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.