Sardines at a junction: Seascape genomics reveals ecological and oceanographic drivers of variation in the NW Mediterranean Sea.

By evaluating genetic variation across the entire genome, one can address existing questions in a novel way while raising new ones. The latter includes how different local environments influence adaptive and neutral genomic variation within and among populations, providing insights into local adaptation of natural populations and their responses to global change. Here, under a seascape genomic approach, ddRAD data of 4609 single nucleotide polymorphisms (SNPs) from 398 sardines (Sardina pilchardus) collected in 11 Mediterranean and one Atlantic site were generated. These were used along with oceanographic and ecological information to detect signals of adaptive divergence with gene flow across environmental gradients. The studied sardines constitute two clusters (FST  = 0.07), a pattern attributed to outlier loci, highlighting putative local adaptation. The trend in the number of days with sea surface temperature above 19°C, a critical threshold for successful sardine spawning, was crucial at all levels of population structuring with implications on the species' key biological processes. Outliers link candidate SNPs to the region's environmental heterogeneity. Our findings provide evidence for a dynamic equilibrium in which population structure is maintained by physical and ecological factors under the opposing influences of migration and selection. This dynamic in a natural system warrants continuous monitoring under a seascape genomic approach that might benefit from a temporal and more detailed spatial dimension. Our results may contribute to complementary studies aimed at providing deeper insights into the mechanistic processes underlying population structuring. Those are key to understanding and predicting future changes and responses of this highly exploited species in the face of climate change.

Benthic Crustacean Digestion Can Modulate the Environmental Fate of Microplastics in the Deep Sea.

Microplastics (MPs) are ubiquitous contaminants of the marine environment, and the deep seafloor is their ultimate sink compartment. Manipulative and field experiments provided evidence of the ingestion of MPs by deep-sea fauna, but knowledge of MPs' fate once ingested still remains scant. We provide evidence of MP partial retention and fragmentation mediated by digestion activity of a Norwegian langoustine, a good bioindicator for MP contamination of the deep sea. We report here that MPs in the intestines were more abundant and significantly smaller (up to 1 order of magnitude in surface) than those in the stomachs. Our results show that the stomach can act as a size-bottleneck for ingested MPs, enhancing the retention of larger particles within the stomach and promoting fragmentation into smaller plastic debris, which is then released in the intestine. Our results provide evidence that the langoustine is responsible for the fragmentation of MPs already accumulated in sediments through its scavenging activity and digestion. These findings highlight the existence of a new peculiar kind of "secondary" MPs, introduced in the environment by biological activities, which could represent a significant pathway of plastic degradation in a secluded and stable environment such as the deep sea.

Mediterranean Bioconstructions Along the Italian Coast.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

I Like This New Me: Unravelling Population Structure of Mediterranean Electric Rays and Taxonomic Uncertainties within Torpediniformes.

The present study focused on the three species of electric rays known to occur in the Mediterranean Sea: Torpedo torpedo, Torpedo marmorata and Tetronarce nobiliana. Correct identification of specimens is needed to properly assess the impact of fisheries on populations and species. Unfortunately, torpedoes share high morphological similarities, boosting episodes of field misidentification. In this context, genetic data was used (1) to identify specimens caught during fishing operations, (2) to measure the diversity among and within these species, and (3) to shed light on the possible occurrence of additional hidden species in the investigated area. New and already published sequences of COI and NADH2 mitochondrial genes were analyzed, both at a small scale along the Sardinian coasts (Western Mediterranean) and at a large scale in the whole Mediterranean Sea. High levels of genetic diversity were found in Sardinian populations, being significantly different from other areas of the Eastern Mediterranean Sea due to the biotic and abiotic factors here discussed. Sardinian torpedoes can hence be indicated as priority populations/areas to be protected within the Mediterranean Sea. Moreover, sequence data confirmed that only the three species occur in the investigated area. The application of several 'species-delimitation' methods found evidence of cryptic species in the three species outside the Mediterranean Sea, as well as in other genera/families, suggesting the urgent need for future studies and a comprehensive revision of the order Torpediniformes for its effective conservation.

To Be, or Not to Be: That Is the Hamletic Question of Cryptic Evolution in the Eastern Atlantic and Mediterranean Raja miraletus Species Complex.

Despite a high species diversity, skates (Rajiformes) exhibit remarkably conservative morphology and ecology. Limited trait variations occur within and between species, and cryptic species have been reported among sister and non-sister taxa, suggesting that species complexes may be subject to stabilising selection. Three sibling species are currently recognised in the Raja miraletus complex: (i) R. miraletus occurring along the Portuguese and Mediterranean coasts, (ii) R. parva in the Central-Eastern Atlantic off West Africa and (iii) R. ocellifera in the Western Indian Ocean off South Africa. In the present study, the genetic variation at mitochondrial and nuclear markers was estimated in the species complex by analysing 323 individuals sampled across most of its geographical distribution area to test the hypothesis that restricted gene flow and genetic divergence within species reflect known climate and bio-oceanographic discontinuities. Our results support previous morphological studies and confirm the known taxonomic boundaries of the three recognised species. In addition, we identified multiple weakly differentiated clades in the Northeastern Atlantic Ocean and Mediterranean, at least two additional cryptic taxa off Senegal and Angola, a pronounced differentiation of ancient South African clades. The hidden genetic structure presented here may represent a valuable support to species' conservation action plans.

Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences.

The evolutionary pathway of Palinuridae (Crustacea, Decapoda) is still controversial, uncertain and unexplored, expecially from a karyological point of view. Here we describe the South African spiny lobster Jasus lalandii karyotype: n and 2n values, heterochromatin distribution, nucleolar organizer region (NOR) location and telomeric repeat structure and location. To compare the genomic and chromosomal organization in Palinuridae we located NORs in Panulirus regius, Palinurus gilchristi and Palinurus mauritanicus: all species showed multiple NORs. In J. lalandii NORs were located on three chromosome pairs, with interindividual polymorphism. In P. regius and in the two Palinurus species NORs were located on two chromosome pairs. In the two last species 45S ribosomal gene loci were also found on B chromosomes. In addition, the nature and location of telomeric repeats were investigated by FISH in J. lalandii, P. gilchristi, P. mauritanicus Palinurus elephas, and P. regius (Palinuridae, Achelata), and in Scyllarus arctus (Scyllaridae, Achelata): all these Achelata species showed the (TTAGG)n pentameric repeats. Furthermore, in J. lalandii these repeats occurred in all the telomeres and in some interstitial chromosomal sites, associated with NORs.

Environmental Status and Geomorphological Characterisation of Seven Black Coral Forests on the Sardinian Continental Shelf (NW Mediterranean Sea).

Marine animal forests are key mesophotic ecosystems that are under threat from increasing natural and human pressures. Despite the fact that various international agreements strive to preserve these fragile ecosystems, the environmental status of the majority of these animal-structured environments is unknown. Assessing their environmental status is the first step needed to monitor these essential habitats' health over time and include them within conservation and protection frameworks, such as the Marine Strategy Framework Directive. Based on Multibeam data and ROV footage, we characterized the geomorphological setting and evaluated the environmental status of seven black coral forests in the centre of the Western Mediterranean Sea, using the Mesophotic Assemblages Conservation Status (MACS) Index. The presence of two antipatharians, Antipathella subpinnata and Leiopathes glaberrima, characterized the seven investigated sites, dwelling on rocky substrate characterized by different environmental drivers (i.e., depth, slope of the substrate, terrain ruggedness, topographic positioning index, and aspect). From the combined evaluation of the associated benthic community status and the anthropogenic impacts affecting it, a "high" and "good" environmental status was assessed for five out of the seven studied black forests, with only two forests classified as having a "moderate" and "poor" status, respectively. Overall, our study showed a site-specific variability of mesophotic black coral forest status, explained by different biological community structures and environmental conditions mainly associated with morphological and anthropogenic factors.

Deep-Dwelling Populations of Mediterranean Corallium rubrum and Eunicella cavolini: Distribution, Demography, and Co-Occurrence.

Corallium rubrum and Eunicella cavolini are two octocorals, reported as co-occurring species in the deep rocky habitats of the Mediterranean Sea with a high hydrodynamic and moderate eutrophication. Their spatial distribution and demography in the deep sea are mainly affected by temperature and direct and indirect anthropogenic activities; however, knowledge of the factors that potentially influence their co-existence is scarce. This paper provides novel data on the distribution and demography of these two species, at depths between 50 and 290 m in the Western Mediterranean Sea, providing insights on their co-occurrence. Both species exhibited the highest population density at deeper sites (>150 m), showing an inverse size-density relation. Density values ranged from 0.03 colonies m-2 to 32 and 80 col. m-2 for yellow gorgonian and red coral, respectively. The two species co-occurred in 13% of the total frames examined, mostly dwelling between 120 and 160 m depth. Distance-based linear modeling (DistLM) emphasized that when co-occurring the variability of the two species' densities were significantly driven by the density-rather than the morphology (i.e., height)-of the other species. We stress the need for further studies to elucidate the possible mutual effects of suspension feeders and to test the role of different environmental factors potentially influencing inter-specific relationships.

Phylogeography of the veined squid, Loligo forbesii, in European waters.

The veined squid, Loligo forbesii Steenstrup, 1856, occurs at the European Shelf areas including the Azores and represents a valuable resource for the European commercial fishery in the North East Atlantic. However, very little is known about its population structure and phylogeography. This lack of knowledge also impedes the development of sustainable fishery management for this species. The present study combined the use of two types of markers that retrieve patterns of gene flow in different time spans; the analysis of 16 nuclear microsatellites and sequencing of the mitochondrial cytochrome oxidase subunit I (COI). Whereas the high mutation rate of microsatellites allows the description of recent patterns of connectivity in species, the lower mutation rate of COI provides phylogeographic patterns on a longer timescale. A total of 347 individuals of L. forbesii were investigated from nearly the entire distribution range of the species, including the North East Atlantic Shelf, the Azores and the Mediterranean. Individuals from the Western and Eastern Mediterranean Sea have never been included in a genetic study before. We were able to analyse COI sequences from all 12 sampling areas and define three clades of L. forbesii. Due to our large sampling area, we are presenting 13 COI-haplotypes that were previously unknown. The microsatellite analysis does not include the Azores but three main clades could be identified at the remaining 11 sampling sites. Low FST values indicate gene flow over large geographical distances. However, the genetically significant differences and an additional slight grouping in the microsatellite structure reveal that geographical barriers seem to influence the population structure and reduce gene flow. Furthermore, both markers provide strong evidence that the observed phylogeographic pattern reflects the geographical history of the Azores and the Mediterranean Sea.

Scattered accumulation hotspots of macro-litter on the seafloor: Insights for mitigation actions.

Marine litter is an ever-increasing problem that demands immediate reduction plans and mitigation actions that should act synergically to efficiently meet ambitious goals. Since the seafloor has been recognized as the major sink for marine debris, the study of litter accumulation dynamics represent a fundamental tool to evaluate possible removal actions. We analysed a 7 years (2013-2019) standardized data series collected along Sardinian fishing grounds through MEDiterranean International Trawl Survey, for which estimates of density and weight of seafloor macro-litter were calculated for over 707 hauls. Results show the absence of any temporal trend in seafloor macro-litter density and weight, but rather indicate a spatial and bathymetric segregation of different litter categories. Our data showed how different sources and physical features of macro-litter items (i.e., plastic and fishing gear, rubber, glass, metals, and cloth) led to spatially segregated accumulation hotspots. We also point out here how the identification of seafloor macro-litter hotspots using aggregated data that include plastic items could obscure the identification of other segregated but yet relevant hotspots of other macro-litter categories accumulated in the marine environment. These hotspots often occurred at shallower depths and closer to coastlines, thus representing potential spots where eventual future litter removal action could be prioritized.

Microsatellite variability of Sardinian pine martens, Martes martes.

The pine marten, Martes martes, is a medium-sized terrestrial carnivore associated with woodland habitats of the western Palearctic region. The present distribution area of the species also includes six islands of the western Mediterranean basin. The origin of these insular populations and their taxonomic status are still debated; their molecular characterization appears relevant for conservation purposes. To describe the genetic variability of the pine martens from Sardinia we characterized 40 insular and 14 Italian individuals at seven nuclear microsatellite loci. The identification of private alleles and the calculated F(ST) value of 0.074 revealed some genetic differentiation between the two populations, which accounts for the high percentages of correct allocation (96.39-98.80%) scored by the genotype assignment test. The presence of two distinct clusters corresponding to Sardinia and mainland Italy was further confirmed by the multivariate Factorial Correspondence Analysis of individual genotypes. Moreover, the genome of the Sardinian individuals bore signs of past demographic fluctuations, i.e. the presence of the monomorphic locus Ma-4, a lower allelic richness and a lower number of private alleles, which may derive from the combination of drift, founder effects, and human overexploitation. Anyway, if such events ever affected the Sardinian population, this is likely to have happened in the past since, according to our microsatellite data, the present-day population does not show evidence of recent bottlenecks or inbreeding, the Wilcoxon sign-rank test and the F(IS) index being not statistically significant (both P > 0.05). Based on this genetic evidence, we advance hypotheses about the distinctiveness of the Sardinian population and its significance for taxonomy and conservation.

Colonization of plastic debris by the long-lived precious red coral Corallium rubrum: New insights on the "plastic benefits" paradox.

Seafloor macrolitter is ubiquitous in world's oceans; still, huge knowledge gaps exist on its interactions with benthic biota. We report here the colonization of plastic substrates by the Mediterranean red coral Corallium rubrum (L. 1758), occurring both in controlled conditions and in the wild at ca. 85 m depth in the Western Mediterranean Sea. Juveniles settled on seafloor macro-litter, with either arborescent or encrusting morphology, ranging from 0.6 to 3.5 mm in basal diameter and 0.2-7.1 years of age, also including a fraction (20%) of potentially sexually mature individuals. In controlled conditions, larvae settled and survived on plastic substrates for >60 days. Our insights show that marine plastic debris can provide favourable substrate for C. rubrum settlement either in controlled conditions or in the wild, suggesting their possible use in restoration activities. However, we pinpoint here that this potential benefit could result in adverse effects on population dynamics.

fshr: a fish sex-determining locus shows variable incomplete penetrance across flathead grey mullet populations.

Whole-genome sequencing data were produced from a single flathead grey mullet female and assembled into a draft genome sequence, whereas publicly available sequence data were used to obtain a male draft sequence. Two pools, each consisting of 60 unrelated individuals, respectively, of male and female fish were analyzed using Pool-Sequencing. Mapping and analysis of Pool-Seq data against the draft genome(s) revealed >30 loci potentially associated with sex, the most promising locus of which, encoding the follicle-stimulating hormone receptor (fshr) and harboring two missense variants, was genotyped on 245 fish from four Mediterranean populations. Genotype data showed that fshr represents a previously unknown sex-determining locus, although the incomplete association pattern between fshr genotype and sex-phenotype, the variability of such pattern across different populations, and the presence of other candidate loci reveal that a greater complexity underlies sex determination in the flathead grey mullet.

First Integrative Morphological and Genetic Characterization of Tremoctopus violaceussensu stricto in the Mediterranean Sea.

An integrative approach based on morphological and genetic analyses was undertaken for the first time to confirm the species identification of Mediterranean samples belonging to the genus Tremoctopus. Sequences of two mtDNA genes (cytochrome c oxidase subunit (COI) and 16S) were generated for the first time from Mediterranean samples. Both the similarity-based identifications and tree-based methods indicated that three females can be identified as Tremoctopus violaceus sensu stricto in agreement with their morphological classifications. All Mediterranean sequences clustered with the sequences of Tremoctopus violaceus from the Gulf of Mexico and were clearly differentiated from the sequences attributed to T. gracilis and T. robsoni. The chromatic pattern of the web and some features of gill filaments, arms formula, stylets, radulae, beaks, and stomach contents were given for all the samples; 105,758, 20,140, and 11,237 oocytes were estimated in the mature, immature, and developing samples, respectively. The presence of four spermatangia inside the cavity of the maturing female suggested the ability of this species to mate before reaching full maturity with more partners. Age investigation using beaks, performed for the first time in T. violaceus and within the genus gave results consistent with the different sizes and maturity conditions of the samples.

Scattered accumulation hotspots of macro-litter on the seafloor: Insights for mitigation actions.

Marine litter is an ever-increasing problem that demands immediate reduction plans and mitigation actions that should act synergically to efficiently meet ambitious goals. Since the seafloor has been recognized as the major sink for marine debris, the study of litter accumulation dynamics represents a fundamental tool to evaluate future removal actions. We analysed a 7 years (2013-2019) standardized data series collected along Sardinian fishing grounds through MEDiterranean International Trawl Survey, for which estimates of density and weight of seafloor macro-litter were calculated over 707 hauls. Results show the absence of any temporal trend in seafloor macro-litter density and weight, but rather indicate a spatial and bathymetric segregation of different litter categories. Our data showed how different sources and physical features of macro-litter items (i.e., plastic and fishing gears, rubber, glass, metal and textile) led to spatially segregated accumulation hotspots. These hotspots often occurred at shallower depths and closer to coastlines, representing spots where future litter removal action could be prioritized. We also point out here how the identification of seafloor macro-litter hotspots using aggregated data that include plastic items could indeed hide the identification of hotspots of other less abundant but yet detrimental macro-litter categories accumulated in the marine environment.

Population structure, demographic history, and selective processes: contrasting evidences from mitochondrial and nuclear markers in the European spiny lobster Palinurus elephas (Fabricius, 1787).

The European spiny lobster Palinurus elephas (Fabricius, 1787) is an ecologically and economically important species inhabiting a wide geographic range that extends from the North-east Atlantic and Azores to the Eastern Mediterranean. We investigated the population structure and evolutionary history of this species by both mitochondrial and microsatellite markers. Ten population samples covering a large part of the species distribution range (three samples from the Atlantic Ocean and seven from the Mediterranean Sea) were analyzed for a portion of the mitochondrial control region and seven polymorphic microsatellite loci. Both markers rejected the hypothesis of panmixia identifying two differentiated gene pools. The control region clearly distinguished the Atlantic and Mediterranean populations in two genetically separated groups. Microsatellites, also revealed two groups roughly associated to the Atlantic-Mediterranean separation, however, the Azores sample did not conform to this geographic scheme. Discrepancy between mitochondrial and nuclear markers emerged also when reconstructing the history of the species. Neutrality tests of the mitochondrial sequences indicated a departure from mutation-drift equilibrium that, combined to the mismatch analysis, pointed toward a sudden population expansion in both Atlantic and Mediterranean gene pools. Unexpectedly, microsatellites did not identify any signal of population expansion neither in the Atlantic pool nor in the Mediterranean one.

Erratum to "A new record of the invasive seaweed Caulerpa cylindracea Sonder in the South Adriatic Sea" [Heliyon 5 (9), (September 2019), e02449].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e02449.].

A new record of the invasive seaweed Caulerpa cylindracea Sonder in the South Adriatic Sea.

The green alga Caulerpa cylindracea Sonder is one of the most infamous and threatening invasive species in the Mediterranean Sea. Since 1985, it started rapidly spreading to all Mediterranean regions causing many ecological changes on natural communities. In the present study, we present an example of this proliferation with the first record in the Marine Protected Area of Tremiti Island (MPATI) in the South Adriatic Sea. Fifteen sites along the coast and 5 different depths have been investigated. Our results provide eveidence of a wide invasion of this pest in three islands, San Domino, San Nicola and Capraia. This study fills a particular data gap in the ongoing biomonitoring of invasive seaweeds in the Mediterranean Sea representing a base line of this invasive species for the MPATI.

European spiny lobster recovery from overfishing enhanced through active restocking in Fully Protected Areas.

Fully protected areas (FPAs) help preserving biodiversity and reversing the global decline of fishery resources. Stocks of the European spiny lobster Palinurus elephas (Fabr. 1787), among the most precious gourmet seafood worldwide, are currently facing a dramatic decline. Previous attempts of recovery based on fishery restrictions or active post-larval restocking in marine reserves provided unsuccessful outcomes. Here we present results of a 5-year restocking program carried through a Collaborative Fishery Research (CFR) project, in three ad-hoc established FPAs replenished using below-legal size wild juveniles. Results showed that Catch per Unit Effort (CPUE) in terms of both density and biomass burst (by ca. 300-700%) just 2 years since FPAs establishment, regardless of location. We also report tangible spillover effects (ca. 30-50% increase in density and biomass CPUE outside the FPAs) by the end of the program. Data from a 15-years lasting monitoring of a pilot FPA established in 1998, where the restocking protocol was conducted and protection kept in force once restocking ceased, demonstrated the persistence in time of restocking' benefits. We foster that creation of FPAs assisted with local restocking under oriented CFR programs can represent an option for the recovery of European spiny lobster stocks from overfishing.

On the presence of Dipturus nidarosiensis (Storm, 1881) in the Central Mediterranean area.

The Norwegian skate Dipturus nidarosiensis (Storm, 1881) has only recently been recorded in the western Mediterranean Sea along the coast of southern Sardinia, off Algeria and the Alboran Sea. The present study confirmed the presence of the species in the Central Mediterranean Sea by identifying morphometric, morphological features and molecular markers. Biological sampling was conducted from 2010 to 2016 on eight specimens collected through commercial landings, offshore observer programmes and scientific surveys in Adriatic and Ionian waters at depths between 320 and 720 m. The total lengths of the specimens (juveniles and adults) ranged from 268 to 1,422 mm, and their body weights ranged from 44.5 to 12,540.0 g. They showed morphometric features that corresponded to those of Norwegian skates in the Northeast Atlantic and the Western Mediterranean. In previous analyses, molecular data were obtained by mitochondrial COI sequences. The haplotype network showed the occurrence of a common haplotype (Hap_1) shared by the individuals from areas in the North Atlantic, Sardinian, Algerian and Spanish Mediterranean Sea areas but not South Africa. The occurrence of individuals in different stages of life (i.e., juveniles, sub-adults and adults) and sexual development (immature and mature) suggested the presence of a species with a permanent reproductive allocation in the deep waters of the Mediterranean, which was exposed to a low level of fishing exploitation. Indeed, the deep depth distribution of the species could be the reason for the absence of information about this species in onshore or offshore fishery data collection programmes and scientific surveys.