Community dynamics and function of algae and bacteria during winter in central European great lakes

Community dynamics and function of algae and bacteria during winter in central European great lakes

JGLR-01494; No. of pages: 9; 4C: Journal of Great Lakes Research xxx (xxxx) xxx Contents lists available at ScienceDirect Journal of Great Lakes Res...

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JGLR-01494; No. of pages: 9; 4C: Journal of Great Lakes Research xxx (xxxx) xxx

Contents lists available at ScienceDirect

Journal of Great Lakes Research journal homepage: www.elsevier.com/locate/jglr

Community dynamics and function of algae and bacteria during winter in central European great lakes George S. Bullerjahn a,⁎, Robert Michael L. McKay a,1, Gábor Bernát b,c, Ondřej Prášil b, Lajos Vörös c, Károly Pálffy c, Nóra Tugyi c, Boglárka Somogyi c a b c

Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA Centre Algatech, Institute of Microbiology, Academy of Sciences of the Czech Republic, Opatovicky mlyn, 379 81 Třeboň, Czech Republic Centre for Ecological Research, Balaton Limnological Institute, Hungarian Academy of Sciences, Klebelsberg Kuno u. 3., 8237 Tihany, Hungary

a r t i c l e

i n f o

Article history: Received 23 January 2019 2 June 2019 Accepted 11 June 2019 Available online xxxx Communicated by Nico Salmaso Keywords: Ice Phytoplankton Bacterial communities Photosynthesis Metagenomics Metabarcoding

a b s t r a c t Abundant phytoplankton and bacteria were identified by microscopy and high-throughput 16S rRNA tag Illumina sequencing of samples from water- and ice phases collected during winter at two central European Great Lakes, Balaton and Fertő (Neusiedlersee). Bacterial reads at all sites were dominated (N85%) by Bacteroidetes and Proteobacteria. Amongst phototrophs, microscopy and 16S sequencing revealed that both phytoplankton and cyanobacteria were represented, with a median of 1500 cyanobacterial sequence reads amongst 13 samples analyzed. The sequence analysis compared replicate Balaton and Fertő ice and water samples with an outgroup from three Hungarian soda lakes. In particular, both water and ice from Fertő contained high contributions from cyanobacteria. Two percent of total reads identified to the level of family in water at Fertő were dominated by a single operational taxonomic unit (OTU) of a cyanobacterium within the Rivulariaceae, which was largely absent from ice. Conversely, ice samples from both lakes yielded an abundant OTU assigned to a Flavobacterium sp. known to be associated with freshwater ice. Principal Coordinates Analysis (PCoA) revealed that the ice communities from all sites were similar to one another, and that the water communities did not cluster together. Fluorescence emission spectra obtained at 77 K confirmed the presence of intact cyanobacteria in Fertő water and ice. Photosynthetic characterization of phototrophs resident in water and ice analyzed by assay of acid-stable photosynthetic H14CO–3 incorporation showed that communities from both phases were photosynthetically active, thus adding to growing recognition of ice-covered lakes as viable habitat for phototrophs. © 2019 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction Balaton (Hungary) is the largest lake in Central Europe with a surface area of 596 km2 and a mean depth of 3.2 m. It has been characterized by relatively stable specific conductivity (SC, 700–800 μS/cm at 25 °C) and pH (8.3–8.6). Moreover, the depth and trophic gradient along the longitudinal axis of the lake involves significantly higher inorganic turbidity and algal biomass in the west basin as compared to the east (Felföldi et al., 2011a; Vörös et al., 2009). Fertő (alias Neusiedlersee) is a wind-exposed, shallow (~ 1.3 m mean depth), eutrophic (annual maximal chlorophyll a concentration: 30–40 μg/L), endorheic steppe lake (SC: 1300–3200 μS/cm; pH: 7.8–9.3), straddling the Austrian/Hungarian border (Löffler, 1979; Somogyi et al., 2010). The total surface area of this

⁎ Corresponding author. E-mail address: [email protected] (G.S. Bullerjahn). 1 Current address: Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON N9B 3P4, Canada.

lake is 309 km2, of which ~55% is covered by Phragmites reeds (Löffler, 1979). As a result of wind-induced sediment resuspension, the open water of the lake is characterized by high inorganic turbidity and typically low Secchi-disk transparency (Löffler, 1979). Both lakes underwent a strong eutrophication during the 1970s–90s, but currently they have a meso-eutrophic character again (Dokulil et al., 2014). In Balaton, the phytoplankton abundance often shows a bimodal annual pattern with a lower spring peak, attributed to diatoms, and a higher summer peak, due to filamentous diazotrophic cyanobacteria and/or planktonic dinoflagellates. In winter, phytoplankton is usually dominated by picoeukaryotes, constituting up to half of the biomass and primary production (Vörös et al., 2009; Somogyi et al., 2016). Small flagellates (e.g. Cryptomonas spp., Rhodomonas spp.) and green algae (e.g. Monoraphidium spp.) are also common members of the winter phytoplankton community (Dokulil et al., 2014). In Fertő, colonial picocyanobacteria and meroplanktonic diatoms dominate the phytoplankton through the whole year; however, chlorophytes can be widespread in winter communities (Somogyi et al., 2009; Dokulil et al., 2014).

https://doi.org/10.1016/j.jglr.2019.07.002 0380-1330/© 2019 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

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G.S. Bullerjahn et al. / Journal of Great Lakes Research xxx (xxxx) xxx

Climate warming has affected Central European lakes resulting in a declining tendency of ice cover duration at a rate of N−5 days per decade (Dokulil et al., 2014; Soja et al., 2014). Balaton and Fertő are usually frozen for 44 ± 26 days, and 73 ± 26 days, respectively, but winters without ice cover can also occur (Soja et al., 2014). A reduction in ice cover duration also involves habitat loss for ice-associated algae and other microorganisms (Beall et al., 2016). For this reason, the study of ice communities is important for understanding the dynamics and functioning of seasonally frozen lakes (Bertilsson et al., 2013). Winter severity manifested through changes in the duration of ice and snow cover patterns may also influence phytoplankton succession as well as nutrient variables and zooplankton biomass in the following summer (Bertilsson et al., 2013; Hampton et al., 2017). Adopting a multifaceted approach including high throughput DNA sequencing and epifluorescence microscopy, we examined partitioning of phytoplankton and bacteria between water- and ice phases in two seasonally ice-covered Great Lakes of central Europe.

Sciences), Tihany in cool boxes for additional sample processing and analysis. Photosynthetically active radiation (PAR, 400–700 nm) in the water column was measured at 0.25 m increments with a Li-COR underwater radiometer, using a flat (2π) quantum sensor. Diminution of light within the ice cover was calculated as the difference between PAR measured above and just below the ice cover according to Somogyi et al., (2016). Water temperature, pH and conductivity of the water samples were measured with a pH 315i portable field meter (WTW, Weilheim, Germany) and a HI9033 portable conductivity field meter (Hanna Instruments, Leighton Buzzard, UK), respectively. Major physicochemical parameters of the water and thawed ice samples are listed in Table 1. Concentrations of chlorophyll a (Chl a), as well as total phosphorus and nitrogen were determined spectrophotometrically (Menzel and Corwin, 1965; Wellburn, 1994; Eaton et al., 1995).

Methods

Nano- and microplankton samples were fixed with Lugol's solution, and their abundance and composition determined using an inverted microscope (Utermöhl, 1958; Krammer and Lange-Bertalot, 1999). Cell volume of the observed taxa was calculated using the formulas of Hillebrand et al. (1999). Total biovolume of the nano- and microplankton was calculated on the basis of cell volume and abundance values. Biomass (wet weight) was estimated from the total biovolume of the fractions assuming a specific mass density of 1.0 g/cm3. The abundance and composition of autotrophic picoplankton (APP) was determined in fresh, unpreserved samples according to MacIsaac and Stockner (1993). Briefly, samples were concentrated on 0.4 μm pore size black celluloseacetate filters (Macherey-Nagel, Düren, Germany), which were subsequently embedded into 50% glycerol on a microscope slide. The slides were examined using an Olympus BX51 epifluorescence microscope (Olympus, Shinjuku, Japan) at 1000× magnification upon blue–violet (U-MWBV2; 400–440 nm) and green (U-MWG2; 510–550 nm)

Study sites and sampling The central European large lakes Balaton and Fertő (Neusiedlersee) were sampled during the period of winter ice cover in February 2016. Onset of ice cover occurred in mid-January. At Balaton, ice and water samples were collected from two different coastal locations at Tihany (46.91415°, 17.89347°) and Keszthely (46.754089o, 17.248807o), whereas Fertő was sampled at Fertőrákos (47.72214°, 16.69194°; see Fig. 1 for sampling locations). Surfaces of optically clear ice samples (5 cm average thickness) were rinsed twice by deionized water immediately following collection in order to minimize the influence of underlying water and recent atmospheric deposition. Samples were transported to the Balaton Limnological Institute (Centre for Ecological Research, Hungarian Academy of

Taxonomic composition and abundance of phytoplankton

Fig. 1. Panel A: Map of Balaton, showing sampling sites at Tihany and Keszthely. Panel B: Map of Fertő/Neusiedlersee.

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

G.S. Bullerjahn et al. / Journal of Great Lakes Research xxx (xxxx) xxx Table 1 Major physicochemical parameters of the collected water and ice samples. Temp, temperature; Sp Cond, Specific conductance; PAR, photosynthetically active radiation; TP, total phosphorus; TN, total nitrogen; TN:TP reflects the molar ratio. PAR refers the percent of light penetrating through ice. Site

Temp

pH

Sp Cond

PAR

Chl a

TP

(μS/cm)

(%)

(μg/L)

(μg/L)

9.3 9.1 9.0 9.0

698 35 679 60

97.5

1.35 6.45 7.75 2.21

31 228 29 41

11 3.4 2 4

9.4 8.8

1280 208

94.4

2.37 1.7

26 22

13 10

(°C) Balaton Tihany Keszthely

Water Ice Water Ice

3.5

Water Ice

0.9

1.6

97.3

TN:TP

Fertő

excitation. Twenty fields (~400 cells) were photographed using a DP71 color camera (Olympus) and APP was counted on the images to avoid fluorescence fading. Picoeukaryotes (EuAPP) emit vivid red light upon blue–violet excitation due to Chl a and show no or only weak fluorescence upon green light excitation. Picocyanobacteria (CyAPP) can be distinguished from picoeukaryotes based on the presence of phycobiliproteins, which emit intense red fluorescence upon excitation with green light. Phycoerythrin-rich picocyanobacteria fluoresce bright yellow, whereas phycocyanin-rich picocyanobacteria show weak red autofluorescence upon blue–violet excitation (MacIsaac and Stockner, 1993). APP abundance was converted to biomass by measuring the dimensions of 50 cells, calculating their biovolume and considering an average density of 1.0 g/cm3.

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High-throughput microbial community analysis For high-throughput microbial community analysis, 100 mL of seston at each sampling time was concentrated in duplicate onto 0.22 μm Sterivex cartridge filters (EMD Millipore, Billerica, MA, USA). The filters were initially air dried and then stored at −80 °C within 2 weeks of collection. DNA was extracted from the cartridges using the PowerWater DNA kit (Qiagen, Germantown, MD, USA) following the manufacturer's instructions, yielding DNA at concentrations exceeding 25 ng/μL. In total, 13 samples were analyzed. Replicate samples from Fertő ice, Fertő water, Keszthely ice and Tihany water were sequenced along with single samples from Keszthely water, Tihany ice and water as well as samples from soda lakes Sósér, Kelemen-szék and Böddi-szék located in the Kiskunság National Park (Danube–Tisza Interfluve, Bács-Kiskun county, Hungary; see Somogyi et al., 2009). DNA was analyzed by the Genomic Services Lab at Hudson Alpha Institute for Biotechnology (Huntsville, AL, USA) where community 16S sequences were obtained following PCR with the primer pair S-D-Bact-0341-b-S-17 and S-DBact-0785-a-A-21 (Klindworth et al., 2013) to amplify the V3-V4 region. Sequencing of the libraries followed on the Illumina MiSeq platform (San Diego, CA, USA) following the Illumina 16S metagenomics sequencing protocol (Illumina, 2014) to generate 300 bp paired-end reads. Bioinformatic analysis was performed using the 16S Metagenomics app (v 1.0.1.0) on the Illumina BaseSpace cloudcomputing platform (Langmead and Nellore, 2018) using ClassifyReads, an algorithm based on the Ribosomal Database Project Classifier described in Wang et al., (2007). ClassifyReads used the Illumina-curated 2013 release of the Greengenes reference taxonomy (DeSantis et al., 2006). Statistical analysis

Low temperature fluorescence emission spectroscopy A 400 mL sample of water (i.e. lake water or ice melt) was filtered through a 47 mm GF/F glass microfiber filter with a nominal pore size of 0.7 μm (Whatman, Maidstone, UK). Moist, oval-shaped fragments (~1 cm × 0.3 cm) were cut using a deformed cork drill and placed into the cavity of a copper finger of a home-made apparatus (Prášil et al., 2009) and were immersed into a transparent Dewar flask filled with liquid nitrogen. 77 K fluorescence emission spectra were recorded using an SM-9000 spectrophotometer (Photon Systems Instruments, Brno, Czech Republic) upon excitation with 457 nm or 534 nm light (20 nm bandwidths) for chl and phycobilin excitation, respectively (Prášil et al., 2009). Three individual spectra of three replicates (9 in total) were recorded, baseline-corrected and averaged.

The 16S Metagenomics app (v 1.0.1.0) on the Illumina BaseSpace cloud-computing platform was used to calculate alpha diversity, which assesses the diversity within each sample, as well as community beta diversity, a measure of diversity between different environments. The latter was calculated using principle coordinates analysis (PCoA) which shows similarity between normalized relative abundance of all samples. The PCoA is generated using classical multidimensional scaling on a Pearson covariance distance matrix generated from per-sample normalized classification abundance vectors. For comparative purposes, PCoA included an outgroup of open water samples collected as part of the winter survey from the nearby alkali soda lakes Sósér, Kelemenszék and Böddi-szék. Results and discussion

Photosynthetic activity

Phytoplankton community composition

Photosynthesis-irradiance (P\\I) curves were recorded by measuring uptake of inorganic 14C by the phytoplankton community under different light intensities (Steemann Nielsen, 1952). Subsamples of 20 mL were distributed into glass vials, preincubated for 1 h in darkness, and after adding 0.084 MBq NaH14CO3 (Institute of Isotopes Budapest, Hungary), were incubated for 2 h in triplicate in a cooled water bath (2.5 °C) under cool white fluorescent illumination of 11, 47, 85, 224, 380, 702, 1214 and 2050 μmol photons/m2/s1. For dark carbon uptake, three vials were incubated in complete darkness. Samples were subsequently filtered through 0.45 μm pore size cellulose-acetate membrane filters and put into HCl vapour to remove remaining inorganic 14C. Filters were dissolved in 10 mL of scintillation mixture, after which radioactivity was measured using a 1211-RACKBETA liquid scintillation counter (LKB Instruments, Mt. Waverley, Australia). P\\I curves were fitted using the model of Eilers and Peeters (1988) with the statistical software R version 3.5.1 (R Core Team, 2018).

At the time of the February 2016 survey, Balaton and Fertő were covered by recently formed thin ice (2–5 cm) with negligible snow cover. Water temperature (0.9–3.5 °C), pH (9–9.4), and specific conductance (679–1280 μS/cm) varied in ranges typical for these lakes during winter (Table 1; Somogyi et al., 2016). We recognize that relying on a single sampling survey to inform our understanding of microbial diversity representative of the winter season carries uncertainty, however, this approach reflects the genuine logistical challenges faced when conducting research during winter on large ice-covered lakes. Phytoplankton biomass was low in both lakes during winter (Table 1) and was an order of magnitude lower than the long-term summer seasonal means (Somogyi et al., 2010; Felföldi et al., 2011). Chl a was measured in both ice- and water phases with no distinct patterns observed (Table 1). At Keszthely, located at the western end of Balaton, the total calculated nanoplankton wet weight biomass determined by microscopy in the water was 112 μg/L with Cryptomonas

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

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ovata and chrysoflagellates dominating this size fraction, out of a total phytoplankton biomass of 1732 μg/L dominated by EuAPP (Fig. 2). Associated with ice, the total wet weight nanoplankton biomass was similar (142 μg/L) but the composition was different; the dominant groups were diatoms and green algae, while cryptophytes and chrysoflagellates were absent. EuAPP again represented the largest biomass fraction, yielding a total of 424 μg/L (Fig. 2). At the Tihany site, the nanoplankton (205 μg/L) dominated total biomass and was composed largely of cryptophytes, dinophytes and centric diatoms, while in the ice (Fig. 2), large benthic diatoms (Nitzschia sp., Gyrosigma macrum, Fragilaria sp.) significantly increased the total biomass (1300 μg/L), a finding consistent with Chl a measured between water and ice phases at this site (Fig. 2, Table 1). In all ice samples, cryptophytes were notably absent. Several recent studies have likewise identified abundant phototrophs associated with ice with uneven partitioning of taxa between ice- and water phases (Bazhenova and Korzhova, 2014; McKay et al., 2015) possibly representative of active recruitment of phytoplankton to the ice through their association with epiphytic ice nucleating bacteria (D'souza et al., 2013). In Fertő, the total nanoplankton biomass was 216 μg/L in the water, with Cryptomonas ovata and Peridinium inconspicuum identified as the dominant species (Fig. 2). EuAPP (85 μg/L) and CyAPP (30 μg/L) contributed the remaining phytoplankton biomass. Associated with ice, the nanoplankton biomass was similar (170 μg/L) with P. inconspicuum dominant; however, the

cryptophytes were absent (Fig. 2). The remainder of total biomass was composed of EuAPP (37 μg/L and CyAPP (16 μg/L). Whereas previous work has shown the dominance of EuAPP in winter water samples (Vörös et al., 2009), we examined the abundance and composition of APP in ice, which was uniformly lower than in the water. The highest abundances (water: 5 × 105 cells/mL, ice: 0.8 × 105 cells/ mL) were found in Balaton at Keszthely, where EuAPP was dominant, as was expected based on prior surveys (Vörös et al., 2009). In the case of Fertő, both groups were present, however CyAPP abundance (water: 6 × 104 cells/mL, ice: 3 × 104 cells/mL) was comparable to EuAPP abundance (water: 4 × 104 cells/mL, ice: 2 × 104 cells/mL). At Tihany (Balaton) APP was negligible both in the water and in the ice, in contrast to a prior study documenting a EuAPPdominated picoplankton fraction averaging 4 × 103 cells/mL (Vörös et al., 2009). The composition and enumeration of the APP fractions at Keszthely are similar to previously reported values of CyAPP and EuAPP for water samples from western Balaton (2.6–11 × 104 cells/ mL CyAPP; 4.0–25 × 104 cells/mL EuAPP; see Somogyi et al., 2016). Regarding the abundance of EuAPP and CyAPP in Fertő, the values reported here fall within those reported previously (Somogyi et al., 2016). Phytoplankton 77 K fluorescence Substantial changes in the composition of phytoplankton collected from different locations and origin (i.e. ice vs. water) should also be manifested in the spectral characteristics of the corresponding samples. Low temperature (77 K) fluorescence emission spectroscopy, which is a useful tool to visualize changes in light harvesting and excitation energy transfer in primary producers (see e.g. Prášil et al., 2009; McKay et al., 2015), revealed notable differences amongst the investigated samples (Fig. 3). Fluorescence emission spectra of phytoplankton collected at Tihany (Balaton) were similar for both ice-melt and water samples, showing a sharp single PS II peak at 685 nm (Fig. 3, top panels), in good agreement with spectral properties of various diatom species (Sang et al., 2006; Lavaud and Lepetit, 2013) and also with the dominance of this taxonomic group at that study site (Fig. 2). In contrast, the higher heterogeneity of the phytoplankton composition in the Keszthely and the Fertő locations was also visible on the corresponding low temperature fluorescence emission spectra. On these latter spectra, there are usually three emission peaks and shoulders with different intensities that are visible: (1) the above-mentioned sharp fluorescence emission peak at 685 nm, (2) a second PS II emission peak/shoulder at around 692–695 nm, and (3) a PS I emission peak at 715 nm (Fig. 3, middle and bottom panels). The intensity of the 695 nm and 715 nm emission relative to the 685 nm one seems to be proportional with the relative abundance of chlorophytes as compared to heterokontophytes (compare Figs. 2 and 3). Besides these, a fourth fluorescence band appears at 620 nm upon 534 nm excitation of the Fertő samples, in good agreement with the content of cyanobacteria in both water and ice, and the presence of cryptomonads in water samples (see Fig. 2 and the section on metagenomic data below). These data differ somewhat from a previous 77 K fluorescence study of ice-covered European reservoirs, in which cyanobacteria partitioned preferentially into the water fraction (McKay et al., 2015). Photosynthetic performance

Fig. 2. Composition of phytoplankton in samples from Balaton and Fertő water and ice determined by microscopy. Panel A: total biomass distribution by taxonomic division/ phylum. Panel B: total biomass showing distribution between autotrophic picoplankton (APP) and nanoplankton. EuAPP: eukaryotic APP; CyAPP: cyanobacterial APP.

The photosynthesis-irradiance (P\\I) curves obtained from the 14C incorporation measurements showed that both the incubated water samples and the ice-melt samples were photosynthetically active (Table 2, Fig. 4.), thus corroborating the 77 K fluorescence data and consistent with work on ice- and water phases from Lake Erie (Twiss et al., 2012; Saxton et al., 2012) and Czech reservoirs (McKay et al., 2015). In the case of Fertő, chlorophyll-specific maximum photosynthetic rate (Pchl m ) of the water and ice-melt samples reached 2.27 and 0.64 μg C/μg

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

G.S. Bullerjahn et al. / Journal of Great Lakes Research xxx (xxxx) xxx

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Fig. 3. Low temperature (77 K) fluorescence emission spectra of intact phototrophic cells excited by two wavelengths of light (457 nm and 534 nm) in water and ice from different locations.

Chl/h, respectively, whereas the corresponding light saturation parameters (Ik) were 25.2 and 29.2 μmol photons/m2/s. The chlorophyllspecific light utilization parameter (αchl) was relatively low for the water (0.0902 [μg C/L/h]/[μmol/m2/s]) and even lower for the ice sample (0.0218 [μg C/L/h]/[μmol/m2/s]). In Balaton, biomass-specific photosynthetic parameters were somewhat different between the two sites

(Table 2, Fig. 4). The values of Ik were considerably higher in the samples from the western basin (Keszthely) with 55.4 and 34.4 μmol photons/m2/s in the water and ice sample, respectively, as compared to those from the eastern site (Tihany). In contrast with Fertő and the Balaton site at Tihany, Pchl m in the water and ice of the Keszthely site was similar (1.53 and 1.52 μg C/μg Chl/h). On the other hand, this

Table 2 Photosynthetic rates and parameters of photosynthesis - irradiance curves. Volumetric rates (vol) are presented alongside rates normalized to Chl a biomass (chl). Pm: maximum photosynthetic rate at light saturation; α: the slope of the P\ \I curve at low irradiances; Ik: irradiance at which photosynthesis becomes light-saturating.

Tihany water Tihany ice Keszthely water Keszthely ice Fertő water Fertő ice

Pvol m (μg C/L/h)

αvol ([μg C/L/h]/[μmol/m2/s])

Ik (μmol/m2/s)

Pchl m (μg C/μg Chl/h)

αchl ([μg C/μg Chl /h]/[μmol/m2/s])

Ik (μmol/m2/s)

3.362 3.284 11.902 3.370 5.385 1.085

3.1657 0.2409 0.2150 0.0980 0.2138 0.0371

1.1 13.6 55.4 34.4 25.2 29.2

2.490 0.535 1.536 1.525 2.272 0.638

2.3343 0.0393 0.0277 0.0444 0.0902 0.0218

1.1 13.6 55.4 34.4 25.2 29.2

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

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G.S. Bullerjahn et al. / Journal of Great Lakes Research xxx (xxxx) xxx

Fig. 4. Photosynthesis-irradiance curves of water samples and melted ice from Balaton and Fertő. The P\ \I curve of Tihany water is not shown due to the limited number of samples tested.

was the only site where αchl of the ice sample (0.0444 [μg C/L/h]/ [μmol/m 2 /s]) exceeded that determined for the water sample (0.0277 [μg C/L/h]/[μmol/m2/s]). By comparison to other wintertime measurements, the highest rates are within the same order of magnitude seen during the winter months in Lake Erie (5.27 μg C/μg Chl/h; see Saxton et al., 2012).

Bacterial community composition Insights into microbial community diversity were drawn from short 16S rRNA amplicon sequencing of spatially-resolved samples collected from ice- and water phases of nearshore sites at Balaton and Fertő. A total of 2.25 × 106 16S amplicon sequences were recovered, and the

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

G.S. Bullerjahn et al. / Journal of Great Lakes Research xxx (xxxx) xxx Table 3 Summary of 16S amplicon sequences, numbers of operational taxonomic units (OTUs; 97% sequence identity) and alpha-diversity estimates from ice- and water phases of central European Great Lakes. Sequencesa

OTUs

(± S.D.)

(± S.D.)

Water Ice Water Ice

233,818 (1.1E4) 231,011 218,482 233,890 (3.9E3)

1052 (66) 1045 781 983 (12)

2.797/2.743 2.587 2.513 3.069/3.015

Water Ice

210,635 (8.2E3) 221,894 (1.7E4)

823 (68) 860 (103)

2.525/2.302 2.946/2.467

Site

Balaton Tihany Keszthely

Fertő a

Shannon index

Reads passing quality filtering.

output from every sample exceeded 2.05 × 105 reads (Table 3). For each sample, N90% of sequences were successfully classified to genus whereas N50% (median: 55%) of sequences were classified to species level. Shannon's diversity index, which combines species richness and abundance into a measure of evenness, did not vary for bacterial communities when analyzed from ice- and water phases aggregated from both lakes (Table 3; two-tailed unpaired t-test, t = 1.6, DF = 8, P = 0.148). Bacterial community profiling by 16S sequence analysis revealed that all samples were dominated by Bacteroidetes and Proteobacteria at the phylum level, with minor and variable contributions of Actinobacteria and Cyanobacteria (Fig. 5). Cyanobacterial reads were present in all samples at a median count of 1500 per sample. As a percentage of total reads, cyanobacteria were most abundant in Fertő ice and water, as well as at Keszthely, in agreement with the 77 K emission spectra showing 620 nm emission peaks indicative of cyanobacterial phycobilins. A single operational taxonomic unit attributed to the Rivulariaceae, a putative Calothrix sp., was the most abundant cyanobacterium identified at Fertő (N3.5% of total reads classified to species level in water), reflecting the presence of members of this family as epiphytes on the Phragmites reeds covering the southern end of the lake (Löffler, 1979). Likewise, this taxon accounted for 2.8% of total 16S reads classified to species level from water at Keszthely. Accounting for elevated cyanobacterial presence in ice at Fertő were CyAPP (classified as Synechcoccus/Cyanobium spp.) which contributed 2.6% of total bacterial reads in ice. CyAPP were also elevated in water samples at Fertő where they contributed 3% of total reads.

Fig. 5. Phylum-level composition of bacterial communites in Balaton and Fertő ice and water.

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A closer examination of the Bacteroidetes and Proteobacteria present showed that Flavobacteria were a consistently dominant class amongst all Balaton and Fertő samples, contributing 62% and 67% of total reads associated with ice from Keszthely and Fertő, respectively while contributing over 40% of reads in water from all sites (Fig. 6). Several OTUs drove the uneven distribution between ice and water within this class, most notably, a single OTU attributed to Flavobacterium frigidarium that contributed N25% of total reads classified to species level in ice of Keszthely (Balaton) and Fertő compared to only 5% of the reads in water. The psychrophile F. frigidarium has been previously isolated from freshwater ice (Foreman et al., 2011). This organism was also identified amongst the abundant Flavobacterium spp. implicated in decomposition of a senescing under-ice bloom of the cyanophyte Aphanizomenon flos-aquae in Lake Stechlin (Bižić-Ionescu et al., 2014), consistent with a role for this taxon in hydrolyzing plant and algal cell wall polysaccharides (Humphry et al., 2001). Notably, the ice is not enriched for the gammaproteobacterial taxa known to be active ice nucleators (D'souza et al., 2013; McKay et al., 2015). Amongst Proteobacteria, Betaproteobacteria and Gammaproteobacteria were the dominant representatives in water samples at all sites; whereas, abundance of the latter declined by 90% in ice at both Keszthely and Fertő (Fig. 6). Two betaproteobacterial OTUs assigned to the genus Janthinobacterium were abundant in both ice- and water phases of the two Balaton sites contributing a mean 14.5% of reads classified to species

Fig. 6. Class-level composition of the Bacteroidetes (top) and Proteobacteria in Balaton and Fertő samples.

Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002

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G.S. Bullerjahn et al. / Journal of Great Lakes Research xxx (xxxx) xxx

Gammaproteobacteria known to be active in ice nucleation (D'souza et al., 2013; McKay et al., 2015). Mechanisms of ice formation in these systems may rely on other particulates, such as fungal spores and pollen, as has been documented previously in freshwaters (Knackstedt et al., 2018).

Acknowledgements We thank Tímea Szabó, Balázs Németh and Anett Kelemen for their technical assistance. G.B. and O.P. were supported by project Algaman (CZ.1.07/2.3.00/20.0203) of the MŠMT, Czech Republic. The research at the Center Algatech is supported by project Algatech plus (NPU I, LO 1416), and by project ALGAMIC (CZ.1.05/2.1.00/19.0392). The work of G.B. was also supported by the Hungarian Academy of Sciences hosted as a distinguished scientist at the Centre for Ecological Research, Balaton Limnological Institute (contract Nr. VK-05/2017). Sequencing was supported by the U.S. National Science Foundation under grant no. DEB-1354707 (R.M.L.M.) Fig. 7. Principal Coordinates Analysis of 16S sequences from Balaton and Fertő samples, color coded for lake and ice vs. water. Note the ice samples cluster together. By comparison, sequences from Hungarian soda lakes (see Somogyi et al., 2009) are distinct from the other freshwater lakes.

in ice and 7% in water. Abundance of these reads was lower in Fertő where they contributed 1.3% and 4.1% of sequences classified to species in ice and water, respectively. Janthinobacterium lividum, one of the two species contributing to the high abundance reported here, is a psychrotroph commonly identified from glacial ice and snow (Miteva, 2008) and was recently cultured from the under-ice surface of Lake Baikal (Bashenkhaeva and Zakharova, 2017). Whereas analysis of alpha diversity did not reveal differences between bacterial communities partitioned to ice- and water phase, Principal Coordinates Analysis (PCoA) of community 16S sequences at the bacterial species level clearly demonstrated that the ice-derived samples clustered together, suggesting a common community composition, whereas the water samples were divergent from one another (Fig. 7). Nonetheless, the freshwater communities were distinct from an outgroup of wintertime bacterial communities from water samples taken from ice-free Hungarian Kiskunsagi alkali (soda pan) lakes located ca. 120 km southeast of Balaton (Fig. 7). Whereas the Fertő and Balaton Keszthely water communities are similar to one another, the Balaton Tihany water community clustered with the ice samples. Conclusions Combining physiological, biophysical and molecular assessment, this study provides a thorough examination at a single point in time of the wintertime phytoplankton and bacteria taxa present in both water and ice of Lakes Balaton and Fertő. Extending previous work on wintertime eukaryotic APP from water samples from these lakes (Somogyi et al., 2016), the results of this study clearly show that wintertime phytoplankton in the ice fractions of Lakes Balaton and Fertő are biologically active, yielding biomass-normalized photosynthetic rates comparable to those measured in wintertime blooms in Lake Erie (Saxton et al., 2012; Twiss et al., 2012). Additionally, the phytoplankton and microbial communities, assessed by microscopy and 16S sequencing, differ from one another, indicating that specific taxa are preferentially partitioned into the ice. Indeed, by PCoA, ice microbial communities from these two very different lakes show similarities not seen in water samples, suggesting that ice formation is selecting for a common community composition. Known psychrophilic bacteria are present that partition into water (Janthinobacterium sp.) and ice (Flavobacterium frigidarium). Nonetheless, ice samples from these lakes are not enriched for

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Please cite this article as: G.S. Bullerjahn, R.M.L. McKay, G. Bernát, et al., Community dynamics and function of algae and bacteria during winter in central European great lakes, Journal of Great Lakes Research, https://doi.org/10.1016/j.jglr.2019.07.002