Ontogenetic allometry of the bluemouth, <Emphasis Type="Italic">Helicolenus dactylopterus dactylopterus</Emphasis> (Teleostei: Scorpaenidae), in the Northeast Atlantic and Mediterranean based on geometric morphometrics

Since the emergence of the ‘microbial loop’ concept, heterotrophic flagellates have received particular attention as grazers in aquatic ecosystems. These microbes have historically been regarded incorrectly as a homogeneous group of bacterivorous protists in aquatic systems. More recently, environmental rDNA surveys of small heterotrophic flagellates in the pelagic zone of freshwater ecosystems have provided new insights. (i) The dominant phyla found by molecular studies differed significantly from those known from morphological studies with the light microscope, (ii) the retrieved phylotypes generally belong to well-established eukaryotic clades, but there is a very large diversity within these clades and (iii) a substantial part of the retrieved sequences cannot be assigned to bacterivorous but can be assigned instead to parasitic and saprophytic organisms, such as zoosporic true fungi (chytrids), fungus-like organisms (stramenopiles), or virulent alveolate parasites (Perkinsozoa and Amoebophrya sp.). All these microorganisms are able to produce small zoospores to assure dispersal in water during their life-cycles. Based on the existing literature on true fungi and fungus-like organisms, and on the more recently published eukaryotic rDNA environmental studies and morphological observations, we conclude that previously overlooked microbial diversity and related ecological potentials require intensive investigation (i) for an improved understanding of the roles of heterotrophic flagellates in pelagic ecosystems and (ii) to properly integrate the concept of ‘the microbial loop’ into modern pelagic microbial ecology.     

Flagellates and heliozoans in the Greenland Sea ice studied alive using light microscopy

The occurrence of flagellates and heliozoans in the Greenland Sea was determined from freshly collected samples and crude cultures established during the expedition ARK XI/2 onboard RV “Polarstern” in autumn 1995. The live material was collected from the water column, new ice, and multi-year ice floes, and examined with light (interference and phase contrast) and epifluorescence microscopy. Photographic and video techniques were utilised for the documentation. The observed general morphology of the cells, swimming motions, feeding behaviour and modes of reproduction assisted in the identification of flagellates. A total of 57 photo- and heterotrophic flagellate taxa, representing cryptophytes, dinoflagellates, haptophytes, chrysophytes, Prasinophyceae, chlorophytes, euglenids, choanoflagellates, kinetoplastids, protists of unknown affinity (Protista incertae sedis), and heliozoans, were found. Diatoms were excluded from this study. Newly forming ice, ice floes and cultures established from the ice samples contained almost twice as many identified flagellate taxa as the water column. In addition to general information on the community structure of flagellates and heliozoans, the light microscopical methods used here provided information on the need for additional microscopy, establishment of cultures, and the suitability of the material for experimental work. Received: 20 June 1996 / Accepted: 1 October 1996     

Marine Heterotrophic Amoebae, Flagellates and Heliozoa From Belize (Central America) and Tenerife (Canary Islands), With Descriptions of New Species, Luffisphaera Bulbochaete N. Sp., L. Longihastis N. Sp., L. Turriformis N. Sp. and Paulinella Intermedia N. Sp.

ABSTRACT. Thirty four taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established from marine samples from Belize (Central America) and Tenerife (Canary Islands). Most species are flagellates drawn from the choanoflagellates, the cryptophyceans, the euglenids, the kinetoplastids, the bicosoecids, the chromulinids, the pedinellids and a variety of laxa of uncertain affinities (Protista incertae sedis). the identity of the thecate choanoflagellates Salpingoeca ringens Kent, 1880, and S. tuba Kent, 1880, is discussed, and four new species of heterotrophic protists are described: one new species of the amoeba genus Paulinella (Paulinella intermedia n. sp.) and three new species of the incertae sedis genus Luffisphaera Belcher & Swale, 1975 ( Luffisphaera bulbochaete n. sp.; L. longihastis n. sp.; L. turriformis n. sp.).     

Ecology of planktonic heterotrophic flagellates. A review.

In aquatic environments heterotrophic flagellates are an important component within the microbial loop and the food web, owing to their involvement in the energy transfer and flux and as an intermediate link between bacteria and primary producers, and greater organisms, such as other protists and metazoan consumers. In the microbial loop heterotrophic flagellates highly contribute to fast biomass and nutrient recycling and to the production in aquatic environments. In fact, these protists consume efficiently viruses, bacteria, cyanobacteria and picophytoplankton, and are grazed mainly by other protists, rotifers and small crustaceans. In this paper the knowledge about these unicellular organisms is reviewed, taking into particular account their ecological relationships and trophic role within the plankton community of marine and freshwater environments.     

Distribution and abundance of uncultured heterotrophic flagellates in the world oceans

Heterotrophic flagellates play fundamental roles in marine ecosystems as picoplankton grazers. This recognized importance contrasts with our ignorance of the taxonomic composition of this functional group, which remains mostly unidentified by microscopical and culturing approaches. Recent molecular marine surveys based on 18S rDNA genes have retrieved many sequences unrelated to cultured organisms and marine stramenopiles were among the first reported uncultured eukaryotes. However, little is known about the organisms corresponding to these sequences. Here we determine the abundance of several marine stramenopile lineages in surface marine waters using molecular probes and fluorescent in situ hybridization. We show that these protists are free-living bacterivorous heterotrophic flagellates. They were widely distributed, occurring in the five world oceans, and accounted for a significant fraction (up to 35%) of heterotrophic flagellates in diverse geographic regions. A single group, MAST-4, represented 9% of cells within this functional assemblage, with the intriguing exception of polar waters where it was absent. MAST-4 cells likely contribute substantially to picoplankton grazing and nutrient re-mineralization in vast areas of the oceans and represent a key eukaryotic group in marine food webs.     

Heterotrophic amoebae,flagellates and heliozoa from Arctic marine waters (North West Territories,Canada and West Greenland)

Summary Thirty eight taxa of heterotrophic protists (amoebae, flagellates and heliozoa) were encountered in cultures established from marine samples from Disko Bay (West Greenland) and Foxe Basin (N.W.T. Canada). Most species are flagellates representing the choanoflagellates, the euglenids, the kinetoplastids, the bicosoecids, the chrysomonads, the pedinellids and a variety of taxa of uncertain affinities (Protista incertae sedis). A new species of thecate choanoflagellate, Salpingoeca intermedia sp. nov., is described, and 4 new types of triangular scales, representing the genus Thaumatomastix Lauterborn, 1899, are presented.     

Pico- and Nanoplankton in Aquatic Ecosystems in the Valley of the Lakes and Great Lakes Depression (Mongolia)

The abundance and biomass of bacterioplankton, phototrophic picoplankton, and heterotrophic nanoflagellates has been determined in lakes, rivers, and reservoirs located in the Valley of the Lakes and Great Lakes Depression (Mongolia). The species richness of the heterotrophic flagellates and their consumption of bacteria are estimated. Pico- and nanoplankton are the most abundant in shallow mineral lakes Orog and Tatsyn and in the freshwater Durgun Reservoir. Heterotrophic nanoflagellates consume 26–92% (on average 66%) of the daily bacterioplankton production. Thus, flagellates are important in the transfer of bacterial carbon to the higher levels of planktonic trophic webs. A total of 30 species and their forms of heterotrophic flagellates from 14 large taxa are identified. The highest species diversity of these protists are found in the Durgun and Taishyr reservoirs.     

The importance of herbivory by protists in lakes of a tropical floodplain system

Inland aquatic ecosystems play a critical role in the global carbon cycle, processing a great fraction of the organic matter coming from terrestrial ecosystems, and the microbial food web is crucial in this process. Thus, we aimed to evaluate whether the food resource of planktonic protozoa comes mainly from small primary producers or heterotrophic bacteria in tropical shallows lakes, assuming the hypothesis that, in general, picocyanobacteria would be the main food resource for protists. We also expected that the autotrophic fraction would be mainly related to protists at the surface of the environments, while the heterotrophic fraction would be more important at the lower strata of the water column. We performed size-fractionation experiments to evaluate the effects of predation of protists on heterotrophic bacteria and picocyanobacteria. We also sampled planktonic organisms at the subsurface and bottom of 20 lakes in a Neotropical floodplain. We found an herbivory preference of heterotrophic flagellates, while ciliates seem to exert a stronger impact on heterotrophic bacteria. We also found no relationship between heterotrophic bacteria and protists in the field data, whereas positive relationships between picocyanobacteria and protists were observed in environments where there was sunlight. Thus, both heterotrophic bacteria and picocyanobacteria were important components in the food webs of tropical shallow lakes. Moreover, the trophic cascade caused by zooplankton predation suggests that protists are efficient in transferring the energy from the base of microbial food webs to higher trophic levels.     

Light and electron microscopical observations on the heterotrophic protist Thaumatomastix salina comb. nov. (syn. Chrysosphaerella salina) and its allies

The marine flagellates presently known as the chrysophytes Chrysosphaerella salina and C. tripus Takahashi & Hara have been refound and studied with light microscopy and whole mounts for electron microscopy. Based on material from Australia and Denmark (the latter from the type locality), C. salina is shown to be a colourless protist related to Reckertia sagittifera Conrad. It is characterized by a combination of flagellar features previously thought to be restricted to Reckertia , i.e. a short anterior flagellum which is scale-clad, and a longer, usually posterior but naked flagellum. The scales on the body are shown to be silicified. The new light microscopical studies have also shown considerable resemblance between C. salina, C. tripus and the genus Thaumatomastix Lauterborn. C. salina and C. tripus are therefore transferred to this genus together with Reckertia and the 2 marine species described since 1980 as belonging to Chrysosphaerella, C. triangulata Balonov and C. patelliformis Takahashi & Hara. Thaumatomastix bipartita sp.nov. is illustrated and described.
Chrysosphaerella appears to be a genus of photosynthetic, colony-forming chrysophytes restricted to freshwater habitats. Thaumatomastix is a genus of heterotrophic protists, usually solitary, which occurs in both marine and freshwater environments. The two genera show little, if any, phylogenetic relationship to each other.     

Protist diversity in suboxic and sulfidic waters of the Black Sea

The oxic-anoxic transition zone of the Black Sea comprises a large suboxic zone as well as anoxic and sulfidic waters. While the prokaryotes and biogeochemical cycles that characterize this zone have been frequently studied, little is known about the diversity or ecology of its microbial eukaryotes. Here, we present the first broad qualitative report of the protist species composition in the Black Sea redoxcline using molecular tools. Fingerprint analysis from the whole redoxcline revealed a complex community structure of metabolically active protists with distinct shifts along the redox gradient. Additionally, 18S rRNA gene clone libraries were used to compare protist species composition of suboxic and sulfidic water layers. Among the ciliates, sequences related to Pleuronema and Strombidium were dominant in both water layers whereas sequences affiliated with anaerobic plagiopylids and Cyclidium were detected only in the sulfidic zone. Among the flagellates, mainly stramenopiles (mostly bicosoecids and chrysophytes) occurred throughout the redoxcline. In the sulfidic zone we found stramenopile sequences but also euglenozoans, jakobids and choanoflagellates that were related to clonal sequences from other anoxic marine habitats, thus indicating the existence of globally distributed groups of anoxic flagellates. Higher species diversity in the sulfidic zone and about twice as many novel sequence types of ciliates and stramenopiles compared with the suboxic layer emphasizes the importance of anoxic, sulfidic waters as habitat for high protist diversity although the function of these organisms is yet unknown.     

Benthic–pelagic coupling: a comparison of the community structure of benthic and planktonic heterotrophic protists in shallow inlets of the southern Baltic

1. The taxonomic composition, abundance and biomass of heterotrophic protists (ciliates, heterotrophic flagellates (HF), rhizopods and actinopods) in the sediment and water column of shallow inlets of the Southern Baltic was studied under a variety of environmental conditions during 1996–1997. A shallow, highly eutrophic station and a deeper, less eutrophic station were compared.
2. Community biomass ranged from 0.12 to 0.34 μg C cm?3 in the water column and from 1.5 to 105 μg C cm?3 in the sediment. Heterotrophic protists dominated zooplankton biomass at both stations (73% and 84% mean contribution), while they were of minor importance within the zoobenthos. Expressed per unit area, benthic biomass contributed a significant part (44% and 49%) to the total heterotrophic protistan community at both stations.
3. Although the methodology for counting ciliates and HF was focussed on a high taxonomic resolution, the results reveal some general trends in the distribution of heterotrophic protists: protozooplankton biomass was dominated by flagellates (80% mean biomass contribution) at the shallow station and by ciliates (73% mean biomass contribution) at the deep station. In the benthos at both stations, ciliates were the dominant protozoans, followed by the hitherto little‐studied rhizopods (25% and 35% mean biomass contribution) and flagellates.
4. The degree of benthic–pelagic coupling differed between taxonomic groups. Benthic and pelagic communities of ciliates showed little taxonomic overlap. In contrast, many heterotrophic flagellate species were found both in the benthos and in the pelagic. These benthic–pelagic species contributed significantly to the biomass of HF in the water column. The planktonic rhizopod community consisted of a subset of those species found in the benthos.
5. The abundance of benthic and pelagic protists was positively correlated at the shallow station, but taxonomic data indicate that the direct exchange between benthic and pelagic communities was only partly responsible.     

A Preliminary Study of Heterotrophic Picoflagellates Using Oligonucleotidic Probes in Lake Pavin

Free-living heterotrophic flagellates have been widely studied as key intermediates in aquatic food webs. However, very few attempts have been made (all in marine systems) to study the smallest cells belonging to these communities. In this study, we report on the quantitative importance and the vertical distribution of heterotrophic picoflagellates during spring in the oligomesotrophic Lake Pavin, using the FISH approach. The rRNA-based probes used, initially designed for two novel lineages of heterotrophic picoflagellates (i.e., the Stramenopiles NS3 and NS4) in marine systems, revealed high abundances (in the order of 10⁴ cells ml⁻¹ and up to about 6 × 10⁴ cells ml⁻¹) of the targeted picoflagellates in Lake Pavin, i.e. one order of magnitude higher than in seawater samples. 8NS39 cells were more often detected than 8NS49 cells, the average contributions of the two populations to the total abundance of heterotrophic flagellates being at 25 and 9%, respectively. However, the probes used were apparently non-specific to the targeted cells, implying that the occurrence of the two novel lineages NS3 and NS4 of Stramenopiles in freshwaters needs to be confirmed. Although, this study clearly confirms the occurrence, in high abundance, of small heterotrophic eukaryotes in Lake Pavin. In a near future, we believe that the availability of microbial clone libraries will allow us to improve our knowledge of the composition and the functional role of small heterotrophic eukaryotes in freshwaters.     

Characterization of Protistan Assemblages in the Ross Sea, Antarctica, by Denaturing Gradient Gel Electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

Cosmopolitan metapopulations of free-living microbial eukaryotes

Metapopulations of macroscopic organisms tend to be geographically restricted, but free-living protists and other microbial eukaryotes present a different picture. Here we show that most organisms smaller than 1 mm occur worldwide wherever their required habitats are realised. This is a consequence of ubiquitous dispersal driven by huge population sizes, and the consequently low probability of local extinction. Organisms larger than 10 mm are much less abundant, and rarely cosmopolitan. The supporting data, together with the discovery that the 1-10 mm size range accommodates a transition from cosmopolitan to regionally-restricted distribution, were derived from extensive inventories of eukaryotic species in a freshwater pond (1278 species), and a shallow marine bay (785 species). All accessible records were examined to establish the extent of global coverage by these species. Some groups of microbial eukaryotes are severely undersampled (e.g. naked amoebae; marine meiofauna in the southern hemisphere) but this fails to weaken evidence that metapopulations of microbial eukaryotes are cosmopolitan.     

Characterization of protistan assemblages in the Ross Sea, Antarctica, by denaturing gradient gel electrophoresis

The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization, but these methods are often inadequate for ecological studies of these communities because most small protists inherently lack adequate taxonomic characters to facilitate their identification at the species level and many protistan species also do not preserve well. We have therefore used a culture-independent approach (denaturing gradient gel electrophoresis [DGGE]) to obtain an assessment of the genetic composition and distribution of protists within different microhabitats (seawater, meltwater or slush on sea-ice floes, and ice) of the Ross Sea, Antarctica. Samples of the same type (e.g., water) shared more of the same bands than samples of different types (e.g., ice versus water), despite being collected from different sites. These findings imply that samples from the same environment have a similar protistan species composition and that the type of microenvironment significantly influences the protistan species composition of these Antarctic assemblages. It should be noted that a large number of bands among the samples within each microhabitat were distinct, indicating the potential presence of significant genetic diversity within each microenvironment. Sequence analysis of selected DGGE bands revealed sequences that represent diatoms, dinoflagellates, ciliates, flagellates, and several unidentified eukaryotes.     

Culturing Bias in Marine Heterotrophic Flagellates Analyzed Through Seawater Enrichment Incubations

The diversity of heterotrophic flagellates is generally based on cultivated strains, on which ultrastructural, physiological, and molecular studies have been performed. However, the relevance of these cultured strains as models of the dominant heterotrophic flagellates in the marine planktonic environment is unclear. In fact, molecular surveys typically recover novel eukaryotic lineages that have refused cultivation so far. This study was designed to directly address the culturing bias in planktonic marine heterotrophic flagellates. Several microcosms were established adding increasing amounts and sources of organic matter to a confined natural microbial community pre-filtered by 3 μm. Growth dynamics were followed by epifluorescence microscopy and showed the expected higher yield of bacteria and heterotrophic flagellates at increased organic matter additions. Moreover, protist diversity analyzed by molecular tools showed a clear substitution in the community, which differed more and more from the initial sample as the organic matter increased. Within this gradient, there was also an increase of sequences related to cultured organisms as well as a decrease in diversity. Culturing bias is partly explained by the use of organic matter in the isolation process, which drives a shift in the community to conditions closer to laboratory cultures. An intensive culturing effort using alternative isolation methods is necessary to allow the access to the missing heterotrophic flagellates that constitute the abundant and active taxa in marine systems.     

Low evolutionary diversification in a widespread and abundant uncultured protist (MAST-4)

Recent culture-independent studies of marine planktonic protists have unveiled a large diversity at all phylogenetic scales and the existence of novel groups. MAST-4 represents one of these novel uncultured lineages, and it is composed of small (~2 μm) bacterivorous eukaryotes that are widely distributed in marine systems. MAST-4 accounts for a significant fraction of the marine heterotrophic flagellates at the global level, playing key roles in the marine ecological network. In this study, we investigated the diversity of MAST-4, aiming to assess its limits and structure. Using ribosomal DNA (rDNA) sequences obtained in this study (both pyrosequencing reads and clones with large rDNA operon coverage), complemented with GenBank sequences, we show that MAST-4 is composed of only five main clades, which are well supported by small subunit and large subunit phylogenies. The differences in the conserved regions of the internal transcribed spacers 1 and 2 (ITS1 and ITS2) secondary structures strongly suggest that these five clades are different biological species. Based on intraclade divergence, ITS secondary structures and comparisons of ITS1 and ITS2 trees, we did not find evidence of more than one species within clade A, whereas as many as three species might be present within other clades. Overall, the genetic divergence of MAST-4 was surprisingly low for an organism with a global population size estimated to be around 10(24), indicating a very low evolutionary diversification within the group.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.