BackgroundHigh-throughput sequencing technologies are lifting major limitations to molecular-based ecological studies of eukaryotic microbial diversity, but analyses of the resulting millions of short sequences remain a major bottleneck for these approaches. Here, we introduce the analytical and statistical framework of sequence similarity networks, increasingly used in evolutionary studies and graph theory, into the field of ecology to analyze novel pyrosequenced V4 small subunit rDNA (SSU-rDNA) sequence data sets in the context of previous studies, including SSU-rDNA Sanger sequence data from cultured ciliates and from previous environmental diversity inventories.ResultsOur broadly applicable protocol quantified the progress in the description of genetic diversity of ciliates by environmental SSU-rDNA surveys, detected a fundamental historical bias in the tendency to recover already known groups in these surveys, and revealed substantial amounts of hidden microbial diversity. Moreover, network measures demonstrated that ciliates are not globally dispersed, but are structured by habitat and geographical location at intermediate geographical scale, as observed for bacteria, plants, and animals.ConclusionsCurrently available ‘universal’ primers used for local in-depth sequencing surveys provide little hope to exhaust the significantly higher ciliate (and most likely microbial) diversity than previously thought. Network analyses such as presented in this study offer a promising way to guide the design of novel primers and to further explore this vast and structured microbial diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0125-5) contains supplementary material, which is available to authorized users.

The Great Oxidation Event (GOE) ∼2.4 billion years ago resulted from the accumulation of oxygen by the ancestors of cyanobacteria [1-3]. Cyanobacteria continue to play a significant role in primary production [4] and in regulating the global marine and limnic nitrogen cycles [5, 6]. Relatively little is known, however, about the evolutionary history and gene content of primordial cyanobacteria [7, 8]. To address these issues, we used protein similarity networks [9], containing proteomes from 48 cyanobacteria as the test group, and reference proteomes from 84 microbes representing four distinct metabolic groups from most reducing to most oxidizing: methanogens, obligate anaerobes (nonmethanogenic), facultative aerobes, and obligate aerobes. These four metabolic groups represent extant bioinformatic proxies for ancient redox chemistries, extending from an anoxic origin through the GOE and ultimately to obligate aerobes [10-13]. Analysis of the network metric degree showed a strong relationship between cyanobacteria and obligate anaerobes, from which cyanobacteria presumably arose, for core functions that include translation, photosynthesis, energy conservation, and environmental interactions. These data were used to reconstruct primordial functions in cyanobacteria that included nine gene families involved in photosynthesis, hydrogenases, and proteins involved in defense from environmental stress. The presence of 60% of these genes in both reaction center I (RC-I) and RC-II-type bacteria may be explained by selective loss of either RC in the evolutionary history of some photosynthetic lineages. Finally, the network reveals that cyanobacteria occupy a unique position among prokaryotes as a hub between anaerobes and obligate aerobes.

The origin of oxygenic photosynthesis in the Archaeplastida common ancestor was foundational for the evolution of multicellular life. It is very likely that the primary endosymbiosis that explains plastid origin relied initially on the establishment of a metabolic connection between the host cell and captured cyanobacterium. We posit that these connections were derived primarily from existing host-derived components. To test this idea, we used phylogenomic and network analysis to infer the phylogenetic origin and evolutionary history of 37 validated plastid innermost membrane (permeome) metabolite transporters from the model plant Arabidopsis thaliana. Our results show that 57% of these transporter genes are of eukaryotic origin and that the captured cyanobacterium made a relatively minor (albeit important) contribution to the process. We also tested the hypothesis that the bacterium-derived hexose-phosphate transporter UhpC might have been the primordial sugar transporter in the Archaeplastida ancestor. Bioinformatic and protein localization studies demonstrate that this protein in the extremophilic red algae Galdieria sulphuraria and Cyanidioschyzon merolae are plastid targeted. Given this protein is also localized in plastids in the glaucophyte alga Cyanophora paradoxa, we suggest it played a crucial role in early plastid endosymbiosis by connecting the endosymbiont and host carbon storage networks. In summary, our work significantly advances understanding of plastid integration and favors a hostcentric view of endosymbiosis. Under this view, nuclear genes of either eukaryotic or bacterial (noncyanobacterial) origin provided key elements of the toolkit needed for establishing metabolic connections in the primordial Archaeplastida lineage.Arabidopsis thaliana | endosymbiosis | evolution | network analysis | symbiont integration