Seascape Genetics of the Atlantic Spotted Dolphin (Stenella frontalis) Based on Mitochondrial DNA.

The Atlantic spotted dolphin (Stenella frontalis) is endemic to tropical, subtropical, and warm temperate waters of the Atlantic Ocean. Throughout its distribution, both geographic distance and environmental variation may contribute to population structure of the species. In this study, we follow a seascape genetics approach to investigate population differentiation of Atlantic spotted dolphins based on a large worldwide dataset and the relationship with marine environmental variables. The results revealed that the Atlantic spotted dolphin exhibits population genetic structure across its distribution based on mitochondrial DNA control region (mtDNA-CR) data. Analyses based on the contemporary landscape suggested, at both the individual and population level, that the population genetic structure is consistent with the isolation-by-distance model. However, because geography and environmental matrices were correlated, and because in some, but not all analyses, we found a significant effect for the environment, we cannot rule out the addition contribution of environmental factors in structuring genetic variation. Future analyses based on nuclear data are needed to evaluate whether local processes, such as social structure and some level of philopatry within populations, may be contributing to the associations among genetic structure, geographic, and environmental distance.

Low mtDNA diversity in a highly differentiated population of spinner dolphins (Stenella longirostris) from the Fernando de Noronha Archipelago, Brazil.

Spinner dolphins (Stenella longirostris, Gray 1828) are widely distributed in tropical waters around the world. Although they occur in large, pelagic groups in the Eastern Tropical Pacific, elsewhere in the Pacific they are found in small and genetically isolated populations associated with islands. This species is considered to be "Least Concern" (LC) by the World Conservation Union (IUCN). To assess genetic diversity and population structure of an island-associated population in the South Atlantic Ocean we surveyed 162 spinner dolphins throughout the Fernando de Noronha Archipelago of the northeast coast of Brazil using ten microsatellite loci and sequencing a 413-bp section of the mitochondrial DNA (mtDNA) control region. Eleven mtDNA haplotypes were identified and haplotype diversity (h) and nucleotide diversity (π) were 0.3747 and 0.0060, respectively. Median-Joining Network revealed the presence of two very divergent haplotypes and F-statistics indicated some heterogeneity between two sampling years. All microsatellite loci were polymorphic (Ho: 0.767; He: 0,764) but, revealed no detectable substructure. We also compared the mtDNA haplotypes from Noronha to 159 haplotypes representing 893 individuals from 14 locations worldwide. We found that the two common haplotypes from the Fernando de Noronha Archipelago were absent in all other populations. These comparisons showed that Noronha spinner dolphins are likely more differentiated than other island populations, suggesting that they form societies with strong site fidelity mediated by females.

Cytochrome b marker reveals an independent lineage of Stenella coeruleoalba in the Gulf of Taranto.

Heterogeneity in geomorphological and hydrographical conditions throughout the Mediterranean Sea could be the driving factors behind the significant differences between putative sub-populations, although the existence of a large panmictic population of striped dolphin Stenella coeruleoalba (Meyen 1833) in this marine region could not be excluded. However, understanding the ecological implications of such genetic differentiation is difficult, as inferences about gene flow are usually made on evolutionary time scales and not along the ecological time frame over which most management and conservation practices are applied. In fact, as stated by the IUCN Red List, in the case of species assessed as vulnerable, the degree of genetic exchange between populations within a biogeographic region and its ecological implications represent a fascinating challenge that should be very deeply explored. This is even more significant in the Gulf of Taranto (Northern Ionian Sea, Central-eastern Mediterranean Sea), where the geomorphological and hydrographic characteristics support the hypothesis of a separated striped dolphin population genetically diverging from its original Mediterranean counterpart. To assess this hypothesis, a genetic analysis was carried out on DNA fragments of the mitochondrial cyt b gene to explore the evolutionary origin of S. coeruleoalba in the investigated area and its genetic diversity in comparison with available sequences from other Mediterranean and Atlantic populations. Results were discussed indicating ecological implications and suggesting conservation objectives. Moreover, a delphinid systematic was also suggested.

Evidence of introgressive hybridization between Stenella coeruleoalba and Delphinus delphis in the Greek Seas.

Natural interspecific hybridization might be more important for the evolutionary history and speciation of animals than previously thought, considering several demographic and life history traits as well as habitat disturbance as factors that promote it. In this aspect, cetaceans comprise an interesting case in which the occurrence of sympatric species in mixed associations provides excellent opportunities for interspecific sexual interaction and the potential for hybridization. Here, we present evidence of natural hybridization for two cetacean species commonly occurring in the Greek Seas (Stenella coeruleoalba and Delphinus delphis), which naturally overlap in the Gulf of Corinth by analyzing highly resolving microsatellite DNA markers and mitochondrial DNA sequences in skin samples from 45 individuals of S. coeruleoalba, 12 D. delphis and three intermediate morphs. Employing several phylogenetic and population genetic approaches, we found 15 individuals that are potential hybrids including the three intermediate morphs, verifying the occurrence of natural hybridization between species of different genera. Their hybrids are fertile and able to reproduce not only with the other hybrids but also with each of the two-parental species. However, current evidence does not allow firm conclusions whether hybridization might constitute a step towards the generation of a new species and/or the swan song of an already existing species (i.e., D. delphis). Given that the focal species form mixed pods in several areas of Mediterranean, this study is an excellent opportunity to understand the mechanisms leading to hybridization in the context of gene flow and urges for the evaluation of the genetic status of common dolphins in the Mediterranean.

The chronicles of the contaminated Mediterranean seas: a story told by the cetaceans' skin genes.

Wild animals in their natural environment could provide a big source of information, but sampling can be very challenging, above all for protected species, like marine mammals. Nevertheless, significant data can be obtained sampling stranded animals right after their death, taking into account proper sampling time and methodology. RNA samples from the skin of 12 individuals including the species Stenella coeruleoalba, Tursiops truncatus, and Grampus griseus were used to test 4 potential gene markers of anthropogenic contaminants exposure. The individuals were sampled in 3 geographic areas: the Adriatic, Ionian and Tyrrhenian seas. Three out of the 4 genes tested showed higher expression in the samples collected from the Adriatic Sea. Minute skin samples tell the story of the specific geographic location where the marine mammal spent its life, thanks to the different impact on gene expression exerted by different contamination levels.

Candidate gene molecular markers as tools for analyzing genetic susceptibility to morbillivirus infection in stranded Cetaceans.

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

Molecular assessment of mating strategies in a population of Atlantic spotted dolphins.

Similar to other small cetacean species, Atlantic spotted dolphins (Stenella frontalis) have been the object of concentrated behavioral study. Although mating and courtship behaviors occur often and the social structure of the population is well-studied, the genetic mating system of the species is unknown. To assess the genetic mating system, we genotyped females and their progeny at ten microsatellite loci. Genotype analysis provided estimates of the minimum number of male sires necessary to account for the allelic diversity observed among the progeny. Using the estimates of male sires, we determined whether females mated with the same or different males during independent estrus events. Using Gerud2.0, a minimum of two males was necessary to account for the genetic variation seen among progeny arrays of all tested females. ML-Relate assigned the most likely relationship between offspring pairs; half or full sibling. Relationship analysis supported the conservative male estimates of Gerud2.0 but in some cases, half or full sibling relationships between offspring could not be fully resolved. Integrating the results from Gerud2.0, ML-Relate with previous observational and paternity data, we constructed two-, three-, and four-male pedigree models for each genotyped female. Because increased genetic diversity of offspring may explain multi-male mating, we assessed the internal genetic relatedness of each offspring's genotype to determine whether parent pairs of offspring were closely related. We found varying levels of internal relatedness ranging from unrelated to closely related (range -0.136-0.321). Because there are several hypothesized explanations for multi-male mating, we assessed our data to determine the most plausible explanation for multi-male mating in our study system. Our study indicated females may benefit from mating with multiple males by passing genes for long-term viability to their young.

Hierarchical population structure and habitat differences in a highly mobile marine species: the Atlantic spotted dolphin.

Recent molecular studies have shown that highly mobile species with continuous distributions can exhibit fine-scale population structure. In this context, we assessed genetic structure within a marine species with high dispersal potential, the Atlantic spotted dolphin (Stenella frontalis). Using 19 microsatellite loci and mitochondrial control region sequences, population structure was investigated in the western North Atlantic, the Gulf of Mexico and the Azores Islands. Analyses of the microsatellite data identified four distinct genetic clusters, which were supported by the control region sequences. The highest level of divergence was seen between two clusters corresponding to previously described morphotypes that inhabit oceanic and shelf waters. The combined morphological and genetic evidence suggests these two lineages are on distinct evolutionary trajectories and could be considered distinct subspecies despite their parapatry. Further analysis of the continental shelf cluster resulted in three groups: animals inhabiting shelf waters in the western North Atlantic, the eastern Gulf of Mexico and the western Gulf of Mexico. Analyses of environmental data indicate the four genetic clusters inhabit distinct habitats in terms of depth and sea surface temperature. Contemporary dispersal rate estimates suggest all of these populations should be considered as distinct management units. Conversely, no significant genetic differentiation was observed between S. frontalis from offshore waters of the western North Atlantic and the Azores, which are separated by approximately 4500 km. Overall, the hierarchical structure observed within the Atlantic spotted dolphin shows that the biogeography of the species is complex because it is not shaped solely by geographic distance.

Multiple populations of pantropical spotted dolphins in Hawaiian waters.

Understanding gene flow and dispersal patterns is important for predicting effects of natural events and anthropogenic activities on animal populations. In Hawaii, most species of odontocetes are managed as single populations. Recent exceptions include false killer whales, spinner dolphins, and common bottlenose dolphins, for which studies have shown fidelity to individual islands or groups of islands. Our study focused on pantropical spotted dolphins. We analyzed mitochondrial control region and 11 microsatellite loci from 101 individuals from 4 areas: Hawaii, Maui/Lanai, Oahu, and Kauai/Niihau. We examined F ST, F' ST, R ST, Jost's D, and ΦST and used TESS to estimate number of populations and assignment probabilities. Our results support genetic differentiation among Hawaii, Maui/Lanai, and Oahu and suggest that pantropical spotted dolphins near Kauai/Niihau are likely transient and in low numbers. Between island regions, F ST for microsatellites ranged from 0.016 to 0.045 and for mtDNA, from 0.011 to 0.282. F ' ST, ranged from 0.098 to 0.262 for microsatellites and 0.019 to 0.415 for mtDNA. R ST and ΦST showed similar results to F ST for microsatellites and mtDNA respectively, and Jost's D fell between F ST and F ' ST. TESS supported 3 populations, and greatest mean assignment probability by island region ranged from 0.50 to 0.72. The private alleles method indicated migration rates among regions from 1.49 to 3.45, and effective population size of the island of Hawaii was estimated to be 220. There was no strong evidence to support sex-biased dispersal or group fidelity. Considering this study in the larger context of other odontocete population studies and studies of connectivity, we suggest genetic differentiation may be mediated by behavior adapted to differing habitat types and niches.

An "ex vivo" model to evaluate toxicological responses to mixtures of contaminants in cetaceans: integumentum biopsy slices.

The need for powerful new tools to detect the effects of chemical pollution, in particular of persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs) on Mediterranean cetaceans led us to develop and apply a suite of sensitive biomarkers for integument biopsies of stranded and free-ranging animals. This multi-response ex vivo method has the aim to detect toxicological effects of contaminant mixtures. In the present study, we applied an ex vivo assay using skin biopsy and liver slices, combining molecular biomarkers [Western blot of Cytochrome P450 1A1 (CYP1A1) and Cytochrome P450 2B (CYP2B)] and gene expression biomarkers (Quantitative real-time PCR of CYP1A1, heat shock protein 70, estrogen receptor alpha and E2F transcription factor) in response to chemical exposure [organochlorines compounds (OCs), polybrominated diphenyl ethers (PBDEs), and PAHs] for stranded Mediterranean Stenella coeruleoalba. The main goal of this experiment was to identify the biomarker and/or a suite of biomarkers that could best detect the presence of a specific class of pollutants (OCs, PBDEs, and PAHs) or a mixture of them. This multi-response biomarker methodology revealed an high sensitivity and selectivity of responses (such as CYP1A and ER α mRNA variations after OCs and PAHs exposure) and could represent a valid future approach for the study of inter- and intra-species sensitivities to various classes of environmental contaminants.

The evolving male: spinner dolphin (Stenella longirostris) ecotypes are divergent at Y chromosome but not mtDNA or autosomal markers.

The susceptibility of the Y chromosome to sexual selection may make this chromosome an important player in the formation of reproductive isolating barriers, and ultimately speciation. Here, we investigate the role of the Y chromosome in phenotypic divergence and reproductive isolation of spinner dolphin (Stenella longirostris) ecotypes. This species contains six known ecotypes (grouped into four subspecies) that exhibit striking differences in morphology, habitat and mating system, despite having adjacent or overlapping ranges and little genetic divergence at previously studied mtDNA and autosomal markers. We examined the phylogeographic structure for all six ecotypes across the species range (n = 261, 17 geographic locations) using DNA sequences from three Y chromosome markers, two maternally inherited mitochondrial (mtDNA) markers, and a biparentally inherited autosomal intron. mtDNA and autosomal analyses revealed low divergence (most Φ(ST) values <0.1) between ecotypes and geographic regions, concordant with previous studies. In contrast, Y intron analyses revealed fixed differences amongst the three most phenotypically divergent groups: S. l. longirostris vs. S. l. roseiventris vs. combined S. l. orientalis/S. l. centroamericana/Tres Marias ecotypes). Another ecotype (whitebelly), previously postulated to be a hybrid between the two phenotypically most divergent ecotypes, had Y haplotypes from both putative parent ecotypes, supporting a hybrid designation. Reduced introgression of the Y chromosome has previously been observed in other organisms ranging from insects to terrestrial mammals, and here we demonstrate this phenomenon in a marine mammal with high dispersal capabilities. These results indicate that reduced introgression of the Y chromosome occurs in a wide taxonomic range of organisms and support the growing body of evidence that rapid evolution of the Y chromosome is important in evolutionary diversification.

Ecotoxicological diagnosis of striped dolphin (Stenella coeruleoalba) from the Mediterranean basin by skin biopsy and gene expression approach.

Mediterranean cetacean odontocetes are exposed to environmental stress, in particular to persistent organic pollutants, polycyclic aromatic hydrocarbons and trace elements. In the present study, the response of "gene-expression biomarkers" was evaluated in Mediterranean striped dolphin (Stenella coeruleoalba) skin biopsies collected in three sampling areas: Pelagos sanctuary (Ligurian sea), Ionian sea, and Strait of Gibraltar. The mRNA levels of five putative biomarker genes (aryl hydrocarbon receptor, E2F-1 transcription factor, cytochrome P450 1A, estrogen receptor 1, and heat shock protein 70) were measured for the first time by quantitative real-time PCR in cetacean skin biopsies. The different responses of most of the genes reflected contamination levels in the three sampling areas. Pelagos sanctuary dolphins appeared to be the most exposed to toxicological stress, having the highest up-regulation of CYP1A and AHR. Moreover, a cluster analysis distinguished the populations on the basis of the gene expression biomarker used in our study, showing different pattern between Mediterranean sea and Strait of Gibraltar. Our results suggest that this molecular approach applied to non-destructive biopsy material is a powerful diagnostic tool for evaluating ecotoxicological impact on cetacean populations.

Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris).

Spinner dolphins (Stenella longirostris) exhibit different social behaviours at two regions in the Hawaiian Archipelago: off the high volcanic islands in the SE archipelago they form dynamic groups with ever-changing membership, but in the low carbonate atolls in the NW archipelago they form long-term stable groups. To determine whether these environmental and social differences influence population genetic structure, we surveyed spinner dolphins throughout the Hawaiian Archipelago with mtDNA control region sequences and 10 microsatellite loci (n = 505). F-statistics, Bayesian cluster analyses, and assignment tests revealed population genetic separations between most islands, with less genetic structuring among the NW atolls than among the SE high islands. The populations with the most stable social structure (Midway and Kure Atolls) have the highest gene flow between populations (mtDNA Phi(ST) < 0.001, P = 0.357; microsatellite F(ST) = -0.001; P = 0.597), and a population with dynamic groups and fluid social structure (the Kona Coast of the island of Hawai'i) has the lowest gene flow (mtDNA 0.042 < Phi(ST) < 0.236, P < 0.05; microsatellite 0.016 < F(ST) < 0.040, P < 0.001). We suggest that gene flow, dispersal, and social structure are influenced by the availability of habitat and resources at each island. Genetic comparisons to a South Pacific location (n = 16) indicate that Hawaiian populations are genetically depauperate and isolated from other Pacific locations (mtDNA 0.216 < F(ST) < 0.643, P < 0.001; microsatellite 0.058 < F(ST) < 0.090, P < 0.001); this isolation may also influence social and genetic structure within Hawai'i. Our results illustrate that genetic and social structure are flexible traits that can vary between even closely-related populations.

Use of immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as new "in vitro" tool to investigate effects of environmental contaminants.

The aim of the present study was to propose the immunofluorescence technique in cultured fibroblasts from Mediterranean cetaceans as a new "in vitro" tool to explore the susceptibility of these marine mammals to different xenobiotic compounds. The cell lines were cultured from integument biopsies of free-ranging and stranded cetaceans (dead within 12h). Using the indirect immunofluorescence assay, we detected endogenous proteins induced by different contaminants. Here we present the method used for qualitative and quantitative evaluation of cytochromes P450 (CYP1A1 and CYP2B) induced by some POPs (DDTs and PCBs) and emerging contaminants (PBDEs) in fibroblast cell cultures of striped dolphin (Stenella coeruleoalba) and bottlenose dolphin (Tursiops truncatus). Immunofluorescence was quantified with a specially designed Olympus macro, DetectIntZ. A major result was the possibility of using this "in vitro" assay to quantify induction of endogenous proteins.

Selection of reliable reference genes for qRT-PCR studies on cetacean fibroblast cultures exposed to OCs, PBDEs, and 17beta-estradiol.

Quantitative real-time PCR (qRT-PCR) represents an effective molecular technique for the detection of mRNA expression in biological samples. Its sensitivity allows the quantification of slight changes in the regulation of gene transcription but is strictly dependent upon the method followed during the normalization procedure. Relative quantification determines changes in the steady-state mRNA levels of genes across multiple samples and it is assessed by comparison with the levels of one or more internal control RNA. In this context, the choice of constitutively expressed control genes, whose transcription is not affected by the contaminants, appears to be fundamental for the reliability of this technique. During this study, fibroblast cell cultures originated from integumentum biopsies, sampled in the cetacean species Stenella coeruleoalba, have been exposed for 6h to increasing concentrations of different mixtures of compounds with endocrine disruptor capacities (EDCs): organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), and 17beta-estradiol. Ten common housekeeping genes have been tested for the expression of their transcripts in exposed cell cultures using qRT-PCR assays and raw data were analyzed with the two Excel applets geNorm and NormFinder. The genes encoding for SDHA, GAPDH and YWHAZ appear to be the most reliable controls, respectively, for the OC, PBDE and 17beta-estradiol treatments. These results clearly show that the transcription of even widely diffused control genes can be regulated by different treatments and underlie the importance of a careful selection of the optimal housekeeping genes in toxicological studies.

Social kin associations and genetic structuring of striped dolphin populations (Stenella coeruleoalba) in the Mediterranean Sea.

We investigated hierarchical patterns of genetic subdivision, and assessed kinship within and between social groups of striped dolphins (Stenella coeruleoalba) in the Tyrrhenian Sea. A total of 165 samples were analysed at eight microsatellite DNA loci, including outgroup samples from the Adriatic, Scotland and Spain for population-level comparisons. We found population genetic structure within the Mediterranean basin, including small but significant differentiation between the Adriatic and Tyrrhenian Seas (FST=0.0047, P=0.008), and between putative 'inshore' and 'offshore' (FST=0.0217, P=0.005) populations in the Tyrrhenian Sea. Assessment of kinship within and among 12 association groups showed higher average kinship for females within than between groups, and smaller groups showed higher average kinship. Comparisons of relatedness for both sexes showed a significant difference between males and females, with females more likely to associate with adult kin. Together these data emphasize the importance of the social cohesion of kin in small groups to the structuring of striped dolphin populations in this environment.

Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies.

BACKGROUND: Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. RESULTS: Ten commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and beta-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization. CONCLUSION: In this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18.