A polyphasic approach to the study of the genus Nitzschia (Bacillariophyta): three new planktonic species from the Adriatic Sea.

The paraphyletic diatom genus Nitzschia comprises over 1000 morphologically distinct pennate taxa, known from the benthos and plankton of freshwater, brackish, and marine environments. The principal diagnostic characters for delimitation of Nitzschia species include valve shape, the position and structure of the raphe, presence/absence and shape of the proximal raphe endings and terminal raphe fissures, areola structure, and specific morphometric features such as cell size, and stria and fibula density. In this study, we isolated 12 diatom strains into culture from samples collected at the surface or greater depths of the southeastern Adriatic Sea. Morphological analyses included LM, SEM, and TEM observations, which, along with specific morphometric features, allowed us to distinguish three new Nitzschia species. These findings were congruent with the results of phylogenetic analyses performed on nuclear-encoded SSU (18S) rDNA and chloroplast-encoded rbcL and psbC genes. One of the new species (Nitzschia dalmatica sp. nov.) formed a lineage within a clade of Bacillariaceae containing members of the Nitzschia sect. Dubiae, which was sister to Psammodictyon. A second lineage was part of a novel clade that is significantly distinct from other Nitzschia species sequenced so far and includes Nitzschia adhaerens sp. nov. and N. cf. adhaerens. A further new species was found, Nitzschia inordinata sp. nov., which appeared as the sister group to the N. adhaerens clade and the conopeoid Nitzschia species in our phylogenetic trees. Our findings contribute to the overall diversity of genus Nitzschia, especially in identifying some deep branches within the Bacillariaceae, and highlight under-scoring of this genus in marine plankton.

Assessment of holographic microscopy for quantifying marine particle size and concentration.

Holographic microscopy has emerged as a tool for in situ imaging of microscopic organisms and other particles in the marine environment: appealing because of the relatively larger sampling volume and simpler optical configuration compared to other imaging systems. However, its quantitative capabilities have so far remained uncertain, in part because hologram reconstruction and image recognition have required manual operation. Here, we assess the quantitative skill of our automated hologram processing pipeline (CCV Pipeline), to evaluate the size and concentration measurements of environmental and cultured assemblages of marine plankton particles, and microspheres. Over 1 million particles, ranging from 10 to 200 µm in equivalent spherical diameter, imaged by the 4-Deep HoloSea digital inline holographic microscope (DIHM) are analyzed. These measurements were collected in parallel with a FlowCam (FC), Imaging FlowCytobot (IFCB), and manual microscope identification. Once corrections for particle location and nonuniform illumination were developed and applied, the DIHM showed an underestimate in ESD of about 3% to 10%, but successfully reproduced the size spectral slope from environmental samples, and the size distribution of cultures (Dunaliella tertiolecta, Heterosigma akashiwo, and Prorocentrum micans) and microspheres. DIHM concentrations (order 1 to 1000 particles ml-1) showed a linear agreement (r 2 = 0.73) with the other instruments, but individual comparisons at times had large uncertainty. Overall, we found the DIHM and the CCV Pipeline required extensive manual correction, but once corrected, provided concentration and size estimates comparable to the other imaging systems assessed in this study. Holographic cameras are mechanically simple, autonomous, can operate at very high pressures, and provide a larger sampling volume than comparable lens-based tools. Thus, we anticipate that these characterization efforts will be rewarded with novel discovery in new oceanic environments.

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.

Dynamics of organic matter in the changing environment of a stratified marine lake over two decades.

The marine lake (Rogoznica Lake), which fluctuates between stratified and holomictic conditions, is a unique environment on the eastern Adriatic coast affected by environmental changes. These changes are reflected in the warming of the water column, the apparent deoxygenation of the epilimnion, and the accumulation of organic matter (OM), toxic sulfide, and ammonium in the anoxic hypolimnion. Since the early 1990s, the volume of anoxic water has increased as the chemocline has moved to the surface water layer. A trend toward enrichment of refractory dissolved organic carbon (DOC) was observed in the anoxic hypolimnion, while a decreasing trend was observed in the oxic epilimnion in the spring DOC. At the same time, the most reactive surface-active fraction of DOC showed the opposite trend. In addition, there is evidence of accumulation of particulate organic carbon (POC) in the water column, followed by an increase in the fraction of POC in total organic carbon (TOC). On a multi-year scale (1996-2020), this work presents a unique time series of the dynamics of OM in the stratified marine system, showing a significant change in its quantity and quality due to climate and environmental variability. DOC-normalized surfactant activity is shown to be a good indicator of environmental change.

Phytoplankton diversity and chemotaxonomy in contrasting North Pacific ecosystems.

Background: Phytoplankton is the base of majority of ocean ecosystems. It is responsible for half of the global primary production, and different phytoplankton taxa have a unique role in global biogeochemical cycles. In addition, phytoplankton abundance and diversity are highly susceptible to climate induced changes, hence monitoring of phytoplankton and its diversity is important and necessary. Methods: Water samples for phytoplankton and photosynthetic pigment analyses were collected in boreal winter 2017, along transect in the North Pacific Subtropical Gyre (NPSG) and the California Current System (CCS). Phytoplankton community was analyzed using light and scanning electron microscopy and photosynthetic pigments by high-performance liquid chromatography. To describe distinct ecosystems, monthly average satellite data of MODIS Aqua Sea Surface temperature and Chlorophyll a concentration, as well as Apparent Visible Wavelength were used. Results: A total of 207 taxa have been determined, mostly comprised of coccolithophores (35.5%), diatoms (25.2%) and dinoflagellates (19.5%) while cryptophytes, phytoflagellates and silicoflagellates were included in the group "others" (19.8%). Phytoplankton spatial distribution was distinct, indicating variable planktonic dispersal rates and specific adaptation to ecosystems. Dinoflagellates, and nano-scale coccolithophores dominated NPSG, while micro-scale diatoms, and cryptophytes prevailed in CCS. A clear split between CCS and NPSG is evident in dendogram visualising LINKTREE constrained binary divisive clustering analysis done on phytoplankton counts and pigment concentrations. Of all pigments determined, alloxanthin, zeaxanthin, divinyl chlorophyll b and lutein have highest correlation to phytoplankton counts. Conclusion: Combining chemotaxonomy and microscopy is an optimal method to determine phytoplankton diversity on a large-scale transect. Distinct communities between the two contrasting ecosystems of North Pacific reveal phytoplankton groups specific adaptations to trophic state, and support the hypothesis of shift from micro- to nano-scale taxa due to sea surface temperatures rising, favoring stratification and oligotrophic conditions.

Phytoplankton pigment dynamics in marine lake fluctuating between stratified and holomictic euxinic conditions.

Biomass dynamics in the marine lake are strongly dependent on seasonal variability in vertical stratification, indicating rapid adaptation of phytoplankton to short-term changes in the water column. A small marine lake (Rogoznica Lake, Croatia), which fluctuates between stably stratified and holomictic euxinic conditions, was used as a model to study the phytoplankton responses to environmental perturbations, in particular the anoxic stress, caused by periodic holomixia. The epilimnion showed significant temporal and vertical variability with a chlorophyll a subsurface maximum with the highest biomass near the chemocline. Fucoxanthin-containing biomass (diatoms) dominated in the epilimnion in colder seasons and was first to recover after holomictic euxinic events. The shift towards the smaller groups prevailed during highly stratified water column conditions in warmer seasons. Results for the hypolimnion were more enigmatic, with high concentrations of alloxanthin, zeaxanthin, and violaxanthin indicating the presence of a viable small-size mixotrophic community under extreme conditions.

Tracing the path of carbon export in the ocean though DNA sequencing of individual sinking particles.

Surface phytoplankton communities were linked with the carbon they export into the deep ocean by comparing 18 S rRNA gene sequence communities from surface seawater and individually isolated sinking particles. Particles were collected in sediment traps deployed at locations in the North Pacific subtropical gyre and the California Current. DNA was isolated from individual particles, bulk-collected trap particles, and the surface seawater. The relative sequence abundance of exported phytoplankton taxa in the surface water varied across functional groups and ecosystems. Of the sequences detected in sinking particles, about half were present in large (>300 µm), individually isolated particles and primarily belonged to taxa with small cell sizes (<50 µm). Exported phytoplankton taxa detected only in bulk trap samples, and thus presumably packaged in the smaller sinking size fraction, contained taxa that typically have large cell sizes (>500 µm). The effect of particle degradation on the detectable 18 S rRNA gene community differed across taxa, and differences in community composition among individual particles from the same location largely reflected differences in relative degradation state. Using these data and particle imaging, we present an approach that incorporates genetic diversity into mechanistic models of the ocean's biological carbon pump, which will lead to better quantification of the ocean's carbon cycle.

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.

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.