A combination of molecular and morphological characteristics was used to investigate populations of wild and farmed gilthead sea bream Sparus aurata from the largest Croatian shellfish production area, Mali Ston Bay. Identification of farmed escapees was performed in the wild in order to evaluate and compare the detection sensitivity of molecular and morphological tools. Using a traditional set of measurements and a truss network system, morphometric trait analysis of gilthead sea bream showed clear body shape differences between individuals from the wild and farmed populations. Microsatellite markers only revealed weak neutral genetic differentiation between farmed and wild samples from Mali Ston Bay (Wright's F ST = 0.019, p > 0.05; Jost's D EST = 0.022), despite the fact that the farmed gilthead sea bream were of Atlantic origin. Using assignment tests, morphological (16%) and molecular (25%) analysis identified farmed escapees in the samples of wild gilthead sea bream in the vicinity of aquaculture sites. Morphological data showed good potential for distinguishing recent escapees in wild populations, since these characteristics are affected by the environment. This study demonstrates the presence of escapees in the local wild population in this region, and suggests the potential for genetic interaction.

In geometric morphometrics, the extent of variation attributable to non-biological causes (i.e. measurement error) is sometimes overlooked. The effects of this variation on downstream statistical analyses are also largely unknown. In particular, it is unclear whether specimen preservation induces substantial variation in shape and whether such variation affects downstream statistical inference. Using a combination of empirical fish body shape data and realistic simulations, we show that preservation introduces substantial artefactual variation and significant non-random error (i.e. bias). Most changes in shape occur when fresh fish are frozen and thawed, whereas a smaller change in shape is observed when frozen and thawed fish are fixed in formalin and transferred to ethanol. Surprisingly, we also show that, in our case, preservation produces only minor effects on three downstream analyses of shape variation: classification using canonical variate analysis, permutation tests of differences in means and computations of differences in mean shape between groups. Even mixing of differently preserved specimens has a relatively small effect on downstream analyses. However, we suggest that mixing fish with different preservation should still be avoided and discuss the conditions in which this practice might be justified.

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In gilthead seabream the number of domesticated individuals increased annually, and escape events occur regularly in the Adriatic Sea. Still there is a lack of population genetic characteristics and evidence of the extent and geographic scale of interbreeding resulting from fish-farm escapees. We screened 1586 individuals using a panel of 21 neutral microsatellite loci in several consecutive years and here report on the medium-scale detection of hybrid and farmed seabream in the natural environment. Wild adults showed a lack of genetic structure within basin and sampling years and reduced connectivity with wild offspring collection, suggesting their temporal residency within the Adriatic. On the contrary, by linking the results of multiannual genetic analyses with the results of coupled hydrodynamic and individual based models (IBM-Ichthyop), we observed a strong connection of wild seabream associated with tuna-aquaculture sites and offspring from the nursery grounds, indicating that the surroundings of tuna sea-cage farms can function as a spawning grounds. The study results present the genetic baseline of wild and farmed strains from the eastern Adriatic Sea, as a first step toward development of a mitigation strategy for fish escapees aimed at controlling further erosion of genetic integrity.

Morphological and ecophysiological traits of wild, farmed and wild farm-associated gilthead seabream Sparus aurata were used to assess the degree of phenotypic adaptation of the species to their respective environments. Geometric morphometrics revealed clear body shape differentiation amongst the 3 types of populations, whereby sig nificant differences were noted in head profile and the anterior-body region of the fish. Morphological resemblance was recorded among 2 gilthead seabream populations associated with tuna farms, indicating that they share the same phenotypic responsiveness pattern. Adaptation in reproductive investment strategy was observed in individuals from farm-associated origin, displaying the highest gonadosomatic index but lowest condition in comparison to their farmed and wild counterparts. Multinomial logistic regression revealed that sex change from male to female with increasing length was more pronounced in farm-associated fish than in farmed and and wild fish. These findings demonstrate a significant influence of the environmental factors inherent to tuna farms on the phenotypic characteristics of gilthead seabream, providing a basis for further research on the ecological effects of tuna farms on local marine populations.

The European flat oyster, Ostrea edulis, is a keystone species suffering major population declines due to overfishing, habitat loss and parasite diseases. Knowledge of its finescale population genetic structure and connectivity, needed for effective conservation, restoration and management, is largely lacking. Along the eastern Adriatic Sea, genotyping of 1178 O. edulis individuals at 12 microsatellite loci was conducted, grouping the sampled populations by geographical origin (North, Middle, South Adriatic), shell-farm association (farmed, farm-impacted, wild oysters) and sampling year (2017, 2018), in order to explore spatio-temporal genetic variation and potential footprint of known human-mediated spat translocation events for aquaculture purpose. Shortterm temporal genetic structuring of O. edulis populations was less pronounced compared to their spatial variability, which showed genetic discontinuity between O. edulis populations from different geographical regions, with the main boundary separating the North from the Middle and South Adriatic, and the weaker one limiting the flow between the Middle and South Adriatic. While the present culture practise and ongoing spat translocation promotes genetic heterogeneity in the investigated farms, reduced genetic diversity and smallest effective populations size of impacted, i.e., farm-associated O. edulis was consistently recorded in all geographical regions. Taken together, the results reflect regional oceanographic features, ongoing spat translocation and intensive harvesting, which might have reduced the wild O. edulis densities below the critical threshold for reproductive success, compromising settlement and favoring unidirectional gene-flow toward higher density farmed O. edulis. Genetic structure of Adriatic O. edulis populations revealed some concerning demographic changes and farm-wild oyster interactions and hence further investigation and management recommendations are given.

A small escape incident of gilthead seabream Sparus aurata tagged with acoustic transmitters (N = 25) from a commercial farm located in a coastal bay of the eastern Adriatic Sea was simulated to enable evaluation of recapture strategies and escapee management. Over a 3 mo monitoring period, tagged individuals showed spatial distribution closely related to fish farms, where 76 and 68% of tagged fish were present at the farm during the second and third weeks post-release respectively. Upon initial release, escaped seabream had a small total home range (0.142 km 2 ) that encompassed the farm site. Short-term residence differed among tagged fish. Few fish (28%) remained in the proximity of the fish farm for longer than 1 mo; most were likely angled or moved outside the acoustic array. Fish that moved elsewhere have the potential to cause substantial ecological and genetic impact, as they likely are able to quickly adapt to natural conditions. Considering the capture impact of the limited recreational fishery on this small-scale escape event, fish recapture by this type of fishery within 2 wk of escape could be feasible and is highly recommended.

This study tests the suitability of the gilthead seabream scales as a proxy for origin selection in wild and anthropogenically pressured environments. Scale morphology and microchemistry were used to discriminate the habitat selection of two wild, farmed and wild farm-associated populations where landmark and outline-based scale morphometrics, trace-element chemistry and scale microstructure characteristics were analysed. The morphometric techniques successfully differentiated between the farmed and wild origin scale phenotypes. Reduced discrimination sensitivity between the wild and wild farm-associated origin was, however, reported. The discrimination based on microchemistry (B, Ba, Mn, K, Sr and Zn) classified the scales with high accuracy according to their origin (wild vs. farmed vs. wild farm-associated) and sampling locations, thus proving itself as a powerful tool in provenance study of gilthead seabream. Disparity in scale microstructure characteristics accounted for radii, circuli and inter-circulus spacing, hence unveiling the differences in growth and environmental conditions between the wild and farmed fish. In brief, scale shape was found to be a potent exploration tool for farmed fish identification, whereas scale microchemistry yielded a good resolution in identifying gilthead seabream membership among different habitats. Considering the importance of this species in aquaculture and fisheries throughout the Mediterranean, more research is needed to assess the usefulness of scales as nonlethal biogeochemical tags.