Cryptophyte and Photosynthetic Picoeukaryote Abundances in the Bransﬁeld Strait during Austral Summer

: A remarkable shift in the species composition and size distribution of the phytoplankton community have been observed in coastal waters along the Antarctic Peninsula over the last three decades. Smaller photoautotrophs such as cryptophytes are becoming more abundant and important for the regional ecosystems. In this study, ﬂow cytometry was used to quantify the smallest phytoplankton in the central Bransﬁeld Strait and explore their distribution across the strait in relation to physical and chemical properties of the two major water masses: the warmer and less saline Transitional Zonal Water with Bellingshausen Sea inﬂuence (TBW), and the cold and salty Transitional Zonal Water with Weddell Sea inﬂuence (TWW). Pico- and nano-phytoplankton clusters were distinguished and enumerated in the cytograms: photosynthetic picoeukaryotes, cryptophytes (about 9 µ m in size), and smaller (3 µ m) nanophytoplankton. It was shown that nanophytoplankton developed higher abundances and biomasses in the warmer and less saline TBW. This biotope was characterized by a more diverse community with a pronounced dominance of Cryptophyta in terms of biomass. The results support the hypothesis that increasing melt-water input can potentially support spatial and temporal extent of cryptophytes. The replacement of large diatoms with small cryptophytes leads to a signiﬁcant shift in trophic processes in favor of the consumers such as salps, which able to graze on smaller prey.


Introduction
Diatoms, haptophytes (predominantly Phaeocystis antarctica), and cryptophytes represent the major taxonomic groups of phytoplankton in the Southern Ocean [1][2][3][4][5]. Summer phytoplankton blooms in coastal waters of the Antarctic Peninsula are usually associated with a shallow upper mixed layer (UML) that keeps phytoplankton under favorable light conditions and better supply of dissolved iron (e.g., [6]). As a rule, diatoms and/or P. antarctica make the largest contribution to the blooms, but the role of cryptophytes has been reported to substantially increase, especially in water areas of melting glaciers [7][8][9]. The mechanisms behind Cryptophyta bloom formation are still poorly understood. Lower water temperatures in the surface layer, narrowing of the UML and a decrease in water salinity appear to favor the development of the phenomenon [9,10].
Publicly available data provide evidence of the transformation of the Antarctic phytoplankton community as a probable consequence of global warming [10]. The transformation covers not only taxonomic composition but also the size structure of the community. The replacement of large diatoms with small cryptophytes leads to a significant shift in trophic processes in favor of the consumers able to graze on smaller prey. Since Antarctic krill is not one of them [11], such a trend gives rise to a well-founded concern. A decrease in the food supply of krill and a substitution of the latter by other zooplankton (such as salps) imply a reduction in the carbon pool available for higher trophic levels by almost 70% [12].
It was shown in a number of experimental studies that Antarctic krill feed on small prey (about 10 µm in size) with low efficiency [13,14], while the optimal size of its food objects is about 50 µm due to the features of the filtration apparatus of euphausiids [13]. This well explains why krill exhibit positive selectivity for diatoms and avoid cryptophytes when feeding on complex prey mixtures [11].
On the contrary, salps, a food competitor of Antarctic krill, are satisfied with a wide range of taxonomic and size composition of phytoplankton prey thus demonstrating much lower feeding selectivity [11]. Against the background of their ongoing expansion to higher latitudes [12,15], their potential ability to utilize the biomass of cryptophytes during phytoplankton bloom periods poses additional threats to Antarctic krill populations, and hence, also for higher consumers including penguins, seals, and whales which feed on krill [16]. Thus, an increasing role of Cryptophyta in the Antarctic waters implies restrictions in food supply for krill and strengthening of its competitor's position.
The phenomenon of increasing cryptophyte abundances in the Antarctic waters have been initially explained by advection [7], sedimentation of large diatoms [17], grazing [2], and tolerance of cryptophytes to lower salinity waters [8]. Recently, Mendes et al. [18] studied the prevalence of cryptophytes in the coastal waters of the western Antarctic Peninsula and hypothesized that the phenomenon can be due to their pigment protection capability and, as a result, an ability to successfully grow under high irradiance exposure in strongly stratified shallow mixing surface layers. In this context, our major objective was to characterize the nanophytoplankton assemblage (2 to 20 µm in size) by flow cytometry (FCM), identify the Cryptophyta component, and analyze their distribution in the water masses of the Bransfield Strait, differing significantly from each other and the rest of the western Antarctic Peninsula shelf by their physical and chemical properties. Special attention was paid to nanophytoplankton of the transitional water mass with Bellingshausen Sea influence, which is warmer and fresher due to melting ice. We tested the hypothesis that an increase in melt-water input can potentially promote the spatial and temporal extent of cryptophytes.
Additionally, we used FCM to enumerate the smallest known component of the phytoplankton community, picophytoplankton (0.2 to 2.0 µm size fraction), which are recognized as an important player in the Southern Ocean ecosystem. In this part of the work, we focused on revealing spatial patterns of picophytoplankton abundance in relation to the water masses and frontal zones in the Bransfield Strait as the frontal features are among the factors other than temperature that control the abundance and distribution of phytoplankton.

Sampling Sites and Methods
The oceanographic and microbiological data were collected on 21 January 2020 over a transect across the central Bransfield Strait (BS) (7 stations, Figure 1) during 79th cruise of the R/V "Akademik Mstislav Keldysh". The length of the transect from Greenwich Island (South Shetland Islands) to the shelf of the Antarctic Peninsula was 93 km. Water samples were taken at 5 to 7 depths from the surface to 190 m, depending on the hydrological structure and chlorophyll a (Chl-a) fluorescence distribution.
The water area sufficiently differs from the rest of the western Antarctic Peninsula shelf by the inflow of cold and saline Weddell Sea shelf water [18,19]. According to Tokarczyk's classification [20], the water masses in the BS are the Transitional Zonal Water with Bellingshausen Sea influence (TBW; water temperature T > 1 • C, salinity S < 34.1 psu), and the Transitional Zonal Water with Weddell Sea influence (TWW; T < 1 • C, S > 34.1 psu). Figure 2 represents water temperature profiles at the stations (6587-6595) along the transect in the BS and the front between TBW and TWW. These waters are separated from each other by a shallow hydrographic front which has a clear surface thermal signature. The 1 • C isotherm serves as a good boundary between TBW and TWW in summer. TBW occupies only a 50-m thick upper layer along the South Shetland Islands whereas the rest of the Bransfield basin is occupied by TWW [21].

Physical and Chemical Oceanographic Data
Temperature, conductivity, and pressure data were collected using a SBE911 CTD attached to a Carrousel system with 24 5-L Niskin bottles for water sampling. Current velocity profiles were measured by a Workhorse Sentinel ADCP. Profile measurements of Chl-a concentration and photosynthetically active radiation (PAR) were conducted using a PUM-200 transparency meter equipped with a Minitracka-II fluorimeter (Chelsey Instruments Ltd., Surrey, UK), and a LI-COR radiometer equipped with two LI-192 sensors.
Dissolved oxygen and nutrients were immediately measured onboard in all of the samples using a modified Winkler method [22] and standard colorimetric methods [23][24][25][26].

Flow Cytometry
Water samples were fixed (2% formaldehyde fin. conc.) and frozen on board in liquid nitrogen for their storage and transportation to a stationary laboratory before flowcytometric analysis. No fractionation was employed with the exception of screening the sample through the 70-µm nylon mesh to avoid clogging the fluidic system of the instrument by larger objects (detritus, microalgae, zooplankton, etc.). Taxonomic composition and abundance of the micro-phytoplankton were not studied.
Two nanophytoplankton clusters were distinguished and enumerated, namely cryptophytes (CP) and other nanophytoplankton (NP). Cryptophytes contain a distinctive set of pigments, the orange fluorescing phycobilins (although there are also colorless members, [27]. These cryptomonads were identified by their bright orange fluorescence while NP did not produce it ( Figure 3). The light microscopy observation of cryptomonad cells confirmed the flow cytometry data. The FL4 and FL2 values measured in individual cells served a measure of their intracellular Chl-a and phycoerythrin (PE) contents, respectively [28]. Picophytoplankton were dominated by photosynthetic picoeukaryotes (PPE) while abundances of picocyanobacteria were extremely low or undetectable. For this reason, we identified and enumerated PPE only, according to Marie et al. [29] (Figure 3). Cell size measurements were calibrated in terms of equivalent spherical diameter (ESD, µm), using a set of standard polystyrene beads (Polysciences, Inc., Warrington, PA, USA) in the size range between 0.5 and 10 µm; calibration curves were obtained for each flow cytometry protocol by plotting the ESD as a function of the FSC signal (coefficient of determination r 2 > 0.8 in all cases). Mean ESD and cell volume were calculated for every cell population from the calibration curves [30]. To allow for a better comparison with earlier published data, nanophytoplankton abundances and biovolumes were converted to cellular carbon (C). Carbon conversion factors were shown to vary depending on taxonomy, cell size, and growth-regulating factors such as light and nutrient availability [31]. In this study, carbon content of pico-and nanophytoplankton was estimated from the mean ESD using the conversion factors of 237 fg C µm -3 [32] and 196.5 fg C µm −3 [33], respectively.

Statistics
Multivariate statistical analysis (cluster analysis and non-metric multi-dimensional scaling, nMDS) of microbiological and hydrological-hydrochemical data collected in the upper 190-m layer was carried out using the PAST3 software [34]. Mapping the spatial distribution of the variables and graph plotting were performed using Surfer v. 10 and Grapher v. 8 software (Golden Software, Inc., Golden, CO, USA), respectively.
In total, 24 biological, hydrological and hydrochemical variables were measured in the study site and investigated by the methods of multivariate statistical analysis ( Table 1)

Description of Oceanographic Features
Vertical distribution of water temperature and salinity across the BS (Figure 4) corresponded to the typical BS circulation system, with the Southern BS Current carrying waters advected from the Weddell Sea and meeting the relatively warm and less saline waters from the western Antarctic Peninsula through the Gerlache Strait Current at the western end of the BS [35,36]. Together they feed into the eastward flowing Bransfield Current, which is a baroclinic jet directed from the southwest to the northeast and transporting TBW along the southern slope of the Southern Shetland Islands [35][36][37][38][39] Table 1.
At the surface, warmer and less saline TBW occupied most of the BS (from station 6587 to 6593, Figure 4). Between stations 6593 and 6594, the 2 water masses were separated from each other in the surface layer by the Bransfield Front [37,38]. Being a conventional boundary between the water masses in the BS [20], the 1 • C isotherm did not deepen below 50 m (station 6587) and also corresponded to the maximum temperature gradient (marked in Figure 2 with empty circles).
The offshore waters of the South Shetland Islands are subject to local heating and meltwater inflow [40], which also affects the oxygen distribution in the northern part of the BS. During the study period, near-surface oxygen concentration reached 8.0 mL L −1 (Table 1, Figure 4), with oxygen saturation ranging between 100% and 104%. High ammonium nitrogen concentrations (1.0 to 3.8 µM) (Figure 4) were apparently associated with both organic matter oxidation as a result of active phytoplankton bloom and runoff of meltwater from the shores of the South Shetland Islands [19]. Concentrations of nitrite nitrogen (about 0.2 µM) also indicated an active decomposition of organic matter against the background of high primary production in TBW [19] (Figure 4) [19,41]. At the same time, meltwater conditions of TBW may also affect bio-optical properties of the surface waters in the BS [42].

Picophytoplankton
Abundance and biomass of PPE averaged 0.87 ± 1.03 × 10 6 cells L −1 and 0.72 ± 0.83 µg C L −1 , respectively (Table 1). They preferred deeper, colder layer below the <1 • C isotherm, thus, demonstrating their association with TWW ( Figure 5). The highest PPE abundance and biomass were observed at 50-m depth at station 6593. In the patch, these values reached 4.18 × 10 6 cells L −1 and 3.5 µg C L −1 , respectively. In the surface layer, PPE were relatively abundant at the front between TWW and TBW.
Patches of the highest CP abundance and biomass were observed in the euphotic layer around the jet of the Bransfield Current (station 6590, surface layer) while in TWW (i.e., below the 1 • C isotherm), cryptophytes were scarce or undetectable, especially in the deeper layers ( Figure 5). The NP demonstrated similar distribution pattern with the highest abundances at 35-50 m depth at station 6591 ( Figure 5). The maximum contribution of CP to the total nanophytoplankton biomass was recorded farther south in TBW (stations 6591 and 6592) at shallow depths (about 10 m) where NP were not abundant ( Figure 5).  Table 1.

Picoeukaryotes
Undetectable levels of prokaryotic picophytoplankton in the study site were not surprising since these microorganisms tend to be scarce or absent in polar region. Their abundance is often inversely related to picoeukaryotes, which are favored by more physically active mixed layers (e.g., [44]). According to the data synthesis by Buitenhuis et al. [45], picoeukaryotes dominate by >75% poleward of 40 • . Thus, in the Antarctic waters, picophytoplankton are mostly represented by picoeukaryotes. Moreover, they may occasionally contribute up to 100% of the autotrophic biomass in subsurface waters of the BS [46].
The picoeukaryote concentrations reported in this study were relatively low compared to the data collected earlier in the summer picophytoplankton community [45][46][47]. In the BS waters, PPE abundance varied from 6 to 14 × 10 6 cells L −1 during summer 2000 [46] that was significantly higher than our estimates. According to Lin et al. [48], the highest PPE summer abundance appeared offshore in the Southwest Atlantic Ocean and changed from 0.2 to 18.2 × 10 6 cells L −1 , with an average of 3.2 × 10 6 cells L −1 . PPE biomass estimates reported in the above study (0.03 to 38.2 µg C L −1 ) also substantially exceeded our results, with neither water temperature nor salinity having significant correlation with the picoeukaryote biomass.
Picoeukaryotes demonstrated statistically significant (p < 0.05) correlation with water temperature (0,35), salinity (-0.39), phosphates (0.41), and total Chl-a (0.45) ( Table 2) but this did not provide any explanation of the low PPE abundance and their patchy distribution in the BS waters. Contrary to expectations from the correlation analysis, the PPE 'patch' was situated in the colder and more saline TWW just below the front between the water masses. This may support a suggestion that the nanophytoplankton over-competed the PPE in TBW, forcing them to stay outside the Cryptophyta bloom. Other factors such as frontal dynamics and light penetration also may be important. In particular, despite picophytoplankton ability to utilize low irradiance [49], they may be light-limited in the Antarctic turbid coastal waters or deeply mixed oceanic waters [50,51].

Cryptophyta
The distribution patterns of Chl-a and the total biomass of nanophytoplankton (TNPB) estimated by flow cytometry were similar, with the highest values at the same stations and depths (Figure 4). This provided evidence that nanophytoplankton was a major, if not dominant, component of the phytoplankton in the study site. To verify this suggestion, we estimated a potential contribution of CP to the total Chl-a, using different conversion factors: 0.2 pg Chl-a cell −1 [52] and 1.5 pg Chl-a cell −1 [53]. In the high Chl-a spot at station 6591 (1.28 µg Chl-a L −1 ), the approximation of CP biomass in terms of Chl-a ranged from 0.31 to as high as 2.32 µg Chl-a L −1 , depending on which conversion factor is used. Additionally, we used the TNPB-to-FLUOR ratio as a measure of the nanophytoplankton contribution to the total phytoplankton biomass. The highest ratio values were observed in TBW with the maximum at station 6592 ( Figure 4). Thus, the nanophytoplankton and CP could potentially dominate phytoplankton in TBW, where the phytoplankton bloom occurred.
The Cryptophyta biomass maximum (about 180 µg C L −1 ) reported in our study is an outstanding characteristic for Antarctic waters. The importance of cryptophytes in different basins of the Southern Ocean has been reported earlier [1,2,[54][55][56][57] but their maximum concentrations were much lower (between 0.3 and 6.0 × 10 6 cells L −1 ) than our estimates. Garibotti et al. [3] have surprisingly revealed peak concentrations in the region of Anvers Island, reaching 369 µg C L −1 , i.e., close to our estimates. Therefore, our results widen the area of Cryptophyta blooms to the central Bransfield Strait and support the conclusion of the above authors that cryptophytes are a distinctive and annually recurrent component of Antarctic phytoplankton. Their blooms are increasingly becoming a peculiarity of the continental shelf west of the Antarctic Peninsula.

Linkage between Nanophytoplankton and Environmental Variables
All of the quantitative characteristics of the nanophytoplankton (CPN, NPN, CPB, NPB, TNPB) demonstrated a significant positive correlation with water temperature (T), dissolved oxygen (DO), and Chl-a fluorescence (FLUOR), while negative correlations were found with salinity (S) and nitrates (NO3) ( Table 2). These environmental variables gradually changed with depth and in the transition from TBW to TWW, that explained well the above dependencies: in general, the nanophytoplankton abundances also decreased from the surface (TBW) to deeper layers (TWW) and from the northern (TBW) to the southern (TWW) stations along the transect. Thus, both CP and NP preferred warmer and fresher TBW, that supported earlier published observations [2,41,51,[57][58][59]. The contribution of CP to the total nanophytoplankton abundance and biomass appeared the highest in TBW ( Figure 5).
Chl-a fluorescence was significantly correlated with the total nanophytoplankton, especially with the NP concentrations (Table 2), most likely due to the higher NP abundance (NPN) and intracellular Chl-a content (NP-CHL) compared with the same characteristics in CP (Table 1). Intracellular Chl-a values in both CP and NP had a positive relationship with the ammonium concentration (NH4). Significant correlations have also been revealed between NP-CHL and Si (0.44), CP-CHL and PO4 (−0.32), CP-CHL and NO3 (−0.41) ( Table 2).
It should be noted that a strong relationship between the nanophytoplankton abundance (CPN, NPN) and water temperature was found over the entire set of data (see the regression lines in Figure 6a,b). However, within each of the biotopes, TBW and TWW, the nature of the relationship was different. The CPN kept strong dependence (r 2 = 0.65) on temperature within TBW but the other links were lost (see the regressions represented with dashed lines in Figure 6a,b). Temperature did not appear to be a key factor controlling the NP abundance.  Table 1.
The CP and NP exhibited significant inter-correlation in terms of their biomass (r 2 = 0.73) but not of their abundance (r 2 = 0.25) (Figure 6c,d) that could provide evidence of their competition for the same resources. Yet again, the linkage between CPB and NPB were strong (r 2 = 0.58) in TBW, and poor (r 2 = 0.31) in TWW.

Comparing Nanophytoplankton from TBW and TWW
Cluster analysis allowed us to classify the nanophytoplankton samples, depending on the community structure (Figure 7a). A total of 4 sample groups were identified, 3 of which (the clusters 2 to 4) were within the TBW biotope (see the scheme of the cluster distribution in the water column in Figure 7b). The sample collected in the surface layer at station 6587 formed the cluster 4, owing to the outstanding microbial characteristics: the highest biomasses and average ESD of CP (178.5 µg C L −1 ; 11.6 µm) and NP (15.7 µg C L −1 ; 3.7 µm). The cluster 3 included 'patches' of nanophytoplankton at stations 6587 and 6591 within the TBW water mass (upper 50 m). In the cluster 2, CPN and CPN% significantly decreased. In the TWW biotope, the community was represented by the cluster 1 with low or undetectable microbial abundances (Figure 7b). Thus, the physical and chemical properties of the water masses deeply influenced the nanophytoplankton structure. The TBW biotope was characterized by much greater cell abundance and by a more diverse community with a pronounced dominance of Cryptophyta in terms of biomass. The ordination of the nanophytoplankton samples by nMDS produced similar results (Figure 7c) however, nMDS did not reveal a clear distinction between the sample clusters 1-4 revealed by the cluster analysis (shown in Figure 7c) in different colors). The clouds of dots corresponding to both the biotopes, TBW (the clusters 2-4) and TWW (the cluster 1), were distributed along the environmental vectors, which indicated a strong environmental control of nanophytoplankton. Distribution of the TBW-TWW fronts, anticyclonic eddies and their thermohaline structures, and nutrients are among the factors, which can control the community across the BS [60][61][62].

Cryptophyta Distribution Pattern in the Bransfield Strait
In general, the spatial distribution patterns of Chl-a fluorescence that we observed in the central BS were well aligned with the earlier long-term observations: the highest Chl-a was usually found close to the South Shetland Islands with the UML depth being negatively related to Chl-a [63]; at the well-stratified sites (TBW), with the euphotic layer varying from 40 to 50 m, the highest Chl-a was at the surface, while the weakly stratified sites (TWW) were associated with the low Chl-a and deeper euphotic layer [64].
However, the spatial patterns of cryptophytes in the coastal areas of the Antarctic Peninsula are more complicated. In the Eastern BS, Gonçalves-Araujo et al. [9] observed a dominance of microplanktonic diatoms and higher Chl-a within shallow upper mixed layers in TBW, close to the South Shetland Islands, while TWW was primarily characterized by lower Chl-a within well-mixed water column and dominated by nanoplanktonic flagellates (including haptophytes and cryptophytes). This contradicted our observations of coincident maxima of the Chl-a fluorescence and the cryptophyte concentration within the TBW surface layer with lower salinity and higher temperature (>1 • C).
According to Schofield et al. [65], peak populations of cryptophytes were observed in the coastal areas of the West Antarctic Peninsula with lower salinity (33.75 to 32.5 psu) and colder water (−1 to 1 • C), while they were absent at higher temperatures (>1 • C) and salinities (>33.75 psu). On the contrary, Mendes et al. [18] provided different data on the distribution of cryptophyte biomass in the Gerlache Strait, which was mostly dependent on the sea surface temperature, rather than salinity. The authors found that cryptophytes were strongly associated with high temperature, water column stability, and Chl-a, and negatively associated with nutrient concentrations, UMLD, and salinity, which was in good agreement with our observations and a few earlier reports [2,51,57,59,64].
An explanation of the phenomenon may consist in early retreat of sea ice, which, together with the increase in sea surface temperature, leads to the development of a shallow mixing layer and strong vertical water column stratification. The latter confines phytoplankton near the surface under high irradiance [66]. Mendes et al. [18] hypothesized that the dominance of cryptophytes in the coastal waters of the Antarctic Peninsula is just associated with their ability to grow under high irradiance exposure due to their pigment protection capability.
Thus, the increase in melt-water input may potentially enlarge the spatial and temporal extent of cryptophytes [8]. Our study supports this suggestion, providing evidence of extremely high cryptophyte biomass within low-salinity, high-temperature TBW. Historical data also show an emerging dominance of cryptophytes and other phytoflagellates in lowsaline coastal waters [43,56,57,[67][68][69][70]. As a consequence of these processes, zooplankton community structure undergoes cascade changes [16,71] leading to an increase in salp abundance and concurrent variations in penguin migration patterns [72]. If this trend persists for a long time, the food web shifts will eventually impact biogeochemical cycling in the Antarctic coastal waters.

Conclusions
In this study, we found higher abundances and biomasses of nanophytoplankton in the warmer and fresher transitional water mass with Bellingshausen Sea influence (TBW). This biotope was characterized by a more diverse community with a pronounced dominance of cryptophytes in terms of biomass (up to 180 µg C L −1 ). Patches of the highest cryptophyte abundance were observed in the euphotic layer around the jet of the Bransfield Current. In the transitional water mass with Weddell Sea influence (TWW), they were scarce or undetectable, especially in the deeper layers. Our results widen the area of Cryptophyta blooms to the central Bransfield Strait and support the conclusion that these small eukaryotes are a distinctive and annually recurrent component of Antarctic phytoplankton.
In the study site, picophytoplankton were represented by picoeukaryotes and characterized by low abundances (about 0.87 × 10 6 cells L −1 ) and patchy distribution. They preferred deeper, colder layer, thus, demonstrating their association with TWW and the frontal zone between the water masses.
Our results support the hypothesis that increasing melt-water input can potentially increase spatial and temporal extent of cryptophytes in the Antarctic region.