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The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

Environmental DNA (eDNA) is an emerging tool for assessing biodiversity and understanding spatial and temporal community patterns and processes, directly from DNA sequencing of environmental samples such as air, water, and sediments. We applied eDNA methods to monitor bony fish communities, detecting as well locally allochthonous species, and to reveal seasonal patterns at two sites in the Venice Lagoon. We analyzed 17 water samples collected over 12 months at two ecologically distinct sites by using available primers for teleosts and High Throughput Illumina sequencing. We identified 1,289 amplicon sequence variants (ASVs) assigned to 62 fish taxa. Most of the species known to inhabit or to enter the Venice Lagoon were detected, with eDNA data reflecting differences in fish communities between the internal (freshwater associated) and the external (sea associated) part of the lagoon. Moreover, seasonal trends of migration have been portrayed, highlighting the most involved species and disclosing possible clashes between migration events and the temporary interruption of sea-lagoon connectivity due to MOSE (MOdulo Sperimentale Elettromeccanico). Of interest, the first-time detection of Oceanic puffer ( Lagocephalus lagocephalus ) DNA in the Venice Lagoon provides evidence of the further northward expansion of this species in the high Adriatic Sea. eDNA successfully profiled fish communities by season and habitat in the Venice Lagoon. Our results support routine application of eDNA to monitor potential ecological consequences of MOSE closures in this World Heritage site.

This study investigates the benthic invertebrate community in the Venice Lagoon using environmental DNA (eDNA) metabarcoding based on superficial water samples. Our objective is to understand if, in a shallow lagoon system, sampling at the surface can provide information on benthic biodiversity, allowing us to establish a baseline for future assessments and to monitor the community's seasonal and spatial variation. eDNA was collected from surface water samples at two sites during nine sampling campaigns from November 2018 to October 2019, and metabarcoding was performed using an available primer pair targeting 16S mitochondrial rDNA of echinoderms, never tested empirically before. Analyses revealed 80 taxonomic units, five not assigned at the species level, belonging not only to the phylum Echinodermata but predominantly assigned to Mollusca, with the majority of the identified species (60 out of 75) representing benthic invertebrates. Several species known to be invasive were detected, some previously recorded in the Venice Lagoon environment and others detected for the first time. Significant spatial differences in species composition were observed between the northern and the southern site of the lagoon. Temporal variation of the benthic community was also observed, mainly due to the distinctiveness of autumn samples, highlighting the dynamic nature of the Venice Lagoon ecosystem. Our results confirm the utility of eDNA for ongoing biodiversity monitoring and management and show that eDNA isolated from superficial water can provide information on the benthic invertebrate community, which might be particularly useful for biodiversity assessment in lagoons, ports, and areas characterized by limited or interdicted access. This study used environmental DNA (eDNA) metabarcoding of superficial water samples to assess benthic invertebrate biodiversity in the Venice Lagoon. Sampling from surface water revealed 80 taxonomic units, mostly benthic species, including several invasive ones, with significant spatial and seasonal variations in community composition. The results demonstrate that eDNA from surface water can effectively monitor benthic biodiversity, providing a valuable tool for ongoing assessments in lagoons and similar environments.

The striped venus Chamelea gallina clam fishery is among the oldest and the largest in the Mediterranean Sea, particularly in the inshore waters of northern Adriatic Sea. The high fishing pressure has lead to a strong stock abundance decline, enhanced by several irregular mortality events. The nearly complete lack of molecular characterization limits the available genetic resources for C. gallina. We achieved the first transcriptome of this species with the aim of identifying an informative set of expressed genes, potential markers to assess genetic structure of natural populations and molecular resources for pathogenic contamination detection. The 454-pyrosequencing of a normalized cDNA library of a pool C. gallina adult individuals yielded 298,494 raw reads. Different steps of reads assembly and filtering produced 36,422 contigs of high quality, one half of which (18,196) were annotated by similarity. A total of 111 microsatellites and 20,377 putative SNPs were identified. A panel of 13 polymorphic transcript-linked microsatellites was developed and their variability assessed in 12 individuals. Remarkably, a scan to search for contamination sequences of infectious origin indicated the presence of several Vibrionales species reported to be among the most frequent clam pathogen's species. Results reported in this study were included in a dedicated database available at http://compgen.bio.unipd.it/chameleabase. This study represents the first attempt to sequence and de novo annotate the transcriptome of the clam C. gallina. The availability of this transcriptome opens new perspectives in the study of biochemical and physiological role of gene products and their responses to large and small-scale environmental stress in C. gallina, with high throughput experiments such as custom microarray or targeted re-sequencing. Molecular markers, such as the already optimized EST-linked microsatellites and the discovered SNPs will be useful to estimate effects of demographic processes and to detect minute levels of population structuring.

Among the main measures adopted to reduce anthropogenic impacts on elasmobranch communities, understanding the ecology of deep-sea sharks is of paramount importance, especially for potentially vulnerable species highly represented in the bycatch composition of commercial fisheries such as the blackmouth catshark Galeus melastomus . In the present work, we unravelled the first indication of population genetic structure of G. melastomus by using a novel and effective panel of nuclear, and polymorphic DNA markers and compared our results with previous findings supporting high genetic connectivity at large spatial scales. Given the lack of species-specific nuclear markers, a total of 129 microsatellite loci (Simple Sequence Repeats, SSRs) were cross-amplified on blackmouth catshark specimens collected in eight geographically distant areas in the Mediterranean Sea and North-eastern Atlantic Ocean. A total of 13 SSRs were finally selected for genotyping, based on which the species exhibited signs of weak, but tangible genetic structure. The clearcut evidence of genetic differentiation of G. melastomus from Scottish waters from the rest of the population samples was defined, indicating that the species is genetically structured in the Mediterranean Sea and adjacent North-eastern Atlantic. Both individual and frequency-based analyses identified a genetic unit formed by the individuals collected in the Tyrrhenian Sea and the Strait of Sicily, distinguished from the rest of the Mediterranean and Portuguese samples. In addition, Bayesian analyses resolved a certain degree of separation of the easternmost Aegean sample and the admixed nature of the other Mediterranean and the Portuguese samples. Here, our results supported the hypothesis that the interaction between the ecology and biology of the species and abiotic drivers such as water circulations, temperature and bathymetry may affect the dispersion of G. melastomus , adding new information to the current knowledge of the connectivity of this deep-water species and providing powerful tools for estimating its response to anthropogenic impacts.

Seascape connectivity studies, informing the level of exchange of individuals between populations, can provide extremely valuable data for marine population biology and conservation strategy definition. Here we used a multidisciplinary approach to investigate the connectivity of the marbled crab ( Pachygrapsus marmoratus ), a high dispersal species, in the Adriatic and Ionian basins. A combination of genetic analyses (based on 15 microsatellites screened in 314 specimens), Lagrangian simulations (obtained with a biophysical model of larval dispersal) and individual-based forward-time simulations (incorporating species-specific fecundity and a wide range of population sizes) disclosed the realized and potential connectivity among eight different locations, including existing or planned Marine Protected Areas (MPAs). Overall, data indicated a general genetic homogeneity, after removing a single outlier locus potentially under directional selection. Lagrangian simulations showed that direct connections potentially exist between several sites, but most sites did not exchange larvae. Forward-time simulations indicated that a few generations of drift would produce detectable genetic differentiation in case of complete isolation as well as when considering the direct connections predicted by Lagrangian simulations.Overall, our results suggest that the observed genetic homogeneity reflects a high level of realized connectivity among sites, which might result from a regional metapopulation dynamics, rather than from direct exchange among populations of the existing or planned MPAs. Thus, in the Adriatic and Ionian basins, connectivity might be critically dependent on unsampled, unprotected, populations, even in species with very high dispersal potential like the marbled crab. Our study pointed out the pitfalls of using wide-dispersing species with broad habitat availability when assessing genetic connectivity among MPAs or areas deserving protection and prompts for the careful consideration of appropriate dispersing features, habitat suitability, reproductive timing and duration in the selection of informative species.

Connectivity between populations shapes the genetic structure of species being crucial for an effective management of environmental resources. Genetic approaches can provide indirect measures of connectivity, allowing the identification of genetically differentiated - unconnected - populations. In this study, we applied a 2b-RAD approach based on hundreds of polymorphic loci to provide the first detailed insight into the population genomics of the Mediterranean mussel Mytilus galloprovincialis in part of its native geographical range. We sampled 19 localities within the Mediterranean and Black Seas, and analyzed a total of 478 samples. We detected strong differences between the two seas, whereas no differences were found between samples from the Western and Central Mediterranean and within Western Mediterranean samples. In the Central Mediterranean a significant differentiation emerged comparing Central Adriatic samples with those from South Adriatic and Ionian Seas. Furthermore, an East-to-West genetic structuring was found in the Central Adriatic Sea, which was not present in the Southern Adriatic and Ionian Seas. These results possibly reflect the local oceanography, with a Middle Adriatic gyre unable to prevent genetic differentiation in this species, and a Southern Adriatic gyre that effectively mixes propagules in Southern areas. In the Black Sea, no signal of genetic structure was found, although samples were spaced at similar distances as in the Adriatic-Ionian area. Genetic connectivity patterns of M. galloprovincialis reveal peculiar species-specific features respect to other species with similar larval duration, suggesting caution in using genetic connectivity data of single species in defining conservation units. We recommend of using genetic connectivity data of many species representing a variety of life history traits, and we call for new investigations using high resolution population genomics, particularly in the Black Sea, to understand if areas separated by hundreds of kilometers can be considered genetically connected as mussels’ data suggest. This information will be critical to ensure “a well-connected system of protected areas” according to Aichi Target 11 of the Convention on Biological Diversity.

Aim The species of the genus Chionodraco (Notothenioidei) are the most abundant icefish on the continental shelf of the Weddell Sea. While previous studies indicated that only Chionodraco hamatus and Chionodraco myersi inhabit the Weddell Sea, the third Chionodraco species, Chionodraco rastrospinosus, was recently sampled in the area. As C. rastrospinosus is supposed to be found only at the Antarctic Peninsula and Scotia Arc, this study aimed at confirming the species classification of C. rastrospinosus by molecular methods and identifying its putative source population. Given the documented evidence of introgression among the three species, we tested whether the newly found C. rastrospinosus shared any genetic variability with the other Chionodraco species. To explain the pattern of distribution of the Chionodraco species, we aimed at estimating the hydrodynamic connectivity between the Antarctic Peninsula and the Weddell Sea. Location Antarctic Peninsula, southern Scotia Arc and the south‐eastern Weddell Sea. Methods We genotyped 19 microsatellites and sequenced the mitochondrial D‐loop for 560 Chionodraco individuals. We simulated the dispersal of more than 3 million drifters (Lagrangian model). Results The molecular analyses support the presence of C. rastrospinosus in the Weddell Sea and its homogeneity with C. rastrospinosus from the Antarctic Peninsula. Bayesian clustering identifies three putative hybrids among C. rastrospinosus and the other congenerics. Lagrangian simulations do not support connectivity driven by the oceanographic features of the Antarctic Peninsula and Weddell Sea via passive larval dispersal only. Main conclusions This study documents, for the first time, the presence of C. rastrospinosus in the Weddell Sea unveiling more biodiversity than previously known in this region. The sympatry of the three Chionodraco species explains the occurrence of occasional, ongoing events of hybridization in the genus. Alternative possible hypotheses need to be tested in future studies about the mechanisms maintaining the interspecific connectivity in Chionodraco spp.

We characterized nine polymorphic microsatellites by an enriched library from the Antarctic silverfish Pleuragramma antarcticum , a key species in the high Antarctic zone of the Southern Ocean. The number of alleles scored ranged from 7 to 39, whereas the observed and expected heterozygosity ranged from 0.4000 to 0.9750 and from 0.3943 to 0.9782, respectively. Cross-amplification was tested in the 3 species of the genus Chionodraco (Channichthyidae). These new microsatellites could potentially be useful in further investigations on P. antarcticum for which many questions on population structure and exposition to global environment change are still open to debate.

The striped venus Chamelea gallina clam fishery is among the oldest and the largest in the Mediterranean Sea, particularly in the inshore waters of northern Adriatic Sea. The high fishing pressure has lead to a strong stock abundance decline, enhanced by several irregular mortality events. The nearly complete lack of molecular characterization limits the available genetic resources for C. gallina. We achieved the first transcriptome of this species with the aim of identifying an informative set of expressed genes, potential markers to assess genetic structure of natural populations and molecular resources for pathogenic contamination detection. The 454-pyrosequencing of a normalized cDNA library of a pool C. gallina adult individuals yielded 298,494 raw reads. Different steps of reads assembly and filtering produced 36,422 contigs of high quality, one half of which (18,196) were annotated by similarity. A total of 111 microsatellites and 20,377 putative SNPs were identified. A panel of 13 polymorphic transcript-linked microsatellites was developed and their variability assessed in 12 individuals. Remarkably, a scan to search for contamination sequences of infectious origin indicated the presence of several Vibrionales species reported to be among the most frequent clam pathogen's species. Results reported in this study were included in a dedicated database available at http://compgen.bio.unipd.it/chameleabase. This study represents the first attempt to sequence and de novo annotate the transcriptome of the clam C. gallina. The availability of this transcriptome opens new perspectives in the study of biochemical and physiological role of gene products and their responses to large and small-scale environmental stress in C. gallina, with high throughput experiments such as custom microarray or targeted re-sequencing. Molecular markers, such as the already optimized EST-linked microsatellites and the discovered SNPs will be useful to estimate effects of demographic processes and to detect minute levels of population structuring.