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Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbor diverse prokaryotic and eukaryotic microbes, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides an inventory of diatoms, bacteria and diatom-associated bacteria originating from loggerhead sea turtles using both metabarcoding and culturing approaches. Amplicon sequencing of the carapace and skin samples chloroplast gene rbcL and 16S rRNA gene detected, in total, 634 diatom amplicon sequence variants (ASVs) and 3661 bacterial ASVs, indicating high diversity. Cultures of putative epizoic and non-epizoic diatoms contained 458 bacterial ASVs and their bacterial assemblages reflected those of their host. Diatom strains allowed for enrichment and isolation of bacterial families rarely observed on turtles, such as Marinobacteraceae, Alteromonadaceae and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed that related diatom genera might retain similar microbial taxa in culture, regardless of the turtle's skin or carapace source. These data provide deeper insights into the sea turtle-associated microbial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations.

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 the shape of the 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.

Diatoms and bacteria are known for being the first colonizers of submerged surfaces including the skin of marine reptiles. Sea turtle carapace and skin harbour diverse prokaryotic and eukaryotic microbial taxa, including several epizoic diatoms. However, the importance of diatom-bacteria associations is hardly investigated in biofilms associated with animal hosts. This study provides a detailed inventory of diatoms, bacteria, and diatom-associated bacteria originating from several loggerhead sea turtles using a combination of metabarcoding and culturing approaches. Carapace and skin samples rbcL and 16S rRNA amplicon sequencing showed a high diversity of diatoms and bacteria, respectively. Cultures of putative epizoic and non-epizoic diatom strains contained from 18 to 101 bacterial amplicon sequence variants (ASVs) and their bacterial assemblages strongly reflected those of their source host. Diatom strains allowed for enrichment and isolation of rare-in-source bacterial families such as Marinobacteraceae, Alteromonadaceae, and Alcanivoracaceae. When accounting for phylogenetic relationships between bacterial ASVs, we observed related diatom genera might retain related microbial taxa in culture, regardless of the source environment. These data provide deeper insights into several levels of sea turtle epizoic diatom and bacterial communities, and reveal the potential of epizoic biofilms as a source of novel microbes and possibly important diatom-bacteria associations.

Our understanding of the importance of microbiomes on large aquatic animals—such as whales, sea turtles and manatees—has advanced considerably in recent years. Recent activity describing epizoic diatoms suggests that epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful attempt to culture putatively obligate epizoic diatoms without their hosts and propose further applications and research avenues in this growing area of study. Analyzing the DNA sequences of these cultures, we found that several unique diatom taxa have independently evolved to occupy in epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. Our discovery that epizoic diatoms can be cultured independently from their hosts raises several questions about the nature of the interaction between these diatom species and their hosts. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Background: Our understanding of the importance of microbiomes on large aquatic animals—such as whales, sea turtles and manatees—has advanced considerably in recent years. Recent activity describing the epizoic diatoms growing on marine vertebrates suggests that these epibiotic diatom communities constitute diverse, polyphyletic, and compositionally stable assemblages that include both putatively obligate epizoic and generalist species. Here, we outline a successful attempt to culture putatively obligate epizoic diatoms without their hosts and propose further applications and research avenues in this growing area of study. Results: We cultured cells of epizoic diatoms from multiple host species sampled in the wild and captivity. Analyzing the DNA sequences of these cultures, we found that several unique diatom taxa have independently evolved to occupy in epibiotic habitats. We created a library of reference sequence data for use in metabarcoding surveys of sea turtle and manatee microbiomes that will further facilitate the use of environmental DNA for studying host specificity in epizoic diatoms and the utility of diatoms as indicators of host ecology and health. Conclusions: Our discovery that epizoic diatoms can be cultured independently from their hosts raises several questions about the nature of the interaction between these diatom species and their hosts. We encourage the interdisciplinary community working with marine megafauna to consider including diatom sampling and diatom analysis into their routine practices.

Background Microbial communities of wild animals are being increasingly investigated to provide information about the hosts’ biology and promote conservation. Loggerhead sea turtles (Caretta caretta) are a keystone species in marine ecosystems and are considered vulnerable in the IUCN Red List, which led to growing efforts in sea turtle conservation by rescue centers around the world. Understanding the microbial communities of sea turtles in the wild and how affected they are by captivity, is one of the stepping stones in improving the conservation efforts. Describing oral and cloacal microbiota of wild animals could shed light on the previously unknown aspects of sea turtle holobiont biology, ecology, and contribute to best practices for husbandry conditions. Results We describe the oral and cloacal microbiota of Mediterranean loggerhead sea turtles by 16S rRNA gene sequencing to compare the microbial communities of wild versus turtles in, or after, rehabilitation at the Adriatic Sea rescue centers and clinics. Our results show that the oral microbiota is more sensitive to environmental shifts than the cloacal microbiota, and that it does retain a portion of microbial taxa regardless of the shift from the wild and into rehabilitation. Additionally, Proteobacteria and Bacteroidetes dominated oral and cloacal microbiota, while Kiritimatiellaeota were abundant in cloacal samples. Unclassified reads were abundant in the aforementioned groups, which indicates high incidence of yet undiscovered bacteria of the marine reptile microbial communities. Conclusions We provide the first insights into the oral microbial communities of wild and rehabilitated loggerhead sea turtles, and establish a framework for quick and non-invasive sampling of oral and cloacal microbial communities, useful for the expansion of the sample collection in wild loggerhead sea turtles. Finally, our investigation of effects of captivity on the gut-associated microbial community provides a baseline for studying the impact of husbandry conditions on turtles’ health and survival upon their return to the wild.

During a survey of the epizoic diatom flora on carapaces of loggerhead sea turtles (Caretta caretta) from the Adriatic Sea, a fairly large population of an unknown marine Planothidium species was observed. Detailed morphological analysis based on scanning electron microscopy observations and comparison with a large number of known Planothidium species, resulted in the description of the new species as Planothidium kaetherobertianum sp. nov. The new species is characterized by a rapheless valve showing a rather broad axial area with irregular, shallow depressions, striae composed of only 1–2 rows of very small areolae. The raphevalve has biseriate striae with a short third row of areolae inserted between the two rows of areolae near the valve margins. The species shows some resemblance with the group of species around Planothidium delicatulum. The new species is illustrated using both LM and SEM observations and compared with other resembling marine and brackish Planothidium species.

Hyalosira gene sequences are divided into two clades within different families. We examined authentic material of Hyalosira (isotype material of H. obtusangula, synonymous with H. delicatula) and voucher specimens of published sequences, and pooled our observations of Hyalosira-like taxa from benthic and epizoic habitats in several parts of the globe. The two molecular clades corresponded to two morphological groups, with Hyalosira obtusangula associated with Grammatophoraceae. We emend the description and provide lectotypification for Hyalosira and propose Placosira to encompass the taxa in the other clade, associated with Rhabdonemataceae. We propose that Hyalosira has uniseriate to triseriate striae, sometimes different on valve face and mantle. Copulae in most species had shallow septa, though in one they were moderately deep. All species had girdle bands bearing two rows of areolae separated by a midrib. We name five new species of Hyalosira. Morphologies of taxa in the Placosira clade were superficially similar to Hyalosira but differed in having areolae with ricae, a single row of areolae on the girdle bands, and tubular rimoportulae on the valve-face-mantle junction. Hyalosira hustedtiana Patrick should revert to its original position in Striatella until the appropriate genus can be determined. We emend Rhabdonematales to encompass Rhabdonemataceae, Grammatophoraceae and Tabellariaceae. (C) 2021 Elsevier GmbH. All rights reserved.

Chrysophyte algae live predominantly in freshwater habitats, therefore marine chrysophyte stomatocysts are less common and poorly documented. Here we report on the occurrence of two unknown chrysophycean stomatocysts in a complex epizoic biofilm formed on a carapace of a loggerhead sea turtle (Caretta caretta) found injured on the southeastern coast of the Adriatic Sea and admitted to the Sea Turtle Rescue Centre in Aquarium Pula, Croatia. Stomatocyst #1 is characterized by a thickened circulus giving the cyst an asymmetrical outlook whereas stomatocyst #2 has a thick, wide, irregularly shaped collar. Detailed morphological investigations based on scanning electron microscopy observations and comparison with known chrysophyte stomatocysts worldwide, showed that both should be described as new to science following the guidelines of the International Statospore Working Group (ISWG).

The current study focuses on four species from the primarily marine diatom genus Craspedostauros that were observed growing attached to numerous sea turtles and sea turtle-associated barnacles from Croatia and South Africa. Three of the examined taxa, C. danayanus sp. nov., C. legouvelloanus sp. nov., and C. macewanii sp. nov., are described based on morphological and, whenever possible, molecular characteristics. The new taxa exhibit characters not previously observed in other members of the genus, such as the presence of more than two rows of cribrate areolae on the girdle bands, shallow perforated septa, and a complete reduction of the stauros. The fourth species, C. alatus, itself recently described from museum sea turtle specimens, is reported for the first time from loggerhead sea turtles rescued in Europe. A 3-gene phylogenetic analysis including DNA sequence data for three sea turtle-associated Craspedostauros species and other marine and epizoic diatom taxa indicated that Craspedostauros is monophyletic and sister to Achnanthes. This study, being based on a large number of samples and animal specimens analyzed and using different preservation and processing methods, provides new insights into the ecology and biogeography of the genus and sheds light on the level of intimacy and permanency in the host-epibiont interaction within the epizoic Craspedostauros species.

Sea turtles are useful sentinels to monitor the dissemination of antimicrobial resistance (AMR) in the marine coastal ecosystems. Forty Gram negative bacteria were isolated from wounds of 52 injured Caretta caretta, living in the Mediterranean Sea. Bacteria were identified using 16S rRNA gene sequencing and tested for susceptibility to 15 antibiotics. In addition, NGS amplicon sequencing was performed to detect the presence of AmpC β-lactamase genes (blaAmpC) and extended-spectrum β-lactamase (ESBL) genes (blaCTX-M, blaSHV, blaTEM). Seventy-five percent of the isolates (30/40 isolates) exhibited multidrug resistance (MDR) phenotypes and 32.5% (13/40 isolates) were confirmed to be positive for at least one gene. The variants of ESBLs genes were blaCTX-M-3, blaTEM-236 and blaSHV-12. Variants of the blaAmpC β-lactamase gene i.e., blaACT-24, blaACT-2, blaACT-17, blaDHA-4 and blaCMY-37, were also detected. In addition, 4 isolates were found simultaneously harboring CTX and AmpC genes while 2 strains harbored 3 genes (blaACT-2+TEM-236+SHV-12, and blaCTX-M-3+ACT- 24+TEM-236).

Bacteria are the most important microorganisms in the world oceans, accounting for up to 75% of the total biomass. They are responsible for fundamental biogeochemical processes and therefore often used as ecological indicators. In this study, bacteria were quantified by flow cytometry and their diversity assessed by High Throughput Sequencing (HTS) in the southern Adriatic Sea. The most abundant bacterial groups were also quantified by qPCR. The samples were collected from the surface to the seabed over a total of 16 different depths at four stations during the late winter BIOTA (BIO-Tracing Adriatic water masses) cruise conducted in March 2016. The investigated area showed unusual water mass properties and was characterized by a shallow mixed layer, which differed from the usual winter convection conditions, typical of middle-altitude ecosystems and important for the seasonal picoplankton dynamics of this area. Heterotrophic bacteria were separated into HNA (relative High Nucleic Acid content) and LNA (Low Nucleic Acid content) subpopulations with abundances up to 1.8 × 105 and 8.8 × 105 cells mL−1, respectively. HNA dominated at offshore stations reaching their maximum at depths below the euphotic zone. The bacterial community was dominated by Alphaproteobacteria, accounting for >40% of the total sequence reads and were mainly represented by the SAR11 clade (90.84%), followed by Marinimicrobia (18% of the total sequence reads), mainly represented by clade SAR406 (8.44%). Distinctive bacterial groups were found in the euphotic layer (Bacteroidetes and Actinobacteria) and aphotic layer samples (Deltaproteobacteria, Marinimicrobia, Chloroflexi, Acidobacteria and Planctomycetes). Results of the qPCR analyses further confirmed HTS results with highest abundances obtained for Alphaproteobacteria, followed by Gammaproteobacteria and Bacteroidetes. The adopted multiple approach, combining different molecular tools, critically supported by optics and flow cytometry, reveal changes in the bacterial assemblages during the unusual thermohaline conditions observed in 2016 in the southern Adriatic Sea.

During a survey of the diatom flora attached to carapaces of loggerhead sea turtles (Caretta caretta) from the Adriatic Sea, a large population of an unknown marine achnanthoid species was observed that could not be assigned to any of the currently described monoraphid diatom genera. Detailed morphological analysis based on scanning electron microscopy observations and comparison with a large number of monoraphid genera such as Madinithidium, Scalariella, Kolbesia and Karayevia, resulted in the description of the new benthic marine genus Majewskaea gen. nov. with the newly described Majewskaea istriaca sp. nov. as generitype. The new genus is characterized by a sternum valve showing a clear recurving ridge surrounding the entire valve margin, the presence of macroareolae closed internally by perforated hymenes, separated by a longitudinal hyaline area, a well-developed sternum and vestigial raphe slits. The raphe valve has a simple straight raphe with bent terminal fissures, a very fine striation pattern composed of macroareolae cut in two by a longitudinal hyaline zone, and a very shallow mantle. The new genus is illustrated using both LM and SEM observations and compared with other resembling marine and brackish Planothidium monoraphid species.

In recent years, biofilm-forming diatoms have received increased attention as sea turtle epibionts. However, most of the research has focused on carapace-associated taxa and communities, while less is known about diatoms growing on sea turtle skin. The current study investigated diatom diversity on the skin of loggerhead sea turtle heads detached from the carcasses found along the Adriatic coast between 1995 and 2004 and stored frozen for a prolonged period of time. By using both light and scanning electron microscopy we have found diatom frustules in 7 out of 14 analysed sea turtle samples. Altogether, 113 diatom taxa were recorded, with a minimum of seven and a maximum of 35 taxa per sample. Eight taxa, Achnanthes elongata, Berkeleya cf. fennica, Chelonicola sp., Licmophora hyalina, Nagumoea sp., Navicula sp., Nitzschia cf. lanceolata, and Poulinea lepidochelicola exceeded 5% of relative abundance in any one sample. The presumably obligately epizoic diatom taxa, A. elongata, Chelonicola sp., and P. lepidochelicola, dominated in six loggerhead samples, contributing up to 97.1% of the total diatom abundance. These observations suggest that on the sea turtle skin highly specialised taxa gain even greater ecological advantage and dominance over the co-occurring benthic forms than in the carapace biofilms. The suitability of frozen sea turtle skin specimens for diatom analysis and limitations of this approach are discussed.

Epizoic diatoms form an important part of micro-epibiota of marine vertebrates such as whales and sea turtles. The present study explores and compares the diversity and biogeography of diatom communities growing on the skin and shell of loggerhead sea turtles (Caretta caretta) from four different localities: Adriatic Sea (Croatia), Ionian Sea (Greece), South Africa and Florida Bay (USA) using both light and scanning electron microscopy. We observed almost 400 diatom taxa belonging to more than 100 genera. Diatom communities from Greece and Croatia showed the highest similarity and were statistically different from those recorded from South Africa and Florida. Part of this variation could be attributed to differences in sampling techniques; however, we believe that geography had an important role. In general, contrary to several previous observations from sea turtles, the presumably exclusively epizoic diatoms contributed less than common benthic taxa to the total diatom flora, which might have been related to the loggerhead feeding behavior. Moreover, skin samples differed from carapace samples in having a distinct diatom composition with a higher proportion of the putative true epizoonts. Our results indicate that epizoic diatom communities differ according to loggerhead geographical location and substrate (skin vs. carapace). The relative abundances of common benthic diatoms and putative exclusive epizoic taxa may inform about sea turtle habitat use or behavior though detailed comparisons among different host species have yet to be performed.

The planktonic diatoms belonging to two genera Chaetoceros and Bacteriastrum , included within the family Chaetocerotaceae, are ecologically important as they represent an important component of the phytoplankton in the coastal regions and are often among bloom-forming taxa. We analysed the chaetocerotacean species composition and abundances in the coastal area of northeastern Adriatic in a biweekly study conducted from September 2008 to October 2009 with the aim of investigating seasonal dynamics and species succession on a fine temporal scale and determining the most important ecological factors influencing their distribution. The study identified seven Chaetoceros and three Bacteriastrum species as major phytoplankton components showing the clear annual succession and two types of blooms (one species/multi species) governed by differing ecological conditions. Autumn bloom was composed of 20 chaetocerotacean species with Chaetoceros contortus and Chaetoceros vixvisibilis alternating in dominance. The summer period was characterized by spreading of freshwater from the Po River up to the eastern coast increasing availability of phosphate which triggered the monospecific Chaetoceros vixvisibilis bloom. We explained the chaetocerotacean dominant species succession pattern by the environmental parameters, with the temperature, salinity and phosphate availability as most important factors driving the species seasonality.

During a survey of the epizoic diatom flora on carapaces of loggerhead sea turtles (Caretta caretta) from the Adriatic Sea, a relatively small population of an unknown amphoroid species was observed, presenting some morphological similarities with the genera Lunella and Catenula. Due to the small size and the lack of distinct structures visible in LM, the observations of its ultrastructure under very high magnification in the scanning electron microscopy (SEM) proved to be critical in taxon delineation and indicated that it should be separated as a new Catenula species. The new species is characterized by very small, dorsiventral valves, striae composed of 1–3 small, marginally placed areolae, a very simple raphe structure lacking terminal raphe fissures, straight simple central raphe endings, the absence of helictoglossa and a distinct central nodule, the latter often discernible in LM. Typical ghost striae, reported to be one of the main morphological features of the genus Catenula, are very faintly present and only visible in the valve interior. The new species is illustrated using both LM and SEM observations and compared with all other known Catenula and Lunella species.

The diatom genus Proschkinia is a common element of biofilms covering diverse substrata in saline inland or shallow coastal environments. It can be distinguished from other naviculoid taxa by its lanceolate valves with a fistula located within the central area and numerous open girdle bands with a U-shaped cross-section and a single row of perforations on the internal side of the fold. Despite this distinct morphology, frustules of Proschkinia are typically weakly silicified and often overlooked when cleaned diatom material is analysed. The current paper describes six new species of Proschkinia: P. browderiana sp. nov., P. lacrimula sp. nov., P. maluszekiana sp. nov., P. sulcata sp. nov., P. torquata sp. nov. and P. vergostriata sp. nov., found in numerous samples of marine organisms, such as sea turtles (including sea turtle museum specimens), sea turtle-associated barnacles and seagrass from across the three oceans. Some of the newly described taxa were found on multiple individuals belonging to different sea turtle species, whereas others were in samples collected from different continents. Molecular phylogenetic analysis indicated that examined Proschkinia strains formed a monophyletic clade, sister to Fistulifera.

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.

Summary Unusual occurrence of planktonic diatom species, Chaetoceros bacteriastroides and Chaetoceros pseudosymmetricus , was noticed in three different marine ecosystems of Adriatic Sea: the Krka Estuary and Telascica Bay in the Central Adriatic, and in southern Adriatic offshore. From 2010 to 2015, these two Chaetoceros species were recorded in heterogeneous environmental conditions and in a very low abundances. Both species are regarded as very rare in world oceans, and consequently knowledge of their distribution and ecology is rather poor. Primarily described from tropical waters and showing Indo-Pacific distribution, C . bacteriastroides and C . pseudosymmetricus findings in Adriatic represent the northernmost records in world's oceans and seas. For C. pseudosymmetricus this is also the first occurrence in European seas. Areal expansion and introduction of new phytoplankton species in the Adriatic Sea might be related to different circulation regimes in the Ionian Sea and the concurrent rise in sea temperature in the Mediterranean in the last decade. Recent investigations have shown that entering currents, of either Atlantic/Western Mediterranean or Eastern Mediterranean origin, modify the composition of the plankton community in the Adriatic by bringing different newcomers.

Neodelphineis indica is a pelagic araphid diatom described from the warm waters of the Indian and Pacific Oceans. These lightly silicified diatoms are characterized with non- aligned striae across the sternum, two rimoportulae and a single, small, unoccluded pore at each end of the valve. Here, we provide new and complementary morphological observations on the species, collected from different locations: the Adriatic Sea, where it was recorded for the first time, and the Pacifi c Ocean from the Mexican coast. The basic morphology described in our specimens confirms previous observations, but we also detected morphological variability in shape, valve length and chain formation. The finding of the species in the Adriatic Sea rises questions as to its presence there, as it may be well considered as an "invasive" species, arriving to the area as a consequence of the "tropicalization" of the Mediterranean 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.

Phytoplankton assemblages and pigment distribution were determined in the transitional Boka Kotorska Bay. Samples were collected four times from three stations in the inner part of the Bay between April 2008 and March 2009. Microscopic analysis revealed that the phytoplankton was composed of marine diatoms, dinoflagellates, cryptophytes, chlorophytes and coccolithophorids. Aside from fucoxanthin as the dominant biomarker pigment, alloxanthin, 19′-hexanoyloxyfucoxanthin, peridinin and chlorophyll b provided indicative contributions. Fucoxanthin and alloxanthin showed significant correlation with the phytoplankton chlorophyll a biomass throughout the investigated period. In November 2008, the diatoms were outcompeted by coccolithophorids, which probably efficiently absorbed nutrients during a period of their reduced supply. Due to the reduced nutrient input, in the summer, the phytoplankton community in the Bay was composed mostly of marine dinoflagellates, cryptophytes, and chlorophytes., Prisutnost zajednica fitoplanktona i rasprostranjenost pigmenata utvrđivani su u tranzicijskim vodama Bokokotorskog zaljeva. Uzorci su prikupljeni četiri puta na tri lokaliteta u unutrašnjosti Zaljeva u periodu između travnja 2008. i ožujka 2009. godine. Mikroskopskom analizom utvrđeno je da fitoplankton čine morske dijatomeje, dinoflagelati, kriptofiti, klorofiti i kokolitoforidi. Pored fukoksantina kao dominantnog biomarkerskog pigmenta, bili su zastupljeni i aloksantin, 19′-heksanoiloksifukoksantin, peridinin i klorofil b‏. Fukoksantin i aloksantin su pokazali značajnu korelaciju sa klorofilom a kao indikatorom biomase fitoplanktona tijekom čitavog perioda ispitivanja. U studenom 2008. godine, dijatomeje su nadjačale kokolitoforide, koji su vjerovatno efikasno apsorbirali nutrijente tijekom perioda u kojem je njihova dostupnost bila smanjena. Kao rezultat smanjenog unosa nutrijenata, u ljetnom periodu, zajednicu fitoplanktona u Bokokotorskom zaljevu sačinjavali su uglavnom morski dinoflagelati, kriptofiti i klorofiti.

Rod Entomoneis Ehrenberg obuhvaća vrste penatnih dijatomeja koje su uglavnom opisane iz bentičkih brakičnih i morskih staništa te ih vrlo rijetko nalazimo u planktonu i slatkim vodama. Ovaj rod dijatomeja karakterizira složena struktura silicijevih ljušturica, sinusoidni uzdignuti kanal rafe, morfološki promjenjive fibule, brojni pojasevi između valvi te česta torzija samih stanica s obzirom na uzdužnu os. U Jadranskom moru nalazimo samo šest vrsta roda Entomoneis, od kojih smo prvu planktonsku vrstu, Entomoneis tenera, otkrile 2015. prilikom terenskog istraživanja južnog Jadrana te objavile opis vrste 2017. godine (Mejdandžić i i sur. 2017). Istraživanja u sklopu projekta Hrvatske zaklade za znanost - „Bio-tracing Adriatic water masses“ (BIOTA) su nastavljena i u ožujku 2016. sa nizom uzorkovanja i mjerenja na području obalnog i otvorenog mora od Dubrovnika do južnojadranske kotline. Za potrebe detaljnijih taksonomskih istraživanja prikupljali su se živi uzorci fitoplanktona s različitih dubina (površina, 30 m, 100 m, 250 m) u kojima smo primijetile nove, do sada nepoznate vrste roda Entomoneis. Pojedinačne stanice iz uzoraka smo izolirale u dvanaest monoklonalnih laboratorijskih kultura na kojima su se provela iscrpna morfološka (svjetlosna i elektronska mikroskopija) i molekularna (tri genska markera i filogenetske analize) istraživanja. Rezultati morfometrije, morfoloških karakteristika i filogenije potvrdili su da se radi o šest novih vrsta za znanost (Sl. 1) koje su nazvane redom: Entomoneis pusilla Bosak & Mejdandžić što znači „vrlo mala“ jer je do sada to najmanja vrsta ovog roda, E. gracilis Mejdandžić & Bosak prema „vitkom” izgledu stanica, E. infula Mejdandžić & Bosak opisujući sedlasti izgled stanica uslijed torzije, E. adriatica Mejdandžić & Bosak koja je ime dobila prema lokalitetu pronalaska, Jadranskom moru, te E. umbratica Mejdandžić & Bosak čije ime znači „živi u sjeni“ jer je pronađena u uzorku uzetom s 250 m dubine. Posljednja novootkrivena vrsta je dobila ime E. vilicicii Bosak & Mejdandžić u čast profesoru emeritusu Damiru Viličiću koji je svojim radom značajno pridonio straživanjima fitoplanktona Jadrana. Svi opisi novih vrsta iz Jadrana napravljeni su u suradnji s kolegama s Biološkog odsjeka PMF-a (Zagreb), Instituta Ruđer Bošković, Centra za forenzična ispitivanja, istraživanja i vještačenja Ivan Vučetić te Sveučilišta u Arkansasu (SAD) te je rad objavljen u prestižnom znanstvenom časopisu Journal of Phycology.

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.

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 100 m 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.

A 275-km-long transversel Northern Adriatic profile from the mouth of the Po River (Italian Adriatic coast) to the Kvarner region (Croatian coastal island area) was investigated in three successive case studies in August 2008, 2009 and 2010. The short Po River pulses in August result in the surface advection of riverine water, nutrients and phytoplankton from the western to the eastern side of the Adriatic. This surface spreading exhibits inter-annual variability depending on the riverine discharge in the preceding period. The Po River discharge pulse in August 2010 in particular resulted in an extraordinary tongue-like advection of riverine water, nutrients, and phytoplankton towards the Eastern Adriatic coast. The phenomenon was detected using both satellite imagery and classical oceanographic measurements. In the advective water, toxic dinoflagellates were most abundant in August 2010, when the influence of the Po was greatest.

The paper reports an unusual response of the microzooplankton community to oceanographic conditions observed during the winter of 2015 in the open southern Adriatic. Record-breaking nauplii abundance of 13,734 ind. m-3 was sampled for the open southern Adriatic by 50-\mum net sampling. This could be explained by (i) warmer-than-usual surface and intermediate ocean temperatures, (ii) higher precipitation that freshened and widened the surface layer, pushing saline Levantine Intermediate Water below a 400-m depth, and (iii) strong wind episodes that transported nutrients from the coastal zone to the open ocean and induced limited vertical mixing. Neritic tintinnids and viable photoautotrophs well below the photic zone support the last possibility. Average seasonal maximum of zooplankton abundance was shifted from spring and early summer to the late winter months. Our results document large and fast variations in production conditions rarely found to occur in oligotrophic waters such as the southern Adriatic Sea.

The morphology of the genus Chaetoceros, one of the most abundant and diverse planktonic diatom groups, was investigated using material collected in the eastern Adriatic Sea from 2006 to 2012. Twenty-seven morphologically distinct species have been identified from both field samples and cultivated strains. Two species, C. bacteriastroides and C. pseudodichaeta, are reported for the first time for the area. Morphometric data, general morphology and ultrastructural characters are presented for each species, based on light and electron microscopy observations, with a special emphasis on species-specific distinctive features. Valve ultrastructure appeared to be characteristic for single species or group of closely related species, especially inside the subgenus Hyalochaete. A collection of appropriate micrographs will facilitate future identification of Chaetoceros species and morphological comparison with material from other geographic areas.

The genus Entomoneis Ehrenberg includes diatoms with structurally complex frustules having a bilobate keel elevated above the valve surface and numerous girdle bands. We describe here a new member of the genus, Entomoneis tenera sp. nov. , a minute species found in the plankton of the south-eastern Adriatic Sea. The description and proposed taxonomic affiliation are based on both morphological observations and molecular analyses obtained from three cultivated strains. The cultures were established from plankton net samples taken during BIOTA (Bio-tracing Adriatic Water Masses) cruise conducted in February–March 2015. In addition to characteristic Entomoneis features such as panduriform cells, often twisted around the apical axis and a raphe with simple endings positioned on the sigmoid keel, morphological characteristics of E. tenera are: (1) very small cells, 16–21 μm long and 5–20 μm wide (2) very lightly silicified, delicate frustules without valve striation discernible in light microscopy, (3) broad lanceolate valves with scalpeliform apices (4) a straight to slightly arcuate junction line. Phylogenetic analyses using SSU, rbc L and psb C supported the position of E . tenera within the Entomoneis genus with a clear separation from the other described species.

Increasing pCO2 is hypothesized to induce shifts in plankton communities towards smaller cells, reduced carbon export rates and increased roles of gelatinous zooplankton. Appendicularians, among the most numerous pan-global ‘gelatinous’ zooplankton, continuously produce filter-feeding houses, shortcutting marine food webs by ingesting submicron particles, and their discarded houses contribute significantly to carbon fluxes. We present a first mesocosm-scale study on the effects of temperature, pCO2 and bloom structures on the appendicularian, Oikopleura dioica. There were effects of temperature and nutrients on phytoplankton communities. No shifts in functional phytoplankton groups, nor changes in particle sizes/morphotypes, known to impact appendicularian feeding, were observed under manipulated pCO2 conditions. However, appendicularian abundance was positively correlated with increased pCO2, temperature and nutrient levels, consistent with hypotheses concerning gelatinous zooplankton in future oceans. This suggests appendicularians will play more important roles in marine pelagic communities and vertical carbon transport under projected ocean acidification and elevated temperature scenarios.

The species composition and size-structure of the phytoplankton community in the Boka Kotorska Bay (SE Adriatic Sea) were analysed with respect to abundance and carbon biomass, together with the physico-chemical parameters, with the aim of evaluating the predefined oligo-mesotrophic status of this transitional water ecosystem. Three stations located in the inner part of the Bay were sampled with seasonal frequency in 2008/2009. Picophytoplankton cells were counted using flow cytometry; nanophytoplankton and microphytoplankton were identified and counted by light microscopy. The relative importance of the picoplankton in the Bay, in terms of both abundance and biomass, during all the investigated seasons emphasized their significance in the phytoplankton community. Picocyanobacteria (Synechococcus) constituted a significant part of the summer assemblages with regard to both abundance (up to 3.38×108 cells L−1) and carbon biomass (up to 73% of total phytoplankton carbon). The contribution of the nanophytoplankton was found to be generally low (

The chain-forming diatom Chaetoceros vixvisibilis is one of the most abundant and frequent diatoms in the northeastern Adriatic Sea. This species had not been previously studied by electron microscopy. Its general morphology is similar to those species allocated in the subgenus Hyalochaete: cells and chains (straight, of variable length, usually 4-8 cells per chain, but can be longer) of delicate appearance, valves thinly silicified, with slightly eccentric annulus and costae radiating from it, a single rimoportula present only at terminal valves, long, thin and delicate setae perforated by tiny poroids with no spines, and one plate-like chloroplast per cell. Setae with no spines are not common among members of Hyalochaete (and the whole genus Chaetoceros), except C. socialis. The most characteristic feature is, however, the resting spores commonly found, which also show morphological variability, from solitary to paired, both valves convex to domed and surface smooth or with small granules, and one to four strong spines often branching dichotomically. Abundances of C. vixvisibilis are positively correlated to the Po River inflow. Maximum abundances (> 10 6 cells L -1 ) were found in the period April-July, in the temperature range between 12 and 16°C, salinity between 33 and 38, when total phosphorus concentration is higher than 0.4 μmol L -1 , but occasionally also in nitrogen limited conditions.

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 2×105 cells l-1 in 50% of samples), and the dinoflagellate Prorocentrum minimum (maximum abundance of 3×105 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.

Two fairly uncommon species of planktonic diatoms of the genus Chaetoceros, C. bacteriastriodes and C. pseudosymmetrycus, were recorded during the study along the South Adriatic transect Dubrovnik-Bari in 2012 and 2015. Species were recorded by observing water samples under inverted microscope. From a total of 89 examined samples unusual Chaetoceros species were recorded in 5 cases in the South Adriatic open waters (to a depth 0-100 m) during winter period. Their abundance did not exceed 102 cellsL-1. Both species have been described from tropical waters, showing Indo- Pacific distribution, and very rarely recorded in the Mediterranean Sea. Areal expansion and introduction of new phytoplankton species might be related to changes in sea circulation in the South Adriatic or ballast water transport. Recent investigations have shown that entering currents, of either Atlantic/Western Mediterranean or Eastern Mediterranean origin, modify the composition of the plankton community in the South Adriatic and each type of current brings different newcomers.

This work describes the dynamics of the autotrophic plankton community in Lim Bay in the north-eastern part of the Adriatic Sea from June 2008 to September 2009. There was an evident shift between microphytoplankton and picophytoplankton domination in summer and autumn periods, while nanophytoplankton contributed negligibly. Picophytoplankton dominated in June 2008 and September 2009 (contributing up to 84 % of the total phytoplankton biomass), while the micro- fraction dominated in September 2008 and June 2009 (contributing up to 97.2 % of the total biomass). The pico- fraction was dominated by Synechococccus in terms of abundance and biomass, with the highest abundances in September of 2009. Picoeukaryotes were not that prominent in terms of abundance or biomass, but they exceeded Synechococcus in terms of biomass in November 2008 and February 2009, and proved to be an important and consistent component of the Lim Bay phytoplankton community. Our results indicate that seasonal variability in the community structure was more affected by specific environmental perturbations occurring in Lim Bay than by availability of nutrients., Ovaj rad opisuje dinamiku autotrofne zajednice u Limskom kanalu, u sjevero-istočnom dijelu Jadranskog mora od lipnja 2008 do rujna 2009. Tijekom ljeta i jeseni jasno je uočena promjena u dominaciji biomase između mikrofitoplanktona i pikofitoplanktona, pri čemu je doprinos nanofitoplanktona bio zanemariv. Pikofitoplankton prevladavao je u lipnju 2008 i rujnu 2009 (do 84% doprinosa ukupnoj fitoplanktonskoj biomasi), dok je mikro-frakcija prevladavala u rujnu 2008 i lipnju 2009 (do 97.2% doprinosa ukupnoj biomasi). Unutar piko-frakcije prevladavale su cijanobakterije roda Synechococcus, kako brojnošću tako i biomasom, s najvećim vrijednostima u rujnu 2009. Međutim, u studenom 2008 i veljači 2009 pikoeukarioti su biomasom premašili populaciju cijanobakterija Synechococcus, čime su se pokazali kao važna komponenta zajednice fitoplanktona Limskog kanala. Rezultati ovog istraživanja su pokazali da je sezonska varijabilnost u strukturi zajednice podložnija utjecaju određenih perturbacija u okolišu nego dostupnosti nutrijenata.