Zrinka Ljubešić

Distribution and diversity of marine picocyanobacteria community: Targeting of Prochlorococcus ecotypes in winter conditions (southern Adriatic Sea)

Adriatic, the northernmost part of the Mediterranean Sea, due its oligotrophy, topography, and hydrology dynamics, and complex circulation patterns, was suggested as an important study site for rapid climatology impacts. Its southern part is mainly oligotrophic and dominated by picophytoplankton, with cyanobacteria as main representatives. Diversity and distribution patterns of different Prochlorococcus ecotypes were investigated by molecular tools and flow cytometry during the winter convection event in the southern Adriatic (BIOTA winter cruise; February/March 2015). Phylogenetic diversity based on clone libraries of the 16S-23S ribosomal DNA ITS region, as well as flow cytometry (histograms of red fluorescence), indicated presence of 2 different Prochlorococcus in the Adriatic. HLI, as a typical clade for Mediterranean Sea, was likewise found to be dominant Prochlorococcus in the Adriatic, followed by less abundant LLI clade. In addition, Prochlorococcus were found to co-occur with diverse Synechococcus population (53% and 47% of obtained ITS sequences, respectively). Different Prochlorococcus ecotypes had similar patterns of vertical distribution, predominantly occupying upper 100m depth layer, but their distribution was clearly affected by the heterogeneity of hydrological conditions, nitrogen concentration and temperature along vertical and horizontal sampling points. Different studies pointed out that, as a consequence of climate changes, serious alteration of biological and ecological patterns are already taking place Therefore, understanding of the distribution and abundance of picophytoplankton in Adriatic, being still limited, is much needed baseline for predicting possible biogeochemical impact of future environmental changes.

Diatoms in an extreme euxinic environment (Rogoznica Lake, eastern Adriatic coast)

Abstract The Rogoznica Lake marine system is a small, karstic, naturally eutrophic and euxinic marine environment. Abundance and temporal distribution of phytoplankton was investigated in relation to environmental conditions in the period from 1998 to 2013. The 36 determined diatoms contributed 90% of the total phytoplankton abundance. The diatom composition is characterized by low species diversity and high single species abundance (up to 107 cells L-1). There were, on average, 2.6 diatom species per sample (maximum 14 diatom species per sample) reflecting extreme environmental conditions. Dominant diatoms Thalassionema nitzschioides, Cyclotella choctawhatcheeana, Dactyliosolen fragilissimus and Chaetoceros curvisetus occurred repeatedly and were alternately dominant in the lake during the whole research period. Some diatoms were dominant only in limited period, like Cyclotella choctawhatcheeana (evident since 2001), and Pseudo-nitzschia spp. (evident in the period 2002 to 2009). It appears that the interplay of environmental conditions such as variability in thermohaline and redox conditions, nutrient and reduced sulphur concentration influence the phytoplankton development and abundance in the lake.

Colonization of diatoms and bacteria on artificial substrates in the northeastern coastal Adriatic Sea

Abstract Every surface that is immersed in seawater becomes rapidly covered with an unavoidable biofilm. Such biofilm formation, also known as fouling, is a complex multistage process and not yet thoroughly investigated. In this study, the succession of diatoms and bacteria was investigated during a one month exposure on an artificial substrate of plexiglass (polymer of methyl methacrylate) mounted above the seafloor at a depth of 5 m. For biofilm analyses, the fouling was investigated using selective agar plates, epifluorescence, light and electronic microscopy, as well as high performance liquid chromatography (HPLC) pigment analysis. During biofilm development, the abundance of all biofilm components increased and reached maximum values after a one month exposure. In the bacterial community, heterotrophic marine bacteria were dominant and reached 1.96 ± 0.79 × 104 colony forming units (CFU) cm-2. Despite the fact that faecal coliforms and intestinal enterococci were detected in the water column, faecal coliforms were not detected in the biofilm and intestinal enterococci appeared after one month of exposure but in the negligible number of 60 ± 10 CFU cm-2. The phototrophic component of the biofilm was dominated by diatoms and reached a concentration of 6.10 × 105 cells cm-2, which was supported by pigment analysis with fucoxanthin as dominant pigment in a concentration up to 110 ng cm-2. The diatom community was dominated by Cylindrotheca closterium and other pennate benthic diatoms. A detailed taxonomic analysis by electronic microscopy revealed 30 different taxa of diatoms. The study confirmed that a plexiglass surface in a marine environment is susceptible to biofouling within 30 days of contact. Furthermore, the co lonization process sequence firstly involved bacteria and cyanobacteria, and secondly diatoms, which together formed a primary biofilm in the sea.

Multigene phylogeny and morphology of newly isolated strain of Pseudo-nitzschia mannii Amato & Montresor (Adriatic Sea)

An increasing number of cryptic and pseudo-cryptic species have been found within many newly described diatom species. To resolve the phylogenetic relationships of the genus Pseudo-nitzschia, molecular markers are being widely used in combination (or separately) with different morphological characters. Sequence analysis of ribosomal DNA markers (18S, ITS and 28S) and morphological analyses of Pseudo-nitzschia mannii strain (CIM_D-4), isolated from the Telašćica Bay (Adriatic Sea), differentiate it from all other currently reported strains of this species.

Bacterial diversity across a highly stratified ecosystem : A salt-wedge Mediterranean estuary

Highly stratified Mediterranean estuaries are unique environments where the tidal range is low and the tidal currents are almost negligible. The main characteristics of these environments are strong salinity gradients and other environmental parameters. In this study, 454 pyrosequencing of the 16S rRNA gene in combination with catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was used to estimate the bacterial diversity across the Krka estuary in February and July 2013. The comparison of the data derived from these two techniques resulted in a significant but weak positive correlation (R=0.28) indicating a substantial difference in the bacterial community structure, depending on the applied method. The phytoplankton bloom observed in February was identified as one of the main factors shaping the bacterial community structure between the two environmentally contrasting sampling months. Roseobacter, Bacteroidetes and Gammaproteobacteria differed substantially between February and July. Typical freshwater bacterial classes (Actinobacteria and Betaproteobacteria) showed strong vertical distribution patterns depending on the salinity gradient. Cyanobacteria decreased in abundance in February due to competition with phytoplankton, while the SAR11 clade increased its abundance in July as a result of a better adaptation toward more oligotrophic conditions. The results provided the first detailed insight into the bacterial diversity in a highly stratified Mediterranean karstic estuary.

Development of periphytic diatoms on different artificial substrates in the Eastern Adriatic Sea

Abstract The settling of diatoms as fouling organisms on a certain substrate is greatly influenced by substrate characteristics and the preferences of a diatom community and diatom species. A distinction among substrates can be made by analysing the specific abundance and composition of diatoms on different substrates. In this study, 11 different artificial substrates were exposed to a marine environment for a period of 30 days. Abundance and taxonomic composition of periphytic diatoms was determined on each of the substrates and on shoots of the marine seagrass Posidonia oceanica. The aim was to compare diatom community structure on different newly colonized surfaces. On all surfaces examined, periphytic diatoms were the pioneering organisms with differences in quantitative and qualitative composition on the different substrates. Taxonomic analysis of diatom communities on the substrates examined revealed 41 diatom taxa, with the dominant genera Cylindrotheca, Amphora, Nitzschia, Cocconeis and Navicula. Given that all the examined artificial substrates were solid materials, differences in the abundance and species composition of diatoms found between the materials point to the substrates’ physical and chemical characteristics as a major influence on the final settling of diatoms. Knowledge from investigating the settlement of fouling organisms on anthropogenic substrates can have future use in management of waste materials that end up in the marine environment.

Winter picoplankton diversity in an oligotrophic marginal sea

Marine picoplankton, unicellular organisms with cell sizes up to 3u202fμm in diameter, numerically dominate marine ecosystems, encompassing Archaea, Bacteria, Eukarya (protists and fungi) as well as viruses. Autotrophic and heterotrophic picoplankton abundance and community composition with a focus on picoeukaryotes (PEs) were investigated in the winter of 2016 at three stations along a coast-to-offshore transect in the southern Adriatic Sea. Abundances were estimated by flow cytometry, while community composition by Illumina High-Throughput Sequencing (HTS) of 16S and 18S rRNA genes. The photosynthetic picoplankton diversity was also investigated by High-Performance Liquid Chromatography (HPLC) of liposoluble pigments. Heterotrophic bacteria and cyanobacteria (Prochlorococcus and Synechococcus) accounted for up to 7u202f×u202f105; 2.3u202f×u202f104 and 2.5u202f×u202f104 cells mL-1, respectively, while photosynthetic picoeukaryotes peaked with 3u202f×u202f103 cells mL-1. Prokaryotes, as revealed by HTS were dominated by Alphaproteobacteria (mainly SAR11, 44.91% of total 16S sequence reads), followed by Gammaproteobacteria (Oceanospirillales and Pseudomonadales, 14.96%), Bacteroidetes (mainly Flavobacteriales, 13%), Cyanobacteria (Prochlorococcus and Synechococcus, 9.52%), Marinimicrobia (SAR406, 7.97%), Deltaproteobacteria (SAR324, 3.83%), Actinobacteria (2.24%) and Chloroflexi (SAR202, 1.90%). Photosynthetic pigment concentrations were very low (12.12 μgL-1 at the most) and taxonomic pigments could be attributed to Prochlorococcus, Synechococcus, Prymnesiophyceae, Bacillariophyceae, Chrysophyceae, and Prasinophyceae. HTS data revealed that PEs were dominated by heterotrophs, such as Syndiniophyceae, parasitic dinoflagellates (79.67% of total 18S sequence reads), Dinophyceae (8.7%) and the radiolarians Collodaria belonging to Sphaerozoidae (22.1%) and Spumellaria (5.0%). On the other hand, photoautotrophs, including Chlorophyta (Mamiellophyceae, Prasinophyceae, Trebouxiophyceae, and Ulvophyceae), Stramenopiles (Bacillariophyta, Chrysophyceae, Dictyochophyceae, Pelagophyceae), photoautotrophic Cryptophyta and some Haptophyta (Prymnesiophyceae), did not exceed 5% of total sequence reads. This study provides the first snapshot of the PEs diversity in oligotrophic euphotic waters of the southern Adriatic Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin.

Morphological diversity and phylogeny of the diatom genus Entomoneis (Bacillariophyta) in marine plankton: six new species from the Adriatic Sea

The diatom genus Entomoneis is known from the benthos and plankton of marine, brackish, and freshwaters. Entomoneis includes diatoms with a bilobate keel elevated above the valve surface, a sigmoid canal raphe, and numerous girdle bands. Owing mostly to the scarcity of molecular data for a diverse set of species, the phylogeny of Entomoneis has not been investigated in depth. The few previous studies that included Entomoneis were focused on broader questions and the available data were from a small number of either unidentified Entomoneis or well‐known species (e.g., E. paludosa). Since the first description of new species combining both molecular and morphological characters (E. tenera), we have continued to cultivate and investigate Entomoneis in the plankton of the Adriatic Sea. Combined multigene phylogeny (SSU rDNA sequences, rbcL, and psbC genes) and morphological observations (LM, SEM and TEM) revealed six new Entomoneis species supported by phylogenetic and morphological data: E. pusilla, E. gracilis, E. vilicicii, E. infula, E. adriatica, and E. umbratica. The most important morphological features for species delineation were cell shape, the degree and mode of torsion, valve apices, the appearance and structure of the transition between keel and valve body, the ultrastructure and the shape of the girdle bands, and the arrangement and density of perforations along the valve and valvocopulae. Our results highlight the underappreciated diversity of Entomoneis and call for a more in‐depth morphological and molecular investigation of this genus especially in planktonic habitats.

Blue Diatoms: Global Phenomenon of ˝Greening˝ in Shellfish and Record of Planktonic Haslea Species in the South Adriatic Sea

Diatoms are unicellular, photoautotrophic eukaryotic microorganisms, often forming colonies and can be found in most aquatic and moist terrestrial habitats. All known diatoms today have specific golden-brown pigment fucoxanthin that masks chlorophylls in diatom plastid, but one genus represents an exception, having the additional specific pigment marennine due to whom the cells appear blue. Blue diatoms from genus Haslea cause a global phenomenon of ˝greening˝ in shellfish (mostly oysters) affecting them in both positive and negative ways. In this study, historical and recent review regarding blue diatoms and physiological and behavioural effect of marennine as well as challenges in shellfish farming from diatom perspective is addressed. This study is also a first record of blue Haslea diatom in the South Adriatic Sea during BIOTA (Bio-tracing Adriatic Water Masses) cruise in March 2016. Investigated blue diatom was cultured in laboratory and morphologically analysed with light microscopy. Diatom investigations are very important for better understanding of the ecology of specific marine area, but also for the economy, aquaculture and tourism. The emergence of green coloured flesh of shellfish in the Adriatic Sea has not been recorded yet, but this finding of the blue diatom from genus Haslea does not rule out this possibility in the future.

Rogoznica Lake, a Euxinic Marine Lake on the Adriatic Coast (Croatia) that Fluctuates Between Anoxic Holomictic and Meromictic Conditions

Rogoznica Lake is a typical example of euxinic marine lake, situated on the eastern Adriatic Coast, Croatia (43°32′ N 15°58′ E). It is a karstic depression with an area of 10.276 m2, filled with seawater with a maximum depth of 15 m. The lake is stratified during the spring–summer season, both thermally and in its salinity. There are oxic upper water layer and anoxic deeper layer. The mixolimnion varies seasonally in depth and thickness for about 1 m due to influence of meteorological conditions (temperature, wind, rainfall), which also affect the dynamics between the water layers. Rogoznica Lake can be considered as a system with both meromictic and holomictic conditions, which alternate seasonally and affect the lake’s ecology. Anoxic water is usually located below 10 m depth, and it is characterized by high concentrations of reduced sulphur species, nutrients and dissolved organic carbon. In anoxic water and laminated sediment, enrichment of trace metals, especially those that accumulate in sulphidic environment (Fe, Mo), is found. According to sedimentary enrichment of Mo (up to 81 mg kg−1), the lake can be classified as typical anoxic and meromictic environment. Extreme ecological conditions, which prevail in the lake, control the phytoplankton and zooplankton activities. Diatoms are the dominant microphytoplankton group, while oligotrich ciliates and copepods, the heterotrophic zooplankton organisms, play an important role and control the food web of the lake, especially in the post-holomictic–anoxic period. Ciliates by grazing on phototrophic sulphur bacteria at the chemocline transfer organic carbon to higher trophic levels, from anoxic to oxic conditions.