Genetic differentiation of Mediterranean horse mackerel (Trachurus mediterraneus) populations as revealed by mtDNA PCR-RFLP analysis

The genetic population structure of Mediterranean horse mackerel, Trachurus mediterraneus , from seven locations throughout the Black, Marmara, Aegean and eastern Mediterranean seas was investigated using restriction fragment length polymorphism (RFLP) analysis of the mtDNA 16S rDNA region. An approximately 2000-bp segment was screened in 280 individuals using six restriction enzymes, resulting in 10 composite haplotypes. The most common haplotype was present in 56.42% individuals; the next most frequent haplotype was present in 22.85% individuals. Average haplotype diversity within samples was moderate (0.38), and nucleotide diversity was low (0.00435). Mean nucleotide divergence for the seven sampling sites was 0.0028. Nucleotide divergence among samples was moderate, with the highest value detected between the Aegean Sea (Izmir) and the eastern Black Sea (Trabzon) populations (0.007055), and the lowest (−0.000043) between the Marmara Sea (Adalar) and the western Black Sea (Sile) populations. In Monte Carlo pairwise comparisons of haplotype frequencies, the Sinop from the middle Black Sea, Trabzon from the eastern Black Sea, and Iskenderun Bay from the north-eastern Mediterranean Sea exhibited highly significant (P   <   0.001) geographical differentiation from each other and from all other populations. Mantel's test indicated that the nucleotide divergence among populations of T. mediterraneus was not significantly associated with their geographical isolation ( r  = −0.2963; P   >   0.05). Consequently, the mtDNA 16S rDNA region provided evidence for the existence of three distinct T. mediterraneus populations (Sinop, Trabzon and Iskenderun Bay) in the Black and north-eastern Mediterranean seas.     

Microsatellite DNA reveals genetically different populations of Atlantic bonito Sarda sarda in the Mediterranean Basin

Microsatellites were used to investigate population genetic structure of Atlantic bonito Sarda sarda from the Black Sea, Marmara Sea, Aegean Sea, north-eastern Mediterranean Sea and Adriatic Sea. Overall average observed heterozygosity was high (0.93). Average observed heterozygosity per locus ranged from 0.79 to 0.98. Pairwise F_ST estimates for all loci between populations ranged from 0 to 0.07626, and significant F_ST values (P < 0.001) were detected between populations; the Blacks Sea and Marmara Sea samples were not significantly different from each other, but significant different from the other samples, and Aegean Sea and north-eastern Mediterranean Sea samples were also not significantly different from each other, but significantly different from all other samples. The Adriatic Sea sample was significant different from all other samples. The Mantel test revealed a significant (P < 0.001, r = 0.68) isolation-by-distance for these 11 populations. Neighbour-joining analysis clustered the Black Sea and Marmara Sea samples together while collections from Aegean Sea and north-eastern Mediterranean Sea were clustered close to each other and far from the others. On the other hand, the Adriatic Sea collection presented very distinctive relationship from the others.     

The taxonomic status of the Black Sea and Marmara Sea populations of the Broadnosed Pipefish Syngnathus cf. argentatus Pallas (Teleostei: Syngnathidae) based on morphological and molecular characters

As the Black Sea and Marmara Sea population of the Broadnosed Pipefish Syngnathus cf. argentatus show some morphological differences from the Mediterranean Sea populations, some authors regard it as an endemic species Syngnathus argentatus Pallas, 1814, while others consider it as a synonym of S. typhle Linnaeus, 1758. The aim of this study is to compare the populations of the Black Sea and Sea of Marmara with the Aegean Sea population, using a combination of morphological and molecular characters, in order to clarify their taxonomic status. Sampling was carried out at three stations in the Black Sea, two in the Sea of Marmara and three in the Aegean Sea, and a total of 24 morphometric and 6 meristic characters were examined. Metric data were analysed by Principal Component Analysis (PCA) and phylogenetic relationships between the populations were analysed using both cytochrome oxidase subunit 1 (COI) gene and cytochrome b (cyt-b) gene sequences. Although constant differences were observed in snout depths between the Black Sea/Marmara Sea and the Aegean Sea populations, other morphological features and genetic analysis did not enable these populations to be differentiated. These findings indicate that S. argentatus is a synonym of S. typhle.     

Genetic structuring of European anchovy (Engraulis encrasicolus) populations through mitochondrial DNA sequences

Mitochondrial DNA sequence variation in 655?bpfragments of the cytochrome oxidase c subunit I gene, known as the DNA barcode, of European anchovy (Engraulis encrasicolus) was evaluated by analyzing 1529 individuals representing 16 populations from the Black Sea, through the Marmara Sea and the Aegean Sea to the Mediterranean Sea. A total of 19 (2.9%) variable sites were found among individuals, and these defined 10 genetically diverged populations with an overall mean distance of 1.2%. The highest nucleotide divergence was found between samples of eastern Mediterranean and northern Aegean (2.2%). Evolutionary history analysis among 16 populations clustered the Mediterranean Sea clades in one main branch and the other clades in another branch. Diverging pattern of the European anchovy populations correlated with geographic dispersion supports the genetic structuring through the Black Sea-Marmara Sea-Aegean Sea-Mediterranean Sea quad.     

The use of otolith shape and chemistry to determine stock structure of Mediterranean horse mackerel Trachurus mediterraneus (Steindachner)

Otolith shape and chemistry of Mediterranean horse mackerel Trachurus mediterraneus were simultaneously used to assess the feasibility of using these natural tags to discriminate populations throughout the Black, Marmara, Aegean and eastern Mediterranean Seas. Otolith shape and chemistry analyses showed a similar pattern of differentiation between T. mediterraneus stocks, revealing a clear discreteness of the middle Black Sea (Sinop) and Aegean Sea (Izmir) samples. Otolith upper side length and width, and Na, K, Mg and Ba, were the morphological traits and trace elements, respectively, differing most among groups. Overall assignment of individuals into their original sample was high for both otolith shape and chemistry. Highest reclassification rate was observed for the south-middle Black Sea and Aegean Sea samples for both analyses. Hierarchical cluster analyses also supported high differentiation of the south-middle Black Sea and Aegean Sea samples for both analyses. Mantel's test revealed that the Euclidean distance both for otolith shape ( r =−0·0917, P > 0·05) and chemistry ( r =−0·1248, P > 0·05) between these populations were not significantly associated with their geographical distances.     

Posidonia oceanica in the Marmara Sea

The seagrass Posidonia oceanica is a stenohaline species endemic to the Mediterranean Sea, where it normally lives at a salinity of between 36.5 and 39.5 ppt. Surveys carried out at the North-eastern distribution limits revealed large beds in the Dardanelles Strait and isolated beds in the Marmara Sea, where the salinity ranges between 21.5 and 28 ppt. Microsatellite analysis of these low-salinity tolerant P. oceanica beds, show different signs of genetic isolation: excess of heterozygosity and a presence of fixed alleles. These particularities are rarely found in the whole distributional range of the species. Moreover, all the populations considered in the analysis have a very low genetic diversity in comparison with most of the meadows sampled throughout the Mediterranean Sea. Taking into consideration the genetic data, rhizome expansion rate and the actual extent of the isolated beds in the Marmara Sea and knowing the reproductive rate and dissemination characteristics of P. oceanica, we hypothesize that the isolated population of the Marmara Sea has been established since the Middle Holocene, before the catastrophic intrusion of brackish water into the Marmara Sea and the strong and persistent flow coming from the Black Sea.     

Extant silicoflagellates from the Northeast Aegean (eastern Mediterranean Sea): Morphologies and double skeletons

Silicoflagellate abundance, vertical distribution and morphology were studied during spring (March 2014) at three sampling stations located in the Northeast Aegean Sea adjacent to the Dardanelles Strait and characterized by a variable influx of cold, low-salinity Black Sea water. The silicoflagellate assemblage was dominated by Dictyocha stapedia and Stephanocha speculum with minor contribution of D. aculeata and Octactis pulchra. While specimens of D. stapedia were represented by the typical morphologies described in other areas of the Mediterranean Sea, populations of S. speculum displayed peculiar characters: they were large, predominantly 7-sided, with a small apical ring as well as apical ring spines, concave basal ring sides and non-rotated apical structure. Some of these features have been described for S. speculum at high latitudes, but the combined characters make these specimens slightly different from the high latitude populations. Similar morphologies have been observed in the western Black Sea, thus we can infer that the peculiar specimens detected in the Northeast Aegean are associated with the influx of Black Sea water masses.     

Contemporary genetic structure and postglacial demographic history of the black scorpionfish,Scorpaena porcus,in the Mediterranean and the Black Seas

Understanding the distribution of genetic diversity in the light of past demographic events linked with climatic shifts will help to forecast evolutionary trajectories of ecosystems within the current context of climate change. In this study, mitochondrial sequences and microsatellite loci were analysed using traditional population genetic approaches together with Bayesian dating and the more recent approximate Bayesian computation scenario testing. The genetic structure and demographic history of a commercial fish, the black scorpionfish, Scorpaena porcus, was investigated throughout the Mediterranean and Black Seas. The results suggest that the species recently underwent population expansions, in both seas, likely concomitant with the warming period following the Last Glacial Maximum, 20 000 years ago. A weak contemporaneous genetic differentiation was identified between the Black Sea and the Mediterranean Sea. However, the genetic diversity was similar for populations of the two seas, suggesting a high number of colonizers entered the Black Sea during the interglacial period and/or the presence of a refugial population in the Black Sea during the glacial period. Finally, within seas, an east/west genetic differentiation in the Adriatic seems to prevail, whereas the Black Sea does not show any structured spatial genetic pattern of its population. Overall, these results suggest that the Black Sea is not that isolated from the Mediterranean, and both seas revealed similar evolutionary patterns related to climate change and changes in sea level.     

Genetically monomorphic invasive populations of the rapa whelk,Rapana venosa

Rapana venosa is a predatory marine gastropod native to the coastal waters of China, Korea, and Japan. Since the 1940s, R. venosa has been transported around the globe and introduced populations now exist in the Black Sea, the Mediterranean Sea, the Adriatic and Aegean seas, off the coasts of France and the Netherlands, in Chesapeake Bay, Virginia, USA, and in the Rio de la Plata between Uruguay and Argentina. This study surveyed variation in two mitochondrial gene regions to investigate the invasion pathways of R. venosa, identify likely sources for introduced populations, and evaluate current hypotheses of potential transportation vectors. Sequence data were obtained for the mitochondrial cytochrome c oxidase I and NADH dehydrogenase subunit 2 gene regions of 178 individuals from eight native locations and 106 individuals from 12 introduced locations. Collections from within the native range displayed very high levels of genetic variation while collections from all introduced populations showed a complete lack of genetic diversity; a single haplotype was common to all introduced individuals. This finding is consistent with the hypothesis that R. venosa was initially introduced into the Black Sea, and this Black Sea population then served as a source for the other secondary invasions by various introduction vectors including ballast water transport. Although non‐native R. venosa populations currently appear to be thriving in their new environments, the lack of genetic variability raises questions regarding the evolutionary persistence of these populations.     

Invasion Pathway of the Ctenophore Mnemiopsis leidyi in the Mediterranean Sea

Gelatinous zooplankton outbreaks have increased globally owing to a number of human-mediated factors, including food web alterations and species introductions. The invasive ctenophore Mnemiopsis leidyi entered the Black Sea in the early 1980s. The invasion was followed by the Azov, Caspian, Baltic and North Seas, and, most recently, the Mediterranean Sea. Previous studies identified two distinct invasion pathways of M. leidyi from its native range in the western Atlantic Ocean to Eurasia. However, the source of newly established populations in the Mediterranean Sea remains unclear. Here we build upon our previous study and investigate sequence variation in both mitochondrial (Cytochrome c Oxidase subunit I) and nuclear (Internal Transcribed Spacer) markers in M. leidyi, encompassing five native and 11 introduced populations, including four from the Mediterranean Sea. Extant genetic diversity in Mediterranean populations (n = 8, N _a = 10) preclude the occurrence of a severe genetic bottleneck or founder effects in the initial colonizing population. Our mitochondrial and nuclear marker surveys revealed two possible pathways of introduction into Mediterranean Sea. In total, 17 haplotypes and 18 alleles were recovered from all surveyed populations. Haplotype and allelic diversity of Mediterranean populations were comparable to populations from which they were likely drawn. The distribution of genetic diversity and pattern of genetic differentiation suggest initial colonization of the Mediterranean from the Black-Azov Seas (pairwise F _ST = 0.001–0.028). However, some haplotypes and alleles from the Mediterranean Sea were not detected from the well-sampled Black Sea, although they were found in Gulf of Mexico populations that were also genetically similar to those in the Mediterranean Sea (pairwise F _ST = 0.010–0.032), raising the possibility of multiple invasion sources. Multiple introductions from a combination of Black Sea and native region sources could be facilitated by intense local and transcontinental shipping activity, respectively.     

Mitochondrial DNA phylogeny and the reconstruction of the population history of a species: the case of the European anchovy (Engraulis encrasicolus)

Analysis of mitochondrial DNA restriction fragment length polymorphism in European anchovy (Engraulis encrasicolus) revealed a large number of mitotypes that form two distinct clusters (phylads). Phylad A consists of one common mitotype and many rare secondary mitotypes that are one mutational step removed from the main type. Nucleotide diversity and number of homoplasious changes are low. Phylad B has a complex pattern of mitotype connectedness, high nucleotide diversity, and a large number of homoplasious changes. It is suggested that the two phylads evolved in isolation from each other and that present coexistence is the result of a secondary contact. Moreover, phylad A has a "star" phylogeny, which suggests that it has evolved in a population that experienced a drastic bottleneck followed by an explosion of size. Phylad A is practically the only phylad present in the Black Sea, with its frequency dropping to 85% in the northern Aegean, and to 40% in the rest of Mediterranean and the Bay of Biscay. The Black Sea is, therefore, the most likely place of origin of phylad A. Molecular data are consistent with a population bottleneck in the Black Sea during the last glaciation event and a subsequent exit of phylad A with the outflow into the Aegean following the ice melting. Phylogenetic analysis of anchovy mtDNA provides a reconstruction of population history in the Mediterranean, which is consistent with the geological information.      

Phylogeography of the Atlanto-Mediterranean sea cucumber Holothuria (Holothuria) mammata: the combined effects of historical processes and current oceanographical pattern

We assessed the genetic structure of populations of the widely distributed sea cucumber Holothuria (Holothuria) mammata Grube, 1840, and investigated the effects of marine barriers to gene flow and historical processes. Several potential genetic breaks were considered, which would separate the Atlantic and Mediterranean basins, the isolated Macaronesian Islands from the other locations analysed, and the Western Mediterranean and Aegean Sea (Eastern Mediterranean). We analysed mitochondrial 16S and COI gene sequences from 177 individuals from four Atlantic locations and four Mediterranean locations. Haplotype diversity was high (H=0.9307 for 16S and 0.9203 for COI), and the haplotypes were closely related (π=0.0058 for 16S and 0.0071 for COI). The lowest genetic diversities were found in the Aegean Sea population. Our results showed that the COI gene was more variable and more useful for the detection of population structure than the 16S gene. The distribution of mtDNA haplotypes, the pairwise F(ST) values and the results of exact tests and amova revealed: (i) a significant genetic break between the population in the Aegean Sea and those in the other locations, as supported by both mitochondrial genes, and (ii) weak differentiation of the Canary and Azores Islands from the other populations; however, the populations from the Macaronesian Islands, Algarve and West Mediterranean could be considered to be a panmictic metapopulation. Isolation by distance was not identified in H. (H.) mammata. Historical events behind the observed findings, together with the current oceanographic patterns, were proposed and discussed as the main factors that determine the population structure and genetic signature of H. (H.) mammata.     

Phylogeography of the common shrimp, Crangon crangon (L.) across its distribution range

The common or brown shrimp Crangon crangon (L.) is a highly abundant and important taxon, both ecologically and commercially, yet knowledge on its population structure and historical biogeography is limited. We studied population genetic structure across the distribution range of this species by sequencing a 388 bp fragment of the cytochrome-c-oxidase I gene for 140 individuals from 25 locations. Strong population structuring and high levels of genetic diversity were observed. Four main phylogroups were uncovered: northeastern Atlantic, western Mediterranean, Adriatic Sea and Black Sea. Gene flow of these shrimp across known oceanographical barriers (e.g., the Strait of Gibraltar and/or Oran-Almeria front, Sicilian Straits, and Turkish Straits) is severely restricted. The oldest and most variable populations currently inhabit the western Mediterranean. The observed absence of structure across the entire northeastern Atlantic shelf is proposed not to be due to gene flow, but to relatively recent colonization following the glacial cycles of the late Pleistocene. Black Sea shrimp are currently disconnected from Mediterranean populations, and colonization is inferred, on the basis of coalescent analysis, to have happened relatively recently, but possibly earlier than 7000 years ago. We postulate the hypothesis that C. crangon survived the last brackish-water (<7 per thousand) period inside the Black Sea and/or one of the adjacent inland seas. We conclude that (1) common shrimp populations from different basins are strongly differentiated, (2) gene flow across basins is probably very limited, and (3) the biogeographic history of the taxon is largely in accordance with the geographic history of its distribution range. This study provides further evidence that high population connectivity of marine species (e.g., by policy makers) should not be assumed.     

Genetic characterization of brown meagre (Sciaena umbra) and Shi Drum (Umbrina cirrosa) populations

This study analyzed population structure of fish, brown meagre (Sciaena umbra) and Shi drum (Umbrina cirrosa) from the Black Sea, the Aegean Sea, and the Mediterranean Sea. The Cytb and 16S rRNA genes of Shi drum and brown meagre fish were sequenced. In brown meagre and Shi drum, 25 and 20 haplotypes, respectively, of Cytb gene were identified; while for 16S rRNA, 4 and 8 haplotypes were identified. Nucleotide diversities of 16S rRNA and Cytb gene sites were found to be 83.8% and 52.6%, respectively, for brown meagre; while that for Shi drum were of 80.5% and 73.6%, respectively. There was a significant relationship between geographic distance and the genetic distance of the fish. Since Shi drum and brown meagre are migratory species, they can migrate between the seas. The lack of barriers among different populations facilitates the gene flow among the populations belonging to different regions. Since there is no information available on Shi drum and brown meagre population genetics, this study may be useful to understand the genetic diversity of these species to assist fishery managers for the management of these resources in terms of conservation and sustainability.     

Biogeographic zonation of the Black Sea and Caspian Sea basin based on Mysid fauna (Crustacea: Mysidacea)

A biogeographic analysis of the Black Sea and Caspian Sea basin and adjacent Aegean Sea and the Sea of Marmara was conducted based on the distribution of 55 mysid species. The Black and Caspian Sea basin proper is inhabited by 35 mysid species belonging to 12 genera, among them 26 species and 3 genera are endemic to the region. Ponto-Caspian and Caspian species are predominant. The explored areas were included into two provinces of the Mediterranean subrealm of the East-Atlantic Subtropical realm and three provinces of the Ponto-Caspian realm.     

Mitochondrial DNA reveals a mosaic pattern of phylogeographical structure in Atlantic and Mediterranean populations of anchovy (Engraulis encrasicolus)

This study extends the geographic coverage of a previous study of mitochondrial DNA restriction fragment length polymorphism in European anchovy. Both studies together include 24 samples representing 17 localities extending from the Black Sea, through the Mediterranean Sea to the eastern Atlantic as far south as Dakar, Senegal. Eighty-eight haplotypes define two clades (A and B) separated by 3.2% sequence divergence. Clade A has a star-like genealogy indicative of a recent population expansion. Clade B has a more complex genealogy, consisting of several haplotypes at intermediate frequencies. The distributions of these clades consist of a mosaic with abrupt changes between some areas and gradients between other areas. Clade A predominates the Black and Aegean seas, but is present throughout the Mediterranean. Unexpectedly, new data show that clade A is also at a high frequency in the Atlantic, from Portugal to at least Senegal. Overall, the level of genetic differentiation among populations is high (F(ST)=0.148, p<0.0001), with the greatest differences between basins. AMOVA reveals four main geographical groups: Atlantic, central Mediterranean, Aegean Sea, and Black Sea. Mismatch distribution clearly indicates historical bottleneck and population expansion for clade A, while for clade B such evidence is equivocal. This difference may reflect a range expansion for both clades, but with higher gene flow (Nm values) between demes for clade A. Both contemporary and historical processes are important in shaping the complex genetic population structure of European anchovy.     

Historic speciation and recent colonization of Eurasian monkey gobies (Neogobius fluviatilis and N. pallasi) revealed by DNA sequences,microsatellites, and morphology

Aim Hidden diversity within an invasive ‘species’ can mask both invasion pathways and confound management goals. We assessed taxonomic status and population structure of the monkey goby Neogobius fluviatilis across Eurasia, comparing genetic variation across its native and invasive ranges. Location Native populations were analysed within the Black and Caspian Sea basins, including major river drainages (Dnieper, Dniester, Danube, Don and Volga rivers), along with introduced locations within the upper Danube and Vistula river systems. Methods DNA sequences and 10 nuclear microsatellite loci were analysed to test genetic diversity and divergence patterns of native and introduced populations; phylogenetic analysis of mtDNA cytochrome b and nuclear RAG‐1 sequences assessed taxonomic status of Black and Caspian Sea lineages. Multivariate analysis of morphology was used to corroborate phylogenetic patterns. Population genetic structure within each basin was evaluated with mtDNA and microsatellite data using F_ST analogues and Bayesian assignment tests. Results Phylogenetic analysis of mitochondrial and nuclear sequences discerned a pronounced genetic break between monkey gobies in the Black and Caspian Seas, indicating a long‐term species‐level separation dating to c. 3 million years. This pronounced separation further was confirmed from morphological and population genetic divergence. Bayesian inference showed congruent patterns of population structure within the Black Sea basin. Introduced populations in the Danube and Vistula River basins traced to north‐west Black Sea origins, a genetic expansion pattern matching that of other introduced Ponto‐Caspian gobiids. Main conclusions Both genetic and morphological data strongly supported two species of monkey gobies that were formerly identified as subspecies: N. fluviatilis in the Black Sea basin, Don and Volga Rivers, and the Kumo‐Manych Depression, and Neogobius pallasi in the Caspian Sea and Volga River delta. Genetic origins of introduced N. fluviatilis populations indicated a common invasion pathway shared with other introduced Ponto‐Caspian fishes and invertebrates.