et al., 2016) and could be subdivided into distinct ESTUs occupying slightly different thermal niches from the current ones, although representative strains to test this hypothesis remain to be isolated. In agreement with the latter hypothesis, Paulsen et al. (2016) measured a positive growth rate of Synechococcus natural populations dominated by clade I in waters as cold as 2 • C in the vicinity of the Svalbard island. Thus, CRD1 ESTUs appear to be strongly outcompeted by their ESTU IA to IVA counterparts at their lower temperature limits. Consistent with this, comparison of their gene content showed that CRD1 ESTUs, including the cold thermotype CRD1B, lack the main adaptation mechanisms reported so far for typical cold thermotypes. Indeed, all CRD1 strains examined in this study (i) exhibit warm-type substitutions in their α and β-phycocyanin subunits, influencing the thermotolerance of this phycobiliprotein (Pittera et al., 2017; Supplementary Figures 3A,B); (ii) possess a different set of desaturase genes, involved in regulation of membrane fluidity (Pittera et al., 2018), than typical warm and cold thermotypes (Supplementary Table 2) and (iii) lack the OCP system, involved in the protection of PSII against photoinactivation, which seemingly plays a key role at low temperature (Kirilovsky, 2007;Six et al., 2021). Still, we cannot exclude that CRD1 strains could use alternative strategies to cope with temperature variations and notably to deal with the generation of reactive oxygen species, known to be generated by a variety of factors including low and high temperature (Nishiyama et al., 2006;Latifi et al., 2009). For instance, all CRD1 strains possess the srxA gene encoding sulfiredoxin catalyzing the reduction of 2-Cys peroxiredoxin involved in H 2 O 2 detoxification (Findlay et al., 2005;Guyet et al., 2020) as well as isiA that, besides its role in increasing the lightharvesting efficiency of PSI under conditions of Fe-limitation, was also shown to provide photoprotection to PSII by dissipating excess light energy under oxidative stress conditions (Yeremenko et al., 2004;Ihalainen et al., 2005;Supplementary Table 2).
The ability of cyanobacteria to grow over a large temperature range largely relies on their capacity to optimize the functioning of their photosynthetic apparatus, notably at low temperature that induces a general slowing down of cell metabolism (Murata et al., 2007;Pittera et al., 2014Pittera et al., , 2017. For this reason, we also compared the photophysiology of CRD1 and clades I-IV strains at three growth temperatures common to all strains. These analyses showed that all three CRD1 strains exhibit a lower growth rate at most temperatures than clade I to IV strains (Figure 1), possibly explaining why they are easily outcompeted by other taxa when Fe is no longer limiting, as observed for instance around the Marquesas Islands (Caputi et al., 2019). Moreover, CRD1 strains also display a lower PSII maximum quantum yield (Figure 2), suggesting that PSII is partially photoinactivated, that is their D1 repair cycle does not fully compensate damage to this protein, even under optimal growth temperatures. Consistent with this, the very high turnover rate of the D1 protein measured in all CRD1 strains indicates that their PSII is much more sensitive to light stress than other strains and can only trigger a moderate increase in R PSII in response to both light and temperature variations, possibly indicating that they are adapted to live deeper in the water column than clades I to IV. This sensitivity could be partially linked to the abovementioned absence of the OCP, potentially reducing their ability to dissipate excess light energy, although it must be noted that the clade II strain A15-62 also lacks the OCP system. Interestingly in this context, all cold thermotypes including the CRD1B strain BIOS-U3-1, possess more copies of the D1:2 isoform (3-6 copies, average: 3.9 ± 1.1) than warm thermotypes (2-3 copies, average: 2.2 ± 0.4), this isoform providing a lower quantum yield but higher PSII resistance to photoinhibition than D1:1 (Supplementary Table 2; Clarke et al., 1993a,b;Campbell et al., 1995;Garczarek et al., 2008). Moreover, A15-62 is one of the only Synechococcus strains to possess two complete copies of the D1:1 isoform, a duplication which could partly explain why, despite the absence of OCP, this atypical clade II strain is able to maintain a high PSII quantum yield over its whole growth temperature range with fairly low D1 repair rates. Interestingly, CRD1 strains also possess a paralog of psbN, which was found to be required for the assembly of the PSII reaction center in Nicotiana tabacum and would play an important role in the D1 repair cycle (Torabi et al., 2014).
Taken together, both comparative genomics and photophysiological analyses highlighted a number of specificities of CRD1 strains compared to their clade I-IV counterparts, rather than them possessing traits distinctive of cold or warm thermotypes. In this context, it is worth noting that although strains representative of ESTUs IVA and CRD1B exhibit a similar fundamental thermal niche in culture, the higher median temperature of CRD1B in the field indicates that it preferentially thrives in temperate waters (about 18 • C, Figure 1), where energetically costly temperature adaptation mechanisms might not be essential. This suggests that for members of the CRD1 clade, adaptation to low-Fe conditions likely prevails over adaptation to temperature variations, and/or that adaptation mechanisms to temperature variations might be more complex and diversified than previously thought. Still, in terms of a realized environmental thermal niche, the occurrence of several CRD1 thermotypes likely explains why the CRD1 clade as a whole occupies most Fe-limited areas, a vast ecosystem constituting about 30% of the world Ocean (Moore et al., 2013;Bristow et al., 2017). A notable exception is the Southern Ocean, for which from the little available data shows that Synechococcus is scarce south of the polar front (Wilkins et al., 2013;Farrant et al., 2016), consistent with the fairly high low-temperature limit (14 • C) of the CRD1B environmental realized niche (Figure 1), while low-Fe availability likely limits the growth of clades I and IV in this area. In contrast, CRD1 growth does not currently appear to be limited by warm temperatures since most oceanic waters display a temperature below 30 • C (Supplementary Figure 2B and Supplementary Table 1). However, one cannot exclude that with global change, some areas of the world Ocean could become warmer than the highest limits determined here for representative strains of CRD1A and C, i.e., 31 and 30 • C, respectively. In this context, it is worth mentioning that in the dataset used for this study, several coastal stations, sampled during the OSD campaign reached 31.5 • C (Supplementary Table 1). Thus, although biogeochemistry global models predict that Synechococcus could be one of the winners of the phytoplankton community in a future world Ocean (Flombaum et al., 2013;Schmidt et al., 2020;Visintini et al., 2021), it might well not be able to survive in the warmest low-Fe areas, an ecological niche that is currently expanding (Polovina et al., 2008). Although a few studies have started to analyze the genomic bases of adaptation of Synechococcus cells to Fe-limitation in the field (Ahlgren et al., 2020;Garcia et al., 2020), further comparative genomic and physiological studies are still needed to decipher the specific capacity of CRD1 clade members to deal with Fe-limitation which should help predict the future distribution and dynamics of Synechococcus taxa in the world Ocean.

DATA AVAILABILITY STATEMENT
The datasets presented in the National Center for Biotechnology Information (NCBI) repository (https://www.ncbi.nlm.nih.gov; PRJNA811120).

AUTHOR CONTRIBUTIONS
MF, HD, and LG designed the experiments. MF, LD, HD, MR, AG, FR-J, TS, LC, and GM collected the samples and performed the physiological measurements. MF and DM ran the flow cytometry analyses. FL isolated several CRD1 strains used in this study. HD, XX, DS, HL, and LG performed sequencing and bioinformatics analyses of metabarcodes. MH, EC, FP, and LG developed and refined the Cyanorak v2.1 database. MF, HD, FP, and LG made the figures. MF, LD, HD, FP, and LG interpreted results. All authors contributed to the preparation of the manuscript, read, and approved the final manuscript.