Typical freshwater and marine bacterial lineages dynamics at salinity between 0 and 4 in the Vistula Lagoon

Highlights

• Marine SAR11-I/II was passively brought to the Lagoon with waters inflowing from the Baltic Sea.

• Freshwater SAR11-IIIb and Limnohabitans are active members of the bacterial community in the Vistula Lagoon.

• Burkholderiales had the highest relative abundance under ice cover in winter.

Abstract

The Vistula Lagoon is an almost closed basin at the southern coast of the Baltic Sea. Its mild salinity gradient provides a unique environment for the coexistence of freshwater and marine bacteria. This study employs catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH) to investigate seasonal dynamics of marine (SAR11 clade I/II) and freshwater (SAR11 clade IIIb (LD12), Limnohabitans clades B, C, D, Burkholderiales (former Betaproteobacteria)) bacterial groups. Samples were collected from below the ice in February, and then monthly from April to October 2011 from three stations with different salinity. The abundance of SAR11 clade I/II strongly positively correlated with salinity, with the highest abundance in autumn at time of inflows of saline waters from the Baltic Sea. Two groups (spring-summer and autumn-winter) were distinguished within this clade, based on a scatter chart between SAR11-I/II abundance and salinity. Salinity explained 69% of the variability of the spring-summer group and 77% of the autumn-winter group. This suggests that the presence of marine SAR11-I/II in the Vistula Lagoon was caused by passive inflow with waters from the open Baltic Sea, and this environment is not suitable for its regular existence. The abundance of the freshwater SAR11 clade IIIb was similar to that of SAR11-I/II. However, it depended on temperature, organic and inorganic phosphorous, and not on salinity, suggesting that SAR11-IIIb found a regular niche in the eutrophic Vistula Lagoon. Burkholderiales positively correlated with temperature, chlorophyll-a, organic and inorganic phosphorous, and heterotrophic nanoflagellates, suggesting that their role in the Vistula Lagoon may be similar to that in lakes. Interestingly, Burkholderiales had the highest relative abundance under ice in February. Finally, the abundance of Limnohabitans clades B, C, D positively depended on temperature, chlorophyll-a and negatively on salinity. This together with positive correlation with Cryptophytes and with heterotrophic nanoflagellates emphasis its importance in this ecosystem. These results show that the conditions in the Vistula Lagoon are rather inappropriate for typical marine bacteria, while freshwater bacteria may find their niches despite slightly elevated salinity.

Introduction

The Baltic Sea is one of the largest brackish water bodies on Earth, and as a whole it can be regarded as a large estuary, due to the saline inflows from the North Sea, and freshwater runoff from over 250 rivers and streams. The salinity of its surface waters ranges from 2 to 30, and is approx. 7–8 in the Baltic Proper, its largest basin, including the southern part of the sea and the Gulf of Gdańsk. The coexistence of marine and freshwater bacterial lineages is well known in the Baltic Sea from sequencing studies (Riemann et al., 2008; Holmfeldt et al., 2009; Herlemann et al., 2011; Dupont et al., 2014; Hugerth et al., 2015; Gołębiewski et al., 2017; Alneberg et al., 2018). However, sequencing data have been recently shown to poorly reflect contribution of specific bacterial lineages to total bacterial abundances (Piwosz et al., 2020). Thus, it is important to apply quantitative microscopic methods, such as catalysed reporter deposition-fluorescence in situ hybridisation (CARD-FISH), to fully understand distribution and dynamics of bacteria in the Baltic Sea (Holmfeld et al., 2009; Piwosz et al., 2013; Ameryk et at., 2014; Herlemann et al., 2014).

The Vistula Lagoon is an inner estuary of the Baltic Proper. It is a shallow (mean depth 2.7 m), well mixed from the surface to the bottom, natural reservoir closed from the sea by a narrow strip of land. Half of the lagoon is located in Russia, with the Strait of Baltiysk being the only connection to the Gulf of Gdańsk and the Baltic Sea. The lagoon is supplied by freshwater from several rivers and streams. The biggest are Pregolya, Pasłęka and Elbląg, since Vistula River was cut off at the end of XIX century. The exchange of water with the Baltic Sea through the narrow Strait of Baltiysk is slow. Water retention time in the lagoon is about 119 days - 8 times longer than in the Gulf of Gdańsk (Witek et al. 2003, 2010). Recently, it has been suggested that in brackish, tide-less estuaries mild salinity gradients may facilitate adaptation to different salinities (Gołębiewski et al., 2017). The Vistula lagoon provides a unique environment for the coexistence of freshwater and marine bacterial clades compared to the open waters of the Baltic Sea and the Gulf of Gdańsk. Longer retention time causes a mild salinity gradient, which allows for the coexistence of freshwater and marine species (at least temporarily), as observed for herring and roach (Kornijów 2018), phytoplankton (Gasiûnaité and Olenina 1998; Nawrocka et al., 2011; Piwosz et al., 2016), and zooplankton (Naumenko 1995; Gasiûnaité and Olenina 1998; Rychter et al., 2011). However, similar information regarding bacterioplankton from the Baltic Sea lagoons is lacking (Nawrocka et al., 2011; Schernewski et al., 2012).

Pelagibacteriales (Alphaproteobacteria) is a group of small bacteria constituting the most abundant heterotrophic pelagic bacterium in the oceans (Brown et al., 2012; Giovannoni 2017). It comprises of three major clades that are typical of different environments: marine clade SAR11–I/II, freshwater clade SAR11-IIIb (known also as LD12), and mesohaline clade SAR11-IIIa (Herlemann et al., 2014). SAR11-I/II is abundant in open and deep parts of the Baltic Sea (26.6% of bacterial numbers at salinity 35.2), but this study investigated only offshore regions (Herlemann et al., 2014). Its contribution to the bacterial community was up to 10% in the coastal waters in the Gulf of Gdańsk (salinity about 7), but it was not actively growing there (Piwosz et al., 2013), and neither was it detected in RNA-based sequencing libraries (Gołębiewski et al., 2017). On the other hand, marine SAR11-I/II was detected in a brackish lagoon (salinity 3–7) of the south-western Atlantic Ocean several months after the intrusions of oceanic waters (Piccini et al., 2006).

The freshwater clade IIIb of SAR11 bacteria is a sister clade to marine SAR11-I/II (Salcher et al., 2011). It occurs as some of the most dominant bacterioplankton in freshwater environments (Henson et al., 2018). Until the recent discovery of clade I in Lake Baikal, clade IIIb was the only known freshwater representative of Pelagibacteriales (Cabello-Yeves et al., 2018). SAR11-IIIb was below detection in open waters along the entire salinity gradient in the Baltic Sea (Herlemann et al., 2014), but reached up to 9% of abundance of bacteria in the coastal waters off the Gulf of Gdańsk, where it showed moderate activity during summer (Piwosz et al., 2013). Moreover, the first and so far the only isolated strain of SAR11-IIIb originates from brackish waters from the coastal lagoon of Lake Borgne near New Orleans, USA with a salinity of 2.39 and temperature of 30.5 °C (Henson et al., 2018).

Limnohabitans (Burkholderiales) are typical planktonic bacteria from freshwater lakes, reservoirs, and streams in temperate regions (Hahn et al., 2010), where they can represent up to 30% of free-living bacteria (Šimek et al., 2010). These bacteria have a lifestyle contrasting to that of the SAR11: they are highly active, grow rapidly on alga-derived substrates, and have very diverse metabolism that includes aerobic anoxygenic phototrophy (Šimek et al., 2010; Kasalický et al. 2013, 2018). Limnohabitans is commonly found in estuaries (Crump and Hobbie 2005; Shaw et al., 2008), and has also been reported to be active in brackish waters of the Gulf of Gdańsk (Piwosz et al., 2013; Gołębiewski et al., 2017).

The objective of this study was to investigate the dynamics of typical marine (SAR11 clade I/II Alphaproteobacteria) and freshwater (SAR11 clade IIIb Alphaproteobacteria, Limnohabitans sp., and Burkholderiales (former Betaproteobacteria)) bacteria in the brackish environment of the Vistula Lagoon over the course of 1 season. We applied the CARD-FISH technique to follow the seasonal dynamics of these bacterial groups at three stations along a salinity gradient from 0.32 to 3.92, and performed statistical analysis to correlate their abundance to environmental conditions. Firstly, we hypothesised to observe contrasting distribution patterns, with freshwater lineages being generally most abundant, in particular at the lowest salinity, and with marine SAR11-I/II increasing with salinity. Secondly, we hypothesised that neutral mixing of fresh and saline water masses would influence the abundance of marine SAR11-I/II, while freshwater SAR11-IIIb and Limnohabitans would be affected by environmental parameters.