Unravelling the relationships among Madrepora Linnaeus, 1758, Oculina Lamark, 1816 and Cladocora Ehrenberg, 1834 (Cnidaria: Anthozoa: Scleractinia).

Despite the widespread use of integrative taxonomic approaches, many scleractinian coral genera and species remain grouped in polyphyletic families, classified as incertae sedis or simply understudied. Oculinidae Gray, 1847 represents a family for which many taxonomic questions remain unresolved, particularly those related to some of the current genera, such as Oculina Lamark, 1816 or recently removed genera, including Cladocora Ehrenberg, 1834 and Madrepora Linnaeus, 1758. Cladocora is currently assigned to the family Cladocoridae Milne Edwards & Haime, 1857 and a new family, Bathyporidae Kitahara, Capel, Zilberberg & Cairns, 2024, was recently raised to accommodate Madrepora . However, the name Bathyporidae is not valid because this was not formed on the basis of a type genus name. To resolve taxonomic questions related to these three genera, the evolutionary relationships are explored through phylogenetic analyses of 18 molecular markers. The results of these analyses support a close relationship between the species Oculina patagonica and Cladocora caespitosa , indicating that these may belong to the same family (and possibly genus), and highlighting the need for detailed revisions of Oculina and Cladocora . By contrast, a distant relationship is found between these two species and Madrepora oculata , with the overall evidence supporting the placement of Madrepora in the resurrected family Madreporidae Ehrenberg, 1834. This study advances our knowledge of coral systematics and highlights the need for a comprehensive review of the genera Oculina , Cladocora and Madrepora .

Whale fall chemosymbiotic communities in a southwest Australian submarine canyon fill a distributional gap.

A whale fall community of chemosymbiotic invertebrates living on cetacean bones has been identified off southwestern Australia during a Remotely Operated Vehicle (ROV) survey at bathyal depths within the Bremer Marine Park, which is part of important marine mammal areas (IMMA) of the Albany Canyon Region. Cetacean bones on the seafloor of the Hood Canyon, consisted of isolated skulls of three species of beaked whales (family Ziphiidae): Mesoplodon cf. layardii, M. grayi, and M. hectori, a few vertebrae, and lower jaws. One of the beaked whale skulls (Mesoplodon cf. layardii) was sampled and found to be intensely colonised by hundreds of specimens of a bathymodilinae mussel ("Adipicola" s.l.). Live polychaetes (Phyllochaetopterus?), skeneimorph gastropods, and amphipods (Seba, Leptamphopus) colonised the skull bone, which represent a later stage (sulfophilic) of carcass decomposition. The reducing sediment below the skull was inhabited by lucinid (Lucinoma) and vesicomyid (Calyptogena) chemosymbiotic bivalves. Additionally, the sediment thanatocoenosis comprised shells of various other chemosymbiotic bivalves, such as Acharax, thyasirids, lucinids, vesicomyids, and limpets, representing the complex ecological turnover phases through time in this whale fall chemosynthetic habitat. With one exception, all bones recovered were colonized by bathymodiolin mussels. This is the first documented case of a chemosynthetic community and associated chemosymbiotic fauna relating to beaked whales, and the first fully documented record of a whale fall community within the Australian Southern Ocean region.

The dawn of the tropical Atlantic invasion into the Mediterranean Sea.

The Mediterranean Sea is a marine biodiversity hotspot already affected by climate-driven biodiversity collapses. Its highly endemic fauna is at further risk if global warming triggers an invasion of tropical Atlantic species. Here, we combine modern species occurrences with a unique paleorecord from the Last Interglacial (135 to 116 ka), a conservative analog of future climate, to model the future distribution of an exemplary subset of tropical West African mollusks, currently separated from the Mediterranean by cold upwelling off north-west Africa. We show that, already under an intermediate climate scenario (RCP 4.5) by 2050, climatic connectivity along north-west Africa may allow tropical species to colonize a by then largely environmentally suitable Mediterranean. The worst-case scenario RCP 8.5 leads to a fully tropicalized Mediterranean by 2100. The tropical Atlantic invasion will add to the ongoing Indo-Pacific invasion through the Suez Canal, irreversibly transforming the entire Mediterranean into a novel ecosystem unprecedented in human history.

Differences in carbonate chemistry up-regulation of long-lived reef-building corals.

With climate projections questioning the future survival of stony corals and their dominance as tropical reef builders, it is critical to understand the adaptive capacity of corals to ongoing climate change. Biological mediation of the carbonate chemistry of the coral calcifying fluid is a fundamental component for assessing the response of corals to global threats. The Tara Pacific expedition (2016-2018) provided an opportunity to investigate calcification patterns in extant corals throughout the Pacific Ocean. Cores from colonies of the massive Porites and Diploastrea genera were collected from different environments to assess calcification parameters of long-lived reef-building corals. At the basin scale of the Pacific Ocean, we show that both genera systematically up-regulate their calcifying fluid pH and dissolved inorganic carbon to achieve efficient skeletal precipitation. However, while Porites corals increase the aragonite saturation state of the calcifying fluid (Ωcf) at higher temperatures to enhance their calcification capacity, Diploastrea show a steady homeostatic Ωcf across the Pacific temperature gradient. Thus, the extent to which Diploastrea responds to ocean warming and/or acidification is unclear, and it deserves further attention whether this is beneficial or detrimental to future survival of this coral genus.

First assessment of anthropogenic impacts in submarine canyon systems off southwestern Australia.

We assessed the anthropogenic impacts on southwestern Australian submarine canyons by quantifying macro-litter discovered during Remotely Operated Vehicle surveys. The study area encompasses the Bremer canyon systems and Perth Canyon. The categories of macro-litter identified by our study are plastic, metal, aluminium, glass, fabric, mixed, derelict fishing gear, and unclassified. The anthropogenic impacts in the canyons explored is minimal, especially in the Bremer canyon systems, whereas Perth Canyon has comparatively more macro-litter, presumably due to intense maritime traffic and nearby urban development. On a global scale, however, the environmental status of southwestern Australian canyons is relatively pristine. This analysis provides a baseline for the monitoring and enduring stewardship of these habitats where lush and diverse biota, including deep-sea corals, thrive.

Genetic Structure of the Endangered Coral Cladocora caespitosa Matches the Main Bioregions of the Mediterranean Sea.

Population connectivity studies are a useful tool for species management and conservation planning, particular of highly threatened or endangered species. Here, we evaluated the genetic structure and connectivity pattern of the endangered coral Cladocora caespitosa across its entire distribution range in the Mediterranean Sea. Additionally, we examined the relative importance of sexual and asexual reproduction in the studied populations and their genetic diversity. A total of 541 individuals from 20 localities were sampled and analysed with 19 polymorphic microsatellite markers. Of the genotyped individuals, 482 (89%) had unique multilocus genotypes. Clonality percentages of the populations varied from 0% (in eight populations) to nearly 69% (in one population from Crete). A heterozygosity deficit and a high degree of inbreeding was the general trend in our data set. Population differentiation in C. caespitosa was characterised by significant pairwise F ST values with lower ones observed at an intraregional scale and higher ones, between populations from different biogeographic regions. Genetic structure analyses showed that the populations are divided according to the three main sub-basins of the Mediterranean Sea: the Western (Balearic, Ligurian and Tyrrhenian seas), the Central (Adriatic and Ionian seas) and the Eastern (Levantine and Aegean seas), coinciding with previously described gene flow barriers. However, the three easternmost populations were also clearly separated from one another, and a substructure was observed for the other studied areas. An isolation-by-distance pattern was found among, but not within, the three main population groups. This substructure is mediated mainly by dispersal along the coastline and some resistance to larval movement through the open sea. Despite the low dispersal ability and high self-recruitment rate of C. caespitosa, casual dispersive events between regions seem to be enough to maintain the species' considerable genetic diversity. Understanding the population connectivity and structure of this endangered scleractinian coral allows for more informed conservation decision making.

Asexual reproduction in bad times? The case of Cladocora caespitosa in the eastern Mediterranean Sea.

We analysed the patterns of genetic variability of eastern Mediterranean populations of the scleractinian coral Cladocora caespitosa, from the Aegean and Levantine seas, using 19 polymorphic microsatellite loci, 11 of which were newly characterized. The observed genetic pattern reflects a scenario of isolation by environment: F ST comparisons showed a higher degree of genetic differentiation between the two Cypriot populations that are separated by only 11 km than between these two Levantine populations and the Aegean population in Greece, which are separated by 1300 km. We hypothesize that local-scale oceanographic factors influenced the dispersal of planulae between the geographically close populations, playing a crucial role in the genetic structure of this coastal coral. Yet, despite being characterized as a species with limited dispersal and high self-recruitment, large-scale migration does eventually occur as first-generation migrants were identified between the most distant populations. In line with previous findings of reproductive plasticity in C. caespitosa, we also found localized differences in reproduction mode (sexual vs. asexual) within a geographically limited context. Several individuals were identified as clones, indicating the predominance of asexual reproduction in one of the Cypriot populations. We interpret this predominance either as a direct response to or as an indirect consequence of perturbations suffered by this C. caespitosa population. These perturbations are caused by unfavourable environmental conditions that threatened local survival, in particular water temperature changes and windstorm swells. Asexual reproduction may be a mechanism used by C. caespitosa to counteract mortality events and recolonize devastated areas, and likely accounts for the occasional high levels of clonality and low levels of genetic diversity. Local adaptations such as these should therefore be considered in conservation and management strategies to maintain and preserve the gene pool of this endangered species. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s00338-020-02040-3).

Chaotic genetic structure and past demographic expansion of the invasive gastropod Tritia neritea in its native range, the Mediterranean Sea.

To better predict population evolution of invasive species in introduced areas it is critical to identify and understand the mechanisms driving genetic diversity and structure in their native range. Here, we combined analyses of the mitochondrial COI gene and 11 microsatellite markers to investigate both past demographic history and contemporaneous genetic structure in the native area of the gastropod Tritia neritea, using Bayesian skyline plots (BSP), multivariate analyses and Bayesian clustering. The BSP framework revealed population expansions, dated after the last glacial maximum. The haplotype network revealed a strong geographic clustering. Multivariate analyses and Bayesian clustering highlighted the strong genetic structure at all scales, between the Black Sea and the Adriatic Sea, but also within basins. Within basins, a random pattern of genetic patchiness was observed, suggesting a superimposition of processes involving natural biological effects (no larval phase and thus limited larval dispersal) and putative anthropogenic transport of specimens. Contrary to the introduced area, no isolation-by-distance patterns were recovered in the Mediterranean or the Black Seas, highlighting different mechanisms at play on both native and introduced areas, triggering unknown consequences for species' evolutionary trajectories. These results of Tritia neritea populations on its native range highlight a mixture of ancient and recent processes, with the effects of paleoclimates and life history traits likely tangled with the effects of human-mediated dispersal.

Late Pleistocene Red Sea Mollusca: 1. Polyplacophora.

This study describes the chiton fauna (Mollusca, Polyplacophora) from the marine Pleistocene coral reef deposits bordering the Red Sea coast and dating to the last interglacial epoch (Marine Isotopic Substage 5e, MIS5e). Twenty-one species were identified, of which only three were previously known from the Pleistocene of the Red Sea (Lucilina sueziensis, Acanthopleura vaillantii and Acanthochitona penicillata). Eight are recorded as fossil for the first time. Six are described as new (Lucilina confusa n. sp., L. aqabaensis n. sp., L. aegyptiaca n. sp., L. colantonii n. sp., "Onithochiton" vandingeneni" n. sp., and Acanthochitona interglacialis n. sp.) and two are assigned at generic level (Callochiton sp. and Craspedochiton sp.). Four of these new species (Lucilina confusa n. sp., L. aegyptiaca n. sp., L. colantonii n. sp. and "Onithochiton" vandingeneni n. sp.) are still living in the Red Sea, bringing to 28 the number of extant species known from the Red Sea. These Pleistocene Red Sea assemblages represent the most diverse chiton fauna yet reported from any interglacial deposit worldwide.

Application of Hyperspectral Imaging to Underwater Habitat Mapping, Southern Adriatic Sea.

Hyperspectral imagers enable the collection of high-resolution spectral images exploitable for the supervised classification of habitats and objects of interest (OOI). Although this is a well-established technology for the study of subaerial environments, Ecotone AS has developed an underwater hyperspectral imager (UHI) system to explore the properties of the seafloor. The aim of the project is to evaluate the potential of this instrument for mapping and monitoring benthic habitats in shallow and deep-water environments. For the first time, we tested this system at two sites in the Southern Adriatic Sea (Mediterranean Sea): the cold-water coral (CWC) habitat in the Bari Canyon and the Coralligenous habitat off Brindisi. We created a spectral library for each site, considering the different substrates and the main OOI reaching, where possible, the lower taxonomic rank. We applied the spectral angle mapper (SAM) supervised classification to map the areal extent of the Coralligenous and to recognize the major CWC habitat-formers. Despite some technical problems, the first results demonstrate the suitability of the UHI camera for habitat mapping and seabed monitoring, through the achievement of quantifiable and repeatable classifications.

A unique and threatened deep water coral-bivalve biotope new to the Mediterranean Sea offshore the Naples megalopolis.

The Gulf of Naples is an example of the most beautiful and biodiverse marine regions of the Mediterranean Sea and of the most impacted areas in terms of industrial activities, large contaminated areas, resource exploitation, infrastructures at sea and maritime transportation. We conducted Remotely Operated Vehicle surveys in the Dohrn Canyon in the Tyrrhenian Sea at approximately 12 NM off Naples metropolitan area, and revealed a hotspot of deep-sea benthic biodiversity of sessile fauna at ca. 400 m depth. The hard bottoms are characterized by a high abundance of charismatic species, such as the habitat forming cold-water corals (CWC) Madrepora oculata, Lophelia pertusa, Desmophyllum dianthus in association with the large size bivalves Acesta excavata and Neopycnodonte zibrowii. This CWC-bivalve co-occurrence represents a novel biotope for the Mediterranean Sea, which coexists with the evidence of severe anthropogenic threats, such as illegal dumping and fishery malpractices that were visually documented during the survey. We recommend the adoption of specific protection measures to preserve these unique deep-sea assemblages showing the uncommon co-existence of such a number of deep-sea species in a single habitat.

Morphological and genetic divergence between Mediterranean and Caribbean populations of Madracis pharensis (Heller 1868) (Scleractinia, Pocilloporidae): too much for one species?

The colonial stony coral genus Madracis is cosmopolitan, lives in shallow and deep water habitats, and includes zooxanthellate, azooxanthellate and facultative symbiotic species. One of its species, Madracis pharensis, has been recorded from the Mediterranean and East Atlantic, where it forms small knobby and facultative zooxanthellate colonies (also named M. pharensis f. pharensis), and from the tropical Caribbean, where it also occurs in a massive and zooxanthellate form (named M. pharensis f. luciphila by some). These two forms have been previously found to host different Symbiodinium species. In this study, species boundaries and phylogenetic relationships between these two Madracis pharensis forms (from the Mediterranean Sea and the Caribbean), M. senaria, and the Indo-west Pacific M. kirbyi were analyzed through an integrated systematics approach, including corallite dimensions, micromorphology and two molecular markers (ITS and ATP8). Significant genetic and morphological differences were found between all the examined Madracis species, and between M. pharensis from the Mediterranean Sea and M. pharensis f. luciphila from the Caribbean in particular. Based on these results, the latter does not represent a zooxanthellate ecomorph of the former but a different species. Its identity remains to be ascertained and its relationship with the Caribbean M. decactis, with which it bears morphologic resemblance, must be investigated in further studies. Overall, the presence of cryptic Madracis species in the Easter and Central Atlantic Ocean remains to be evaluated.

Paleochannel and beach-bar palimpsest topography as initial substrate for coralligenous buildups offshore Venice, Italy.

We provide a model for the genesis of Holocene coralligenous buildups occurring in the northwestern Adriatic Sea offshore Venice at 17-24 m depth. High-resolution geophysical surveys and underwater SCUBA diving reconnaissance revealed meandering shaped morphologies underneath bio-concretionned rocky buildups. These morphologies are inferred to have been inherited from Pleistocene fluvial systems reactivated as tidal channels during the post- Last Glacial Maximum transgression, when the study area was a lagoon protected by a sandy barrier. The lithification of the sandy fossil channel-levee systems is estimated to have occurred at ca. 7 cal. ka BP, likely due to the interaction between marine and less saline fluids related to onshore freshwater discharge at sea through a sealed water-table. The carbonate-cemented sandy layers served as nucleus for subsequent coralligenous buildups growth.

Diversity and Distribution of Prokaryotes within a Shallow-Water Pockmark Field.

Pockmarks are crater-like depression on the seafloor associated with hydrocarbon ascent through muddy sediments in continental shelves around the world. In this study, we examine the diversity and distribution of benthic microbial communities at shallow-water pockmarks adjacent to the Middle Adriatic Ridge. We integrate microbial diversity data with characterization of local hydrocarbons concentrations and sediment geochemistry. Our results suggest these pockmarks are enriched in sedimentary hydrocarbons, and host a microbial community dominated by Bacteria, even in deeper sediment layers. Pockmark sediments showed higher prokaryotic abundance and biomass than surrounding sediments, potentially due to the increased availability of organic matter and higher concentrations of hydrocarbons linked to pockmark activity. Prokaryotic diversity analyses showed that the microbial communities of these shallow-water pockmarks are unique, and comprised phylotypes associated with the cycling of sulfur and nitrate compounds, as well as numerous know hydrocarbon degraders. Altogether, this study suggests that shallow-water pockmark habitats enhance the diversity of the benthic prokaryotic biosphere by providing specialized environmental niches.

Merging scleractinian genera: the overwhelming genetic similarity between solitary Desmophyllum and colonial Lophelia.

BACKGROUND: In recent years, several types of molecular markers and new microscale skeletal characters have shown potential as powerful tools for phylogenetic reconstructions and higher-level taxonomy of scleractinian corals. Nonetheless, discrimination of closely related taxa is still highly controversial in scleractinian coral research. Here we used newly sequenced complete mitochondrial genomes and 30 microsatellites to define the genetic divergence between two closely related azooxanthellate taxa of the family Caryophylliidae: solitary Desmophyllum dianthus and colonial Lophelia pertusa. RESULTS: In the mitochondrial control region, an astonishing 99.8 % of nucleotides between L. pertusa and D. dianthus were identical. Variability of the mitochondrial genomes of the two species is represented by only 12 non-synonymous out of 19 total nucleotide substitutions. Microsatellite sequence (37 loci) analysis of L. pertusa and D. dianthus showed genetic similarity is about 97 %. Our results also indicated that L. pertusa and D. dianthus show high skeletal plasticity in corallum shape and similarity in skeletal ontogeny, micromorphological (septal and wall granulations) and microstructural characters (arrangement of rapid accretion deposits, thickening deposits). CONCLUSIONS: Molecularly and morphologically, the solitary Desmophyllum and the dendroid Lophelia appear to be significantly more similar to each other than other unambiguous coral genera analysed to date. This consequently leads to ascribe both taxa under the generic name Desmophyllum (priority by date of publication). Findings of this study demonstrate that coloniality may not be a robust taxonomic character in scleractinian corals.

Antarctic ice sheet sensitivity to atmospheric CO2 variations in the early to mid-Miocene.

Geological records from the Antarctic margin offer direct evidence of environmental variability at high southern latitudes and provide insight regarding ice sheet sensitivity to past climate change. The early to mid-Miocene (23-14 Mya) is a compelling interval to study as global temperatures and atmospheric CO2 concentrations were similar to those projected for coming centuries. Importantly, this time interval includes the Miocene Climatic Optimum, a period of global warmth during which average surface temperatures were 3-4 °C higher than today. Miocene sediments in the ANDRILL-2A drill core from the Western Ross Sea, Antarctica, indicate that the Antarctic ice sheet (AIS) was highly variable through this key time interval. A multiproxy dataset derived from the core identifies four distinct environmental motifs based on changes in sedimentary facies, fossil assemblages, geochemistry, and paleotemperature. Four major disconformities in the drill core coincide with regional seismic discontinuities and reflect transient expansion of grounded ice across the Ross Sea. They correlate with major positive shifts in benthic oxygen isotope records and generally coincide with intervals when atmospheric CO2 concentrations were at or below preindustrial levels (∼280 ppm). Five intervals reflect ice sheet minima and air temperatures warm enough for substantial ice mass loss during episodes of high (∼500 ppm) atmospheric CO2 These new drill core data and associated ice sheet modeling experiments indicate that polar climate and the AIS were highly sensitive to relatively small changes in atmospheric CO2 during the early to mid-Miocene.

Development of Microsatellite Markers in the Deep-Sea Cup Coral Desmophyllum dianthus by 454 Sequencing and Cross-Species Amplifications in Scleractinia Order.

Microsatellite loci were isolated for the first time for the deep-sea coral Desmophyllum dianthus, using 454 GS-FLX Titanium pyrosequencing. We developed conditions for amplifying 24 markers in 10 multiplex reactions. Three to 16 alleles per locus were detected across 25 samples analyzed from Santa Maria di Leuca coral province (Mediterranean Sea). For the 24 polymorphic loci, observed and expected heterozygosities ranged from 0.211 to 0.880 and 0.383 to 0.910, respectively; 3 loci deviated from Hardy-Weinberg equilibrium, after null allele and sequential Holm-Bonferroni corrections. These newly isolated microsatellites are very useful genetic markers that provide data for future conservation strategies. Cross-amplification of these microsatellites, tested in 46 coral species, representing 40 genera, and 10 families of the phylum Cnidaria, produced informative allelic profiles for 1 to 24 loci. The utility of extending analyses to cross-species amplifications is also discussed.

Calcification is not the Achilles' heel of cold-water corals in an acidifying ocean.

Ocean acidification is thought to be a major threat to coral reefs: laboratory evidence and CO2 seep research has shown adverse effects on many coral species, although a few are resilient. There are concerns that cold-water corals are even more vulnerable as they live in areas where aragonite saturation (Ωara ) is lower than in the tropics and is falling rapidly due to CO2 emissions. Here, we provide laboratory evidence that net (gross calcification minus dissolution) and gross calcification rates of three common cold-water corals, Caryophyllia smithii, Dendrophyllia cornigera, and Desmophyllum dianthus, are not affected by pCO2 levels expected for 2100 (pCO2  1058 µatm, Ωara 1.29), and nor are the rates of skeletal dissolution in D. dianthus. We transplanted D. dianthus to 350 m depth (pHT 8.02; pCO2  448 µatm, Ωara 2.58) and to a 3 m depth CO2 seep in oligotrophic waters (pHT 7.35; pCO2  2879 µatm, Ωara 0.76) and found that the transplants calcified at the same rates regardless of the pCO2 confirming their resilience to acidification, but at significantly lower rates than corals that were fed in aquaria. Our combination of field and laboratory evidence suggests that ocean acidification will not disrupt cold-water coral calcification although falling aragonite levels may affect other organismal physiological and/or reef community processes.

Desmophyllum dianthus (Esper, 1794) in the scleractinian phylogeny and its intraspecific diversity.

The cosmopolitan solitary deep-water scleractinian coral Desmophyllum dianthus (Esper, 1794) was selected as a representative model species of the polyphyletic Caryophylliidae family to (1) examine phylogenetic relationships with respect to the principal Scleractinia taxa, (2) check population structure, (3) test the widespread connectivity hypothesis and (4) assess the utility of different nuclear and mitochondrial markers currently in use. To carry out these goals, DNA sequence data from nuclear (ITS and 28S) and mitochondrial (16S and COI) markers were analyzed for several coral species and for Mediterranean populations of D. dianthus. Three phylogenetic methodologies (ML, MP and BI), based on data from the four molecular markers, all supported D. dianthus as clearly belonging to the "robust" clade, in which the species Lophelia pertusa and D. dianthus not only grouped together, but also shared haplotypes for some DNA markers. Molecular results also showed shared haplotypes among D. dianthus populations distributed in regions separated by several thousands of kilometers and by clear geographic barriers. These results could reflect limited molecular and morphological taxonomic resolution rather than real widespread connectivity. Additional studies are needed in order to find molecular markers and morphological features able to disentangle the complex phylogenetic relationship in the Order Scleractinia and to differentiate isolated populations, thus avoiding the homoplasy found in some morphological characters that are still considered in the literature.

The fingerprint of chemosymbiosis: origin and preservation of isotopic biosignatures in the nonseep bivalve Loripes lacteus compared with Venerupis aurea.

Endosymbionts in marine bivalves leave characteristic biosignatures in their host organisms. Two nonseep bivalve species collected in Mediterranean lagoons, thiotrophic symbiotic Loripes lacteus and filter-feeding nonsymbiotic Venerupis aurea, were studied in detail with respect to generation and presence of such signatures in living animals, and the preservation of these signals in subfossil (late Pleistocene) sedimentary shells. Three key enzymes from sulfur oxidation (APS-reductase), CO(2) fixation (RubisCO) and assimilation of nitrogen [glutamine synthetase (GS)] were detected by immunofluorescence in the bacterial symbionts of Loripes. In Loripes, major activity was derived from GS of the symbionts whereas in Venerupis the host GS is active. In search of geologically stable biosignatures for thiotrophic chemosymbiosis that might be suitable to detect such associations in ancient bivalves, we analyzed the isotopic composition of shell lipids (δ(13)C) and the bulk organic matrix of the shell (δ(13)C , δ(15)N , δ(34)S). In the thiotrophic Loripes, δ(13)C values were depleted compared with the filter-feeding Venerupis by as much as 8.5‰ for individual fatty acids, and 4.4‰ for bulk organic carbon. Likewise, bulk δ(15)N and δ(34)S values were more depleted in recent thiotrophic Loripes. Whereas δ (34)S values were found to be unstable over time, the combined δ(15)N and δ(13)C values in organic shell extracts revealed a specific signature for chemosymbiosis in recent and subfossil specimens.