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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Environ Microbiol Rep. 2020 Dec 27;13(2):126–137. doi: 10.1111/1758-2229.12916

Conserved bacterial genomes from two geographically isolated peritidal stromatolite formations shed light on potential functional guilds

Samantha C Waterworth a, Eric W Isemonger b, Evan R Rees a, Rosemary A Dorrington b, Jason C Kwan a,#
PMCID: PMC8408775  NIHMSID: NIHMS1732479  PMID: 33369160

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 which are 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.

INTRODUCTION

Stromatolites are organo-sedimentary structures that date back between 3.4 and 3.8 billion years, forming the oldest fossils of living organisms on Earth (Dupraz et al., 2009; Nutman et al., 2016, 2019). The study of extant stromatolite analogs may help to elucidate the biological mechanisms that led to the formation and evolution of their ancient ancestors. The biogenicity of stromatolites has been studied extensively in the hypersaline and marine formations of Shark Bay, Australia, and Exuma Cay, The Bahamas, respectively (Mobberley et al., 2015; Centeno et al., 2016; Gleeson et al., 2016; Ruvindy et al., 2016; Warden et al., 2016; White et al., 2016; Khodadad and Foster, 2012; Babilonia et al., 2018; Wong et al., 2018; Chen et al., 2020). The presence of Archaea has been noted in several microbial mats and stromatolite systems (Casaburi et al., 2016; Balci et al., 2018; Medina-Chávez et al., 2019; Chen et al., 2020), particularly in the stromatolites of Shark Bay, where they are hypothesized to potentially fulfill the role of nitrifiers and hydrogenotrophic methanogens (Wong et al., 2017). Studies of freshwater microbialites in Mexico found that there were significantly more genes associated with phosphate uptake and metabolism than in other communities associated with fresh or marine waters (Breitbart et al., 2009) and that nitrogen fixation by heterocystous cyanobacteria, sulfur-reducing bacteria, and purple sulfur bacteria was important to the formation of these particular stromatolites (Falcón et al., 2007).

In the current study, we sought to investigate the metagenomes, at a genome-resolved level, of two geographically isolated (~2.8 km apart), peritidal, stromatolite formations at Cape Recife and Schoenmakerskop, on the eastern coast of South Africa. The stromatolite formations at Cape Recife and Schoenmakerskop are exposed to both fresh and marine water that has little dissolved inorganic phosphate and decreasing levels of dissolved inorganic nitrogen moving from freshwater to marine-influenced formations (Rishworth et al., 2017a). These formations are morphologically and structurally similar to fossilized phosphatic stromatolites dating back to the Great Oxygenation Event at the Paleoproterozoic (Büttner et al., 2020). The stromatolite formations at Cape Recife and Schoenmakerskop have been extensively characterized with respect to their physical structure, nutrient, and chemical environment (Perissinotto et al., 2014; Rishworth et al., 2016, 2017b, 2019; Dodd et al., 2018) and they experience regular shifts in salinity due to tidal overtopping and groundwater seepage (Rishworth et al., 2019). Stromatolites are impacted by fluctuating environmental pressures caused by periodic inundation by seawater, which affects the nutrient concentrations, temperature and chemistry of the system (Rishworth et al., 2016). These formations are characterized by their proximity to the ocean, where stromatolites in the upper formations receive freshwater from the inflow seeps, middle formation stromatolites withstand a mix of freshwater seepage and marine over-topping, and lower formations are in closest contact with the ocean (Perissinotto et al., 2014). The stromatolite formations at Cape Recife and Schoenmakerskop are exposed to both fresh and marine water that has little dissolved inorganic phosphate and decreasing levels of dissolved inorganic nitrogen (Cape Recife: 82–9 µM, Schoenmakerskop: 424–14 µM) moving from freshwater to marine influenced formations (Rishworth et al., 2017a). Microbial communities within these levels therefore likely experience distinct environmental pressures, including fluctuations in salinity and dissolved oxygen (Rishworth et al., 2016). While carbon predominantly enters these systems through cyanobacterial carbon fixation, it is unclear how other members of the stromatolite-associated bacterial consortia influence mineral stratification resulting from the cycling of essential nutrients such as nitrogen, phosphorus, and sulfur.

However, not much is known about the microbial assemblage of these peritidal formations. It is hypothesized that the growth of peritidal stromatolites at these sites is promoted by decreased levels of wave action, higher water alkalinity, decreased levels of salinity, and decreased calcite and aragonite saturation (Dodd et al., 2018). Here, we investigate the metabolic potential of the microbes associated with these stromatolites and their potential role in stromatolite formation.

RESULTS AND DISCUSSION

Stromatolite formations were classified according to their tidal proximity as defined in previous studies, as “Upper” (freshwater inflow), “Middle”, and “Lower” (marine-influenced) (Perissinotto et al., 2014; Rishworth et al., 2016, 2017b). Samples for this study were collected at low tide from the upper stromatolites at Cape Recife and Schoenmakerskop in January and April 2018 for comparisons over time and geographic space. Additional samples were collected in April 2018 from middle and lower formations for extended comparison across the two sites (Fig. 1).

Figure 1.

Figure 1.

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Stromatolites were collected from four different points at two different sites in January and April 2017. Aerial view of sampling locations within the Schoenmakerskop site (A) and the Cape Recife site (B) are represented by orange stars. The relative elevation of the same sampling sites relative to the ocean are presented in panels C - D. A simplified schematic of delineation of “Upper”, “Middle” and “Lower” formations at each site are provided in panels E - F. Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

Preliminary assessment of the structure of bacterial communities in these samples using 16S rRNA amplicon sequencing (BioProject PRJNA574289) showed that microbial communities from the upper formations were distinct from those in the middle and lower formations (Fig. S1), but that all communities were dominated by Cyanobacteria with lesser, but notable abundances of Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria (Fig. 2A). Shotgun metagenomic sequencing was performed for all ten sites (BioProject PRJNA612530), and binning of the metagenomic data resulted in a total of 183 bacterial genome bins (Table S1). (Please see Suppl. Experimental Procedures and https://github.com/samche42/Strom_Brief_report for full experimental details). The phylogenetic distribution and relative abundance thereof were approximately congruent with the 16S rRNA community profiles of each site, where Cyanobacteria were consistently dominant (36 – 89%) while Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia were less abundant but notable bacterial classes (Fig. 2B). Furthermore, scrutiny of all contigs from Cape Recife and Schoenmakerskop showed that none of the datasets comprised more than 1.5% archaeal genes. Two low-quality archaeal bins were retrieved from each of the lower formations and were classified as Woesearchaeales (order) and Nitrosoarchaeum (genus), respectively. The coverage of these genomes is among the lowest in each of the lower formation samples (Table S1) and would suggest a low numerical abundance of these Archaea, and it is likely that this kingdom is less important in the stromatolites found at Cape Recife and Schoenmakerskop. These results are largely in agreement with similar studies investigating freshwater microbialites from various regions of Mexico, and freshwater microbialites in Clinton Creek, Canada, which were dominated by Cyanobacteria and Proteobacteria but included less than 2% archaeal sequence (Breitbart et al., 2009; Centeno et al., 2012; White et al., 2015; Yanez-Montalvo et al., 2020) and is in contrast to communities present in hypersaline stromatolites, where Archaea comprise a large proportion of the microbial community (Wong et al., 2017, 2018). To identify bins common to both Cape Recife and Schoenmakerskop, we calculated pairwise average nucleotide identity (ANI) between all binned genomes using ANIcalculator (Varghese et al., 2015) and defined conserved bins as sharing more than 97% ANI in two or more of the sampled regions (Table S2). Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. Bins classified within the Acaryochloris and Hydrococcus genera were conserved across upper formations, and seven bins were conserved across the middle formations, which were classified within the Rivularia genus and Phormidesmiaceae and Spirulinaceae families (Fig. 2C). Acaryochloris species have been identified as dominant oxygenic photoautotrophs and may promote carbonate mineralization through photosynthetically induced alkalinization in microbialite formations in the cold, fresh waters of Pavilion Lake in British Columbia, Canada (Chan et al., 2014; Russell et al., 2014; White et al., 2016). Hydrococcus species have not been reported as dominant in any stromatolite-like formations to our knowledge. However, a metagenomic study of microbialites in the Alchichica crater lake, Mexico, reported the presence of Pleurocapsa-like cyanobacteria surrounded by aragonite using scanning electron microscopy and provided evidence to suggest that Pleurocapsales were responsible for the biomineralization of aragonite (Saghaï et al., 2015). As Hydrococcus is within the Pleurocapsales order, it is possible that this conserved bin may be performing a similar function.

Figure 2.

Figure 2.

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Distribution and abundance of bacterial taxa in stromatolite formations A) based on 16S rRNA gene fragment amplicon libraries. Phylogenetic classification and average relative abundance (n=3) of dominant phyla in different sample sites indicated that all stromatolite samples are dominated by Cyanobacteria with notable abundances of Bacteroidetes, Alpha- and Gammaproteobacteria. OTUs are shown in relative abundance and colored according to their phylum classification. B) Taxonomic classification of putative genome bins in stromatolites collected from Schoenmakerskop and Cape Recife. Coverage per genome has been used as a proxy for abundance (normalized to approximate genome size) and used to scale the size of individual genome bars, expressed as a percentage of the sum of all genome bin coverages. The coverage of conserved (etched lines) and non-conserved (solid colors) bacterial genomes is indicated. The taxonomic classification of each genome is indicated by color. C) Taxonomic classifications of conserved bacterial species in each of the samples. All bins were classified using GTDB-Tk (Chaumeil et al., 2019). Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

Finally, Rivularia spp. have been identified as key members in several stromatolite and microbialite formations, including freshwater microbialites in Pavilion Lake, Canada (White et al., 2016), Bacalar Lagoon, Mexico (Yanez-Montalvo et al., 2020), Vai Lahi Lake, Tonga (Kempe and Kazmierczak, 2012), Plateau de Langres, France (Caudwell et al., 2001), in microbialites formed in alkaline Lake Alchichica, Mexico (Tavera and Komárek, 1996; Kaźmierczak et al., 2011), and in marine “microstromatolites” from The Bahamas, Yugoslavia, and Australia (Golubic and Campbell, 1981). Filamentous “sister taxa” of Rivularia: Calothrix and Dicothrix, were similarly dominant in formations found in Highborne Cay (Myshrall et al., 2010; Reid et al., 2011). A study of the stromatolites found in hypersaline lakes in Laguna Negra, Argentina, showed the formations were dominated by Rivularia spp. (Shalygin et al., 2018), which aided in accretion through the secretion of exopolymeric substances which trapped both inorganic matter and living organisms (such as diatoms) that contribute to stromatolite formation (Mlewski et al., 2018). Recent investigations into the micro-morphological features of the Cape Recife stromatolites (Büttner et al., 2020) revealed the presence of filamentous microbes, and provided evidence to suggest that they played a key role in mineralization through the production of EPS. It is possible that the observed filamentous bacteria may be the conserved and dominant putative Rivularia bins identified here, and they may be contributing to the formation of the stromatolites. Furthermore, the crusts and tunnel lining surrounding these filaments have been shown to include biogenic phosphatic deposits (Büttner et al., 2020), and therefore it may be possible that these filamentous bacteria are responsible for the production of the phosphatic deposits.

KEGG analysis of the metagenomic datasets revealed a high abundance of genes encoding phosphate transport (pstSCAB), phosphate uptake regulation (phoURBP), alkaline phosphatases (phoADX), and phosphonate metabolism (phnCDEFGHIJKLM) (Fig. 3). Overall gene abundances indicated the negligible presence of canonical dissimilatory sulfate reduction/oxidation via aprAB and dsrAB encoded enzymes or genes associated with sulfonate metabolism (Fig. 3). Genes associated with assimilatory nitrate reduction (narB, nirA) were the most prevalent markers of nitrogen metabolism. All sites included several genes associated with dissimilatory nitrate reduction, where cytoplasmic NADH-dependent nitrate reductase nirBD was numerically dominant relative to periplasmic cytochrome c nitrate reductase nrfAH (Fig. 3).

Figure 3.

Figure 3.

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The abundance of select KEGG-annotated metabolic genes relative to total gene abundance per sample site (annotated on contigs > 3000 bp). Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

The functional potential of each bin was assessed through KEGG annotation of genes, and gene abundance was counted and calculated as a relative percentage of total gene count per sample. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. As genomes investigated here were of high purity, we were confident that copy number was not an artifact of contamination. However, to ensure that this was true, we identified whether gene copies were present on the same contigs, or if found on different contigs, that the classification of the contig matched that of other copies within that bin (Table S3). Using this approach, we found that among conserved bins, those classified as Acaryochloris and Hydrococcus species in the upper formations included a relatively high abundance of assimilatory sulfate reduction and sulphonate metabolism, respectively (Fig. 4). Reduction of sulfate has previously been shown to promote the precipitation of carbonates in the form of micritic crusts in Bahamian and Australian stromatolites (Reid et al., 2000; Wong et al., 2018) and it has been suggested that microbial cycling of sulfur played an important role in ancient Australian stromatolites (Bontognali et al., 2012; Allen, 2016). Seep waters feeding the stromatolite formations at both Cape Recife and Schoenmakerskop have relatively high levels of sulfate (Dodd et al., 2018), and it is possible that the Hydrococcus and Acaryochloris species represented by these conserved bins may utilize this nutrient and influence the redox potential of their environment. Despite the relatively high levels of sulfate in the water, only genomes representative of a Thioiploca sp. from the Schoenmakerskop lower formation and a Desulfobacula sp. from the Schoenmakerskop middle formation included all genes required for dissimilatory sulfate reduction (Fig. S2). This suggests that calcite formation in these peritidal stromatolites may be influenced by processes other than dissimilatory sulfate reduction, in contrast to stromatolite formations in the Cayo Coco lagoonal network, Highborne Cay, and Eleuthera Island in The Bahamas (Visscher et al., 2000; Dupraz et al., 2004; Pace et al., 2018).

Figure 4.

Figure 4.

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Summary of phosphate, nitrogen and sulfate transport and metabolism genes in conserved bins in Schoenmakerskop and Cape Recife upper, middle, and lower stromatolite formations. Relative gene abundance was calculated as a percentage of total genes per sample. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. The phylogenetic tree of conserved bins was created using JolyTree (Criscuolo, 2019) with a sketch size of 10 000, and the heatmap was created in iTol (Letunic and Bork, 2019). Bins from Schoenmakerskop stromatolites are indicated with orange font, and bins from Cape Recife stromatolites are indicated with purple font. Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper (April), ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April). Bins were taxonomically assigned to their deepest classification as per the GTDB-Tk taxonomic classification. Numbers following underscores relate to the number of rounds of clustering performed before that bin was finalized.

The reduction of nitrogen, nitrates, and nitrites can lead to calcite precipitation (Rodriguez-Navarro et al., 2003; Wei et al., 2015; Konopacka-Łyskawa et al., 2017; Wong et al., 2018; Lee and Park, 2019), and the released NH3 can react with CO2 and H2O, to form 2NH4+ + CO32- (Konopacka-Łyskawa et al., 2017). Therefore, bacteria that can fix nitrogen or produce ammonia from nitrates/nitrites could potentially promote the growth of stromatolites (Visscher and Stolz, 2005). We found that several of the genomic bins carried the genes associated with ferredoxin-dependent assimilatory nitrate reduction (nirA-narB genes) (Moreno-Vivián et al., 1999), nitrogen fixation (nifDHK genes), and dissimilatory nitrite reduction (nirBD genes) (Griffith, 2016), all of which result in the production of ammonia (Fig. S2). We noted a particularly high abundance of nirA-narB genes in conserved bins classified as Rivularia sp. (Fig. 4), which are the dominant species in the middle formations. Finally, genes associated with nitrogen fixation (nifDHK) were identified in several bins and were most abundant in non-conserved Blastochloris species (Fig. S2). Previous studies of nitrogen-cycling in microbialites in karstic and soda lakes in Mexico and Cuba found a large abundance of genes involved in denitrification, which originated from Alphaproteobacteria, as well as genes involved in nitrogen fixation where the majority of nifH transcripts (nitrogen fixation) were from bacteria within the Nostocales order (Alcántara-Hernández et al., 2017). However, the karstic and soda lakes were low in dissolved inorganic nitrogen (DIN) (Alcántara-Hernández et al., 2017), contrary to environments found in Cape Recife and Schoenmakerskop, which are relatively high in DIN, ranging from 95–450 mM at the two sites (Rishworth et al., 2016). We could detect very few genes associated with denitrification in our metagenomes, and nif genes were primarily associated with Blastochloris species (Alphaproteobacteria). As Rivularia are traditionally observed to be heterocystous nitrogen-fixers, we were surprised that we did not find any nif genes in the conserved Rivularia genomes. We additionally looked for alternative nitrogen fixation operons (nifB, fdxN, nifS, and nifU genes) (Mulligan and Haselkorn, 1989), but only identified nifB and nifS genes in the Rivularia bins. It is possible that these bins are incorrectly classified and may be a different species of the Nostocales order, and additional microscopy will be required for definitive classification. We further searched the non-binned bacterial contigs, contigs classified at the kingdom level as “Archaea” and “Unclassified”, and “small” (<3000 bp) contigs for nif genes (Fig. 5). We found a total of 20 nif genes in these “non-binned” datasets. Upon alignment against the NCBI nr database, found that none of these 20 genes were homologous with genes from any Nostocales spp. Studies conducted around the freshwater microbialites of Cuatro Ciénegas, where DIN levels are relatively high (Breitbart et al., 2009), found genes associated with nitrate reduction, ammonia assimilation, and nitrogen fixation, as well as isotopic evidence for the assimilatory reduction of nitrates and nitrites but could not find isotopic signatures of nitrogen fixation (Breitbart et al., 2009). As the freshwater environment of Cuatro Ciénegas microbialites is somewhat similar to that found in the upper and middle formations at Cape Recife and Schoenmakerskop, it is possible that a similar trend may be found here, where nitrogen fixation is less important and the assimilatory reduction of nitrates and nitrites by conserved bins may play an important role in stromatolites.

Figure 5.

Figure 5.

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The relative abundance of phosphate, nitrogen, and sulfate transport and metabolism genes annotated on contigs from the unclustered (A), Archaea (B), and “Unclassified” (C) bins, as well as all contigs smaller than 3000 bp in length (D). Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

Similarly, the bins that were classified as Rivularia spp. included the greatest abundance of genes associated with phosphate uptake, alkaline phosphatases, and phosphonate metabolism (Fig. 4, Fig. S2). The stromatolites at Cape Recife and Schoenmakerskop experience limited inorganic phosphate availability (Rishworth et al., 2016, 2017a, 2018; Dodd et al., 2018) and the high abundance of genes encoding phosphate-metabolizing enzymes may be indicative of how stromatolite communities cope with low dissolved inorganic phosphorus in their environment as suggested for microbialite assemblages in Lake Alchichica, Mexico where a similar over-representation of phosphate-associated genes was observed (Breitbart et al., 2009; Valdespino-Castillo et al., 2014). These overrepresented genes were hypothesized to play an important role in controlling carbonate precipitation through the production of stored polyphosphate, which chelates metals, such as calcium (Breitbart et al., 2009; Valdespino-Castillo et al., 2014). However, phylotyping of alkaline phosphatase genes in microbialites from Lake Alchichica suggested that Alphaproteobacteria were the possible hosts of these genes (Valdespino-Castillo et al., 2017), in contrast to the cyanobacterial Rivularia spp. as shown here. Phosphatic structures have been observed in Cape Recife stromatolites (Büttner et al., 2020) and due to their structural similarity to filamentous bacteria observed in tunnels lined with phosphatic deposits, and their abundant genetic potential for phosphate metabolism, the conserved Rivularia bins may contribute to the formation of these macrostructures.

To confirm that we had not inadvertently missed any important functional genes, we also annotated genes from contigs that were unclustered (i.e., not clustered into genome bins), small contigs (<3000 bp in length), all contigs in the “Archaea” and “unclassified” kingdom bins (Fig. 5). There was no specific category of predicted gene function that was hidden in these genes (e.g., dissimilatory sulfate reduction genes were confirmed as absent, rather than overlooked), but we did note an abundance of alkaline phosphatases in the “small contigs”. A total of 373, 477, and 627 additional copies of phoA, phoD, and phoX respectively were identified, which indicated that there was even more genetic potential for inorganic phosphate production in these systems than previously realized, and further validates the hypothesis that phosphate metabolism may be important to these stromatolite formations.

Finally, we searched for genes associated with photosystem I, photosystem II, the cytochrome b6/f complex, and photosynthetic electron transport (Fig. S3) to identify genomes that may perform photosynthesis. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. A previous study of “core” photosynthetic genes among cyanobacterial genomes found that photosystem I genes: psaA - psaF, psaL, and psaM, and photosystem II genes: psbA, psbB, psbC, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbO, psbP, psbT, psbW, and psbX, and cytochrome b6 genes: petA - petD, petG, petM, and petN, were present in all genomes investigated (Mulkidjanian et al., 2006). We found a wide distribution of these genes among most of the cyanobacterial bins detected here, with particular abundance noted in the conserved Rivularia and Pleurocapsa bins (Fig. S3). Cyanobacterial bins with few to no photosynthetic genes (e.g. ARU_4_12, RM1_405_57, SM2_5_0 among others) were noted as being of low quality, and absence of these genes may not be reflective of the bacterium’s true genetic potential). As photosynthesis is well known to drive precipitation of carbonates (Kamennaya et al., 2012; Shiraishi, 2012; Zhu and Dittrich, 2016), this wide distribution and abundance of genes would suggest that photosynthesis may drive precipitation of calcium carbonate in this system, as observed in several other formations including microbial mats, stromatolites, microbialites (Reid et al., 2000; Dupraz and Visscher, 2005; Visscher and Stolz, 2005; Baumgartner et al., 2006; Kazmierczak and Kempe, 2006; Houghton et al., 2014; Casaburi et al., 2016). While there was a slight increased abundance of photosynthetic genes in Rivularia and Pleurocapsa bins, the wide distribution of the genes in several bins would suggest that carbonate precipitation is a result of microbial cumulative photosynthetic effort.

CONCLUSIONS

This pilot study has provided a glimpse into these unique, extant, stromatolite formations. In the Schoenmakerskop and Cape Recife stromatolite communities, there is extensive genetic potential for phosphate metabolism in conserved bins, classified as Rivularia spp., that likely contributes to the scavenging of phosphate and playing a role in the chelation of calcium ions and aid in the control of carbonate precipitation. It is also likely that this overrepresentation of phosphate-associated genes is responsible for the accumulation of phosphatic structures in the Cape Recife stromatolite formations, and the genetic evidence here would suggest that similar mineral structures could be found in the Schoenmakerskop formations. Additionally, precipitation of calcium carbonate may be driven by photosynthesis, as there is an abundance of photosynthesis-associated genes present in the metagenomes. Genes involved in the cycling of sulfur and nitrogen were also identified in conserved bins and may influence the redox potential of the stromatolite environment. Finally, the low abundance of genes originating from Archaea would suggest that these taxa are not particularly vital to this peritidal system.

Supplementary Material

Figure S1

Figure S1. The relative abundance of phosphate, nitrogen, and sulfate transport and metabolism genes in all genome bins from Schoenmakerskop and Cape Recife stromatolite formations. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. The phylogenetic tree of conserved bins was created using JolyTree (Criscuolo, 2019) with a sketch size of 10 000, and the heatmap was created in iTol (Letunic and Bork, 2019). Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

Figure S2

Figure S2. The relative abundance of genes associated with bacterial photosynthesis in all genome bins from Schoenmakerskop and Cape Recife stromatolite formations. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. The phylogenetic tree of conserved bins was created using JolyTree (Criscuolo, 2019) with a sketch size of 10 000, and the heatmap was created in iTol (Letunic and Bork, 2019). Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

Supplementary-Experimental Procedure
Tabe S1

Table S1. Characteristics and taxonomic classifications of genomes bins from the upper, middle, and lower stromatolite formations from Cape Recife and Schoenmakerskop. Taxonomic classification is provided in both GTDB-Tk format and equivalent NCBI classifications.

Table S2

Table S2. Conserved bacterial species are defined by shared ANI greater than 97% in stromatolite formations from Cape Recife and Schoenmakerskop. Shared ANI is provided of bin A relative to bin B, and bin B relative to bin A.

Figure S3
Table S3

Table S3. Summary of gene copies of phosphate, nitrogen, and sulfate transport and metabolism genes annotated on contigs clustered into genome bins from Schoenmakerskop and Cape Recife stromatolite formations.

ORIGINALITY-SIGNIFICANCE.

A collection of peritidal stromatolites found off the coast of Port Elizabeth, South Africa, have recently been shown to be morphologically and structurally similar to fossilized phosphatic stromatolites from the Paleoproterozoic era. This report is, to our knowledge, the first detailed examination of microbial genomes associated with this type of modern stromatolite.

ACKNOWLEDGEMENTS

The authors acknowledge Caro Damarjanan, who conducted a pilot study that informed this work. We also wish to thank Karthik Anantharaman (University of Wisconsin-Madison) for his helpful critique.

The authors acknowledge funding from the Gordon and Betty Moore Foundation (Grant number 6920) (awarded to R.A.D and J.C.K.), and grants awarded to R.A.D by the South African National Research Foundation (UID: 87583 and 109680). E.I.W was supported by a South African NRF Ph.D. fellowship. Development of Autometa in J.C.K.’s laboratory and contributions by E.R.R. was supported by the U.S. National Science Foundation (DBI-1845890). This research was performed in part using the computer resources and assistance of the UW-Madison Center for High Throughput Computing (CHTC) in the Department of Computer Sciences. The CHTC is supported by UW-Madison, the Advanced Computing Initiative, the Wisconsin Alumni Research Foundation, Wisconsin Institutes for Discovery, and the National Science Foundation and is an active member of the Open Science Grid, which is supported by the National Science Foundation and the U.S. Department of Energy’s Office of Science. The authors also acknowledge the Center for High-Performance Computing (CHPC, South Africa) for providing computing facilities for bioinformatics data analysis.

REFERENCES

  1. Alcántara-Hernández RJ, Valdespino PM, Centeno CM, Alcocer J, and Falcón LI (2017) Genetic diversity associated to N cycle pathways in microbialites from Lake Alchichica, Mexico http://www.int-res.com/articles/ame2016/78/a078p121.pdf. 78: 121–133. [Google Scholar]
  2. Allen JF (2016) A proposal for formation of Archaean stromatolites before the advent of oxygenic photosynthesis. Front Microbiol 7: 1784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Babilonia J, Conesa A, Casaburi G, Pereira C, Louyakis AS, Reid RP, and Foster JS (2018) Comparative metagenomics provides insight into the ecosystem functioning of the Shark Bay stromatolites, Western Australia. Front Microbiol 9: 1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balci N, Gunes Y, Sertcetin E, and Kurt MA (2018) Non-cyanobacterial community shape the stromatolites of Lake Salda , SW, Turkey [Google Scholar]
  5. Baumgartner LK, Reid RP, Dupraz C, Decho AW, Buckley DH, Spear JR, et al. (2006) Sulfate reducing bacteria in microbial mats: Changing paradigms, new discoveries. Sediment Geol 185: 131–145. [Google Scholar]
  6. Bontognali TRR, Sessions AL, Allwood AC, Fischer WW, Grotzinger JP, Summons RE, and Eiler JM (2012) Sulfur isotopes of organic matter preserved in 3.45-billion-year-old stromatolites reveal microbial metabolism. Proc Natl Acad Sci U S A 109: 15146–15151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Breitbart M, Hoare A, Nitti A, Siefert J, Haynes M, Dinsdale E, et al. (2009) Metagenomic and stable isotopic analyses of modern freshwater microbialites in Cuatro Ciénegas, Mexico. Environ Microbiol 11: 16–34. [DOI] [PubMed] [Google Scholar]
  8. Büttner SH, Isemonger EW, Isaacs M, van Niekerk D, Sipler RE, and Dorrington RA (2020) Living phosphatic stromatolites in a low-phosphorus environment: Implications for the use of phosphorus as a proxy for phosphate levels in paleo-systems. Geobiology [DOI] [PubMed] [Google Scholar]
  9. Casaburi G, Duscher AA, Pamela Reid R, and Foster JS (2016) Characterization of the stromatolite microbiome from Little Darby Island, The Bahamas using predictive and whole shotgun metagenomic analysis. Environ Microbiol 18: 1452–1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caudwell C, Lang J, and Pascal A (2001) Lamination of swampy-rivulets Rivularia haematites stromatolites in a temperate climate. Sediment Geol 143: 125–147. [Google Scholar]
  11. Centeno CM, Legendre P, Beltrán Y, Alcántara-Hernández RJ, Lidström UE, Ashby MN, and Falcón LI (2012) Microbialite genetic diversity and composition relate to environmental variables. FEMS Microbiol Ecol 82: 724–735. [DOI] [PubMed] [Google Scholar]
  12. Centeno CM, Mejía O, and Falcón LI (2016) Habitat conditions drive phylogenetic structure of dominant bacterial phyla of microbialite communities from different locations in Mexico. Rev Biol Trop 64: 1057–1065. [DOI] [PubMed] [Google Scholar]
  13. Chan OW, Bugler-Lacap DC, Biddle JF, Lim DS, McKay CP, and Pointing SB (2014) Phylogenetic diversity of a microbialite reef in a cold alkaline freshwater lake. Can J Microbiol 60: 391–398. [DOI] [PubMed] [Google Scholar]
  14. Chaumeil P-A, Mussig AJ, Hugenholtz P, and Parks DH (2019) GTDB-Tk: A toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen R, Wong HL, Kindler GS, MacLeod FI, Benaud N, Ferrari BC, and Burns BP (2020) Discovery of an abundance of biosynthetic gene clusters in Shark Bay microbial mats. Front Microbiol 11: 1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Criscuolo A (2019) A fast alignment-free bioinformatics procedure to infer accurate distance-based phylogenetic trees from genome assemblies. Riogrande Odontol 5: e36178. [Google Scholar]
  17. Dodd C, Anderson CR, Perissinotto R, du Plooy SJ, and Rishworth GM (2018) Hydrochemistry of peritidal stromatolite pools and associated freshwater inlets along the Eastern Cape coast, South Africa. Sediment Geol 373: 163–179. [Google Scholar]
  18. Dupraz C, Reid RP, Braissant O, Decho AW, Norman RS, and Visscher PT (2009) Processes of carbonate precipitation in modern microbial mats. Earth-Sci Rev 96: 141–162. [Google Scholar]
  19. Dupraz C and Visscher PT (2005) Microbial lithification in marine stromatolites and hypersaline mats. Trends Microbiol 13: 429–438. [DOI] [PubMed] [Google Scholar]
  20. Dupraz C, Visscher PT, Baumgartner LK, and Reid RP (2004) Microbe-mineral interactions: Early carbonate precipitation in a hypersaline lake (Eleuthera Island, Bahamas) Sedimentology 51: 745–765. [Google Scholar]
  21. Falcón LI, Cerritos R, Eguiarte LE, and Souza V (2007) Nitrogen fixation in microbial mat and stromatolite communities from Cuatro Cienegas, Mexico. Microb Ecol 54: 363–373. [DOI] [PubMed] [Google Scholar]
  22. Gleeson DB, Wacey D, Waite I, O’Donnell AG, and Kilburn MR (2016) Biodiversity of living, non-marine, thrombolites of Lake Clifton, Western Australia. Geomicrobiol J 33: 850–859. [Google Scholar]
  23. Golubic S and Campbell SE (1981) Biogenically formed aragonite concretions in marine Rivularia. In Phanerozoic Stromatolites Springer Berlin Heidelberg, pp. 209–229. [Google Scholar]
  24. Griffith JC (2016) Insights into the soil microbial communities in New Zealand indigenous tussock grasslands.(Thesis, Doctor of Philosophy) University of Otago. Retrieved from http://hdl.handle.net/10523/6906 [Google Scholar]
  25. Houghton J, Fike D, Druschel G, Orphan V, Hoehler TM, and Des Marais DJ (2014) Spatial variability in photosynthetic and heterotrophic activity drives localized δ13C org fluctuations and carbonate precipitation in hypersaline microbial mats. Geobiology 12: 557–574. [DOI] [PubMed] [Google Scholar]
  26. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, and Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9.: [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kamennaya NA, Ajo-Franklin CM, Northen T, and Jansson C (2012) Cyanobacteria as biocatalysts for carbonate mineralization. Minerals 2: 338–364. [Google Scholar]
  28. Kazmierczak J and Kempe S (2006) Genuine modern analogues of Precambrian stromatolites from caldera lakes of Niuafo’ou Island, Tonga. Naturwissenschaften 93: 119–126. [DOI] [PubMed] [Google Scholar]
  29. Kaźmierczak J, Kempe S, Kremer B, López-García P, Moreira D, and Tavera R (2011) Hydrochemistry and microbialites of the alkaline crater lake Alchichica, Mexico. Facies 57: 543–570. [Google Scholar]
  30. Kempe S and Kazmierczak J (2012) Terrestrial Analogues for Early Planetary Oceans: Niuafo’ou Caldera lakes (Tonga) and their geology, water chemistry, and stromatolites. In Life on Earth and other Planetary Bodies Hanslmeier A, Kempe S, and Seckbach J (eds). Dordrecht: Springer Netherlands, pp. 195–234. [Google Scholar]
  31. Khodadad CLM and Foster JS (2012) Metagenomic and metabolic profiling of nonlithifying and lithifying stromatolitic mats of Highborne Cay, The Bahamas. PLoS One 7: e38229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Konopacka-Łyskawa D, Kościelska B, Karczewski J, and Gołąbiewska A (2017) The influence of ammonia and selected amines on the characteristics of calcium carbonate precipitated from calcium chloride solutions via carbonation. Mater Chem Phys 193: 13–18. [Google Scholar]
  33. Lee YS and Park W (2019) Enhanced calcium carbonate-biofilm complex formation by alkali-generating Lysinibacillus boronitolerans YS11 and alkaliphilic Bacillus sp. AK13. AMB Express 9: 49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Letunic I and Bork P (2019) Interactive Tree Of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Res 47: 256–259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Medina-Chávez NO, Viladomat-Jasso M, Olmedo-Álvarez G, Eguiarte LE, Souza V, and De la Torre-Zavala S (2019) Diversity of Archaea domain in Cuatro Cienegas Basin: Archaean domes. bioRxiv 766709. [Google Scholar]
  36. Mlewski EC, Pisapia C, Gomez F, Lecourt L, Soto Rueda E, Benzerara K, et al. (2018) Characterization of pustular mats and related Rivularia-rich laminations in oncoids from the Laguna Negra lake (Argentina). Front Microbiol 9: 996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mobberley JM, Khodadad CLM, Visscher PT, Reid RP, Hagan P, and Foster JS (2015) Inner workings of thrombolites: spatial gradients of metabolic activity as revealed by metatranscriptome profiling. Sci Rep 5: 12601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moreno-Vivián C, Cabello P, Martínez-Luque M, Blasco R, and Castillo F (1999) Prokaryotic nitrate reduction: Molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol 181: 6573–6584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mulkidjanian AY, Koonin EV, Makarova KS, Mekhedov SL, Sorokin A, Wolf YI, et al. (2006) The cyanobacterial genome core and the origin of photosynthesis. Proc Natl Acad Sci U S A 103: 13126–13131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mulligan ME and Haselkorn R (1989) Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon. J Biol Chem 264: 19200–19207. [PubMed] [Google Scholar]
  41. Myshrall KL, Mobberley JM, Green SJ, Visscher PT, Havemann SA, Reid RP, and Foster JS (2010) Biogeochemical cycling and microbial diversity in the thrombolitic microbialites of Highborne Cay, Bahamas. Geobiology 8: 337–354. [DOI] [PubMed] [Google Scholar]
  42. Nutman AP, Bennett VC, Friend CRL, Van Kranendonk MJ, and Chivas AR (2016) Rapid emergence of life shown by discovery of 3,700-million-year-old microbial structures. Nature 537: 535–538. [DOI] [PubMed] [Google Scholar]
  43. Nutman AP, Bennett VC, Friend CRL, Van Kranendonk MJ, Rothacker L, and Chivas AR (2019) Cross-examining Earth’s oldest stromatolites: Seeing through the effects of heterogeneous deformation, metamorphism and metasomatism affecting Isua (Greenland) ∼3700 Ma sedimentary rocks. Precambrian Res 331: 105347. [Google Scholar]
  44. Pace A, Bourillot R, Bouton A, Vennin E, Braissant O, Dupraz C, et al. (2018) Formation of stromatolite lamina at the interface of oxygenic-anoxygenic photosynthesis. Geobiology 16: 378–398. [DOI] [PubMed] [Google Scholar]
  45. Perissinotto R, Bornman TG, Steyn P-P, Miranda NAF, Dorrington RA, Matcher GF, et al. (2014) Tufa stromatolite ecosystems on the South African south coast. S Afr J Sci 110: 01–08. [Google Scholar]
  46. Reid RP, Gaspar APL, Bowlin EM, Custals L, and Andres MS (2011) Microbialites and sediments: A 2-year record of burial and exposure of stromatolites and thrombolites at Highborne Cay Bahamas. In STROMATOLITES: Interaction of Microbes with Sediments Tewari V and Seckbach J (eds). Dordrecht: Springer Netherlands, pp. 407–425. [Google Scholar]
  47. Reid RP, Visscher PT, Decho AW, Stolz JF, Bebout BM, Dupraz C, et al. (2000) The role of microbes in accretion, lamination and early lithification of modern marine stromatolites. Nature 406: 989–992. [DOI] [PubMed] [Google Scholar]
  48. Rishworth GM, Cawthra HC, Dodd C, and Perissinotto R (2019) Peritidal stromatolites as indicators of stepping-stone freshwater resources on the Palaeo-Agulhas Plain landscape. Quat Sci Rev 105704. [Google Scholar]
  49. Rishworth GM, van Elden S, Perissinotto R, Miranda NAF, Steyn P-P, and Bornman TG (2016) Environmental influences on living marine stromatolites: Insights from benthic microalgal communities. Environ Microbiol 18: 503–513. [DOI] [PubMed] [Google Scholar]
  50. Rishworth GM, Perissinotto R, Bird MS, and Pelletier N (2018) Grazer responses to variable macroalgal resource conditions facilitate habitat structuring. R Soc Open Sci 5: 171428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rishworth GM, Perissinotto R, Bird MS, Strydom NA, Peer N, Miranda NAF, and Raw JL (2017) Non-reliance of metazoans on stromatolite-forming microbial mats as a food resource. Sci Rep 7: 42614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Rishworth GM, Perissinotto R, Bornman TG, and Lemley DA (2017) Peritidal stromatolites at the convergence of groundwater seepage and marine incursion: Patterns of salinity, temperature and nutrient variability. J Mar Syst 167: 68–77. [Google Scholar]
  53. Rodriguez-Navarro C, Rodriguez-Gallego M, Ben Chekroun K, and Gonzalez-Muñoz MT (2003) Conservation of ornamental stone by Myxococcus xanthus-induced carbonate biomineralization. Appl Environ Microbiol 69: 2182–2193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Russell JA, Brady AL, Cardman Z, Slater GF, Lim DSS, and Biddle JF (2014) Prokaryote populations of extant microbialites along a depth gradient in Pavilion Lake, British Columbia, Canada. Geobiology 12: 250–264. [DOI] [PubMed] [Google Scholar]
  55. Ruvindy R, White RA 3rd, Neilan BA, and Burns BP (2016) Unravelling core microbial metabolisms in the hypersaline microbial mats of Shark Bay using high-throughput metagenomics. ISME J 10: 183–196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Saghaï A, Zivanovic Y, Zeyen N, Moreira D, Benzerara K, Deschamps P, et al. (2015) Metagenome-based diversity analyses suggest a significant contribution of non-cyanobacterial lineages to carbonate precipitation in modern microbialites. Front Microbiol 6: 797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shalygin S, Pietrasiak N, Gomez F, Mlewski C, Gerard E, and Johansen JR (2018) Rivularia halophila sp. nov. (Nostocales, Cyanobacteria): The first species of Rivularia described with the modern polyphasic approach. Eur J Phycol 53: 537–548. [Google Scholar]
  58. Shiraishi F (2012) Chemical conditions favoring photosynthesis-induced CaCO3 precipitation and implications for microbial carbonate formation in the ancient ocean. Geochim Cosmochim Acta 77: 157–174. [Google Scholar]
  59. Tavera R and Komárek J (1996) Cyanoprokaryotes in the volcanic lake of Alchichica, Puebla State, Mexico. archiv_algolstud 83: 511–538. [Google Scholar]
  60. Valdespino-Castillo PM, Alcántara-Hernández RJ, Alcocer J, Merino-Ibarra M, Macek M, and Falcón LI (2014) Alkaline phosphatases in microbialites and bacterioplankton from Alchichica soda lake, Mexico. FEMS Microbiol Ecol 90: 504–519. [DOI] [PubMed] [Google Scholar]
  61. Valdespino-Castillo PM, Alcántara-Hernández RJ, Merino-Ibarra M, Alcocer J, Macek M, Moreno-Guillén OA, and Falcón LI (2017) Phylotype dynamics of bacterial P utilization genes in microbialites and bacterioplankton of a monomictic endorheic lake. Microb Ecol 73: 296–309. [DOI] [PubMed] [Google Scholar]
  62. Varghese NJ, Mukherjee S, Ivanova N, Konstantinidis KT, Mavrommatis K, Kyrpides NC, and Pati A (2015) Microbial species delineation using whole genome sequences. Nucleic Acids Res 43: 6761–6771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Visscher PT, Pamela Reid R, and Bebout BM (2000) Microscale observations of sulfate reduction: Correlation of microbial activity with lithified micritic laminae in modern marine stromatolites. Geology 28: 919–922. [Google Scholar]
  64. Visscher PT and Stolz JF (2005) Microbial mats as bioreactors: Populations, processes, and products. In Geobiology: Objectives, Concepts, Perspectives Noffke N (ed). Amsterdam: Elsevier, pp. 87–100. [Google Scholar]
  65. Warden JG, Casaburi G, Omelon CR, Bennett PC, Breecker DO, and Foster JS (2016) Characterization of microbial mat microbiomes in the modern thrombolite ecosystem of Lake Clifton, Western Australia using shotgun metagenomics. Front Microbiol 7: 1064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wei S, Cui H, Jiang Z, Liu H, He H, and Fang N (2015) Biomineralization processes of calcite induced by bacteria isolated from marine sediments. Braz J Microbiol 46: 455–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. White RA 3rd, Power IM, Dipple GM, Southam G, and Suttle CA (2015) Metagenomic analysis reveals that modern microbialites and polar microbial mats have similar taxonomic and functional potential. Front Microbiol 6: 966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. White RA, Chan AM, Gavelis GS, Leander BS, Brady AL, Slater GF, et al. (2016) Metagenomic analysis suggests modern freshwater microbialites harbor a distinct core microbial community. Front Microbiol 6.: [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wong HL, Visscher PT, White RA III, Smith D-L, Patterson MM, and Burns BP (2017) Dynamics of archaea at fine spatial scales in Shark Bay mat microbiomes. Sci Rep 7: 46160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wong HL, White RA 3rd, Visscher PT, Charlesworth JC, Vázquez-Campos X, and Burns BP (2018) Disentangling the drivers of functional complexity at the metagenomic level in Shark Bay microbial mat microbiomes. ISME J 12: 2619–2639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Yanez-Montalvo A, Gómez-Acata S, Águila B, Hernández-Arana H, and Falcón LI (2020) The microbiome of modern microbialites in Bacalar Lagoon, Mexico. PLoS One 15: e0230071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zhu T and Dittrich M (2016) Carbonate precipitation through microbial activities in the natural environment, and their potential in biotechnology: A review. Front Bioeng Biotechnol 4: 4. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Figure S1. The relative abundance of phosphate, nitrogen, and sulfate transport and metabolism genes in all genome bins from Schoenmakerskop and Cape Recife stromatolite formations. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. The phylogenetic tree of conserved bins was created using JolyTree (Criscuolo, 2019) with a sketch size of 10 000, and the heatmap was created in iTol (Letunic and Bork, 2019). Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

NIHMS1732479-supplement-Figure_S1.eps (1.2MB, eps)
Figure S2

Figure S2. The relative abundance of genes associated with bacterial photosynthesis in all genome bins from Schoenmakerskop and Cape Recife stromatolite formations. Please refer to the Suppl. Materials and Methods for additional details and find all scripts used here: https://github.com/samche42/Strom_Brief_report. The phylogenetic tree of conserved bins was created using JolyTree (Criscuolo, 2019) with a sketch size of 10 000, and the heatmap was created in iTol (Letunic and Bork, 2019). Abbreviations are as follows: JSU: Schoenmakerskop Upper (Jan), JRU: Cape Recife Upper (Jan), ASU: Schoenmakerskop Upper April, ARU: Cape Recife Upper (April), SM1: Schoenmakerskop Middle 1 (April), SM2: Schoenmakerskop Middle 2 (April), RM1: Cape Recife Middle 1 (April), RM2: Cape Recife Middle 1 (April), SL: Schoenmakerskop Lower (April), RL: Cape Recife Lower (April).

NIHMS1732479-supplement-Figure_S2.eps (4.2MB, eps)
Supplementary-Experimental Procedure
NIHMS1732479-supplement-Supplementary-Experimental_Procedure.docx (19.1KB, docx)
Tabe S1

Table S1. Characteristics and taxonomic classifications of genomes bins from the upper, middle, and lower stromatolite formations from Cape Recife and Schoenmakerskop. Taxonomic classification is provided in both GTDB-Tk format and equivalent NCBI classifications.

NIHMS1732479-supplement-Tabe_S1.xlsx (52.2KB, xlsx)
Table S2

Table S2. Conserved bacterial species are defined by shared ANI greater than 97% in stromatolite formations from Cape Recife and Schoenmakerskop. Shared ANI is provided of bin A relative to bin B, and bin B relative to bin A.

NIHMS1732479-supplement-Table_S2.xlsx (37.8KB, xlsx)
Figure S3
NIHMS1732479-supplement-Figure_S3.eps (4.4MB, eps)
Table S3

Table S3. Summary of gene copies of phosphate, nitrogen, and sulfate transport and metabolism genes annotated on contigs clustered into genome bins from Schoenmakerskop and Cape Recife stromatolite formations.

NIHMS1732479-supplement-Table_S3.xlsx (225.9KB, xlsx)

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