Genetic evidence for plastic reproductive philopatry and matrotrophy in blacktip reef sharks (Carcharhinus melanopterus) of the Moorea Island (French Polynesia).

The exploitation of sharks and the degradation of their habitats elevate the urgency to understand the factors that influence offspring survival and ultimately shark reproductive success. We monitored and sampled blacktip reef sharks (Carcharhinus melanopterus) in nursery habitats of Moorea Island (French Polynesia), to improve knowledge on shark reproductive behavior and biology. We sampled fin clips and morphometrics from 230 young-of-the-year sharks and used microsatellite DNA markers to process parentage analysis to study the reproductive philopatric behavior in female sharks and the matrotrophy within litters. These traits are driving the success of the local replenishment influencing selection through birth site and maternal reserves transmitted to pups. Parentage analysis revealed that some female sharks changed their parturition areas (inter-seasonally) while other female sharks came back to the same site for parturition, providing evidence for a plastic philopatric behavior. Morphometrics showed that there was no significant relationship between body condition indices and nursery locations. However, similarities and differences in body condition were observed between individuals sharing the same mother, indicating that resource allocation within some shark litters might be unbalanced. Our findings further our understanding of the reproductive biology and behavior that shape shark populations with the aim to introduce these parameters into future conservation strategies.

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.

Telomere DNA length regulation is influenced by seasonal temperature differences in short-lived but not in long-lived reef-building corals.

Telomeres are environment-sensitive regulators of health and aging. Here,we present telomere DNA length analysis of two reef-building coral genera revealing that the long- and short-term water thermal regime is a key driver of between-colony variation across the Pacific Ocean. Notably, there are differences between the two studied genera. The telomere DNA lengths of the short-lived, more stress-sensitive Pocillopora spp. colonies were largely determined by seasonal temperature variation, whereas those of the long-lived, more stress-resistant Porites spp. colonies were insensitive to seasonal patterns, but rather influenced by past thermal anomalies. These results reveal marked differences in telomere DNA length regulation between two evolutionary distant coral genera exhibiting specific life-history traits. We propose that environmentally regulated mechanisms of telomere maintenance are linked to organismal performances, a matter of paramount importance considering the effects of climate change on health.

Diversity of the Pacific Ocean coral reef microbiome.

Coral reefs are among the most diverse ecosystems on Earth. They support high biodiversity of multicellular organisms that strongly rely on associated microorganisms for health and nutrition. However, the extent of the coral reef microbiome diversity and its distribution at the oceanic basin-scale remains to be explored. Here, we systematically sampled 3 coral morphotypes, 2 fish species, and planktonic communities in 99 reefs from 32 islands across the Pacific Ocean, to assess reef microbiome composition and biogeography. We show a very large richness of reef microorganisms compared to other environments, which extrapolated to all fishes and corals of the Pacific, approximates the current estimated total prokaryotic diversity for the entire Earth. Microbial communities vary among and within the 3 animal biomes (coral, fish, plankton), and geographically. For corals, the cross-ocean patterns of diversity are different from those known for other multicellular organisms. Within each coral morphotype, community composition is always determined by geographic distance first, both at the island and across ocean scale, and then by environment. Our unprecedented sampling effort of coral reef microbiomes, as part of the Tara Pacific expedition, provides new insight into the global microbial diversity, the factors driving their distribution, and the biocomplexity of reef ecosystems.

Ecology of Endozoicomonadaceae in three coral genera across the Pacific Ocean.

Health and resilience of the coral holobiont depend on diverse bacterial communities often dominated by key marine symbionts of the Endozoicomonadaceae family. The factors controlling their distribution and their functional diversity remain, however, poorly known. Here, we study the ecology of Endozoicomonadaceae at an ocean basin-scale by sampling specimens from three coral genera (Pocillopora, Porites, Millepora) on 99 reefs from 32 islands across the Pacific Ocean. The analysis of 2447 metabarcoding and 270 metagenomic samples reveals that each coral genus harbored a distinct new species of Endozoicomonadaceae. These species are composed of nine lineages that have distinct biogeographic patterns. The most common one, found in Pocillopora, appears to be a globally distributed symbiont with distinct metabolic capabilities, including the synthesis of amino acids and vitamins not produced by the host. The other lineages are structured partly by the host genetic lineage in Pocillopora and mainly by the geographic location in Porites. Millepora is more rarely associated to Endozoicomonadaceae. Our results show that different coral genera exhibit distinct strategies of host-Endozoicomonadaceae associations that are defined at the bacteria lineage level.

Open science resources from the Tara Pacific expedition across coral reef and surface ocean ecosystems.

The Tara Pacific expedition (2016-2018) sampled coral ecosystems around 32 islands in the Pacific Ocean and the ocean surface waters at 249 locations, resulting in the collection of nearly 58 000 samples. The expedition was designed to systematically study warm-water coral reefs and included the collection of corals, fish, plankton, and seawater samples for advanced biogeochemical, molecular, and imaging analysis. Here we provide a complete description of the sampling methodology, and we explain how to explore and access the different datasets generated by the expedition. Environmental context data were obtained from taxonomic registries, gazetteers, almanacs, climatologies, operational biogeochemical models, and satellite observations. The quality of the different environmental measures has been validated not only by various quality control steps, but also through a global analysis allowing the comparison with known environmental large-scale structures. Such publicly released datasets open the perspective to address a wide range of scientific questions.

Host transcriptomic plasticity and photosymbiotic fidelity underpin Pocillopora acclimatization across thermal regimes in the Pacific Ocean.

Heat waves are causing declines in coral reefs globally. Coral thermal responses depend on multiple, interacting drivers, such as past thermal exposure, endosymbiont community composition, and host genotype. This makes the understanding of their relative roles in adaptive and/or plastic responses crucial for anticipating impacts of future warming. Here, we extracted DNA and RNA from 102 Pocillopora colonies collected from 32 sites on 11 islands across the Pacific Ocean to characterize host-photosymbiont fidelity and to investigate patterns of gene expression across a historical thermal gradient. We report high host-photosymbiont fidelity and show that coral and microalgal gene expression respond to different drivers. Differences in photosymbiotic association had only weak impacts on host gene expression, which was more strongly correlated with the historical thermal environment, whereas, photosymbiont gene expression was largely determined by microalgal lineage. Overall, our results reveal a three-tiered strategy of thermal acclimatization in Pocillopora underpinned by host-photosymbiont specificity, host transcriptomic plasticity, and differential photosymbiotic association under extreme warming.

Integrative omics framework for characterization of coral reef ecosystems from the Tara Pacific expedition.

Coral reef science is a fast-growing field propelled by the need to better understand coral health and resilience to devise strategies to slow reef loss resulting from environmental stresses. Key to coral resilience are the symbiotic interactions established within a complex holobiont, i.e. the multipartite assemblages comprising the coral host organism, endosymbiotic dinoflagellates, bacteria, archaea, fungi, and viruses. Tara Pacific is an ambitious project built upon the experience of previous Tara Oceans expeditions, and leveraging state-of-the-art sequencing technologies and analyses to dissect the biodiversity and biocomplexity of the coral holobiont screened across most archipelagos spread throughout the entire Pacific Ocean. Here we detail the Tara Pacific workflow for multi-omics data generation, from sample handling to nucleotide sequence data generation and deposition. This unique multidimensional framework also includes a large amount of concomitant metadata collected side-by-side that provide new assessments of coral reef biodiversity including micro-biodiversity and shape future investigations of coral reef dynamics and their fate in the Anthropocene.

Endogenous viral elements reveal associations between a non-retroviral RNA virus and symbiotic dinoflagellate genomes.

Endogenous viral elements (EVEs) offer insight into the evolutionary histories and hosts of contemporary viruses. This study leveraged DNA metagenomics and genomics to detect and infer the host of a non-retroviral dinoflagellate-infecting +ssRNA virus (dinoRNAV) common in coral reefs. As part of the Tara Pacific Expedition, this study surveyed 269 newly sequenced cnidarians and their resident symbiotic dinoflagellates (Symbiodiniaceae), associated metabarcodes, and publicly available metagenomes, revealing 178 dinoRNAV EVEs, predominantly among hydrocoral-dinoflagellate metagenomes. Putative associations between Symbiodiniaceae and dinoRNAV EVEs were corroborated by the characterization of dinoRNAV-like sequences in 17 of 18 scaffold-scale and one chromosome-scale dinoflagellate genome assembly, flanked by characteristically cellular sequences and in proximity to retroelements, suggesting potential mechanisms of integration. EVEs were not detected in dinoflagellate-free (aposymbiotic) cnidarian genome assemblies, including stony corals, hydrocorals, jellyfish, or seawater. The pervasive nature of dinoRNAV EVEs within dinoflagellate genomes (especially Symbiodinium), as well as their inconsistent within-genome distribution and fragmented nature, suggest ancestral or recurrent integration of this virus with variable conservation. Broadly, these findings illustrate how +ssRNA viruses may obscure their genomes as members of nested symbioses, with implications for host evolution, exaptation, and immunity in the context of reef health and disease.

Pervasive tandem duplications and convergent evolution shape coral genomes.

BACKGROUND: Over the last decade, several coral genomes have been sequenced allowing a better understanding of these symbiotic organisms threatened by climate change. Scleractinian corals are reef builders and are central to coral reef ecosystems, providing habitat to a great diversity of species. RESULTS: In the frame of the Tara Pacific expedition, we assemble two coral genomes, Porites lobata and Pocillopora cf. effusa, with vastly improved contiguity that allows us to study the functional organization of these genomes. We annotate their gene catalog and report a relatively higher gene number than that found in other public coral genome sequences, 43,000 and 32,000 genes, respectively. This finding is explained by a high number of tandemly duplicated genes, accounting for almost a third of the predicted genes. We show that these duplicated genes originate from multiple and distinct duplication events throughout the coral lineage. They contribute to the amplification of gene families, mostly related to the immune system and disease resistance, which we suggest to be functionally linked to coral host resilience. CONCLUSIONS: At large, we show the importance of duplicated genes to inform the biology of reef-building corals and provide novel avenues to understand and screen for differences in stress resilience.

Characterization of 25 new microsatellite markers for the green turtle (Chelonia mydas) and cross-species amplification in other marine turtle species.

BACKGROUND: The green sea turtle, Chelonia mydas, is a migratory species with a strong natal homing behavior leading to a complex population structure worldwide. The species has suffered severe declines in local populations; it is therefore crucial to understand its population dynamics and genetic structure to adopt appropriate management policies. Here, we describe the development of 25 new microsatellite markers specific to C. mydas and suitable for these analyses. METHODS AND RESULTS: They were tested on 107 specimens from French Polynesia. An average allelic diversity of 8 alleles per locus was reported and observed heterozygosity ranged from 0.187 to 0.860. Ten loci were significantly deviant from the Hardy-Weinberg equilibrium, and 16 loci showed a moderate to high level of linkage disequilibrium (4-22%). The overall Fis was positive (0.034, p-value < 0.001), and sibship analysis revealed 12 half- or full-sibling dyads, suggesting possible inbreeding in this population. Cross-amplification tests were performed on two other marine turtle species, Caretta caretta and Eretmochelys imbricata. All loci successfully amplified on these two species, though 1 to 5 loci were monomorphic. CONCLUSION: These new markers will not only be relevant for further analyses on the population structure of the green turtle and the two other species, but they will also be invaluable for parentage studies, for which a high number of polymorphic loci are necessary. This can provide important insight into male reproductive behavior and migration, an aspect of sea turtle biology that is of critical importance for the conservation of the species.

Characterization of 35 new microsatellite markers for the blacktip reef shark (Carcharhinus melanopterus) and cross-species amplification in eight other shark species.

BACKGROUND: Shark species are overfished at a global scale, as they are poached for the finning industry or are caught as bycatch. Efficient conservation measures require fine-scale spatial and temporal studies to characterize shark habitat use, infer migratory habits, analyze relatedness, and detect population genetic differentiation. Gathering these types of data is costly and time-consuming, especially when it requires collection of shark tissue samples. METHODS AND RESULTS: Genetic tools, such as microsatellite markers, are the most economical sampling method for collecting genetic data, as they enable the estimation of genetic diversity, population structure and parentage relationships and are thus an efficient way to inform conservation strategies. Here, a set of 45 microsatellite loci was tested on three blacktip reef shark (Carcharhinus melanopterus) populations from three Polynesian islands: Moorea, Morane and Tenararo. The set was composed of 10 previously published microsatellite markers and 35 microsatellite markers that were developed specifically for C. melanopterus as part of the present study. The 35 novel and 10 existing loci were cross-amplified on eight additional shark species (Carcharhinus amblyrhynchos, C. longimanus, C. sorrah, Galeocerdo cuvier, Negaprion acutidens, Prionacea glauca, Rhincodon typus and Sphyrna lewini). These species had an average of 69% of successful amplification, considered if at least 50% of the individual samples being successfully amplified per species and per locus. CONCLUSIONS: This novel microsatellite marker set will help address numerous knowledge gaps that remain, concerning genetic stock identification, shark behavior and reproduction via parentage analysis.

Investigating population dynamics from parentage analysis in the highly endangered fan mussel Pinna nobilis.

Understanding dispersal patterns is a major focus for conservation biology as it influences local survival and resilience in case of local disturbance, particularly for sessile species. Dispersal can be assessed through parentage analyses by estimating family structure and self-recruitment. This study documents the family structure of a pelagic spawner, Pinna nobilis, which is facing a major crisis that threatens its survival as most of its populations have been decimated by a parasite, Haplosporidium pinnae. In this context, we focused on a single population (Peyrefite, Banyuls-sur-mer, France) where 640 individuals were sampled in 2011, 2015, and 2018 and genotyped for 22 microsatellite markers. Genetic diversity was high and homogeneous among years, with mean allele numbers ranging between 13.6 and 14.8 and observed heterozygosities (H o) between 0.7121 and 0.7331. Low, but significant, genetic differentiations were found between 2011-2015 and 2015-2018. A parentage analysis described 11 clusters, including one prevailing, and revealed that 46.9% of individuals were involved in half-sib relationships, even between years, suggesting that source populations were recurrent year after year. There were few individuals resampled between years (30 in 2015 and 14 in 2018), indicating a rapid turnover. Considering the large number of half-sib relationships but the low number of relations per individual, we conclude that P. nobilis exhibit homogeneous reproductive success. Self-recruitment was not detected, making this population highly vulnerable as replenishment only relies on connectivity from neighboring populations. In the context of the pandemic caused by H. pinnae, these results will have to be considered when choosing a location to reintroduce individuals in potential future rescue plans.

Genetic homogeneity of the critically endangered fan mussel, Pinna nobilis, throughout lagoons of the Gulf of Lion (North-Western Mediterranean Sea).

The fan mussel, Pinna nobilis, endemic to the Mediterranean Sea, is a critically endangered species facing mass mortality events in almost all of its populations, following the introduction of the parasite Haplosporidium pinnae. Such a unique pandemic in a marine organism, which spreads rapidly and with mortality rates reaching up to 100%, could lead to the potential extinction of the species. Only few regions, involving lagoon habitats, remain healthy throughout the entire Mediterranean Sea. This study describes the genetic structure of P. nobilis across the Gulf of Lion, including confined locations such as lagoons and ports. A total of 960 samples were collected among 16 sites distributed at 8 localities, and then genotyped using 22 microsatellite markers. Genetic diversity was high in all sites with mean allele numbers ranging between 10 and 14.6 and with observed heterozygosities (Ho) between 0.679 and 0.704. No genetic differentiation could be identified (FST ranging from 0.0018 to 0.0159) and the percentages of related individuals were low and similar among locations (from 1.6 to 6.5%). Consequently, all fan mussels, over the entire coastline surveyed, including those in the most geographically isolated areas, belong to a large genetically homogeneous population across the Gulf of Lion. Considering the ongoing mass mortality context, this result demonstrates that almost all of the genetic diversity of P. nobilis populations is still preserved even in isolated lagoons, which might represent a refuge habitat for the future of the species.

Natural hybridization between pen shell species: Pinna rudis and the critically endangered Pinna nobilis may explain parasite resistance in P. nobilis.

Recently, Pinna nobilis pen shells population in Mediterranean Sea has plummeted due to a Mass Mortality Event caused by an haplosporidian parasite. In consequence, this bivalve species has been included in the IUCN Red List as "Critically Endangered". In the current scenario, several works are in progress to protect P. nobilis from extinction, being identification of hybrids (P. nobilis x P. rudis) among survivors extremely important for the conservation of the species.Morphological characteristics and molecular analyses were used to identify putative hybrids. A total of 10 individuals of each species (P. nobilis and P. rudis) and 3 doubtful individuals were considered in this study. The putative hybrids showed shell morphology and mantle coloration intermingled exhibiting both P. nobilis and P. rudis traits. Moreover, the analyses of 1150 bp of the 28S gene showed 9 diagnostic sites between P. rudis and P. nobilis, whereas hybrids showed both parental diagnostic alleles at the diagnostic loci. Regarding the multilocus genotypes from the 8 microsatellite markers, the segregation of two Pinna species was clearly detected on the PCoA plot and the 3 hybrids showed intermediate positions.This is the first study evidencing the existence of hybrids P. nobilis x P. rudis, providing molecular methodology for a proper identification of new hybrids. Further studies testing systematically all parasite-resisting isolated P. nobilis should be undertaken to determine if the resistance is resulting from introgression of P. rudis into P. nobilis genome and identifying aspects related to resistance.

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.

Characterization of 25 new microsatellite markers for the fin whale (Balaenoptera physalus) and cross-species amplification in other cetaceans.

Cetaceans are large mammals widely distributed on Earth. The fin whale, Balaenoptera physalus, is the second largest living animal. In the 20th century, commercial whaling reduced its global population by 70%, and in the Mediterranean Sea not only was their overall population depleted but the migration between the Mediterranean Sea and the Atlantic Ocean was reduced. Previous genetic studies identified isolation between these two regions, with a limited gene-flow between these adjacent populations based on nuclear and mitochondrial markers. However, only limited information exists for the Mediterranean population as genetic diversity and abundance trends are still unknown. In this study, 39 highly polymorphic microsatellite markers were tested, including 25 markers developed de novo together with 14 markers previously published. An average allelic diversity of 8.3 alleles per locus was reported, ranging from 3 to 15 alleles per locus, for B. physalus. Expected heterozygosity was variable among loci and ranged from 0.34 to 0.91. Only two markers in the new set were significantly deviant from the Hardy Weinberg equilibrium. Cross-species amplification was tested in four other cetacean species. A total of 27 markers were successfully amplified in the four species (Balaenoptera acutorostrata, Megaptera novaeangliae, Physeter macrocephalus and Globicephala melas). A multivariate analysis on the multilocus genotypes successfully discriminated the five species. This new set of microsatellite markers will not only provide a useful tool to identify and understand the genetic diversity and the evolution of the B. physalus population, but it will also be relevant for other cetacean species, and will allow further parentage analyses. Eventually, this new set of microsatellite markers will provide critical data that will shed light on important biological data within a conservation perspective.

A framework for in situ molecular characterization of coral holobionts using nanopore sequencing.

Molecular characterization of the coral host and the microbial assemblages associated with it (referred to as the coral holobiont) is currently undertaken via marker gene sequencing. This requires bulky instruments and controlled laboratory conditions which are impractical for environmental experiments in remote areas. Recent advances in sequencing technologies now permit rapid sequencing in the field; however, development of specific protocols and pipelines for the effective processing of complex microbial systems are currently lacking. Here, we used a combination of 3 marker genes targeting the coral animal host, its symbiotic alga, and the associated bacterial microbiome to characterize 60 coral colonies collected and processed in situ, during the Tara Pacific expedition. We used Oxford Nanopore Technologies to sequence marker gene amplicons and developed bioinformatics pipelines to analyze nanopore reads on a laptop, obtaining results in less than 24 h. Reef scale network analysis of coral-associated bacteria reveals broadly distributed taxa, as well as host-specific associations. Protocols and tools used in this work may be applicable for rapid coral holobiont surveys, immediate adaptation of sampling strategy in the field, and to make informed and timely decisions in the context of the current challenges affecting coral reefs worldwide.

Genetic connectivity of lionfish (Pterois volitans) in marine protected areas of the Gulf of Mexico and Caribbean Sea.

Lionfish (Pterois volitans) have rapidly invaded the tropical Atlantic and spread across the wider Caribbean in a relatively short period of time. Because of its high invasion capacity, we used it as a model to identify the connectivity among nine marine protected areas (MPAs) situated in four countries in the Gulf of Mexico and the Caribbean Sea. This study provides evidence of local genetic differentiation of P. volitans in the Gulf of Mexico and the Caribbean Sea. A total of 475 lionfish samples were characterized with 12 microsatellites, with 6-20 alleles per locus. Departures from Hardy-Weinberg equilibrium (HWE) were found in 10 of the 12 loci, all caused by heterozygous excess. Moderate genetic differentiation was observed between Chiriviche, Venezuela and Xcalak, México localities (F ST = 0.012), and between the Los Roques and the Veracruz (F ST = 0.074) sites. STRUCTURE analysis found that four genetic entities best fit our data. A unique genetic group in the Gulf of Mexico may imply that the lionfish invasion unfolded both in a counterclockwise manner in the Gulf of Mexico. In spite of the notable dispersion of P. volitans, our results show some genetic structure, as do other noninvasive Caribbean fish species, suggesting that the connectivity in some MPAs analyzed in the Caribbean is limited and caused by only a few source individuals with subsequent genetic drift leading to local genetic differentiation. This indicates that P. volitans dispersion could be caused by mesoscale phenomena, which produce stochastic connectivity pulses. Due to the isolation of some MPAs from others, these findings may hold a promise for local short-term control of by means of intensive fishing, even in MPAs, and may have regional long-term effects.