Telonemia are one of the oldest identified marine protists that for most part of their history have been recognized as a distinct incertae sedis lineage. Today, their evolutionary proximity to the SAR supergroup (Stramenopiles, Alveolates, and Rhizaria) is firmly established. However, their ecological distribution and importance as a natural predatory flagellate, especially in freshwater food webs, still remain unclear. To unravel the distribution and diversity of the phylum Telonemia in freshwater habitats, we examined over a thousand freshwater metagenomes from all over the world. In addition, to directly quantify absolute abundances, we analyzed 407 samples from 97 lakes and reservoirs using Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). We recovered Telonemia 18S rRNA gene sequences from hundreds of metagenomic samples from a wide variety of habitats, indicating a global distribution of this phylum. However, even after this extensive sampling, our phylogenetic analysis did not reveal any new major clades, suggesting current molecular surveys are near to capturing the full diversity within this group. We observed excellent concordance between CARD-FISH analyses and estimates of abundances from metagenomes. Both approaches suggest that Telonemia are largely absent from shallow lakes and prefer to inhabit the colder hypolimnion of lakes and reservoirs in the Northern Hemisphere, where they frequently bloom, reaching 10%-20% of the total heterotrophic flagellate population, making them important predatory flagellates in the freshwater food web.
- Klíčová slova
- CARD-FISH, Telonemia, freshwater lakes, metagenomics, microbial food webs, predatory flagellate,
- MeSH
- biodiverzita MeSH
- fylogeneze * MeSH
- hybridizace in situ fluorescenční * MeSH
- jezera mikrobiologie parazitologie MeSH
- metagenom MeSH
- metagenomika MeSH
- RNA ribozomální 18S * genetika MeSH
- sladká voda * mikrobiologie parazitologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- RNA ribozomální 18S * MeSH
BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.
- MeSH
- Bacteria MeSH
- Eukaryota genetika MeSH
- metagenom * MeSH
- plankton MeSH
- sladká voda MeSH
- viry * genetika MeSH
- voda MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- voda MeSH
Morphology-based microscopic approaches are insufficient for a taxonomic classification of bacterivorous heterotrophic nanoflagellates (HNF) in aquatic environments since their cells do not display reliably distinguishable morphological features. This leads to a considerable lack of ecological insights into this large and taxonomically diverse functional guild. Here, we present a combination of fluorescence in situ hybridization followed by catalyzed reporter deposition (CARD-FISH) and environmental sequence analyses which revealed that morphologically indistinguishable, so far largely cryptic and uncultured aplastidic cryptophytes are ubiquitous and prominent protistan bacterivores in diverse freshwater ecosystems. Using a general probe for Cryptophyceae and its heterotrophic CRY1 lineage, we analyzed different water layers in 24 freshwater lakes spanning a broad range of trophic states, sizes and geographical locations. We show that bacterivorous aplastidic cryptophytes and the CRY1 lineage accounted for ca. 2/3 and ¼ of total HNF, respectively, in both epilimnetic and hypolimnetic samples. These heterotrophic cryptophytes were generally smaller and more abundant than their chloroplast-bearing counterparts. They had high uptake rates of bacteria, hinting at their important roles in channeling carbon flow from prokaryotes to higher trophic levels. The worldwide ubiquity of Cryptophyceae and its CRY1 lineage was supported by 18S rRNA gene sequence analyses across a diverse set of 297 freshwater metagenomes. While cryptophytes have been considered to be mainly plastidic "algae", we show that it is the aplastidic counterparts that contribute considerably to bacterial mortality rates. Additionally, our results suggest an undiscovered diversity hidden amongst these abundant and morphologically diverse aplastidic cryptophytes.
Heterotrophic nanoflagellates (HNF) and ciliates are major protistan planktonic bacterivores. The term HNF, however, describes a functional guild only and, in contrast to the morphologically distinguishable ciliates, does not reflect the phylogenetic diversity of flagellates in aquatic ecosystems. Associating a function with taxonomic affiliation of key flagellate taxa is currently a major task in microbial ecology. We investigated seasonal changes in the HNF and ciliate community composition as well as taxa-specific bacterivory in four hypertrophic freshwater lakes. Taxa-specific catalyzed reporter deposition-fluorescence in situ hybridization probes assigned taxonomic affiliations to 51%-96% (average ±SD, 75 ± 14%) of total HNF. Ingestion rates of fluorescently labelled bacteria unveiled that HNF contributed to total protist-induced bacterial mortality rates more (56%) than ciliates (44%). Surprisingly, major HNF bacterivores were aplastidic cryptophytes and their Cry1 lineage, comprising on average 53% and 24% of total HNF abundance and 67% and 21% of total HNF bacterivory respectively. Kinetoplastea were important consumers of bacteria during summer phytoplankton blooms, reaching 38% of total HNF. Katablepharidacea (7.5% of total HNF) comprised mainly omnivores, with changing contributions of bacterivorous and algivorous phylotypes. Our results show that aplastidic cryptophytes, accompanied by small omnivorous ciliate genera Halteria/Pelagohalteria, are the major protistan bacterivores in hypertrophic freshwaters.
Marine bacterioplankton represent a diverse assembly of species differing largely in their abundance, physiology, metabolic activity, and role in microbial food webs. To analyze their sensitivity to bottom-up and top-down controls, we performed a manipulation experiment where grazers were removed, with or without the addition of phosphate. Using amplicon-reads normalization by internal standard (ARNIS), we reconstructed growth curves for almost 300 individual phylotypes. Grazer removal caused a rapid growth of most bacterial groups, which grew at rates of 0.6 to 3.5 day-1, with the highest rates (>4 day-1) recorded among Rhodobacteraceae, Oceanospirillales, Alteromonadaceae, and Arcobacteraceae. Based on their growth response, the phylotypes were divided into three basic groups. Most of the phylotypes responded positively to both grazer removal as well as phosphate addition. The second group (containing, e.g., Rhodobacterales and Rhizobiales) responded to the grazer removal but not to the phosphate addition. Finally, some clades, such as SAR11 and Flavobacteriaceae, responded only to phosphate amendment but not to grazer removal. Our results show large differences in bacterial responses to experimental manipulations at the phylotype level and document different life strategies of marine bacterioplankton. In addition, growth curves of 130 phylogroups of aerobic anoxygenic phototrophs were reconstructed based on changes of the functional pufM gene. The use of functional genes together with rRNA genes may significantly expand the scientific potential of the ARNIS technique. IMPORTANCE Growth is one of the main manifestations of life. It is assumed generally that bacterial growth is constrained mostly by nutrient availability (bottom-up control) and grazing (top-down control). Since marine bacteria represent a very diverse assembly of species with different metabolic properties, their growth characteristics also largely differ accordingly. Currently, the growth of marine microorganisms is typically evaluated using microscopy in combination with fluorescence in situ hybridization (FISH). However, these laborious techniques are limited in their throughput and taxonomical resolution. Therefore, we combined a classical manipulation experiment with next-generation sequencing to resolve the growth dynamics of almost 300 bacterial phylogroups in the coastal Adriatic Sea. The analysis documented that most of the phylogroups responded positively to both grazer removal and phosphate addition. We observed significant differences in growth kinetics among closely related species, which could not be distinguished by the classical FISH technique.
- Klíčová slova
- aerobic anoxygenic phototrophs, amplicon sequencing, bacterioplankton, grazing, growth curves, growth rate, manipulation experiment, phosphorus limitation, top-down control,
- Publikační typ
- časopisecké články MeSH
Bacteria are an active and diverse component of pelagic communities. The identification of main factors governing microbial diversity and spatial distribution requires advanced mathematical analyses. Here, the bacterial community composition was analysed, along with a depth profile, in the open Adriatic Sea using amplicon sequencing of bacterial 16S rRNA and the Neural gas algorithm. The performed analysis classified the sample into four best matching units representing heterogenic patterns of the bacterial community composition. The observed parameters were more differentiated by depth than by area, with temperature and identified salinity as important environmental variables. The highest diversity was observed at the deep chlorophyll maximum, while bacterial abundance and production peaked in the upper layers. The most of the identified genera belonged to Proteobacteria, with uncultured AEGEAN-169 and SAR116 lineages being dominant Alphaproteobacteria, and OM60 (NOR5) and SAR86 being dominant Gammaproteobacteria. Marine Synechococcus and Cyanobium-related species were predominant in the shallow layer, while Prochlorococcus MIT 9313 formed a higher portion below 50 m depth. Bacteroidota were represented mostly by uncultured lineages (NS4, NS5 and NS9 marine lineages). In contrast, Actinobacteriota were dominated by a candidatus genus Ca. Actinomarina. A large contribution of Nitrospinae was evident at the deepest investigated layer. Our results document that neural network analysis of environmental data may provide a novel insight into factors affecting picoplankton in the open sea environment.
- MeSH
- biodiverzita * MeSH
- mikrobiota * MeSH
- neuronové sítě (počítačové) * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Středozemní moře MeSH
Phagotrophic protists are key players in aquatic food webs. Although sequencing-based studies have revealed their enormous diversity, ecological information on in situ abundance, feeding modes, grazing preferences, and growth rates of specific lineages can be reliably obtained only using microscopy-based molecular methods, such as Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). CARD-FISH is commonly applied to study prokaryotes, but less so to microbial eukaryotes. Application of this technique revealed that Paraphysomonas or Spumella-like chrysophytes, considered to be among the most prominent members of protistan communities in pelagic environments, are omnipresent but actually less abundant than expected, in contrast to little known groups such as heterotrophic cryptophyte lineages (e.g., CRY1), cercozoans, katablepharids, or the MAST lineages. Combination of CARD-FISH with tracer techniques and application of double CARD-FISH allow visualization of food vacuole contents of specific flagellate groups, thus considerably challenging our current, simplistic view that they are predominantly bacterivores. Experimental manipulations with natural communities revealed that larger flagellates are actually omnivores ingesting both prokaryotes and other protists. These new findings justify our proposition of an updated model of microbial food webs in pelagic environments, reflecting more authentically the complex trophic interactions and specific roles of flagellated protists, with inclusion of at least two additional trophic levels in the nanoplankton size fraction. Moreover, we provide a detailed CARD-FISH protocol for protists, exemplified on mixo- and heterotrophic nanoplanktonic flagellates, together with tips on probe design, a troubleshooting guide addressing most frequent obstacles, and an exhaustive list of published probes targeting protists.
BACKGROUND: Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. RESULTS: Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7-101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. CONCLUSIONS: Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future. Video abstract.
- Klíčová slova
- Freshwater bacterioplankton, Long-read amplicon sequencing, Microdiversity, PacBio, Phylogeography, Ribosomal internal transcribed spacers,
- MeSH
- biodiverzita * MeSH
- fylogeneze MeSH
- fylogeografie * MeSH
- plankton klasifikace genetika izolace a purifikace MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA metody MeSH
- sladká voda * MeSH
- vodní organismy klasifikace genetika izolace a purifikace MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Japonsko MeSH
- Názvy látek
- RNA ribozomální 16S MeSH
Heterotrophic nanoflagellates (HNF) are considered as major planktonic bacterivores, however, larger HNF taxa can also be important predators of eukaryotes. To examine this trophic cascading, natural protistan communities from a freshwater reservoir were released from grazing pressure by zooplankton via filtration through 10- and 5-µm filters, yielding microbial food webs of different complexity. Protistan growth was stimulated by amendments of five Limnohabitans strains, thus yielding five prey-specific treatments distinctly modulating protistan communities in 10- versus 5-µm fractions. HNF dynamics was tracked by applying five eukaryotic fluorescence in situ hybridization probes covering 55-90% of total flagellates. During the first experimental part, mainly small bacterivorous Cryptophyceae prevailed, with significantly higher abundances in 5-µm treatments. Larger predatory flagellates affiliating with Katablepharidacea and one Cercozoan lineage (increasing to up to 28% of total HNF) proliferated towards the experimental endpoint, having obviously small phagocytized HNF in their food vacuoles. These predatory flagellates reached higher abundances in 10-µm treatments, where small ciliate predators and flagellate hunters also (Urotricha spp., Balanion planctonicum) dominated the ciliate assemblage. Overall, our study reports pronounced cascading effects from bacteria to bacterivorous HNF, predatory HNF and ciliates in highly treatment-specific fashions, defined by both prey-food characteristics and feeding modes of predominating protists.
- Klíčová slova
- Cercozoa, Cryptophyceae, Katablepharidacea, bacterivorous and predatory flagellates, ciliates, freshwater microbial food webs,
- MeSH
- Cercozoa * MeSH
- Cryptophyta MeSH
- hybridizace in situ fluorescenční MeSH
- potravní řetězec * MeSH
- sladká voda MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phytoplankton is a key component of aquatic microbial communities, and metabolic coupling between phytoplankton and bacteria determines the fate of dissolved organic carbon (DOC). Yet, the impact of primary production on bacterial activity and community composition remains largely unknown, as, for example, in the case of aerobic anoxygenic phototrophic (AAP) bacteria that utilize both phytoplankton-derived DOC and light as energy sources. Here, we studied how reduction of primary production in a natural freshwater community affects the bacterial community composition and its activity, focusing primarily on AAP bacteria. The bacterial respiration rate was the lowest when photosynthesis was reduced by direct inhibition of photosystem II and the highest in ambient light condition with no photosynthesis inhibition, suggesting that it was limited by carbon availability. However, bacterial assimilation rates of leucine and glucose were unaffected, indicating that increased bacterial growth efficiency (e.g., due to photoheterotrophy) can help to maintain overall bacterial production when low primary production limits DOC availability. Bacterial community composition was tightly linked to light intensity, mainly due to the increased relative abundance of light-dependent AAP bacteria. This notion shows that changes in bacterial community composition are not necessarily reflected by changes in bacterial production or growth and vice versa. Moreover, we demonstrated for the first time that light can directly affect bacterial community composition, a topic which has been neglected in studies of phytoplankton-bacteria interactions.IMPORTANCE Metabolic coupling between phytoplankton and bacteria determines the fate of dissolved organic carbon in aquatic environments, and yet how changes in the rate of primary production affect the bacterial activity and community composition remains understudied. Here, we experimentally limited the rate of primary production either by lowering light intensity or by adding a photosynthesis inhibitor. The induced decrease had a greater influence on bacterial respiration than on bacterial production and growth rate, especially at an optimal light intensity. This suggests that changes in primary production drive bacterial activity, but the effect on carbon flow may be mitigated by increased bacterial growth efficiencies, especially of light-dependent AAP bacteria. Bacterial activities were independent of changes in bacterial community composition, which were driven by light availability and AAP bacteria. This direct effect of light on composition of bacterial communities has not been documented previously.
- Klíčová slova
- AAP community composition, aerobic anoxygenic phototrophic bacteria, bacterial community composition, phytoplankton-bacteria coupling,
- MeSH
- aerobní bakterie růst a vývoj metabolismus MeSH
- ekosystém * MeSH
- fotosyntéza MeSH
- fototrofní procesy * MeSH
- fyziologie bakterií MeSH
- mikrobiota * MeSH
- mořská voda mikrobiologie MeSH
- sladká voda mikrobiologie MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
