Regional and local environmental conditions do not shape the response to warming of a marine habitat-forming species.

The differential response of marine populations to climate change remains poorly understood. Here, we combine common garden thermotolerance experiments in aquaria and population genetics to disentangle the factors driving the population response to thermal stress in a temperate habitat-forming species: the octocoral Paramuricea clavata. Using eight populations separated from tens of meters to hundreds of kilometers, which were differentially impacted by recent mortality events, we identify 25 °C as a critical thermal threshold. After one week of exposure at this temperature, seven of the eight populations were affected by tissue necrosis and after 30 days of exposure at this temperature, the mean % of affected colonies increased gradually from 3 to 97%. We then demonstrate the weak relation between the observed differential phenotypic responses and the local temperature regimes experienced by each population. A significant correlation was observed between these responses and the extent of genetic drift impacting each population. Local adaptation may thus be hindered by genetic drift, which seems to be the main driver of the differential response. Accordingly, conservation measures should promote connectivity and control density erosion in order to limit the impact of genetic drift on marine populations facing climate change.

Evidence for a genetic sex determination in Cnidaria, the Mediterranean red coral (Corallium rubrum).

Sexual reproduction is widespread among eukaryotes, and the sex-determining processes vary greatly among species. While genetic sex determination (GSD) has been intensively described in bilaterian species, no example has yet been recorded among non-bilaterians. However, the quasi-ubiquitous repartition of GSD among multicellular species suggests that similar evolutionary forces can promote this system, and that these forces could occur also in non-bilaterians. Studying sex determination across the range of Metazoan diversity is indeed important to understand better the evolution of this mechanism and its lability. We tested the existence of sex-linked genes in the gonochoric red coral (Corallium rubrum, Cnidaria) using restriction site-associated DNA sequencing. We analysed 27 461 single nucleotide polymorphisms (SNPs) in 354 individuals from 12 populations including 53 that were morphologically sexed. We found a strong association between the allele frequencies of 472 SNPs and the sex of individuals, suggesting an XX/XY sex-determination system. This result was confirmed by the identification of 435 male-specific loci. An independent test confirmed that the amplification of these loci enabled us to identify males with absolute certainty. This is the first demonstration of a GSD system among non-bilaterian species and a new example of its convergence in multicellular eukaryotes.

Genomic resources and their influence on the detection of the signal of positive selection in genome scans.

Genome scans represent powerful approaches to investigate the action of natural selection on the genetic variation of natural populations and to better understand local adaptation. This is very useful, for example, in the field of conservation biology and evolutionary biology. Thanks to Next Generation Sequencing, genomic resources are growing exponentially, improving genome scan analyses in non-model species. Thousands of SNPs called using Reduced Representation Sequencing are increasingly used in genome scans. Besides, genome sequences are also becoming increasingly available, allowing better processing of short-read data, offering physical localization of variants, and improving haplotype reconstruction and data imputation. Ultimately, genome sequences are also becoming the raw material for selection inferences. Here, we discuss how the increasing availability of such genomic resources, notably genome sequences, influences the detection of signals of selection. Mainly, increasing data density and having the information of physical linkage data expand genome scans by (i) improving the overall quality of the data, (ii) helping the reconstruction of demographic history for the population studied to decrease false-positive rates and (iii) improving the statistical power of methods to detect the signal of selection. Of particular importance, the availability of a high-quality reference genome can improve the detection of the signal of selection by (i) allowing matching the potential candidate loci to linked coding regions under selection, (ii) rapidly moving the investigation to the gene and function and (iii) ensuring that the highly variable regions of the genomes that include functional genes are also investigated. For all those reasons, using reference genomes in genome scan analyses is highly recommended.

Highly contrasted responses of Mediterranean octocorals to climate change along a depth gradient.

Climate change has a strong impact on marine ecosystems, including temperate species. Analysing the diversity of thermotolerance levels within species along with their genetic structure enables a better understanding of their potential response to climate change. We performed this integrative study on the Mediterranean octocoral Eunicella cavolini, with samples from different depths and by means of a common garden experiment. This species does not host photosynthetic Symbiodinium, enabling us to focus on the cnidarian response. We compared the thermotolerance of individuals from 20 m and 40 m depths from the same site and with replicates from the same colony. On the basis of an innovative statistical analysis of necrosis kinetics and risk, we demonstrated the occurrence of a very different response between depths at this local scale, with lower thermotolerance of deep individuals. Strongly thermotolerant individuals were observed at 20 m with necrosis appearing at higher temperatures than observed in situ. On the basis of nine microsatellite loci, we showed that these marked thermotolerance differences occur within a single population. This suggests the importance of acclimatization processes in adaptation to these different depths. In addition, differences between replicates demonstrated the occurrence of a variability of response between fragments from the same colony with the possibility of an interaction with a tank effect. Our results provide a basis for studying adaptation and acclimatization in Mediterranean octocorals in a heterogeneous environment.

The red coral (Corallium rubrum) transcriptome: a new resource for population genetics and local adaptation studies.

The question of species survival and evolution in heterogeneous environments has long been a subject for study. Indeed, it is often difficult to identify the molecular basis of adaptation to contrasted environments, and nongenetic effects increase the difficulty to disentangle fixed effects, such as genetic adaptation, from variable effects, such as individual phenotypic plasticity, in adaptation. Nevertheless, this question is also of great importance for understanding the evolution of species in a context of climate change. The red coral (Corallium rubrum) lives in the Mediterranean Sea, where at depths ranging from 5 to 600 m, it meets very contrasted thermal conditions. The shallowest populations of this species suffered from mortality events linked with thermal anomalies that have highlighted thermotolerance differences between individuals. We provide here a new transcriptomic resource, as well as candidate markers for the study of local adaptation. We sequenced the transcriptome of six individuals from 5 m and six individuals from 40 m depth at the same site of the Marseilles bay, after a period of common garden acclimatization. We found differential expression maintained between the two depths even after common garden acclimatization, and we analysed the polymorphism pattern of these samples. We highlighted contigs potentially implicated in the response to thermal stress, which could be good candidates for the study of thermal adaptation for the red coral. Some of these genes are also involved in the response to thermal stress in other corals. Our method enables the identification of candidate loci of local adaptation useful for other nonmodel organisms.

PCR survey of 50 introns in animals: cross-amplification of homologous EPIC loci in eight non-bilaterian, protostome and deuterostome phyla.

Exon Primed Intron Crossing (EPIC) markers provide molecular tools that are susceptible to be variable within species while remaining amplifiable by PCR using potentially universal primers. In this study we tested the possibility of obtaining PCR products from 50 EPIC markers on 23 species belonging to seven different phyla (Porifera, Cnidaria, Arthropoda, Nematoda, Mollusca, Annelida, Echinodermata) using 70 new primer pairs. A previous study had identified and tested those loci in a dozen species, including another phylum, Urochordata (Chenuil et al., 2010). Results were contrasted among species. The best results were achieved with the oyster (Mollusca) where 28 loci provided amplicons susceptible to contain an intron according to their size. This was however not the case with the other mollusk Crepidula fornicata, which seems to have undergone a reduction in intron number or intron size. In the Porifera, 13 loci appeared susceptible to contain an intron, a surprisingly high number for this phylum considering its phylogenetic distance with genomic data used to design the primers. For two cnidarian species, numerous loci (24) were obtained. Ecdysozoan phyla (arthropods and nematodes) proved less successful than others as expected considering reports of their rapid rate of genome evolution and the worst results were obtained for several arthropods. Some general patterns among phyla arose, and we discuss how the results of this EPIC survey may give new insights into genome evolution of the study species. This work confirms that this set of EPIC loci provides an easy-to-use toolbox to identify genetic markers potentially useful for population genetics, phylogeography or phylogenetic studies for a large panel of metazoan species. We then argue that obtaining diploid sequence genotypes for these loci became simple and affordable owing to Next-Generation Sequencing development. Species surveyed in this study belong to several genera (Acanthaster, Alvinocaris, Aplysina, Aurelia, Crepidula, Eunicella, Hediste, Hemimysis, Litoditis, Lophelia, Mesopodopsis, Mya, Ophiocten, Ophioderma, Ostrea, Pelagia, Platynereis, Rhizostoma, Rimicaris), two of them, belonging to the family Vesicomydae and Eunicidae, could not be determined at the genus level.

Phylogeography of the red coral (Corallium rubrum): inferences on the evolutionary history of a temperate gorgonian.

The red coral Corallium rubrum (Cnidaria, Octocorallia) is an exploited, long-lived sessile species from the Mediterranean Sea and the adjacent coastline in the Atlantic Ocean. Surveys of genetic variation using microsatellites have shown that populations of C. rubrum are characterized by strong differentiation at the local scale but a study of the phylogeography of this species was still lacking. Here, we used seven polymorphic microsatellite loci, together with sequence data from an intron of the elongation factor 1 (EF1) gene, to investigate the genetic structure of C. rubrum across its geographical range in the western Mediterranean Sea and in the Adriatic Sea. The EF1 sequences were also used to analyse the consequences of demographic fluctuations linked with past environmental change. Clustering analysis with microsatellite loci highlighted three to seven genetic groups with the distinction of North African and Adriatic populations; this distinction appeared significant with AMOVA and differentiation tests. Microsatellite and EF1 data extended the isolation by distance pattern previously observed for this species at the western Mediterranean scale. EF1 sequences confirmed the genetic differentiation observed between most samples with microsatellites. A statistical parsimony network of EF1 haplotypes provided no evidence of high sequence divergence among regions, suggesting no long-term isolation. Selective neutrality tests on microsatellites and EF1 were not significant but should be interpreted with caution in the case of EF1 because of the low sample sizes for this locus. Our results suggest that recent Quaternary environmental fluctuations had a limited impact on the genetic structure of C. rubrum.

From global to local genetic structuring in the red gorgonian Paramuricea clavata: the interplay between oceanographic conditions and limited larval dispersal.

Defining the scale of connectivity among marine populations and identifying the barriers to gene flow are tasks of fundamental importance for understanding the genetic structure of populations and for the design of marine reserves. Here, we investigated the population genetic structure at three spatial scales of the red gorgonian Paramuricea clavata (Cnidaria, Octocorallia), a key species dwelling in the coralligenous assemblages of the Mediterranean Sea. Colonies of P. clavata were collected from 39 locations across the Mediterranean Sea from Morocco to Turkey and analysed using microsatellite loci. Within three regions (Medes, Marseille and North Corsica), sampling was obtained from multiple locations and at different depths. Three different approaches (measures of genetic differentiation, Bayesian clustering and spatially explicit maximum-difference algorithm) were used to determine the pattern of genetic structure. We identified genetic breaks in the spatial distribution of genetic diversity, which were concordant with oceanographic conditions in the Mediterranean Sea. We revealed a high level of genetic differentiation among populations and a pattern of isolation by distance across the studied area and within the three regions, underlining short effective larval dispersal in this species. We observed genetic differentiation among populations in the same locality dwelling at different depths, which may be explained by local oceanographic conditions and which may allow a process of local adaptation of the populations to their environment. We discuss the implications of our results for the conservation of the species, which is exposed to various threats.

Fine-scale genetic structure and inferences on population biology in the threatened Mediterranean red coral, Corallium rubrum.

Identifying microevolutionary processes acting in populations of marine species with larval dispersal is a challenging but crucial task because of its conservation implications. In this context, recent improvements in the study of spatial genetic structure (SGS) are particularly promising because they allow accurate insights into the demographic and evolutionary processes at stake. Using an exhaustive sampling and a combination of image processing and population genetics, we highlighted significant SGS between colonies of Corallium rubrum over an area of half a square metre, which sheds light on a number of aspects of its population biology. Based on this SGS, we found the mean dispersal range within sites to be between 22.6 and 32.1 cm, suggesting that the surveyed area approximately corresponded to a breeding unit. We then conducted a kinship analysis, which revealed a complex half-sib family structure and allowed us to quantify the level of self-recruitment and to characterize aspects of the mating system of this species. Furthermore, significant temporal variations in allele frequencies were observed, suggesting low genetic drift. These results have important conservation implications for the red coral and further our understanding of the microevolutionary processes acting within populations of sessile marine species with a larval phase.

Genetic survey of shallow populations of the Mediterranean red coral [Corallium rubrum (Linnaeus, 1758)]: new insights into evolutionary processes shaping nuclear diversity and implications for conservation.

Combined action from over-harvesting and recent mass mortality events potentially linked to ongoing climate changes has led to new concerns for the conservation of shallow populations (5-60 m) of Corallium rubrum, an octocorallian that is mainly found in the Mediterranean Sea. The present study was designed to analyse population structure and relationships at different spatial scales (from 10s of meters to 100s of kilometres) with a focus on dispersal pattern. We also performed the first analysis of the distribution of genetic diversity using a comparative approach between regional-clusters and samples. Forty populations dwelling in four distinct regions between 14 and 60 m in depth were genotyped using 10 microsatellites. Our main results indicate (i) a generalized pair-sample differentiation combined with a weak structure between regional-clusters; (ii) the occurrence of isolation by distance at the global scale, but also within two of the three analysed regional-clusters; (iii) a high level of genetic diversity over the surveyed area with a heterogeneous distribution from regional-cluster to sample levels. The evolutionary consequences of these results are discussed and their management implications are provided.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).

Natural and artificial secondary contact in brown trout (Salmo trutta, L.) in the French western Pyrenees assessed by allozymes and microsatellites.

Analysis of allozyme polymorphism in brown trout (Salmo trutta) populations from south-western France shows that two genetically differentiated wild forms (characterised by the LDH-C1*100 and 90 alleles) introgress in this area. As allozymes could not evaluate the impact of stocking in the Atlantic basin, microsatellites have been necessary to detect the influence of hatchery fish and to confirm that the observed structure was natural. Microsatellites confirm the distinctness of the two wild forms based on allozyme loci. This situation provides a new example of secondary contact for this species in the Atlantic basin, with various levels of mixing being seen between the two population groups. The origin of these forms is discussed in the light of previous studies concerning modern and ancestral Atlantic trout (Hamilton et al, 1989) and lineages stemming from different glacial refuges (Garcia Marin et al, 1999; Weiss et al, 2000). This local analysis provides new insights in defining the evolutionary history of this species and confirms the important role of glaciation events in this history.

Genetic structure of brown trout (Salmo trutta, L.) populations from south-western France: data from mitochondrial control region variability.

The genetic relationships between several French Atlantic populations of brown trout were analysed using polymerase chain reaction-single-strand conformation polymorphism and sequencing of two parts of the mitochondrial control region. Ten mitochondrial haplotypes were identified, separated by a small number of mutations. The distribution of these haplotypes confirmed our hypothesis of the existence of two genetically well-differentiated groups of populations in this area, which has already been suggested using nuclear markers. Nuclear and mitochondrial markers, however, show different patterns of genetic differentiation: some populations which belong to one group according to allozyme results appear to be part of the other when using mitochondrial DNA. The origin of these different forms is discussed; they seem to belong to the Atlantic grouping. Nevertheless, the hypothesis of an ancestral and a modern form suggested for northern populations does not appear valid in this particular case.