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100Effective identification of species using short DNA fragments (DNA barcoding and DNA 101 metabarcoding) requires reliable sequence reference libraries of known taxa. Both 102 taxonomically comprehensive coverage and content quality are important for sufficient 103 accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are 104 particularly important if molecular identification tools are to be implemented in biomonitoring 105 and reports in the context of the EU Water Framework Directive (WFD) and the Marine 106Strategy Framework Directive (MSFD). We analysed gaps in the two most important 107 reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a 108 focus on the taxa most frequently used in WFD and MSFD. Our analyses show that 109 coverage varies strongly among taxonomic groups, and among geographic regions. In 110 general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, 111 caddisflies and vascular plants) are well represented in the barcode libraries, while others 112 have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also 113 found that species monitored in several countries often are represented by barcodes in 114 reference libraries, while species monitored in a single country frequently lack sequence 115 records. A large proportion of species (up to 50%) in several taxonomic groups are only 116represented by private data in BOLD. Our results have implications for the future strategy to 117 fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the 118 monitoring of European aquatic biota under the WFD and MSFD. For example, missing 119 species relevant to monitoring in multiple countries should be prioritized. We also discuss 120 why a strategy for quality control and quality assurance of barcode reference libraries is 121 needed and recommend future steps to ensure full utilization of metabarcoding in aquatic 122 biomonitoring. 123 124

Mass appearances of the toxic dinoflagellate genus Ostreopsis are known to cause dangerous respiratory symptoms in humans exposed to aerosols. The outbreaks can appear in shallow marine waters of temperate regions around the globe. We followed a massive bloom event on a public beach on the northern Adriatic coast near Rovinj, Croatia. We identified the responsible species and the produced toxins as well as the dynamics of the event with respect to environmental conditions. Ostreopsis cf. ovata appeared in masses from September through October 2010 on a public beach near Rovinj, Croatia but stayed undetected by public health organizations. Respiratory symptoms were observed whenever humans were exposed to substrate samples containing large numbers of Ostreopsis cells. During the mass abundance of O. cf. ovata also exposure to the aerosols on the beach evoked respiratory symptoms in humans. Our measurements showed high cell abundances and high toxin contents with a stable relative contribution of putative Palytoxin and Ovatoxins a-e. Artificial beach structures proved to dramatically reduce settling of the observed Ostreopsis biofilm. Blooms like those reported herein have a high potential to happen undetected with a high potential of affecting the health of coastal human populations. Increased monitoring efforts are therefore required to understand the ecology and toxicology of those bloom events and reduce their negative impact on coastal populations.

The northern Adriatic (NA) is a favorable basin for studying the adaptive strategies of plankton to a variety of conditions along the steep gradients of environmental parameters over the year. Earlier studies identified phosphorus (P)-limitation as one of the key stresses within the NA that shape the biological response in terms of biodiversity and metabolic adjustments. A wide range of reports supports the notion that P-limitation is a globally important phenomenon in aquatic ecosystems. In this study P stress of marine microphytoplankton was determined at species level along a trophic gradient in the NA. In P-limitation all species with considerable contributions to the diatom community expressed alkaline phosphatase activity (APA), compared to only a few marginal dinoflagellate species. Nevertheless, APA expressing species did not always dominate the phytoplankton community, suggesting that APA is also an important strategy for species to survive and maintain active metabolism outside of their mass abundances. A symbiotic relationship could be supposed for diatoms that did not express APA themselves and probably benefited from APA expressed by attached bacteria. APA was not expressed by any microphytoplankton species during the autumn when P was not limiting, while most of the species did express APA during the P-limitation. This suggests that APA expression is regulated by orthophosphate availability. The methods employed in this study allowed the microscopic detection of APA for each microphytoplankton cell with simultaneous morphologic/taxonomic analysis. This approach uncovered a set of strategies to compete in P-limited conditions within the marine microphytoplankton community. This study confirms the role of P-limitation as a shaping factor in marine ecosystems.

This paper deals with the precise identification of species of Pseudo-nitzschia, focusing on those which are a potential source of domoic acid, from the Krka River estuary of the Central Adriatic Sea. Domoic acid was measured in phytoplankton net samples and shellfish collected in the winter and early spring of 2011 and 2012. Domoic acid was only detected in early March 2011, both in plankton net samples and shellfish extracts, during a Pseudo-nitzschia species bloom. The measured concentrations of particulate domoic acid (DA) in filtered concentrated seawater varied from 3.1˗6.2 ng DA ml-1. In shellfish sample DA concentration was 0.2 μg g-1. Species belonging to the Pseudo-nitzschia delicatissima complex were more common than those from the Pseudo-nitzschia seriata complex. Morphological analyses by electron microscopy revealed the presence of three potentially toxic species: P. calliantha, P. pseudodelicatissima and P. pungens, and one non-toxic species: P. subfraudulenta. However, P. calliantha and P. pseudodelicatissima dominated during the March 2011 bloom. This study presents the first evaluation of particulate domoic acid along the Eastern Adriatic Sea and the first record of the presence of P. calliantha, P. pseudodelicatissima, P. pungens and P. subfraudulenta in the Krka River estuary.

Coccolithophores are unicellular calcifying eukaryotes with a complex life‐cycle; they are important primary producers, and major drivers of global biogeochemical cycles. The majority of research on coccolithophores has been focused on open ocean waters, while the knowledge of their roles in coastal ecosystems is limited. Early 20th century studies of the Adriatic coast produced major taxonomic papers describing half of the projected diversity of coccolithophores. Afterwards, there have been only scarce records of their diversity and ecology in the Adriatic, and coastal systems in general. We aimed to assess coccolithophore diversity and species succession, and closely examine their ecological preferences in the coastal area of the northern Adriatic. We recorded coccolithophores throughout the year at 100% frequency, documented 52 taxa, abundances as high as 2.4 × 105 cells L−1, and noted the winter domination over the phytoplankton community. Out of 52 observed coccolithophore taxa, 31 were heterococcolithophorids and 21 holococcolithophorids. Moreover, seven pairs of heterococcolith and holococcolith phases were noted, and two strategies of temporal separation of life‐cycle phases were observed. We report ecological preferences of those life‐cycle pairs and give in situ evidence that holo‐ and heterococcolith phases are widening the specie's ecological niches. This expansion allows the species to exploit a wider range of ecological conditions and enables phenotypic plasticity, important in response to changes in the environment. With this study we emphasize that coastal areas are highly productive for coccolithophore studies. Our results indicate that a shift in research effort on coccolithophores in the coastal waters is needed.

Summary The extent of DMSP demethylation has been hypothesized to depend on DMSP availability and bacterial sulfur demand, which might lead to niche differentiation of the demethylating bacterial community. In this study, we determined DMSP concentrations in marine snow and the ambient water over a seasonal cycle and linked DMSP concentrations to the abundance of bacteria harbouring the demethylation dmdA gene in the Adriatic Sea. In marine snow, DMSP concentrations were up to four times higher than in the ambient water and three times higher in marine snow in summer than in winter. The average dmdA:recA gene ratio over the sampling period was 0.40 ± 0.24 in marine snow and 0.48 ± 0.21 in the ambient water. However, at the subclade level, differences in the demethylating bacterial community of marine snow and the ambient water were apparent. Seasonal patterns of potentially demethylating bacteria were best visible at the oligotype level. In the ambient water, the SAR116 and the OM60/NOR5 clade were composed of oligotypes that correlated to high DMSP concentrations, while oligotypes of the Rhodospirillales correlated to low DMSP concentrations. Our results revealed a pronounced seasonal variability and spatial heterogeneity in DMSP concentrations and the associated demethylating bacterial community.

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