Probing environmental DNA reveals circum-Baltic presence and diversity of chlorophyll a/b-containing filamentous cyanobacteria (genus Prochlorothrix)

Hydrobiologia (2014) 736:165–177 DOI 10.1007/s10750-014-1903-8

PRIMARY RESEARCH PAPER

Probing environmental DNA reveals circum-Baltic presence and diversity of chlorophyll a/b-containing filamentous cyanobacteria (genus Prochlorothrix) Nataliya Velichko • Svetlana Averina Olga Gavrilova • Natalia Ivanikova • Alexander V. Pinevich

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Received: 20 October 2013 / Revised: 28 April 2014 / Accepted: 29 April 2014 / Published online: 21 May 2014 Ó Springer International Publishing Switzerland 2014

Abstract Cyanobacteria that possess phycobilisomes, light-harvesting antenna, have been well studied. In contrast, more rare green cyanobacteria (four genera/five species) that instead make use of chlorophyll–protein complex are poorly studied. In particular, the genus Prochlorothrix is represented by a small environmental DNA database and reports of only two cultured species from Northern Europe. In this work, marine and freshwater habitats of Northwestern Russia were investigated. PCR with Prochlorothrix 16S rRNA gene specific primers, FISH analysis with a Prochlorothrix 16S rRNA-targeted probe, Prochlorothrix culture isolation, and phylogenetic analysis of Prochlorothrix diversity were carried out. We identified Prochlorothrix 16S rDNA in samples from the St. Petersburg region and corroborated this finding by FISH. Attempts to isolate PCRand FISH-detected Prochlorothrix strains were unsuccessful. Phylogenetic analysis revealed that the Prochlorothrix 16S rDNA sequences identified were very

Handling editor: Stefano Amalfitano

Electronic supplementary material The online version of this article (doi:10.1007/s10750-014-1903-8) contains supplementary material, which is available to authorized users. N. Velichko  S. Averina  O. Gavrilova  N. Ivanikova  A. V. Pinevich (&) Department of Microbiology, Faculty of Biology, St. Petersburg State University, St. Petersburg, Russia e-mail: [email protected]

similar and formed a single cluster with high bootstrap support. Some of these sequences represent environmental strains of the species Prochlorothrix hollandica and P. scandica, while the others belong to new Prochlorothrix species or even to a new Prochlorothrix-related genus. Our results suggest a broader distribution and greater diversity in Prochlorothrix than previously thought. Keywords Prochlorothrix  Cyanobacteria  Environmental DNA  16S rRNA gene  The rare biosphere  Endemism  Uncultured bacteria

Introduction Cyanobacteria are one of the most abundant living organisms on Earth. They withstand a multitude of environmental stresses and thus occupy all permissive niches, except for darkened, thermal ([80°C), and acidic (pH \ 5) environments. This is facilitated by their unique ability for oxygenic photosynthesis often coupled with aerobic nitrogen fixation, hence their paramount importance in global autotrophic metabolism. Most cyanobacteria, including the simple plastids in red algae, use phycobiliproteins as accessory pigments. The latter is aggregated in phycobilisomes, which along with chlorosomes of (non-) sulfur green bacteria, are membrane-attached light-harvesting antennae. A minor cyanobacterial subgroup (five

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species from four genera) makes use of membraneembedded chlorophyll–protein antennae instead of phycobilisomes. Three unicellular genera—Acaryochloris, Prochloron, and Prochlorococcus, and the filamentous genus Prochlorothrix (Partensky & Garczarek, 2003; Pinevich et al., 2012) comprise this group and are jointly known as the prochlorophytes (Lewin, 1981) and green cyanobacteria (Pinevich et al., 2012). Acaryochloris marina and Prochloron didemni possess multi-chlorophyll light-harvesting antennae and participate in obligate symbiosis with ascidians. These organisms are poorly studied because of methodical problems imposed by their lifestyle. In contrast, Prochlorococcus marinus, which inhabits the oceanic euphotic zone in low-to-mid latitudes, has received considerable attention as a generally recognized prokaryote of global ecological significance (Partensky et al., 1999). The genus Prochlorothrix is represented by two closely related species—P. hollandica (Burger-Wiersma et al., 1989) and P. scandica (Skulberg, 2008; Pinevich et al., 2012); both of which possess chlorophyll a/b-containing light-harvesting antennae. Successful isolation of these cyanobacteria was permitted by eutrophication outbursts in Lake Loosdrecht, The Netherlands and Lake Ma¨laren, Sweden, respectively (Burger-Wiersma et al., 1989; Skulberg, 2008). No other representatives of this genus have been obtained in culture since then. Light microscopy-based exposure of Prochlorothrix is hampered by its likeness to the more abundant phycobiliprotein-containing cyanobacteria Leptolyngbya, Limnothrix, and Pseudanabaena (Geiß et al., 2003). Additionally, because of the very low chlorophyll b:a ratio in Prochlorothrix, successful detection of this cyanobacterium through pigment analysis depends on highly sensitive tools that are not commonly available (Pinevich et al., 2012). An alternative means to study Prochlorothrix diversity and distribution is based on the detection and comparison of PCR-amplified 16S rRNA gene sequences in environmental DNA. So far, the sequences have only been sporadically reported for (i) Lake Loosdrecht, The Netherlands (Zwart et al., 1998); (ii) Darss-Zingst Estuary, Baltic Sea, Germany (Geib et al., 2003); (iii) La Rocha Bay, Atlantic Ocean, Uruguay (Piccini et al., 2006); (iv) Lake Charles and Lake Pontchartrain, Louisiana, USA (Amaral-Zettler

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et al., 2008); (v) Gulf of Finland, Baltic Sea, Russia (Pinevich et al., 2012); and (vi) Lake Baikal, Eastern Siberia, Russia (GenBank accession numbers JQ700556.1 and JQ700557.1). Maximum likelihood similarities of Prochlorothrix 16S rRNA gene sequences deposited in GenBank to date are shown in Table S1 (see Online Resource 1). Although European PCR-based findings of Prochlorothrix, except for those in Lake Loosdrecht, associate themselves with only two Baltic Sea near-shore point localities (Table S1), there is no discernable reason for the restricted distribution seen in this cyanobacterium. Any new reports of Prochlorothrix, either from environmental DNA or cultured strains, would shed more light on the eco-geographic distribution and genetic diversity of this rare cyanobacterium. Proof of presence, along with demonstration of absence, should be discussed in light of Prochlorothrix physiology. The aims of this study were (1) to search for Prochlorothrix in 17 sampling sites mainly within Northwestern Russia using PCR with primers specifically targeted to Prochlorothrix 16S rDNA; (2) to substantiate the PCR-witnessed presence of Prochlorothrix by FISH-visualization using the specifically designed 16S rRNA-targeted oligonucleotide probe; (3) to isolate cultured strains from Prochlorothrixpositive sampling sites; and (4) to perform phylogenetic analysis on the Prochlorothrix 16S rRNA gene to elucidate the genetic diversity and taxonomy of this cyanobacterium.

Materials and methods Sampling Water samples (c. 5 l) were collected from 2010 to 2012 from (i) Gulf of Finland, Russia; (ii) 3 small lakes near St. Petersburg; (iii) Lake Ladoga, Lake Chudskoe, and Lake Ilmen; (iv) 3 small lakes on the Karelia Isthmus; v) Lake Saimaa, Finland; and (vi) Kaliningradskyi Bay and Kurshskyi Bay, Baltic Sea, Russia (Fig. 1). The sampling period, May–June, was chosen as it is when cyanobacterial blooms begin. The limnological characteristics of the sampling sites for clones 26.5, 26.6, 9.4, and 9.6 (sources of amplicons most similar to the Prochlorothrix 16S rRNA gene) are presented in Table 1.

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Fig. 1 Map of the Baltic Sea region showing sampling sites including those numbered 6 and 8 (Kaliningrad area, Russia). The inset is a map of sampling sites 1–5, 7, 9–14, 16, and 17 (St. Petersburg area, Russia) and sampling site 15 (Lake Saimaa, Finland). Empty circles = Prochlorothrix absent; filled circles = Prochlorothrix present; filled circles with clone numbers in parentheses (sampling sites 5 and 14) represent amplicons with most similarity to Prochlorothrix 16S rRNA gene obtained

Table 1 Sampling record with local limnological features Clone number

Sampling site

Median depth (m)

Water temp (°C)

Salinity (%)

pH

Total N (lg l-1)

Total P (lg l-1)

26.5 and 26.6

Gulf of Finland (59°550 5300 N, 29°370 5600 E)

6

18–20

2–6

6.8–7.5

830

27–83

9.4 and 9.6

Lake Borisovskoye (60°350 5400 N, 30°000 4100 E)

3

18

0.045

6.8

600

100

a

Total Ca (mg l-1)

9.6

45

O2 (lg l-1)

Org. C (mg l-1)

7–17

10

11.4

a

Not determined

Isolation of environmental DNA Microbial plankton was collected by vacuum filtration of 5 l-samples through 0.45-lm-pore-size Sartorius membrane filters (Germany), re-suspended in TE buffer (10-mM Tris–HCl, pH 7.5, and 1-mM Na2EDTA) and collected by centrifugation at 7,0009g for 10 min at ambient temperature. Cells (* 100 mg wet wt) were disrupted with 3-ml lysis cocktail (0.1-M Tris–HCl, pH 8.5, 20-mM Na2EDTA, 1.5-M NaCl, 2% cetyltrimethylammonium bromide,

0.2% 2-mercaptoethanol, and 2-lg ml-1 Proteinase K (Sigma, USA)) for 3 h at 60°C with continuous stirring. Cell lysate was purged of proteins by emulsifying with an equal volume of chloroform/isoamyl alcohol (24:1 vol/vol) at -20°C for 30 min. After centrifugation at 10,0009g for 15 min, the supernatant was mixed with cold isopropyl alcohol (2:3 vol/vol). The DNA pellet was washed twice with 80% ethanol and re-dissolved in 50 ll of double distilled water. DNA preparations were treated with 50-lg ml-1 RNase (Fermentas, Lithuania) at 37°C for 30 min.

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Fig. 2 Gel electrophoresis demonstrating the Prochlorothrix 16S rRNA gene-targeted primer specificity and separation of the environmental DNA amplicons. (A) Lanes 1–11: Anabaena sp. CALU 786, Calothrix sp. CALU 790, Oscillatoria sp. CALU 657, Lyngbya sp. CALU 725, Leptolyngbya sp. CALU 1171, Anabaena sp. CALU 458, Pseudanabaena sp. CALU 1079, Nostoc sp. CALU 1088, Synechococcus sp. CALU 670,

Synechocystis sp. CALU 891, and P. hollandica SAG 10.89 DNA templates, respectively; lane M = marker DNA. (B) Lanes 1–17: environmental DNA templates from sampling sites 1–17; lanes 18 and 19, P. scandica NIVA-8/90 and P. hollandica SAG 10.89 DNA templates; lane 20 = blank control; lane M = marker DNA

PCR conditions

specificity was confirmed through a control test with P. hollandica SAG 10.89 (PCC 9006) DNA template along with templates from ten common cyanobacteria, both filamentous and unicellular ones (Fig. 2A).

Purified environmental DNA, P. hollandica SAG 10.89 (PCC 9006) and P. scandica NIVA-8/90 genomic DNA were used as PCR templates. Primers for the amplification of Prochlorothrix 16S rRNA gene sequences (16S-Pctx-fw 50 -CTTAGCGGCGGACGG GTGAG and 16S-Pctx-rev 50 -GGTGTGACGGGCGG TGTGTA) were designed in the program OLIGO version 7.50. For this purpose, the P. hollandica SAG 10.89 16S rRNA gene sequence and the most similar sequences from GenBank were used. The PCR mixture (25 ll) contained 5 ll buffer (500-mM KCl, 150-mM Tris–HCl, pH 8.8, 0.1-mM Na2EDTA, 0.1% Tween20, and 0.5% glycerol), 6.25-lM MgCl2, 50–100 ng of DNA, 40-nM dNTP mixture, 0.5 lM of each primer, and 1–1.5-U ml-1 recombinant Taq polymerase (Fermentas, Lithuania). PCR was performed in an MJ MiniTM Research Thermocycler (BioRad, USA) with initial DNA denaturation at 94°C (5 min) and 35 subsequent cycles of DNA melting at 94°C for 30 s, primer annealing at 57°C for 40 s, and DNA synthesis at 72°C for 3 min. The 1,200 bp 16S rRNA gene amplicons were separated on 1% agarose gels (Fermentas, Lithuania) in TBE buffer (89-mM Tris, 89-mM boric acid, pH 8.0, 2.5-mM Na2EDTA) and purified using a QIAEX II Kit (Qiagen, Germany). Prochlorothrix 16S rRNA gene-targeted primer

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Clone library preparation Purified 16S DNA-amplicons were cloned in the pTZ57R plasmid using an InsT/ATM clone Kit (Fermentas, Lithuania). For the ligation reaction, a 0.5-pM DNA fragment was incubated with 0.2 pM of the plasmid and 5-U ll-1 T4 DNA ligase (Fermentas, Lithuania) at 22°C for 30 min. A ligase mixture containing *15 ng of the vector was used to transform chemically competent E. coli XL1-blue cells that were plated on solid Luria-Bertani medium containing IPTG/X-Gal and incubated at 37°C for 12 h. Cells from transformed colonies were treated with a QIAprep Spin Kit (Qiagen, Germany) to isolate plasmids containing 1,200-bp inserts of the PCR amplicons. Gene sequencing The cloned 16S rDNA fragments were sequenced on an ABI PrismÒ 3500xl Analyzer (Applied Biosystems, USA) using the universal bacterial primers M13/pUC (M13F 50 -GTAAAACGACGGCCAG and M13R 50 -CAGGAAACAGCTATGAC) and a BigDyeÒ

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Sequencing Kit 3.1 (Applied Biosystems, USA) according to manufacturer’s protocol. FISH The cyanobacteria containing Prochlorothrix 16S rRNA genes (sampling sites 5 and 14) were visualized in corresponding environmental samples by FISH analysis. An oligonucleotide probe specifically targeted to Prochlorothrix 16S rRNA was designed using ARB software (Ludwig et al., 2004). For this purpose, about 40,000 cyanobacterial 16S rRNA sequences, including those of Prochlorothrix, were exported from the SILVA database (http://www.arb-silva.de). Their alignment elicited conservative loci discriminating Prochlorothrix from other cyanobacteria. Successfully designed probes were tested with ARB and ProbeBase software (Loy et al., 2007). One of these probes proved not only most similar, without any mismatches, to P. hollandica SAG 10.89, P. scandica NIVA-8/90, and uncultured clones (LD16 (AJ007866) and LD22 (AJ006285)) 16S rDNA sequences, but also demonstrated appropriate thermodynamic DG°T values for oligo–target duplex and high hybridization efficiency. The designed Prochlorothrix-specific 18-meric PCTX622 oligonucleotide probe was 30 labeled with a ROX (6-carboxyl-X-rhodamine) reference dye (Invitrogen, USA). According to the BLAST analysis, the PCTX622 sequence CCATCGCTCTCC CACAGT is complementary to positions 300 to 317 on the 16S rRNA gene in clones 26.5 and 26.6, positions 144 to 161 in clones 9.4 and 9.6, positions 467 to 484 in P. scandica NIVA-8/90, positions 526 to 543 in P. hollandica SAG 10.89, and positions 539 to 556 in clones LD16 and LD22. Hybridization conditions were optimized as recommended for a standard FISH protocol (Amann & Fuchs, 2008). To check the desired specificity, samples from P. hollandica SAG 10.89, P. scandica NIVA-8/90, Anabaena sp. CALU 786 (St. Petersburg University Culture Collection), Leptolyngbya sp. CALU 1171, Limnothrix sp. CALU 1714, Oscillatoria sp. CALU 657, Plectonema sp. CALU 485, Pseudanabaena sp. CALU 1716, Synechococcus sp. CALU 670, and Synechocystis sp. CALU 891 batch cultures were used. Preparations for FISH were fixed with 2% p-formaldehyde at 4°C for 3–5 h and washed with PBS buffer (15-mM K, Na– phosphate, pH 7.3). After centrifugation at 10,0009g for 15 min, cell material was permeabilized

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with 2-lg ml-1 Proteinase K (Sigma, USA) and dehydrated in a 50–96% ethanol series. The slides were then incubated at 46°C for 6 h in a hybridization camera with a PCTX622 probe in hybridization buffer (25-mM Tris–HCl, pH 9.0, 15-mM NaCl, and 0.2% SDS) containing 35% formamide. Any probe excess was eliminated through two washings in buffer (20mM Tris–HCl, pH 8.0, 15-mM NaCl, 0.01% SDS, and 5-mM Na2EDTA). The FISH preparations were observed in a Leica TCS-SP5 confocal microscope (Leica Microsytems GmbH, Germany) under a fluorescence excitation beam of 588 nm, in the ROX maximum fluorescence emission band (608 nm). Culture isolation and microscopic monitoring Ten ml-aliquots of cotton-filtered water samples (see above) were mixed with 30 ml of modified BG-11 medium (Pinevich et al., 2012) containing 250-lg ml-1 cycloheximide (Serva, Germany) to inhibit eukaryotic translation; the samples were then cultured for approximately a month in 50-ml Erlenmeyer flasks, without stirring, at ambient temperature and under continuous illumination with 10-lE photon m-2 s-1 cool-white luminescent light. The presumed Prochlorothrix filaments can be discriminated from their phycobilisome-containing counterparts through morphological characters (see: Geiß et al., 2003). For this purpose, enrichment culture preparations were examined in Leica 1000 microscope equipped with the DFC 480 camera add-on (Leica Microsystems GmbH). Phylogenetic analysis Sequence alignment and neighbor-joining tree reconstruction were conducted in the ClustalW and MEGA 5.05 programs, respectively (Larkin et al., 2007; Tamura et al., 2007). Nucleotide sequences GenBank accession numbers for the Prochlorothrix 16S rRNA gene sequences obtained in this work (clones 9.4, 9.6, 26.5, and 26.6) are KF207539, KF207540, KF207541, and KF207542, respectively. Table S1 has been added to the BioFresh database (www.freshwaterbiodiversity.eu).

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Results Since most European Prochlorothrix environmental sequences were discovered near megalopolises, we searched in the lakes of St. Petersburg’s environs and in the Gulf of Finland. The expanded set of sampling sites in Russia included the Baltic Sea southern shore, three large lakes in St. Petersburg’s neighboring districts, three small lakes on the Karelia Isthmus, and Lake Saimaa in Finland (Fig. 1). Amplification of Prochlorothrix 16S rRNA gene fragments PCR of the environmental, P. scandica NIVA-8/90, and P. hollandica SAG 10.89 DNA templates generated amplicons of 1,200 bp. Only a few of the sampling sites (4 of 17) proved Prochlorothrixpositive (Fig. 2B). In particular, the 1,200 bp Prochlorothrix 16S rRNA gene fragments were amplified from environmental DNA collected in the near-shore zone of the southern Gulf of Finland (Fig. 2B, lanes 5 and 13; sampling sites 5 and 13, respectively), in a small lake near St. Petersburg (lane 9), and in Lake Borisovskoye, Karelia Isthmus (lane 14). However, no Prochlorothrix amplicons were obtained for the samples from a small lake in Karelia Isthmus (lane 1), two small lakes near St. Petersburg (lanes 2 and 4), Lake Ilmen (lane 3), Kaliningradskyi Bay (lane 6), the near-shore zone in the Northern Gulf of Finland (lanes 7 and 16), Kurshskyi Bay (lane 8), Lake Chudskoe (lane 10), Lake Ladoga (lanes 11 and 12), Lake Saimaa (lane 15), and a second lake in Karelia Isthmus (lane 17). The 1,200 bp-DNA fragments (Fig. 2B, lanes 5, 9, 13, and 14) were cloned in the pTZ57R vector. Sixty 16S rRNA gene fragments were sequenced. Only four of them (clones 26.5, 26.6, 9.4, and 9.6) fulfilled the requirements of reliable reading quality, sufficiently large size (c. 900–1,100 bp), and high similarity (C94%) to Prochlorothrix 16S rRNA gene fragments reported in previous studies. FISH-visualization of Prochlorothrix Uncultured bacteria with 16S rRNA genes highly similar to those of Prochlorothrix (sampling sites 5 and 14; clones 26.5 and 26.6, and 9.4 and 9.6, respectively) were visualized by FISH analysis using

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the ROX-labeled probe PCTX622. Relatively thin (*1 lm), long ([100 lm), and wavy filaments similar to Prochlorothrix, particularly because of the absence of intercellular constrictions, were observed in the FISH photomicrographs of environmental samples from Lake Borisovskoye (Fig. 3B) and the Gulf of Finland southern shore (Fig. 3C). The brilliant light emitted by these filaments indicated a high amount of target ribosomes, while negligible fluorescence of the accompanying microorganisms confirmed the probe specificity. Thus, the PCR-witnessed presence of Prochlorothrix in samples from the St. Petersburg region was substantiated by direct visualization. Isolation and enrichment of Prochlorothrix strains In culture collections, Prochlorothrix (Pinevich et al., 2012) represents non-branching, immotile flexible filaments, or trichomes, composed of numerous nondifferentiated, elongated-cylindrical cells 7–10 9 1.5–2 lm in size. Unfortunately, we were unable to isolate Prochlorothrix strains from the water samples collected in course of this work. No filaments with Prochlorothrix morphology and cell dimensions were found in the enriched samples under light microscopy. Additionally, no colonies with apple-green pigmentation, which discriminates Prochlorothrix from phycobiliprotein-containing cyanobacteria (Skulberg, 2008), were observed on solid medium inoculated with material from the batch cultures. Thus, representatives of the Prochlorothrix genus detected by PCR and FISH analysis belong to the uncultured bacteria. Prochlorothrix phylogeny The data on Prochlorothrix diversity obtained in this work were analyzed in respect of phylogenetic relationships among individual strains and their joint position on the evolutionary tree. For dendrogram reconstruction, complete mutual coverage of all aligned sequences was required; therefore, only the shortest matching sequences were chosen for comparison. In particular, the minimum length sequence in analysis (533 bases) corresponded to the 16S rRNA gene fragment from Darss-Zingst Estuary (Geib et al., 2003). Among the references for sequence alignment were Prochlorothrix environmental 16S rDNA, P.

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Fig. 3 FISH (left) and phase contrast (right) photomicrographs. (A) Sample from P. hollandica SAG 10.89 batch culture. (B) Environmental sample from Lake Borisovskoye (sampling site 14; see: Fig. 1, inset). (C) Environmental sample from near-shore zone of the southern Gulf of Finland (sampling site 5; see: Fig. 1, inset)

hollandica SAG 10.89, P. scandica NIVA-8/90, Prochloron didemni, Synechococcus sp., Synechocystis sp. 16S rDNA sequences, and eight 16S rDNA sequences of eight filamentous strains from the genus Pseudanabaena. In the neighbor-joining tree constructed with the 533 bp 16S rRNA gene fragments, Prochlorothrix sequences comprised a single cluster with high bootstrap support (Fig. 4). This assemblage is closely related to Leptolyngbya, Limnothrix, and Pseudanabaena, which based on the polyphasic approach, are members of the order Pseudanabaenales (Koma´rek, 2006). Moreover, the Prochlorothrix cluster was relatively distant from Microcoleus, Oscillatoria, Planktothrix, and Trichodesmium; based on a polyphasic re-evaluation of cyanobacterial genera, all of them belong to the order Oscillatoriales (Koma´rek, 2006). Within the Prochlorothrix cluster, most sequences were distributed among three groups (Fig. 4). The first group, with the exception of clone GF1-26 (Baltic Sea), encompasses freshwater sequences, in particular the clones 9.4 (920 bases) and 9.6 (930 bases). The second group comprises marine sequences, i.e., clones 26.5 (1,112 bases) and 26.6 (1,110 bases). The third group consists of freshwater/marine sequences found in lakes where

temporary sea water incursions had occurred (Piccini et al., 2006; Amaral-Zettler et al., 2008). Outside these groups stand the freshwater sequences DP10.2.29 (Lake Dongping) and LD7 (Lake Loosdrecht), the marine sequence AP19 (Daya Bay), and the DarssZingst freshwater/marine sequence (Geiß et al., 2004). Taxonomic implications for Prochlorothrix The evaluation of similarity between Prochlorothrix 16S rDNA, particularly the environmental sequences, may have a taxonomic outcome. A BLAST comparison of clones 26.5 and 26.6 with P. hollandica SAG 10.89 and P. scandica NIVA-8/90 16S rDNA (which are 99% mutually similar; Pinevich et al., 2012) revealed 99% similarity. Therefore, according to the commonly accepted C3% dissimilarity criterion of species demarcation (Stackebrandt & Goebel, 1994), both environmental strains should be assigned to either P. hollandica or P. scandica. Furthermore, a BLAST comparison of clones 9.4 and 9.6 revealed only 94% similarity to P. hollandica SAG 10.89 and P. scandica NIVA-8/90, indicating a new species of the genus Prochlorothrix or even a new Prochlorothrixrelated genus. However, the genetic divergence between cultured Prochlorothrix species (Pinevich

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Fig. 4 Rooted tree obtained by phylogeny analysis (neighbor-joining method) of 533 cyanobacterial 16S rDNA nucleotide fragments. Bootstraps for 1,000 replications are shown at nodes

et al., 2012) as well as between PCR-detected environmental Prochlorothrix strains was surprisingly low (see Table S1; Fig. 4).

are usually abundant and easy to culture. Correspondingly, information on Prochlorothrix geographic distribution and genetic diversity is limited despite it being two decades since its initial discovery (Pinevich et al., 2012).

Discussion Prochlorothrix, a member of the rare biosphere The scarcity of Prochlorothrix DNA in the environment and the cultured strains make this cyanobacterium stand apart from other members of Pseudanabaena group (see: Koma´rek, 2006), which

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In agreement with the rare biosphere concept (Sogin et al., 2006; Pedro´s-Alio´, 2012), Prochlorothrix exemplifies (evolutionary) unsuccessful bacterium

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with its fragmentary distribution and low population density. Attempts to screen the rare biosphere based on 16S rDNA PCR records have encountered some method-dependent restrictions. First, DNA undersampling: confirmation of a bacterium’s presence depends on the completeness of template extraction from small resident populations, resting forms, and transitory objects. In the case of Prochlorothrix, sample size is most important—this cyanobacterium accumulates only in the course of sporadic water blooms (Skulberg, 2008; Pinevich et al., 2012). Second, the PCR-based data are affected by polymerase and sequencing errors. Finally, heterogeneous DNA templates are often selectively amplified. If a 16S rRNA gene region (in our case, positions 1 to 1,200) is highly variable, conservative primers may not hybridize with some of the environmental DNA sequences. However, all known Prochlorothrix 16S rRNA are highly similar (see Table S1; Fig. 4).

Prochlorothrix demonstrates endemism tendency In this work, despite the multitude and variety of sampling sites only a few tested positive for the presence of Prochlorothrix, and even sites in close proximity produced opposing results (Fig. 1). Thus, our hypothesis on the fragmentary geographic distribution of Prochlorothrix remains valid (Pinevich et al., 2012). In fact, Prochlorothrix tends to endemism as demonstrated by its restricted geographic range. It is important to note that the ubiquity of prokaryotes in general (according to Baas Becking’s formula; De Wit & Bouvier, 2006) cannot be (dis) proved at this point. Endemism per se, or association with a single habitat, is also questionable (Allison et al., 1992). Rather, prokaryotes demonstrate limitations of detectable development according to the space-and-time matrix of non-permissive niches. The endemism tendency has been reported in bacteria and archaea (Rippka et al., 1974; Whitaker et al., 2003; Ivanikova et al., 2007; Sa´nchez-Baracaldo et al., 2008; Haverkamp et al., 2009; Caravati et al., 2010; Jasser et al., 2010). Another example of cyanobacteria exhibiting the endemism tendency is Prochlorococcus marinus ecotypes that inhabit the oceanic euphotic zone in the lowto-mid latitudes (Partensky et al., 1999).

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Eutrophication dependence in Prochlorothrix To date, Prochlorothrix has not been recorded in oligotrophic lakes. In contrast, a gas vesicle containing P. hollandica was discovered during a routine study of water blooms in the Loosdrecht lake system, The Netherlands (Burger-Wiersma et al., 1989). These polymictic and highly eutrophic (*60-mg m-3 chlorophyll a) water pools of *2-m depth originate from a flooded turf pit, with phytoplankton being mostly represented by the phycobiliprotein-containing filamentous cyanobacteria Limnothrix and Pseudanabaena (Zwart et al., 2005). Prochlorothrix was also observed in the shallow Lake Tjeukemeer, The Netherlands (van Liere et al., 1989). Finally, P. hollandica has been identified via fluorescence microscopy in other eutrophic Dutch lakes (pers. comm. J. van der Does; van Liere et al., 1989). A second representative of the Prochlorothrix genus, P. scandica, was isolated from Lake Ma¨laren (1,140 km2, depth *13 m) near Stockholm, Sweden (Skulberg, 2008). Based on these reports, Prochlorothrix should be sought in eutrophic lakes occupied by cyanobacteria (van Liere et al., 1989). Prochlorothrix population size increases during eutrophication outbursts caused by waste water from large cities (Burger-Wiersma et al., 1989). However, the incidental detection of environmental DNA and even more fortuitous isolation of Prochlorothrix strains resulted from water blooms that rarely associate with this cyanobacterium. Thus, the discovery of Prochlorothrix sequences 26.5 and 26.6 might have been facilitated by a water bloom caused by St. Petersburg’s waste water and local efflux from the Bolshaya Izhora agricultural settlement in the vicinity of the southern Gulf of Finland. Moreover, Prochlorothrix sequences 9.4 and 9.6 originated from a summer bloom in running-water from Lake Borisovskoye (0.2–0.8 9 2.3 km), which lies close the same name settlement in Karelia Isthmus. The actual reason for Prochlorothrix growth fluctuations during eutrophication outbursts is unknown; the enrichment of heterotrophic satellites, in particular of helper bacteria (see below), may be involved. In small polymictic lakes, Prochlorothrix never becomes a dominant member of the plankton community. P. hollandica growth rates amount to 30–40% that of phycobiliprotein-containing filamentous cyanobacteria (0.02–0.14 day-1; Pel et al., 2004). The

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latter dominates and even suppresses Prochlorothrix in its natural environment. Prochlorothrix ecology: temperature range The optimum growth temperature for P. hollandica PCC 9006 and P. scandica NIVA-8/90 is 20 and 24°C, respectively (Skulberg, 2008). However, these species can grow at lower temperatures in the wild. Similar to many phycobiliprotein-containing filamentous cyanobacteria, Prochlorothrix occupies a climatic zone characterized by strong seasonal changes in temperature and episodic formation of ice cover during winter. For instance, when P. hollandica PCC 9006 was discovered in July 1984 in Lake Loosdrecht the water temperature was 18°C (Burger-Wiersma et al., 1989), whereas in April 1998, it was only 8°C (Zwart et al., 2005). As a rule, mass development of Prochlorothrix in this lake occurred at the end of summer when water temperature was 15–18°C (Post & Bullerjahn, 1994). Water sampling in Lake Ma¨laren, the site of P. scandica NIVA-8/90 detection, was performed in October 1990. During this period, the temperature was 10–13°C throughout the water column due to intensive mixing (Skulberg, 2008). Prochlorothrix ecology: salinity response Prochlorothrix is stenohaline (Burger-Wiersma et al., 1989), inhabits low-salinity waters, and is sensitive to even small increases in salinity. Cultured strains are non-tolerant to osmotic shock at more than 6 PSU (Geiß et al., 2004; Pinevich et al., 2012). P. hollandica PCC 9006 is inhibited by 25-mM NaCl, with complete arrest at 100 mM or at sea water equivalent salinity (Burger-Wiersma et al., 1989). Our observations on P. hollandica PCC 9006 and P. scandica NIVA-8/90 (Pinevich et al., unpublished data) have confirmed the negative effect of 50–100-mM NaCl. In both species, increased salinity provoked morphological changes: cells became elongated and filaments became zigzag shaped. The cyanobacteria degraded after a week of growth at [200-mM NaCl. Prochlorothrix sensitivity to high salinity is primarily due to low-level synthesis of compatible organic solutes, with salt adaptation varying in response to locally available ions. In particular, P. hollandica is resistant to 170-mM NaCl in a semi-synthetic medium supplied with water from Darss-Zingst Estuary; under these conditions, an

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upshift in accumulated sucrose was recorded (Bergmann et al., 2008). Since Prochlorothrix is absent from large freshwater lakes (Lake Ladoga, Lake Ilmen, and Lake Chudskoe; this work), it is clearly unable to withstand extremely low or zero salinity. The optimum environment for Prochlorothrix is brackish waters with a metastable salt gradient, such as Darss-Zingst Estuary. This polymictic and eutrophic water body consists of a chain of lagoons and is separated from the Baltic Sea by the Frischland/Darss Peninsula; it is fed by the Prerow River and supports opposing gradients of dissolved organic matter and salinity (Geiß et al., 2004). Perhaps a metastable pattern of both biotic and abiotic factors favors the development of this cyanobacterium. The presence of Prochlorothrix rDNA in the Baltic Sea, e.g., in the Gulf of Finland (this work), may be explained by temporal salt gradients. Similarly, Prochlorothrix rRNA gene fragments detected in North American lakes accumulated after the passage of Hurricanes Katrina and Rita, which caused mixing of fresh and sea water (Amaral-Zettler et al., 2008). Prochlorothrix adaptation to medium salinity also explains the discovery of the DQ450184 sequence (Piccini et al., 2006) in the brackish part of South American La Rocha Bay. Alternately, the emergence of Prochlorothrix 16S rDNA in brackish coastal waters and in the offshore Baltic Sea (Geiß et al., 2003) may be down to accidental invasions from the mainland. Thus, the crucial aspects of Prochlorothrix eco-physiology are (i) peak development during eutrophication outbursts; (ii) relatively low growth temperature; and (iii) stenohalinity with a preference for metastable salt gradients. Prochlorothrix, a mostly uncultured bacterium Although Prochlorothrix environmental DNA was detected by PCR and visualized by FISH, all attempts to isolate corresponding cultured strains were unsuccessful. A general failure to cultivate Prochlorothrix and other, as yet unrecognized, filamentous green cyanobacteria can be easily explained by ineffective enrichments. Presumably, co-culturing of Prochlorothrix, at its sub-threshold population density, with numerous and well adapted Leptolyngbya, Limnothrix, Oscillatoria, and Pseudanabaena would result in the elimination of this cyanobacterium. Besides, Prochlorothrix has only been effectively cultured in epibiotic

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symbiosis with heterotrophic helper bacteria (Morris et al., 2008). In contrast, axenization procedures resulted in growth decay (Schyns et al., 1997; Pinevich et al., 2012). Prochlorothrix, though principally represented by environmental DNA, is not necessarily a non-culturable bacterium. By definition, non-culturability means the dependence on a metastable natural environment that is hard to reproduce or even impossible to establish in artificial media (McDougald et al., 1998; Jørgensen & Gallardo, 1999; Gray & Head, 2001; Zinder & Salyers, 2005). In other words, non-culturable bacteria cannot withstand laboratory-born stresses. Nevertheless, tenacious efforts to domesticate presumed non-culturable bacteria are sometimes successful. For instance, Prochlorococcus marinus, which was long considered a non-culturable cyanobacterium (Partensky et al., 1999), has been successfully brought into culture (Scanlan & West, 2006; Zinser et al., 2009). Similar endeavors with Prochlorothrix should be continued.

Conclusions The Prochlorothrix 16S rDNA results corroborated by FISH analysis suggest a broader dispersal/diversity of the chlorophyll a/b-containing filamentous cyanobacteria than previously thought. However, we are forced to conclude that this green cyanobacterium is rare, both locally and globally. The low numbers of individuals coupled with a patchy distribution pattern confirm Prochlorothrix’s membership in the rare biosphere, and its tendency toward endemism. Moreover, rare PCRbased detection was not supported by strain isolation; therefore, with few exceptions, Prochlorothrix remains an uncultured bacterium. The scarcity of Prochlorothrix environmental DNA and corresponding cultured strains make this green cyanobacterium stand apart from other members of the Pseudanabaena group, which are usually abundant and easily cultured. Evidently, Prochlorothrix is an evolutionarily disadvantaged microorganism, although its eco-physiological background is unknown. For example, habitat restriction in Prochlorothrix does not necessarily arise from dependence on chlorophyll a/b-light-harvesting antennae, which do not absorb short-wavelength red light very well. The reason that this green cyanobacterium is so scarce may be because of other

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physiological pitfalls, in particular, the absence of effective osmotic adaptation. Acknowledgments The authors gratefully acknowledge N. Lee (Laboratory of Microbial Systems Ecology, Department of Microbiology, Technical University of Munich, Munich, Germany) for assistance with FISH protocol development. The authors are indebted to the team at Genomic Technologies and Cell Biology (Agricultural Microbiology Institute, Russian Academy of Sciences, St. Petersburg, Russia) for DNA sequencing, and to the team at Chromas (St. Petersburg State University) for technical support with light microscopy. The authors also thank Sindbad Karimi (St. Petersburg State University) who read the manuscript and made many valuable corrections and suggestions. The paper went through several rounds of peer review and the reviewers’ comments are gratefully acknowledged. This work was financed in part by St. Petersburg State University Research Project No. 1.37.88.2011.

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