Rosaluz Tavera

Prokaryotic and Eukaryotic Community Structure in Field and Cultured Microbialites from the Alkaline Lake Alchichica (Mexico)

The geomicrobiology of crater lake microbialites remains largely unknown despite their evolutionary interest due to their resemblance to some Archaean analogs in the dominance of in situ carbonate precipitation over accretion. Here, we studied the diversity of archaea, bacteria and protists in microbialites of the alkaline Lake Alchichica from both field samples collected along a depth gradient (0–14 m depth) and long-term-maintained laboratory aquaria. Using small subunit (SSU) rRNA gene libraries and fingerprinting methods, we detected a wide diversity of bacteria and protists contrasting with a minor fraction of archaea. Oxygenic photosynthesizers were dominated by cyanobacteria, green algae and diatoms. Cyanobacterial diversity varied with depth, Oscillatoriales dominating shallow and intermediate microbialites and Pleurocapsales the deepest samples. The early-branching Gloeobacterales represented significant proportions in aquaria microbialites. Anoxygenic photosynthesizers were also diverse, comprising members of Alphaproteobacteria and Chloroflexi. Although photosynthetic microorganisms dominated in biomass, heterotrophic lineages were more diverse. We detected members of up to 21 bacterial phyla or candidate divisions, including lineages possibly involved in microbialite formation, such as sulfate-reducing Deltaproteobacteria but also Firmicutes and very diverse taxa likely able to degrade complex polymeric substances, such as Planctomycetales, Bacteroidetes and Verrucomicrobia. Heterotrophic eukaryotes were dominated by Fungi (including members of the basal Rozellida or Cryptomycota), Choanoflagellida, Nucleariida, Amoebozoa, Alveolata and Stramenopiles. The diversity and relative abundance of many eukaryotic lineages suggest an unforeseen role for protists in microbialite ecology. Many lineages from lake microbialites were successfully maintained in aquaria. Interestingly, the diversity detected in aquarium microbialites was higher than in field samples, possibly due to more stable and favorable laboratory conditions. The maintenance of highly diverse natural microbialites in laboratory aquaria holds promise to study the role of different metabolisms in the formation of these structures under controlled conditions.

Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria.

Significance Cyanobacteria are known to promote the precipitation of Ca-carbonate minerals by the photosynthetic uptake of inorganic carbon. This process has resulted in the formation of carbonate deposits and a fossil record of importance for deciphering the evolution of cyanobacteria and their impact on the global carbon cycle. Though the mechanisms of cyanobacterial calcification remain poorly understood, this process is invariably thought of as extracellular and the indirect by-product of metabolic activity. Here, we show that contrary to common belief, several cyanobacterial species perform Ca-carbonate biomineralization intracellularly. We observed at least two phenotypes for intracellular biomineralization, one of which shows an original connection with cell division. These findings open new perspectives on the evolution of cyanobacterial calcification. Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria.

Steady state of phytoplankton assemblage in the tropical Lake Catemaco (Mexico)

Phytoplankton of the tropical lake Catemaco (Veracruz, Mexico) showed similar species composition during samplings from 1993 to 1995. There were two small dominant cyanobacterial species Cylindrospermopsis catemaco Kom.-Legn. et Tavera and Cylindrospermopsis philippinensis (Taylor) Kom., and a group of larger algae and cyanobacteria that were always present, however in smaller numbers: Aulacoseira granulata (Ehr.) Simons morphotype curvata, A. cf. italica (Ehr.) Simons mf. curvata, Fragilaria construens (Her.) Grun., Achnanthes minutissima Kütz., Planktolyngbya circumcreta (G.S.West) Anagn. et Kom., Chroococcus microscopicus Kom.-Legn. et Cronberg. Moreover we found several other scarcely present species. The percentage of total biomass of the two dominant species of Cylindrospermopsis varied between 34 and 81%, but they accounted for 80 to 95% of abundance. Apart from geomorphological features and climate conditions, biological variables played an important role. Fish-stock was formed by filter-feeding native herbivorous species of fish Dorosoma petenense (Günther), Bramocharax caballeroi (Contreras et Rivera), Astyanax mexicanus (Filippi), and an introduced, also herbivore Oreochromis niloticus (L.). Feeding activity of fish removed large species of algae and cyanobacteria as well as detrital remnants and zooplankton from the water. Smaller, inedible cyanobacteria remained in the water and formed the stable portion of the phytoplankton, dominant both in biomass and abundance. CANOCO analysis of samples and species variability demonstrated results of competition between two species of Cylindrospermopsis: steady state during the dominance of C. catemaco lasting probably for the whole year 1993 (one dry and one wet season) and steady state during the dominance of C. philippinensis in 1994 and 1995. According to the functional classification of phytoplankton suggested by Reynolds et al. (2002), Catemaco dominant assemblage would belong to the functional group SN.

Specific carbonate–microbe interactions in the modern microbialites of Lake Alchichica (Mexico)

The role of microorganisms in microbialite formation remains unresolved: do they induce mineral precipitation (microbes first) or do they colonize and/or entrap abiotic mineral precipitates (minerals first)? Does this role vary from one species to another? And what is the impact of mineral precipitation on microbial ecology? To explore potential biogenic carbonate precipitation, we studied cyanobacteria–carbonate assemblages in modern hydromagnesite-dominated microbialites from the alkaline Lake Alchichica (Mexico), by coupling three-dimensional imaging of molecular fluorescence emitted by microorganisms, using confocal laser scanning microscopy, and Raman scattering/spectrometry from the associated minerals at a microscale level. Both hydromagnesite and aragonite precipitate within a complex biofilm composed of photosynthetic and other microorganisms. Morphology and pigment-content analysis of dominant photosynthetic microorganisms revealed up to six different cyanobacterial morphotypes belonging to Oscillatoriales, Chroococcales, Nostocales and Pleurocapsales, as well as several diatoms and other eukaryotic microalgae. Interestingly, one of these morphotypes, Pleurocapsa-like, appeared specifically associated with aragonite minerals, the oldest parts of actively growing Pleurocapsa-like colonies being always aragonite-encrusted. We hypothesize that actively growing cells of Pleurocapsales modify local environmental conditions favoring aragonite precipitation at the expense of hydromagnesite, which precipitates at seemingly random locations within the biofilm. Therefore, at least part of the mineral precipitation in Alchichica microbialites is most likely biogenic and the type of biominerals formed depends on the nature of the phylogenetic lineage involved. This observation may provide clues to identify lineage-specific biosignatures in fossil stromatolites from modern to Precambrian times.

Atelomixis as a possible driving force in the phytoplankton composition of Zirahuen, a warm-monomictic tropical lake

The present work reviews our current understanding of the limnology of Lake Zirahuén and extends this knowledge through an analysis of the phytoplankton, illustrating phenomena that typify monomictic tropical lakes. The analysis reinforces the postulate that atelomixis determines the variation in phytoplankton composition of deep tropical lakes with a monomictic mixing regime. Similarly, it is proposed that an incomplete or partial atelomixis, generating a highly dynamic mixing layer, is a force that possibly drives the selection of algal groups, like the Desmidiaceae, with a high surface area to volume ratio. These organisms, characteristic of tropical lakes, though found in the deep epilimnetic layer, receive sufficient irradiance to support their high photosynthetic rate; their high population density survive thanks to a constant supply of nutrients. Lake Zirahuén is a particularly relevant case for Mexico, since the low calcium concentration and the trophic level of the lake provide an explanation for the otherwise uncommon presence of desmids in other Mexican lakes, even in lakes that exhibit atelomictic phenomena.

Formation of low-T hydrated silicates in modern microbialites from Mexico and implications for microbial fossilization

Microbialites are organo-sedimentary rocks found in abundance throughout the geological record back to ~3.5 Ga. Interpretations of the biological and environmental conditions under which they formed rely on comparisons with modern microbialites. Therefore, a better characterization of diverse modern microbialites is crucial to improve such interpretations. Here, we studied modern microbialites from three Mexican alkaline crater lakes: Quechulac, La Preciosa and Atexcac. The geochemical analyses of water solutions showed that they were supersaturated to varying extents with several mineral phases, including aragonite, calcite, hydromagnesite, as well as hydrated Mg-silicates. Consistently, X-ray diffraction and Fourier transform infrared spectroscopy analyses revealed that microbialites are composed of a diversity of mineral phases including aragonite and sometimes calcite, hydromagnesite, and more interestingly, a poorly-crystalline hydrated silicate phase. Coupling of scanning electron microscopy with energy dispersive X-ray spectrometry microanalyses on polished sections showed that this latter phase is abundant, authigenic, magnesium-rich and sometimes associated with iron and manganese. This mineral phase is similar to kerolite, a hydrated poorly crystalline talc-like phase (Mg3Si4O10(OH)2·nH2O). Diverse microfossils were permineralized by this silicate phase. Some of them were imaged in 3D by FIB-tomography showing that their morphologically was exquisitely preserved down to the few nm-scale. The structural and chemical features of these fossils were further studied using a combination of transmission electron microscopy and scanning transmission X-ray microscopy at the carbon and magnesium K-edges and iron L2,3-edges. These results showed that organic carbon is pervasively associated with kerolite. Overall, it is suggested that the poorly-crystalline hydrated magnesium-rich silicate forms in many alkaline lakes and has a strong potential for fossilization of microbes and organic matter. Moreover, the frequent occurrence of such an authigenic silicate phase in modern lacustrine microbialites calls for a reappraisal of its potential presence in ancient rocks.

Metagenome-based diversity analyses suggest a significant contribution of non-cyanobacterial lineages to carbonate precipitation in modern microbialites

Cyanobacteria are thought to play a key role in carbonate formation due to their metabolic activity, but other organisms carrying out oxygenic photosynthesis (photosynthetic eukaryotes) or other metabolisms (e.g., anoxygenic photosynthesis, sulfate reduction), may also contribute to carbonate formation. To obtain more quantitative information than that provided by more classical PCR-dependent methods, we studied the microbial diversity of microbialites from the Alchichica crater lake (Mexico) by mining for 16S/18S rRNA genes in metagenomes obtained by direct sequencing of environmental DNA. We studied samples collected at the Western (AL-W) and Northern (AL-N) shores of the lake and, at the latter site, along a depth gradient (1, 5, 10, and 15 m depth). The associated microbial communities were mainly composed of bacteria, most of which seemed heterotrophic, whereas archaea were negligible. Eukaryotes composed a relatively minor fraction dominated by photosynthetic lineages, diatoms in AL-W, influenced by Si-rich seepage waters, and green algae in AL-N samples. Members of the Gammaproteobacteria and Alphaproteobacteria classes of Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacterial taxa, followed by Planctomycetes, Deltaproteobacteria (Proteobacteria), Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi. Community composition varied among sites and with depth. Although cyanobacteria were the most important bacterial group contributing to the carbonate precipitation potential, photosynthetic eukaryotes, anoxygenic photosynthesizers and sulfate reducers were also very abundant. Cyanobacteria affiliated to Pleurocapsales largely increased with depth. Scanning electron microscopy (SEM) observations showed considerable areas of aragonite-encrusted Pleurocapsa-like cyanobacteria at microscale. Multivariate statistical analyses showed a strong positive correlation of Pleurocapsales and Chroococcales with aragonite formation at macroscale, and suggest a potential causal link. Despite the previous identification of intracellularly calcifying cyanobacteria in Alchichica microbialites, most carbonate precipitation seems extracellular in this system.

16S rDNA-based analysis reveals cosmopolitan occurrence but limited diversity of two cyanobacterial lineages with contrasted patterns of intracellular carbonate mineralization

Cyanobacteria are mainly thought to induce carbonate precipitation extracellularly via their photosynthetic activity combined with the nucleation potential of exopolymeric substances. The discovery in microbialites of the alkaline lake Alchichica (Mexico) of Candidatus Gloeomargarita lithophora, a cyanobacterium forming large amounts of intracellular Mg-Ca-Sr-Ba carbonate spherules, showed that intracellular biomineralization in cyanobacteria is also possible. A second cyanobacterium isolated from the same environment, Candidatus Synechococcus calcipolaris G9, has been recently shown to also form intracellular calcium carbonates at the cell poles, a capability shared by all cultured species of the Thermosynechococcus clade, to which it belongs. To explore the diversity of these two distant cyanobacterial lineages representing two different patterns of intracellular calcification, we designed specific primers against their 16S rRNA genes and looked for their occurrence in a wide variety of samples. We identified the presence of members of the Gloeomargarita and Thermosynechococcus/S. calcipolaris lineages in microbialites collected from Lake Alchichica and three other neighboring Mexican lakes. The two clades also occurred in karstic areas and in some thermophilic or hypersaline microbial mats collected in South America and/or Southern Europe. Surprisingly, the within-group diversity in the two clades was low, especially within the S. calcipolaris clade, with all 16S rRNA gene sequences retrieved sharing more than 97% identity. This suggests that these clades are composed of a limited number of operational taxonomic units (OTUs) with cosmopolitan distribution. Moreover, scanning electron microscopy coupled with energy dispersive x-ray spectrometry showed the presence of intracellularly calcifying Gloeomargarita-like cyanobacteria in fresh samples where this clade was relatively abundant, suggesting that these cyanobacteria do precipitate carbonates intracellularly under natural conditions.

Multifaceted approach for the analysis of the phototrophic microbial community in a freshwater recreational area of Xochimilco, México

The freshwater microbial community in a recreational area of Xochimilco, México was investigated and compared based on spatial (three different sites) and temporal (dry and rainy seasons) environmental variables. Many of the 16S- and 18S rRNA gene sequences recovered by DGGE fingerprinting analysis were related to phototrophic microbial phylotypes of known identity. Our genetic and morphological analysis indicated the ubiquitous presence of the microeukaryotic green algae Desmodesmus-Scenedesmus spp. and of the unicellular cyanobacteria Cyanobium spp. as the most representative populations in the samples. While 18S rRNA-DGGE fingerprinting analysis revealed a homogeneous community composition across sites and seasons, the 16S rRNA showed significant differences between localities and seasons. None of the cyanobacteria species with potential to produce toxins were identified across the investigated samples. Correlations between biotic and abiotic variables evidenced an important difference between the dry and the rainy season, with a greater consistency in data from the rainy season. According to Principal Component Analysis (PCA), a strong relation between inorganic nitrogen, species richness, and subaquatic irradiance determines environmental variability in Xochimilco. Complementary and relevant data in results obtained from microscopy, fingerprinting, and statistical analysis applied in ecology indicate that a multifaceted approach to the study of microbial communities is necessary to accomplish a comprehensive scientific framework and to generate proper management strategies.

Phytoplankton composition and biomass in a shallow monomictic tropical lake

Lake Zempoala was studied throughout 16 months in 1996–1997. It is a shallow monomictic lake situated at 2800 masl at the Neovolcanic Belt, well within the Mexican tropical zone. Most of the phytoplankton species in this lake may be characterized as temperate, according to their geographical distribution. A break down in phytoplankton biomass was observed before the lakes circulation, and open to question if a clear-water phase could be present in a tropical lake.