Authors: Bettina E Schirrmeister (corresponding author) [1]; Alexandre Antonelli [2,3]; Homayoun C Bagheri [1]
Background
Cyanobacteria are oxygenic phototrophic prokaryotes from which chloroplasts, the light harvesting organelles in plants, evolved. Some are able to convert atmospheric nitrogen into a form usable for plants and animals. During Earth history, cyanobacteria have raised atmospheric oxygen levels starting approximately 2.45 - 2.22 billion years ago and provided the basis for the evolution of aerobic respiration [1, 2, 3, 4, 5, 6, 7]. Cyanobacteria have also evolved extensive morphological diversity. Various patterns of cell organization exist, ranging from single-celled to differentiated multicellular forms with branching patterns. Species of this phylum occupy various habitats. They can be found in marine, freshwater or terrestrial environments, ranging from polar to tropical climate zones. Based on their morphology, they have been divided into five sections [8, 9] (Table 1). Sections I and II comprise single-celled bacteria, whereas sections III to V comprise multicellular forms. The latter sections are distinguished according to their level of organization. Section III is multicellular and undifferentiated, sections IV and V are multicellular and differentiated. The latter have the ability to produce heterocysts for nitrogen fixation and akinetes (climate-resistant resting cells). In addition, species in section V have the ability to branch in multiple dimensions.
Table 1 caption: Subset of cyanobacterial taxa used for the analyses with GenBank accession numbers for 16S rDNA sequences [see PDF for image]
Different interpretations of multicellularity are currently used [10, 11, 12]. For cyanobacteria, characterization of multicellularity has been described in previous studies [13, 14, 15, 16]. Cell to cell adhesion, intercellular communication, and for more complex species, terminal cell differentiation seem to be three essential processes that define multicellular, prokaryotic organisms on this planet [16]. Some forms of complexity found in several multicellular eukaryotes are not present in prokaryotes, but simple forms of multicellularity can be identified in three sections of the phylum cyanobacteria. Multicellular patterns comprise basic filamentous forms as found for section III, as well as more complex forms involving terminal differentiation, present in sections IV and V. In eukaryotes, multicellular complexity ranges from what is comparable to cyanobacteria to cases with up to 55 cell types as estimated for higher invertebrates such as arthropods or molluscs [17]. Considering that cyanobacterial sections III, IV and V resemble some of the first forms of multicellular filaments on Earth, knowing when and how these shapes evolved would further our understanding of complex life forms.
Some of the oldest body fossils unambiguously identified as cyanobacteria have been found in the Kasegalik and McLeary Formations of the Belcher Subgroup, Canada, and are evaluated to be between 1.8 billion and 2.5 billion years old [6, 18]. Studies from ~ 2.0 billion year old formations [18, 19] contain both unicellular and multicellular morphotypes of cyanobacteria. Cyanobacteria certainly existed as early as 2.32 billion years ago, if one accepts the assumption that they were responsible for the rapid accumulation of oxygen levels, known as the "Great Oxygenation Event" [1, 2, 3, 5, 7]. Multicellular fossils belonging to the cyanobacteria are well known from the late Precambrian [12, 20, 21] and possibly already existed 2.32 billion years ago. Other microbe-like multicellular filaments even older than 3.0 billion years have been found several times [22, 23, 24, 25, 26]. Some of the latter fossils are morphologically similar to species from the cyanobacterial order Oscillatoriales [27, 28], but no clear evidence has been adduced yet. Although biogenicity of some of the oldest fossils has been questioned [29, 30], a large variety of bacteria including anoxic phototrophs already existed by the time cyanobacteria evolved oxygenic photosynthesis [26]. Though impressive for prokaryotes, the fragmentary fossil record alone is not sufficient to disentangle the origin of cyanobacteria and their morphological phenotypes. Therefore, additional methods such as phylogenetic analysis provide a promising possibility to gather further clues on the evolution of such a complex phylum.
Phylogenetic analyses of cyanobacteria have gained in quantity over the past 20 years [4, 31, 32, 33, 34, 35, 36, 37, 38, 39]. These studies have shown that morphological characterization does not necessarily reflect true relationships between taxa, and possibly none of the five traditional morphological sections is monophyletic. Similar morphologies must have evolved several times independently, but details on this morphological evolution are scarce. Analyses assessing characteristics of cyanobacterial ancestors [37, 39] provide not only fundamental information on the history of cyanobacteria, but also on the evolution of life forms in the Archean Eon.
If one studies phylogenetic relationships based on protein coding genes in bacteria, it is possible to encounter the outcome of horizontal gene transfer (HGT) [40]. This issue is not as problematic for ribosomal DNA [41]. Nonetheless, the problem could be potentially reduced by analyzing datasets of concatenated conserved genes. Identification of these genes for phylogenetic analyses is not without difficulty, and requires in an ideal case comparison of complete genome data [42]. In cyanobacteria, many phylogenetic studies have concentrated on specific clades or smaller subsets of known species in this diverse phylum [39, 43, 44, 45, 46, 47, 48]. Therefore the genomic data presently available are strongly biased towards certain groups. In particular, genomic studies in cyanobacteria have emphasized marine species from Section I. Marine microphytoplankton (Synechococcus and Prochlorococcus) are a particularly well studied group [43, 45, 47, 48], reflected by 19 sequenced genomes out of 41 cyanobacterial genomes sequenced to date (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi, accessed in January 2011). From species belonging to section III only two genomes (Trichodesmium erythraeum and Arthrospira platensis) are known. For sections IV (four genomes known) and V (no genomes known) molecular data are rare or missing. As genomic data accumulate, promising phylogenomic approaches to cyanobacteria are being established [37, 38, 39, 47]. Despite these advances, it is at present difficult to obtain sequences other then 16S rDNA to cover a representative sample of species from all five sections.
The aim of this paper is to use molecular phylogenetic methods to address the evolutionary history of cyanobacteria and the evolution of multicellularity. For this purpose, we established a phylogeny based on 16S rDNA sequences belonging to 1,254 cyanobacterial taxa. From that phylogeny we sampled 58 cyanobacterial taxa that represent all main clades obtained and all five sections described by Castenholz et al. [8, 9], and feature a 1:1 ratio of unicellular to multicellular species. We used several methods to reconstruct the morphological evolution of ancestral lineages, and compared our results to known fossil data. Since the fossil record is inconclusive on the timing and taxonomic position of multicellular cyanobacteria, our study provides independent evidence on the first appearance and evolution of multicellularity among the ancestors of living cyanobacteria.
Results and Discussion
Phylogenetic analysis
Phylogenetic analyses of all identified cyanobacteria
To infer the evolution of multicellularity in cyanobacteria we carried out several phylogenetic analyses. To ensure a correct taxon-sampling, a phylogeny containing 1,254 16S rDNA sequences of cyanobacteria obtained from GenBank was reconstructed (Figure 1). Cyanobacterial morphotypes were assigned to four groups (A-D) which correlate to the five sections described by Castenholz et al. [9]. Using this nomenclature, sub-groups in the phylogeny were assigned to one of the four different morphological groups (A-D) according to their dominant shape. In total 14 sub-groups were identified for the phylogenetic tree. Five sub-groups consist of unicellular species from section I (A1-A5), two sub-groups are composed of single celled section II bacteria (B1, B2), four sub-groups are made up of multicellular species belonging to section III (C1-C4) and two sub-groups cover differentiated species from section IV and V (D1-D2). One sub-group contains both species from section I and III and is therefore designated as AC1. The phylogeny further contains six chloroplast genomes from the eukaryotic phyla Glaucophyta, Rhodophyta and the division Chlorophyta. Chloroplast sequences branch close to the bases and form a sister group to the cyanobacterial sub-groups mentioned. Furthermore six different Eubacteria were included in the phylogeny. They appear to form a distinct outgroup to the cyanobacteria and chloroplasts.
Figure 1: Phylogenetic tree of 1,254 cyanobacterial species. Maximum likelihood phylogram of cyanobacteria, based on GTR+G+I substitution model. Six eubacterial species form an outgroup. The ingroup contains 1,254 cyanobacterial strains and six different chloroplast sequences. Bootstrap values (> 50%) calculated from 100 re-samplings are displayed at the nodes. Colors define major morphological characters in the groups. Yellow are single-celled cyanobacteria of section I; orange single-celled from section II; green are multicellular, undifferentiated cyanobacteria from section III; blue are multicellular and differentiated bacteria from section IV; and pink from section V. Sections as described by Castenholz 2001 [9]. Different sub-groups (AC1;A1-A5;B1, B2;C1-C4;D1-D2) are defined for the phylogeny. Nomenclature of groups correlates with morphological sections as illustrated in the legend. From these sub-groups taxa were sampled for further analyses. A complete list with species included in the analysis can be found in Additional File 7. [see PDF for image]
Phylogenetic analyses to identify an outgroup
Rooted and unrooted phylogenetic analyses reconstructed with maximum likelihood and Bayesian inference and based on 16S rRNA gene sequences of 27 eubacterial species, including 5 cyanobacteria revealed congruent results. Cyanobacteria form a monophyletic group. Figure 2 shows the unrooted Bayesian consensus tree which supports cyanobacterial monophyly with posterior probabilities (PP)/bootstrap values (BV) of 1.0/100%. Phylogenetic trees constructed with an archaean outgroup support cyanobacterial monophyly with PP/BV of 1.0/98% (Additional File 1). In both cases, Plantomyces brasilienses and Chlamydia trachomatis, both gram negative bacteria, form a sister group to the cyanobacteria. This does not agree with other studies [49, 50, 51, 52], where Deinococcus-thermus was suggested to be the closest eubacterial relative to cyanobacteria. These discrepancies may be due to a lack of information when solely using 16S rRNA gene sequences for such distant relations. Furthermore, our results confirm the basal position of Gloeobacter violaceus, closest to the rest of the eubacteria, as found elsewhere [51]. This supports previous findings which state that Gloeobacter violaceus diverged very early from cyanobacteria living today [32, 33, 53, 54]. Gloeobacter shows differences in cell structure and metabolism that clearly distinguish it from the rest of extant cyanobacteria [55, 56]. It lacks thylacoid membranes and many genes from …
