Marine picophytoplankton (here defined as autotrophic cells <2 μm in diameter) is a diverse group consisting of picocyanobacteria (Prochlorococcus, Synechococcus, and Cyanobium), and photosynthetic picoeukaryotes (PPE). In oligotrophic systems, they account for more than 50% of the total chlorophyll a (Chl a) (Peña et al., 1990; Agawin et al., 2000; Durand et al., 2001) and can be responsible for most of the primary production (Magazzu and Decembrini, 1995; Uitz et al., 2010; Rii et al., 2016). Their success in low-nutrient environments is mainly explained by their small size, which provides them with a high surface-to-volume ratio, resulting in a competitive advantage for nutrient uptake compared to larger phytoplankton cells (Partensky et al., 1999; Pittera et al., 2014). Picophytoplankton are also abundant in eutrophic waters (Caroppo, 2015), in both coastal (Calvo-Díaz et al., 2004; Morán, 2007) and estuarine systems (Paerl et al., 2020; Sathicq et al., 2020), suggesting that they have a significant role in carbon cycling and ecosystem functions. Understanding which factors control picophytoplankton biomass is important for determining their role in the marine food web.

In the Baltic Sea, an estuary-like semi-enclosed large brackish water body, picophytoplankton are a key component of the phytoplankton community and reports of their contribution to Chl a, as a proxy for biomass, ranges from 15 to 85% (Sondergaard et al., 1991; Stal et al., 1999, 2003; Ohlendieck et al., 2000; Tamm et al., 2018). Baltic Sea picophytoplankton consists of the simplest cyanoprokaryotes Synechococcus and Cyanobium (hereafter referred to as picocyanobacteria; Flombaum et al., 2013; Celepli et al., 2017) and PPE. Cyanobium is a genera closely related to Synechococcus which has typically been associated with freshwater (Komárek et al., 1999; Callieri and Stockner, 2002; Sánchez-Baracaldo et al., 2005). The large contribution and diversity of picocyanobacteria in the Baltic Sea have recently been recognized (Stal et al., 2003; Herlemann et al., 2011; Bertos-Fortis et al., 2016; Hu et al., 2016; Celepli et al., 2017), but there is currently no systematic monitoring of picocyanobacteria in the Baltic Sea Proper. Coastal areas in the Baltic Sea are dynamic, and nutrient runoff has been related to increase in picocyanobacteria abundances (Lagus et al., 2007); however, abundance measurements in coastal areas remain scarce. Likewise, the PPE community is thought to be diverse and important in coastal ecosystems, but their distribution and abundance in the Baltic Sea are largely unknown (Kuosa, 1991; Tamm et al., 2018). Although knowledge of the picophytoplankton in the Baltic Sea is increasing, information about the ecological role, community composition, and distribution of picophytoplankton, especially in coastal areas, is lacking.

Picocyanobacteria can be divided into two populations based on the presence of the two phycobiliprotein pigments phycoerythrin (PE), and phycocyanin (PC) (Stomp et al., 2007; Liu et al., 2014; Tamm et al., 2018). PE-rich picocyanobacteria (PE-rich) strains are adapted to blue and green light absorption, and phycocyanin-rich picocyanobacteria (PC-rich) strains are adapted to red light absorption. Consequently, PE-rich are better adapted to low-turbidity waters (generally open waters); meanwhile, PC-rich are better adapted to turbid waters (generally coastal waters) (Campbell and Carpenter, 1987; Vörös et al., 1998; Stomp et al., 2007; Rajaneesh et al., 2015). The Baltic Sea picocyanobacteria community is dominated by a unique PE pigment cluster (Mazur-Marzec et al., 2013; Larsson et al., 2014; Tamm et al., 2018). However, PC-rich populations have been observed to have similar contributions to the picocyanobacterial community as PE-rich in areas with higher turbidity such as the Gulf of Finland, and some coastal areas (Stomp et al., 2007; Haverkamp et al., 2008). PE-rich and PC-rich frequently co-occur in estuarine environments, and recent studies from diverse coastal areas suggest that physio-ecological adaptations to nutrients and temperature affect their distribution and seasonality (Rajaneesh et al., 2015; Jiang et al., 2016; Paerl et al., 2020).

Picophytoplankton seasonal dynamics are determined by temperature, light, nutrient concentration (Otero-Ferrer et al., 2018; Hunter-Cevera et al., 2019; Fowler et al., 2020), and biotic factors (Ohlendieck et al., 2000; Ploug et al., 2011). The few studies focusing on picophytoplankton seasonality in the Baltic Sea suggest that their community composition and abundance have a strong seasonal variation (Kuosa, 1991; Bertos-Fortis et al., 2016). Maximum picocyanobacteria cell concentrations of 10⁵–10⁶ cells mL^–1 have been observed during summer, coinciding with high-temperature and low-nutrient conditions (Kuosa, 1991; Albertano et al., 1997; Hajdu et al., 2007; Tamm et al., 2018). Peak cell abundances for PPE of up to 10³ cells mL^–1 have been reported during the autumn in a non-stratified water column (Kuosa, 1991). Thus, the dynamics and relative importance of picocyanobacteria and PPE along different seasons are expected to vary. In a global perspective, PPE appear to be better adapted to low temperatures and NO₃ availability while picocyanobacteria is generally favored by high temperatures and NH₄ availability (Glibert et al., 2016; Otero-Ferrer et al., 2018). Picocyanobacteria preference of NH₄ over NO₃ has been observed in laboratory experiments on isolates (Moore et al., 2002; Tan et al., 2019). Similar observations from natural populations have also been reported from bulk populations in nutrient addition bioassays during summer phytoplankton blooms (Stal et al., 1999; Kuuppo et al., 2003; Lagus et al., 2007). This was also confirmed by recent studies at the single-cell level (Berthelot et al., 2018; Klawonn et al., 2019). In the Baltic Sea, the occurrence of picocyanobacteria during late summer has been linked to the presence of dinitrogen (N₂)-fixing cyanobacterial blooms (Ohlendieck et al., 2000; Stal and Walsby, 2000; Ploug et al., 2011), suggesting that interactions with larger phytoplankton may be an important factor driving seasonal dynamics of picophytoplankton. However, disentangling the nutrient effect from other factors such as temperature is necessary to understand the seasonal changes in picophytoplankton community composition.

This study aims to explore the abundance, apparent net growth rate, and nutrient limitation of three populations of picophytoplankton, i.e., PE-rich, PC-rich, and PPE, over an annual cycle at a coastal sampling station located in the Baltic Sea Proper. Weekly field sampling was conducted in March to December in 2018, and a series of nutrient addition bioassays were performed to assess how nutrient limitation drives picophytoplankton growth. This study provides high-resolution data on the dynamics of picophytoplankton and nutrient controls during different seasons. The results show that the response and contribution to total biomass of the different populations vary with nutrients and season and highlight the importance of picophytoplankton to the total phytoplankton community throughout the year.

Picophytoplankton have a competitive advantage for nutrient uptake in oligotrophic environments where they contribute significantly to the total Chl a (Peña et al., 1990; Agawin et al., 2000; Durand et al., 2001). However, several observations have also pointed out the ecological relevance of picophytoplankton in coastal and eutrophic environments (Morán, 2007; Caroppo, 2015; Pulina et al., 2017; Paerl et al., 2020). Information about the seasonal abundance of picophytoplankton in the Baltic Sea is limited. As a consequence, picophytoplankton biomass contribution is frequently estimated using Chl a fractionation (Sondergaard et al., 1991; Stal et al., 1999, 2003; Ohlendieck et al., 2000; Tamm et al., 2018) or not included in the calculations (Uusitalo et al., 2013; Olofsson et al., 2020). At the K-station during 2018, picocyanobacteria were present throughout the year, with maximum abundances during summer (4.7 × 10⁵ cells mL^–1). These numbers were comparable to other observations in the Baltic Sea Proper during summer (1.5 × 10⁵ to ∼5.5 × 10⁵ cells mL^–1) (Albertano et al., 1997; Mazur-Marzec et al., 2013), suggesting that picocyanobacteria abundances at the coast are as high as in offshore locations. PE-rich cell abundance during spring at <10°C was notably higher than previous reports in the Baltic Sea and other temperate ecosystems (Kuosa, 1991; Hunter-Cevera et al., 2019). Similar abundances have also been recorded in the southern Baltic Proper (Mazur-Marzec et al., 2013) in line with observations of positive growth of Synechococcus at <2°C (Paulsen et al., 2016), suggesting that picocyanobacteria is adapted to low temperatures in the Baltic Sea. PPE abundance reached 1.1 × 10⁵ cells mL^–1 during spring, around two orders of magnitude higher than the maximum abundances reported from the Gulf of Finland (Kuosa, 1991), and one order of magnitude higher than the maximum observed in other estuaries (Rajaneesh et al., 2015; Mohan et al., 2016). The current study (K-station) highlights the contribution of picocyanobacteria and PPE to the total phytoplankton biomass in the estuarine and eutrophic coastal Baltic Sea over an annual cycle.

Picophytoplankton is composed of multiple populations spanning diverse physiological adaptations and ranges (Rajaneesh et al., 2015; Tamm et al., 2018). Recent studies, in other estuarine and coastal areas, have separated Synechococcus/Cyanobium into pigment-based populations (PE-rich and PC-rich), suggesting differences in distribution patterns between the groups (Mitbavkar et al., 2015; Li et al., 2019; Paerl et al., 2020). In this study, the three populations, PE-rich, PC-rich, and PPE, had significant differences in regard to (1) seasonal dynamics, (2) biomass contribution, (3) temperature regimes, and (4) nutrient limitation. These results emphasize the importance of high-resolution studies of ecological relevant populations in order to understand the dynamics of the genetic and physiologically diverse picophytoplankton (Not et al., 2009; Bertos-Fortis et al., 2016).

Seasonal variations in the PE-rich and PC-rich contributions to the picocyanobacteria community have previously been observed in tropical and subtropical estuaries (Liu et al., 2014; Rajaneesh et al., 2015; Jiang et al., 2016; Li et al., 2019). At the K-station, PE-rich and PC-rich abundances increased during spring and peaked during early summer. During this period, PE-rich dominated the picocyanobacteria community (up to 99%). This was consistent with previous observations of PE-rich dominance in the Baltic Sea during the spring–summer period (Mazur-Marzec et al., 2013; Larsson et al., 2014; Tamm et al., 2018). During the autumn, PE-rich abundance declined, resulting in PC-rich dominance. The reduction in irradiance from summer to autumn can cause a transition from PE-rich to PC-rich (Stomp et al., 2007). However, other environmental factors such as nutrient concentration may also influence the picocyanobacterial community composition (Mitbavkar et al., 2015). PPE increase in abundance is thought to be related to low temperatures and high nutrient concentration (Kuosa, 1991; Otero-Ferrer et al., 2018). Our observations showed that PPE abundances peaked during spring, at 11–15°C. The low PPE abundances during autumn contrast with the peak abundances observed by Kuosa (1991) during the same period. These patterns could be explained by the long-lasting summer of 2018 (Humborg et al., 2019), which could have shifted PPE favorable temperatures to later in the autumn while concurrent light limitation may have restricted PPE growth (Fowler et al., 2020).

The biomass estimates at the K-station confirmed that the pico-fraction (<2 μm) is a major contributor to the total phytoplankton biomass, in line with previous recordings in the Baltic Sea based on Chl a measurements (Sondergaard et al., 1991; Stal et al., 1999, 2003; Ohlendieck et al., 2000; Tamm et al., 2018), and can dominate the phytoplankton community during spring, early summer, and autumn. Resolving the biomass estimates into the three populations reveal that on an annual basis, PPE was the main contributor to the phytoplankton community except during the bloom of N₂-fixers at the end of July (>18°C). This highlights the importance of PPE in coastal environments (Worden et al., 2004). A high contribution of both PE-rich and PC-rich co-occur with high temperatures, in line with previous studies (Stal et al., 2003). A community composition shift from PPE to picocyanobacteria can have large impacts on the microbial food web and should be systematically included in future phytoplankton biomass studies and carbon flux models (Schmidt et al., 2020).

Range adaptation to temperature showed different patterns among the picophytoplankton groups. The highest apparent net growth rates were observed during spring and beginning of summer at 10°C and 17–19°C for PE-rich and at 11–15°C for PPE. The temperature range for PC-rich was 13–16°C, as the highest apparent net growth rates were during autumn. The apparent net growth rates of picocyanobacteria were in line with previous observations in temperate ecosystems (Table 2). Similarly to the high-resolution growth dynamic study by Hunter-Cevera et al. (2016), in this Baltic Sea study, apparent net growth showed no increase at >16–17°C. However, higher apparent net growth rates of Synechococcus (up to 1.84 day^–1) have been reported in warmer climate (Table 2). Thus, an increase in temperature due to climate change might result in an increase of Synechococcus growth at higher latitudes (Stawiarski et al., 2016). The apparent growth calculations did not account for grazing or viral lysis. Grazing by ciliates and flagellates (2–20 μm) is considered an important top-down control on picophytoplankton (Grinienë et al., 2016). However, some observations have reported that grazing fails to control picocyanobacterial blooms, particularly in nutrient-rich environments (Kuuppo et al., 2003; Samuelsson, 2003). On the other hand, viral lysis can also influence picophytoplankton growth (Tsai et al., 2015) but may be a minor factor, particularly for PPE (Tsai et al., 2018).

The bioassays showed that NH₄ was the preferred form of nitrogen for picophytoplankton. The preference of NH₄ over NO₃ for Synechococcus/Cyanobium has been extensively documented (Moore et al., 2002; Berthelot et al., 2018; Klawonn et al., 2019; Tan et al., 2019). This study shows that both PE-rich and PC-rich can use NO₃ and NH₄ at low temperature but showed preference for NH₄ at higher temperatures (>15°C–17°C). This is in line with the effect of temperature on nitrogen assimilation enzymatic pathways (Glibert et al., 2016). The assimilation of NH₄ generally occurs through the GS-GOGAT pathway, while NO₃ uptake depends on the enzyme nitrate reductase (NR). GS-GOGAT is positively correlated with temperature, while NR is negatively correlated (Glibert et al., 2016). As a result, NH₄ assimilation will be higher than NO₃ assimilation at high temperatures. Thus, the uptake of NH₄ is an advantageous adaptation for Synechococcus/Cyanobium to compete with other NO₃ specialists such as diatoms under nitrogen limitation during the warm periods (Glibert et al., 2016). It should be noted that our results shows that Baltic Sea PE-rich were better adapted to high temperatures than PC-rich, and as a consequence PE-rich may benefit more from NH₄ uptake. In coastal areas and shallow water ecosystems (<50 m depth), NH₄ from benthic or riverine origin can be the main nitrogen source for the phytoplankton community (Herbert, 1999; Klawonn et al., 2019), which could benefit PE-rich at high temperatures. In line with the observations by Berthelot et al. (2018), PPE growth increased in NH₄ addition treatments during nitrogen limitation. However, nutrient additions outside of the temperature range resulted in significant reductions of the apparent net growth rates of PPE. This was likely because nutrient addition in a nutrient-limited system can favor competitors better adapted for high temperatures than PPE such as PE-rich.

This study provides an annual high-resolution description of picophytoplankton abundance and dynamics in the coastal Baltic Sea Proper. It also investigates apparent net growth rates and nutrient limitation of three functional picophytoplankton groups. In this study, PE-rich, PC-rich, and PPE showed different seasonal dynamics defined by different temperature ranges and nutrient limitation. To understand the dynamics of genotypes, future research should include the molecular diversity within each group. This study shows, for the first time, the contribution and importance of picophytoplankton over a full annual cycle and situates PPE as one of the most important components of the phytoplankton community in terms of biomass especially during spring and early summer. The results further suggest that in eutrophic coastal systems where NH₄ is the main nitrogen species (agricultural landscape), PE-rich will be favored over PPE. This effect could be further magnified during earlier and more extensive blooms of N₂-fixing cyanobacteria that are projected as a consequence of global warming (Neumann et al., 2012; Andersson et al., 2015). Such events could favor PE-rich over PC-rich and PPE, leading to picophytoplankton community shifts having consequences on the contribution to the total carbon biomass in coastal areas.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

CL, HF, and JA conceived the study. JA and HF conducted the field sampling and bioassay experiments. JA performed the laboratory and data analysis and plotted the figures. JA, CL, and HF wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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