Sunčica Bosak

Composition and annual cycle of phytoplankton assemblages in the northeastern Adriatic Sea

Phytoplankton abundance and seasonality in the northeastern Adriatic Sea were directly related to the annual regime of the Po River discharge in the period 2002–2007. In 2005, 2006, and 2007, there was a shift in the timing of the annual Po River maximum discharge and of phytoplankton maxima from spring to autumn. In addition, the eastern coastal area was greatly influenced by oligotrophic karstic rivers and the Eastern Adriatic Current. Dominant phytoplankton taxa were defined on the basis of abundance and frequency of appearance. Such an approach may help in the comparison of phytoplankton communities within different environments. The dominant taxa were the diatoms Cerataulina pelagica, Chaetoceros socialis, Chaetoceros vixvisibilis and Pseudo-nitzschia spp., which appeared at maximum abundances )106 cells l-1 in more than 20% of samples. Among other phytoplankton, the most common was the coccolithophorid Emiliania huxleyi (maximum abundance of 2=10 5 cells l -1 in 50% of samples), and the dinoflagellate Prorocentrum minimum (maximum abundance of 3=10 5 cells l -1 in 12% of samples). The present state of the phytoplankton community may provide valuable information for evaluation of possible future environmental changes in the shallow Mediterranean ecosystem.

A Novel Type of Colony Formation in Marine Planktonic Diatoms Revealed by Atomic Force Microscopy

Diatoms have evolved a variety of colonial life forms in which cells are connected by organic threads, mucilage pads or silicate structures. In this study, we provide the first description of a novel strategy of colony formation among marine planktonic diatoms. Bacteriastrum jadranum forms loose but regular chains with distinct heterovalvate terminal cells. The colonial cells and their siliceous projections, the setae, are not in direct contact; instead, they are enclosed within the optically transparent organic matrix. This cell jacket structure was detected by staining procedure with Alcian Blue, which showed that the polysaccharides are predominant matrix constituents and revealed that the jacket reaches the span of the setae. The scanning electron microscopy (SEM) observations showed distinguishable fibrillar network firmly associated with cells. Using atomic force microscopy (AFM), we were able to visualise and characterise the cell jacket structure at molecular resolution. At nanoscale resolution, the cell jacket appears as a cross-linked fibrillar network organised into a recognisable structure. The circular patches of self-repeating pattern (hexagonal pores with openings of 8–100 nm) are connected through thicker surrounding fibrils and reinforced by branching fibrils. The pore-forming fibrils within the patches are only 0.6–1.6 nm high, the surrounding fibrils connecting patches are 2.0–2.8 nm high, and the branching fibrils are considerably wider but not higher than 4.0 nm. The discovered polysaccharide fibrillar network is highly organised and delicately structured with a monomolecular fibril height of 0.6 nm. We conclude that the Bacteriastrum polysaccharide jacket represents an essential part of the cell, as the conjunction of the polymer network with the frustule appears to be extremely tight and such specific and unique patterns have never been found in self-assembled polysaccharide gel networks, which are usually encountered in the marine environment.

Phytoplankton distribution across the southeast Adriatic continental and shelf slope to the west of Albania (spring aspect)

We present the first insight to the oceanography of the southeastern Adriatic Sea, where coastal water influenced by Albanian rivers comes into contact with the inflowing oligotrophic Eastern Adriatic Current (Ionian Surface Water and Levantine Intermediate Water). A distinct plankton distribution was observed on each side of the shelf break hydrographic boundary in May 2009, during gradual warming of the surface waters. The prochlorophytes accumulated along the nutricline above the shelf and continental slope. The phosphorus limited inshore waters were dominated by a small diatom Chaetoceros circinalis, dinoflagellates, cryptophytes, autotrophic picoplankton, and heterotrophic nanoplankton. The offshore surface layer was characterized by bigger nanoplankton (coccolithophorids, green flagellates). Low nutrient concentrations influence relatively low productivity not only above the Albanian shelf but also further to the north along the Montenegrine and Croatian coastal Adriatic Sea.

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.

Defining centric diatoms of most relevant phytoplankton functional groups in deep karst lakes

Phytoplankton in karst lakes is characterized by co-occurrence of chrysophytes (Chrysophyceae), dinoflagellates (Dinophyceae), and diatoms (Bacillariophyta) as the most diverse and abundant group. Using Reynolds functional approach in ecological phytoplankton investigations makes karstic lakes comparable and facilitates interpretation of their responses to changing environmental conditions. Accurate taxonomic identification to species level, based on precise resolution of specific characteristics by electron microscopy, is essential in order to correctly associate species into coda. This paper defines the most abundant centric diatoms and coexisting species in the phytoplankton of karst lakes in Croatia as a contribution to better description of phytoplankton functional groups dominated by centric diatoms. Several representatives for diatom-based Reynolds functional groups of four lakes in Croatia were described in this paper: Pantocsekiella costei and Stephanocostis chantaica for group A, Pantocsekiella ocellata and Cyclotella distinguenda for group B, and Stephanodiscus neoastraea for group C, together with their ecological preferences which clearly correspond to pertaining functional classification. Selected and coexisting functional groups define natural, oligo- to mesotrophic karst deep lake systems.

Morphology and phylogeny of four species from the genus Bacteriastrum (Bacillariophyta)

Abstract: Four colonial species of the multipolar centric diatom genus Bacteriastrum – B. furcatum, B. hyalinum, B. jadranum and B. mediterraneum – were investigated using field samples from the Adriatic Sea and cultured strains obtained from these samples. Morphology and frustule ultrastructure were examined using light and electron microscopy, and nuclear-encoded large-subunit (LSU) rDNA sequences were obtained from the strains. The description of B. jadranum has been emended based on a novel mechanism of colony formation in Bacteriastrum in which cells are joined through an organic cell jacket which holds together cells in chains. Descriptions for two of the three other species were supplemented; vegetative cells of B. hyalinum have short Y spinules on the fused part of the setae and T-shaped outgrowths, and the spores of this species possess a ring of puncta and a granular surface on the exterior side of the primary valve mantle. Specific for B. furcatum is the location of pores scattered around the setae bases. No unique ultrastructural character was found for B. mediterraneum. Phylogenetic results grouped the sequences of the four species in a clade in which B. furcatum (section Sagittata) and B. hyalinum (section Isomorpha) formed a clade as sister to a clade with B. jadranum (section Isomorpha) and B. mediterraneum (section Sagittata). Apparently, neither section was monophyletic, and the supposed defining features of isomorphic terminal setae in Isomorpha and dimorphic ones in Sagittata were not synapomorphies. Nonetheless, the genus Bacteriastrum itself remained monophyletic.

Morphological study of Chaetoceros wighamii Brightwell (Chaetocerotaceae, Bacillariophyta) from Lake Vrana, Croatia

Abstract Chaetoceros wighamii Brightwell is a planktonic diatom species originally described from brackish waters. Since its original description, the species has been reported in a wide variety of habitats, ranging from freshwater to marine. Varying descriptions exist in the taxonomic literature and several taxa have been considered as synonyms, including freshwater species Chaetoceros amanita. In this study we provide morphological and ultrastructural information on a cultured strain isolated from freshwater sample collected in the Lake Vrana (Vransko jezero) in Croatia, in April 2011. The cells form short and robust chains with very narrow apertures, often partially occluded by silica membranes. Other distinctive features observable in light microscopy are the shape and orientation of the setae which are very long, straight and robust, diverging in various directions from the chain axis and the single parietal chloroplast extending from valve to valve. Distinct ultrastructural characteristics are the absence of processes either in intercalary or terminal valves and the ornamentation of the valve face with densely distributed ribs spreading from an irregular eccentric hyaline area without a clearly defined annulus. The outer surface of the terminal valve is ornamented with small spines and setae are composed of fl at longitudinal filaments interconnected with short bars and ornamented with small spines tightly arranged around the setae. Our description agrees well with that reported for the freshwater morphotypes of C. wighamii (syn. C. amanita) and contributes for a reliable distinction of this intriguing taxon from similar morphotypes. This finding supports the interpretation of Chaetoceros wighamii as a freshwater/brackish species and represents the first report of a Chaetoceros species in lacustrine environment in Croatia and possibly in any Central European habitats.

Winter picoplankton diversity in an oligotrophic marginal sea

Marine picoplankton, unicellular organisms with cell sizes up to 3 μm 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 7 × 105; 2.3 × 104 and 2.5 × 104 cells mL-1, respectively, while photosynthetic picoeukaryotes peaked with 3 × 103 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.