Integrating depth-dependent protist dynamics and microbial interactions in spring succession of a freshwater reservoir.

BACKGROUND: Protists are essential contributors to eukaryotic diversity and exert profound influence on carbon fluxes and energy transfer in freshwaters. Despite their significance, there is a notable gap in research on protistan dynamics, particularly in the deeper strata of temperate lakes. This study aimed to address this gap by integrating protists into the well-described spring dynamics of Rímov reservoir, Czech Republic. Over a 2-month period covering transition from mixing to established stratification, we collected water samples from three reservoir depths (0.5, 10 and 30 m) with a frequency of up to three times per week. Microbial eukaryotic and prokaryotic communities were analysed using SSU rRNA gene amplicon sequencing and dominant protistan groups were enumerated by Catalysed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). Additionally, we collected samples for water chemistry, phyto- and zooplankton composition analyses. RESULTS: Following the rapid changes in environmental and biotic parameters during spring, protistan and bacterial communities displayed swift transitions from a homogeneous community to distinct strata-specific communities. A prevalence of auto- and mixotrophic protists dominated by cryptophytes was associated with spring algal bloom-specialized bacteria in the epilimnion. In contrast, the meta- and hypolimnion showcased a development of a protist community dominated by putative parasitic Perkinsozoa, detritus or particle-associated ciliates, cercozoans, telonemids and excavate protists (Kinetoplastida), co-occurring with bacteria associated with lake snow. CONCLUSIONS: Our high-resolution sampling matching the typical doubling time of microbes along with the combined microscopic and molecular approach and inclusion of all main components of the microbial food web allowed us to unveil depth-specific populations' successions and interactions in a deep lentic ecosystem.

In the right place, at the right time: the integration of bacteria into the Plankton Ecology Group model.

BACKGROUND: Planktonic microbial communities have critical impacts on the pelagic food web and water quality status in freshwater ecosystems, yet no general model of bacterial community assembly linked to higher trophic levels and hydrodynamics has been assessed. In this study, we utilized a 2-year survey of planktonic communities from bacteria to zooplankton in three freshwater reservoirs to investigate their spatiotemporal dynamics. RESULTS: We observed site-specific occurrence and microdiversification of bacteria in lacustrine and riverine environments, as well as in deep hypolimnia. Moreover, we determined recurrent bacterial seasonal patterns driven by both biotic and abiotic conditions, which could be integrated into the well-known Plankton Ecology Group (PEG) model describing primarily the seasonalities of larger plankton groups. Importantly, bacteria with different ecological potentials showed finely coordinated successions affiliated with four seasonal phases, including the spring bloom dominated by fast-growing opportunists, the clear-water phase associated with oligotrophic ultramicrobacteria, the summer phase characterized by phytoplankton bloom-associated bacteria, and the fall/winter phase driven by decay-specialists. CONCLUSIONS: Our findings elucidate the major principles driving the spatiotemporal microbial community distribution in freshwater ecosystems. We suggest an extension to the original PEG model by integrating new findings on recurrent bacterial seasonal trends. Video Abstract.

High-resolution metagenomic reconstruction of the freshwater spring bloom.

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.

Cryptic and ubiquitous aplastidic cryptophytes are key freshwater flagellated bacterivores.

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 » 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.

Trophic flexibility of marine diplonemids - switching from osmotrophy to bacterivory.

Diplonemids are one of the most abundant groups of heterotrophic planktonic microeukaryotes in the world ocean and, thus, are likely to play an essential role in marine ecosystems. So far, only few species have been introduced into a culture, allowing basic studies of diplonemid genetics, morphology, ultrastructure, metabolism, as well as endosymbionts. However, it remains unclear whether these heterotrophic flagellates are parasitic or free-living and what are their predominant dietary patterns and preferred food items. Here we show that cultured diplonemids, maintained in an organic-rich medium as osmotrophs, can gradually switch to bacterivory as a sole food resource, supporting positive growth of their population, even when fed with a low biovolume of bacteria. We further observed remarkable differences in species-specific feeding patterns, size-selective grazing preferences, and distinct feeding strategies. Diplonemids can discriminate between low-quality food items and inedible particles, such as latex beads, even after their ingestion, by discharging them in the form of large waste vacuoles. We also detected digestion-related endogenous autofluorescence emitted by lysosomes and the activity of a melanin-like material. We present the first evidence that these omnipresent protists possess an opportunistic lifestyle that provides a considerable advantage in the generally food resource-limited marine environments.

CARD-FISH and prey tracer techniques reveal the role of overlooked flagellate groups as major bacterivores in freshwater hypertrophic shallow lakes.

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.

CARD-FISH in the Sequencing Era: Opening a New Universe of Protistan Ecology.

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.

Recovery of freshwater microbial communities after extreme rain events is mediated by cyclic succession.

Small lakes and ponds occupy an enormous surface area of inland freshwater and represent an important terrestrial-water interface. Disturbances caused by extreme weather events can have substantial effects on these ecosystems. Here, we analysed the dynamics of nutrients and the entire plankton community in two flood events and afterwards, when quasi-stable conditions were established, to investigate the effect of such disturbances on a small forest pond. We show that floodings result in repeated washout of resident organisms and hundredfold increases in nutrient load. Despite this, the microbial community recovers to a predisturbance state within two weeks of flooding through four well-defined succession phases. Reassembly of phytoplankton and especially zooplankton takes up to two times longer and features repetitive and adaptive patterns. Release of dissolved nutrients from the pond is associated with inflow rates and community recovery, and returns to predisturbance levels before microbial compositions recover. Our findings shed light on the mechanisms underlying functional resilience of small waterbodies and are relevant to global change-induced increases in weather extremes.

A freshwater radiation of diplonemids.

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Japanese deep freshwater lakes. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and catalysed reporter deposition-fluorescent in situ hybridization (CARD-FISH). We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to a marine clade, which are mainly isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single-cell genomes of marine diplonemids show they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.

Cascading effects in freshwater microbial food webs by predatory Cercozoa, Katablepharidacea and ciliates feeding on aplastidic bacterivorous cryptophytes.

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.

Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations.

High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards.IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Correction to: Hunters or farmers? Microbiome characteristics help elucidate the diet composition in an aquatic carnivorous plant.

AbstractFollowing publication of the original article [1], the author reported an error in Fig. 3.

Hunters or farmers? Microbiome characteristics help elucidate the diet composition in an aquatic carnivorous plant.

BACKGROUND: Utricularia are rootless aquatic carnivorous plants which have recently attracted the attention of researchers due to the peculiarities of their miniaturized genomes. Here, we focus on a novel aspect of Utricularia ecophysiology-the interactions with and within the complex communities of microorganisms colonizing their traps and external surfaces. RESULTS: Bacteria, fungi, algae, and protozoa inhabit the miniature ecosystem of the Utricularia trap lumen and are involved in the regeneration of nutrients from complex organic matter. By combining molecular methods, microscopy, and other approaches to assess the trap-associated microbial community structure, diversity, function, as well as the nutrient turn-over potential of bacterivory, we gained insight into the nutrient acquisition strategies of the Utricularia hosts. CONCLUSIONS: We conclude that Utricularia traps can, in terms of their ecophysiological function, be compared to microbial cultivators or farms, which center around complex microbial consortia acting synergistically to convert complex organic matter, often of algal origin, into a source of utilizable nutrients for the plants.

Hidden in plain sight-highly abundant and diverse planktonic freshwater Chloroflexi.

BACKGROUND: Representatives of the phylum Chloroflexi, though reportedly highly abundant in the extensive deep water habitats of both marine (SAR202 up to 30% of total prokaryotes) and freshwater (CL500-11 up to 26% of total prokaryotes), remain uncultivated and uncharacterized. There are few metagenomic studies on marine Chloroflexi representatives, while the pelagic freshwater Chloroflexi community is largely unknown except for a single metagenome-assembled genome of CL500-11. RESULTS: Here, we provide the first extensive examination of the community composition of this cosmopolitan phylum in a range of pelagic habitats (176 datasets) and highlight the impact of salinity and depth on their phylogenomic composition. Reconstructed genomes (53 in total) provide a perspective on the phylogeny, metabolism, and distribution of three novel classes and two family-level taxa within the phylum Chloroflexi. We unraveled a remarkable genomic diversity of pelagic freshwater Chloroflexi representatives that thrive not only in the hypolimnion as previously suspected, but also in the epilimnion. Our results suggest that the lake hypolimnion provides a globally stable habitat reflected in lower species diversity among hypolimnion-specific CL500-11 and TK10 clusters in distantly related lakes compared to a higher species diversity of the epilimnion-specific SL56 cluster. Cell volume analyses show that the CL500-11 are among the largest prokaryotic cells in the water column of deep lakes and with a biomass to abundance ratio of two they significantly contribute to the deep lake carbon flow. Metabolic insights indicate participation of JG30-KF-CM66 representatives in the global cobalamin production via cobinamide to cobalamin salvage pathway. CONCLUSIONS: Extending phylogenomic comparisons to brackish and marine habitats suggests salinity as the major influencer of the community composition of the deep-dwelling Chloroflexi in marine (SAR202) and freshwater (CL500-11) habitats as both counterparts thrive in intermediate brackish salinity; however, freshwater habitats harbor the most phylogenetically diverse community of pelagic Chloroflexi representatives that reside both in epi- and hypolimnion.

Determining lineage-specific bacterial growth curves with a novel approach based on amplicon reads normalization using internal standard (ARNIS).

The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir Rímov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers' removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.

Cryptophyta as major bacterivores in freshwater summer plankton.

Small bacterivorous eukaryotes play a cardinal role in aquatic food webs and their taxonomic classification is currently a hot topic in aquatic microbial ecology. Despite increasing interest in their diversity, core questions regarding predator-prey specificity remain largely unanswered, e.g., which heterotrophic nanoflagellates (HNFs) are the main bacterivores in freshwaters and which prokaryotes support the growth of small HNFs. To answer these questions, we fed natural communities of HNFs from Rímov reservoir (Czech Republic) with five different bacterial strains of the ubiquitous betaproteobacterial genera Polynucleobacter and Limnohabitans. We combined amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) targeting eukaryotic 18 S rRNA genes to track specific responses of the natural HNF community to prey amendments. While amplicon sequencing provided valuable qualitative data and a basis for designing specific probes, the number of reads was insufficient to accurately quantify certain eukaryotic groups. We also applied a double-hybridization technique that allows simultaneous phylogenetic identification of both predator and prey. Our results show that community composition of HNFs is strongly dependent upon prey type. Surprisingly, Cryptophyta were the most abundant bacterivores, although this phylum has been so far assumed to be mainly autotrophic. Moreover, the growth of a small lineage of Cryptophyta (CRY1 clade) was strongly stimulated by one Limnohabitans strain in our experiment. Thus, our study is the first report that colorless Cryptophyta are major bacterivores in summer plankton samples and can play a key role in the carbon transfer from prokaryotes to higher trophic levels.

Aerobic Anoxygenic Photosynthesis Is Commonly Present within the Genus Limnohabitans.

The genus Limnohabitans (Comamonadaceae, Betaproteobacteria) is a common and a highly active component of freshwater bacterioplanktonic communities. To date, the genus has been considered to contain only heterotrophic species. In this study, we detected the photosynthesis genes pufLM and bchY in 28 of 46 strains from three Limnohabitans lineages. The pufM sequences obtained are very closely related to environmental pufM sequences detected in various freshwater habitats, indicating the ubiquity and potential importance of photoheterotrophic Limnohabitans in nature. Additionally, we sequenced and analyzed the genomes of 5 potentially photoheterotrophic Limnohabitans strains, to gain further insights into their phototrophic capacity. The structure of the photosynthesis gene cluster turned out to be highly conserved within the genus Limnohabitans and also among all potentially photosynthetic Betaproteobacteria strains. The expression of photosynthetic complexes was detected in a culture of Limnohabitans planktonicus II-D5T using spectroscopic and pigment analyses. This was further verified by a novel combination of infrared microscopy and fluorescent in situ hybridization.IMPORTANCE The data presented document that the capacity to perform anoxygenic photosynthesis is common among the members of the genus Limnohabitans, indicating that they may have a novel role in freshwater habitats.

Strain-specific consumption and transformation of alga-derived dissolved organic matter by members of the Limnohabitans-C and Polynucleobacter-B clusters of Betaproteobacteria.

We investigated changes in quality and quantity of extracellular and biomass-derived organic matter (OM) from three axenic algae (genera Rhodomonas, Chlamydomonas, Coelastrum) during growth of Limnohabitans parvus, Limnohabitans planktonicus and Polynucleobacter acidiphobus representing important clusters of freshwater planktonic Betaproteobacteria. Total extracellular and biomass-derived OM concentrations from each alga were approximately 20 mg l-1 and 1 mg l-1 respectively, from which up to 9% could be identified as free carbohydrates, polyamines, or free and combined amino acids. Carbohydrates represented 54%-61% of identified compounds of the extracellular OM from each alga. In biomass-derived OM of Rhodomonas and Chlamydomonas 71%-77% were amino acids and polyamines, while in that of Coelastrum 85% were carbohydrates. All bacteria grew on alga-derived OM of Coelastrum, whereas only Limnohabitans strains grew on OM from Rhodomonas and Chlamydomonas. Bacteria consumed 24%-76% and 38%-82% of all identified extracellular and biomass-derived OM compounds respectively, and their consumption was proportional to the concentration of each OM compound in the different treatments. The bacterial biomass yield was higher than the total identifiable OM consumption indicating that bacteria also utilized other unidentified alga-derived OM compounds. Bacteria, however, also produced specific OM compounds suggesting enzymatic polymer degradation or de novo exudation.

The Limnohabitans Genus Harbors Generalistic and Opportunistic Subtypes: Evidence from Spatiotemporal Succession in a Canyon-Shaped Reservoir.

We studied the diversity of Limnohabitans using reverse line blot hybridization with Limnohabitans lineage-specific probes in the freshwater canyon-shaped Rímov reservoir (Czech Republic). To examine the succession of distinct lineages, we performed (i) a study of an intensive spring sampling program at the lacustrine part of the Rímov reservoir (from ice melt through a phytoplankton peak to the clear-water phase), and (ii) a seasonal study (April to November) when the occurrence of distinct Limnohabitans lineages was related to the inherent longitudinal heterogeneity of the reservoir. Significant spatiotemporal changes in the compositions of distinct Limnohabitans lineages allowed for the identification of "generalists" that were always present throughout the whole season as well as "specialists" that appeared in the reservoir only for limited periods of time or irregularly. Our results indicate that some phytoplankton groups, such as cryptophytes or cyanobacteria, and zooplankton composition were the major factors modulating the distribution and dynamics of distinct Limnohabitans lineages. The highest Limnohabitans diversity was observed during the spring algal bloom, whereas the lowest was during the summer cyanobacterial bloom. The microdiversity also markedly increased upstream in the reservoir, being highest at the inflow, and thus likely reflecting strong influences of the watershed.IMPORTANCE The genus Limnohabitans is a typical freshwater bacterioplankton and is believed to play a significant role in inland freshwater habitats. This work is unique in detecting and tracing different closely related lineages of this bacterial genus in its natural conditions using the semiquantitative reverse line blot hybridization method and in discovering the factors influencing the microdiversity, subtype alternations, and seasonality.