Organomineralization processes in freshwater stromatolites: a living example from eastern Patagonia

Living stromatolites have been mostly described within shallow marine and (hyper)saline lacustrine environments. Southernmost South America lacks detailed investigations of these (organo)sedimentary buildups, particularly in regions experiencing extreme and variable environmental conditions. Here, we report and describe living freshwater stromatolites in the Maquinchao region, north‐western Patagonia, Argentina. Fossil stromatolites characterized by globular and cauliflower shapes are also present in a continuous palaeoshoreline of a former lake at an altitude of 830 m, whereas their living counterparts only occur in the calm waters of sheltered or meandering sections of the Maquinchao River. The living stromatolites and their host waters have been sampled and studied using various chemical and microscopic techniques to better constrain the environmental versus biological factors controlling their development. Our results indicate that today stromatolites only proliferate in freshwater when Ca2+ levels are high. A microscopic inspection of the living stromatolite mat indicates stronger photosynthetic activity in the upper green layer associated with crypto/microcrystalline calcite (nanoglobules) compared to the lower beige‐white biofilm. This biofilm contains more low‐Mg calcite (rhombohedra) precipitates, which can form millimetre‐sized aggregates in the underlying anoxic layer. Although sulphate‐reducing bacteria are living in the entire mat, they appear more abundant and widely distributed in the lower beige‐white layer and are always associated with Mg calcite. Low salinity and low‐turbidity water along with microbial (photosynthetic and heterotrophic) activity are the most important factors promoting low‐Mg calcite precipitation in the Maquinchao Basin. These conditions are very different from those proposed for recently described lacustrine stromatolites at high altitude in the subtropical and tropical Andes as well as in Chilean Patagonia. Hence, all these observations in modern freshwater stromatolites show the importance of geomicrobiological studies in identifying proxies of the hydrological conditions prevailing during their formation.


INTRODUCTION
Stromatolites constitute some of the oldest evidence for life on Earth (Hofmann et al., 1999). Commonly defined as laminated benthic organosedimentary structures built by the trapping and binding and/or precipitation of minerals via microbial processes (See Riding, 2011), it is no longer clear that all stromatolites represent biogenic structures (Grotzin- This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ger & Rothman, 1996;McLoughlin et al., 2008). They can display a variety of morphologies, including columnar, club, spheroidal, domal, nodular or irregular shape (Logan & Cebulski, 1970). The formation of these organosedimentary structures is a consequence of both microbially induced and microbially influenced mineralization (Burne & Moore, 1987;Trichet & D efarge, 1995;Dupraz et al., 2009) in which cyanobacteria are of primary importance. Due to the variety of surface environments where they occur, their assignment to particular environmental conditions of formation is not always straightforward. Studies of Bahamian stromatolites, for instance, find that lithified interiors beneath surface mat communities are modified by endolithic microbes . Based on the morphology, marine living stromatolites have been interpreted as analogues of fossil stromatolites. Today, living stromatolites occur in a few shallow marine and mostly (hyper)saline lacustrine environments. In southernmost South America there is a particular scarcity of detailed studies of lacustrine stromatolites in regions subject to extreme and variable environmental conditions. Lately, thrombolites and stromatolites have been identified in Chilean southern Patagonian lakes and used as indicators of palaeoclimate (Solari et al., 2010). To the best of our knowledge, however, no geomicrobiological investigations of the processes governing carbonate precipitation have been conducted. Previous studies on African freshwater stromatolites have shown that they are often distributed along lake shorelines. Thus, they can be used as excellent indicators of hydrological changes in palaeolakes improving the reconstruction of continental palaeoclimates (Casanova & Hillaire-Marcel, 1993). While it is clear that some stromatolites are built in response to microbial forcing Awramik & Grey, 2005), results of very recent studies have shown that the frequency of lamina formation is more closely related to regional climate forcing (Andersen et al., 2011;Petryshyn et al., 2012;). Yet, both aspects can be related and even complementary.
Recent freshwater stromatolites from the Maquinchao Basin (north-western Patagonia, Argentina) were studied in order to determine the processes involved in their formation and accretion, while relating these to environmental parameters during their development. Understanding the actual processes governing the formation of these modern microbialites aids in the interpretation of their fossil counterparts formed in the same basin during the last deglaciation and the early Holocene.

STUDY AREA
Laguna Cari-Laufquen is a closed basin (41°8 0 47″ S, 69°27 0 37″ W, ca 786 m asl; Fig. 1) located in a tectonic depression surrounded by basalt plateaus of Mesozoic to Tertiary age in northern Patagonia, Argentina (Coira, 1979). Presently, the lake system consists of two separate water bodies locally known as Laguna Cari-Laufquen Chica (LCLC; 2Á5 9 1Á5 km, averaging 3 m water depth in January, 2011), which is a permanent lake body with pH 8Á7 (Schwalb et al., 2002) and a sodium bicarbonate concentration of 230 ppm (Galloway et al., 1988); and Laguna Cari-Laufquen Grande (LCLG; 4 9 2 km, average 2 m depth in January, 2011), an ephemeral lake of brackish water with pH 8Á8 (Schwalb et al., 2002) and solute concentration of 4000 ppm (Galloway et al., 1988). LCLC sits at 825 m above sea-level, and periodically flows towards the larger basin through the Maquinchao River (Fig. 1). A 20 years series of meteorological data show a mean annual air temperature of 9Á0°C ranging from 16Á1°C to 1Á7°C in Austral summer and winter seasons, respectively, whereas annual precipitation averages 31Á6 cm over the same time interval (source: Argentinean Meteorological Survey). Previous investigations have shown evidence of former lake-level high stands during which both lakes LCLG and LCLC were merged to form a large palaeolake in the late Pleistocene (Del Valle et al., 1993. This lake encompassed more than 1500 km 2 and was at least 70 m deep during the pluvial phase maximum (Gonz alez Bonorino & Rabassa, 1973;Volkheimer, 1973;Coira, 1979;Galloway et al., 1988;Tatur et al., 2000;Ariztegui et al., 2008). Several palaeoshorelines indicate higher lake levels than today (Fig. 1). They have been previously dated at ca 19 ka (Galloway et al., 1988) and between 14 ka and 10-8 ka BP (Bradbury et al., 2001). Furthermore, lacustrine sediments outcropping within the Maquinchao Basin document a well-defined Late Pleistocene pluvial episode (Whatley & Cuminsky, 1999;Cusminsky et al., 2011) followed by a prograding sedimentary sequence. Stromatolites, also called 'tufa' by Cartwright et al. (2011), are widely distributed in the Laguna Cari-Laufquen often tracing the palaeoshorelines. They display a variable structure ranging from cauliflower to more globular with a nucleus of various compositions depending on their location. Fossil stromatolites showing a globular aspect can form plurimetric complexes and are present in a continuous palaeoshoreline at an altitude of 830 m (Figs 1, 2A and 3), dated at ca 22Á0 ka BP (Cartwright et al., 2011). Globular carbonate laminated structures most commonly surround a basaltic nucleus and do not exhibit any growth preferential axis or other evidence of hydrodynamic conditions.

Samples
Growing microbial bodies showing a globular structure similar to fossil stromatolites have been found in the Maquinchao River and are the subject of this study (Figs 2B and 4). Five living stromatolites attached to their substrate (basaltic pebbles) were sampled in the field in January, 2011 (Austral summer) when they were subaerially exposed, preserved in sterile 50-ml plastic tubes and stored at 4°C. The biofilms were subsampled by manual separation into a ca 1 mm thick surface sample (green biofilm) and an underlying sample taken from ca 2 to 3 mm below the surface (beige-white biofilm). Each layer was observed by optical microscopy under natural light (with prior ultrasonic for 30 sec) and air-dehydrated for scanning electron microscopy (SEM), while the entire stromatolite mat was observed using confocal laser scanning microscopy (CLSM).
Surface water samples were taken in five distinct areas as follows ( Fig. 1): (1) The LCLC outflow towards the Maquinchao River; (2) LCLC shore area; (3) LCLG shore area; (4) groundwater stream close to a vegetated area on the LCLG shore; and (5) sheltered section of the Maquinchao River containing living stromatolites. Today, the maximum 3 m depth of both lakes results in a water column which is continuously mixed making sampling at different depths unnecessary. Physicochemical data, that is, temperature, pH, salinity and conductivity were measured at all sites before sampling ( Fig. 3A and B).

Methods
Twenty millilitre of each water sample was filtered (0Á2 lm nylon filters) and acidified (1% v/v concentrated HCl) for subsequent elemental analysis by inductively coupled plasmaoptical emission spectrometry (ICP-OES, Varian Vista MPX, ETH Z€ urich). The extraction was performed on two to four independently incubated samples. Water samples were analysed for their stable isotope composition (dD and d 18 O, referenced to Vienna Standard Mean Ocean Water (VSMOW) at the Alfred Wegener Institute for Marine and Polar Research (AWI) Potsdam, Germany. A Finnigan MAT Delta-S mass spectrometer equipped with two equilibration units was used for the online determination of hydrogen and oxygen isotopic compositions. The external errors for standard measurements of hydrogen and oxygen are better than 0Á8& and 0Á1&, respectively (Meyer et al., 2000).
Various microscopic techniques were used to analyse the stromatolite structure down to the nanoscale: optical microscopy (Axioscope, ETH Z€ urich) under transmitted light, CLSM (Centre Technologique des Microstructures, Universit e Lyon 1, France), SEM (Jeol Zeiss Supra 50 VP, University of Zurich) on platinum coated samples prior to fixation with glutaraldehyde, and transmission electron microscopy (TEM, Phillips CM100, Centre Technologique des Microstructures, Universit e Lyon 1, France) after staining sectioned samples (Amann et al., 1995). Investi-gation of stained samples and control materials was carried out using a Zeiss LSM 510 META. Staining procedures for fluorescence in situ hybridization (FISH) were modified from Decho & Kawaguchi (1999). Samples were stained with the 16S rRNA probe SRB385 (CGGCGTCGCTGCGTCAGG) labelled with cyanine 3 to image sulphate-reducing bacteria (SRB). This oligoprobe targets SRB of the y-proteobacteria (Amann et al., 1990). Photosynthetic organisms and carbonate minerals were visualized by fluorescence. Unstained green biofilms were excited by the 633 nm laser line with the fluorescence detected above 650 nm, while the unstained beige/white biofilms were excited by the 543 nm laser line with fluorescence detected using a 560 nm long pass filter. Both biofilms were excited by a different wavelength in order to distinguish the autofluorescence of ostracods from that of cyanobacteria in the green biofilm. Autofluorescence from filamentous cyanobacteria in the unstained biofilms has been spectrally separated from the red-shifted autofluorescent calcite and indicated by the development of an artificial green colour. The Cy3-labelled probe SRB385 was excited by the 488 nm laser line with the fluorescence detected between 500 and 555 nm. CLSM Image analyses were performed using Image J and Imaris software version 5Á6.
Samples from fossil and living stromatolites were ground to an ultrafine powder in an agate mortar before being placed in a silicon wafer and a plastic sample holder for X-Ray diffraction analyses (XRD). They were analysed in a Bruker, AXS D8 Advance device, equipped with a Lynx-eye super-speed detector using Cu-K radiation and an anti-scattering slit of 20 mm, while rotating the sample. The sample patterns were recorded from 5°t o 85°2h in steps of 0Á004°, 1 sec counting time per step.

Water chemistry
The water chemistry of LCLG and LCLC, as well as that of the Maquinchao River varies by location. January, 2011 (Austral summer) was particularly hot and dry and the Maquinchao River was partially cut from LCLC being almost entirely fed by groundwater while flowing towards LCLG. This situation implies differential evaporation between the two lakes and the river (e.g. LCLC was almost dry) and consequently supports their different chemistry. Both shore and outflow water of LCLC display temperatures above 33°C while they are around 27°C at the other sites (3, 4 and 5; Fig. 5A). The pH in the water column ranged from 8Á1 (where stromatolites are living and in the Maquinchao River) to 9-9Á4 in both lakes. Salinity was fairly constant in most of the sampled sites indicating brackish conditions (1Á3-12Á5&). However, the riverine waters in which stromatolites are presently growing display more freshwater conditions (0Á7&).
Major and minor ions show some differences between the lakes and the other sites. Waters are dominated by Na with concentrations ranging from 150 up to 4750 ppm, while Ca and Mg concentrations average 19 and 68 ppm, respectively. For cations, the major elemental abundances in both lakes were Na + ≫ Mg 2+ > K + > Ca 2+ > Si 4+ , whereas the relative abundances were Na + ≫ Ca 2+ > Mg 2+ > K + > Si 4+ at the other sites (Table 1; Fig. 5B). Sulphur displays similar concentrations to Na and is likely present as SO 2À 4 (Schwalb et al., 2002). The Mg concentration was as high as 100 ppm and 200 ppm in LCLG and LCLC, respectively, whereas it was less than 25 ppm at the other sites. The highest Ca concentration (42 ppm) was measured where stromatolites are presently living, while minimum values were found in both lakes (around 7 ppm). Silicon concentrations of 3 or 4 ppm in both lakes contrasted with values reaching 20 ppm in the river and groundwater. The Mg:Ca ratio is typically greater than 15 and 25 in both lakes, whereas the other sites display ratios lower than 1. Figure 6 shows that LCLG and LCLC samples are characterized by 18 O-enriched waters (9Á53 and 5Á82&), whereas the groundwater and Maquinchao River sites display depleted values (À10& and À87& for d 18 O and dD, respectively). Lake waters are enriched isotopically compared to those reported by Schwalb et al. (2002) and Cartwright et al. (2011) which is consistent with the comparatively lower water levels of our sampling year. How- ever, the lake waters as a whole form a well-defined dDd 18 O mixing line with river-supplied inflow (Craig et al., 1974). This line is geometrically coincident with and statistically identical to a regression line computed from the lake data alone (r = 0Á97), indicating that lake waters evolved from the same primary water sources. Groundwater and Maquinchao River waters plot along the global meteoric water line, implying very low to non-evaporation. Despite the lack of in situ physicochemical measurements throughout the year, the existing meteorological data for the area suggest contrasting seasonal changes. It appears, however, that the biota is adapting to these conditions through time.

Living stromatolites sample description
Five stromatolite samples were collected from the Maquinchao River during mid-January 2011. Living stromatolites are found in several protected areas of the river such as the quiet water of meanders (Fig. 4A). Waters are less turbid here than in either of the lakes and contain abundant green slime between the stromatolites (Fig. 4B-D). These carbonate buildups display a globular morphology ( Fig. 7) with a thin (ca 1 mm thick) outermost biofilm composed of a green layer overlying an undulating beige-white layer, varying in thickness between 4 and 9 mm (Fig. 4B-D).
A series of XRD analyses in both the green and beigewhite layers showed that low-Mg calcite is the main mineral phase (70%) with a few detrital quartz grains and halite, which is an artefact of the sample processing. No crystalline Mg-Si minerals have been found. Similarly, low-Mg calcite is the dominant mineral phase in the fossil stromatolites displaying a Mg/Ca ratio of about 0Á03.

Upper green layer of the stromatolite mat
The green layer is composed of mainly filamentous and coccoid bacteria (Fig. 8A) with little granular amorphous organic matter (OM) (white arrow in Fig. 8A). These  bacteria are mostly photosynthetic as shown by their green colour under natural light and red autofluorescent pigments due to chlorophyll and phycobiliproteins ( Fig. 8A and C), in addition to the presence of Spirulina (data not shown). Their abundance is supported by the strong autofluorescence in the first millimetre of the mat compared to the deeper layers (Fig. 8C). Microcrystalline low-Mg calcite is widely observed associated with cyanobacteria as characterized by the mixed, that is, yellow colour. The blue-fluorescing SRB are co-localized with the red-fluorescing cyanobacteria indicating that SRB are living in oxic conditions (Fig. 8E). Microbial communities are trapped with diatoms within an extracellular polymeric substances (EPS) matrix (Fig. 9A). Diatoms are partially to entirely dissolved (Fig. 9B) when completely trapped by EPS. Organic filaments (Fig. 9C) and cryptocrystalline low-Mg calcite are embedded within EPS. Mineralized EPS are Ca-enriched (Fig. 9D) compared to low-Mg calcite (Fig. 9E). The latter is always associated with low Si content (from likely dissolved diatoms), while no Al was detected (Fig. 9E). Micro/cryptocrystalline low-Mg calcites are either characterized by nanoglobules (Fig. 9F) or arranged as platelets, closely associated with EPS suggesting an in situ growth. This low-Mg calcite crystal arrangement creates a nanoporosity (Fig. 9F). Examination by TEM confirms that this biofilm is mainly composed of cyanobacteria as shown by multiple rows of tightly stacked thylakoids, that is, membranes where photosynthesis occurs (Fig. 10A), indicating healthy, active photoautotrophic organisms (Stolz, 1991). Extracellular polymeric substances were distributed as nanofilaments creating a mesh-like network that is not restricted to the surface of the cells (Fig. 10B). Other microorganisms such as rod-shaped Gram-negative bacteria have also been identified (Fig. 10C), probably SRB.

Lower beige-white layer of the stromatolite mat
The beige-white biofilm displays more granular amorphous organic matter (AOM) compared to the green biofilm (Fig. 8B) and an association of coccoid cyanobacteria and colourless dead filaments (Fig. 8B). Some filaments were also photosynthetic as shown by their red autofluorescence (Fig. 8D) but most were colourless in contrast to the green colour they displayed in the overlying biofilm. Green, autofluorescent, low-Mg calcite crystals occur locally in conjunction with cyanobacteria ( Fig. 8D) but are more abundant when associated with SRB (Fig. 8F) in this layer than they are in the overlying green one. Extracellular polymeric substances and microcrystalline low-Mg calcite form a complex matrix (Fig. 11A and B). These crystals occur as stacks of platelets which combine further to form rhom- bohedra (Fig. 11F). The surface of the platelets is characterized by a nanoglobular structure (Fig. 11C) connected by nanofilaments of EPS (Fig. 11C). These crystals, consisting of low-Mg calcite, were associated with a small amount of silicon possibly resulting from diatom dissolution (Fig. 11D). The ultrastructure of this biofilm consists primarily of dying cyanobacteria surrounded by substantial fibrillar EPS (Fig. 10D) together with the remains of partially dissolved diatoms. Large quantities of fibrillar EPS can form aggregates containing several cells (Fig. 10D). The abundance of empty cyanobacterial sheathes along with the increased density of the collapsed sheath material are indicative of dying cyanobacteria (Fig. 10E) and indicate early to advanced stages of degradation (Stolz et al., 2001). Ample virus-like particles have been found, sometimes icosahedral in shape (Pacton et al., 2014; Fig. 10F).

DISCUSSION
Environmental conditions constraining stromatolite occurrence in the Maquinchao Basin: implications for palaeohydrological reconstructions Clear differences in water composition have been observed between the presently growing stromatolites and the other sampled sites. These differences allow the envi-ronmental parameters controlling stromatolite growth to be identified and, thus, permit the conditions of carbonate precipitation in their fossil counterparts to be estimated. Firstly, the alkaline pH throughout the different sampling sites, that is, 8Á1-9Á4, indicates that this parameter is not a determining factor for the presence of stromatolites. Secondly, the living stromatolites appear to prefer freshwater rather than the more turbid and brackish lake waters. Thirdly, calcium is the most significant element in the freshwater sites, contrasting with the brackish waters of both present-day lakes where Mg and K dominate (Fig. 5B). Similarly to the high-altitude volcanic lakes of Patagonia, the water of both lakes contains essentially all inorganic nutrients, and at much higher concentrations (except for Ca), than the Maquinchao River, making it unlikely that the input of these chemicals is critical for stromatolite development (Farias et al., 2013). Although nutrient availability has not been investigated in detail, turbidity is one of the most important factors constraining stromatolite development in the Maquinchao Basin. It is widely accepted that the presence of dissolved Mg 2+ inhibits the precipitation of calcite favouring the precipitation of aragonite, especially with a Mg 2+ :Ca 2+ ratio >12 (Bischoff & Fyfe, 1968;M€ uller et al., 1972;Berner, 1975). Therefore, the palaeolake in which the fossil stromatolites were living most probably had a lower Mg 2+ :Ca 2+ ratio than today and was thus closer in composition to the Maquinchao River. At present, the Maquinchao region is one of the driest in the world but both lake basins show geomorphological evidence (palaeo-shorelines) of major fluctuations in the water balance since the last deglaciation. According to Bradbury et al. (2001) the large Cari-Laufquen palaeolake experienced several high stands during the Late Pleistocene and Early Holocene. These lacustrine basins had low productivity and were less saline and turbid than today. Lakes fed by rivers draining volcanic terrains of dominantly basic composition, coupled with groundwater inflow could create a set of conditions involving high CO 2 input and carbonate alkalinity, high dissolved silica, and high levels of Mg, Ca and Na (Wright, 2012). According to Valero-Garc es et al. (2001) Quaternary lacustrine travertines and stromatolites from high-altitude Andean lakes in north-western Argentina were probably formed during low lake levels, when conditions were more favourable for cyanobacterial growth than for plant development. These authors suggested that grazing of microbial mats could be another factor preventing stromatolite formation as microbial mats form the basis of benthic food webs that include metazoan consumers such as gastropods, microcrustaceans, insect larvae and vertebrates (Elser et al., 2005). The geochemical and microscopic studies of the Cari-Laufquen stromatolites presented here clearly indicate that, in the Maquinchao Basin, stromatolites only proliferate under dominantly freshwater conditions corresponding to meander-like fluviatile environments. Although the effect of seasonal variations on stromatolite development has yet to be studied, cold periods might reduce microbial metabolic activity and subsequent carbonate precipitation (Pl ee et al., 2008). Thus, living stromatolite mats provide a proxy to reconstruct the prevailing environmental conditions influencing growth of their fossil counterparts in the same basin. These results agree with the presence of fossil stromatolites along the late Pleistocene palaeoshorelines.

Stromatolites: the role of microbial communities in carbonate precipitation
There are several significant differences in macroscopic characteristics and species composition between the two biofilm layers constituting the stromatolites. Firstly, both the green and beige/white biofilms contain filamentous and coccoid cyanobacteria, diatoms and other heterotrophs, but in different relative abundances. We observed an increase in dead photosynthetic microbes in the underlying beige/white biofilm as shown by their colourless aspect (Grilli Caiola et al., 1993) along with an increase in SRB and other heterotrophs. Examination by TEM of the beige/white biofilm confirms the increased degradation of cyanobacterial cells as well as degradation of EPS into fine fibres. The latter is associated with an increase in granular AOM suggesting OM degradation of microbial origin (Pacton et al., 2011). This associattion might be due to the aerobic respiration and/or sulphate reduction of microorganisms. Such SRB are known not only to survive oxic conditions but also to thrive in the presence of cyanobacteria (Canfield & Des Marais, 1991;Dupraz & Visscher, 2005;Baumgartner et al., 2006) resulting in the partial degradation of EPS and low molecular weight organic carbon (LMW-OC) (Decho, 2000;Glunk et al., 2011). Virus-like particles have also been identified in both biofilms, free within EPS, but appeared more abundant in the beige/white biofilm. It is known that viruses are the most abundant biological entities throughout marine and terrestrial ecosystems (Suttle, 2005), and more specifically in modern microbial mats (Desnues et al., 2008;Pacton et al., 2014). As they have been found in the biofilm associated with degraded cells, we suggest that they may have been involved in the infection of these cells. It is known that viruses represent the largest fraction of nanosized particles that can undergo degradation or, under specific conditions, mineralization processes (Pacton et al., 2014). Viruses have been found as important mineralized entities in microbial mats (Pacton et al., 2014;De Wit et al., 2015) although an alternative origin such as membrane vesicles (MVs) cannot be ruled out. The latter share morphological and chemical similarities and could outnumber viral particles in sea water samples, especially when they are quantified using epifluorescence microscopy (Forterre et al., 2013;Biller et al., 2014). However, according to Soler et al. (2015), the abundance of MVs could also be overestimated as they could contain viral genomes. Further studies are required to better discriminate MVs from true viruses in environmental samples. Although there is no mineralogical difference between the carbonates of both biofilm layers (low-Mg calcite), crystal morphologies are quite distinctive. Low-Mg calcite crystals in the outermost green biofilm are either characterized by nanoglobules (probably mineralized viruses) or arranged as platelets associated with Si and EPS. The platelets within the beige/white biofilm tend to form euhedral rhombohedra that are often coated or partially filling the pore spaces of the biofilm. The EPS show an enrichment in Si-Ca-Mg elements suggesting that they are mineralized as amorphous Mg-Si ( am Mg-Si) phases associated with low-Mg calcite (Fig. 11D). Amorphous Mg-Si precipitates have previously been reported in numerous environments, always associated with microbial activity (Arp et al., 2003;Souza-Egipsy et al., 2005;Benzerara et al., 2010;Pacton et al., 2012). It has also been demonstrated that am Mg-Si enhances fossilization of microbes, and is commonly found in the rock record. The authigenesis of am Mg-Si phases is, however, poorly understood, with previous work suggesting that am Mg-Si layers in microbialites replace a primary mineral phase (Arp et al., 2003). In contrast, this data favour a primary origin for these am Mg-Si phases. The close association between carbonate crystals, EPS and am Mg-Si suggests that the precipitation is microbially mediated. The microbially mediated precipitation pathways are called organomineralization processes and are defined as (i) biologically induced mineralization resulting from the interaction between biological activity and the environment; and (ii) biologically influenced mineralization, which is a passive mineralization of organic matter (biogenic or abiogenic in origin), influencing crystal morphology and composition (Dupraz et al., 2009).
In the green biofilm, most of the mineral phases appear as a complex of Mg-Si and Ca characterized by nanoglobules closely related to EPS. Initially the negatively charged functional groups in fresh EPS bind Ca 2+ and Mg 2+ , inhibiting carbonate precipitation (Dupraz et al., 2009). Nanoglobules of EPS could be the first step towards mineralization as EPS have been reported to be permineralized as amorphous Mg-Si phases ( am Mg-Si) before being calcified (Souza-Egipsy et al., 2005;Pacton et al., 2012). In the underlying beige/white layer, sulphate reduction and possible respiration would increase availability of Ca 2+ , promoting carbonate precipitation as rhombohedra (Dupraz & Visscher, 2005). The EPS act as a template for carbonate precipitation arranging platelets into rhombohedra suggesting an EPS-influenced organomineralization. As SRB have been shown to be highly active in lithified zones of microbial mats Dupraz et al., 2004), they might have been involved in lithification of stromatolites. However, SRB and other heterotrophs could have reduced EPS by breaking down exopolymers and inhibiting precipitation in the upper green layer (Arp et al., 2012). This phenomenon is illustrated by the degradation of nanofilaments of EPS into fine fibres. Further investigation of the fossil stromatolites is required in order to decipher the biotic from the abiotic contribution to their formation.
Although cyanobacteria primarily contribute to stromatolite morphogenesis, the metabolic activity of heterotrophic bacteria such as SRB is also significant in microbial mats (Canfield & Des Marais, 1991;Des Marais, 2003;Dupraz et al., 2004). The globular morphology of these stromatolites likely reflects specific microbial responses to their environment, which may result from differential growth, migration of microbes with respect to environmental conditions such as light, O 2 , etc. or a combination of both (Andersen et al., 2011).

CONCLUSIONS
Modern stromatolites from the Maquinchao Basin (Argentina) are presently formed by microbial communities including filamentous and coccoid cyanobacteria, non-photosynthetic microorganisms capable of respiration and/or sulphate reduction, and diatoms. Combining water chemistry and microscopy data enabled the optimum conditions for stromatolite growth to be constrained.
Unlike recently described stromatolites at high altitude in the subtropical and tropical Andes, a combination of low salinity and turbidity, relatively high Ca and microbial (photosynthetic and heterotrophic) activity appear to be some of the most important factors promoting low-Mg calcite formation in the Maquinchao Basin. Our results further indicate that carbonate formation is mediated by organomineralization processes in which EPS play a fundamental role. Freshwater stromatolites appear to be a useful proxy for changing hydrological conditions. They also highlight the significant role of modern geomicrobiological studies in understanding the mechanisms and environmental conditions underlying their formation.