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The increasing frequency and intensity of heatwaves driven by climate change significantly impact microbial communities in freshwater habitats, particularly eukaryotic microorganisms. Heterotrophic nanoflagellates are important bacterivorous grazers and play a crucial role in aquatic food webs, influencing the morphological and taxonomic structure of bacterial communities. This study investigates the responses of three flagellate taxa to heatwave conditions through single-strain and mixed culture experiments, highlighting the impact of both biotic and abiotic factors on functional redundancy between morphologically similar protist species under thermal stress. Our results indicate that temperature can significantly impact growth and community composition. However, density-dependent factors also had a significant impact. In sum, stabilizing effects due to functional redundancy may be pronounced as long as density-dependent factors play a minor role and can be overshadowed when flagellate abundances increase.

Sequencing of amplified DNA is the first step towards the generation of Amplicon Sequence Variants (ASVs) or Operational Taxonomic Units (OTUs) for biodiversity assessment and comparative analyses of environmental communities and microbiomes. Notably, the rapid advancements in sequencing technologies have paved the way for the growing utilization of third-generation long-read approaches in recent years. These sequence data imply increasing read lengths, higher error rates, and altered sequencing chemistry. Likewise, methods for amplicon classification and reference databases have progressed, leading to the expansion of taxonomic application areas and higher classification accuracy. With Natrix, a user-friendly and reducible workflow solution, processing of prokaryotic and eukaryotic environmental Illumina sequences using 16S or 18S is possible. Here, we present an updated version of the pipeline, Natrix2, which incorporates VSEARCH as an alternative clustering method with better performance for 16S metabarcoding approaches and mothur for taxonomic classification on further databases, including PR2, UNITE and SILVA. Additionally, Natrix2 includes the handling of Nanopore reads, which entails initial error correction and refinement of reads using Medaka and Racon to subsequently determine their taxonomic classification.

Abstract Microbial communities in freshwater streams play an essential role in ecosystem functioning via biogeochemical cycling. Yet, the impacts of treated wastewater influx into stream ecosystems on microbial strain diversity remain mostly unexplored. Here, we coupled full‐length 16S ribosomal RNA gene Nanopore sequencing and strain‐resolved metagenomics to investigate the impact of treated wastewater on a mesocosm system (AquaFlow) run with restored river water. Over 10 days, community Bray–Curtis dissimilarities between treated and control mesocosm decreased (0.57 ± 0.058 to 0.26 ± 0.046) based on ribosomal protein S3 gene clustering, finally converging to nearly identical communities. Similarly, strain‐resolved metagenomics revealed a high diversity of bacteria and viruses after the introduction of treated wastewater; these microbes also decreased over time resulting in the same strain clusters in control and treatment at the end of the experiment. Specifically, 39.2% of viral strains detected in all samples were present after the introduction of treated wastewater only. Although bacteria present at low abundance in the treated wastewater introduced additional antibiotic resistance genes, signals of naturally occurring ARG‐encoding organisms resembled the resistome at the endpoint. Our results suggest that the previously stressed freshwater stream and its microbial community are resilient to a substantial introduction of treated wastewater.

Majda and colleagues employ a comparative genomic approach to investigate the effect of nutrient and carbon shortage on chrysophyte evolution. Their results indicate that heterotrophic taxa have reduced genome size and higher GC content compared to phototrophic taxa.

Abstract Background Sequencing of marker genes amplified from environmental samples, known as amplicon sequencing, allows us to resolve some of the hidden diversity and elucidate evolutionary relationships and ecological processes among complex microbial communities. The analysis of large numbers of samples at high sequencing depths generated by high throughput sequencing technologies requires efficient, flexible, and reproducible bioinformatics pipelines. Only a few existing workflows can be run in a user-friendly, scalable, and reproducible manner on different computing devices using an efficient workflow management system. Results We present Natrix, an open-source bioinformatics workflow for preprocessing raw amplicon sequencing data. The workflow contains all analysis steps from quality assessment, read assembly, dereplication, chimera detection, split-sample merging, sequence representative assignment (OTUs or ASVs) to the taxonomic assignment of sequence representatives. The workflow is written using Snakemake, a workflow management engine for developing data analysis workflows. In addition, Conda is used for version control. Thus, Snakemake ensures reproducibility and Conda offers version control of the utilized programs. The encapsulation of rules and their dependencies support hassle-free sharing of rules between workflows and easy adaptation and extension of existing workflows. Natrix is freely available on GitHub ( https://github.com/MW55/Natrix ) or as a Docker container on DockerHub ( https://hub.docker.com/r/mw55/natrix ). Conclusion Natrix is a user-friendly and highly extensible workflow for processing Illumina amplicon data.

Species of the ciliate genus Urotricha are key players in freshwater plankton communities. In the pelagial of lakes, about 20 urotrich species occur throughout an annual cycle, some of which play a pivotal role in aquatic food webs. For example, during the phytoplankton spring bloom, they consume a remarkable proportion of the algal production. In ecological studies, urotrich ciliates are usually merely identified to genus rank and grouped into size classes. This is unsatisfying considering the distinct autecological properties of individual species and their specific spatial and temporal distribution patterns. As a basis for future research, we characterized in detail four common urotrich morphotypes, i.e., specimens identified as U. furcata and tentatively as U. agilis, U. pseudofurcata, and U. castalia, using state-of-the-art methods. We used an integrative polyphasic approach, in which morphological studies (in vivo observation, silver staining methods, scanning electron microscopy) were linked with a molecular approach exploiting four different gene fragments as taxonomic DNA barcodes with different resolution potential (SSU rDNA, ITS-1, ITS-2, hypervariable V4 and V9 regions of the SSU rDNA). We shed light on the diversity of urotrich ciliates as well as on their global distribution patterns, and annual cycles. Additionally, we coupled individual species occurrences and environmental parameters, and subsequently modeled the distribution and occurrence, using logistic regressions. Furthermore, for one strain putatively identified as U. castalia, we ascertained the optimal cultivation media and food preferences. Thereby, our comprehensive view on these important freshwater ciliates that frequently occur in environmental high throughput sequencing datasets worldwide will allow future studies to better exploit protistan plankton data from lakes.

The surveillance of wastewater for the Covid-19 virus during this unprecedented pandemic and mapped to the distribution and magnitude of the infected in the population near real-time exemplifies the importance of tracking rapidly changing trends of pathogens or public health problems at a large scale. The rising trends of antimicrobial resistance (AMR) with multidrug-resistant pathogens from the environmental water have similarly gained much attention in recent years. Wastewater-based epidemiology from water samples has shown that a wide range of AMR-related genes is frequently detected. Albeit sewage is treated before release and thus, the abundance of pathogens should be significantly reduced or even pathogen-free, several studies indicated the contrary. Pathogens are still measurable in the released water, ultimately entering freshwaters, such as rivers and lakes. Furthermore, socio-economic and environmental factors, such as chemical industries and animal farming nearby, impact the presence of AMR. Many bacterial species from the environment are intrinsically resistant and also contribute to the resistome of freshwater lakes. This study collected the most extensive standardized freshwater data set from hundreds of European lakes and conducted a comprehensive multi-omics analysis on antimicrobial resistance from these freshwater lakes. Our research shows that genes encoding for AMR against tetracyclines, cephalosporins, and quinolones were commonly identified, while for some, such as sulfonamides, resistance was less frequently present. We provide an estimation of the characteristic resistance of AMR in European lakes, which can be used as a comprehensive resistome dataset to facilitate and monitor temporal changes in the development of AMR in European freshwater lakes.

Abstract Biogeography in Europe is known to be crucially influenced by the large mountain ranges serving as biogeographical islands for cold‐adapted taxa and geographical barriers for warm‐adapted taxa. While biogeographical patterns are well‐known for plants and animals in Europe, we here investigated diversity and distribution patterns of protist freshwater communities on a European scale (256 lakes) in the light of the well‐studied post‐glacial distribution patterns of macroorganisms. Thus, our study compared 43 alpine protist communities of lakes located in the Alps, Carpathians, Pyrenees, and the Sierra Nevada with that of surrounding lowland lakes. We verified altitudinal diversity gradients of freshwater protists with decreasing richness and diversity across altitudes similar to those observed for plants and animals. Alpine specialists and generalists could be identified differing significantly in richness and diversity, but hardly in occurrence and proportions of major taxonomic groups. High proportions of region‐specific alpine specialists indicate an increased occurrence of distinct lineages within each mountain range and thus, suggested either separated glacial refugia or post‐glacial diversification within mountain ranges. However, a few alpine specialists were shared between mountain ranges suggesting a post‐glacial recolonization from a common lowland pool. Our results identified generalists with wide distribution ranges and putatively wide tolerance ranges toward environmental conditions as main drivers of protist diversification (specification) in alpine lakes, while there was hardly any diversification in alpine specialists.

Abstract Background Microbes are essentail components of all ecosystems because they drive many biochemical processes and act as primary producers. In freshwater ecosystems, the biodiversity in and the composition of microbial communities can be used as indicators for environmental quality. Recently, some environmental features have been identified that influence microbial ecosystems. However, the impact of human action on lake microbiomes is not well understood. This is, in part, due to the fact that environmental data is, albeit theoretically accessible, not easily available. Results In this work, we present SEDE-GPS, a tool that gathers data that are relevant to the environment of an user-provided GPS coordinate. To this end, it accesses a list of public and corporate databases and aggregates the information in a single file, which can be used for further analysis. To showcase the use of SEDE-GPS, we enriched a lake microbial ecology sequencing dataset with around 18,000 socio-economic, climate, and geographic features. The sources of SEDE-GPS are public databases such as Eurostat, the Climate Data Center, and OpenStreetMap, as well as corporate sources such as Twitter. Using machine learning and feature selection methods, we were able to identify features in the data provided by SEDE-GPS that can be used to predict lake microbiome alpha diversity. Conclusion The results presented in this study show that SEDE-GPS is a handy and easy-to-use tool for comprehensive data enrichment for studies of ecology and other processes that are affected by environmental features. Furthermore, we present lists of environmental, socio-economic, and climate features that are predictive for microbial biodiversity in lake ecosystems. These lists indicate that human action has a major impact on lake microbiomes. SEDE-GPS and its source code is available for download at http://SEDE-GPS.heiderlab.de

Polynucleobacter (Burkholderiaceae, Betaproteobacteria) and Limnohabitans (Comamonadaceae, Betaproteobacteria) are abundant freshwater bacteria comprising large genetic and taxonomic diversities, with species adapted to physico-chemically distinct types of freshwater systems. The relative importance of environmental drivers, i.e., physico-chemistry, presence of microeukaryotes and geographic position for the diversity and prevalence has not been investigated for both taxa before. Here, we present the first pan-European study on this topic, comprising 255 freshwater lakes. We investigated Limnohabitans and Polynucleobacter using an amplicon sequencing approach of partial 16S rRNA genes along environmental gradients. We show that physico-chemical factors had the greatest impact on both genera. Analyses on environmental gradients revealed an exceptionally broad ecological spectrum of operational taxonomic units (OTUs). Despite the coarse resolution of the genetic marker, we found OTUs with contrasting environmental preferences within Polynucleobacter and Limnohabitans subclusters. Such an ecological differentiation has been characterized for PnecC and LimC before but was so far unknown for less well studied subclusters such as PnecA and PnecB. Richness and abundance of OTUs are geographically clustered, suggesting that geographic diversity patterns are attributable to region-specific physico-chemical characteristics (e.g., pH and temperature) rather than latitudinal gradients or lake sizes.

Protists influence ecosystems by modulating microbial population size, diversity, metabolic outputs and gene flow. In this study we used eukaryotic ribosomal amplicon diversity from 218 European freshwater lakes sampled in August 2012 to assess the effect of mountain ranges as biogeographic barriers on spatial patterns and microbial community structure in European freshwaters. The diversity of microbial communities as reflected by amplicon clusters suggested that the eukaryotic microbial inventory of lakes was well-sampled at the European and at the local scale. Our pan-European diversity analysis indicated that biodiversity and richness of high mountain lakes differed from that of lowland lakes. Further, the taxon inventory of high-mountain lakes strongly contributed to beta-diversity despite a low taxon inventory. Even though ecological factors, in general, strongly affect protist community pattern, we show that geographic distance and geographic barriers significantly contribute to community composition particularly for high mountain regions which presumably act as biogeographic islands. However, community composition in lowland lakes was also affected by geographic distance but less pronounced as in high mountain regions. In consequence protist populations are locally structured into distinct biogeographic provinces and community analyses revealed biogeographic patterns also for lowland lakes whereby European mountain ranges act as dispersal barriers in particular for short to intermediate distances whereas the effect of mountain ranges levels off on larger scale.

Abstract Background High-throughput sequencing (HTS) technologies are increasingly applied to analyse complex microbial ecosystems by mRNA sequencing of whole communities, also known as metatranscriptome sequencing. This approach is at the moment largely limited to prokaryotic communities and communities of few eukaryotic species with sequenced genomes. For eukaryotes the analysis is hindered mainly by a low and fragmented coverage of the reference databases to infer the community composition, but also by lack of automated workflows for the task. Results From the databases of the National Center for Biotechnology Information and Marine Microbial Eukaryote Transcriptome Sequencing Project, 142 references were selected in such a way that the taxa represent the main lineages within each of the seven supergroups of eukaryotes and possess predominantly complete transcriptomes or genomes. From these references, we created an annotated microeukaryotic reference database. We developed a tool called TaxMapper for a reliably mapping of sequencing reads against this database and filtering of unreliable assignments. For filtering, a classifier was trained and tested on each of the following: sequences of taxa in the database, sequences of taxa related to those in the database, and random sequences. Additionally, TaxMapper is part of a metatranscriptomic Snakemake workflow developed to perform quality assessment, functional and taxonomic annotation and (multivariate) statistical analysis including environmental data. The workflow is provided and described in detail to empower researchers to apply it for metatranscriptome analysis of any environmental sample. Conclusions TaxMapper shows superior performance compared to standard approaches, resulting in a higher number of true positive taxonomic assignments. Both the TaxMapper tool and the workflow are available as open-source code at Bitbucket under the MIT license: https://bitbucket.org/dbeisser/taxmapper and as a Bioconda package: https://bioconda.github.io/recipes/taxmapper/README.html .

Background Most protist lineages consist of members with diverging features e.g. different modes of nutrition and adaptations for life in different habitat types and climatic zones. The nutritional mode is particularly variable in chrysophytes and they are therefore an excellent model group to study the core genes and metabolic pathways of a functionally diverse lineage. The objective of our study is the identification of the joint genetic repertoire expressed in closely related chrysophytes as well as the extent of variation on species and strain level. Therefore, we investigated the transcriptomes of six strains belonging to four species of Ochromonadales. We performed analyses on metabolic pathway level as well as on sequence level. Results We could identify 1,574 core genes shared between all six investigated strains of Ochromonadales. Most of these core genes were affiliated with the primary metabolism. Phylogenetic analysis of 166 protein-coding core genes supported a close relation of Poteriospumella lacustris and Poterioochromonas malhamensis and resolved for more than 50% of investigated genes the relationship of strains affiliated with the species P. lacustris. Further, we found diverging phylogenetic patterns for genes interacting with the environment. Conclusions In Ochromonadales, a functionally diverse lineage, the core transcriptome represents only a minor part of the individual transcriptomes. But this small fraction of genes comprises the basal metabolism essential for life in several protist lineages. Phylogenetic analyses of these genes indicate a similar degree of conservation as observed for genes coding for ribosomal proteins.

We studied the impact of protist grazing and exudation on the growth and transcriptomic response of the prokaryotic prey species Polynucleobacter asymbioticus. Different single- and multi-species communities of chrysophytes were used to determine a species-specific response to the predators and the effect of chrysophyte diversity. We sequenced the mRNA of Pn. asymbioticus in communities with three single chrysophyte species (Chlorochromonas danica, Poterioochromonas malhamensis and Poteriospumella lacustris) and all combinations. The molecular responses of Pn. asymbioticus significantly changed in the presence of predators with different trophic modes and combinations of species. In the single-species samples we observed significant differences related to the relative importance of grazing and exudation in the protist-bacteria interaction, i.e., to the presence of either the heterotrophic Ps. lacustris or the mixotrophic C. danica. When grazing dominates the interaction, as in the presence of Ps. lacustris, genes acting in stress response are up-regulated. Further genes associated with transcription and translation are down-regulated indicating a reduced growth of Pn. asymbioticus. In contrast, when the potential use of algal exudates dominates the interaction, genes affiliated with iron transport are up-regulated. Rapid phototrophic growth of chrysophytes, with a high demand on soluble iron, could thus lead to iron-limitation and cause changes in the iron metabolism of Pn. asymbioticus. Additionally, we observe a benefit for Pn. asymbioticus from a more diverse protistan community, which could be due to shifts in the relative importance of phototrophy in the mixotrophic chrysophytes when competing for food with other species. Our study highlights the importance of biotic interactions and the specificity of such interactions, in particular the differential effect of grazing and algal exudation in the interaction of bacteria with mixotrophic protists.

Chrysophytes are a large group of heterotrophic, phototrophic, or even mixotrophic protists that are abundant in aquatic as well as terrestrial environments. Although much is known about chrysophyte biology and ecology, it is unknown if they are sexual or not. Here we use available transcriptomes of 18 isolates of 15 putatively asexual species to inventory the presence of genes used in meiosis. Since we were able to detect a set of nine meiosis-specific and 29 meiosis-related genes shared by the chrysophytes, we conclude that they are secretively sexual and therefore should be investigated further using genome sequencing to uncover any missed genes from the transcriptomes.

Freshwater systems are characterized by an enormous diversity of eukaryotic protists and prokaryotic taxa. The community structures in different lakes are thereby influenced by factors such as habitat size, lake chemistry, biotic interactions, and seasonality. In our study, we used high throughput 454 sequencing to study the diversity and temporal changes of prokaryotic and eukaryotic planktonic communities in three Austrian lakes during the ice-free season. In the following year, one lake was sampled again with a reduced set of sampling dates to observe reoccurring patterns. Cluster analyses (based on SSU V9 (eukaryotic) and V4 (prokaryotic) OTU composition) grouped samples according to their origin followed by separation into seasonal clusters, indicating that each lake has a unique signature based on OTU composition. These results suggest a strong habitat-specificity of microbial communities and in particular of community patterns at the OTU level. A comparison of the prokaryotic and eukaryotic datasets via co-inertia analysis (CIA) showed a consistent clustering of prokaryotic and eukaryotic samples, probably reacting to the same environmental forces (e.g., pH, conductivity). In addition, the shifts in eukaryotic and bacterioplanktonic communities generally occurred at the same time and on the same scale. Regression analyses revealed a linear relationship between an increase in Bray–Curtis dissimilarities and elapsed time. Our study shows a pronounced coupling between bacteria and eukaryotes in seasonal samplings of the three analyzed lakes. However, our temporal resolution (biweekly sampling) and data on abiotic factors were insufficient to determine if this was caused by direct biotic interactions or by reacting to the same seasonally changing environmental forces.

Aquatic environments are heavily impacted by human activities including climate warming and the introduction of xenobiotics. Due to the application of silver nanoparticles as bactericidal agent the introduction of silver into the environment strongly has increased during the past years. Silver ions affect the primary metabolism of algae, in particular photosynthesis. Mixotrophic algae are an interesting test case as they do not exclusively rely on photosynthesis which may attenuate the harmful effect of silver. In order to study the effect of silver ions on mixotrophs, cultures of the chrysophyte Poterioochromonas malhamensis were treated in a replicate design in light and darkness with silver nitrate at a sub-lethal concentration. At five time points samples were taken for the identification and quantitation of proteins by mass spectrometry. In our analysis, relative quantitative protein mass spectrometry has shown to be a useful tool for functional analyses in conjunction with transcriptome reference sequences. A total of 3,952 proteins in 63 samples were identified and quantified, mapping to 4,829 transcripts of the sequenced and assembled transcriptome. Among them, 720 and 104 proteins performing various cellular functions were differentially expressed after eight days in light versus darkness and after three days of silver treatment, respectively. Specifically pathways of the energy and primary carbon metabolism were differentially affected by light and the utilization of expensive reactions hints to an energy surplus of P. malhamensis under light conditions. The excess energy is not invested in growth, but in the synthesis of storage metabolites. The effects of silver were less explicit, observable especially in the dark treatments where the light effect could not mask coinciding but weaker effects of silver. Photosynthesis, particularly the light harvesting complexes, and several sulphur containing enzymes were affected presumably due to a direct interference with the silver ions, mainly affecting energy supply.

Background Chrysophytes are protist model species in ecology and ecophysiology and important grazers of bacteria-sized microorganisms and primary producers. However, they have not yet been investigated in detail at the molecular level, and no genomic and only little transcriptomic information is available. Chrysophytes exhibit different trophic modes: while phototrophic chrysophytes perform only photosynthesis, mixotrophs can gain carbon from bacterial food as well as from photosynthesis, and heterotrophs solely feed on bacteria-sized microorganisms. Recent phylogenies and megasystematics demonstrate an immense complexity of eukaryotic diversity with numerous transitions between phototrophic and heterotrophic organisms. The question we aim to answer is how the diverse nutritional strategies, accompanied or brought about by a reduction of the plasmid and size reduction in heterotrophic strains, affect physiology and molecular processes. Results We sequenced the mRNA of 18 chrysophyte strains on the Illumina HiSeq platform and analysed the transcriptomes to determine relations between the trophic mode (mixotrophic vs. heterotrophic) and gene expression. We observed an enrichment of genes for photosynthesis, porphyrin and chlorophyll metabolism for phototrophic and mixotrophic strains that can perform photosynthesis. Genes involved in nutrient absorption, environmental information processing and various transporters (e.g., monosaccharide, peptide, lipid transporters) were present or highly expressed only in heterotrophic strains that have to sense, digest and absorb bacterial food. We furthermore present a transcriptome-based alignment-free phylogeny construction approach using transcripts assembled from short reads to determine the evolutionary relationships between the strains and the possible influence of nutritional strategies on the reconstructed phylogeny. We discuss the resulting phylogenies in comparison to those from established approaches based on ribosomal RNA and orthologous genes. Finally, we make functionally annotated reference transcriptomes of each strain available to the community, significantly enhancing publicly available data on Chrysophyceae. Conclusions Our study is the first comprehensive transcriptomic characterisation of a diverse set of Chrysophyceaen strains. In addition, we showcase the possibility of inferring phylogenies from assembled transcriptomes using an alignment-free approach. The raw and functionally annotated data we provide will prove beneficial for further examination of the diversity within this taxon. Our molecular characterisation of different trophic modes presents a first such example.

Chrysophyceae, also known as golden algae, contain characteristic, three-dimensional biomineralized silica structures. Their chemical composition and microscopic structure was studied. By high-temperature conversion of the skeleton of Mallomonas caudata and Synura petersenii into elementary silicon by magnesium vapour, nanostructured defined replicates were produced which were clearly seen after removal of the formed magnesium oxide with acid.

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches ("AmpliconDuo filter"). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

BACKGROUND:The loss of photosynthesis has occurred often in eukaryotic evolution, even more than its acquisition, which occurred at least nine times independently and which generated the evolution of the supergroups Archaeplastida, Rhizaria, Chromalveolata and Excavata. This secondary loss of autotrophic capability is essential to explain the evolution of eukaryotes and the high diversity of protists, which has been severely underestimated until recently. However, the ecological and evolutionary scenarios behind this evolutionary "step back" are still largely unknown. METHODOLOGY/PRINCIPAL FINDINGS:Using a dynamic model of heterotrophic and mixotrophic flagellates and two types of prey, large bacteria and ultramicrobacteria, we examine the influence of DOC concentration, mixotroph's photosynthetic growth rate, and external limitations of photosynthesis on the coexistence of both types of flagellates. Our key premises are: large bacteria grow faster than small ones at high DOC concentrations, and vice versa; and heterotrophic flagellates are more efficient than the mixotrophs grazing small bacteria (both empirically supported). We show that differential efficiency in bacteria grazing, which strongly depends on cell size, is a key factor to explain the loss of photosynthesis in mixotrophs (which combine photosynthesis and bacterivory) leading to purely heterotrophic lineages. Further, we show in what conditions an heterotroph mutant can coexist, or even out-compete, its mixotrophic ancestor, suggesting that bacterivory and cell size reduction may have been major triggers for the diversification of eukaryotes. CONCLUSIONS/SIGNIFICANCE:Our results suggest that, provided the mixotroph's photosynthetic advantage is not too large, the (small) heterotroph will also dominate in nutrient-poor environments and will readily invade a community of mixotrophs and bacteria, due to its higher efficiency exploiting the ultramicrobacteria. As carbon-limited conditions were presumably widespread throughout Earth history, such a scenario may explain the numerous transitions from phototrophy to mixotrophy and further to heterotrophy within virtually all major algal lineages. We challenge prevailing concepts that affiliated the evolution of phagotrophy with eutrophic or strongly light-limited environments only.