INTRODUCTION

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The current species diversity of the cyanobacterial radiation comprises several lineages of phyletically (morphologically and genetically) related organisms (26). An intriguing question is whether this reflects stability of cyanobacterial characters or whether the phyletic similarities originate from relatively recent common ancestors. Analyses of precambrian microfossils (superficially, hardly distinguishable from recent cyanobacteria) support the view of retention of cyanobacterial properties (1, 11, 28). However, on the basis of molecular data, a 2-billion-year-old mutual ancestor for prokaryotes has been suggested (5), implying that the similarities between the earliest records of cyanobacteria and present-day species do not reflect homologies but rather indicate analogies. In this context, the phyletically clustered groups may reflect a relatively recent divergence of the modern species.

In this work we have addressed, by molecular evolutionary studies, the mechanisms responsible for conserving or homogenizing phyletical characters within groups of cyanobacteria. We investigated the evolutionary rates and origins for two genomic regions, by analyzing strains both within and among groups of phyletically related organisms. This was done by comparative analysis of the small-subunit rRNA-encoding DNA (16S rDNA), which is conserved by the RNA function (37), and the rbcLX region with both conserved and less conserved elements. The rbcLX region contains an intergenic spacer (with no identified functional units), the gene rbcX with a possible chaperonin-like function (18), and the 3' end of rbcL (encoding the highly conserved D-ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit [LSU]) (23). We analyzed a data set consisting of four phyletically clustered cyanobacterial strain categories, as inferred from microscopic observations and 16S rDNA analysis (26, 31). The data set includes the Microcystis category (16 strains), consisting of unicellular organisms, the Tychonema (6 strains) and Planktothrix (10 strains) categories, which contain multicellular, filamentous organisms, and the Nostoc category (12 strains), which includes both morphologically and genetically slightly divergent organisms (26, 34). The strains in this last category share among other features the ability of cellular differentiation to produce heterocysts with nitrogenase activity.

Our sequence data suggest an evolutionary model involving several events of gene transfer between phyletically closely related organisms but not between less related organisms. We propose that this gene transfer has led to the observed sequence homogeneity for the groups of related organisms and that exchange of genetic material stabilizes the function and structure of proteins encoded by neutrally evolving genes. Our gene transfer model may explain the similarity between the fossil and the recent species.

	MATERIALS AND METHODS

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Sample preparation. The organisms investigated are listed in Table 1. The majority of the strains were isolated at the Norwegian Institute for Water Research (33). The strains were grown in laboratory cultures as previously described by Rudi et al. (26).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structures of the 16S rDNA (A) and rbcLX (B) regions. The primers used for PCR amplification and sequencing are indicated with arrows. Two of the strains (Nostoc sp. strain NIVA-CYA 308 and N. commune) contain an approximately 155-nt insertion between rbcX and rbcS.

Phylogenetic reconstruction. The sequences were aligned either manually or by using the multisequence alignment algorithm PILEUP (14) in the Wisconsin package version 8.1 for UNIX (Genetic Computer Group, Madison, Wis.). Phylogenetic trees were constructed with the neighbor-joining method (27) or based on parsimony analysis (9), using the Phylogeny Inference Package (PHYLIP; version 3.5) developed by J. Felsenstein (Department of Genetics, University of Washington, Seattle), and the package Phylogenetic Analysis Using Parsimony (PAUP; version 3.1.1) developed by D. L. Swofford (Illinois Natural History Survey, Champaign). The Kimura two-parameter model with a transversion:transition weight of 2:1 (16) was applied to compute the nucleotide distance matrix, and Dayhoff's PAM 001 matrix (4) was used for calculating the amino acid distance matrix for the neighbor-joining analysis. Bootstrap analysis (8) constituted the basis for inferring confidence of the branch points in the phylogenetic trees. Consensus trees were constructed from 500 bootstrap replicates.

Phyletic structure of the nucleotide alignments. The tree length distribution based on the informative sites for the phylogenetic trees was analyzed by use of either the exhaustive (for 11 taxa or less) or the random tree (for more than 11 taxa) option in the PAUP package. We used the third moment statistics (g1) implemented in this package to determine the randomization value of the informative sites. This value was used to evaluate the phyletic information, according to the criteria given by Hillis and Huelsenbeck (15).

Models for estimating nucleotide substitution distributions. For nucleotide alignments with randomized phyletic structures, the observed frequency distributions of nucleotide substitutions in the alignments were compared to the expected distribution from alignments randomized by independent substitutions and to the distribution given that each of the informative sites in the alignment is due to single substitutions (phyletically informative substitutions).

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	RESULTS

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We have sequenced 16S rDNA and rbcLX regions from a total of 44 strains belonging to the cyanobacterial categories Microcystis, Planktothrix, Tychonema, and Nostoc (Fig. 1). This sequence information was used to construct phylogenetic trees and for the comparison of the evolutionary patterns of the regions analyzed.

16S rDNA. In the 16S rDNA region (Fig. 1A), the sequence divergence between species belonging to different categories varies from 0.1 to 0.15 nucleotide substitutions per position. For the most divergent strains in the Nostoc category, the sequence divergence is about 0.05 nucleotide substitutions per position. This category, however, consists of three genetically distinct lineagesNostoc lineage I, II, and III. Strains NIVA-CYA 142, 266/1, 83/1, 103, 267/4, 281/1 belong to Nostoc lineage I, strains Nostoc commune, Nostoc flagelliforme, and NIVA-CYA 124, 194, and 308 belong to Nostoc lineage II, and strain NIVA-CYA 246 belongs to Nostoc lineage III. There are only 0.005 nucleotide substitutions per position among the most divergent strains within both the Microcystis and the Planktothrix category, while the strains in the Tychonema category are all monomorphic.

rbcL and rbcX genes. The investigated strains show an overall homogeneous structure of the rbcLX region (Fig. 1B). The rbcL gene is relatively conserved (0.2 nucleotide substitutions per position), while the intergenic region as well as the gene rbcX are variable, with 0.3 nucleotide substitutions per position when species belonging to different categories are compared. There are no significant differences in codon usage for the strains within each clustered group. The sequence divergence for the rbcL and the rbcX gene is only 0.002 nucleotide substitutions per position within the Tychonema and Planktothrix categories. There are about 0.01 nucleotide substitutions per position between the most divergent isolates in the Microcystis category. The Nostoc category shows the same three genetically clustered lineages as for 16S rDNA, with approximately 0.05 nucleotide substitutions per position for the most divergent strains within each lineage. On the other hand, for species belonging to different lineages, the sequence divergence is on average 0.1 and 0.2 nucleotide substitutions per position for rbcL and rbcX, respectively.

Evolutionary rates for the 16S rDNA and rbcLX regions. The evolutionary rates for the 16S rDNA and rbcLX regions were calculated within and between Nostoc lineages I and II. Within Nostoc lineages I and II, the sequence divergence in the rbcX region is only 2 to 2.5 times that of the 16S rDNA divergence. However, the sequence divergence between lineages I and II displays a 4.5- to 35-fold-higher difference in the rbcX region compared to 16S rDNA (Fig. 2). The spacer between rbcL and rbcX for Nostoc lineage I and II has a variation in length from 70 to 132 bp. Within the lineages, the sequence divergence is in the same range as for rbcX, both for the spacer and for the rbcL gene, while between the lineages the divergence is on average 1.5 and 0.3 times the variation in rbcX for the spacer and rbcL, respectively (results not shown). For the rbcL gene, only 0 to 5% of the mutations are replacement substitutions. This indicates that most of the replacement substitutions are eliminated by selection. The rbcX gene, with 20 to 50% replacement substitutions, is also constrained by function, but more mutations are tolerated here. The spacer region, however, displaying the highest evolutionary rate, has probably a low functional constraint.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   rbcX (A) and rDNA (B) distance trees for Nostoc lineages I, II, and III. The trees were built with the neighbor-joining method (27) from the PHYLIP package, using distance matrices derived from the Kimura two-parameter model (16). Numbers at the nodes indicate the percentage of 500 bootstrap trees (8) in which the cluster descending from the node was found. Abbreviations for strains used: AL 1, Anabaena lemmermannii NIVA-CYA 266/1; AL 2, A. lemmermannii NIVA-CYA 83/1; AL 3, A. lemmermannii NIVA-CYA 281/1; AS, Anabaena sp. strain NIVA-CYA 267/4; AF, Aphanizonenon flos-aquae NIVA-CYA 142; AG, Aphanizomenon gracile NIVA-CYA 103; NS 1, Nostoc sp. strain NIVA-CYA 124; NS 2, Nostoc sp. strain NIVA-CYA 194; NS 3, Nostoc sp. strain NIVA-CYA 308; NS 4, Nostoc sp. strain NIVA-CYA 246; NC, N. commune; NF, N. flagelliforme.

Comparison of the 16S rDNA and rbcLX phylogenies for the genetically distinct clusters of strains. For this comparison, we used the translated rbcL and rbcX sequences, because the amino acid sequence displays suitable polymorphism for phyletic reconstruction (and the nucleotide sequence has too high variation). Comparison of the phylogeny based on 16S rDNA and the translated (amino acid) sequence of rbcX for the six genetically distinct clusters of strains defined in this work shows the same clustering of strains (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Comparison of distance trees for the rbcX amino acid sequence (A) and 16S rDNA nucleotide sequence (B) for the six evolutionary distinct clusters described in this work. The strains used in the phylogenetic analysis were selected to include all of the genotypes. The distance matrix for the amino acid sequences was calculated by using Dayhoff's PAM 001 matrix (4), while the 16S rDNA distance matrix was calculated with the Kimura two-parameter model (16). The trees were built by using the neighbor-joining algorithm (27) from the PHYLIP package. The numbering at nodes indicates the percentage of that branch in 500 bootstrap trees (8).

Comparison of the 16S rDNA and rbcLX phylogenies for the genetically clustered strains. There is low accordance, using both the neighbor-joining method (Fig. 2) and the maximum parsimony analysis (Fig. 4), between the 16S rDNA phylogeny and the rbcLX phylogeny for isolates within Nostoc lineages I and II. The only Nostoc strains which are clustered in both the 16S rDNA and rbcLX trees are Anabaena flos-aquae NIVA-CYA 83/1 (AL 2) and 266/1 (AL 1) (Fig. 2 and 4). The clustering of organisms is highly congruent when results from the two phylogenetic methods are compared (compare Fig. 2 and 4). This is exemplified by the strains Aphanizomenon flos-aquae NIVA-CYA 142 (AF) and Aphanizomenon gracile NIVA-CYA 103 (AG), for which 16S rDNAs are grouped with 84% bootstrap support by the neighbor-joining method (Fig. 2B) and with 96% bootstrap support by the parsimony analysis (90% identity for the informative sites [Fig. 2B and 4B]). In contrast to rDNA, the rbcLX locus reveals a phylogeny where the strains are relatively distantly related (43% identity for the informative sites [Fig. 2A and 4A]). This suggests that the rDNA and rbcLX loci do not have the same evolutionary pattern. The only difference between the parsimony and neighbor-joining analyses is the location of Nostoc sp. strain NIVA-CYA 194 (NS 2) for the rbcLX region (Fig. 2A and 4A). However, when only the strains in Nostoc lineage II are considered in the neighbor-joining analysis, the branching pattern is congruent with that of the parsimony analysis (results not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Maximum parsimony tree for Nostoc lineages I and II, displaying the rbcLX locus (A) and the 16S rDNA locus (B). Congruent trees were built both with the DNAPARS program from the software package PHYLIP and by the exhaustive tree search in the software package PAUP (9). The evolutionary distances between the strains are not shown. Informative sites used in the reconstruction are shown for each branch. The numbering at the nodes indicate the percentage of 500 bootstrap trees in which the cluster descending from the node was found with the DNAPARS program. The strict consensus are shown for all trees, except the 16S rDNA tree for Nostoc lineage I, where the majority rule tree is shown. Strain abbreviations are as for Fig. 2.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Strict consensus maximum parsimony tree for the Microcystis group, displaying the rbcLX locus. Congruent trees were built with the DNAPARS program from the software package PHYLIP and by the branch and bound tree search in the software package PAUP (9). The evolutionary distances between the strains are not indicated. Informative sites used in the reconstruction are shown for each branch. The numbering at the nodes indicate the percentage of 500 bootstrap trees in which the cluster descending from the node was found with the DNAPARS program.

Phyletic structure of the 16S rDNA and rbcLX nucleotide alignments. Using the exhaustive and the random tree search in the program package PAUP, we determined the tree length distribution of the data. The exhaustive tree search was used for data consisting of 11 or fewer taxa, while the random tree search with 100,000 replicates was used for more than 11 taxa.

Nucleotide substitution distribution for the rbcLX locus. The observed distribution of nucleotide substitutions for the rbcLX locus was compared to the distribution expected for random independent substitutions and to the expected distribution for phyletically informative substitutions (see Materials and Methods). As inferred from Fig. 6, the observed distributions resemble the distribution based on the assumption of phyletically informative substitutions. Thus, the inference from this is that each separate informative site is phyletically informative (from the definition of a single substitution), while the combination of these sites in phylogenetic reconstruction resembles randomly generated data (Table 2).

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Analysis of the frequency distribution of substitutions in the rbcLX alignment for Nostoc lineage I (A), Planktothrix (B), Microcystis (C), and Nostoc lineage II (D). The frequency distributions of positions in the alignments, where the number of taxa with 1 up to n/2 nucleotide differences from the consensus sequence for each subgroup of taxa, were determined for a model based on independent substitutions (black bars), phyletically informative substitutions (gray bars), and observed distribution (white bars). These distributions were calculated as described in Materials and Methods.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Mosaic structure of the informative sites in the rbcLX alignment for Nostoc lineage I. Blocks of sequences with similar phyletic affiliations are boxed. Informative sites 1 and 2 are in the rbcL region, while the other informative sites are in the rbcX region. Strain abbreviations are as for Fig. 2.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Data structure of the rbcLX locus. Assuming that the sites in the spacer between the rbcL and rbcX genes and the third codon position in protein coding genes are neutral, we estimated that there are less than 13.8% polymorphic neutral sites in the rbcLX region for the clustered groups. Based on these observations, we find it unlikely that our data are subjected to errors caused by mutational saturation, which would require 75% probability of nucleotide change (15). The randomization values for the rbcLX locus (Table 2) are quite intriguing because only 10% phylogenetic signal is usually sufficient to skew the tree length distribution beyond the 95% confidence limit of the random distribution (15).

Possible mechanisms for exchange of genetic material. Transformation is so far the only known biological mechanism for exchange of chromosomal DNA in cyanobacteria (19). For incorporating DNA into the genome, homologous recombination is a ubiquitous mechanism in eubacteria (19). For homologous recombination in Escherichia coli, there is a sharp decline in the number of recombinants with decreasing sequence identity. Compared to the frequencies for a fragment with 100% similarity in a 400-bp region, there were 42- and 300-fold decreases of cointegrate formation for fragments with 90% and 65 to 70% identity, respectively (30). Accordingly, stable exchange of genetic information solitarily within closely related groups of cyanobacteria observed in this work can be explained by homologous recombination frequencies (20).

Models for explaining the sequence homogeneity within the phyletically clustered groups of organisms. The most restrictive explanation for the sequence homogeneity within the clustered groups of organisms is that each cluster has evolved relatively recently from their common ancestors. However, recent common ancestors cannot explain the differences in relative evolutionary rates within, as opposed to among, the genetically clustered groups for rbcX compared to 16S rDNA. In Nostoc lineages I and II, the rbcX divergence between, relative to within the clusters, is 3 to 3.5, while for 16S rDNA this ratio is 0.2 to 1.5 (this effect is not as profound for the rbcL locus, because LSU is highly conserved by function). Presupposing a recent common ancestor, one should expect the opposite result of what we observed (i.e., lower ratios for rbcX compared to 16S rDNA). This is because the neutral sites in protein-coding sequences are relatively rapidly saturated by mutations, leading to a slower evolutionary rate for distantly related species than for closely related species (17). rRNA, on the other hand, has a high degree of functional constancy, resulting in a relative clock-like evolutionary rate (i.e., similar rates for closely and distantly related strains) (37).

Macroevolution of cyanobacterial characters. In the fossil records, stasisevolutionary lineages that persist for long periods without change of macroevolutionary (morphological) charactersis common. This led to the theory about punctuated equilibrium, stating that macroevolutionary characters are stable for long time periods, interrupted by short periods of their abrupt changes (12). However, for molecular characters (amino and nucleic acid sequences), the comparison between the time of divergence and the molecular divergence for different species gave rise to the neutral theory of molecular evolution (17). This theory, in contrast, states that the evolution of molecular characters is gradual and clock-like. Although not currently widely accepted, a view of macroevolutionphyletic gradualismis that morphological characters also can evolve in a gradual fashion (29).

Preservation of macroevolutionary characters by exchange of genetic material for neutrally evolving genes. The work of Stulp (34) has shown that organisms belonging to Nostoc lineage I can occupy different distinct ecological niches, which indicates that they may have diverged by selection for some characters while still exchanging genetic material at the rbcLX locus. Although not thoroughly investigated, this is probably also the case for the other phyletically clustered groups of organisms defined in this work.

Morphological and molecular clustering of modern cyanobacteria. Finally, we suggest that the morphological clustering of the living cyanobacteria reflects stability through structure and function of relevant characters. The DNA sequence clustering is a result of sequence homogenization and concerted evolution by exchange of genetic material for neutrally evolving genes. Those conclusions are in accordance with both the theory of punctuated equilibrium and the neutral theory of molecular evolution.