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RESEARCH ARTICLE

A novel phylogenetic clade of picocyanobacteria from the Mazurian lakes (Poland) reects the early ontogeny of glacial lakes
1 Iwona Jasser1, Adriana Krolicka & Anna Karnkowska-Ishikawa2
1

Microbial Ecology Department, Institute of Botany, University of Warsaw, Warsaw, Poland; and 2Department of Plant Systematics and Geography, Institute of Botany, University of Warsaw, Warsaw, Poland

Correspondence: Iwona Jasser, Microbial Ecology Department, Institute of Botany, University of Warsaw, ul. Miecznikowa 1, 02096 Warsaw, Poland. Tel.: 148 22 554 1444; fax: 148 22 554 1413; e-mail: jasser.iwona@biol.uw.edu.pl Received 6 May 2010; revised 3 August 2010; accepted 3 October 2010. Final version published online 9 November 2010. DOI:10.1111/j.1574-6941.2010.00990.x Editor: Patricia Sobecky Keywords picocyanobacteria phylogeny; lake ontogeny; 16S rRNA gene; cpcBA.

Abstract The community of picocyanobacteria inhabiting the Great Mazurian Lakes system (comprising lakes ranging from mesotrophic to hypertrophic) is dominated by phycoerythrin-rich cells, which outnumber phycocyanin-rich cells, even in hypertrophic lakes. The genetic diversity and phylogeny of 43 strains of picocyanobacteria isolated from four Mazurian lakes were studied by analyzing the nucleotide sequences of the 16S rRNA gene and cpcBA-IGS operon. Phylogenetic analyses assigned some of the strains to several previously described clusters (Groups A, B, C, E and I) and revealed the existence of a novel clade, Group M (Mazurian), which exhibited a low level of similarity to the other clusters. Both phycocyanin and phycoerythrin picocyanobacteria were assigned to this clade based on an analysis of the 16S rRNA gene. The cpcBA sequence analysis assigned only phycocyanin strains to Group M, whereas the phycoerythrin strains from the M ribogroup were assigned to Groups B and E. We hypothesize that Group M originally contained only phycocyanin picocyanobacteria. The phycoerythrin found in strains belonging to ribogroup M seems to have been acquired through horizontal gene transfer as an adaptation to the changing environment early in the ontogeny of these glacial lakes.

MICROBIOLOGY ECOLOGY

Introduction
Picocyanobacteria, rst discovered three decades ago, inhabit various types of aquatic environment in terms of chemistry, trophic status and underwater light climate, and play a very important role as primary producers in both marine and freshwater ecosystems (Callieri & Stockner, 2002). Depending on the trophic status and light dominating the water column, different groups of picocyanobacteria prevail. Oligo- and mesotrophic clearwater lakes are usually dominated by orange-uorescing, phycoerythrin-rich picocyanobacteria, which efciently absorb the blue/green light prevailing in these types of waters. Red-uorescing, phycocyanin-rich picocyanobacteria, which optimally use red light, are dominant in more productive ecosystems (V or os et al., 1998). Historically, picocyanobacteria were classied into two genera: Synechococcus and Synechocystis (Waterbury et al., 1986; Andreoli et al., 1989; Ernst et al., 1995). Their discrimination was based on cell size, shape and division type. In the late 1990s, Kom arek and colleagues
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rek, 1996; Koma rek et al., 1999) proposed a new (Koma systematics of picocyanobacteria based on cytomorphological, biochemical and molecular features. According to this approach, all freshwater picocyanobacteria belonging to the so-called Synechococcus-type were divided into three genera: Synechococcus, Cyanobium and Cyanobacterium. Molecular techniques such as denaturing gradient gel electrophoresis analysis and the sequencing of amplied genes have created novel opportunities for the taxonomic and phylogenetic analysis of picoplanktonic autotrophs ja ` , 2004). These methods (Becker et al., 2002; Zeidner & Be have permitted in situ studies of community composition and the seasonal dynamics of picocyanobacteria without the need to isolate the studied organisms. The cultivation of picocyanobacterial strains has also received more attention following the introduction of new isolation methods (Crosbie et al., 2003b). Several recent studies have examined the diversity and phylogenetic relationships of picocyanobacteria inhabiting various environments. The results of 16S rRNA gene analyses, which depict the phylogenetic relationship
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between sequences, are considered too conservative to reveal recent differences between picocyanobacterial isolates. Thus, the analyses of less well-conserved genes, such as the phycocyanin cpcBA and phycoerythrin cpeBA operons and the internal transcribed spacer region between the 16S and 23S rRNA genes, have been carried out (Crosbie et al., 2003a; Ernst et al., 2003; Haverkamp et al., 2008). It has been suggested that these analyses may be used to track possible adaptation to the environment (Ernst et al., 2003; Six et al., 2007). The goal of this study was to characterize the genetic diversity and phylogeny of picocyanobacterial strains isolated from selected Mazurian lakes. The Mazurian region, located in north-eastern Poland, comprises about 2000 lakes of glacial origin. Several of them, including the two largest lakes in Poland, are interconnected, forming a waterway called the Great Mazurian Lakes system, which extends for about 100 km north to south. The lakes represent a gradient of trophic status from mesotrophy to hypertrophy, with numerical trophic state index (TSI) values ranging from 40 to 4 70 (Carlson, 1977), and a mean chlorophyll a content ranging & Siuda, 2006). There from 3.3 to 87.3 mg chl a L1 (Chrost are both shallow and deep lakes (mean depth between 1.1 and 14 m, maximum from 3 to 50 m), with most developing stable thermal stratication of the water column during the summer. In this environment, phycoerythrin picocyanobacteria prevail, even in the most eutrophic and hypertrophic lakes, accounting, on average, for 4 90% of picocyanobacterial abundance in less productive lakes and 5560% in hypertrophic ones. These results were obtained in a survey of 15 lakes of the Great Mazurian Lakes system and Lake Majcz Wielki in 2006, and were conrmed in more extensive studies of selected lakes conducted in 2007 (Jasser et al., 2010). The unusual predominance of the phycoerythrin phenotype in the productive lakes is intriguing, and poses the question as to how diverse the picocyanobacterial community in this system actually is. Another issue is how closely related (phylogenetically) are the phycoerythrin and phycocyanin phenotypes occurring in these lakes to known strains and clades? We addressed these questions by sequencing selected amplied molecular markers of isolates obtained from some of the lakes (Jasser et al., 2010).

The specic aims of our study were (1) to characterize the genetic diversity of isolated picocyanobacterial strains by nucleotide sequence analysis of 16S rRNA and cpcBA-IGS genes and (2) to describe and compare the phylogenetic relationships of these strains with already known groups.

Materials and methods


Study site and methods
A total of 43 monoclonal picocyanobacterial isolates (13 phycoerythrin and 30 phycocyanin) obtained using two isolation methods (Jasser et al., 2010) were used for the genetic and phylogenetic analyses. The strains were isolated from four Mazurian lakes in north-eastern Poland (541N and 221E) in 2006. Three of the lakes are interconnected and situated within the Great Mazurian Lakes system, whereas the fourth, Lake Majcz Wielki, is located in the same area, but connected to the system by a river. The studied lakes exemplify various kinds of glacial lake. Lake Mikoajskie and niardwy, Lake Bedany represent gully lakes, whereas Lake S the largest lake in Poland, and Lake Majcz Wielki represent kettle-type glacial lakes. The lakes were formed by ice sheets in the late Pleistocene during the Pomeranian phase of Vistulian glaciations (Bajkiewicz-Grabowska, 1989; Wacnik, 2009). At present, two of the lakes are relatively low producing (mesotrophic Lake Majcz Wielki and meso-eutrophic niardwy), whereas the other two are eutrophic (Lake Lake S Mikoajskie and Lake Bedany). All four lakes are characterized by low background water turbidity and the predominance of phycoerythrin-rich picocyanobacteria (Table 1). Both phycoerythrin and phycocyanin strains were isolated from each of the lakes; however, most of the phycoerythrin isolates were obtained from the eutrophic lakes, and most of the phycocyanin isolates came from the mesotrophic lake. The picocyanobacterial isolates were analyzed microscopically and molecularly to verify their taxonomic purity (Jasser et al., 2010). All strains were cultivated in BG11 medium in a plant growth chamber at a constant temperature of 20 1C. Cool white (4000 K, Osram Lumilux, Munich, Germany) uorescent tubes provided light at 16 mmol photons m2 s1 in a 14/10 h day/night cycle.

Table 1. Basic physical, chemical and biological characteristics of the lakes from which picocyanobacteria were isolated in 2006 Lakes Majcz Wielki niardwy S Bedany Mikoajskie Area (ha) 160 11 340 941 498 Mean depth (m) 6.0 5.8 10.0 11.2 Maximum depth (m) 16.5 23.4 46.0 25.9 Chlorophyll a (mg L1) 5.1 14.0 27.2 28.1 SD (m) 4.0 2.2 1.6 1.6 Carlsons index (TSI) 43.3 52.5 58.1 58.3 KPAR 0.61 0.72 1.06 1.04 KBG (484) (m1) 0.69 0.53 0.66 0.59 PE (%) 95 88 88 87

KPAR light attenuation coefcient estimated from vertical light proles (PAR range, 400700 nm). KBG (484) background turbidity at 484 nm measured according to the method of Stomp et al. (2007).

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Pigment analysis
The in vivo absorbance spectra of the pigments of whole cells were analyzed using a Hitachi U-1800 spectrophotometer (Tokyo, Japan). The spectra were recorded between 380 and 750 nm at 1 nm intervals with 4 nm slits.

DNA extraction and PCR amplification


DNA was extracted from 2 mL cultures of the isolated strains using a commercial kit (EURx, Gdansk, Poland) designed for the extraction of genomic DNA from bacteria (Jasser et al., 2010). Amplication of the cpcBA-IGS region of the phycocyanin operon (Robertson et al., 2001) for direct sequencing was performed using the DNA from 39 isolates with the specic primers cpcBF(UFP) and cpcAR(URP). A fragment of 16S rRNA gene was amplied from the DNA of 43 isolates using the primers 16S5_F (Scheldman et al., ` re et al., 2000). PCR products were 1999) and B23S5_R (Lepe puried using a kit (EURx) and then directly sequenced in both directions with the original PCR primers using an ABI (3130xl) capillary sequencer.

addition for 10 replicates, as well as with TBR branch swapping and MULTREES on. Bootstrap support for specic nodes (Felsenstein, 1985) was estimated with default options using 100 replications. Bayesian analyses (BA) were performed and the values of their model parameters were estimated using MRBAYES 3.1.2 software (Ronquist & Huelsenbeck, 2003). Two independent analyses were run with three heated and one cold chain (temperature parameter 0.1) for 3 000 000 generations, discarding the rst 25% of trees. Models of sequence evolution for the ML, NJ and BA methods, and their parameter values for the ML and NJ methods, were estimated using MODELTEST 3.7 (Posada & Crandall, 1998). For 16S rRNA gene alignment, the TrN1I1G model and a general time-reversible (GTR1I1G) model were used. For cpcBA-IGS alignment, a GTR1I1G model was used for the rst codon, a SYM1I1G model for the second and a TVM1G model for the third. The sequences from strains PCC 6301, PCC 7002 and PCC 7942 were used to root the trees (Ernst et al., 2003; Haverkamp et al., 2008).

Sequence accession numbers, alignments and phylogenetic analyses


The 16S rRNA gene and cpcBA-IGS nucleotide sequences of picocyanobacterial isolates from the Mazurian lakes were submitted to the DDBJ/EMBL/GenBank databases with the following accession numbers: FJ63765FJ763789, GQ130142GQ130156 and FJ763795FJ763832. The GenBank accession numbers for all the DNA sequences reported here and used for phylogenetic analyses are shown in Supporting Information, Table S1. Sequence alignments produced using CLUSTALX 1.83 (Thompson et al., 1997) with default options were manually edited. The cpcBA-IGS alignment was edited according to the primary structure of the protein using the GENETIC DATA ENVIRONMENT (2.3) software (Smith et al., 1994). Areas that could not be aligned unambiguously were excluded from further analysis. For phylogenetic analyses, a 16S rRNA gene dataset of 1341 characters in the alignment of 85 sequences and a cpcBA-IGS dataset of 388 characters in the alignment of 68 sequences were generated. A partitioned dataset was used for the cpcBA-IGS alignment, with three partitions (three codon positions). Neighbor joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) analyses were performed using PAUP version 4.0b10 for Macintosh OS X (Swofford, 2002). To identify the best tree in MP analysis, the heuristic search option was used with MULTREES, tree-bisection-reconnection (TBR) branch swapping, with ACCTRAN optimization and random addition, for 10 replicates. The single ML tree was obtained by a heuristic search using a random stepwise
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Results
Pigment analyses
The pigment patterns of the 43 studied strains represented two main phycobilin pigment types: I and II. Type I strains (30 isolates) carried only phycocyanin and appeared blue/ green in culture, with a maximum absorbance peak at around 630635 nm. All phycoerythrin strains exhibited a type II pigment absorption pattern, with maximum absorbance at 570 nm. No maxima between 490 and 495 nm (the phycourobilin peak) were detected for any of the strains.

The 16S rRNA gene region


The molecular identication of the 43 strains by 16S rRNA gene nucleotide sequence analysis revealed several clusters of picocyanobacteria. The genetic distance between individual strains was up to 3.9%, whereas the maximum genetic distance between all analyzed strains was 7.1%. Further analysis demonstrated that the phycoerythrin and phycocyanin cyanobacteria isolated from the studied lakes belonged to four previously described clades and one novel clade (Fig. 1). The existence of this new clade, named the Mazurian clade (M), was conrmed in four different phylogenetic analyses: BA = 0.93, ML = 71, NJ = 62 and MP = 61. Both phycoerythrin and phycocyanin picocyanobacteria, isolated from the eutrophic Lake Mikoajskie and the mesotrophic Lake Majcz Wielki, respectively, were assigned to this clade. The similarity between the 16S rRNA gene nucleotide sequences within this clade varied between 99.1% and 100%. The rest of the isolated phycoerythrin
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Fig. 1. Phylogenetic tree of the 16S rRNA gene nucleotide sequences obtained by Bayesian inference (model: GTR1I1G). Terminal branches display strain names and GenBank accession numbers (sequences determined in this study are shown in bold) and pigment group (, phycoerythrin rich; , phycocyanin rich). Numbers at the nodes show posterior probabilities ( 4 0.75, rst number) of the tree bipartitions, as well as the bootstrap values obtained by ML ( 4 50%, second number) and NJ ( 4 50%, third number) analyses (model: GTR1I1G), and MP analysis ( 4 50%, fourth number). Clades not present in particular analyses or without substantial support are indicated by .

picocyanobacteria clustered, with a high bootstrap support and posterior probability, with Group B subalpine cluster I and Group E. The strains assigned to Group B came from three of the studied lakes (mesotrophic, meso-eutrophic and eutrophic) and shared high sequence similarity (up to 99.8%) with Synechococcus rubescens (SAG 3.81) and isolate MW 10#1 from Lake Mondsee in Austria. One phycoery c

thrin isolate, BE0807D, could be assigned to Group E. This strain was isolated from the second eutrophic lake, Bedany, and the sequence similarity to the strain PS 717 from Lake Biwa was 99.5%. The majority of the remaining phycocyanin picocyanobacteria were found to convene with Group A, the Cyanobium gracile cluster. Group A was supported by only three
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analytical methods (BA = 0.83, NJ = 78, MP = 80), and the 16S rRNA gene nucleotide sequences exhibited a similarity of between 98.4% and 100%. The strains grouped in this cluster were obtained from all four sampled lakes. A few other phycocyanin isolates, originating from the eutrophic Lake Bedany, exhibited high sequence similarity (up to 98.6%) to strains JJ27STR (GenBank accession number: AM710383) and JJM10D4 (GenBank accession number: AM710359) obtained from lakes and reservoirs in the Czech Republic. This group was tentatively named Group Cz. The afliation of the isolates from Lake Bedany to this cluster was robust, as conrmed using three methods (BA = 0.96, NJ = 59, MP = 90). The position of one of the isolated phycocyanin strains, MA0607K, was not resolved (Fig. 1).

The phycocyanin operon


Comparison of the cpcBA operon (IGS excluded) sequences of 39 of the Mazurian strains and those available in GenBank showed that the isolates were clustered according to pigment phenotype. The isolated picocyanobacteria exhibited genetic distances between strains of up to 23.7%, whereas the maximum genetic distance between all analyzed strains was 29.6%. The cpcBA phylogenetic analysis (Fig. 2) revealed that in most cases, the isolates were assigned to the same clusters as those determined by the 16S rRNA gene analysis, i.e. Groups A, B, E and the newly established Group M. A few strains, however, fell into different clades. Specically, 10 isolates, including nine from the mesotrophic Lake Majcz

Fig. 2. Phylogenetic tree of the cpcBA-IGS sequences obtained by Bayesian inference (models for partitions are listed in Materials and methods). Terminal branches display strain names and GenBank accession numbers (sequences determined in this study are shown in bold), pigment group (, phycoerythrin; , phycocyanin) and IGS length. Numbers at the nodes show posterior probabilities ( 4 0.75, rst number) of the tree bipartitions, as well as the bootstrap values obtained by ML ( 4 50%, second number), NJ ( 4 50%, third number) and MP ( 4 50%, fourth number) analyses. Clades not present in particular analyses or without substantial support are indicated by .

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Wielki and one from the eutrophic Lake Mikoajskie, were grouped in the Mazurian cluster (ML = 61, NJ = 99, MP = 100). According to the phycocyanin operon analysis all the isolates from Group M were phycocyanin, whereas phycoerythrin strains were clustered within other groups. Interestingly, in the GenBank databases, we identied one isolate grouping with our phycocyanin strains in Group M. It was a red-colored strain, PS 724 (AF223436), from the eutrophic Lake Tsukui in Japan (Robertson et al., 2001), which shared 96.697.2% similarity to our isolates. In the cpcBA phylogeny, most of the remaining phycocyanin isolates fell into Group A, as in the analysis of the 16S rRNA gene. However, in the cpcBA-derived tree, Group A received support from only two of the analyses (NJ = 92, MP = 82). A few phycocyanin isolates were assigned to groups different from those determined by analysis of the 16S rRNA gene (Figs 1 and 2). Among them were strains from Lake Bedany, which were assigned to the Czech ribogroup (Group Cz), and, according to the cpcBA-inferred tree, formed a strongly supported clade. This clade seemed to be closely related to Group C, even though the posterior probability in the BA was only 0.79. The position of phycocyanin isolate MA0607K remained unresolved. In the cpcBA phylogeny, the phycoerythrin picocyanobacteria that had been grouped in clade M following analysis of the 16S rRNA gene were clustered, with strong support within Groups B and E. Group B was formed by two sister clades: one including isolates BO 8807 from Lake Constance and PS 714 from Lough Neagh in Northern Ireland and the second containing MH 305 and MH 307 from Mondsee in Austria. Mazurian strains assigned to ribogroup B belonged to the rst sister clade. Two of our isolates fell within the second sister clade: one from Lake Mikoajskie, MI0607I, which clustered in Group M according to the 16S rRNA gene-inferred tree, and one from Lake Bedany, BE0807B (sequence not present in the 16S rRNA gene analysis). In most cases, the IGS length was stable within the phylogenetic clusters, especially those with high bootstrap support, and varied between 38 and 99 bp. The exception was Group A, with strains generally characterized by an IGS length of 44 or 45 bp, but where one strain, PCC 7009, had a length of 60 bp (Fig. 2).

Discussion
The predominance of phycoerythrin-rich cells in the community of picocyanobacteria in the Mazurian lakes and Lake Majcz Wielki (Jasser et al., 2010) seems to contradict both the widely accepted rule concerning the composition of picocyanobacteria communities in waters of different trophic levels and the predominance of phycocyanin in nutrient-rich waters (V or os et al., 1998; Callieri & Stockner, 2002). It was established previously that phycoerythrin
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cyanobacteria generally predominate in oligo- and mesotrophic deep lakes, in which blue and green light dominate the underwater light climate, whereas phycocyanin strains do so in shallow, eutrophic and colored lakes, in which red light penetrates the deepest (V or os et al., 1998). In this study, the gradual increase in the proportion of phycocyanin-rich cells in the picocyanobacteria community was found to be paralleled by an increase in trophic status accompanied by a change in the green to red light ratio. These changes in the underwater light spectrum were later attributed to the background turbidity (Stomp et al., 2007). Thus, according to a model proposed by Stomp et al. (2007), phycoerythrin-rich cells dominate in clearwater, low producing lakes, whereas phycocyanin-rich ones prevail in turbid, eutrophic lakes. In waters of intermediate turbidity, the coexistence of phycoerythrin and phycocyanin strains is expected, with both phenotypes exploiting various light niches. The Mazurian lakes are characterized by high chlorophyll a content and high TSI values, placing them within the mesotrophic and highly eutrophic lakes. On the other hand, background turbidity values [KBG (484)] calculated according to the algorithm of Stomp et al. (2007) are low, being closer to the level of prealpine lakes than productive, lowland lakes, which explains the predominance of phycoerythrin strains in the picocyanobacteria community of these lakes (Jasser et al., 2010). However, the diversity and phylogeny of the picocyanobacteria thriving in this unique environment have not been recognized until now. The analysis of phycoerythrin and phycocyanin picocyanobacteria isolated from selected Mazurian lakes revealed the considerable phenotypic and genetic diversity of these organisms. Although more phycocyanin (30) than phycoerythrin (13) strains were isolated from the Mazurian lakes, this cannot be used to draw any conclusions on the ecological role and importance of these picocyanobacterial strains in these ecosystems. Indeed, the higher number of phycocyanin strains taken into culture is at odds with the actual predominance of phycoerythrin in these lakes. This anomaly is due to the fact that opportunistic species may be cultivated when isolating cyanobacteria from the environment, so that the observed diversity might not reect the authentic relationships between strains (Everroad & Wood, 2006). However, despite the limited number of isolates, our analyses have provided some intriguing results on the phenotypic and genetic diversity of Mazurian lake picocyanobacteria. All phycoerythrin and phycocyanin isolates had similar spectral phenotypes within the given pigment phenotypic groups. None of the strains contained phycourobilin, which is understandable, given the trophic status and morphometry of the studied lakes, nor did they exhibit chromatic adaptation in green and red light (data not shown). This allowed us to assign the strains to cyanobacterial Group I, which are incapable of altering their
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absorbance characteristics in response to light changes (Bhaya et al., 2000). A genetic distance of up to 3.9% between the isolated picocyanobacteria was determined by analysis of their 16S rRNA gene nucleotide sequences. This is compared with 4.9% between all nonmarine sequences used in our analysis, which is in agreement with the value found by Crosbie et al. (2003a). The nucleotide sequences encoding the phycocyanin operon showed considerably more variation (up to 26%). These two complementary phylogenetic analyses established that some of the picocyanobacteria isolated from the Mazurian lakes exhibited a cosmopolitan diversity, while others appeared unique, occurring neither in cultures nor in environmental clone libraries. The phylogenetic trees inferred from the 16S rRNA gene nucleotide sequences allowed us to assign most strains to Groups A, B, D and E, representing some of the clades described previously by Crosbie et al. (2003a) and Ernst et al. (2003). A few of the phycocyanin isolates, originating from one of the eutrophic lakes, were closely related to phycocyanin isolates from Czech reservoirs of various trophic levels, which we provisionally called Group Cz. However, there was relatively low support for Group A, suggesting that this group is not stable. All phylogenetic analyses conrmed the existence of one new clade, named Group M (Mazurian). This cluster included phycoerythrin and phycocyanin phenotypes originating from two lakes of different trophic status (the mesotrophic Majcz Wielki and the eutrophic Mikoajskie) and was characterized by various levels of sequence similarity. The isolates did not cluster with any other known group or solitary strains or sequences. The cpcBA phylogenetic analysis, reecting the variability of phycobilin-encoding genes, revealed that Group M contained only strains of the phycocyanin phenotype. The phycoerythrin isolates belonging to ribogroup M clustered with Groups B and E and did not form any separate, new clade. Unexpectedly, this analysis assigned a phycoerythrin strain, PS 724, from the eutrophic Lake Tsukui in Japan to Group M. Crosbie et al. (2003a) showed that this strain had an ambiguous, unresolved position, branching with no support at the bottom of Group D containing phycocyanin strains from Japanese lakes. In our analysis, the red-colored PS 724 again clustered only with green isolates, this time originating from the Mazurian lakes. Its IGS length was 97 bp, which matched that of the other members of Group M. Whether the position of this strain is the outcome of lateral gene transfer, as suggested for marine picocyanobacteria by Six et al. (2007), or proof of a close phylogenetic relationship, is not known. The analysis of the phycocyanin operon also highlighted the strong correlation between the length of the IGS of the isolated strains and the phylogenetic groups into which they fall (Jasser et al., 2010). Such a relationship has been
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reported previously (Robertson et al., 2001; Crosbie et al., 2003a). However, according to our results, only wellsupported clades are characterized by a stable IGS length. On the contrary, clades with lower support, such as the C. gracile cluster, group strains with a variable IGS length. This strengthens the notion, based on low support in the analysis of 16S rRNA gene, that the Group A is not well established. Although the topology of the two phylogenetic trees was similar, with most of the strains falling into the same groups, we have not performed a closer comparison by creating concatenated nucleotide sequences of the 16S rRNA gene and cpcBA operon as proposed by Haverkamp et al. (2009). In our view, using a combined tree to reveal any congruency between the two phylogenies does not seem justied, because they concern genes evolving differently over dissimilar timescales (Six et al., 2007). The presence and absence of phycoerythrin strains within clusters of closely related picocyanobacteria and the consequent incongruence between the phylogenetic trees derived from the 16S rRNA gene and cpcBA analyses has been discussed previously (Crosbie et al., 2003a; Ernst et al., 2003; Haverkamp et al., 2008). The cpcBA phylogenetic analysis, which allowed us to examine the pigment-based diversity of isolated strains, revealed that Group M could be further characterized as a phycocyanin-rich group of picocyanobacteria. Ernst et al. (2003) hypothesized that the presence of the phycoerythrin and phycocyanin phenotypes in closely related strains suggests that all the ancestral strains of these picocyanobacteria contained both phycocyanin and phycoerythrin, and that some strains had lost the ability to produce phycoerythrin during recent evolution. However, they also proposed the opposite, i.e. that some strains could have acquired this ability during independent adaptive radiations by mutation or horizontal gene transfer. In the case of our isolates belonging to Group M, we hypothesize that these strains were originally green, phycocyanin-rich picocyanobacteria, characterized by a stable and much longer IGS than all other isolates, and that some members of this group could have acquired the phycoerythrin-encoding genes by horizontal gene transfer. Such an evolutionary scenario has been proposed by Six et al. (2007) for marine picocyanobacteria. These authors stated that although genes encoding a phycobilisome core evolve together with the core genome (represented by the 16S rRNA gene), the rods evolve independently and show rapid adaptation to light niches. They suggested that the mechanism behind this process is the lateral transfer of phycobilisome rod genes between Synechococcus strains via cyanophages or by natural transformation. The recent analysis of 19 complete genomes of Synechococcus and Prochlorococcus marinus and nine genomes of cyanophages by Zhaxybayeva et al. (2009) has provided support for the
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idea that horizontal (lateral) gene transfer is important in shaping the diversity of picocyanobacterial genomes. Our hypothesis that some phycocyanin strains from ribogroup M could have acquired phycoerythrin is supported by two arguments. The rst is related to the glacial origin of the Mazurian lakes. Although glacial lakes are thought to progress through their ontogeny according to Lindemans (1942) evolutionary sequence from oligotrophy via mesotrophy to eutrophy, it is often overlooked that at their origin, such lakes went through a phase of high organic matter content and high productivity (Whiteside, 1983). Numerous paleolimnological studies suggest that early in their histories, glacial lakes received a high input of suspended inorganic and organic matter from the melting ice cap and/or due to erosion of the sparsely vegetated drainage area (Sarmaja-Korjonen et al., 2006; Szeroczynska et al., 2007). Thus, in their early ontogenic stages, the lakes are believed to have undergone periods characterized by high productivity and poor light conditions, as reected by the high content of organic matter in their sediments and the presence of diatom species that are adapted to low light conditions in their sediment remnants (Bigler et al., 2003). Although we do not have paleolimnological data for the studied lakes, a high organic carbon content and productivity in the early Holocene, and a subsequent decrease in productivity have been described for one of the Mazurian lakes, Lake Kortowskie (Rybak & Rybak, 1985). Thus, assuming the occurrence of high trophic status and poor light conditions at the origin of the studied Mazurian lakes, we propose that the picocyanobacteria forming Group M might have evolved as strains containing only phycocyanin in their phycobilisomes. Later in the lakes ontogeny during oligotrophication, these strains could have acquired the phycoerythrin genes from phycoerythrin strains of Synechococcus. The phycoerythrin, in turn, might have allowed them to adapt to a less productive environment with better light penetration. This hypothesis is further supported by the fact that in the cpcBA phylogenetic analysis, the phycoerythrin strains belonging to the M ribogroup did not form a separate new clade, but were assigned to two different cosmopolitan phycoerythrin clades: Groups B and E, from which they could acquire genes encoding phycoerythrin. In this respect, the outcome of both phylogenetic analyses could support the concept of adaptive radiation (Ernst et al., 2003; Six et al., 2007). The absence in cultures and in environmental clone libraries of nucleotide sequences identical to the 16S rRNA gene clustering with Group M suggests the existence of endemic species in the Mazurian lakes and, as a consequence, the possibility of geographic boundaries for picocyanobacteria. Challenging Finlays (2002) widely accepted hypothesis on the cosmopolitan distribution of microbial organisms, Ernst et al. (2003) suggested the existence of
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geographical boundaries for such organisms. Unique nucleotide sequences, not closely related to cosmopolitan Synechococcus clades, were found by Ivanikova et al. (2007) in Lake Superior and by Haverkamp et al. (2009) in the Baltic Sea. Following their isolation of picocyanobacteria forming two new phylogenetic clusters from the East China Sea and East Sea, Choi & Noh (2009) proposed that despite the generally ubiquitous distribution of Synechococcus species, unseen boundaries present even in oceans enable the development of endemic species of picocyanobacteria. The problem of endemism in prokaryotic populations was addressed by Whitaker et al. (2003) and Whitaker (2006). Aided by high-resolution genetic and genomic tools, these authors found proof of allopatric speciation of microorganisms and endemism in geographically isolated locations. In addition, Pommier et al. (2007) identied strong evidence of endemism in bacterial communities in the marine environment. Thus, it seems that the Mazurian lakes, which at their origin were characterized by isolation in space and time, and, which, as glacial lakes, are fed mainly by underground water and precipitation (Bajkiewicz-Grabowska, 2008), could at some point in their history have provided conditions for the development and maintenance of endemic ecotypes adapted to the local environment. In conclusion, the results of the present study and a previous study (Jasser et al., 2010) have revealed the high diversity of the picocyanobacterial communities in the Mazurian lakes studied, which comprise cosmopolitan strains and also apparently unique and, as it seems now, possibly endemic genotypes. The phylogeny and genetic and phenotypic diversity of the isolated picocyanobacteria might reect lake ontogeny, as well as the adaptation of Synechococcus to the changing environment.

Acknowledgements
This research was supported by the Polish Ministry of Science and Higher Education (grants N304 015 31/0535 and N304 016 32/0959). We thank the Microbial Ecology Department team for help in collecting samples, and Anna Lajos V Hillbricht-Ilkowska, Ryszard Chrost, or os and two anonymous referees for their critical comments, which helped improve the manuscript.

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Supporting Information
Additional Supporting Information may be found in the online version of this article: Table S1. The GenBank accession numbers for all the 16S rRNA gene and cpcBA-IGS nucleotide sequences used in the phylogenetic analyses (sequences determined in this study are shown in bold), together with pigment group (phycoerythrin-rich, phycocyanin-rich or ND not determined), strain name, clade assignment following 16S rRNA gene and cpcBA-IGS analyses (U ungrouped, SA II subalpine cluster II), and isolation details (country/continent, ecosystem/lake). Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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