Summary Stromatolites are complex microbial mats that form lithified layers. Fossilized stromatolites are the oldest evidence of cellular life on Earth, dating back over 3.4 billion years. Modern stromatolites are relatively rare but may provide clues about the function and evolution of their ancient counterparts. In this study, we focus on peritidal stromatolites occurring at Cape Recife and Schoenmakerskop on the southeastern South African coastline, the former being morphologically and structurally similar to fossilized phosphatic stromatolites formations. Using assembled shotgun metagenomic analysis, we obtained 183 genomic bins, of which the most dominant taxa were from the Cyanobacteria phylum. We identified functional gene sets in genomic bins conserved across two geographically isolated stromatolite formations, which included relatively high copy numbers of genes involved in the reduction of nitrates and phosphatic compounds. Additionally, we found little evidence of Archaeal species in these stromatolites, suggesting that they may not play an important role in peritidal stromatolite formations, as proposed for hypersaline formations.

In the geological record, fossil phosphatic stromatolites date back to the Great Oxidation Event in the Paleoproterozoic, but living phosphatic stromatolites have not been described previously. Here, we report on cyanobacterial stromatolites in a supratidal freshwater environment at Cape Recife, South African southern coast, precipitating Ca carbonate alternating with episodes of Ca phosphate deposition. In their structure and composition, the living stromatolites from Cape Recife closely resemble their fossilized analogues, showing phosphatic zonation, microbial casts, tunnel structures and phosphatic crusts of biogenic origin. The microbial communities appear to be also similar to those proposed to have formed fossil phosphatic stromatolites. Phosphatic domains in the material from Cape Recife are spatially and texturally associated with carbonate precipitates, but form distinct entities separated by sharp boundaries. Electron Probe Micro‐Analysis shows that Ca/P ratios and the overall chemical compositions of phosphatic precipitates are in the range of octacalcium phosphate, amorphous tricalcium phosphate and apatite. The coincidence in time of the emergence of phosphatic stromatolites in the fossil record with a major episode of atmospheric oxidation led to the assumption that at times of increased oxygen release the underlying increased biological production may have been linked to elevated phosphorus availability. The stromatolites at Cape Recife, however, form in an environment where ambient phosphorus concentrations do not exceed 0.28 μM, one to two orders of magnitude below the previously predicted minimum threshold of >5 μM for biogenic phosphate precipitation in paleo‐systems. Accordingly, we contest the previously proposed suitability of phosphatic stromatolites as a proxy for high ambient phosphate concentrations in supratidal to shallow ocean settings in earth history.

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13Stromatolites are complex microbial mats that form lithified layers and ancient forms are 14 the oldest evidence of life on earth, dating back over 3.4 billion years. Their emergence 15 aligns with the oxygenation of the Earth's atmosphere and insight into these ancient 16 structures would shed light on the earliest days of Earth. Modern stromatolites are 17 relatively rare but may provide clues about the function and evolution of their ancient 18 counterparts. Previous studies have assessed microbial diversity and overall functional 19 potential but not at a genome-resolved level. In this study, we focus on peritidal 20 stromatolites occurring at Cape Recife and Schoenmakerskop on the southeastern 21 South African coastline. We identify functional gene sets in bacterial species conserved 22 across two geographically distinct stromatolite formations and show that these bacteria 23 may promote carbonate precipitation through the reduction of sulfur and nitrogenous 24 compounds and produce calcium ions that are predicted to play an important role in 25 promoting lithified mats. We propose that abundance of extracellular alkaline 26 phosphatases, in combination with the absence of transport regulatory enzymes, may 27 lead to the precipitation of phosphatic deposits within these stromatolites. We conclude 28 that the cumulative effect of several conserved bacterial species drives accretion in 29 these two stromatolite formations. 30 31 3

Evidence of life on earth dates back more than 3.4 billion years in the form of lithified layers of complex microbial mats known as microbialites, which are ubiquitous in the fossil record. Modern, extant microbialites are comparatively rare but have been documented globally. Multi-faceted, molecular investigations are required to understand community structure and function, factors that influence formation and growth, and how modern microbialite microbial communities differ from those in non-lithifying microbial mats. In this study we selected living, layered microbialites (stromatolites) in a peritidal environment near Schoenmakerskop in the Eastern Cape of South Africa as a study site for conducting a spatial survey to map the composition and small molecule production of the microbial communities across the system. Substrate cores and water samples were collected from nine sampling stations ranging from the upper point of the freshwater inflow to the lower marine interface where tidal overtopping takes place. Substrate cores provided material for parallel analyses of microbial community diversity by 16S rRNA gene amplicon sequencing and metabolomics using LC-MS2. Species diversity was correlated with metabolite diversity between the nine sampling stations and prominent specialized metabolites were targeted for characterization. A new series of cyclic hexadepsipeptides, named ibhayipeptolides, was most abundant in substrate cores of submerged microbialites, and we predict that this large molecular family is produced by cyanobacteria belonging to the family Geitlerinemaceae. These data contribute knowledge about, and facilitate future targeted studies of, specialized metabolite function and biosynthesis in microbialite communities.