Quotas regulation is necessary but not sufficient to mitigate the impact of SCUBA diving in a highly visited marine protected area.

When effectively managed, Marine Protected Areas (MPAs) can produce wide ecosystem benefits that can foster, directly and indirectly, local economies. Tourism is one of the sectors mainly benefited by the effect of conservation. SCUBA diving represents an important tourism activity, especially in the context of MPAs, where it is one of the few activities often fostered rather than limited, for its capacity to integrate environmental and socio-economic sustainability. However, SCUBA diving can also produce negative impacts on the environment when tourism frequentation exceeds a sustainable threshold, these potentially generating negative effects on the sector itself. In this study, we (1) investigated the impact of SCUBA diving in one of the most frequented diving areas of the Mediterranean Sea (Cabo de Palos - Islas Hormigas marine reserve), and (2) assessed the potential benefits over time related to the adoption of a regulation change for the diving activity (i.e., formally adoption of diving quotas). Specifically, we compared demographic (density of alive and dead colonies) and morphometric (height, width and complexity) characteristics of the false coral (Myriapora truncata) between dived and fully protected (non-dived) locations over four diving seasons (one before and three after the change in diving quotas). The density of alive colonies of the false coral was, on average, six times lower in dived locations compared to controls, highlighting a clear impact of SCUBA diving (consistent over time). Colonies were also significantly smaller in dived locations. The diving quotas produced a significant reduction of the ratio dead/total colonies in the dived locations soon after their adoption, but these benefits disappeared over the following years, possibly due to a gradual decline in operators' and divers' observance and concern, rather than an increasing number of dives. This suggests that the adoption of effective regulations is crucial for the environmental sustainability of diving tourism in protected areas and can provide positive effects, but an effort is needed to ensure that compliance is consistent over time, and that low-impact diving practices are adopted by this important recreational sector.

An integrated assessment of the Good Environmental Status of Mediterranean Marine Protected Areas.

Local, regional and global targets have been set to halt marine biodiversity loss. Europe has set its own policy targets to achieve Good Environmental Status (GES) of marine ecosystems by implementing the Marine Strategy Framework Directive (MSFD) across member states. We combined an extensive dataset across five Mediterranean ecoregions including 26 Marine Protected Areas (MPAs), their reference unprotected areas, and a no-trawl case study. Our aim was to assess if MPAs reach GES, if their effects are local or can be detected at ecoregion level or up to a Mediterranean scale, and which are the ecosystem components driving GES achievement. This was undertaken by using the analytical tool NEAT (Nested Environmental status Assessment Tool), which allows an integrated assessment of the status of marine systems. We adopted an ecosystem approach by integrating data from several ecosystem components: the seagrass Posidonia oceanica, macroalgae, sea urchins and fish. Thresholds to define the GES were set by dedicated workshops and literature review. In the Western Mediterranean, most MPAs are in good/high status, with P. oceanica and fish driving this result within MPAs. However, GES is achieved only at a local level, and the Mediterranean Sea, as a whole, results in a moderate environmental status. Macroalgal forests are overall in bad condition, confirming their status at risk. The results are significantly affected by the assumption that discrete observations over small spatial scales are representative of the total extension investigated. This calls for large-scale, dedicated assessments to realistically detect environmental status changes under different conditions. Understanding MPAs effectiveness in reaching GES is crucial to assess their role as sentinel observatories of marine systems. MPAs and trawling bans can locally contribute to the attainment of GES and to the fulfillment of the MSFD objectives. Building confidence in setting thresholds between GES and non-GES, investing in long-term monitoring, increasing the spatial extent of sampling areas, rethinking and broadening the scope of complementary tools of protection (e.g., Natura 2000 Sites), are indicated as solutions to ameliorate the status of the basin.

Environmental DNA effectively captures functional diversity of coastal fish communities.

Robust assessments of taxonomic and functional diversity are essential components of research programmes aimed at understanding current biodiversity patterns and forecasting trajectories of ecological changes. Yet, evaluating marine biodiversity along its dimensions is challenging and dependent on the power and accuracy of the available data collection methods. Here we combine three traditional survey methodologies (underwater visual census strip transects [UVCt], baited underwater videos [BUV] and small-scale fishery catches [SSFc]), and one novel molecular technique (environmental DNA metabarcoding [eDNA]-12S rRNA and cytochrome oxidase subunit 1 [COI]) to investigate their efficiency and complementarity in assessing fish diversity. We analysed 1,716 multimethod replicates at a basin scale to measure the taxonomic and functional diversity of Mediterranean fish assemblages. Taxonomic identities were investigated at species, genus and family levels. Functional identities were assessed using combinations of morphological, behavioural and trophic traits. We show that: (a) SSFc provided the higher taxonomic diversity estimates followed by eDNA, and then UVCt and BUV; (b) eDNA was the only method able to gather the whole spectrum of considered functional traits, showing the most functionally diversified and least redundant fish assemblages; and (c) the effectiveness of eDNA in describing functional structure reflected its lack of selectivity towards any considered functional trait. Our findings suggest that the reach of eDNA analysis stretches beyond taxon detection efficiency and provides new insights into the potential of metabarcoding in ecological studies.

Improving marine protected area governance through collaboration and co-production.

Marine protected areas (MPAs) socio-ecological effectiveness depends on a number of management and governance elements, among which stakeholder engagement and community support play key roles. Collaborative conservation initiatives that engage stakeholders in action research and knowledge co-production processes can enhance management and governance of MPAs. To design effective strategies aimed at reconciling biodiversity conservation and management of sustainable human uses, it is key to assess how local communities respond to such initiatives and identify the set of contextual factors, institutional, local and individual, potentially affecting these responses. This paper presents the approach and results of one such initiative, spanning 6 EU countries and 11 MPAs in the Mediterranean Sea, focusing on small-scale fishers as key MPA users. Through a collaborative project, managers and fishers agreed upon specific governance interventions (e.g. increasing stakeholder engagement, engaging fishers in monitoring activities, reducing fishing efforts) to be implemented in each MPA for one year. Structured surveys queried: MPA managers on the MPA context, governance structure, feasibility and effectiveness of the tested interventions; and small-scale fishers on their perceptions of the impact of the tested interventions on a set of 9 socio-ecological variables (e.g. amount of fish caught, level of participation in decision-making, support for the MPA). Results revealed that the interventions tested were relatively feasible, effective and cost-effective. Fishers reported positive perceptions of the interventions for the 9 variables considered, especially for level of support for the MPA and for those associated with aspects of governance. Proportional odds models highlighted perceived effects are maximized under certain institutional, local and individual circumstances (e.g. old MPAs, small fisher communities, and fishers with a high proportion of income from fisheries). Findings highlight that employing good governance processes involving stakeholders may rapidly generate improved local support for conservation and provide insights for potential leverage points upon which to act to maximize perceived effectiveness and enhance support toward MPAs.

Ocean acidification affects somatic and otolith growth relationship in fish: evidence from an in situ study.

Ocean acidification (OA) may have varied effects on fish eco-physiological responses. Most OA studies have been carried out in laboratory conditions without considering the in situ pCO2/pH variability documented for many marine coastal ecosystems. Using a standard otolith ageing technique, we assessed how in situ ocean acidification (ambient, versus end-of-century CO2 levels) can affect somatic and otolith growth, and their relationship in a coastal fish. Somatic and otolith growth rates of juveniles of the ocellated wrasse Symphodus ocellatus living off a Mediterranean CO2 seep increased at the high- pCO2 site. Also, we detected that slower-growing individuals living at ambient pCO2 levels tend to have larger otoliths at the same somatic length (i.e. higher relative size of otoliths to fish body length) than faster-growing conspecifics living under high pCO2 conditions, with this being attributable to the so-called 'growth effect'. Our findings suggest the possibility of contrasting OA effects on fish fitness, with higher somatic growth rate and possibly higher survival associated with smaller relative size of otoliths that could impair fish auditory and vestibular sensitivity.

Sandy bottoms have limited species richness but substantially contribute to the regional coastal fish ß-diversity: A case study of the Central Mediterranean Sea.

Global marine biodiversity loss impairs entire ecosystems and their stability. Robust biodiversity estimates are key to inform policies and management strategies, and need to consider the contribution of diverse habitats, including those for which estimates of biodiversity are scattered or totally absent. This study assessed the fish diversity associated with three main coastal habitats (rocky bottoms, Posidonia oceanica meadows, sandy bottoms), and their role in shaping the overall coastal fish diversity, also in relation to potential environmental and anthropogenic drivers affecting patterns of fish diversity in coastal areas. Using underwater visual census, we sampled 62 sites distributed on the three habitats, for a total of 496 replicates. We assessed the contribution of each habitat to ß-diversity, divided into Local Contribution to ß-diversity (LCBD), a comparative indicator of the contributions to ß-diversity of each habitat, and Species Contribution to ß-diversity (SCBD), which measures the relative importance of each species in affecting ß-diversity. Finally, we modelled species diversity in relation to potential environmental and anthropogenic drivers. Overall, 72 species were recorded, with the highest species richness observed on rocky bottoms (56 species, 16 unique to this habitat), followed by P. oceanica (38 species, 0 unique) and sandy bottoms (32 species, 14 unique). Sandy bottom assemblages had a significantly higher contribution to LCBD than P. oceanica meadows and rocky bottoms, and two of the five species with the highest contribution to SCBD are exclusively associated with sandy bottoms. Finally, sea surface temperature, sea surface salinity, and habitat were highlighted as significant predictors of species richness. Our findings, aside from highlighting the environmental drivers of coastal fish diversity in the Mediterranean Sea, unravel the potential key role of sandy bottoms in contributing to overall coastal fish diversity and can inform conservation planning.

Study of Inter- and Intra-varietal Genetic Variability in Grapevine Cultivars.

Vitis vinifera includes a large number of cultivars that are further distinguished in biotypes and clones, and it is actually hard to differentiate them, even through complex molecular techniques. In this work, the plant materials of 56 putative Sangiovese and 14 putative Montepulciano biotypes, two of the most widespread black-berried Italian cultivars, were collected in different wine-growing areas of Italy distributed in 13 regions, from north to south. Firstly, the samples were analyzed using SSR markers to have proper varietal identification. According to the results, the genotypes belonged to three different cultivars: Sangiovese, Sanforte, and Montepulciano. Subsequently, the samples were investigated using AFLP, SAMPL, M-AFLP, and I-SSR molecular markers to estimate their intra-varietal genetic variability. The DNA marker-based method used turned out to be performing to bring out the geographic differences among the biotypes screened, and it can therefore be considered as a powerful tool available for all the grapevine varieties.

Small-scale fisheries catch more threatened elasmobranchs inside partially protected areas than in unprotected areas.

Elasmobranchs are heavily impacted by fishing. Catch statistics are grossly underestimated due to missing data from various fishery sectors such as small-scale fisheries. Marine Protected Areas are proposed as a tool to protect elasmobranchs and counter their ongoing depletion. We assess elasmobranchs caught in 1,256 fishing operations with fixed nets carried out in partially protected areas within Marine Protected Areas and unprotected areas beyond Marine Protected Areas borders at 11 locations in 6 Mediterranean countries. Twenty-four elasmobranch species were recorded, more than one-third belonging to the IUCN threatened categories (Vulnerable, Endangered, or Critically Endangered). Catches per unit of effort of threatened and data deficient species were higher (with more immature individuals being caught) in partially protected areas than in unprotected areas. Our study suggests that despite partially protected areas having the potential to deliver ecological benefits for threatened elasmobranchs, poor small-scale fisheries management inside Marine Protected Areas could hinder them from achieving this important conservation objective.

Use of Lagrangian simulations to hindcast the geographical position of propagule release zones in a Mediterranean coastal fish.

The study of organism dispersal is fundamental for elucidating patterns of connectivity between populations, thus crucial for the design of effective protection and management strategies. This is especially challenging in the case of coastal fish, for which information on egg release zones (i.e. spawning grounds) is often lacking. Here we assessed the putative location of egg release zones of the saddled sea bream (Oblada melanura) along the south-eastern coast of Spain in 2013. To this aim, we hindcasted propagule (egg and larva) dispersal using Lagrangian simulations, fed with species-specific information on early life history traits (ELTs), with two approaches: 1) back-tracking and 2) comparing settler distribution obtained from simulations to the analogous distribution resulting from otolith chemical analysis. Simulations were also used to assess which factors contributed the most to dispersal distances. Back-tracking simulations indicated that both the northern sector of the Murcia region and some traits of the North-African coast were hydrodynamically suitable to generate and drive the supply of larvae recorded along the coast of Murcia in 2013. With the second approach, based on the correlation between simulation outputs and field results (otolith chemical analysis), we found that the oceanographic characteristics of the study area could have determined the pattern of settler distribution recorded with otolith analysis in 2013 and inferred the geographical position of main O. melanura spawning grounds along the coast. Dispersal distance was found to be significantly affected by the geographical position of propagule release zones. The combination of methods used was the first attempt to assess the geographical position of propagule release zones in the Mediterranean Sea for O. melanura, and can represent a valuable approach for elucidating dispersal and connectivity patterns in other coastal species.

The "Tracked Roaming Transect" and distance sampling methods increase the efficiency of underwater visual censuses.

Underwater visual census (UVC) is the most common approach for estimating diversity, abundance and size of reef fishes in shallow and clear waters. Abundance estimation through UVC is particularly problematic in species occurring at low densities and/or highly aggregated because of their high variability at both spatial and temporal scales. The statistical power of experiments involving UVC techniques may be increased by augmenting the number of replicates or the area surveyed. In this work we present and test the efficiency of an UVC method based on diver towed GPS, the Tracked Roaming Transect (TRT), designed to maximize transect length (and thus the surveyed area) with respect to diving time invested in monitoring, as compared to Conventional Strip Transects (CST). Additionally, we analyze the effect of increasing transect width and length on the precision of density estimates by comparing TRT vs. CST methods using different fixed widths of 6 and 20 m (FW3 and FW10, respectively) and the Distance Sampling (DS) method, in which perpendicular distance of each fish or group of fishes to the transect line is estimated by divers up to 20 m from the transect line. The TRT was 74% more time and cost efficient than the CST (all transect widths considered together) and, for a given time, the use of TRT and/or increasing the transect width increased the precision of density estimates. In addition, since with the DS method distances of fishes to the transect line have to be estimated, and not measured directly as in terrestrial environments, errors in estimations of perpendicular distances can seriously affect DS density estimations. To assess the occurrence of distance estimation errors and their dependence on the observer's experience, a field experiment using wooden fish models was performed. We tested the precision and accuracy of density estimators based on fixed widths and the DS method. The accuracy of the estimates was measured comparing the actual total abundance with those estimated by divers using FW3, FW10, and DS estimators. Density estimates differed by 13% (range 0.1-31%) from the actual values (average = 13.09%; median = 14.16%). Based on our results we encourage the use of the Tracked Roaming Transect with Distance Sampling (TRT+DS) method for improving density estimates of species occurring at low densities and/or highly aggregated, as well as for exploratory rapid-assessment surveys in which divers could gather spatial ecological and ecosystem information on large areas during UVC.

Ocean acidification does not impair predator recognition but increases juvenile growth in a temperate wrasse off CO2 seeps.

Fish behavioural effects under Ocean Acidification (OA) rely on changes expected to occur in brain function, which can be reversed by gabazine, a GABA-A antagonist. Here, using standard two-channel choice flume, we assessed OA effects on the predator recognition ability of both gabazine-treated and -untreated Symphodus ocellatus post-settlers living off CO2 seeps in the Mediterranean Sea. To estimate the post-settlers background predation risk we evaluated the density of their predator in the wild and through otolith aging techniques we assessed their post-settlement growth. Results showed that: 1) post-settlers predator recognition was unaffected under OA; 2) post-settlers living in elevated CO2 were on average 15% bigger in size than those from ambient conditions. Our results support fish behavioural tolerance to OA, potentially mediated by pre-exposure to high-risk predation levels, and speculate that by increasing body size, juvenile fish might more efficiently avoid their predators.

Genetic variability and geographic typicality of Italian former Prosecco grape variety using PCR-derived molecular markers.

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 80 Italian Prosecco accessions coming from Prosecco DOC area (north-east area of Italy). The studied samples include genotypes from Veneto and Friuli Venezia Giulia region. In order to verify the varietal identity of the samples, analyses based on 22 SSR loci were performed, and two grape varieties were found: Prosecco tondo and Prosecco lungo. In addition to microsatellite analysis, intra-varietal variability study was performed using AFLP, SAMPL, ISSR, and M-AFLP molecular markers. This molecular approach could discriminate different Prosecco tondo accessions coming from Treviso hills, from Veneto plain, from Friuli Venezia Giulia region, and from Padua hills (Serprina samples). As concerning Prosecco lungo variety, it was possible to discriminate molecularly the accessions from Veneto region and those from Friuli Venezia Giulia region. The molecular analysis allowed a distinction of the Prosecco genotypes on the basis of their geographic origins with plant-specific markers able to differentiate all Prosecco accessions. In this paper, the studied grape variety is termed Prosecco and not Glera (which is the present name) because the sampled vineyards were established many years ago when the name of the variety was Prosecco.

A strategy to investigate the intravarietal genetic variability in Vitis vinifera L. for clones and biotypes identification and to correlate molecular profiles with morphological traits or geographic origins.

Grapevine is the most economically important and widely cultivated fruit crop in the world. Molecular markers have been used on Vitis vinifera to distinguish among both varieties and clones. Microsatellites are used to fingerprint varieties and several other techniques, reported in many papers, are used to analyze the differences among clones, but it is not available in the literature as a well defined strategy to screen a large number of Vitis cultivars. In fact, it is often necessary to use different techniques to investigate the genetic variability in different grapevine varieties and a proposed technique is used to study a cultivar, which is often not suitable for either the study of another cultivar or compare the genetic relationship among various cultivars. We describe here a strategy used for the analysis of several grapevine cultivars to describe a universal method to obtain DNA polymorphisms of Vitis vinifera genotypes from the same cultivar by using amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), microsatellites AFLP (M-AFLP), and ISSR molecular markers. The strategy here adopted permitted both to identify different biotypes (i.e., Primitivo), accessions (i.e., Garnacha tinta), and clones (i.e., Callet, Manto Negro, Moll) among the variability of same variety and to correlate the genetic differences to their geographical origins (i.e., Garnacha tinta; Malvasia nera di Brindisi/Lecce) or morphological traits (i.e., Malvasia of Candia). Here is also described the application of the protocol that allows to highlight the genetic variability accumulated during centuries of cultivations and selections of the same variety in different environments by vine growers.

Study of intra-varietal genetic variability in grapevine cultivars by PCR-derived molecular markers and correlations with the geographic origins.

The genetic grapevine intravarietal variability will be analyzed by PCR-derived marker systems. In particular, the object of the investigation will be the clonal variations of Malvasia nera di Brindisi/Lecce, Negroamaro and Primitivo, also known as Zinfandel, which are three grapevine varieties cultivated in Apulia region (Italy). In order to assess varietal identity of the samples, 132 DNA tests were performed by amplifying 16 SSR loci. The study of the intravarietal variability was performed using AFLPs, SAMPLs, ISSRs, and M-AFLPs. The application of the above-mentioned techniques allowed both to discriminate all genotypes of the three cultivars and to distinguish the accessions of each cultivar sampled from different geographic cultivation areas. Furthermore, the study of biotypes cultivated in different geographical environments of Salento (i.e., Apulia region) allowed important correlations between molecular marker variability and phenotypic traits. These results are suggesting both to focus our attention on the effects of the environment on the genotype and to consider, as a practical consequence, the importance of preserving autochthon grapevine biotypes found in different areas to truly preserve the richness of the germplasm. Thus, more accurate DNA studies give new information that can be extremely useful to the vine nurseries for the correct choice (i.e., supported by more accurate intravarietal variability analysis) of the grape multiplication materials.

Inter- and intra-varietal genetic variability in Malvasia cultivars.

The DNA molecular analyses together with ampelography, ampelometry, and biochemistry are essential for grapevine identification and investigation of genetic differences among the Vitis vinifera L. cultivars and clones. Ten Malvasia cultivars (i.e., Istrian Malvasia; M. delle Lipari; M. bianca di Candia; M. di Candia Aromatica; M. del Lazio; M. bianca lunga, also known as Malvasia del Chianti; M. nera di Brindisi/Lecce; M. di Casorzo; M. di Schierano, and M. nera di Bolzano) were analyzed using molecular approaches to study the genetic inter-varietal variability. Thirty Istrian Malvasia genotypes (i.e., 8 Italian clones, such as ISV 1, ISV F6, VCR 4, VCR 113, VCR 114, VCR 115, ERSA 120, ERSA 121, and 22 autochthonous grapevine accessions grown in Istrian Peninsula, Croatia) were investigated to evaluate the morphological and genetic intra-varietal variability. DNA analysis allowed discrimination of all Malvasia genotypes at molecular level using AFLP, SAMPL, and M-AFLP markers. Italian clones and autochthonous Croatian accessions of Istrian Malvasia were grouped according to their different geographic origins. These results showed the great genetic variability of Malvasia genotypes suggesting the need for the preservation of autochthonous grapevine biotypes found on different areas to approve the correct choice and selection of the grape multiplication materials.

Clones identification and genetic characterization of Garnacha grapevine by means of different PCR-derived marker systems.

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 53 Garnacha accessions coming from Italy, France and Spain. The samples studied include 28 Italian accessions (named Tocai rosso in Vicenza area; Alicante in Sicily and Elba island; Gamay perugino in Perugia province; Cannonau in Sardinia), 19 Spanish accessions of different types (named Garnacha tinta, Garnacha blanca, Garnacha peluda, Garnacha roja, Garnacha erguida, Garnacha roya) and 6 French accessions (named Grenache and Grenache noir). In order to verify the varietal identity of the samples, analyses based on 14 simple sequence repeat (SSR) loci were performed. The presence of an additional allele at ISV3 locus (151 bp) was found in four Tocai rosso accessions and in a Sardinian Cannonau clone, that are, incidentally, chimeras. In addition to microsatellite analysis, intravarietal variability study was performed using AFLP, SAMPL and M-AFLP molecular markers. AFLPs could discriminate among several Garnacha samples; SAMPLs allowed distinguishing few genotypes on the basis of their geographic origin, whereas M-AFLPs revealed plant-specific markers, differentiating all accessions. Italian samples showed the greatest variability among themselves, especially on the basis of their different provenance, while Spanish samples were the most similar, in spite of their morphological diversity.

Normal Auger spectra of Br in alkali bromide molecules.

Molecular Auger electron spectra following the bromine 3d ionization in gas-phase alkali bromides and in HBr were studied both experimentally and theoretically. The AES for HBr and CsBr were measured using photoexcitation, and for LiBr, NaBr, and KBr by using electron impact. These results are compared with the theoretical spectra from nonrelativistic ab initio calculations and one-center approximation and with the spectra of Br(-), computed with the multiconfiguration Dirac-Fock method.