J. Limnol., 68(2): 385-395, 2009 DOI: 10.3274/JL09-68-2-21

Characterization of photoautotrophic picoplankton assemblages in turbid, alkaline lakes of the Carpathian Basin (Central Europe) Tamás FELFÖLDI*, Boglárka SOMOGYI1), Károly MÁRIALIGETI and Lajos VÖRÖS1) Department of Microbiology, Eötvös Loránd University, Pázmány Péter s. 1/c., H-1117 Budapest, Hungary 1) Balaton Limnological Research Institute of the HAS, Klebelsberg Kuno u. 3., H-8237 Tihany, Hungary *e-mail corresponding author: [email protected]

ABSTRACT The photoautotrophic picoplankton (PPP) of ten shallow, hyposaline soda lakes located in three different geographical regions in the Carpathian Basin (Central Europe) was characterized. These lakes, which frequently dry out completely, are extremely rich in PPP. Epifluorescence microscopy was applied to determine picocyanobacterial and picoeukaryotic cell abundance and PCR-based molecular techniques (denaturing gradient gel electrophoresis and cloning with phylospecies delineation) to identify the members of PPP. Most of these lakes were eu- and hypertrophic with varying contribution of picocyanobacteria to the total PPP cell number. We found an unusually high PPP abundance with peaks of 8.16 × 106 cells mL-1 for picoeukaryotes and 1.78 × 107 cells mL-1 for picocyanobacteria. The majority of the retrieved PPP sequences belonged to picocyanobacteria (nonmarine Synechococcus/ Cyanobium), while others showed similarity to eukaryotic algal plastids (close to Trebouxiophycean isolates). Molecular analysis revealed significant genetic diversity in the PPP fraction of these lakes and showed that the closest relatives of our picocyanobacterial clones were recovered from different habitats, indicating seemingly no correlation between the 'saline' ecotypes and their phylogenetic position. Our results also confirmed that PPP might exploit different aquatic ecosystems and be successful even in the case of abrupt changes of environmental parameters (in our case, salinity). According to our knowledge, this is the first survey focusing on the identification of the PPP community members in turbid and alkaline lakes with extraordinarily high picoplankton productivity. Key words: soda lake, photoautotrophic picoplankton, epifluorescence microscopy, PCR-based molecular techniques

1. INTRODUCTION Shallow, turbid soda lakes are very characteristic of the Carpathian Basin. These are mostly intermittent shallow, alkaline pans that frequently dry out completely by the end of the summer. Their salinity varies from hypo- to mesosaline ranges in accordance with the season and water level (Schmidt & Fehér 2001; Schmidt 2003). Algological investigations of Hungarian soda lakes and Lake Fertő (Neusiedlersee) were intensive in the last century, which resulted in an exhaustive long list of species with limited information about pico-sized (SO42-

Vojvodina (Serbia)

2 Lake Slano Kopovo* 3 Lake Rusanda*

45°37' 20°12' 45°31' 20°17'

1.04 1.70

30 20

2 4

8.9 9.3

Na+ Na+

Cl-> SO42->HCO3SO42-> HCO3->Cl-

46°44' 46°46' 46°49' 46°46' 46°33' 46°28' 46°50'

0.10 0.01 1.20 1.17 0.50 0.70 1.00

22 60-100 Cl-

4 Lake Kis-Sós* 5 Kastély pond 6 Kelemen-szék pan Kiskunság (Hungary) 7 Böddi-szék pan 8 Büdös-szék pan 9 Lake Fehér 10 Zab-szék pan

19°59' 19°08' 19°11' 19°08' 20°02' 20°37' 19°10'

Tab. 2. List of investigated samples with their phytoplankton biomass and PPP abundance collected from different soda lakes in the Carpathian Basin. *: sample was cloned. Lake Lake Fertő Lake Slano Kopovo Lake Rusanda Lake Kis-Sós Kastély Pond

Kelemen-szék Pan

Böddi-szék Pan

Büdös-szék Pan Lake Fehér

Zab-szék Pan

Sampling date

28 Apr 2004 23 Apr 2005 23 Apr 2005 23 Apr 2005 16 Mar 2004 4 Jul 2004 17 Oct 2004 1 May 2003 17 Oct 2004 7 Jul 2005 26 Apr 2004 4 Jul 2004 3 Sep 2004 17 Oct 2004 23 Apr 2005 7 Jul 2005 3 Sep 2004 17 Oct 2004 7 Jul 2005 27 May 2004 1 May 2003 4 Jun 2003* 15 Sep 2004 17 Oct 2004 7 Jul 2005

Temperature Conductivity Chlorophyll-a (°C)

(µS cm-1)

(µg L-1)

16 15 15 11 11 28 10 19 9 28 12 30 22 8 16 28 24 11 29 21 19 21 24 8 28

2500 3220 10400 1845 5390 5220 5580 4670 7950 6300 4100 9050 14370 16480 6710 9700 4410 4700 3700 3190 4150 13890 5160 6640 4100

31 6.0 2.0 3.0 68 32 94 24 23 7.6 9.0 81 94 57 120 81 44 34 11 81 30 120 11 27 14

tion was determined spectrofluorimetrically according to Wetzel & Likens (1991). The abundance and composition of the PPP was determined from fresh, unpreserved samples. Aliquots of 0.5-3 mL were filtered through black polycarbonate filters with 0.4 µm pore-size. The filters were placed on microscopic slides and were embedded into 50% glycerol. The slides were examined with an Optiphot 2 epifluorescence microscope (Nikon, Japan) with 1000× magnification using blue-violet (BV-2A) and green (G2A) excitation light. Following the routine enumeration protocol for identifying PPP types (picocyanobacteria and picoeukaryotic algae), first the picophytoplankton

Abundance of Abundance of picocyanobacteria picoeukaryotes (104 cells mL-1) (104 cells mL-1) 309 168 7.3 0.1 466 356 760 399 10 32 0.1 1032 1783 623 213 519 22.7 107 30 0.1 928 23 461 2.1 36