Spatiotemporal Variations in Antarctic Protistan Communities Highlight Phytoplankton Diversity and Seasonal Dominance by a Novel Cryptophyte Lineage.

The Andvord fjord in the West Antarctic Peninsula (WAP) is known for its productivity and abundant megafauna. Nevertheless, seasonal patterns of the molecular diversity and abundance of protistan community members underpinning WAP productivity remain poorly resolved. We performed spring and fall expeditions pursuing protistan diversity, abundance of photosynthetic taxa, and the connection to changing conditions. 18S rRNA amplicon sequence variant (ASV) profiles revealed diverse predatory protists spanning multiple eukaryotic supergroups, alongside enigmatic heterotrophs like the Picozoa. Among photosynthetic protists, cryptophyte contributions were notable. Analysis of plastid-derived 16S rRNA ASVs supported 18S ASV results, including a dichotomy between cryptophytes and diatom contributions previously reported in other Antarctic regions. We demonstrate that stramenopile and cryptophyte community structures have distinct attributes. Photosynthetic stramenopiles exhibit high diversity, with the polar diatom Fragilariopsis cylindrus, unidentified Chaetoceros species, and others being prominent. Conversely, ASV analyses followed by environmental full-length rRNA gene sequencing, electron microscopy, and flow cytometry revealed that a novel alga dominates the cryptophytes. Phylogenetic analyses established that TPG clade VII, as named here, is evolutionarily distinct from cultivated cryptophyte lineages. Additionally, cryptophyte cell abundance correlated with increased water temperature. Analyses of global data sets showed that clade VII dominates cryptophyte ASVs at Southern Ocean sites and appears to be endemic, whereas in the Arctic and elsewhere, Teleaulax amphioxeia and Plagioselmis prolonga dominate, although both were undetected in Antarctic waters. Collectively, our studies provide baseline data against which future change can be assessed, identify different diversification patterns between stramenopiles and cryptophytes, and highlight an evolutionarily distinct cryptophyte clade that thrives under conditions enhanced by warming. IMPORTANCE The climate-sensitive waters of the West Antarctic Peninsula (WAP), including its many fjords, are hot spots of productivity that support multiple marine mammal species. Here, we profiled protistan molecular diversity in a WAP fjord known for high productivity and found distinct spatiotemporal patterns across protistan groups. Alongside first insights to seasonal changes in community structure, we discovered a novel phytoplankton species with proliferation patterns linked to temperature shifts. We then examined evolutionary relationships between this novel lineage and other algae and their patterns in global ocean survey data. This established that Arctic and Antarctic cryptophyte communities have different species composition, with the newly identified lineage being endemic to Antarctic waters. Our research provides critical knowledge on how specific phytoplankton at the base of Antarctic food webs respond to warming, as well as information on overall diversity and community structure in this changing polar environment.

Community structure of photosynthetic picoeukaryotes differs in lakes with different trophic statuses along the middle-lower reaches of the Yangtze River.

Photosynthetic picoeukaryotes (PPEs) play an important role in aquatic ecosystem functioning. There is still a relative lack of information on freshwater PPEs, especially in eutrophic lakes. We used a combination of flow cytometric sorting and pyrosequencing to investigate the PPEs community structure in more than 20 mesotrophic and eutrophic lakes along the middle-lower reaches of the Yangtze River in China. The abundance of PPEs ranged between 2.04 × 103 and 5.92 × 103 cells mL-1. The contribution of PPEs to total picophytoplankton abundance was generally higher in eutrophic lakes than in mesotrophic lakes. The sequencing results indicated that the Shannon diversity of PPEs was significantly higher in mesotrophic lakes than in eutrophic lakes. At the class level, PPEs were mainly dominated by three taxonomic groups, including Cryptophyceae, Coscinodiscophyceae and Chlorophyceae, and 15 additional known phytoplankton classes, including Synurophyceae, Dinophyceae, Chrysophyceae, Trebouxiophyceae and Prymnesiophyceae, were identified. Coscinodiscophyceae dominated in the most eutrophic lakes, while Chrysophyceae, Dinophyceae and other classes of PPEs were more abundant in the mesotrophic lakes. We also observed several PPEs operational taxonomic units, and those affiliated with Cyclotella atomus, Chlamydomonas sp. and Poterioochromonas malhamensis tended to be more prevalent in the eutrophic lakes. The canonical correspondence analysis and Mantel analysis highlighted the importance of environmental parameters as key drivers of PPEs community composition.

Differences in planktonic microbial communities associated with three types of macrophyte stands in a shallow lake.

Little is known about how various substances from living and decomposing aquatic macrophytes affect the horizontal patterns of planktonic bacterial communities. Study sites were located within Lake Kolon, which is a freshwater marsh and can be characterised by open-water sites and small ponds with different macrovegetation (Phragmites australis, Nymphea alba and Utricularia vulgaris). Our aim was to reveal the impact of these macrophytes on the composition of the planktonic microbial communities using comparative analysis of environmental parameters, microscopy and pyrosequencing data. Bacterial 16S rRNA gene sequences were dominated by members of phyla Proteobacteria (36%-72%), Bacteroidetes (12%-33%) and Actinobacteria (5%-26%), but in the anoxic sample the ratio of Chlorobi (54%) was also remarkable. In the phytoplankton community, Cryptomonas sp., Dinobryon divergens, Euglena acus and chrysoflagellates had the highest proportion. Despite the similarities in most of the measured environmental parameters, the inner ponds had different bacterial and algal communities, suggesting that the presence and quality of macrophytes directly and indirectly controlled the composition of microbial plankton.

Use of Highly Specific Molecular Markers Reveals Positive Correlation between Abundances of Mesodinium cf. major and Its Preferred Prey, Teleaulax amphioxeia, During Red Water Blooms in the Columbia River Estuary.

In a previous study, Teleaulax amphioxeia-the preferred prey of Mesodinium in the Columbia River estuary-were undetectable within intense annual blooms, suggesting blooms are prey-limited or prey are acquired outside of bloom patches. We used a novel molecular approach specifically targeting the prey (i.e., Unique Sequence Element [USE] within the ribosomal RNA 28S D2 regions of T. amphioxeia nucleus and nucleomorph) in estuarine water samples acquired autonomously with an Environmental Sample Processor integrated within a monitoring network (ESP-SATURN). This new approach allowed for both more specific detection of the prey and better constraint of sample variability. A positive correlation was observed between abundances of M. cf. major and T. amphioxeia during bloom periods. The correlation was stronger at depth (> 8.2 m) and weak or nonexistent in the surface, suggesting that predator-prey dynamics become uncoupled when stratification is strong. We confirmed exclusive selectivity for T. amphioxeia by M. cf. major and observed the incorporation of the prey nucleus into a 4-nuclei complex, where it remained functionally active. The specific biomarker for T. amphioxeia was also recovered in M. cf. major samples from a Namibian coastal bloom, suggesting that a specific predator-prey relationship might be widespread between M. cf. major and T. amphioxeia.

Polymetallic nodules, sediments, and deep waters in the equatorial North Pacific exhibit highly diverse and distinct bacterial, archaeal, and microeukaryotic communities.

Concentrated seabed deposits of polymetallic nodules, which are rich in economically valuable metals (e.g., copper, nickel, cobalt, manganese), occur over vast areas of the abyssal Pacific Ocean floor. Little is currently known about the diversity of microorganisms inhabiting abyssal habitats. In this study, sediment, nodule, and water column samples were collected from the Clarion-Clipperton Zone of the Eastern North Pacific. The diversities of prokaryote and microeukaryote communities associated with these habitats were examined. Microbial community composition and diversity varied with habitat type, water column depth, and sediment horizon. Thaumarchaeota were relatively enriched in the sediments and nodules compared to the water column, whereas Gammaproteobacteria were the most abundant sequences associated with nodules. Among the Eukaryota, rRNA genes belonging to the Cryptomonadales were relatively most abundant among organisms associated with nodules, whereas rRNA gene sequences deriving from members of the Alveolata were relatively enriched in sediments and the water column. Nine operational taxonomic unit (OTU)s were identified that occur in all nodules in this dataset, as well as all nodules found in a study 3000-9000 km from our site. Microbial communities in the sediments had the highest diversity, followed by nodules, and then by the water column with <1/3 the number of OTUs as in the sediments.

A New Heterotrophic Cryptomonad: Hemiarma marina n. g., n. sp.

We report a new heterotrophic cryptomonad Hemiarma marina n. g., n. sp. that was collected from a seaweed sample from the Republic of Palau. In our molecular phylogenetic analyses using the small subunit ribosomal RNA gene, H. marina formed a clade with two marine environmental sequences, and the clade was placed as a sister lineage of the freshwater cryptomonad environmental clade CRY1. Alternatively, in the concatenated large and small subunit ribosomal RNA gene phylogeny, H. marina was placed as a sister lineage of Goniomonas. Light and electron microscopic observations showed that H. marina shares several ultrastructural features with cryptomonads, such as flattened mitochondrial cristae, a periplast cell covering, and ejectisomes that consist of two coiled ribbon structures. On the other hand, H. marina exhibited unique behaviors, such as attaching to substrates with its posterior flagellum and displaying a jumping motion. H. marina also had unique periplast arrangement and flagellar transitional region. On the basis of both molecular and morphological information, we concluded that H. marina should be treated as new genus and species of cryptomonads.

Impacts of forestry planting on primary production in upland lakes from north-west Ireland.

Planted forests are increasing in many upland regions worldwide, but knowledge about their potential effects on algal communities of catchment lakes is relatively unknown. Here, the effects of afforestation were investigated using palaeolimnology at six upland lake sites in the north-west of Ireland subject to different extents of forest plantation cover (4-64% of catchment area). (210)Pb-dated sediment cores were analysed for carotenoid pigments from algae, stable isotopes of bulk carbon (δ(13)C) and nitrogen (δ(15)N), and C/N ratios. In lakes with >50% of their catchment area covered by plantations, there were two- to sixfold increases in pigments from cryptophytes (alloxanthin) and significant but lower increases (39-116%) in those from colonial cyanobacteria (canthaxanthin), but no response from biomarkers of total algal abundance (ß-carotene). In contrast, lakes in catchments with <20% afforestation exhibited no consistent response to forestry practices, although all lakes exhibited fluctuations in pigments and geochemical variables due to peat cutting and upland grazing prior to forest plantation. Taken together, patterns suggest that increases in cyanobacteria and cryptophyte abundance reflect a combination of mineral and nutrient enrichment associated with forest fertilization and organic matter influx which may have facilitated growth of mixotrophic taxa. This study demonstrates that planted forests can alter the abundance and community structure of algae in upland humic lakes of Ireland and Northern Ireland, despite long histories of prior catchment disturbance.

Complex communities of small protists and unexpected occurrence of typical marine lineages in shallow freshwater systems.

Although inland water bodies are more heterogeneous and sensitive to environmental variation than oceans, the diversity of small protists in these ecosystems is much less well known. Some molecular surveys of lakes exist, but little information is available from smaller, shallower and often ephemeral freshwater systems, despite their global distribution and ecological importance. We carried out a comparative study based on massive pyrosequencing of amplified 18S rRNA gene fragments of protists in the 0.2-5 µm size range in one brook and four shallow ponds located in the Natural Regional Park of the Chevreuse Valley, France. Our study revealed a wide diversity of small protists, with 812 stringently defined operational taxonomic units (OTUs) belonging to the recognized eukaryotic supergroups (SAR--Stramenopiles, Alveolata, Rhizaria--Archaeplastida, Excavata, Amoebozoa, Opisthokonta) and to groups of unresolved phylogenetic position (Cryptophyta, Haptophyta, Centrohelida, Katablepharida, Telonemida, Apusozoa). Some OTUs represented deep-branching lineages (Cryptomycota, Aphelida, Colpodellida, Tremulida, clade-10 Cercozoa, HAP-1 Haptophyta). We identified several lineages previously thought to be marine including, in addition to MAST-2 and MAST-12, already detected in freshwater, MAST-3 and possibly MAST-6. Protist community structures were different in the five ecosystems. These differences did not correlate with geographical distances, but seemed to be influenced by environmental parameters.

Rhinomonas nottbecki n. sp. (cryptomonadales) and molecular phylogeny of the family Pyrenomonadaceae.

The cryptomonad Rhinomonas nottbecki n. sp., isolated from the Baltic Sea, is described from live and fixed cells studied by light, scanning, and transmission electron microscopy together with sequences of the partial nucleus- and nucleomorph-encoded 18S rRNA genes as well as the nucleus-encoded ITS1, 5.8S, ITS2, and the 5'-end of the 28S rRNA gene regions. The sequence analyses include comparison with 43 strains from the family Pyrenomonadaceae. Rhinomonas nottbecki cells are dorsoventrally flattened, obloid in shape; 10.0-17.2 µm long, 5.5-8.1 µm thick, and 4.4-8.8 µm wide. The inner periplast has roughly hexagonal plates. Rhinomonas nottbecki cells resemble those of Rhinomonas reticulata, but the nucleomorph 18S rRNA gene of R. nottbecki differs by 2% from that of R. reticulata, while the ITS region by 11%. The intraspecific variability in the ITS region of R. nottbecki is 5%. In addition, the predicted ITS2 secondary structures are different in R. nottbecki and R. reticulata. The family Pyrenomonadaceae includes three clades: Clade A, Clade B, and Clade C. All Rhinomonas sequences branched within the Clade C, while the genus Rhodomonas is paraphyletic. The analyses suggest that the genus Storeatula is an alternating morphotype of the genera Rhinomonas and Rhodomonas and that the family Pyrenomonadaceae includes some species that were described multiple times, as well as novel species.

Antarctic mixotrophic protist abundances by microscopy and molecular methods.

Protists are traditionally described as either phototrophic or heterotrophic, but studies have indicated that mixotrophic species, organisms that combine both strategies, can have significant impacts on prey populations in marine microbial food webs. While estimates of active mixotroph abundances in environmental samples are determined microscopically by fluorescent particle ingestion, species identification is difficult. We developed SYBR-based qPCR strategies for three Antarctic algal species that we identified as mixotrophic. This method and traditional ingestion experiments were applied to determine the total mixotroph abundance in Antarctic water samples, to ascertain the abundance of known mixotrophic species, and to identify environmental variables that impact the distribution and abundance of these species. Despite differences in sampling locations and years, mixotroph distribution was strongly influenced by season. Environmental variables that best explained variation in the individual mixotroph species abundances included temperature, oxygen, date, fluorescence, conductivity, and latitude. Phosphate was identified as an additional explanatory variable when nutrients were included in the analysis. Utilizing culture-based grazing rates and qPCR abundances, the estimated summed impact on bacterial populations by the three mixotrophs was usually < 2% of the overall mixotrophic grazing, but in one sample, Pyramimonas was estimated to contribute up to 80% of mixotrophic grazing.

Microbial and chemical profile of a ponds system for the treatment of landfill leachate.

The present study describes the behavior of spatio-temporal variation of parameters and microbial profile of a pilot stabilization ponds system, consisted of three serial ponds, for the treatment of landfill leachate. Bacterial diversity was determined through molecular techniques (FISH, PCR and phylogenic analysis), while the phytoplankton community was evaluated through optical microscopy and quantified by the Sedgewick-Rafter chamber. Physicochemical parameters were also evaluated. The ponds system presented the following removal efficiency: 56% for TCOD; 83% for SBOD5 and 82% for N-NH4(+). Moreover, the analysis of chlorophyll a and DO showed stratification in the mass of water by the vertical profile. The analysis of the phytoplankton community showed a low number of species, with a predominance of Chlamydomonas sp. and presence of Cryptomonas sp. in lower density. The bacterial diversity analysis showed the presence of Planctomycetales, Verrucomicrobiales, some Desulfovibionaceae sulfate-reducing bacteria and Pseudomonas sp.

Taxonomy and phylogeny of a new kleptoplastidal dinoflagellate, Gymnodinium myriopyrenoides sp. nov. (Gymnodiniales, Dinophyceae), and its cryptophyte symbiont.

A new kleptoplastidal dinoflagellate, Gymnodinium myriopyrenoides sp. nov., was described using light microscopy, electron microscopy and phylogengetic analysis based on partial LSU rDNA sequences. Cells were dorsiventrally flattened, elongate-elliptical in ventral view. There was no displacement of the cingulum encircling the anterior part of the cell. The cingulum was curved posteriorly at the terminal junction with the sulcus. The sulcus was generally narrow but expanded in the posterior end. The epicone possessed an apical groove made of one and one-half counterclockwise revolutions. Phylogenetic analysis based on LSU rDNA showed that the sequence of G. myriopyrenoides was included in the Gymnodiniales sensu stricto clade and had special affinities with the species Amphidinium poecilochroum and Gymnodinium acidotum, which also harbor kleptochloroplasts. Phylogenetic analysis based on plastid-encoded SSU rDNA and ultrastructural observations suggested that the symbionts of G. myriopyrenoides were cryptophytes of the genus Chroomonas or Hemiselmis. Organelles including the nucleus, the nucleomorph, mitochondria, Golgi bodies and large chloroplasts remained in the cytoplasm of the symbionts, but not the periplast, ejectosomes or flagellar apparatus. The symbiotic level of G. myriopyrenoides was estimated to be a relatively early stage in the unarmored kleptoplastidal dinoflagellates.

High-level congruence of Myrionecta rubra prey and Dinophysis species plastid identities as revealed by genetic analyses of isolates from Japanese coastal waters.

We analyzed cryptophyte nucleomorph 18S rRNA gene sequences retained in natural Myrionecta rubra cells and plastid 16S rRNA gene and psbA sequences retained in natural cells of several Dinophysis species collected from Japanese coastal waters. A total of 715 nucleomorph sequences obtained from 134 M. rubra cells and 564 plastid 16S rRNA gene and 355 psbA sequences from 71 Dinophysis cells were determined. Almost all sequences in M. rubra and Dinophysis spp. were identical to those of Teleaulax amphioxeia, suggesting that M. rubra in Japanese coastal waters preferentially ingest T. amphioxeia. The remaining sequences were closely related to those of Geminigera cryophila and Teleaulax acuta. Interestingly, 37 plastid 16S rRNA gene sequences, which were different from T. amphioxeia and amplified from Dinophysis acuminata and Dinophysis norvegica cells, were identical to the sequence of a D. acuminata cell found in the Greenland Sea, suggesting that a widely distributed and unknown cryptophyte species is also preyed upon by M. rubra and subsequently sequestered by Dinophysis. To confirm the reliability of molecular identification of the cryptophyte Teleaulax species detected from M. rubra and Dinophysis cells, the nucleomorph and plastid genes of Teleaulax species isolated from seawaters were also analyzed. Of 19 isolates, 16 and 3 clonal strains were identified as T. amphioxeia and T. acuta, respectively, and no sequence variation was confirmed within species. T. amphioxeia is probably the primary source of prey for M. rubra in Japanese coastal waters. An unknown cryptophyte may serve as an additional source, depending on localities and seasons.

Morphology, ultrastructure, and small subunit rDNA phylogeny of the marine heterotrophic flagellate Goniomonas aff. amphinema.

Marine goniomonads have a worldwide distribution but ultrastructural information has not been available so far. An isolate of the heterotrophic marine nanoflagellate Goniomonas (G. aff. amphinema) from North Wales (UK) has been studied, providing information on its morphology and cellular structure using video, electron, laser scanning confocal microscopy (LSCM), and atomic force microscopy. Here, we describe a new feature, a granular area, potentially involved in particle capture and feeding. The binding of the lectin wheat germ agglutinin to the granular area of cells with discharged ejectisomes indicates the adhesive nature of this novel feature. The presence of a microtubular intracellular cytopharynx, apparently also used for feeding, has been revealed by LSCM. The small subunit rRNA gene of the isolate has been sequenced (1,788 bp). Phylogenetic results corroborate significant genetic divergence within the marine members of Goniomonas. This work highlights the need for integrated morphological, ultrastructural, and molecular investigation when describing and studying heterotrophic nanoflagellates.

Diversification of unicellular eukaryotes: cryptomonad colonizations of marine and fresh waters inferred from revised 18S rRNA phylogeny.

The cryptomonads is a well-defined lineage of unicellular eukaryotes, composed of several marine and freshwater groups. However, the evolutionary relationships among these groups are unclear due to conflicting inferences between morphological and molecular phylogenies. Here, we have inferred the evolutionary relationships among marine and freshwater species in order to better understand the importance of the marine-freshwater boundary on the historical diversification patterns of cryptomonads. We have constructed improved molecular phylogenies by taking into account rate variation both across sites and across sequences (covarion substitutions), and by analysing the vast majority of publicly available cryptomonad 18S rRNA sequences and related environmental phylotypes. The resulting phylogenies included 55 sequences, and revealed two novel freshwater cryptomonad clades (CRY1 and CRY2) and a large hidden diversity of cryptomonads. CRY1 was placed deeply within the cryptomonad phylogeny together with all the major freshwater lineages (i.e. Goniomonas and Cryptomonas), while CRY2 was placed within a lineage of marine species identified as Plagioselmis-like with the aid of a new sequence generated from a cultured species. The inferred phylogenies suggest only few successful marine-freshwater transitions over the history of cryptomonads. Most of the transitions seem to have occurred from marine to fresh waters, but re-colonizations of marine habitats have also taken place. This implies that the differences in the biogeophysical conditions between marine and fresh waters constitute a substantial barrier for the cross-colonization of these environments by cryptomonads.

Development of a denaturing high-performance liquid chromatography method for detection of protist parasites of metazoans.

Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-pairing technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63 degrees C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.