Documenting the Occurrence of Microbial Carbonate Deposits in Puerto Rico

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Rodríguez-Colón, Wilson R. Ramírez-Martínez, Aliyah M. Chabrier-Alpi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8570072/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Microbial carbonate deposits, including microbial mats and microbialites, record coupled biological, geochemical, and sedimentary processes, providing insight into biosedimentary dynamics through Earth’s history and serving as analogs for ancient and potentially extraterrestrial environments. This study presents the first documentation of lithifying microbial systems in Puerto Rico, identified within three proximal coastal lagoons (Salinetas, Vernales, and Providencia) along the southwestern coast of the Guánica municipality. These lagoons define a continuum of lithification, ranging from non-lithifying hypersaline mats to fully lithified microbialite deposits. An integrated field, geochemical, petrographic, and microbial ecological approach was used to characterize deposit morphology, mineralogy, and microbial community structure. At Salinetas, elevated salinity, low pH, and evaporitic conditions correspond to halite- and gypsum-dominated mats hosting halophilic Archaea and anoxygenic phototrophs, with minimal carbonate precipitation. In Vernales, carbonate precipitates closely associated with microbial filaments and EPS indicate enhanced organomineralization. Providencia hosts extensive lithified deposits displaying composite stromatolitic and thrombolitic fabrics, localized occurrences of high-Mg carbonate phases, and microbial communities enriched in sulfate-reducing bacteria relative to the other lagoons. Collectively, variations in hydrology, salinity, substrate, and microbial community composition correspond to differing degrees of lithification across a small coastal region, establishing southwestern Puerto Rico as a natural laboratory for investigating microbialite formation and biosignature preservation in coastal carbonate systems. Earth and environmental sciences/Biogeochemistry Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Earth and environmental sciences/Ocean sciences Earth and environmental sciences/Solid earth sciences Microbial Carbonates Microbial Mats Microbialites Puerto Rico Guánica Mg-carbonates Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Modern microbial carbonate mineral deposits, including microbial mats and microbialites, provide valuable insights into the geobiological evolution of Earth [ 1 ] . Microbialites, including laminated stromatolites and clotted thrombolites, form through the lithification of microbial mats via sediment trapping, binding, and in situ organomineralization (i.e., microbially induced mineralization) [ 2 , 3 ] . Although microbialites were widespread during the Precambrian, with the oldest examples dating to ca. 3.5 Ga, their abundance declined markedly through the Phanerozoic [ 1 , 4 ] . Today, unlithified microbial mats (vertically laminated biofilms composed of diverse microbial communities) are common in shallow aquatic environments globally, yet only a subset undergoes sustained lithification, making modern microbialites comparatively rare [ 5 ] . This disparity highlights a fundamental and unresolved question: why do some microbial mats lithify while others remain unlithified under broadly similar environmental conditions? Because modern microbialites preserve coupled biological, mineralogical, and geochemical signatures, they also serve as valuable analogs for interpreting ancient biosedimentary systems and potential biosignatures in extraterrestrial settings, including Mars (e.g., [ 6 , 7 ] ). Previous research from microbialite-forming environments has improved understanding of mechanisms governing organomineralization within these systems [ 8 – 16 ] . These processes reflect complex interactions among microbial community composition, metabolic activity, extracellular polymeric substances (EPS), and physicochemical conditions such as salinity, pH, mineral saturation state, and water chemistry, as well as broader environmental influences operating across geomorphic and sedimentary contexts [ 3 ] . Despite these advances, the relative importance and interaction of microbial processes, aqueous geochemistry, and environmental forcing remain incompletely resolved. Continued characterization of lithifying microbial systems, particularly in new or understudied settings, is therefore necessary to better understand why some microbial mats undergo lithification while others remain unlithified. Several microbial carbonate environments have been studied within coastal lagoons across the circum-Caribbean region, including sites in the Bahamas (e.g., Storr Lake, San Salvador Island, and Big Pond, Eulethera Island) [ 8 , 17 ] , Turks and Caicos (e.g., Windsor Point Salt Pond) [ 18 ] , Venezuela (e.g., Laguna Pirata, Los Roques Archipelago) [ 19 ] , Cuba (e.g., Cayo Cocos and Sabana-Camagüey Archipielago) [ 20 , 21 ] , and Bonaire (e.g., GotoMeer Basin) [ 22 ] . In Puerto Rico, previous studies have focused on non-lithifying benthic and ephemeral microbial mats in hypersaline lagoons in Cabo Rojo [ 23 , 24 ] . However, actively lithifying microbial mats and microbialite deposits have not previously been described from other regions of the island. This study documents, for the first time, the occurrence, internal structure, mineralogy, and microbial community composition of lithifying microbial deposits in Puerto Rico. This work focuses on three coastal lagoons in southwest Guánica Municipality: Salinas Vernales (characterized by calcifying microbial mats), Salinas Salinetas (where microbial mats show minimal to no lithification), and Laguna Providencia (hosting fully developed microbialites). Together, these lagoons represent a spectrum of lithification states and composition, offering a valuable opportunity to examine the environmental and microbial factors associated with microbial mat lithification and microbialite development in dynamic coastal settings. Geologic Setting and Field Site Description The study sites are three coastal lagoons near the town of Guánica, along the southwestern coast of Puerto Rico (Fig. 1 a). The bedrock surrounding the lagoons is composed primarily of a Miocene-age unit named Ponce Limestone, a bioclastic packstone–wackestone unit that is partially dolomitized [ 25 , 26 ] (Fig. 1 b). Dolomitization in Ponce Limestone is dated to ca. 10.21–6.83 Ma [ 26 ] , with dolomite-rich outcrops occurring immediately adjacent to the lagoon margins. Active tectonism has been documented in the region via the Punta Montalva fault (Fig. 1 b), a left-lateral strike-slip fault system (trending ca. 110˚-115˚) that passes ca. 150–200 m north of the lagoons, with notable activity during a 6.4 M w seismic event in January 2020 [ 27 , 28 ] . Quaternary beach deposits isolating the lagoons from the Caribbean Sea are composed of modern coral and calcareous algal bioclasts (e.g., Halimeda sp. ) and lithoclasts of Ponce Limestone [ 25 ] . The studied lagoons have mesosaline to hypersaline conditions most of the year and have been historically important for salt production in the area, due to relatively low annual rainfall in the southwest of Puerto Rico, ranging from 600 to 1,000 mm, and mean temperatures exceeding 26°C [ 29 , 30 ] . Salinas Vernales, the smallest lagoonal system ( ca. 15,330 m²), comprises small, abandoned artificial solar salterns ( ca. 0.2–0.35 m depth) adjacent to Playa de Los Almendros (Fig. 1 b-c). Construction of the salterns is evidenced by subdivided crystallizer ponds, stoned dike walkways, and anthropogenic debris in the area (Fig. 1 c-d; Supplementary Fig. S1 a online). It is unclear whether a natural pond existed prior to the development of the salterns; however, historical aerial imagery shows that the southernmost pond (hereafter SV1) did not exist before the 1930s. Salinas Salinetas is an intermediate-sized natural hypersaline system ( ca. 41,380 m²) with minimal human modification (Fig. 1 f). Historical documentation shows, however, that this system was also used for salt production, although in a lower capacity than the others from the area. This system comprises three interconnected ponds, positioned between Salinas Vernales and Laguna Providencia, and adjacent to Playa La Jungla (Fig. 1 b). Laguna Providencia is the largest ( ca. 0.25 km²), deepest lagoon ( ca. 1.2–1.3 m depth south, ca. 0.8 m north) in the study area (Fig. 1 i). The lagoon contains several anthropogenic modifications, including an artificial canal cut along the southwestern margin ( ca. 150 m long) constructed between the late 19th and early 20th centuries, and abandoned solar salterns along the eastern side near Playa Santa neighborhood (Fig. 1 b). Results Physicochemical Field Measurements, Distribution, and Macro Morphology of Microbial Deposits Salinas Vernales. Calcifying microbial mats in Salinas Vernales were restricted to SV1, which is bordered by mangroves and covers ca. 1,600 m² of the lagoonal system (Fig. 1 c; Supplementary Fig. S1 a online). Preliminary aqueous measurements from SV1 indicate slightly alkaline conditions (pH ca. 8.3) with low salinity ( ca. 25.4 ppt) and conductivity ( ca. 39.4 mS), contrasting with higher salinity ( ca. 78.5 ppt) and lower pH ( ca. 6.8) in the adjacent northern pond (hereafter SV2), despite similar alkalinity values (Table 1 ). Microbial deposits in SV1 occur as isolated hemispheroidal mounds, coalescent mound clusters, and laterally continuous crusts up to ca. 15 m in length (Fig. 1 d; Supplementary Fig. S1 a online). Most deposits preferentially developed on hardened calcrete substrates, whereas areas lacking firm substrate were dominated by organic-rich ooze and lacked microbial mats. Polygonal and flat microbial mats occurred locally near pond margins, including zones with small pinnacle-like protrusions (Supplementary Fig. S1 e online). In contrast, adjacent crystallizer ponds to the north (SV2–SV6) hosted only sparse, desiccated biofilms and lacked comparable deposits. A representative mat from the eastern margin of SV1 (hereafter SV1-E) was selected for petrographic, microscopic, and microbial diversity analyses (Fig. 1 e). Table 1 Physicochemical data collected across the lagoons. Lagoon Sample Name pH Temperature (˚C) Specific Conductivity (mS) Salinity (ppt) Alkalinity (meq L − 1 ) Salinas Vernales (SV1-SV2) SV1 8.34 30.4 39.4 25.4 3.1 SV2 6.78 34.4 111.3 78.5 3.1 Salinetas (SS) SS-NW 5.32 34.5 110.3 77.8 3.2 Providencia (LP) LP19 7.33 32.3 80.6 55.3 2.4 LP19-Flow 7.1 37.2 30.6 19.3 3.9 Salinas Salinetas. The northwestern pond (hereafter SS-NW) hosted the most extensive microbial mat development within Salinas Salinetas, with pink-pigmented mats forming atop evaporitic mineral crusts across ca. 3,750 m² (Fig. 1 f–g; Supplementary Fig. S2a–c online). Waters in SS-NW were strongly saline and slightly acidic (salinity ca. 77.8 ppt; pH ca. 5.3; Table 1 ). Although petrographic analyses were not conducted on these deposits, SS-NW mats were analyzed for microbial community composition. Other ponds within Salinas Salinetas contained only sparse or discontinuous microbial mats, including small chip-like mats on shell fragments and mats enriched in detrital Halimeda bioclasts (Supplementary Fig. S2 online). Laguna Providencia. The most spatially extensive and lithified microbial deposits were documented in Laguna Providencia, where microbialites form a west-east-trending buildup extending ca. 350 m across the central sector of the western lagoon (Fig. 1 i; Supplementary Fig. S3a online), with only sparse, small microbialites occurring along limited portions of the northern and southern shores. Surface waters exhibited near-neutral to slightly alkaline pH ( ca. 7.1–7.3), moderate salinity ( ca. 55 ppt), and low alkalinity ( ca. 2.4 meq L⁻¹), whereas a localized inflow from the Ponce Limestone showed lower salinity ( ca. 19 ppt) and higher alkalinity ( ca. 3.9 meq L⁻¹; Table 1 ). Drone-derived orthomosaics and field observations identify two dominant microbialite growth forms within the buildup: laterally continuous clustered microbialite accumulations ( ca. 1–2 m) and more isolated mound-like structures (Supplementary Fig. S3b-c, e-f online). Clustered forms are more prevalent toward the western portion of the buildup, whereas isolated structures dominate toward the eastern margin and former crystallizer zones. Microbialite heads decrease in size from west to east, and locally form linear to curvilinear alignments several meters in length along the southern portion of the buildup (Supplementary Fig. S3d online). Preservation ranges from partially lithified to sub-fossilized structures, with some microbialites supporting active pustular mats at their upper surfaces (Fig. 1 k; Supplementary Fig. S4a). A representative microbialite from the western buildup (hereafter LP19) was selected for petrographic, mineralogical, and microbial analyses. Internal Structure, Microfabrics, and Mineralogy Salinas Vernales (SV1-E). Microbial mat from Salinas Vernales (SV1-E) exhibits clear vertical stratification, with thin pigmented surface layers grading into darker basal zones (Fig. 1 e; Supplementary Fig. S1 d,f). The mat fabric consists of alternating micritic and biofilm-rich laminae (Fig. 2 a-b), locally incorporating micritic peloids and spherulitic grains ( ca. 50–400 µm) with micritic nuclei and fibrous to botryoidal carbonate overgrowths. Sparse detrital components, including Halimeda spp. fragments and bivalve material, are present but volumetrically minor (Supplementary Fig. S5a online). SEM imaging shows abundant microbial filaments embedded within EPS matrices, closely associated with nanogranular to subhedral micritic precipitates, including small rhombohedral and trigonal aggregates attached directly to filaments (Fig. 3 a-b; Supplementary Fig. S6a-b online). Larger spherulitic grains display fibrous internal textures and radial acicular growth. XRD analyses indicate that SV1-E mats are dominated by aragonite and high-Mg calcite ( ca. 16.6 mol% MgCO₃), with Mg content increasing with depth (Fig. 4 a). Salinas Salinetas (SS-NW). Microbial mats from Salinas Salinetas (SS-NW) display laminated fabrics broadly similar to those observed in SV1, but are distinguished by the presence of prominent pink pigmented layers above and below the crust and a lack of carbonate lithification (Fig. 1 g-h). No carbonate precipitates were observed within these mats; instead, evaporitic minerals dominate, including gypsum and a thick halite crust that serves as the primary substrate (Fig. 1 g; Supplementary Fig. S2b). SEM observations reveal embedded gypsum crystals, pennate diatom frustules, and dispersed microbial filaments within the mat matrix (Fig. 3 c-d). XRD analyses confirm gypsum and halite as the dominant mineral phases, with only minor aragonite and high-Mg calcite ( ca. 27.1 mol% MgCO₃) detected in basal layers (Fig. 4 b). Laguna Providencia (LP19). The Laguna Providencia microbialite displays composite fabrics and extensive calcification. At the surface, pustular microbial mats form a thin crust composed of pigmented layers overlying a darker organic-rich zone, which in turn overlies lithified interior intervals and a basal layer (Fig. 1 k). Sub-fossilized portions are dominated by thrombolitic textures in upper regions, characterized by clustered mesoclots, whereas deeper intervals contain irregular stromatolitic laminae (Supplementary Fig. S4 online). Spherulitic grains are abundant throughout the microbialite, occurring as small forms ( ca. 10–25 µm) within microbial laminations in surface mats and as larger spherulites associated ( ca. 250 µm) with EPS-rich zones and occasional bioclasts (Fig. 2 c-d; Supplementary Fig. S5b-d online). Petrographic analyses show thrombolitic clots composed of spherulitic peloids, microspar, and acicular carbonate overgrowths (Fig. 2 e), whereas stromatolitic intervals display irregular micrite-rich laminae (Fig. 2 f). Fluorescence microscopy reveals strong signals in spherulite nuclei with weaker fluorescence along rims and overgrowths (Supplementary Fig. S5c–f online). Mineralogical staining further indicates pervasive Mg-calcite within spherulitic peloids and micritic laminae, with minor aragonite cements localized to rims and void-filling phases (Supplementary Fig. S5g online). SEM observations document spherulites with dense micritic nuclei and outward botryoidal aragonite growth, along with centric diatom valves, framboidal pyrite, putative amorphous Mg-silicates in deeper layers, and locally developed aragonite needles associated with organic matter (Fig. 3 e–f; Supplementary Fig. S6c–d online) XRD analyses indicate a mineralogical progression from surface gypsum to aragonite and high-Mg calcite in intermediate layers, with the black organic-rich zones hosting the most diverse assemblages, including low-Mg calcite, high-Mg calcite, and very high-Mg calcite (VHMC; ca. 38.8 mol% MgCO₃), commonly associated with loosely consolidated gray sediment (Fig. 4 c; Fig. 5 ). Stable Isotope Geochemistry Carbonate δ¹³C values from LP19 range from − 4.6‰ to -1.6‰, and δ¹⁸O values from + 0.9‰ to + 1.4‰ (Fig. 5 ; Supplementary Table S1 online). Dolomitic samples from nearby Ponce Limestone samples (Supplementary Fig. S7 online) display δ¹³C values between − 0.81‰ and + 1.38‰ and relatively enriched δ¹⁸O values from + 1.05‰ to + 1.96‰ (Supplementary Table S1 online). In contrast, calcitic Ponce Limestone samples show the most depleted compositions, with δ¹³C values from − 9.79‰ to -6.31‰ and δ¹⁸O values from − 4.90‰ to -3.53‰. Beach sediments exhibit δ¹³C of -0.08‰ and moderately depleted δ¹⁸O (-2.59‰). Microbial Community Composition Alpha diversity analyses revealed pronounced differences in richness and evenness across lagoons and mat layers (Supplementary Table S2). Microbialites from Laguna Providencia exhibited the highest diversity, with bottom and brown layers yielding over 1,200 observed ASVs and Shannon indices > 5.5, whereas the basal layer of Salinas Salinetas showed markedly reduced diversity (Observed = 348 ± 313; Shannon = 2.61 ± 1.34). Surface layers from SV1-E and SS-NW displayed intermediate diversity (Shannon ca. 4.9). Venn analyses indicate substantial lagoon-specific ASV pools, with only 316 ASVs shared across all systems and limited pairwise overlap, particularly between SV1-E and SS-NW (Fig. 6 a). Beta-diversity analyses reveal clear clustering by lagoon (Fig. 6 b), with tight grouping of LP19 samples and distinct ordination space occupied by SV1-E and SS-NW. These differences are statistically significant (PERMANOVA: pseudo-F = 5.08, R² = 0.31, p = 0.001), indicating strong spatial structuring of microbial communities across lagoons. Salinas Vernales (SV1-E). Bacterial taxa dominated upper mat layers (64.69–98.47%) and decreased with depth, whereas Archaea exhibited the opposite trend (1.53–35.31%; Supplementary Table S2). Surface crust and green layers were dominated by Pseudomonadota (20.67–35.38%), followed by Planctomycetota (10.40-12.02%), Bacteroidota (7–11%), and Spirochaetota (3.65–7.36%) (Fig. 6 c). Chloroflexota (order Aggregatilineales ) were enriched in crust and black layers (18.27% and 13.52%). Cyanobacteria (primarily Desertifilaceae ) occurred in the crust (1.17–8.23%; Supplementary Fig. S8a online). Thermodesulfobacteriota ranged from 4.15% in the green layer to 9.07% in the black layer. Sulfate-reducing bacteria (SRB) included Desulfomonilaceae (enriched in red layers) and Desulfohalobiaceae (more abundant at depth; Supplementary Fig. S8c online). Archaeal groups included Nanoarchaeota (1.02–3.94%) and Hadaarchaeota (up to 2.66%). Methanogenic Archaea ( Methanobacteriota ), particularly unclassified Methanofastidiosales , peaked in the black layer (up to 14.03%; Supplementary Fig. S8d online). Salinas Salinetas (SS-NW). Prokaryotic communities in SS-NW were dominated by Bacteria (54.10-98.14%), but the microbial mats from this lagoon exhibited the highest relative abundance of Archaea (1.86–45.90%; Supplementary Table S2 online). Pseudomonadota and Rhodothermota dominated surface crust and green layers (18.31–20.77% and 27.50-32.44%, respectively; Fig. 6 c). Cyanobacteria ranged from 2.56–5.91% and included Rubidibacteraceae and Geitlerinemaceae (Supplementary Fig. S8a online). Purple non-sulfur bacteria (PNSB; genus Rhodovibrio ) were also abundant in crust and green layers (8.78–11.63%; Supplementary Fig. S8b online). Deeper pink and black layers were enriched in Acetothermia (6.38–40.41%), Acidobacteriota (36.20%), Actinomycetota (9.87%), and Bacillota (5.47%). Archaea were dominated by Halobacteriota (up to 28.07% in the crust), and methanogens, especially Methanofastidiosales , were more abundant here than in other lagoons, reaching 43.15% in the black layer (Supplementary Fig. S8d online). Laguna Providencia (LP19). The microbialite in Laguna Providencia showed the highest overall diversity but lacked clear vertical stratification (Fig. 6 c). Bacterial relative abundance decreased modestly with depth (84.66% to 64.31%), while Archaea increased (15.34–9.70% in crust to 24.10-44.98% at depth; Supplementary Table S2 online). Planctomycetota were consistently abundant (13.47–26.15%), Pseudomonadota were most represented in the crust (13.48–19.19%), and Chloroflexota ( Aggregatilineales ) increased slightly with depth (9.26–10.19%). Cyanobacteria were present but low in abundance (1.52–2.03%), represented by Rubidibacteraceae (Supplementary Fig. S8a online). SRB included Desulfohalobiaceae (up to 6.33% in brown layers) and Desulfatiglandaceae (up to 3.06%; Supplementary Fig. S8c online). Archaea included Nanoarchaeota (4.52–6.64%), Hadaarchaeota (2.94–7.63%), and methanogenic taxa such as Methanofastidiosales (up to 18.04% in black layers; Supplementary Fig. S8d online). Discussion The three lagoonal systems examined in this study define a clear continuum of microbial carbonate development, ranging from non-lithifying hypersaline mats at Salinas Salinetas, through partially calcifying mats at Salinas Vernales, to fully lithified microbialites at Laguna Providencia. Although some dataset here represents a single temporal snapshot, the combined sedimentological, petrographic, and microbial evidence indicates that lithification potential varies markedly over short spatial scales within a shared coastal setting. Across all systems, microbial mats develop on physically stable substrates, indicating that substrate availability influences deposit distribution; however, substrate stability alone is insufficient to explain carbonate lithification, as demonstrated by the non-lithifying mats developed on halite crusts in Salinas Salinetas. Instead, the strongest distinction across the lithification continuum corresponds to differences in microbial community structure and diversity, which increase from Salinas Salinetas to Salinas Vernales and are highest in the lithified microbialites of Laguna Providencia. These differences likely reflect variations in microbial metabolism operating within permissive hydrological and geochemical contexts, as well as differences in relative system maturity, collectively governing whether microbial mats remain unlithified, initiate calcification, or develop into lithified microbialites. Non-Lithifying Microbial Deposits in Salinas Salinetas The absence of lithification in microbial deposits from Salinas Salinetas reflects strong geochemical and hydrological constraints that overwhelm biological influences during the observed conditions. In SS-NW, microbial mats are restricted to persistently submerged areas and colonize thick evaporitic crusts that dominate the benthic substrate (Supplementary Fig. S2b). High salinity and moderate alkalinity indicate a hydrologically restricted basin dominated by evaporative concentration (Table 1 ). Historical satellite and aerial imagery show that SS-NW frequently undergoes isolation and seasonal desiccation, and sampling near the end of the dry season (June 2019) likely captured conditions of intensified evaporation and gypsum-to-halite supersaturation. Under such conditions, carbonate dissolution likely exceeds precipitation, effectively precluding sustained lithification in this system. Although microbial communities in SS-NW are well developed, their role in carbonate formation appears secondary to these physicochemical constraints. Surface layers are dominated by halophilic Archaea and PNSB (e.g., Rhodovibrio sp. ), consistent with the observed pink pigmentation and a shift toward anoxygenic phototrophy under hypersaline conditions. Similar salinity-driven transitions toward anoxygenic phototrophy have been documented in other hypersaline microbial mats [ 31 ] . While Cyanobacteria are present, the dominance of anoxygenic phototrophs and sulfide-oxidizing taxa likely promotes net carbonate dissolution through CO₂ and proton release [ 32 , 33 ] . Minor occurrences of high-Mg calcite and aragonite in basal layers (Fig. 4 b) may reflect localized, transient supersaturation, potentially linked to methanogenic activity, which can increase alkalinity by consuming organic acids and CO₂ [ 34 , 35 ] . However, the absence of lithification across most of the Salinas Salinetas system, including other basins, indicates that any carbonate precipitation is spatially restricted, short-lived, and insufficient to overcome basin-scale evaporative and geochemical controls. Calcifying Microbial Mats in Salinas Vernales Unlike the non-lithifying mats at Salinas Salinetas, SV1 hosts actively calcifying microbial mats under physicochemical conditions more favorable for carbonate precipitation. Field measurements indicate lower salinity and more alkaline conditions in SV1 relative to SV2, which exhibits geochemical characteristics more similar to Salinas Salinetas (Table 1 ). These comparatively lower salinities may support more diverse and metabolically active microbial communities, including taxa and guilds directly involved in organomineralization, thereby enhancing lithification potential. Although short-term dilution from rainfall, surface runoff, or localized subsurface inputs may contribute to this contrast, the proximity of SV1 to the shoreline and lack of persistent freshwater surface inflow indicate a system fundamentally influenced by seawater. Over time, evaporation likely concentrates ions to levels permissive of carbonate supersaturation; however, the consistently lower salinities observed at SV1 relative to SV2 suggest that additional hydrological inputs, potentially including subsurface water sources, warrant targeted geochemical and hydrological investigation. Under these conditions, calcifying microbial mats preferentially colonize hard calcrete surfaces (Supplementary Fig. S1 a-b online). The distribution of hemispheroidal mounds in central areas and polygonal mats along pond margins likely reflects gradients in water depth and exposure frequency (Supplementary Fig. S1 a online), similar to patterns described from the Turks and Caicos [ 36 ] . Polygonal textures likely formed during episodes of shallow inundation and periodic exposure, whereas hemispheroidal mounds represent more mature structures developed under longer-lived submergence, although confirmation of this sequence will require targeted geomorphic and hydrological analyses. Microscopically, SV1-E contains abundant micritic carbonate closely associated with EPS (Figs. 2 a, 3 a), including trigonal crystals comparable to those described from Lagoa Vermelha, Brazil [ 16 ] , Big Pond [ 17 ] , and Mérantaise River, France [ 37 ] . SEM imaging reveals small ( ca. 5 µm) spherulites nucleating on or adjacent to microbial filaments (Fig. 3 b), consistent with organomineralization either within porewaters or directly on EPS or filament surfaces [ 3 ] . Larger spherulites with micritic nuclei and sparry aragonite rims (Fig. 2 b) resemble carbonate grains from other microbial calcifying systems ( [e.g., 16,17,38,39] ). The micritic nuclei likely reflect microbial micrite or partially micritized detrital particles incorporated during mat accretion. These observations indicate that carbonate production in SV1 is predominantly authigenic and microbially mediated or early diagenetic overgrowths, with minor contributions from detrital material, similar to textures documented in Abu Dhabi sabkha mats, where diagenetic aragonite rims overgrow earlier micritic nuclei, and in microbialites from the Great Salt Lake, USA, where organomineralization dominates early stages of precipitation [ 39 ] . Microbial community patterns in SV1-E are consistent with a biogenic contribution to carbonate precipitation. Filamentous Cyanobacteria (represented in this case by Desertifilaceae ) dominate the crust and green layers and are known to promote carbonate nucleation by generating EPS during photosynthetic CO₂ uptake [ 35 ] . Carbon fixation through photosynthesis can increase alkalinity, and at the same time, EPS acts as a trap for sediment particles and nucleation sites, provided that its cation-binding capacity does not inhibit mineral formation [ 3 ] . SEM imaging of filamentous structures within the green layer (Supplementary Fig. S6a online) provides visual support for their presence. Cyanobacteria are also well-documented contributors to microbial deposit morphotypes, and their elevated relative abundance in SV1-E compared to the other lagoons may help explain documented morphologies such as the observed pinnacle-like structures in some of the microbial mats (Supplementary Fig. S1 e online). Deeper layers also host SRB (e.g., Desulfohalobiaceae , Desulfomonilaceae ) and methanogens (e.g., Methanofastidiosales ), whose metabolism of organic matter increases HCO₃⁻, further promoting carbonate supersaturation [ 35 ] . Although we cannot directly quantify the relative contributions of these guilds, the combined presence of filamentous Cyanobacteria, SRB, and methanogens suggests multiple metabolic and EPS-mediated pathways that likely contribute to the micritic and spherulitic textures observed in SV1. Microbialite Deposits in Laguna Providencia Among the three studied lagoons, Laguna Providencia exhibits the most extensive lithified microbial deposits and, to our knowledge, represents the first formally documented microbialite buildup in Puerto Rico (Supplementary Fig. S3 online). This greater extent and degree of lithification likely reflect a longer period of microbialite development relative to Salinas Vernales and Salinas Salinetas, as well as differences in present-day physicochemical conditions, a higher microbial community diversity, and local accommodation space. SfM-derived DEM data indicate that the western and northern sectors of the lagoon correspond to shallow, low-gradient zones consistent with laterally extensive microbialite accretion, whereas a steeper slope along the southern margin may have limited microbialite development in that direction (Supplementary Fig. S3a-i online). Similar slope-controlled zonation has been documented in lake microbialites from Laguna Pozo Bravo, Argentina [ 40 ] , and the Great Salt Lake [ 41 ] . Sedimentary records from Laguna Providencia (results not yet published) further indicate a complex paleoenvironmental evolution and a longer-lived lagoonal system, providing increased time for sustained microbialite accretion and maturation [ 42 ] . The central-lagoon position and localized distribution of the microbialite buildup, including meter-scale linear to curvilinear alignments, suggest a more complex hydrological history. Observations of low-salinity, high-alkalinity flows entering the lagoon from the Ponce Limestone (Table 1 ) raise the possibility of localized groundwater inputs or subtle tectonic influences associated with the nearby Punta Montalva fault [ 42 ] , although additional hydrological and geochemical data are required to evaluate these interpretations. Internally, sub-fossilized microbialites in Laguna Providencia exhibit composite mesostructures, with stromatolitic textures at the base and thrombolitic fabrics toward the top (Supplementary Fig. S4 online). Such vertical changes in fabrics likely reflect temporal shifts in environmental conditions and microbial mat structure. Comparable composite microbialites have been described in modern systems, including Laguna Pozo Bravo [ 40 ] and Shark Bay, Australia [ 43 ] , where fluctuating water levels, hydrodynamic energy, and microbial succession produce vertically stacked stromatolitic-thrombolitic sequences. In this case, the stromatolitic intervals were likely produced by filament-rich microbial mats capable of sediment trapping, binding, and in-situ organomineralization, similar to those observed in SV1. In contrast, thrombolitic textures appear consistent with coccoid-dominated pustular mats, such as those currently found at the surface of the microbialites. Genomic data support this interpretation, as pustular crust layers in LP19 show a higher relative abundance of coccoid cyanobacterial families such as Rubidibacteraceae compared to SV1-E, where filamentous cyanobacteria dominate. Shifts between filamentous and coccoid cyanobacteria have been linked to the formation of stromatolitic versus thrombolitic morphotypes in other lithifying mats [ 43 , 44 ] . One point to consider is that Cyanobacteria occur at relatively low abundance in LP19 compared to other microbial groups, a pattern also observed in SV1-E and SS-NW, but to varying degrees. Similar discrepancies between observed mat morphologies and cyanobacterial sequence abundances have been reported in microbialite studies of Laguna Bacala, México [ 45 ] , and Storr’s Lake [ 46 ] . These studies note that primer bias, differential DNA extraction efficiency, and cell-wall-dependent lysis rates can lead to underrepresentation of Cyanobacteria in amplicon datasets. Although filamentous cyanobacterial morphologies are readily observed microscopically in SV1-E (Supplementary Fig. S6a online), reconciling the low cyanobacterial signal in LP19 will require targeted microscopy, taxonomic imaging, and possibly alternative molecular approaches. Another aspect to consider is the vertical structure of the sampled microbialite. Pustular mats occur only in the surficial crust, whereas deeper black microbial assemblages occur beneath a middle-lithified horizon. This configuration may explain the limited vertical stratification observed in the 16S dataset (Fig. 6 c) and suggests that the lithified substrate, rather than the actively growing microbial mat, exerts a strong influence on current community distribution. Additional spatial and seasonal sampling across the Laguna Providencia microbialite buildup will be needed to determine whether this pattern is consistent throughout the lagoon or reflects localized conditions at the sampled site. Spherulites present within the pustular microbial mats associated with the thrombolitic microbialites (Fig. 2 c-d) transition downward into clotted spherulitic textures in sub-fossilized sections (Fig. 2 e; Supplementary Fig. S5c-d online), occasionally enclosing bioclasts (Supplementary Fig. S5b). These clotted fabrics, composed of spherulites and other unidentified peloidal grains embedded in acicular cements, closely resemble textures reported from microbialites in Lagoa Vermelha [ 16 ] , Rottnest Island in Western Australia [ 38 ] , Cuatro Cienagas in Mexico [ 47 ] , Lake Salda in Turkey [ 48 ] , and throughout the geologic record [ 1 ] . Microscopic examination of the deeper stromatolitic layers also revealed subtle sub-laminations composed of micritic and micropeloidal bands (Fig. 2 f). The increased lithification within both stromatolitic and thrombolitic fabrics likely reflects microbial activity, potentially linked to EPS degradation following burial [ 17 , 49 ] , and diagenetic processes commonly associated with the alteration of porewaters during burial, which can promote early cementation [ 39 , 50 ] . In the stromatolitic intervals, these sub-laminations may reflect successive microbial biofilms, similar to those observed in SV1-E, that underwent progressive infilling by micritic precipitates. Within thrombolitic intervals, fluorescence microscopy and mineralogical staining together indicate organic-rich spherulite nuclei that likely served as initial nucleation sites for Mg-calcite micrite, followed by later aragonite precipitation forming less fluorescent rims and void-filling cements (Supplementary Fig. S5e-g) [ 16 ] . The development of these complex fabrics may therefore reflect the cumulative influence of a diverse, metabolically heterogeneous microbial community capable of sustaining multiple organomineralization and diagenetic pathways over time. These observations are in agreement with a paragenetic sequence in which Mg-calcite micrite precipitated first within organic-rich nuclei, where coccoid mats and later microbial activity during burial may have contributed to nucleation (e.g., [ 16 , 38 ] ), and aragonite then precipitated during early diagenesis. The occurrence of framboidal pyrite within LP19 (Fig. 3 f) could provide direct evidence of localized anoxia and active sulfur cycling, potentially linking sulfate-reduction activity, for example, to these paragenetic changes [ 16 , 51 ] . Although the contemporary microbial community may not represent the assemblages responsible for lithification, genomic data reveal significant populations of SRB, particularly Desulfovermiculus and Desulfatiglans , within the brown and black layers of LP19 relative to SV1-E and SS-NW (Supplementary Fig. S8c online). Putative Authigenic Mg-Carbonate Phases in Microbialites The occurrence of VHMC confined to the black layer in LP19 is an intriguing result (Fig. 4 c; Fig. 5 ). VHMC, together with high- and low-Mg calcite phases, was detected immediately beneath the middle-lithified crust within unlithified gray mud associated with the black-layer horizon and is not prominent in other LP19 microbialite layers (Fig. 4 c). This stratigraphic confinement indicates that Mg-carbonate formation was restricted to a discrete microenvironment distinct from the overlying lithified fabrics. X-ray diffraction patterns show that this VHMC lacks distinct 101 and 015 reflections and exhibits only weak 021 ordering, consistent with poorly ordered proto-dolomite rather than fully ordered dolomite (Fig. 5 ) [ 52 ] . Although some ordering peaks may be obscured by aragonite signals, the Laguna Providencia microbialite VHMC remains mineralogically distinct from the ordered dolomite of the adjacent Ponce Limestone [ 26 ] . Low-temperature dolomite formation remains a long-standing challenge in sedimentary geochemistry due to kinetic barriers to Mg incorporation under Earth-surface conditions (the “dolomite problem”) [ 53 ] . Increasing evidence from modern environments indicates that microbial activity (particularly sulfate reduction) can facilitate the formation of poorly ordered Mg-carbonate precursors that act as transient intermediates toward dolomite, as documented in microbial deposits from Lagoa Vermelha [ 54 ] , the Great Salt Lake [ 9 ] , the Abu Dhabi sabkhas [ 55 ] , and Petukhovskoe soda lake, Russia [ 15 ] . In LP19, the occurrence of VHMC within organic-rich gray sediment coincides with elevated SRB abundances relative to the other lagoons, consistent with microbially influenced organomineralization pathways reported in these systems. The presence of amorphous Mg-silicate phases within this horizon (Supplementary Fig. S6c online) suggests an additional formation pathway, whereby silicate- or clay-related precursors contribute to Mg incorporation and nucleation, in agreement with models from other microbial and evaporitic environments [ 55 , 56 ] . Mineralogical, stratigraphic, and isotopic distinctions collectively support a predominantly authigenic origin for the VHMC (Fig. 5 ). Although both the microbialites and the Ponce Limestone exhibit relatively enriched δ¹⁸O values, carbonates from the Ponce Limestone record a multi-stage diagenetic history, with secondary dolomite and calcite displaying δ¹³C-δ¹⁸O compositions that remain isotopically distinct from the gray sediment hosting VHMC [ 26 , 57 ] . In contrast, depleted δ¹³C values in the microbialites are consistent with carbonate precipitation influenced by organic-matter degradation under reduced conditions [ 8 , 17 ] . Attempts to isolate the VHMC using dilute acetic acid resulted in dissolution, consistent with a poorly ordered and metastable Mg-carbonate rather than an ordered dolomite phase. Nevertheless, the isotopic signal likely reflects a mixed carbonate assemblage, and in the absence of porewater geochemistry or direct evidence of microbial nucleation, detrital input from Ponce Limestone dolomite cannot be excluded; accordingly, the VHMC occurrence should be regarded as suggestive rather than conclusive. Concluding Remarks This study documents the first known occurrence of actively lithifying microbial mats and microbialites in Puerto Rico, expanding the inventory of microbial carbonate systems within the Caribbean and providing new insight into why some microbial mats undergo lithification while others do not. The three lagoons examined represent distinct positions along a lithification continuum shaped by the interaction of microbial community composition with physicochemical and hydrological context. In Salinas Salinetas, hypersaline and acidic conditions correspond to extensive but non-lithifying mats; in Salinas Vernales, more favorable conditions support active organomineralization, including micritic precipitation and spherulite development; and in Laguna Providencia, lithified microbialites preserve composite stromatolitic and thrombolitic fabrics and localized high-Mg carbonate phases indicative of microbially influenced early diagenesis. Together, these systems indicate that while substrate stability influences the spatial distribution of microbial deposits, sustained carbonate lithification is most closely associated with the development of diverse and compositionally distinct microbial communities operating within permissive hydrological and geochemical settings, potentially amplified over longer periods of system maturity. Although porewater chemistry, metabolic rates, and temporal variability were not directly quantified, the close spatial coexistence of non-lithifying, partially lithifying, and fully lithified deposits within a small coastal region establishes the Guánica lagoons as a valuable natural laboratory for investigating microbial carbonate formation and biosignature preservation. In addition, the occurrence of poorly ordered Mg-carbonate phases within the Laguna Providencia microbialites bears directly on the low-temperature dolomite problem, indicating that microbialite-hosted environments can generate transient Mg-carbonate precursors distinct from detrital or diagenetic dolomite in adjacent carbonate units and providing a framework for distinguishing biogenic vs abiotic signals in both terrestrial and putative extraterrestrial carbonate environments (e.g., [ 6 ] ). Given the uniqueness and vulnerability of these coastal systems, coordinated protective measures and educational initiatives are warranted to limit anthropogenic impacts. Responsible management of these lagoons is essential not only to safeguard a unique coastal ecosystem in Puerto Rico and the Caribbean, but also to preserve globally relevant analogs for understanding Earth’s early biosphere and the search for life beyond our planet. Methods Fieldwork and Sample Collection The microbial deposits described in this study were first documented during exploratory fieldwork in 2017, with initial observations focusing on the calcifying microbial mats from Salinas Vernales and non-lithifying microbial mats in the southeastern pond of Salinas Salinetas (SS-SE). Subsequent fieldwork in 2019 and 2020 expanded documentation to include the other systems in Salinas Salinetas (SS-C and SS-NW) and the microbialites in Laguna Providencia. The microbial community datasets presented here primarily derive from the 2019 fieldwork. All samples collected in 2019 were secured in sterile sampling bags, stored in a fridge during fieldwork, transported under sterile conditions to the Microbial Geochemistry Laboratory at the University of Kansas, and stored at -80°C until further analysis. Samples were dissected and divided based on pigmentation differences across each microbial deposit. Microbialite buildup observations and the nomenclature of the microbialite structures were denominated following the terminology established by Grey and Awramik [ 58 ] . Ponce Limestone isotope data were added from Padilla-Montalvo [ 26 ] (Supplementary Table S1 online), alongside additional Ponce Limestone and beach sand samples collected for this study. Water temperature, pH, conductivity, and salinity were measured directly in the field using a handheld multiparameter probe (AquaRead Ltd.). For alkalinity analyses, surface water samples were collected in the field, filtered through 0.20 µm syringe filters, and total alkalinity was determined by acid titration to a pH endpoint of 4.3 shortly after collection. Drone Imagery Orthomosaic and multispectral drone imagery were acquired over the three lagoon systems and surrounding landscapes during the 2019 field season. Visible-spectrum (RGB) imagery was collected using a DJI Phantom 4 Pro V2 unmanned aerial vehicle equipped with a 20-megapixel RGB camera (1-inch, 2.54 cm CMOS sensor), with flight missions designed to provide complete spatial coverage of each lagoon and adjacent areas relevant to hydrologic and geomorphic interpretation. All flights were conducted using automated flight plans generated in DroneDeploy (version 2.202.0), incorporating a minimum of 80% forward overlap and 70% sidelap to ensure robust image matching and photogrammetric reconstruction. To minimize distortion associated with off-nadir viewing and refraction at the air-water interface, only the central, near-nadir portions of individual images were used during reconstruction. RGB and multispectral imagery were processed into georeferenced orthomosaics (GeoTIFF format) using standard structure-from-motion (SfM) workflows in DroneDeploy and Agisoft Metashape Professional (version 2.0.1), including image alignment and point-cloud generation, with no major processing artifacts observed. A relative digital surface model (DSM) was derived from the RGB imagery, representing combined topographic and shallow bathymetric variability across lagoon basins and surrounding terrain. To facilitate qualitative interpretation of bathymetric and topographic gradients, the DSM was visualized using a false-color depth ramp, with warmer colors (red) indicating relatively shallower areas and cooler colors (green) indicating relatively deeper areas (Supplementary Fig. S4a–i). Petrography and X-Ray Diffraction For petrographic analysis, select samples were impregnated with bio-epoxy and prepared as thin sections at the KU Department of Geology Rock and Thin Section Preparation Laboratory. Petrographic analysis was conducted using an Olympus BX53M optical microscope equipped with a motorized stage (©Marzhauser Wezlar) and an X-Cite 120Q for fluorescence illumination. Thin sections were analyzed under an Olympus BX53M optical microscope, and the microbialite fabrics and petrographic features were described following terminology established by Grey and Awramik [ 58 ] , and Flügel [ 59 ] . Mineralogical characterization was performed across various depths. Each underwent treatment with 30% Hydrogen Peroxide (H 2 O 2 ), air drying, and was ground for Powder X-ray diffraction analysis (PXRD). Analysis was performed using a Bruker D2 Phaser powder x-ray diffractometer (CoKᾱ radiation) equipped with a 1D mode Lynxeye detector. The analysis spanned a 2θ angle range of 5˚ to 90˚ at 0.03˚ increments every 0.3 s. A 24.6 mm x 1.0 mm Zero Diffraction Silica crystal plate (© MTI Corp) was inserted into the standard D2 Phaser sample discs and coated with grease to hold the powder. In some samples, ©FischerBrand Sodium Chloride [NaCl] was added, and the halite [200] peak 2Q was used for peak alignment of calcite and dolomite [104] reflection displacements [ 60 ] . Scan results were processed (change to CuKᾱ spectra, background fitting, and alignment) and normalized for comparison using the R “powdR” package for XRD analysis [ 61 ] . The mole percentage of MgCO3 content in the Mg-carbonate precipitates was quantified using the Rietveld method (with the equation Mg% = 100 – (333.33x -911.99) [ 60 ] . Scanning Electron Microscopy and EDS Microbial mats and Microbialite fragments were fixed in 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 30 min, rinsed twice in buffer, and post-fixed in 1% osmium tetroxide for 1 h. Samples were dehydrated in a graded ethanol series (70%, 95%, and 100%) and dried using either critical point drying (CPD; Bal-Tec K850 Critical Point Drier) or hexamethyldisilazane (HMDS) substitution. After drying, samples were mounted on 12.7 mm aluminum stubs with conductive silver paint. Some samples were sputter-coated with ~ 10 nm of iridium or gold using a Quorum 150RS sputter coater, while others were analyzed without coating. For uncoated preparations, variable pressure SEM was used, or the samples were treated with Hitachi ionic liquid (diluted at 10–15%) HILEM© to improve surface conductivity. In addition, selected microbialite samples from Laguna Providencia were examined directly from polished petrographic thin sections without coating. Conventional imaging was performed with a Hitachi S-4700 cold field emission Scanning Electron Microscope (SEM), upper and lower secondary electron detectors and an Oxford Instruments X-Max 150 EDX detector. Images were collected at 5–10 kV accelerating voltage, 5–10 µA emission current, condenser lens at 2, and 9–12 mm working distance. Energy-dispersive X-ray spectroscopy (EDS) was carried out at 10 kV using Aztec software (Oxford Instruments) to map the distribution of major elements within precipitates and biofilms. Variable pressure imaging was conducted with a Hitachi FlexSEM 1000 II SEM at 30 Pa chamber pressure, 5 kV accelerating voltage, and 10 mm working distance, also coupled to an Oxford EDX system. The critical point drier, the sputter coater, and the imaging on the S-4700 SEM/EDS were performed at the Microscopy and Analytical Imaging Research Resource Core Laboratory, University of Kansas. Imaging on the FlexSEM was performed at the Nanofabrication Facility, University of Kansas. DNA extraction, sequencing, and processing Microbial DNA was extracted in duplicate from each depth interval using the DNeasy PowerSoil Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. Extracted DNA was used for PCR amplification of prokaryotic communities (Bacteria and Archaea). Primers 515F-Y [ 62 ] and 806R-Y [ 63 ] were used to target the V4 hypervariable region of the 16S rRNA gene. PCR reactions included a mock community composed of bacterial and archaeal taxa in known concentrations as a quality control. Amplicon sizes were verified by gel electrophoresis. PCR products were purified using AmpureXP beads (Beckman Coulter, Brea, CA, USA), and dual-indexed libraries were prepared with Illumina Nextera® XT v2 indices (Illumina, San Diego, CA, USA) following the Illumina 16S Metagenomic Sequencing Library Preparation protocol. The pooled libraries were sequenced on an Illumina MiSeq platform using a paired-end 300-cycle kit at the University of Kansas Genome Sequencing Core (Lawrence, KS). The raw sequencing data for this dataset were processed using Quantitative Insights into Microbial Ecology2 (QIIME2–amplicon-2024.10) [ 64 ] . Demultiplexed reads were trimmed using the cutadapt plugin, where primer sequences were removed, and the DADA2 plugin was used to denoise and dereplicate sequences, infer ASVs, and filter chimeras. Taxonomy was assigned with the SILVA v138.2 reference database [ 65 ] . The data were normalized using cumulative-sum scaling (CSS) implemented in the R (v3.6.3) package metagenomeSeq [ 66 ] . The normalized dataset was further analyzed in R using the phyloseq package for community composition and diversity metrics, with additional analyses and visualizations performed using R packages ggplot2 and supporting packages such as dplyr and tidyr [ 67 , 68 ] . Alpha diversity metrics, including Observed Species, Shannon, and Simpson indices, were calculated to assess microbial diversity using the estimate_richness function in the phyloseq R package [ 67 ] . To analyze beta diversity, Beta diversity was assessed using Bray–Curtis dissimilarities calculated from normalized abundance data in phyloseq. Principal Coordinates Analysis (PCoA) was performed with the ordinate function, and ordination plots were generated in ggplot2 [ 67 , 68 ] . Differences in microbial community composition across lagoons and layers were statistically evaluated using Permutational Multivariate Analysis of Variance (PERMANOVA) in the vegan package [ 69 ] . PERMANOVA was conducted with the adonis2 function, using Bray–Curtis dissimilarities as the response variable and sample grouping as the explanatory variable, with 999 permutations. Results are reported as the proportion of variance explained (R²) and associated p-values. Venn diagrams were constructed with the ggvenn package and were based on the number of shared and unique amplicon sequence variants (ASVs) to visualize overlap in microbial community composition among lagoons and depositional layers [ 70 ] . Declarations Funding This research was supported by internal research funds from the University of Kansas. Drone and sUAS hardware, software, and data analysis were partially supported by Grant No. 2646 from the Unconventional Energy Center at Colorado Mesa University. Compelling Interests Authors declared no compelling interests. Author Contribution B.J.R.C. and J.A.R. conceived the study and designed the field campaign. B.J.R.C. led all field investigations and conducted the mineralogical, petrographic, XRD, SEM, stable isotope, and microbial ecology analyses; processed and analyzed all sequencing datasets; prepared all figures; and wrote the manuscript. W.R.R.M. contributed to field sampling, geological interpretation, and review of sedimentological components. A.M.C.A. assisted with fieldwork, site documentation, and field observations. C.R.V. contributed to interpretation of microbial community patterns and reviewed the microbial ecology results. N.M.R. provided SEM imaging support and technical guidance for microscopy analyses and reviewed the manuscript. G.S.B. acquired and processed drone and sUAS datasets. Y.H. generated the raw 16S rRNA gene sequencing data and provided technical support for genomic data acquisition. B.S.M.S. provided oversight of the microbial genomics component and supervised sequencing workflows. J.A.R. supervised the project as principal investigator, provided funding and logistical support, contributed to field activities, and substantially revised the manuscript. All authors reviewed and approved the final manuscript. Acknowledgement The authors thank Darien López Ocasio and the Puerto Rico Department of Natural and Environmental Resources for granting access and research permits for Laguna Providencia. Fieldwork in the Playa La Jungla region was made possible through access provided by Robert Viqueira (R.I.P.), Jorge Viqueira, Gretchen Marcial, Miguel Santiago, and the team at Protectores de Cuencas, Inc. Bryan Rodríguez-Colón, Aliyah Chabrier-Alpi, and Wilson Ramírez-Martínez gratefully acknowledge the assistance of Miguel Jordán during field campaigns, and Iremar Fernández and James Padilla-Montalvo for support during field activities and collection of Ponce Limestone samples. B. Rodríguez-Colón, J. A. Roberts, and N. Martínez-Rivera thank Eduardo Rosa-Molinar, former director of the Microscopy and Analytical Imaging Laboratory at the University of Kansas, for guidance on microscopy workflows and data processing. The authors also thank Marina Suárez and Robert Goldstein for insights on isotope geochemistry, Pike Holman for thin-section preparation, and Bruce Barnett and Mohammed Elshenawy for stable isotope data acquisition. This research was supported by internal research funds from the University of Kansas. Drone and sUAS hardware, software, and data analysis were partially supported by Grant No. 2646 from the Unconventional Energy Center at Colorado Mesa University. Data Availability Raw 16S amplicon sequencing data are available on Figshare[71]. All bioinformatics and statistical analysis scripts used in this study are openly available on GitHub at https://bit.ly/PRGeoMicroSciRep. All remaining data supporting the findings of this study are included within the manuscript and its Supplementary Information. Drone and sUAS imagery are available upon request. References Riding, R. Microbial carbonates: the geological record of calcified bacterial-algal mats and biofilms. 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A., Ramirez-Martinez, P. I. & Padilla-Montalvo, W. R. J.A. Documenting Microbialite Deposits and Unraveling Their Paleoenvironmental Origins in a Coastal Lagoon, Southwestern Puerto Rico. American Geophysical Union Fall Meeting , Asbtract PP32A-06 (2024). Jahnert, R. J. & Collins, L. B. Controls on microbial activity and tidal flat evolution in Shark Bay, Western Australia. Sedimentology 60 , 1071–1099. https://doi.org/https://doi.org/10.1111/sed.12023 (2013). Shiraishi, F. et al. Cyanobacterial exopolymer properties differentiate microbial carbonate fabrics. Sci. Rep. 7 , 11805. https://doi.org/10.1038/s41598-017-12303-9 (2017). Johnson, D. B., Beddows, P. A., Flynn, T. M. & Osburn, M. R. Microbial diversity and biomarker analysis of modern freshwater microbialites from Laguna Bacalar. Mexico Geobiology . 16 , 319–337. https://doi.org/10.1111/gbi.12283 (2018). Paul, V. G., Wronkiewicz, D. J., Mormile, M. R. & Foster, J. S. Mineralogy and Microbial Diversity of the Microbialites in the Hypersaline Storr's Lake, the Bahamas. Astrobiology 16 , 282–300. https://doi.org/10.1089/ast.2015.1326 (2016). Chacón-Baca, E. et al. The generation of a clotted peloidal micrite fabric by endolithic cyanobacteria in recent thrombolites from Cuatro Cienegas, northern Mexico. Sedimentology 71 , 2290–2313. https://doi.org/https://doi.org/10.1111/sed.13215 (2024). Gunes, Y., Sekerci, F., Avcı, B., Ettema, Thijs, J. G. & Balci, N. Morphological and Microbial Diversity of Hydromagnesite Microbialites in Lake Salda: A Mars Analog Alkaline Lake. Geobiology 22, e12619 (2024). https://doi.org/https://doi.org/10.1111/gbi.12619 Visscher, P. T. et al. Formation of lithified micritic laminae in modern marine stromatolites (Bahamas); the role of sulfur cycling. Am. Mineral. 83 , 1482–1493. https://doi.org/10.2138/am-1998-11-1236 (1998). Ge, Y. Extensive Early Marine Seafloor Cementation in a Modern Epeiric Sea Induced by Seawater Properties and a Shallow Redox Boundary Below the Seafloor. Geochemistry, Geophysics, Geosystems 23, e2022GC010444 (2022). https://doi.org/https://doi.org/10.1029/2022GC010444 Marin-Carbonne, J. et al. Early precipitated micropyrite in microbialites: a time capsule of microbial sulfur cycling. Geochemical Perspect. Lett. 21 , 7–12. https://doi.org/10.7185/geochemlet.2209 (2022). Gregg, J. M., Bish, D. L., Kaczmarek, S. E., Machel, H. G. & Hollis, C. Mineralogy, nucleation and growth of dolomite in the laboratory and sedimentary environment: A review. Sedimentology 62 , 1749–1769. https://doi.org/10.1111/sed.12202 (2015). Roberts, J. A. The problem with dolomite. Nat. Geosci. 17 , 716–716. https://doi.org/10.1038/s41561-024-01490-6 (2024). Vasconcelos, C. & McKenzie, J. A. Microbial mediation of modern dolomite precipitation and diagenesis under anoxic conditions (Lagoa Vermelha, Rio de Janeiro, Brazil). J. Sediment. Res. 67 , 378–390. https://doi.org/10.1306/d4268577-2b26-11d7-8648000102c1865d (1997). Bontognali, T. R. R. et al. Dolomite formation within microbial mats in the coastal sabkha of Abu Dhabi (United Arab Emirates). Sedimentology 57 , 824–844. https://doi.org/10.1111/j.1365-3091.2009.01121.x (2010). Hobbs, F. W. C., Fang, Y., Lebrun, N., Yang, Y. & Xu, H. Co-precipitation of primary dolomite and Mg-rich clays in Deep Springs Lake, California. Sedimentology 71, 1363–1383 (2024). https://doi.org/https://doi.org/10.1111/sed.13176 Ortega-Ariza, D., Franseen, E. K., Santos-Mercado, H., Ramírez-Martínez, W. R. & Core-Suárez, E. E. Strontium Isotope Stratigraphy for Oligocene-Miocene Carbonate Systems in Puerto Rico and the Dominican Republic: Implications for Caribbean Processes Affecting Depositional History. J. 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Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat. Microb. Ecol. 75 , 129–137 (2015). Bolyen, E. et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 37 , 852–857. https://doi.org/10.1038/s41587-019-0209-9 (2019). Quast, C. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 41 https://doi.org/10.1093/nar/gks1219 (2013). Paulson, J. N., Stine, O. C., Bravo, H. C. & Pop, M. Differential abundance analysis for microbial marker-gene surveys. Nat. Methods . 10 , 1200–1202. https://doi.org/10.1038/nmeth.2658 (2013). McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One . 8 , e61217. https://doi.org/10.1371/journal.pone.0061217 (2013). Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer, 2009). Dixon, P. VEGAN, a package of R functions for community ecology. J. Veg. Sci. 14 , 927–930 (2003). Yan, L. & ggvenn Draw Venn Diagram by 'ggplot2’, R package version 0.1.19. (2025). https://doi.org/https://github.com/yanlinlin82/ggvenn Rodriguez-Colon, B. J. Raw 16S Amplicon Sequencing 2019 Dataset from Microbial Mats and Microbialites in Southwestern Puerto Rico. figshare. (2025). https://doi.org/10.6084/m9.figshare.30826205 Additional Declarations No competing interests reported. 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\u003cstrong\u003e(e)\u003c/strong\u003erepresentative sample collected and dissected for analysis. \u003cstrong\u003e(f–h)\u003c/strong\u003eSalinas Salinetas (17.94663° N, -66.96545° W): \u003cstrong\u003e(f)\u003c/strong\u003e Drone image of the three interconnected ponds from 2019; \u003cstrong\u003e(g)\u003c/strong\u003e field photograph of the pink halophilic microbial mats from northwestern pond (named SS-NW); \u003cstrong\u003e(h)\u003c/strong\u003erepresentative sample collected and dissected. \u003cstrong\u003e(i–k)\u003c/strong\u003e Laguna Providencia (17.94309° N, -66.95929° W): \u003cstrong\u003e(i)\u003c/strong\u003e Drone image of the lagoon from 2019 showing the location of the microbialite buildup; \u003cstrong\u003e(j)\u003c/strong\u003e field photograph from the western sector where a sample (named LP19) was collected; \u003cstrong\u003e(k)\u003c/strong\u003erepresentative microbialite sample LP19 dissected for petrographic and mineralogical analysis.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/5dad47e1804f902419cfcd86.png"},{"id":100111469,"identity":"58ecef6b-f8ec-4700-86ff-540fe5533e5c","added_by":"auto","created_at":"2026-01-13 06:41:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1723976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePetrographic features of calcifying microbial mats in Salinas Vernales and microbialites from Laguna Providencia. (a–b)\u003c/strong\u003eTransmitted-light micrographs showing laminated microbial mat layers in Salinas Vernales (SV1) with micritic bands (light blue arrows) and interbedded organic-rich zones. Peloidal micrite and spherulitic grains \u003cstrong\u003e(b)\u003c/strong\u003e occur within EPS-rich laminae. The section shown in \u003cstrong\u003e(b)\u003c/strong\u003e was stained with Alizarin Red S and potassium ferricyanide to differentiate carbonate phases, showing red to pink tones in Mg-calcite bands and unstained aragonitic regions. \u003cstrong\u003e(c–d)\u003c/strong\u003e Detail of laminated mat region \u003cstrong\u003e(c)\u003c/strong\u003e and close-up \u003cstrong\u003e(d)\u003c/strong\u003e from active microbial mats in Laguna Providencia (LP) under cross-polarized light showing microbially mediated spherulitic aggregates nucleating within the EPS matrix. \u003cstrong\u003e(e–f)\u003c/strong\u003e Cross-polarized images from LP showing thrombolitic mesoclots composed of clustered spherulitic micro-peloids surrounded by micritic and acicular overgrowths (e), and alternating stromatolitic laminae outlined by red dashed lines (f).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/9640c6252e3fef92246f60f8.png"},{"id":100365101,"identity":"2bdd8799-2830-4080-b5e3-a5eda98a0960","added_by":"auto","created_at":"2026-01-16 07:54:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1533927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM) images showing microfabrics and mineral associations in microbial deposits. (a)\u003c/strong\u003eSEM micrographs of calcifying microbial mats in SV1E showing grains directly associated with EPS (letter e, green arrow). Some of the crystals show intertwined growth (blue arrow), while others have trigonal morphologies (red arrows). Insets in (a) display representative EDS spectra confirming Ca–Mg carbonate composition of these precipitates. \u003cstrong\u003e(b)\u003c/strong\u003e Small spherulite (letter s) directly growing from a filamentous microbial structure (letter f, green arrows) \u003cstrong\u003e(c-d)\u003c/strong\u003e Within the non-lithifying hypersaline mat in SS-NW showing euhedral gypsum crystals (letters Gp) embedded within the mat matrix \u003cstrong\u003e(c)\u003c/strong\u003eand \u003cstrong\u003e(d) \u003c/strong\u003ediatom (letter d; orange arrow) frustules coated by fine detrital and organic material. \u003cstrong\u003e(e–f)\u003c/strong\u003e Across the clustered thrombolitic microbialites fabrics from LP, spherulitic structures (letter s) with radiating aragonite needles were observed (red arrows). \u003cstrong\u003e(e)\u003c/strong\u003e Framboidal pyrite (letters Py, red circle), large carbonate grains (letter c, red arrow), and diatom fragments (letter d, orange arrow) within micritic carbonate matrices. Elemental maps in (f) confirm the spatial association of S and Fe with the pyrite.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/4641848c33bd6144d8533eea.png"},{"id":100111467,"identity":"fd2d7a92-615e-4237-804b-2798aab94260","added_by":"auto","created_at":"2026-01-13 06:41:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":361054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eX-ray diffraction (XRD) patterns of microbial mat and microbialite layers from southwestern Puerto Rico. (a) \u003c/strong\u003eIn SV1-E, green and black mat layers showing dominant aragonite (A) and high-Mg calcite (MC) with minor gypsum (G) and halite (H). \u003cstrong\u003e(b) \u003c/strong\u003eMinerology of microbial mat crust and black layer from SS-NW was dominated by gypsum (G) and halite (H), with subordinate aragonite (A) and traces of high-Mg calcite (MC) in the black layers. \u003cstrong\u003e(c) \u003c/strong\u003eIn LP19, the crust, intermediate, and basal layers show progressive mineralogical differentiation from aragonite-rich crusts to Mg-enriched carbonate phases in deeper zones. Very high-Mg calcite (VHMC) peaks occur exclusively in the black lithified layers and were not detected in the bottom layers.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/900ed199bb1efe1a6be9fd55.png"},{"id":100366989,"identity":"af927470-5125-4a68-934c-3707fb59397b","added_by":"auto","created_at":"2026-01-16 07:56:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":881389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStable isotope and mineralogy for Mg-carbonate formation within microbialites from Laguna Providencia (LP). (Top left) \u003c/strong\u003eField photograph of a microbialite showing the loose gray sediment hosted within the black layer, where Mg-enriched carbonate phases were sampled. \u003cstrong\u003e(Top right)\u003c/strong\u003e δ¹³C and δ¹⁸O values (‰ VPDB) of carbonates from microbialites (green symbols) compared with dolomitized and calcitic facies of the Ponce Limestone (purple and red circles)\u003csup\u003e[26]\u003c/sup\u003e, and beach sediment for reference (light blue cross-squares). \u003cstrong\u003e(Bottom)\u003c/strong\u003e Representative XRD patterns comparing the microbialite black layer (LP19) with Ponce Limestone and adjacent beach sand for reference. The microbialite contains abundant very high-Mg calcite (VHMC, or proto-dolomite) and aragonite (A), whereas the dolomitized Ponce Limestone shows ordered dolomite (D) reflections, shown across the dashed lines. Beach sand contained minerology coming from bioclastic sediment and lithoclasts from the Ponce Limestone. Miller indices (hkl) for the minerals were identified and included.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/06d97cbf13738ba90607691d.png"},{"id":100111472,"identity":"15f15634-0863-4b48-b4d5-b2c00377c7f3","added_by":"auto","created_at":"2026-01-13 06:41:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":517205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCommunity Composition of Microbial Deposits in Guánica, PR. (a) \u003c/strong\u003eVenn diagram showing the distribution of unique and shared ASVs among the three sampled sites. Numbers indicate the count of ASVs detected exclusively in each site or shared across multiple sites. \u003cstrong\u003e(b) \u003c/strong\u003eBeta diversity Principal Coordinates Analysis (PCoA) of CSS-normalized microbial community composition across samples based on Bray-Curtis dissimilarity. Each point represents a sample colored by sample deposit and shaped by layers. Dashed ellipses show the approximate 95% confidence regions around the centroid of each site group, visualizing the dispersion and clustering of communities. PERMANOVA explains approximately 31% of the total variance in microbial community composition (Pseudo-F = 5.08, R² = 0.31, p = 0.001, 999 permutations), indicating significant compositional differences among lagoons. \u003cstrong\u003e(c) \u003c/strong\u003eRelative abundance of major microbial phyla across sample layers.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/10051cbf975dd8097db043ee.png"},{"id":107352900,"identity":"6c12d798-49f6-40e3-88c7-57297db93c05","added_by":"auto","created_at":"2026-04-20 16:18:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7284164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/74164e8a-a856-4fa1-a670-47633c70ea7e.pdf"},{"id":100111479,"identity":"5121b825-c9b9-40de-9e02-0b4e93a79e7f","added_by":"auto","created_at":"2026-01-13 06:41:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":26762384,"visible":true,"origin":"","legend":"","description":"","filename":"RodriguezColonetalSciRepSupInfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-8570072/v1/3bf9f8470b129ba2ab127daf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Documenting the Occurrence of Microbial Carbonate Deposits in Puerto Rico","fulltext":[{"header":"Introduction","content":"\u003cp\u003eModern microbial carbonate mineral deposits, including microbial mats and microbialites, provide valuable insights into the geobiological evolution of Earth\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Microbialites, including laminated stromatolites and clotted thrombolites, form through the lithification of microbial mats via sediment trapping, binding, and in situ organomineralization (i.e., microbially induced mineralization)\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Although microbialites were widespread during the Precambrian, with the oldest examples dating to \u003cem\u003eca.\u003c/em\u003e 3.5 Ga, their abundance declined markedly through the Phanerozoic\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Today, unlithified microbial mats (vertically laminated biofilms composed of diverse microbial communities) are common in shallow aquatic environments globally, yet only a subset undergoes sustained lithification, making modern microbialites comparatively rare \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. This disparity highlights a fundamental and unresolved question: why do some microbial mats lithify while others remain unlithified under broadly similar environmental conditions? Because modern microbialites preserve coupled biological, mineralogical, and geochemical signatures, they also serve as valuable analogs for interpreting ancient biosedimentary systems and potential biosignatures in extraterrestrial settings, including Mars (e.g., \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003ePrevious research from microbialite-forming environments has improved understanding of mechanisms governing organomineralization within these systems \u003csup\u003e[\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. These processes reflect complex interactions among microbial community composition, metabolic activity, extracellular polymeric substances (EPS), and physicochemical conditions such as salinity, pH, mineral saturation state, and water chemistry, as well as broader environmental influences operating across geomorphic and sedimentary contexts\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Despite these advances, the relative importance and interaction of microbial processes, aqueous geochemistry, and environmental forcing remain incompletely resolved. Continued characterization of lithifying microbial systems, particularly in new or understudied settings, is therefore necessary to better understand why some microbial mats undergo lithification while others remain unlithified.\u003c/p\u003e \u003cp\u003eSeveral microbial carbonate environments have been studied within coastal lagoons across the circum-Caribbean region, including sites in the Bahamas (e.g., Storr Lake, San Salvador Island, and Big Pond, Eulethera Island)\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, Turks and Caicos (e.g., Windsor Point Salt Pond)\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, Venezuela (e.g., Laguna Pirata, Los Roques Archipelago)\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e, Cuba (e.g., Cayo Cocos and Sabana-Camag\u0026uuml;ey Archipielago)\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, and Bonaire (e.g., GotoMeer Basin)\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In Puerto Rico, previous studies have focused on non-lithifying benthic and ephemeral microbial mats in hypersaline lagoons in Cabo Rojo\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. However, actively lithifying microbial mats and microbialite deposits have not previously been described from other regions of the island.\u003c/p\u003e \u003cp\u003eThis study documents, for the first time, the occurrence, internal structure, mineralogy, and microbial community composition of lithifying microbial deposits in Puerto Rico. This work focuses on three coastal lagoons in southwest Gu\u0026aacute;nica Municipality: Salinas Vernales (characterized by calcifying microbial mats), Salinas Salinetas (where microbial mats show minimal to no lithification), and Laguna Providencia (hosting fully developed microbialites). Together, these lagoons represent a spectrum of lithification states and composition, offering a valuable opportunity to examine the environmental and microbial factors associated with microbial mat lithification and microbialite development in dynamic coastal settings.\u003c/p\u003e\n\u003ch3\u003eGeologic Setting and Field Site Description\u003c/h3\u003e\n\u003cp\u003eThe study sites are three coastal lagoons near the town of Gu\u0026aacute;nica, along the southwestern coast of Puerto Rico (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The bedrock surrounding the lagoons is composed primarily of a Miocene-age unit named Ponce Limestone, a bioclastic packstone\u0026ndash;wackestone unit that is partially dolomitized\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Dolomitization in Ponce Limestone is dated to \u003cem\u003eca.\u003c/em\u003e 10.21\u0026ndash;6.83 Ma\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, with dolomite-rich outcrops occurring immediately adjacent to the lagoon margins. Active tectonism has been documented in the region via the Punta Montalva fault (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), a left-lateral strike-slip fault system (trending \u003cem\u003eca.\u003c/em\u003e 110˚-115˚) that passes \u003cem\u003eca.\u003c/em\u003e 150\u0026ndash;200 m north of the lagoons, with notable activity during a 6.4 M\u003csub\u003ew\u003c/sub\u003e seismic event in January 2020\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Quaternary beach deposits isolating the lagoons from the Caribbean Sea are composed of modern coral and calcareous algal bioclasts (e.g., \u003cem\u003eHalimeda sp.\u003c/em\u003e) and lithoclasts of Ponce Limestone\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The studied lagoons have mesosaline to hypersaline conditions most of the year and have been historically important for salt production in the area, due to relatively low annual rainfall in the southwest of Puerto Rico, ranging from 600 to 1,000 mm, and mean temperatures exceeding 26\u0026deg;C\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSalinas Vernales, the smallest lagoonal system (\u003cem\u003eca.\u003c/em\u003e 15,330 m\u0026sup2;), comprises small, abandoned artificial solar salterns (\u003cem\u003eca.\u003c/em\u003e 0.2\u0026ndash;0.35 m depth) adjacent to Playa de Los Almendros (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c). Construction of the salterns is evidenced by subdivided crystallizer ponds, stoned dike walkways, and anthropogenic debris in the area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea online). It is unclear whether a natural pond existed prior to the development of the salterns; however, historical aerial imagery shows that the southernmost pond (hereafter SV1) did not exist before the 1930s. Salinas Salinetas is an intermediate-sized natural hypersaline system (\u003cem\u003eca.\u003c/em\u003e 41,380 m\u0026sup2;) with minimal human modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Historical documentation shows, however, that this system was also used for salt production, although in a lower capacity than the others from the area. This system comprises three interconnected ponds, positioned between Salinas Vernales and Laguna Providencia, and adjacent to Playa La Jungla (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Laguna Providencia is the largest (\u003cem\u003eca.\u003c/em\u003e 0.25 km\u0026sup2;), deepest lagoon (\u003cem\u003eca.\u003c/em\u003e 1.2\u0026ndash;1.3 m depth south, \u003cem\u003eca.\u003c/em\u003e 0.8 m north) in the study area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). The lagoon contains several anthropogenic modifications, including an artificial canal cut along the southwestern margin (\u003cem\u003eca.\u003c/em\u003e 150 m long) constructed between the late 19th and early 20th centuries, and abandoned solar salterns along the eastern side near Playa Santa neighborhood (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePhysicochemical Field Measurements, Distribution, and Macro Morphology of Microbial Deposits\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSalinas Vernales.\u003c/em\u003e Calcifying microbial mats in Salinas Vernales were restricted to SV1, which is bordered by mangroves and covers \u003cem\u003eca.\u003c/em\u003e 1,600 m\u0026sup2; of the lagoonal system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea online). Preliminary aqueous measurements from SV1 indicate slightly alkaline conditions (pH \u003cem\u003eca.\u003c/em\u003e 8.3) with low salinity (\u003cem\u003eca.\u003c/em\u003e 25.4 ppt) and conductivity (\u003cem\u003eca.\u003c/em\u003e 39.4 mS), contrasting with higher salinity (\u003cem\u003eca.\u003c/em\u003e 78.5 ppt) and lower pH (\u003cem\u003eca.\u003c/em\u003e 6.8) in the adjacent northern pond (hereafter SV2), despite similar alkalinity values (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Microbial deposits in SV1 occur as isolated hemispheroidal mounds, coalescent mound clusters, and laterally continuous crusts up to \u003cem\u003eca.\u003c/em\u003e 15 m in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea online). Most deposits preferentially developed on hardened calcrete substrates, whereas areas lacking firm substrate were dominated by organic-rich ooze and lacked microbial mats. Polygonal and flat microbial mats occurred locally near pond margins, including zones with small pinnacle-like protrusions (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee online). In contrast, adjacent crystallizer ponds to the north (SV2\u0026ndash;SV6) hosted only sparse, desiccated biofilms and lacked comparable deposits. A representative mat from the eastern margin of SV1 (hereafter SV1-E) was selected for petrographic, microscopic, and microbial diversity analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical data collected across the lagoons.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLagoon\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemperature (˚C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpecific Conductivity (mS)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSalinity (ppt)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAlkalinity\u003c/p\u003e \u003cp\u003e(meq L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSalinas Vernales\u003c/p\u003e \u003cp\u003e(SV1-SV2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSV1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSV2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e111.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e78.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalinetas\u003c/p\u003e \u003cp\u003e(SS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSS-NW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e110.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e77.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProvidencia\u003c/p\u003e \u003cp\u003e(LP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e80.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLP19-Flow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSalinas Salinetas.\u003c/em\u003e The northwestern pond (hereafter SS-NW) hosted the most extensive microbial mat development within Salinas Salinetas, with pink-pigmented mats forming atop evaporitic mineral crusts across \u003cem\u003eca.\u003c/em\u003e 3,750 m\u0026sup2; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef\u0026ndash;g; Supplementary Fig. S2a\u0026ndash;c online). Waters in SS-NW were strongly saline and slightly acidic (salinity \u003cem\u003eca.\u003c/em\u003e 77.8 ppt; pH \u003cem\u003eca.\u003c/em\u003e 5.3; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although petrographic analyses were not conducted on these deposits, SS-NW mats were analyzed for microbial community composition. Other ponds within Salinas Salinetas contained only sparse or discontinuous microbial mats, including small chip-like mats on shell fragments and mats enriched in detrital \u003cem\u003eHalimeda\u003c/em\u003e bioclasts (Supplementary Fig. S2 online).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLaguna Providencia.\u003c/em\u003e The most spatially extensive and lithified microbial deposits were documented in Laguna Providencia, where microbialites form a west-east-trending buildup extending \u003cem\u003eca.\u003c/em\u003e 350 m across the central sector of the western lagoon (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei; Supplementary Fig. S3a online), with only sparse, small microbialites occurring along limited portions of the northern and southern shores. Surface waters exhibited near-neutral to slightly alkaline pH (\u003cem\u003eca.\u003c/em\u003e 7.1\u0026ndash;7.3), moderate salinity (\u003cem\u003eca.\u003c/em\u003e 55 ppt), and low alkalinity (\u003cem\u003eca.\u003c/em\u003e 2.4 meq L⁻\u0026sup1;), whereas a localized inflow from the Ponce Limestone showed lower salinity (\u003cem\u003eca.\u003c/em\u003e 19 ppt) and higher alkalinity (\u003cem\u003eca.\u003c/em\u003e 3.9 meq L⁻\u0026sup1;; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDrone-derived orthomosaics and field observations identify two dominant microbialite growth forms within the buildup: laterally continuous clustered microbialite accumulations (\u003cem\u003eca.\u003c/em\u003e 1\u0026ndash;2 m) and more isolated mound-like structures (Supplementary Fig. S3b-c, e-f online). Clustered forms are more prevalent toward the western portion of the buildup, whereas isolated structures dominate toward the eastern margin and former crystallizer zones. Microbialite heads decrease in size from west to east, and locally form linear to curvilinear alignments several meters in length along the southern portion of the buildup (Supplementary Fig. S3d online). Preservation ranges from partially lithified to sub-fossilized structures, with some microbialites supporting active pustular mats at their upper surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek; Supplementary Fig. S4a). A representative microbialite from the western buildup (hereafter LP19) was selected for petrographic, mineralogical, and microbial analyses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInternal Structure, Microfabrics, and Mineralogy\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eSalinas Vernales (SV1-E).\u003c/em\u003e Microbial mat from Salinas Vernales (SV1-E) exhibits clear vertical stratification, with thin pigmented surface layers grading into darker basal zones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee; Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed,f). The mat fabric consists of alternating micritic and biofilm-rich laminae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b), locally incorporating micritic peloids and spherulitic grains (\u003cem\u003eca.\u003c/em\u003e 50\u0026ndash;400 \u0026micro;m) with micritic nuclei and fibrous to botryoidal carbonate overgrowths. Sparse detrital components, including \u003cem\u003eHalimeda spp.\u003c/em\u003e fragments and bivalve material, are present but volumetrically minor (Supplementary Fig. S5a online). SEM imaging shows abundant microbial filaments embedded within EPS matrices, closely associated with nanogranular to subhedral micritic precipitates, including small rhombohedral and trigonal aggregates attached directly to filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b; Supplementary Fig. S6a-b online). Larger spherulitic grains display fibrous internal textures and radial acicular growth. XRD analyses indicate that SV1-E mats are dominated by aragonite and high-Mg calcite (\u003cem\u003eca.\u003c/em\u003e 16.6 mol% MgCO₃), with Mg content increasing with depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSalinas Salinetas (SS-NW).\u003c/em\u003e Microbial mats from Salinas Salinetas (SS-NW) display laminated fabrics broadly similar to those observed in SV1, but are distinguished by the presence of prominent pink pigmented layers above and below the crust and a lack of carbonate lithification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-h). No carbonate precipitates were observed within these mats; instead, evaporitic minerals dominate, including gypsum and a thick halite crust that serves as the primary substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg; Supplementary Fig. S2b). SEM observations reveal embedded gypsum crystals, pennate diatom frustules, and dispersed microbial filaments within the mat matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). XRD analyses confirm gypsum and halite as the dominant mineral phases, with only minor aragonite and high-Mg calcite (\u003cem\u003eca.\u003c/em\u003e 27.1 mol% MgCO₃) detected in basal layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLaguna Providencia (LP19).\u003c/em\u003e The Laguna Providencia microbialite displays composite fabrics and extensive calcification. At the surface, pustular microbial mats form a thin crust composed of pigmented layers overlying a darker organic-rich zone, which in turn overlies lithified interior intervals and a basal layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). Sub-fossilized portions are dominated by thrombolitic textures in upper regions, characterized by clustered mesoclots, whereas deeper intervals contain irregular stromatolitic laminae (Supplementary Fig. S4 online).\u003c/p\u003e \u003cp\u003eSpherulitic grains are abundant throughout the microbialite, occurring as small forms (\u003cem\u003eca.\u003c/em\u003e 10\u0026ndash;25 \u0026micro;m) within microbial laminations in surface mats and as larger spherulites associated (\u003cem\u003eca.\u003c/em\u003e 250 \u0026micro;m) with EPS-rich zones and occasional bioclasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d; Supplementary Fig. S5b-d online). Petrographic analyses show thrombolitic clots composed of spherulitic peloids, microspar, and acicular carbonate overgrowths (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), whereas stromatolitic intervals display irregular micrite-rich laminae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Fluorescence microscopy reveals strong signals in spherulite nuclei with weaker fluorescence along rims and overgrowths (Supplementary Fig. S5c\u0026ndash;f online). Mineralogical staining further indicates pervasive Mg-calcite within spherulitic peloids and micritic laminae, with minor aragonite cements localized to rims and void-filling phases (Supplementary Fig. S5g online). SEM observations document spherulites with dense micritic nuclei and outward botryoidal aragonite growth, along with centric diatom valves, framboidal pyrite, putative amorphous Mg-silicates in deeper layers, and locally developed aragonite needles associated with organic matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u0026ndash;f; Supplementary Fig. S6c\u0026ndash;d online) XRD analyses indicate a mineralogical progression from surface gypsum to aragonite and high-Mg calcite in intermediate layers, with the black organic-rich zones hosting the most diverse assemblages, including low-Mg calcite, high-Mg calcite, and very high-Mg calcite (VHMC; \u003cem\u003eca.\u003c/em\u003e 38.8 mol% MgCO₃), commonly associated with loosely consolidated gray sediment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eStable Isotope Geochemistry\u003c/h3\u003e\n\u003cp\u003eCarbonate δ\u0026sup1;\u0026sup3;C values from LP19 range from \u0026minus;\u0026thinsp;4.6\u0026permil; to -1.6\u0026permil;, and δ\u0026sup1;⁸O values from +\u0026thinsp;0.9\u0026permil; to +\u0026thinsp;1.4\u0026permil; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online). Dolomitic samples from nearby Ponce Limestone samples (Supplementary Fig. S7 online) display δ\u0026sup1;\u0026sup3;C values between \u0026minus;\u0026thinsp;0.81\u0026permil; and +\u0026thinsp;1.38\u0026permil; and relatively enriched δ\u0026sup1;⁸O values from +\u0026thinsp;1.05\u0026permil; to +\u0026thinsp;1.96\u0026permil; (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online). In contrast, calcitic Ponce Limestone samples show the most depleted compositions, with δ\u0026sup1;\u0026sup3;C values from \u0026minus;\u0026thinsp;9.79\u0026permil; to -6.31\u0026permil; and δ\u0026sup1;⁸O values from \u0026minus;\u0026thinsp;4.90\u0026permil; to -3.53\u0026permil;. Beach sediments exhibit δ\u0026sup1;\u0026sup3;C of -0.08\u0026permil; and moderately depleted δ\u0026sup1;⁸O (-2.59\u0026permil;).\u003c/p\u003e\n\u003ch3\u003eMicrobial Community Composition\u003c/h3\u003e\n\u003cp\u003eAlpha diversity analyses revealed pronounced differences in richness and evenness across lagoons and mat layers (Supplementary Table S2). Microbialites from Laguna Providencia exhibited the highest diversity, with bottom and brown layers yielding over 1,200 observed ASVs and Shannon indices\u0026thinsp;\u0026gt;\u0026thinsp;5.5, whereas the basal layer of Salinas Salinetas showed markedly reduced diversity (Observed\u0026thinsp;=\u0026thinsp;348\u0026thinsp;\u0026plusmn;\u0026thinsp;313; Shannon\u0026thinsp;=\u0026thinsp;2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34). Surface layers from SV1-E and SS-NW displayed intermediate diversity (Shannon ca. 4.9). Venn analyses indicate substantial lagoon-specific ASV pools, with only 316 ASVs shared across all systems and limited pairwise overlap, particularly between SV1-E and SS-NW (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Beta-diversity analyses reveal clear clustering by lagoon (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), with tight grouping of LP19 samples and distinct ordination space occupied by SV1-E and SS-NW. These differences are statistically significant (PERMANOVA: pseudo-F\u0026thinsp;=\u0026thinsp;5.08, R\u0026sup2; = 0.31, p\u0026thinsp;=\u0026thinsp;0.001), indicating strong spatial structuring of microbial communities across lagoons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSalinas Vernales (SV1-E).\u003c/em\u003e Bacterial taxa dominated upper mat layers (64.69\u0026ndash;98.47%) and decreased with depth, whereas Archaea exhibited the opposite trend (1.53\u0026ndash;35.31%; Supplementary Table S2). Surface crust and green layers were dominated by \u003cem\u003ePseudomonadota\u003c/em\u003e (20.67\u0026ndash;35.38%), followed by \u003cem\u003ePlanctomycetota\u003c/em\u003e (10.40-12.02%), \u003cem\u003eBacteroidota\u003c/em\u003e (7\u0026ndash;11%), and \u003cem\u003eSpirochaetota\u003c/em\u003e (3.65\u0026ndash;7.36%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). \u003cem\u003eChloroflexota\u003c/em\u003e (order \u003cem\u003eAggregatilineales\u003c/em\u003e) were enriched in crust and black layers (18.27% and 13.52%). Cyanobacteria (primarily \u003cem\u003eDesertifilaceae\u003c/em\u003e) occurred in the crust (1.17\u0026ndash;8.23%; Supplementary Fig. S8a online). \u003cem\u003eThermodesulfobacteriota\u003c/em\u003e ranged from 4.15% in the green layer to 9.07% in the black layer. Sulfate-reducing bacteria (SRB) included \u003cem\u003eDesulfomonilaceae\u003c/em\u003e (enriched in red layers) and \u003cem\u003eDesulfohalobiaceae\u003c/em\u003e (more abundant at depth; Supplementary Fig. S8c online). Archaeal groups included \u003cem\u003eNanoarchaeota\u003c/em\u003e (1.02\u0026ndash;3.94%) and \u003cem\u003eHadaarchaeota\u003c/em\u003e (up to 2.66%). Methanogenic Archaea (\u003cem\u003eMethanobacteriota\u003c/em\u003e), particularly unclassified \u003cem\u003eMethanofastidiosales\u003c/em\u003e, peaked in the black layer (up to 14.03%; Supplementary Fig. S8d online).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSalinas Salinetas (SS-NW).\u003c/em\u003e Prokaryotic communities in SS-NW were dominated by Bacteria (54.10-98.14%), but the microbial mats from this lagoon exhibited the highest relative abundance of Archaea (1.86\u0026ndash;45.90%; Supplementary Table S2 online). \u003cem\u003ePseudomonadota\u003c/em\u003e and \u003cem\u003eRhodothermota\u003c/em\u003e dominated surface crust and green layers (18.31\u0026ndash;20.77% and 27.50-32.44%, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Cyanobacteria ranged from 2.56\u0026ndash;5.91% and included \u003cem\u003eRubidibacteraceae\u003c/em\u003e and \u003cem\u003eGeitlerinemaceae\u003c/em\u003e (Supplementary Fig. S8a online). Purple non-sulfur bacteria (PNSB; genus \u003cem\u003eRhodovibrio\u003c/em\u003e) were also abundant in crust and green layers (8.78\u0026ndash;11.63%; Supplementary Fig. S8b online). Deeper pink and black layers were enriched in \u003cem\u003eAcetothermia\u003c/em\u003e (6.38\u0026ndash;40.41%), \u003cem\u003eAcidobacteriota\u003c/em\u003e (36.20%), \u003cem\u003eActinomycetota\u003c/em\u003e (9.87%), and \u003cem\u003eBacillota\u003c/em\u003e (5.47%). Archaea were dominated by \u003cem\u003eHalobacteriota\u003c/em\u003e (up to 28.07% in the crust), and methanogens, especially \u003cem\u003eMethanofastidiosales\u003c/em\u003e, were more abundant here than in other lagoons, reaching 43.15% in the black layer (Supplementary Fig. S8d online).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLaguna Providencia (LP19).\u003c/em\u003e The microbialite in Laguna Providencia showed the highest overall diversity but lacked clear vertical stratification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Bacterial relative abundance decreased modestly with depth (84.66% to 64.31%), while Archaea increased (15.34\u0026ndash;9.70% in crust to 24.10-44.98% at depth; Supplementary Table S2 online). \u003cem\u003ePlanctomycetota\u003c/em\u003e were consistently abundant (13.47\u0026ndash;26.15%), \u003cem\u003ePseudomonadota\u003c/em\u003e were most represented in the crust (13.48\u0026ndash;19.19%), and \u003cem\u003eChloroflexota\u003c/em\u003e (\u003cem\u003eAggregatilineales\u003c/em\u003e) increased slightly with depth (9.26\u0026ndash;10.19%). Cyanobacteria were present but low in abundance (1.52\u0026ndash;2.03%), represented by \u003cem\u003eRubidibacteraceae\u003c/em\u003e (Supplementary Fig. S8a online). SRB included \u003cem\u003eDesulfohalobiaceae\u003c/em\u003e (up to 6.33% in brown layers) and \u003cem\u003eDesulfatiglandaceae\u003c/em\u003e (up to 3.06%; Supplementary Fig. S8c online). Archaea included \u003cem\u003eNanoarchaeota\u003c/em\u003e (4.52\u0026ndash;6.64%), \u003cem\u003eHadaarchaeota\u003c/em\u003e (2.94\u0026ndash;7.63%), and methanogenic taxa such as \u003cem\u003eMethanofastidiosales\u003c/em\u003e (up to 18.04% in black layers; Supplementary Fig. S8d online).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe three lagoonal systems examined in this study define a clear continuum of microbial carbonate development, ranging from non-lithifying hypersaline mats at Salinas Salinetas, through partially calcifying mats at Salinas Vernales, to fully lithified microbialites at Laguna Providencia. Although some dataset here represents a single temporal snapshot, the combined sedimentological, petrographic, and microbial evidence indicates that lithification potential varies markedly over short spatial scales within a shared coastal setting. Across all systems, microbial mats develop on physically stable substrates, indicating that substrate availability influences deposit distribution; however, substrate stability alone is insufficient to explain carbonate lithification, as demonstrated by the non-lithifying mats developed on halite crusts in Salinas Salinetas. Instead, the strongest distinction across the lithification continuum corresponds to differences in microbial community structure and diversity, which increase from Salinas Salinetas to Salinas Vernales and are highest in the lithified microbialites of Laguna Providencia. These differences likely reflect variations in microbial metabolism operating within permissive hydrological and geochemical contexts, as well as differences in relative system maturity, collectively governing whether microbial mats remain unlithified, initiate calcification, or develop into lithified microbialites.\u003c/p\u003e\n\u003ch3\u003eNon-Lithifying Microbial Deposits in Salinas Salinetas\u003c/h3\u003e\n\u003cp\u003eThe absence of lithification in microbial deposits from Salinas Salinetas reflects strong geochemical and hydrological constraints that overwhelm biological influences during the observed conditions. In SS-NW, microbial mats are restricted to persistently submerged areas and colonize thick evaporitic crusts that dominate the benthic substrate (Supplementary Fig. S2b). High salinity and moderate alkalinity indicate a hydrologically restricted basin dominated by evaporative concentration (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Historical satellite and aerial imagery show that SS-NW frequently undergoes isolation and seasonal desiccation, and sampling near the end of the dry season (June 2019) likely captured conditions of intensified evaporation and gypsum-to-halite supersaturation. Under such conditions, carbonate dissolution likely exceeds precipitation, effectively precluding sustained lithification in this system.\u003c/p\u003e \u003cp\u003eAlthough microbial communities in SS-NW are well developed, their role in carbonate formation appears secondary to these physicochemical constraints. Surface layers are dominated by halophilic Archaea and PNSB (e.g., \u003cem\u003eRhodovibrio sp.\u003c/em\u003e), consistent with the observed pink pigmentation and a shift toward anoxygenic phototrophy under hypersaline conditions. Similar salinity-driven transitions toward anoxygenic phototrophy have been documented in other hypersaline microbial mats\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. While Cyanobacteria are present, the dominance of anoxygenic phototrophs and sulfide-oxidizing taxa likely promotes net carbonate dissolution through CO₂ and proton release\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Minor occurrences of high-Mg calcite and aragonite in basal layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) may reflect localized, transient supersaturation, potentially linked to methanogenic activity, which can increase alkalinity by consuming organic acids and CO₂\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. However, the absence of lithification across most of the Salinas Salinetas system, including other basins, indicates that any carbonate precipitation is spatially restricted, short-lived, and insufficient to overcome basin-scale evaporative and geochemical controls.\u003c/p\u003e\n\u003ch3\u003eCalcifying Microbial Mats in Salinas Vernales\u003c/h3\u003e\n\u003cp\u003eUnlike the non-lithifying mats at Salinas Salinetas, SV1 hosts actively calcifying microbial mats under physicochemical conditions more favorable for carbonate precipitation. Field measurements indicate lower salinity and more alkaline conditions in SV1 relative to SV2, which exhibits geochemical characteristics more similar to Salinas Salinetas (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These comparatively lower salinities may support more diverse and metabolically active microbial communities, including taxa and guilds directly involved in organomineralization, thereby enhancing lithification potential. Although short-term dilution from rainfall, surface runoff, or localized subsurface inputs may contribute to this contrast, the proximity of SV1 to the shoreline and lack of persistent freshwater surface inflow indicate a system fundamentally influenced by seawater. Over time, evaporation likely concentrates ions to levels permissive of carbonate supersaturation; however, the consistently lower salinities observed at SV1 relative to SV2 suggest that additional hydrological inputs, potentially including subsurface water sources, warrant targeted geochemical and hydrological investigation.\u003c/p\u003e \u003cp\u003eUnder these conditions, calcifying microbial mats preferentially colonize hard calcrete surfaces (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-b online). The distribution of hemispheroidal mounds in central areas and polygonal mats along pond margins likely reflects gradients in water depth and exposure frequency (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea online), similar to patterns described from the Turks and Caicos\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Polygonal textures likely formed during episodes of shallow inundation and periodic exposure, whereas hemispheroidal mounds represent more mature structures developed under longer-lived submergence, although confirmation of this sequence will require targeted geomorphic and hydrological analyses.\u003c/p\u003e \u003cp\u003eMicroscopically, SV1-E contains abundant micritic carbonate closely associated with EPS (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), including trigonal crystals comparable to those described from Lagoa Vermelha, Brazil\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, Big Pond\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, and Mérantaise River, France\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. SEM imaging reveals small (\u003cem\u003eca.\u003c/em\u003e 5 µm) spherulites nucleating on or adjacent to microbial filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), consistent with organomineralization either within porewaters or directly on EPS or filament surfaces\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Larger spherulites with micritic nuclei and sparry aragonite rims (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) resemble carbonate grains from other microbial calcifying systems (\u003csup\u003e[e.g., 16,17,38,39]\u003c/sup\u003e). The micritic nuclei likely reflect microbial micrite or partially micritized detrital particles incorporated during mat accretion. These observations indicate that carbonate production in SV1 is predominantly authigenic and microbially mediated or early diagenetic overgrowths, with minor contributions from detrital material, similar to textures documented in Abu Dhabi sabkha mats, where diagenetic aragonite rims overgrow earlier micritic nuclei, and in microbialites from the Great Salt Lake, USA, where organomineralization dominates early stages of precipitation\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicrobial community patterns in SV1-E are consistent with a biogenic contribution to carbonate precipitation. Filamentous Cyanobacteria (represented in this case by \u003cem\u003eDesertifilaceae\u003c/em\u003e) dominate the crust and green layers and are known to promote carbonate nucleation by generating EPS during photosynthetic CO₂ uptake\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Carbon fixation through photosynthesis can increase alkalinity, and at the same time, EPS acts as a trap for sediment particles and nucleation sites, provided that its cation-binding capacity does not inhibit mineral formation \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. SEM imaging of filamentous structures within the green layer (Supplementary Fig. S6a online) provides visual support for their presence. Cyanobacteria are also well-documented contributors to microbial deposit morphotypes, and their elevated relative abundance in SV1-E compared to the other lagoons may help explain documented morphologies such as the observed pinnacle-like structures in some of the microbial mats (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee online). Deeper layers also host SRB (e.g., \u003cem\u003eDesulfohalobiaceae\u003c/em\u003e, \u003cem\u003eDesulfomonilaceae\u003c/em\u003e) and methanogens (e.g., \u003cem\u003eMethanofastidiosales\u003c/em\u003e), whose metabolism of organic matter increases HCO₃⁻, further promoting carbonate supersaturation\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Although we cannot directly quantify the relative contributions of these guilds, the combined presence of filamentous Cyanobacteria, SRB, and methanogens suggests multiple metabolic and EPS-mediated pathways that likely contribute to the micritic and spherulitic textures observed in SV1.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicrobialite Deposits in Laguna Providencia\u003c/h2\u003e \u003cp\u003eAmong the three studied lagoons, Laguna Providencia exhibits the most extensive lithified microbial deposits and, to our knowledge, represents the first formally documented microbialite buildup in Puerto Rico (Supplementary Fig. S3 online). This greater extent and degree of lithification likely reflect a longer period of microbialite development relative to Salinas Vernales and Salinas Salinetas, as well as differences in present-day physicochemical conditions, a higher microbial community diversity, and local accommodation space. SfM-derived DEM data indicate that the western and northern sectors of the lagoon correspond to shallow, low-gradient zones consistent with laterally extensive microbialite accretion, whereas a steeper slope along the southern margin may have limited microbialite development in that direction (Supplementary Fig. S3a-i online). Similar slope-controlled zonation has been documented in lake microbialites from Laguna Pozo Bravo, Argentina\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, and the Great Salt Lake\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Sedimentary records from Laguna Providencia (results not yet published) further indicate a complex paleoenvironmental evolution and a longer-lived lagoonal system, providing increased time for sustained microbialite accretion and maturation\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. The central-lagoon position and localized distribution of the microbialite buildup, including meter-scale linear to curvilinear alignments, suggest a more complex hydrological history. Observations of low-salinity, high-alkalinity flows entering the lagoon from the Ponce Limestone (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) raise the possibility of localized groundwater inputs or subtle tectonic influences associated with the nearby Punta Montalva fault\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, although additional hydrological and geochemical data are required to evaluate these interpretations.\u003c/p\u003e \u003cp\u003eInternally, sub-fossilized microbialites in Laguna Providencia exhibit composite mesostructures, with stromatolitic textures at the base and thrombolitic fabrics toward the top (Supplementary Fig. S4 online). Such vertical changes in fabrics likely reflect temporal shifts in environmental conditions and microbial mat structure. Comparable composite microbialites have been described in modern systems, including Laguna Pozo Bravo\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e and Shark Bay, Australia\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e, where fluctuating water levels, hydrodynamic energy, and microbial succession produce vertically stacked stromatolitic-thrombolitic sequences. In this case, the stromatolitic intervals were likely produced by filament-rich microbial mats capable of sediment trapping, binding, and in-situ organomineralization, similar to those observed in SV1. In contrast, thrombolitic textures appear consistent with coccoid-dominated pustular mats, such as those currently found at the surface of the microbialites. Genomic data support this interpretation, as pustular crust layers in LP19 show a higher relative abundance of coccoid cyanobacterial families such as \u003cem\u003eRubidibacteraceae\u003c/em\u003e compared to SV1-E, where filamentous cyanobacteria dominate. Shifts between filamentous and coccoid cyanobacteria have been linked to the formation of stromatolitic versus thrombolitic morphotypes in other lithifying mats\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne point to consider is that Cyanobacteria occur at relatively low abundance in LP19 compared to other microbial groups, a pattern also observed in SV1-E and SS-NW, but to varying degrees. Similar discrepancies between observed mat morphologies and cyanobacterial sequence abundances have been reported in microbialite studies of Laguna Bacala, México\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, and Storr’s Lake\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. These studies note that primer bias, differential DNA extraction efficiency, and cell-wall-dependent lysis rates can lead to underrepresentation of Cyanobacteria in amplicon datasets. Although filamentous cyanobacterial morphologies are readily observed microscopically in SV1-E (Supplementary Fig. S6a online), reconciling the low cyanobacterial signal in LP19 will require targeted microscopy, taxonomic imaging, and possibly alternative molecular approaches.\u003c/p\u003e \u003cp\u003eAnother aspect to consider is the vertical structure of the sampled microbialite. Pustular mats occur only in the surficial crust, whereas deeper black microbial assemblages occur beneath a middle-lithified horizon. This configuration may explain the limited vertical stratification observed in the 16S dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec) and suggests that the lithified substrate, rather than the actively growing microbial mat, exerts a strong influence on current community distribution. Additional spatial and seasonal sampling across the Laguna Providencia microbialite buildup will be needed to determine whether this pattern is consistent throughout the lagoon or reflects localized conditions at the sampled site.\u003c/p\u003e \u003cp\u003eSpherulites present within the pustular microbial mats associated with the thrombolitic microbialites (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d) transition downward into clotted spherulitic textures in sub-fossilized sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee; Supplementary Fig. S5c-d online), occasionally enclosing bioclasts (Supplementary Fig. S5b). These clotted fabrics, composed of spherulites and other unidentified peloidal grains embedded in acicular cements, closely resemble textures reported from microbialites in Lagoa Vermelha\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, Rottnest Island in Western Australia\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, Cuatro Cienagas in Mexico\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e, Lake Salda in Turkey\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, and throughout the geologic record\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Microscopic examination of the deeper stromatolitic layers also revealed subtle sub-laminations composed of micritic and micropeloidal bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The increased lithification within both stromatolitic and thrombolitic fabrics likely reflects microbial activity, potentially linked to EPS degradation following burial\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e, and diagenetic processes commonly associated with the alteration of porewaters during burial, which can promote early cementation\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. In the stromatolitic intervals, these sub-laminations may reflect successive microbial biofilms, similar to those observed in SV1-E, that underwent progressive infilling by micritic precipitates. Within thrombolitic intervals, fluorescence microscopy and mineralogical staining together indicate organic-rich spherulite nuclei that likely served as initial nucleation sites for Mg-calcite micrite, followed by later aragonite precipitation forming less fluorescent rims and void-filling cements (Supplementary Fig. S5e-g)\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The development of these complex fabrics may therefore reflect the cumulative influence of a diverse, metabolically heterogeneous microbial community capable of sustaining multiple organomineralization and diagenetic pathways over time. These observations are in agreement with a paragenetic sequence in which Mg-calcite micrite precipitated first within organic-rich nuclei, where coccoid mats and later microbial activity during burial may have contributed to nucleation (e.g., \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e), and aragonite then precipitated during early diagenesis. The occurrence of framboidal pyrite within LP19 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef) could provide direct evidence of localized anoxia and active sulfur cycling, potentially linking sulfate-reduction activity, for example, to these paragenetic changes\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Although the contemporary microbial community may not represent the assemblages responsible for lithification, genomic data reveal significant populations of SRB, particularly \u003cem\u003eDesulfovermiculus\u003c/em\u003e and \u003cem\u003eDesulfatiglans\u003c/em\u003e, within the brown and black layers of LP19 relative to SV1-E and SS-NW (Supplementary Fig. S8c online).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePutative Authigenic Mg-Carbonate Phases in Microbialites\u003c/h2\u003e \u003cp\u003eThe occurrence of VHMC confined to the black layer in LP19 is an intriguing result (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). VHMC, together with high- and low-Mg calcite phases, was detected immediately beneath the middle-lithified crust within unlithified gray mud associated with the black-layer horizon and is not prominent in other LP19 microbialite layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This stratigraphic confinement indicates that Mg-carbonate formation was restricted to a discrete microenvironment distinct from the overlying lithified fabrics. X-ray diffraction patterns show that this VHMC lacks distinct 101 and 015 reflections and exhibits only weak 021 ordering, consistent with poorly ordered proto-dolomite rather than fully ordered dolomite (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Although some ordering peaks may be obscured by aragonite signals, the Laguna Providencia microbialite VHMC remains mineralogically distinct from the ordered dolomite of the adjacent Ponce Limestone\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLow-temperature dolomite formation remains a long-standing challenge in sedimentary geochemistry due to kinetic barriers to Mg incorporation under Earth-surface conditions (the “dolomite problem”)\u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. Increasing evidence from modern environments indicates that microbial activity (particularly sulfate reduction) can facilitate the formation of poorly ordered Mg-carbonate precursors that act as transient intermediates toward dolomite, as documented in microbial deposits from Lagoa Vermelha\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e, the Great Salt Lake\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, the Abu Dhabi sabkhas\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e, and Petukhovskoe soda lake, Russia\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In LP19, the occurrence of VHMC within organic-rich gray sediment coincides with elevated SRB abundances relative to the other lagoons, consistent with microbially influenced organomineralization pathways reported in these systems. The presence of amorphous Mg-silicate phases within this horizon (Supplementary Fig. S6c online) suggests an additional formation pathway, whereby silicate- or clay-related precursors contribute to Mg incorporation and nucleation, in agreement with models from other microbial and evaporitic environments\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMineralogical, stratigraphic, and isotopic distinctions collectively support a predominantly authigenic origin for the VHMC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although both the microbialites and the Ponce Limestone exhibit relatively enriched δ¹⁸O values, carbonates from the Ponce Limestone record a multi-stage diagenetic history, with secondary dolomite and calcite displaying δ¹³C-δ¹⁸O compositions that remain isotopically distinct from the gray sediment hosting VHMC\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. In contrast, depleted δ¹³C values in the microbialites are consistent with carbonate precipitation influenced by organic-matter degradation under reduced conditions\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Attempts to isolate the VHMC using dilute acetic acid resulted in dissolution, consistent with a poorly ordered and metastable Mg-carbonate rather than an ordered dolomite phase. Nevertheless, the isotopic signal likely reflects a mixed carbonate assemblage, and in the absence of porewater geochemistry or direct evidence of microbial nucleation, detrital input from Ponce Limestone dolomite cannot be excluded; accordingly, the VHMC occurrence should be regarded as suggestive rather than conclusive.\u003c/p\u003e \u003c/div\u003e "},{"header":"Concluding Remarks","content":"\u003cp\u003eThis study documents the first known occurrence of actively lithifying microbial mats and microbialites in Puerto Rico, expanding the inventory of microbial carbonate systems within the Caribbean and providing new insight into why some microbial mats undergo lithification while others do not. The three lagoons examined represent distinct positions along a lithification continuum shaped by the interaction of microbial community composition with physicochemical and hydrological context. In Salinas Salinetas, hypersaline and acidic conditions correspond to extensive but non-lithifying mats; in Salinas Vernales, more favorable conditions support active organomineralization, including micritic precipitation and spherulite development; and in Laguna Providencia, lithified microbialites preserve composite stromatolitic and thrombolitic fabrics and localized high-Mg carbonate phases indicative of microbially influenced early diagenesis. Together, these systems indicate that while substrate stability influences the spatial distribution of microbial deposits, sustained carbonate lithification is most closely associated with the development of diverse and compositionally distinct microbial communities operating within permissive hydrological and geochemical settings, potentially amplified over longer periods of system maturity.\u003c/p\u003e\u003cp\u003eAlthough porewater chemistry, metabolic rates, and temporal variability were not directly quantified, the close spatial coexistence of non-lithifying, partially lithifying, and fully lithified deposits within a small coastal region establishes the Guánica lagoons as a valuable natural laboratory for investigating microbial carbonate formation and biosignature preservation. In addition, the occurrence of poorly ordered Mg-carbonate phases within the Laguna Providencia microbialites bears directly on the low-temperature dolomite problem, indicating that microbialite-hosted environments can generate transient Mg-carbonate precursors distinct from detrital or diagenetic dolomite in adjacent carbonate units and providing a framework for distinguishing biogenic vs abiotic signals in both terrestrial and putative extraterrestrial carbonate environments (e.g.,\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e). Given the uniqueness and vulnerability of these coastal systems, coordinated protective measures and educational initiatives are warranted to limit anthropogenic impacts. Responsible management of these lagoons is essential not only to safeguard a unique coastal ecosystem in Puerto Rico and the Caribbean, but also to preserve globally relevant analogs for understanding Earth’s early biosphere and the search for life beyond our planet.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eFieldwork and Sample Collection\u003c/h2\u003e\u003cp\u003eThe microbial deposits described in this study were first documented during exploratory fieldwork in 2017, with initial observations focusing on the calcifying microbial mats from Salinas Vernales and non-lithifying microbial mats in the southeastern pond of Salinas Salinetas (SS-SE). Subsequent fieldwork in 2019 and 2020 expanded documentation to include the other systems in Salinas Salinetas (SS-C and SS-NW) and the microbialites in Laguna Providencia. The microbial community datasets presented here primarily derive from the 2019 fieldwork. All samples collected in 2019 were secured in sterile sampling bags, stored in a fridge during fieldwork, transported under sterile conditions to the Microbial Geochemistry Laboratory at the University of Kansas, and stored at -80°C until further analysis. Samples were dissected and divided based on pigmentation differences across each microbial deposit. Microbialite buildup observations and the nomenclature of the microbialite structures were denominated following the terminology established by Grey and Awramik\u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Ponce Limestone isotope data were added from Padilla-Montalvo\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online), alongside additional Ponce Limestone and beach sand samples collected for this study. Water temperature, pH, conductivity, and salinity were measured directly in the field using a handheld multiparameter probe (AquaRead Ltd.). For alkalinity analyses, surface water samples were collected in the field, filtered through 0.20 µm syringe filters, and total alkalinity was determined by acid titration to a pH endpoint of 4.3 shortly after collection.\u003c/p\u003e\u003ch2\u003eDrone Imagery\u003c/h2\u003e\u003cp\u003eOrthomosaic and multispectral drone imagery were acquired over the three lagoon systems and surrounding landscapes during the 2019 field season. Visible-spectrum (RGB) imagery was collected using a DJI Phantom 4 Pro V2 unmanned aerial vehicle equipped with a 20-megapixel RGB camera (1-inch, 2.54 cm CMOS sensor), with flight missions designed to provide complete spatial coverage of each lagoon and adjacent areas relevant to hydrologic and geomorphic interpretation.\u003c/p\u003e\u003cp\u003eAll flights were conducted using automated flight plans generated in DroneDeploy (version 2.202.0), incorporating a minimum of 80% forward overlap and 70% sidelap to ensure robust image matching and photogrammetric reconstruction. To minimize distortion associated with off-nadir viewing and refraction at the air-water interface, only the central, near-nadir portions of individual images were used during reconstruction.\u003c/p\u003e\u003cp\u003eRGB and multispectral imagery were processed into georeferenced orthomosaics (GeoTIFF format) using standard structure-from-motion (SfM) workflows in DroneDeploy and Agisoft Metashape Professional (version 2.0.1), including image alignment and point-cloud generation, with no major processing artifacts observed. A relative digital surface model (DSM) was derived from the RGB imagery, representing combined topographic and shallow bathymetric variability across lagoon basins and surrounding terrain. To facilitate qualitative interpretation of bathymetric and topographic gradients, the DSM was visualized using a false-color depth ramp, with warmer colors (red) indicating relatively shallower areas and cooler colors (green) indicating relatively deeper areas (Supplementary Fig. S4a–i).\u003c/p\u003e\u003ch2\u003ePetrography and X-Ray Diffraction\u003c/h2\u003e\u003cp\u003eFor petrographic analysis, select samples were impregnated with bio-epoxy and prepared as thin sections at the KU Department of Geology Rock and Thin Section Preparation Laboratory. Petrographic analysis was conducted using an Olympus BX53M optical microscope equipped with a motorized stage (©Marzhauser Wezlar) and an X-Cite 120Q for fluorescence illumination. Thin sections were analyzed under an Olympus BX53M optical microscope, and the microbialite fabrics and petrographic features were described following terminology established by Grey and Awramik \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e, and Flügel \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMineralogical characterization was performed across various depths. Each underwent treatment with 30% Hydrogen Peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), air drying, and was ground for Powder X-ray diffraction analysis (PXRD). Analysis was performed using a Bruker D2 Phaser powder x-ray diffractometer (CoKᾱ radiation) equipped with a 1D mode Lynxeye detector. The analysis spanned a 2θ angle range of 5˚ to 90˚ at 0.03˚ increments every 0.3 s. A 24.6 mm x 1.0 mm Zero Diffraction Silica crystal plate (© MTI Corp) was inserted into the standard D2 Phaser sample discs and coated with grease to hold the powder. In some samples, ©FischerBrand Sodium Chloride [NaCl] was added, and the halite [200] peak 2Q was used for peak alignment of calcite and dolomite [104] reflection displacements\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Scan results were processed (change to CuKᾱ spectra, background fitting, and alignment) and normalized for comparison using the R “powdR” package for XRD analysis\u003csup\u003e[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]\u003c/sup\u003e. The mole percentage of MgCO3 content in the Mg-carbonate precipitates was quantified using the Rietveld method (with the equation Mg% = 100 – (333.33x -911.99)\u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eScanning Electron Microscopy and EDS\u003c/h2\u003e\u003cp\u003eMicrobial mats and Microbialite fragments were fixed in 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 30 min, rinsed twice in buffer, and post-fixed in 1% osmium tetroxide for 1 h. Samples were dehydrated in a graded ethanol series (70%, 95%, and 100%) and dried using either critical point drying (CPD; Bal-Tec K850 Critical Point Drier) or hexamethyldisilazane (HMDS) substitution. After drying, samples were mounted on 12.7 mm aluminum stubs with conductive silver paint. Some samples were sputter-coated with ~ 10 nm of iridium or gold using a Quorum 150RS sputter coater, while others were analyzed without coating. For uncoated preparations, variable pressure SEM was used, or the samples were treated with Hitachi ionic liquid (diluted at 10–15%) HILEM© to improve surface conductivity. In addition, selected microbialite samples from Laguna Providencia were examined directly from polished petrographic thin sections without coating.\u003c/p\u003e\u003cp\u003eConventional imaging was performed with a Hitachi S-4700 cold field emission Scanning Electron Microscope (SEM), upper and lower secondary electron detectors and an Oxford Instruments X-Max 150 EDX detector. Images were collected at 5–10 kV accelerating voltage, 5–10 µA emission current, condenser lens at 2, and 9–12 mm working distance. Energy-dispersive X-ray spectroscopy (EDS) was carried out at 10 kV using Aztec software (Oxford Instruments) to map the distribution of major elements within precipitates and biofilms. Variable pressure imaging was conducted with a Hitachi FlexSEM 1000 II SEM at 30 Pa chamber pressure, 5 kV accelerating voltage, and 10 mm working distance, also coupled to an Oxford EDX system. The critical point drier, the sputter coater, and the imaging on the S-4700 SEM/EDS were performed at the Microscopy and Analytical Imaging Research Resource Core Laboratory, University of Kansas. Imaging on the FlexSEM was performed at the Nanofabrication Facility, University of Kansas.\u003c/p\u003e\u003ch2\u003eDNA extraction, sequencing, and processing\u003c/h2\u003e\u003cp\u003eMicrobial DNA was extracted in duplicate from each depth interval using the DNeasy PowerSoil Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol. Extracted DNA was used for PCR amplification of prokaryotic communities (Bacteria and Archaea). Primers 515F-Y\u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e and 806R-Y\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e were used to target the V4 hypervariable region of the 16S rRNA gene. PCR reactions included a mock community composed of bacterial and archaeal taxa in known concentrations as a quality control. Amplicon sizes were verified by gel electrophoresis. PCR products were purified using AmpureXP beads (Beckman Coulter, Brea, CA, USA), and dual-indexed libraries were prepared with Illumina Nextera® XT v2 indices (Illumina, San Diego, CA, USA) following the Illumina 16S Metagenomic Sequencing Library Preparation protocol. The pooled libraries were sequenced on an Illumina MiSeq platform using a paired-end 300-cycle kit at the University of Kansas Genome Sequencing Core (Lawrence, KS).\u003c/p\u003e\u003cp\u003eThe raw sequencing data for this dataset were processed using Quantitative Insights into Microbial Ecology2 (QIIME2–amplicon-2024.10)\u003csup\u003e[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e. Demultiplexed reads were trimmed using the cutadapt plugin, where primer sequences were removed, and the DADA2 plugin was used to denoise and dereplicate sequences, infer ASVs, and filter chimeras. Taxonomy was assigned with the SILVA v138.2 reference database\u003csup\u003e[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. The data were normalized using cumulative-sum scaling (CSS) implemented in the R (v3.6.3) package metagenomeSeq\u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e. The normalized dataset was further analyzed in R using the phyloseq package for community composition and diversity metrics, with additional analyses and visualizations performed using R packages ggplot2 and supporting packages such as dplyr and tidyr \u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlpha diversity metrics, including Observed Species, Shannon, and Simpson indices, were calculated to assess microbial diversity using the estimate_richness function in the phyloseq R package\u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e. To analyze beta diversity, Beta diversity was assessed using Bray–Curtis dissimilarities calculated from normalized abundance data in phyloseq.\u0026nbsp;Principal Coordinates Analysis (PCoA) was performed with the ordinate function, and ordination plots were generated in ggplot2\u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]\u003c/sup\u003e. Differences in microbial community composition across lagoons and layers were statistically evaluated using Permutational Multivariate Analysis of Variance (PERMANOVA) in the vegan package\u003csup\u003e[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]\u003c/sup\u003e. PERMANOVA was conducted with the adonis2 function, using Bray–Curtis dissimilarities as the response variable and sample grouping as the explanatory variable, with 999 permutations. Results are reported as the proportion of variance explained (R²) and associated p-values. Venn diagrams were constructed with the ggvenn package and were based on the number of shared and unique amplicon sequence variants (ASVs) to visualize overlap in microbial community composition among lagoons and depositional layers\u003csup\u003e[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by internal research funds from the University of Kansas. Drone and sUAS hardware, software, and data analysis were partially supported by Grant No. 2646 from the Unconventional Energy Center at Colorado Mesa University.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompelling Interests\u003c/h2\u003e \u003cp\u003eAuthors declared no compelling interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.J.R.C. and J.A.R. conceived the study and designed the field campaign. B.J.R.C. led all field investigations and conducted the mineralogical, petrographic, XRD, SEM, stable isotope, and microbial ecology analyses; processed and analyzed all sequencing datasets; prepared all figures; and wrote the manuscript. W.R.R.M. contributed to field sampling, geological interpretation, and review of sedimentological components. A.M.C.A. assisted with fieldwork, site documentation, and field observations. C.R.V. contributed to interpretation of microbial community patterns and reviewed the microbial ecology results. N.M.R. provided SEM imaging support and technical guidance for microscopy analyses and reviewed the manuscript. G.S.B. acquired and processed drone and sUAS datasets. Y.H. generated the raw 16S rRNA gene sequencing data and provided technical support for genomic data acquisition. B.S.M.S. provided oversight of the microbial genomics component and supervised sequencing workflows. J.A.R. supervised the project as principal investigator, provided funding and logistical support, contributed to field activities, and substantially revised the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Darien L\u0026oacute;pez Ocasio and the Puerto Rico Department of Natural and Environmental Resources for granting access and research permits for Laguna Providencia. Fieldwork in the Playa La Jungla region was made possible through access provided by Robert Viqueira (R.I.P.), Jorge Viqueira, Gretchen Marcial, Miguel Santiago, and the team at Protectores de Cuencas, Inc. Bryan Rodr\u0026iacute;guez-Col\u0026oacute;n, Aliyah Chabrier-Alpi, and Wilson Ram\u0026iacute;rez-Mart\u0026iacute;nez gratefully acknowledge the assistance of Miguel Jord\u0026aacute;n during field campaigns, and Iremar Fern\u0026aacute;ndez and James Padilla-Montalvo for support during field activities and collection of Ponce Limestone samples. B. Rodr\u0026iacute;guez-Col\u0026oacute;n, J. A. Roberts, and N. Mart\u0026iacute;nez-Rivera thank Eduardo Rosa-Molinar, former director of the Microscopy and Analytical Imaging Laboratory at the University of Kansas, for guidance on microscopy workflows and data processing. The authors also thank Marina Su\u0026aacute;rez and Robert Goldstein for insights on isotope geochemistry, Pike Holman for thin-section preparation, and Bruce Barnett and Mohammed Elshenawy for stable isotope data acquisition. This research was supported by internal research funds from the University of Kansas. Drone and sUAS hardware, software, and data analysis were partially supported by Grant No. 2646 from the Unconventional Energy Center at Colorado Mesa University.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eRaw 16S amplicon sequencing data are available on Figshare[71]. All bioinformatics and statistical analysis scripts used in this study are openly available on GitHub at https://bit.ly/PRGeoMicroSciRep. All remaining data supporting the findings of this study are included within the manuscript and its Supplementary Information. Drone and sUAS imagery are available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRiding, R. Microbial carbonates: the geological record of calcified bacterial-algal mats and biofilms. \u003cem\u003eSedimentology\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 179\u0026ndash;214 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurne, R., Moore, L. \u0026amp; Microbialites Organosedimentary Deposits of Benthic Microbial Communities. \u003cem\u003ePalaios\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 241\u0026ndash;254 (1987).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDupraz, C. et al. Processes of carbonate precipitation in modern microbial mats. \u003cem\u003eEarth Sci. 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Raw 16S Amplicon Sequencing 2019 Dataset from Microbial Mats and Microbialites in Southwestern Puerto Rico. \u003cem\u003efigshare.\u003c/em\u003e (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.6084/m9.figshare.30826205\u003c/span\u003e\u003cspan address=\"10.6084/m9.figshare.30826205\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microbial Carbonates, Microbial Mats, Microbialites, Puerto Rico, Guánica, Mg-carbonates","lastPublishedDoi":"10.21203/rs.3.rs-8570072/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8570072/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicrobial carbonate deposits, including microbial mats and microbialites, record coupled biological, geochemical, and sedimentary processes, providing insight into biosedimentary dynamics through Earth\u0026rsquo;s history and serving as analogs for ancient and potentially extraterrestrial environments. This study presents the first documentation of lithifying microbial systems in Puerto Rico, identified within three proximal coastal lagoons (Salinetas, Vernales, and Providencia) along the southwestern coast of the Gu\u0026aacute;nica municipality. These lagoons define a continuum of lithification, ranging from non-lithifying hypersaline mats to fully lithified microbialite deposits. An integrated field, geochemical, petrographic, and microbial ecological approach was used to characterize deposit morphology, mineralogy, and microbial community structure. At Salinetas, elevated salinity, low pH, and evaporitic conditions correspond to halite- and gypsum-dominated mats hosting halophilic Archaea and anoxygenic phototrophs, with minimal carbonate precipitation. In Vernales, carbonate precipitates closely associated with microbial filaments and EPS indicate enhanced organomineralization. Providencia hosts extensive lithified deposits displaying composite stromatolitic and thrombolitic fabrics, localized occurrences of high-Mg carbonate phases, and microbial communities enriched in sulfate-reducing bacteria relative to the other lagoons. Collectively, variations in hydrology, salinity, substrate, and microbial community composition correspond to differing degrees of lithification across a small coastal region, establishing southwestern Puerto Rico as a natural laboratory for investigating microbialite formation and biosignature preservation in coastal carbonate systems.\u003c/p\u003e","manuscriptTitle":"Documenting the Occurrence of Microbial Carbonate Deposits in Puerto Rico","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 06:41:19","doi":"10.21203/rs.3.rs-8570072/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-23T07:40:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-20T18:10:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T20:28:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95976307652430012640699880943088600101","date":"2026-01-23T13:44:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74135188948370737030934988747053252939","date":"2026-01-23T12:35:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-23T11:44:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-19T06:47:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-13T08:39:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-13T08:38:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-10T18:30:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ec4a5a15-62a9-4766-8520-833608a72a13","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":61011252,"name":"Earth and environmental sciences/Biogeochemistry"},{"id":61011253,"name":"Biological sciences/Ecology"},{"id":61011254,"name":"Earth and environmental sciences/Ecology"},{"id":61011255,"name":"Earth and environmental sciences/Environmental sciences"},{"id":61011256,"name":"Biological sciences/Microbiology"},{"id":61011257,"name":"Earth and environmental sciences/Ocean sciences"},{"id":61011258,"name":"Earth and environmental sciences/Solid earth sciences"}],"tags":[],"updatedAt":"2026-04-20T16:18:49+00:00","versionOfRecord":{"articleIdentity":"rs-8570072","link":"https://doi.org/10.1038/s41598-026-47709-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-13 15:59:14","publishedOnDateReadable":"April 13th, 2026"},"versionCreatedAt":"2026-01-13 06:41:19","video":"","vorDoi":"10.1038/s41598-026-47709-x","vorDoiUrl":"https://doi.org/10.1038/s41598-026-47709-x","workflowStages":[]},"version":"v1","identity":"rs-8570072","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8570072","identity":"rs-8570072","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}