Innovative approaches to accurately assess the effectiveness of biocide-based treatments to fight biodeterioration of Cultural Heritage monuments.

The development of diagnostic methods to accurately assess the effects of treatments on lithobiont colonization remains a challenge for the conservation of Cultural Heritage monuments. In this study, we tested the efficacy of biocide-based treatments on microbial colonization of a dolostone quarry, in the short and long-term, using a dual analytical strategy. We applied a metabarcoding approach to characterize fungal and bacterial communities over time, integrated with microscopy techniques to analyze the interactions of microorganisms with the substrate and evaluate the effectiveness. These communities were dominated by the bacterial phyla Actinobacteriota, Proteobacteria and Cyanobacteria, and the fungal order Verrucariales, which include taxa previously reported as biodeteriogenic agents and observed here associated with biodeterioration processes. Following the treatments, changes over time in the abundance profiles depend on taxa. While Cyanobacteriales, Cytophagales and Verrucariales decreased in abundance, other groups, such as Solirubrobacteriales, Thermomicrobiales and Pleosporales increased. These patterns could be related not only to the specific effects of the biocide on the different taxa, but also to different recolonization abilities of those organisms. The different susceptibility to treatments could be associated with the inherent cellular properties of different taxa, but differences in biocide penetration to endolithic microhabitats could be involved. Our results demonstrate the importance of both removing epilithic colonization and applying biocides to act against endolithic forms. Recolonization processes could also explain some of the taxon-dependent responses, especially in the long-term. Taxa showing resistance, and those benefiting from nutrient accumulation in the form of cellular debris following treatments, may have an advantage in colonizing treated areas, pointing to the need for long-term monitoring of a wide range of taxa. This study highlights the potential utility of combining metabarcoding and microscopy to analyze the effects of treatments and design appropriate strategies to combat biodeterioration and establish preventive conservation protocols.

Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits.

Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars.

Inhabited subsurface wet smectites in the hyperarid core of the Atacama Desert as an analog for the search for life on Mars.

The modern Martian surface is unlikely to be habitable due to its extreme aridity among other environmental factors. This is the reason why the hyperarid core of the Atacama Desert has been studied as an analog for the habitability of Mars for more than 50 years. Here we report a layer enriched in smectites located just 30 cm below the surface of the hyperarid core of the Atacama. We discovered the clay-rich layer to be wet (a phenomenon never observed before in this region), keeping a high and constant relative humidity of 78% (aw 0.780), and completely isolated from the changing and extremely dry subaerial conditions characteristic of the Atacama. The smectite-rich layer is inhabited by at least 30 halophilic species of metabolically active bacteria and archaea, unveiling a previously unreported habitat for microbial life under the surface of the driest place on Earth. The discovery of a diverse microbial community in smectite-rich subsurface layers in the hyperarid core of the Atacama, and the collection of biosignatures we have identified within the clays, suggest that similar shallow clay deposits on Mars may contain biosignatures easily reachable by current rovers and landers.

Raman imaging of microbial colonization in rock-some analytical aspects.

Raman imaging allows one to obtain spatially resolved chemical information in a nondestructive manner. Herein, we present analytical aspects of effective in situ and in vivo Raman imaging of algae and cyanobacteria from within their native rock habitats. Specifically, gypsum and halite inhabited by endolithic communities from the hyperarid Atacama Desert were analyzed. Raman imaging of these phototrophic colonization reveals a pigment composition within the aggregates that helps in understanding some of their adaptation strategies to survive in this harsh polyextreme environment. The study is focused on methodical aspects of Raman imaging acquisition and subsequent data processing. Point imaging is compared with line imaging in terms of their image quality, spatial resolution, spectral signal-to-noise ratio, time requirements, and risk of laser-induced sample alteration. The roles of excitation wavelength, exposure time, and step size of the imaging grid on successful Raman imaging results are also discussed. Graphical abstract.

Haloterrigena sp. Strain SGH1, a Bacterioruberin-Rich, Perchlorate-Tolerant Halophilic Archaeon Isolated From Halite Microbial Communities, Atacama Desert, Chile.

An extreme halophilic archaeon, strain SGH1, is a novel microorganism isolated from endolithic microbial communities colonizing halites at Salar Grande, Atacama Desert, in northern Chile. Our study provides structural, biochemical, genomic, and physiological information on this new isolate living at the edge of the physical and chemical extremes at the Atacama Desert. SGH1 is a Gram-negative, red-pigmented, non-motile unicellular coccoid organism. Under the transmission electron microscope, strain SGH1 showed an abundant electro-dense material surrounding electron-lucent globular structures resembling gas vacuoles. Strain SGH1 showed a 16S rRNA gene sequence with a close phylogenetic relationship to the extreme halophilic archaea Haloterrigena turkmenica and Haloterrigena salina and has been denominated Haloterrigena sp. strain SGH1. Strain SGH1 grew at 20-40°C (optimum 37°C), at salinities between 15 and 30% (w/v) NaCl (optimum 25%) and growth was improved by addition of 50 mM KCl and 0.5% w/v casamino acids. Growth was severely restricted at salinities below 15% NaCl and cell lysis is avoided at a minimal 10% NaCl. Maximal concentrations of magnesium chloride and sodium or magnesium perchlorates that supported SGH1 growth were 0.5 and 0.15M, respectively. Haloterrigena sp. strain SGH1 accumulates bacterioruberin (BR), a C50 xanthophyll, as the major carotenoid. Total carotenoids in strain SGH1 amounted to nearly 400 µg BR per gram of dry biomass. Nearly 80% of total carotenoids accumulated as geometric isomers of BR: all-trans-BR (50%), 5-cis-BR (15%), 9-cis-BR (10%), 13-cis-BR (4%); other carotenoids were dehydrated derivatives of BR. Carotenogenesis in SGH1 was a reversible and salt-dependent process; transferring BR-rich cells grown in 25% (w/v) NaCl to 15% (w/v) NaCl medium resulted in depigmentation, and BR content was recovered after transference and growth of unpigmented cells to high salinity medium. Methanol extracts and purified BR isomers showed an 8-9-fold higher antioxidant activity than Trolox or ß-carotene. Both, plasma membrane integrity and mitochondrial membrane potential measurements under acute 18-h assays showed that purified BR isomers were non-toxic to cultured human THP-1 cells.

Differential Colonization and Succession of Microbial Communities in Rock and Soil Substrates on a Maritime Antarctic Glacier Forefield.

Glacier forefields provide a unique chronosequence to assess microbial or plant colonization and ecological succession on previously uncolonized substrates. Patterns of microbial succession in soils of alpine and subpolar glacier forefields are well documented but those affecting high polar systems, including moraine rocks, remain largely unexplored. In this study, we examine succession patterns in pioneering bacterial, fungal and algal communities developing on moraine rocks and soil at the Hurd Glacier forefield (Livingston Island, Antarctica). Over time, changes were produced in the microbial community structure of rocks and soils (ice-free for different lengths of time), which differed between both substrates across the entire chronosequence, especially for bacteria and fungi. In addition, fungal and bacterial communities showed more compositional consistency in soils than rocks, suggesting community assembly in each niche could be controlled by processes operating at different temporal and spatial scales. Microscopy revealed a patchy distribution of epilithic and endolithic lithobionts, and increasing endolithic colonization and microbial community complexity along the chronosequence. We conclude that, within relatively short time intervals, primary succession processes at polar latitudes involve significant and distinct changes in edaphic and lithic microbial communities associated with soil development and cryptogamic colonization.

Response of Endolithic Chroococcidiopsis Strains From the Polyextreme Atacama Desert to Light Radiation.

Cyanobacteria exposed to high solar radiation make use of a series of defense mechanisms, including avoidance, antioxidant systems, and the production of photoprotective compounds such as scytonemin. Two cyanobacterial strains of the genus Chroococcidiopsis from the Atacama Desert - which has one of the highest solar radiation levels on Earth- were examined to determine their capacity to protect themselves from direct photosynthetically active (PAR) and ultraviolet radiation (UVR): the UAM813 strain, originally isolated from a cryptoendolithic microhabitat within halite (NaCl), and UAM816 strain originally isolated from a chasmoendolithic microhabitat within calcite (CaCO3). The oxidative stress induced by exposure to PAR or UVR + PAR was determined to observe their short-term response, as were the long-term scytonemin production, changes in metabolic activity and ultrastructural damage induced. Both strains showed oxidative stress to both types of light radiation. The UAM813 strain showed a lower acclimation capacity than the UAM816 strain, showing an ever-increasing accumulation of reactive oxygen species (ROS) and a smaller accumulation of scytonemin. This would appear to reflect differences in the adaptation strategies followed to meet the demands of their different microhabitats.

Ultrastructural and biochemical analyses reveal cell wall remodelling in lichen-forming microalgae submitted to cyclic desiccation-rehydration.

BACKGROUND AND AIMS: One of the most distinctive features of desiccation-tolerant plants is their high cell wall (CW) flexibility. Most lichen microalgae can tolerate drastic dehydration-rehydration (D/R) conditions; however, their mechanisms of D/R tolerance are scarcely understood. We tested the hypothesis that D/R-tolerant microalgae would have flexible CWs due to species-specific CW ultrastructure and biochemical composition, which could be remodelled by exposure to cyclic D/R. METHODS: Two lichen microalgae, Trebouxia sp. TR9 (TR9, adapted to rapid D/R cycles) and Coccomyxa simplex (Csol, adapted to seasonal dry periods) were exposed to no or four cycles of desiccation [25-30 % RH (TR9) or 55-60 % RH (Csol)] and 16 h of rehydration (100 % RH). Low-temperature SEM, environmental SEM and freeze-substitution TEM were employed to visualize structural alterations induced by D/R. In addition, CWs were extracted and sequentially fractionated with hot water and KOH, and the gel permeation profile of polysaccharides was analysed in each fraction. The glycosyl composition and linkage of the main polysaccharides of each CW fraction were analysed by GC-MS. KEY RESULTS: All ultrastructural analyses consistently showed that desiccation caused progressive cell shrinkage and deformation in both microalgae, which could be rapidly reversed when water availability increased. Notably, the plasma membrane of TR9 and Csol remained in close contact with the deformed CW. Exposure to D/R strongly altered the size distribution of TR9 hot-water-soluble polysaccharides, composed mainly of a ß-3-linked rhamnogalactofuranan and Csol KOH-soluble ß-glucans. CONCLUSIONS: Cyclic D/R induces biochemical remodelling of the CW that could increase CW flexibility, allowing regulated shrinkage and expansion of D/R-tolerant microalgae.

Fundamental drivers for endolithic microbial community assemblies in the hyperarid Atacama Desert.

In hyperarid deserts, endolithic microbial communities colonize the rocks' interior as a survival strategy. Yet, the composition of these communities and the drivers promoting their assembly are still poorly understood. We analysed the diversity and community composition of endoliths from four different lithic substrates - calcite, gypsum, ignimbrite and granite - collected in the hyperarid zone of the Atacama Desert, Chile. By combining microscopy, mineralogy, spectroscopy and high throughput sequencing, we found these communities to be highly specific to their lithic substrate, although they were all dominated by the same four main phyla, Cyanobacteria, Actinobacteria, Chloroflexi and Proteobacteria. Our finding indicates a fine scale diversification of the microbial reservoir driven by substrate properties. The data suggest that the overall rock chemistry and the light transmission properties of the substrates are not essential drivers of community structure and composition. Instead, we propose that the architecture of the rock, i.e., the space available for colonization and its physical structure, linked to water retention capabilities, is ultimately the driver of community diversity and composition at the dry limit of life.

Endolithic microbial habitats as refuges for life in polyextreme environment of the Atacama Desert.

The extremely harsh conditions of hyperarid deserts are a true challenge for microbial life. Microorganisms thriving in such polyextreme environments are fascinating as they can tell us more about life, its strategies and its boundaries than other groups of organisms. The Atacama Desert (North Chile) holds two world records of extreme environmental characteristics: the lowest rainfall and greatest surface ultraviolet radiation and total solar irradiance ever measured on Earth. Despite these limiting conditions for life, we recently identified several remarkable examples of endolithic habitats colonized by phototrophic and heterotrophic microorganisms in the hyperarid core of the Atacama Desert.

Discovery of carotenoid red-shift in endolithic cyanobacteria from the Atacama Desert.

The biochemical responses of rock-inhabiting cyanobacteria towards native environmental stresses were observed in vivo in one of the Earth's most challenging extreme climatic environments. The cryptoendolithic cyanobacterial colonization, dominated by Chroococcidiopsis sp., was studied in an ignimbrite at a high altitude volcanic area in the Atacama Desert, Chile. Change in the carotenoid composition (red-shift) within a transect through the cyanobacteria dominant microbial community (average thickness ~1 mm) was unambiguously revealed in their natural endolithic microhabitat. The amount of red shifted carotenoid, observed for the first time in a natural microbial ecosystem, is depth dependent, and increased with increasing proximity to the rock surface, as proven by resonance Raman imaging and point resonance Raman profiling. It is attributed to a light-dependent change in carotenoid conjugation, associated with the light-adaptation strategy of cyanobacteria. A hypothesis is proposed for the possible role of an orange carotenoid protein (OCP) mediated non-photochemical quenching (NPQ) mechanism that influences the observed spectral behavior. Simultaneously, information about the distribution of scytonemin and phycobiliproteins was obtained. Scytonemin was detected in the uppermost cyanobacteria aggregates. A reverse signal intensity gradient of phycobiliproteins was registered, increasing with deeper positions as a response of the cyanobacterial light harvesting complex to low-light conditions.

Structure and temporal dynamics of the bacterial communities associated to microhabitats of the coral Oculina patagonica.

Corals are known to contain a diverse microbiota that plays a paramount role in the physiology and health of holobiont. However, few studies have addressed the variability of bacterial communities within the coral host. In this study, bacterial community composition from the mucus, tissue and skeleton of the scleractinian coral Oculina patagonica were investigated seasonally at two locations in the Western Mediterranean Sea, to further understand how environmental conditions and the coral microbiome structure are related. We used denaturing gradient gel electrophoresis in combination with next-generation sequencing and electron microscopy to characterize the bacterial community. The bacterial communities were significantly different among coral compartments, and coral tissue displayed the greatest changes related to environmental conditions and coral health status. Species belonging to the Rhodobacteraceae and Vibrionaceae families form part of O. patagonica tissues core microbiome and may play significant roles in the nitrogen cycle. Furthermore, sequences related to the coral pathogens, Vibrio mediterranei and Vibrio coralliilyticus, were detected not only in bleached corals but also in healthy ones, even during cold months. This fact opens a new view onto unveiling the role of pathogens in the development of coral diseases in the future.

Raman imaging in geomicrobiology: endolithic phototrophic microorganisms in gypsum from the extreme sun irradiation area in the Atacama Desert.

The Raman imaging method was successfully applied for mapping the distribution of biomolecules (e.g., pigments) associated with cryptoendolithic and hypoendolithic microorganisms, as well as the inorganic host mineral matrix that forms the habitat for the biota. To the best of our knowledge, this is the first comprehensive study in the field of geomicrobiology based on this technique. The studied microbial ecosystem was located nearly 3000 m above sea level within the driest desert on Earth, the Atacama in Chile. Enhancement of carotenoid Raman signal intensity close to the surface was registered at different areas of endolithic colonization dominated by algae, with cyanobacteria present as well. This is interpreted as an adaptation mechanism to the excessive solar irradiation. On the other hand, cyanobacteria synthesize scytonemin as a passive UV-screening pigment (found at both the hypoendolithic and cryptoendolithic positions). The distribution of the scytonemin Raman signal was mapped simultaneously with the surrounding mineral matrix. Thus, mapping was done of the phototrophic microorganisms in their original microhabitat together with the host rock environment. Important information which was resolved from the Raman imaging dataset of the host rock is about the hydration state of Ca-sulfate, demonstrated on the presence of gypsum (CaSO4·2H2O) and the absence of both anhydrite (CaSO4) and bassanite (CaSO4·1/2H2O). Obtaining combined "in situ" simultaneous information from the geological matrix (inorganic) together with the microbial biomolecules (organic) is discussed and concluded as an important advantage of this technique. We discuss how selection of the laser wavelength (785 and 514.5-nm) influences the Raman imaging results.

Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments.

Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates- chasmoendolithic versus cryptoendolithic - might be an essential element in determining the colonization and the diversity of the microbial communities in endolithic substrates at the dry limit for life.

Adaptation strategies of endolithic chlorophototrophs to survive the hyperarid and extreme solar radiation environment of the Atacama Desert.

The Atacama Desert, northern Chile, is one of the driest deserts on Earth and, as such, a natural laboratory to explore the limits of life and the strategies evolved by microorganisms to adapt to extreme environments. Here we report the exceptional adaptation strategies of chlorophototrophic and eukaryotic algae, and chlorophototrophic and prokaryotic cyanobacteria to the hyperarid and extremely high solar radiation conditions occurring in this desert. Our approach combined several microscopy techniques, spectroscopic analytical methods, and molecular analyses. We found that the major adaptation strategy was to avoid the extreme environmental conditions by colonizing cryptoendolithic, as well as, hypoendolithic habitats within gypsum deposits. The cryptoendolithic colonization occurred a few millimeters beneath the gypsum surface and showed a succession of organized horizons of algae and cyanobacteria, which has never been reported for endolithic microbial communities. The presence of cyanobacteria beneath the algal layer, in close contact with sepiolite inclusions, and their hypoendolithic colonization suggest that occasional liquid water might persist within these sub-microhabitats. We also identified the presence of abundant carotenoids in the upper cryptoendolithic algal habitat and scytonemin in the cyanobacteria hypoendolithic habitat. This study illustrates that successful lithobiontic microbial colonization at the limit for microbial life is the result of a combination of adaptive strategies to avoid excess solar irradiance and extreme evapotranspiration rates, taking advantage of the complex structural and mineralogical characteristics of gypsum deposits-conceptually called "rock's habitable architecture." Additionally, self-protection by synthesis and accumulation of secondary metabolites likely produces a shielding effect that prevents photoinhibition and lethal photooxidative damage to the chlorophototrophs, representing another level of adaptation.

In situ metabolism in halite endolithic microbial communities of the hyperarid Atacama Desert.

The Atacama Desert of northern Chile is one of the driest regions on Earth, with areas that exclude plants and where soils have extremely low microbial biomass. However, in the driest parts of the desert there are microorganisms that colonize the interior of halite nodules in fossil continental evaporites, where they are sustained by condensation of atmospheric water triggered by the salt substrate. Using a combination of in situ observations of variable chlorophyll fluorescence and controlled laboratory experiments, we show that this endolithic community is capable of carbon fixation both through oxygenic photosynthesis and potentially ammonia oxidation. We also present evidence that photosynthetic activity is finely tuned to moisture availability and solar insolation and can be sustained for days, and perhaps longer, after a wetting event. This is the first demonstration of in situ active metabolism in the hyperarid core of the Atacama Desert, and it provides the basis for proposing a self-contained, endolithic community that relies exclusively on non-rainfall sources of water. Our results contribute to an increasing body of evidence that even in hyperarid environments active metabolism, adaptation, and growth can occur in highly specialized microhabitats.

Microbial diversity and the presence of algae in halite endolithic communities are correlated to atmospheric moisture in the hyper-arid zone of the Atacama Desert.

The Atacama Desert is one of the oldest and driest deserts in the world, and its hyper-arid core is described as 'the most barren region imaginable'. We used a combination of high-throughput sequencing and microscopy methods to characterize the endolithic microbial assemblages of halite pinnacles (salt rocks) collected in several hyper-arid areas of the desert. We found communities dominated by archaea that relied on a single phylotype of Halothece cyanobacteria for primary production. A few other phylotypes of salt-adapted bacteria and archaea, including Salinibacter, Halorhabdus, and Halococcus were major components of the halite communities, indicating specific adaptations to the unique halite environments. Multivariate statistical analyses of diversity metrics clearly separated the halite communities from that of the surrounding soil in the Yungay area. These analyses also revealed distribution patterns of halite communities correlated with atmospheric moisture. Microbial endolithic communities from halites exposed to coastal fogs and high relative humidity were more diverse; their archaeal and bacterial assemblages were accompanied by a novel algae related to oceanic picoplankton of the Mamiellales. In contrast, we did not find any algae in the Yungay pinnacles, suggesting that the environmental conditions in this habitat might be too extreme for eukaryotic photosynthetic life.

Distribution of scytonemin in endolithic microbial communities from halite crusts in the hyperarid zone of the Atacama Desert, Chile.

Scytonemin, a UV-screening molecule produced by certain Cyanobacteria to protect against harmful UV radiation, was studied in endolithic cyanobacterial colonies in the halite crust from one of the driest places on Earth - the hyperarid zone of the Atacama Desert. The distribution of the pigment within the evaporitic crust was studied in detail by various independent analytical methods: Raman spectroscopy (including Raman imaging); advanced microscopic observations (fluorescence microscopy, confocal laser scanning microscopy, low-temperature scanning electron microscopy); and spectrophotometric analyses. The differences in scytonemin biosynthesis were mapped within the colonized interior layers, which can be divided into scytonemin-rich and scytonemin-poor zones. A 532 nm laser for excitation proved to be an ideal excitation source with which to observe the relative content of scytonemin within a particular cell aggregate, as well as between different cell aggregates; based on the scytonemin/carotenoid Raman signal intensity ratio of selected corroborative bands for these two compounds. Significantly, scytonemin was found to accumulate within a decayed biomass in the surface portions of the halite crust. These were found to be highly enriched in both the absolute scytonemin content (as documented by UV/VIS spectrophotometry) and its content relative to other pigments associated with the cyanobacterial cells (e.g. carotenoids and chlorophyll).

Eukarya associated with the stony coral Oculina patagonica from the Mediterranean Sea.

Oculina patagonica is a putative alien scleractinian coral from the Southwest Atlantic that inhabits across the Mediterranean Sea. Here, we have addressed the diversity of Eukarya associated with this coral and its changes related to the environmental conditions and coral status. A total of 46 colonies of O. patagonica were taken from Alicante coast (Spain) and Pietra Ligure coast (Italy) and analyzed using denaturing gradient gel electrophoresis (DGGE) of the small-subunit 18S rRNA and 16S plastid rRNA genes, internal transcribed spacer region 2 (ITS 2) analyses, and electron microscopy. Our results show that Eukarya and plastid community associated to O. patagonica change with environmental conditions and coral status. Cryptic species, which can be difficult to identify by optical methods, were distinguished by 18S rRNA gene DGGE: the barnacle Megatrema anglicum, which was detected at two locations, and two boring sponges related to Cliona sp. and Siphonodictyon coralliphagum detected in samples from Tabarca and Alicante Harbour, respectively. Eukaryotic phototrophic community from the skeletal matrix of healthy corals was dominated by Ochrosphaera sp. while bleached corals from the Harbour and Tabarca were associated to different uncultured phototrophic organism. Differences in ultrastructural morphologies of the zooxanthellae between healthy and bleached corals were observed. Nevertheless, no differences were found in Symbiodinium community among time, environments, coral status and location, showing that O. patagonica hosted only one genotype of Symbiodinium belonging to clade B2. The fact that this clade has not been previously detected in other Mediterranean corals and is more frequent in the tropical Western Atlantic, is a new evidence that O. patagonica is an alien species in the Mediterranean Sea.