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Probing the evolution, ecology and physiology of marine protists using transcriptomics

David A. Caron1, Harriet Alexander2, Andrew E. Allen3,4, John M. Archibald5,6, E. Virginia Armbrust7, Charles Bachy8, Callum J. Bell9, Arvind Bharti10, Sonya T. Dyhrman11, Stephanie M. Guida9, Karla B. Heidelberg1, Jonathan Z. Kaye12, Julia Metzner12, Sarah R. Smith4 and Alexandra Z. Worden6,8 Abstract | Protists, which are single-celled eukaryotes, critically influence the ecology and chemistry of marine ecosystems, but genome-based studies of these organisms have lagged behind those of other microorganisms. However, recent transcriptomic studies of cultured species, complemented by meta-omics analyses of natural communities, have increased the amount of genetic information available for poorly represented branches on the tree of eukaryotic life. This information is providing insights into the adaptations and interactions between protists and other microorganisms and macroorganisms, but many of the genes sequenced show no similarity to sequences currently available in public databases. A better understanding of these newly discovered genes will lead to a deeper appreciation of the functional diversity and metabolic processes in the ocean. In this Review, we summarize recent developments in our understanding of the ecology, physiology and evolution of protists, derived from transcriptomic studies of cultured strains and natural communities, and discuss how these novel large-scale genetic datasets will be used in the future.

Phototrophy Marine protists are a phylogenetically broad group of chloroplasts but also consume prey. Protists typically A nutritional mode that eukaryotic organisms that are capable of existence as reproduce through mitotic division rather than through involves the use of light for the single cells, although many form colonies that exhibit sexual reproduction, which enables populations to double production of organic carbon coordinated behaviour. The term protist is largely histor- in a few hours to a few days. Their rapid growth rates ena- and the acquisition of energy. ical in nature, at one time referring to a kingdom-level ble them to contribute to important ecosystem functions: 1 Phytoplankton distinction in Whittaker’s tree of life , but still commonly protists have roles in primary production, as partners in Planktonic protists that use applied to all eukaryotes that are not plants, multi­cellular various symbiotic relation­ships and, at multiple trophic phototrophy as their nutritional animals or fungi. Most protists are microscopic, but levels, as links between the small animals that prey on mode. The term has ecological collectively these species span more than five orders of them and the vast numbers of bacteria, archaea, protists importance but little (FIG. 1) phylogenetic importance magnitude in size, display a myriad of morphologies, and even some metazoa that they consume . These because the behaviour occurs behaviours and nutritional modes, and have pivotal eco- links in the food web are essential for the biogeochemi- across many lineages of logical roles in marine ecosystems2,3. Protistan species cal cycles of the ocean, including the transport of carbon protists. are the most abundant eukaryotes in marine pelagic to the deep ocean. communities and constitute a substantial portion of the Despite the fundamental roles of protists in marine total biomass in the plankton. ecosystems, genome-based studies of these species Correspondence to D.A.C. Nutritional modes of protists range from pure have lagged behind those of other marine organ- Department of Biological photo­trophy among many phytoplankton to the pure isms. However, in the past few decades, a lot of DNA Sciences, University of hetero­trophy of species traditionally called protozoa. sequence information has become available for proti- Southern California, Protozoa generally derive their nutrition from the inges- stan marker genes4, transcriptomes5, genomes (almost 3616 Trousdale Parkway, 6 metatranscriptomes5 Los Angeles, California tion of bacteria, archaea or other eukaryotes, but some 20 complete genomes in total) , and 7 90089, USA. are parasites (or more accurately, parasitoids) of other even metagenomes . Analyses of these data have altered [email protected] protists and metazoa. In addition, many protists com- our understanding of protistan diversity, phylogeny, doi:10.1038/nrmicro.2016.160 bine photo­trophy and heterotrophy in a single species evolution and physiology, and enriched our knowledge Published online 21 Nov 2016 (mixotrophy), such as phytoflagellates that have of various ecological interactions.

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Author addresses A few large culture-based and field-based projects have begun to address this disparity. The Marine 1 Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, Microbial Eukaryote Transcriptome Sequencing Los Angeles, California 90089, USA. 2Department of Population Health and Reproduction, School of Veterinary Medicine, Project (MMETSP) released nearly 680 transcriptome 1 Shields Avenue, University of California Davis, Davis, California 95616, USA. assemblies of pure cultures of protists in 2014, repre- 3Microbial and Environmental Genomics, J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, senting isolates from much of the world’s oceans and a San Diego, California 92037, USA. few freshwater ecosystems (BOX 1). The MMETSP was 4Scripps Institution of Oceanography, Integrative Oceanography Division, University of California San Diego, 9500 Gilman Drive, La Jolla, San Diego, California 92093, USA. conceived to facilitate comparative transcriptomics of 5Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper environmentally important microbial eukaryotes. The Medical Building, 5850 College Street, Halifax, Nova Scotia B3H 4R2, Canada. dataset increased ~50‑fold the amount of information 6Canadian Institute for Advanced Research, 180 Dundas Street W, Toronto, Ontario M5G 1Z8, available on gene expression from free-living protists, Canada. and doubled the number of protistan lineages for which 7School of Oceanography, University of Washington, 1503 NE Boat Street, Seattle, 5 Washington 98195, USA. genomic studies have been conducted . Subsequent 17 8Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, work has continued to improve the gene assemblies . California 95039, USA. Concomitantly, two field programmes (the Tara Oceans 9National Center for Genome Resources, 2935 Rodeo Park Drive East, Santa Fe, and Malaspina expeditions)4,18 have circumnavigated the New Mexico 87505, USA. 10Syngenta Biotechnology, 629 Davis Drive, Research Triangle Park, North Carolina 27709, USA. planet collecting samples to provide an enormous sam- 11Department of Earth and Environmental Sciences and the Lamont-Doherty Earth Observatory, ple set for the investigation of the abundances, morph­ Columbia University, 61 Route 9W, Palisades, New York 10964, USA. ologies, genetic diversity and ecological significance of 12Gordon and Betty Moore Foundation, 1661 Page Mill Road, Palo Alto, California 94304, USA. marine protists. These studies highlight the importance of marine protists, demonstrate the genetic and physio­ The historical partitioning of eukaryotes into king- logical potentials that underpin their activities and doms based on morphological characteristics, multi- provide new insights into the evolution of these species. cellularity or trophic mode1 has been challenged and Heterotrophy changed in recent years by information on genetic Insights into protistan phylogeny and evolution A nutritional mode that relatedness. The resulting framework for a molecular Modern protistan phylogeny. Our view of the evolution- involves the use of preformed organic matter for the taxonomy for protists has raised questions regarding ary relationships among protistan lineages has changed acquisition of carbon and the biogeography of these taxa and the adequacy of the substantially during the past few decades (FIG. 2). The energy. classical morphological species concept for protists8–10. Whittaker system1, which prevailed for two decades, Direct sequencing of rRNA genes from environmental grouped organisms into five kingdoms: Monera, Protista, Protozoa samples has revealed a huge protistan diversity, includ- Plantae, Fungi and Animalia. Eukaryotes that can exist as Protists that are not 11,12 photosynthetic, but are instead ing new lineages , and has stimulated hypotheses single cells were contained within the kingdom Protista. dependent on the ingestion of regarding the protistan rare biosphere — diverse taxa Protists were further organized according to morphology preformed organic matter present at low relative abundances in virtually all nat- and nutritional preferences, a scheme that was practical (usually prey) for their ural ecosystems — which may play important parts in for biologists of the time but artificially separated some nutrition. This older term is still in use; ‘heterotrophic protists’ the evolution of eukaryotes as well as in the stability closely related taxa that have different nutritional behav- 13–15 is used synonymously. and functional resilience of microbial communities . iours (for example, photosynthetic and heterotrophic Importantly, studies of protistan gene expression have dinoflagellates). Parasitoids begun to provide unprecedented insight into how The adoption of ultrastructural and DNA sequence Protists that exhibit a parasitic protists respond to environmental cues, competi- information eventually undermined Whittaker’s lifestyle, infecting other protists 19 or multicellular organisms. tion, predation and symbiotic interactions with other scheme . The ‘new’ system derived from this infor- Parasitoids are typically microorganisms. mation placed all eukaryotes into a single domain, the differentiated from parasites In this Review, we summarize recent developments in Eukaryota, with the animals, plants and fungi repre- by the fact that parasitoids, the characterization of protistan gene expression, discuss sented as minor branches among a huge diversity of unlike parasites, always kill 20 their hosts. how transcriptomic studies are revealing novel insights protists . Nearly 15 years of reorganization followed, into protistan biology, and explain how these findings during which all eukaryotes were arranged into ‘super- Metazoa will influence our understanding of ocean ecology groups’, a scheme that is believed to more accurately Multicellular, eukaryotic and biogeochemistry. reflect evolutionary history. The scheme continues to organisms (animals) that have undergo numerous minor revisions to resolve the root differentiated cells and tissues. Filling in the blanks and branching order of various protistan groups (FIG. 2). Mixotrophy Compared with other microorganisms, microbial The result is a domain of eukaryotic life, the domain A nutritional mode in which an eukaryotes have been the ‘poor cousins’ of the omics Eukarya, that bears little resemblance to Whittaker’s individual cell can use both revolution6, in large part owing to their larger and more scheme at the broadest phylogenetic groupings, although inorganic and preformed 16 organic sources of carbon complex genomes . The eukaryotic genomes that have many of the protistan phyla remained intact throughout 21,22 and nutrients for growth. In been sequenced to date are dominated by metazoan the reorganization (FIG. 2). Reorganization within and protists, mixotrophy is model organisms, a handful of microorganisms that among some of the supergroups, such as the division generally accomplished by parasitize humans (for example, Plasmodium spp., of amoeboid protists into the Amoebozoa and Rhizaria23, combining heterotrophy Trypanosoma spp. and Giardia lamblia) and a few photo­ and further grouping of some of the supergroups into with a photosynthetic ability acquired through chloroplasts, synthetic marine microbial eukaryotes. Consequently, higher order ‘clusters’ (for example, the grouping of the kleptoplastidy or harbouring DNA sequence databases that are devoted to free-living, Stramenopiles, Alveolata and Rhizaria supergroups into endosymbiotic algae. ecologically important protists remain sparse. the SAR cluster)24 continue to refine the structure of the

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Primary production µm) The photosynthetic production of organic carbon, carried out Mesoplankton

by a wide variety of protists, (200–2,000 macroalgae and plants.

Metatranscriptomes µm) Collections of all the transcriptomes (all RNA

transcripts) present in (20–200 Microplankton communities of microorganisms; a metatranscriptome of a community is derived from

RNA extraction and purification, µm) trophic level Size and trophic reverse transcription of RNA to cDNA and sequencing of the (2–20 Nanoplankton resulting cDNA.

Metagenomes

Collections of all the DNA µm) present in communities of

microorganisms, representing (0.2–2 Picoplankton all the genetic potential of the communities. The metagenome Phototrophic Mixotrophic Heterotrophic Parasitoid Bacteria of a community can be used to Protists reconstruct the genomes of the individual species comprising Figure 1 | Ecological and biogeochemical roles of protists in the marine plankton. Protists are an important part of that community, thus assigning living biomass in oceanic ecosystems and are central to a wide array of food web processesNature and biogeochemicalReviews | Microbiology cycles. specific metabolic roles to Species of phototrophic, heterotrophic and mixotrophic protists span more than three orders of magnitude in size and those taxa. several trophic levels at, and near, the base of the food web. Complex predator–prey relationships that involve protists make photosynthetically produced organic material (red arrows) and bacterial biomass (black arrows) available to Dinoflagellates higher trophic levels and also remineralize a substantial fraction of this material back to inorganic nutrients and carbon Members of a major, dioxide to support new primary production. Recent environmental gene surveys suggest that protistan parasitoids are flagellated protistan lineage 112 (the class Dinophyta, in the also important players in marine food webs, as they prey on microplanktonic and mesoplanktonic species (blue supergroup Alveolata) dashed arrows). In addition to the myriad of predator–prey relationships, the interactions of protists with other containing phototrophic, microorganisms and multicellular organisms include a wide range that are not depicted here (involving competition, heterotrophic and mixotrophic commensalism and mutualism). (kleptoplastidic) species. Numerous photosynthetic and mixotrophic dinoflagellates domain, whereas relationships among other protistan tertiary endosymbiotic events then spread the so‑called are harmful and produce toxic 25,26 algal blooms that have groups remain unresolved at this time . These uncer- primary chloroplasts of red and green algae to an array 31,32 traditionally been called tainties notwithstanding, the basic topology within the of organisms , including euglenids, diatoms, haptophytes red tides. eukaryotic tree has finally begun to stabilize26. and dinoflagellates. The construction of ‘phylogenomic’ rRNA genes are slow to evolve and so have been frameworks for understanding the relationships between Amoebozoa used extensively to redefine the high-level phylogenetic photosynthetic and heterotrophic lineages, and how A protist supergroup that includes many small amoeboid relationships among eukaryotes. Studies are continu- chloroplasts have been gained and lost, has been instru- forms and the slime moulds. ing, but now with the use of multi-gene datasets, which mental for documenting gene transfer events between are necessary to increase resolution; much of this work symbiont and host33. Transcriptome-based studies have Rhizaria relies heavily on transcriptomic datasets because of the played a key part in this process. For example, gene A large protist supergroup that includes ecologically present scarcity of sequenced genomes for microbial discovery surveys have revealed the presence of novel 27 34 important, large amoeboid eukaryotes. Grant and Katz , for example, used tran- chloroplast genes in heterotrophic organisms . forms (radiolaria and scriptomic data that encompassed >15,000 characters Algae that have secondary chloroplasts are among foraminifera) and the Cercozoa. (including concatenated sequences of ribosomal genes the most genetically and biologically complex cells and >200 protein-coding genes) to establish the phylo­ known. The cryptophyte Guillardia theta and the Stramenopiles chlorarachniophyte A diverse protist supergroup of genetic placement of several species of amoebae within Bigelowiella natans are two unrelated 31 phototrophic, heterotrophic the Amoebozoa. algal species that both have secondary chloroplasts , and mixotrophic (phagotrophic but they are unusual in that the nucleus of the second- phytoflagellates) species Symbiosis, endosymbiosis and organelle acquisition. ary endosymbiont persists in the form of a vestigial characterized by the presence nucleomorph (REF. 35) of two flagella of unequal The evolution of mitochondria and chloroplasts from ‘ ’ . The nuclear genomes of both length and structure in their bacteria through endosymbiosis were pivotal events in organisms have now been sequenced, and the data sup- motile life stages. The the history of the eukaryotic cell. Present-day eukaryotes plemented with transcriptomic sequence information36. supergroup includes the brown are thought to have diversified from a mito­chondrion- Both organisms are predicted to have >21,000 intron- algae, the chrysophytes, the bearing ancestor28,29, and chloroplasts evolved from rich nuclear genes. Genes from B. natans showed a level diatoms and other important groups. The term is generally cyanobacterial endosymbionts in a common ances- of alternative intron splicing that exceeded that seen in used synonymously with tor that is shared by red algae, green algae (and their genes from more complex organisms, such as the model heterokonts. land plant relatives) and glaucophytes30. Secondary and plant Arabidopsis thaliana, and on par with the level seen

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Box 1 | The Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) The Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) was a global community effort to augment the available DNA sequence information on ecologically relevant protists and provide a much needed reference dataset for environmental metagenomic and metatranscriptomic studies. The overall goal was to substantially increase the sequence datasets from cultured, well-curated protistan species to provide a resource for gene annotation throughout the Eukarya and for the interpretation of metagenomic and metatranscriptomic datasets from diverse marine ecosystems. Supported by the Gordon and Betty Moore Foundation, the project directly involved nearly 70 laboratories engaged in protistan research, and more than 200 investigators worldwide participated by submitting material for sequencing. The project entailed the sequencing and assembly of 678 transcriptomes that encompassed 210 unique genera, 305 species and 396 strains of protists, and the results were released to the public in June 2014. Depicted in the Alveolata figure are the geographical origins of approximately half of the strains, indicating the degree of global coverage. The A supergroup that includes MMETSP sequence datasets are publicly available through the iMicrobe and NCBI BioProject websites, and recently three important groups of re‑annotated datasets are available at the MMETSP re-assemblies website. A list of publications that resulted from the protists: the dinoflagellates, project is available at the Gordon and Betty Moore Foundation MMETSP webpage. An interactive version of the map the ciliates and the parasitic indicating the locations from which strains were obtained is available on the iMicrobe site. A list of MMETSP participants apicomplexans. The defining and their affiliations are provided (Supplementary information S1 (box)). characteristic of the Alveolata is the presence of alveoli (flattened vesicles beneath the cell wall).

Glaucophytes (Also known as glaucocystophytes). Members of the class Glaucocystophyceae, a small algal clade that is grouped together with the green algae and land plants in the supergroup Archaeplastida.

Euglenids Members of the phylum Euglenida (in the supergroup Excavata). Free-living euglenids include phototrophic, heterotrophic and mixotrophic species, and a few notable parasites of animals.

Diatoms Members of the phylum Nature Reviews | Microbiology Bacillariophyta (in the supergroup Stramenopiles), in genes expressed in the human cerebral cortex36. The fundamental for understanding the evolution of com- a clade that is characterized role of such rampant alternative splicing is unclear and plex life41. Coral–dinoflagellate (Symbiodinium spp.) by siliceous cell walls. is being investigated. symbioses have become a model system for studying Haptophytes One particularly exciting example of ‘evolution metazoan–­protist symbiosis. Transcriptomic approaches Members of an algal group that in action’ is the enigmatic rhizarian Paulinella (and the recent sequencing of the Symbiodinium encompasses prymnesiophytes chromatophora, for which transcriptomic analyses kawagutii nuclear genome42) have become fundamen- (supergroup unresolved), have revealed that at least three genes in the nuclear tal tools for unravelling the role of the dinoflagellates in including the bloom-forming 43 species of the genus genome encode subunits of photosystem I in the these associations . Approximately half of the dinoflag- Phaeocystis as well as globally chromatophore — the recently established endosymbi- ellate genes do not match genes from any other organ- and biogeochemically ont of P. chromatophora37,38. Transcripts from these genes ism, and a small percentage of these genes are regulated important forms such as the are translated in the host cytoplasm and the resultant transcriptionally. Moreover, biochemical complementa- coccolithophorids, which often proteins are targeted back to the chromatophore39. The rity between symbiont and host seems to indicate host– bear calcium carbonate plates (coccoliths). chromatophore thus qualifies as a cyanobacterium- symbiont co‑evolution, implying that the interaction derived photosynthetic organelle of recent origin that involves an energetic cost efficiency, which may explain Cryptophyte has evolved completely independently of classical the ecological success of the association. A member of the class chloro­plasts40. Transcriptomic studies of such transi- Progress towards understanding the associations Cryptophyta (supergroup unresolved); this is a group of tional states of organelle acquisition are providing a between rhizarians and photosynthetic protists, which 44,45 small, flagellated protists that greater understanding of the underlying interactions are exceptionally abundant in the ocean plankton , has contains mostly photosynthetic between endosymbionts and their hosts41. been slow because of challenges in culturing most host forms but also mixotrophic A myriad of endosymbiotic associations, such as species. Culture-independent transcriptomic approaches and heterotrophic species. those that involve large rhizarians and photosynthetic provide a way around this obstacle. For example, the The photosynthetic species (FIG. 3a) have plastids that contain protists , evolved more recently than the origin comparison of transcriptomes from four rhizarians phycobiliprotein pigments and of mitochondria and chloroplasts. Understanding how (two polycystines, an acantharian and a phaeodarian) are derived from red algae. these associations are established and maintained is revealed the presence of genes that encode c‑type lectins

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Red algae Archaeplastida Green plants Alveolates

Excavates

Stramenopiles Fungi SAR Opisthokonts

Animals Rhizaria Amoebozoa Burki, 2014

Supergroups Other hypotheses Archaeplastida Excavata Rhizaria SAR Amoebozoa Stramenopiles SAR Alveolates Chromalveolata

Opisthokonta Plantae Bikoma

Adl et al., 2012 Excavata

Rhizaria Unresolved lineages Amoebozoa Alveolates Unikonta Archaeplastida Opisthokonta

Tekle et al., 2009 Stramenopiles

Chromalveolata Amoebozoa Plantae

Rhizaria Discicristates Amoebozoa Excavates Excavata Opisthokonts Baldauf, 2008 Opisthokonta

Chromophytes Eukaryota Oomycetes Simpson & Roger, 2004 Diatoms Ciliates Fungi Dinoflagellates Apicomplexans Archaea Eucarya Euryarchaeota Animals Crenarchaeota Animals Fungi Plants and Bacteria Plants green algae Eubacteria Protists

Woese et al., 1990 Plantae Fungi Animalia

Sogin, 1991 Archaebacteria

Protista

Monera Whittaker, 1969

Nature Reviews | Microbiology

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Chlorarachniophyte in the polycystines and acantharian, which harbour the same eukaryotic supergroup as green algae and land A member of a group of dinoflagellate endosymbionts, but not in the phaeodar- plants) and in algae from other supergroups, specifically algae (Chlorarachniophyceae) ian, which is symbiont-free46. These proteins may play a in other stramenopiles (beyond diatoms) and in crypto- that are often mixotrophic. part in establishing the endosymbiosis because they are phytes53. Some of these new findings may simply be the These species are also often amoeboid in form and are known to be involved in cell–cell interactions in other result of expanding the datasets from taxa with larger placed in the supergroup species. Other transcriptomic studies have been carried genomes, given the focus of early genome sequenc- Rhizaria within the Cercozoa, out on rhizarians that retain the functional chloroplasts ing projects on widespread taxa with small genomes together with large amoeboid of ingested diatom prey; these studies have investigated (in which gene loss is often substantial)54. forms, such as radiolaria and how plastid function is maintained and the possibility of foraminifera. horizontal gene transfer between host and prey47. Insights into protistan physiology and ecology Nucleomorph Transcriptomic data are now available for a greatly A vestigial nucleus that is Algal phytochromes. Transcriptomics has also trans- expanded array of protistan taxa, and this has facil- associated with plastids in formed our view of evolution within and among algal itated investigations of gene expression in protists, in some protists. They are derived from the engulfment and groups that have sequenced genomes, such as green particular for many important primary producers, and, reduction of a eukaryotic algae (also known as chlorophytes) and glaucophytes. to a lesser extent, for a few heterotrophic and mixo­ endosymbiont. A recent example is the discovery of widespread sen- trophic species. Such studies are beginning to elucidate sory proteins that react to changes in light. In plants, the specific physiological responses of these protists to Chromatophore phytochromes are involved in numerous processes, environmental cues and are identifying the genes that (In this Review:) An endosymbiotic cyanobacterium including seed germination, stem elongation and repro- are associated with specific nutritional strategies. New 48 with a reduced genome. duction . Until recently, these organelles were thought gene discovery within various microalgal lineages in In the protist Paulinella to have limited distributions among other eukaryotes, particular has moved forwards rapidly, resulting in a chromatophora, this structure being mainly restricted to fungi and stramenopiles (in substantial increase in the number of known protist is considered an early stage of 49–51 chloroplast acquisition. particular, diatoms) ; they were found in a single protein families and a clearer understanding of their prasinophyte genome (Micromonas pusilla)52 but were evolutionary origins and metabolic functions55. Plastid absent from the other prasinophyte genomes studied One of several types of and also from the genomes of other studied green algae, Microalgal responses to environmental changes. cyanobacterium-derived, such as the model alga Chlamydomonas reinhardtii. Microalgae have crucial roles in the fixation and export double-membrane-bound organelles that are present However, increased transcriptomic coverage of envi- of carbon from surface waters to the deep ocean, and in many protists; an example ronmental lineages has revealed phytochromes in the they form the base of food webs in the most produc- is the chloroplast. majority of the prasinophyte groups (that is, in species tive regions of the ocean56. Global distribution patterns from six of the seven accepted clades), indicating that of microalgae can be inferred from satellite-derived they have a broader distribution in green algae than assessments of chlorophyll and other pigments57, but had been realized previously. Transcriptomic data also individual species and taxonomic groups exhibit unique revealed their presence in glaucophytes (which are in metabolic capabilities that cannot be represented in these larger-scale data. This results in predictive models ◀ Figure 2 | The rapidly changing landscape of protistan phylogeny. of primary production and ocean biogeochemistry that DNA sequence information and ultrastructural detail provided by electron microscopy lack resolution. Consequently, early transcriptomic and have substantially changed our view of the evolutionary relationships among protistan genomic studies have focused strongly on these species groups. The system proposed by Whittaker1, which prevailed for nearly three decades, with unique capacities and are now bridging this knowl- grouped eukaryotes into four kingdoms (animals, plants, fungi and protists). The scheme edge gap by providing insights into their capabilities and of Woese et al.19 redefined the highest-level organization into domains of life and placed their responses to nutrient availability (which is a major all eukaryotic species within the domain Eukarya. Subsequent revisions (the Sogin factor determining their activities)58–60. 20 system ) placed the animals, plants and fungi as only minor evolutionary branches Green algae are one of the few non-parasitic proti- among an enormous diversity of single-celled (protistan) eukaryotic taxa. The basic, stan groups with several sequenced genomes, which is redefined structure of the domain emerged approximately a decade ago (Simpson and a favourable consequence of their small genome sizes Roger21), with eukaryotes organized into several supergroups. The composition of these supergroups and their evolutionary relationships to one another have begun to stabilize and relative ease of culture. Genomic datasets have in recent years (Baldauf22), but new details within many protistan groups, the placement been instrumental for the alignment and identification of many ‘orphan’ lineages and the relationship of supergroups to each other continue to of transcriptomic data, which, in turn, have been used be proposed and tested (Tekle et al.145 and Adl et al.25). Our present understanding of to characterize the response of green algae to environ- protistan evolution (Burki26) differs substantially from the scheme proposed by Whittaker. mental factors. Approaches are now quite advanced for Studies of protistan evolution now incorporate large amounts of genomic and some freshwater taxa, such as C. reinhardtii, which has transcriptomic information27. Whittaker system adapted from Whittaker, R. H. New become a model system for cellular functions and bio- concepts of kingdoms of organisms. Science 163, 150–160 (1969). Reprinted with technological applications. For example, transcriptomic, permission from AAAS. Woese scheme reproduced with permission from REF. 146, quantitative proteomic and metabolomic data were National Academy of Sciences. Sogin system adapted with permission from REF. 20, combined for three strains of C. reinhardtii to docu- Elsevier. Simpson and Roger scheme adapted with permission from REF. 21, Elsevier. 61 Baldauf system adapted with permission from REF. 22, Wiley. Scheme of Tekle et al. ment their response to nitrogen starvation . There were adapted with permission from REF. 25, Wiley. System of Adl et al. adapted from Tekle, Y. I., conserved responses across all strains, which exhibited Wegener Parfrey, L. & Katz, L. A. Molecular data are transforming hypotheses on the a differential expression of thousands of genes, with a origin and diversification of eukaryotes. Bioscience, 2009, 59, 6, 471–481, by permission of prioritization for the maintenance of respiration over Oxford University Press on behalf of the American Institute of Biological Sciences. Burki photosynthesis, and a decreased expression of nitrogen- scheme adapted with permission from REF. 26, Cold Spring Harbor Laboratory Press. rich proteins. Studies of synchronous cultures of

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a Figure 3 | Symbiosis and mixotrophy among protists. Many varieties of mixotrophic nutrition and endosymbiosis exist among protists. a | Symbiotic relationships between heterotrophic and photosynthetic protists are extremely Planktonic common among many large rhizarians in the oceanic foraminiferan plankton147. The hosts acquire free-living photosynthetic (Globigerinoides spp.) protists during ontogeny and convert them to intracellular symbionts, as shown here for planktonic foraminiferan hosts and photosynthetic dinoflagellates. Many of these associations involve species-specific relationships between host and endosymbiont. b | Prey capture (mostly bacteria, but also other microorganisms) by photosynthetic protists Free-living photosynthetic In hospice dinoflagellates (Pelagodinium spp.) dinoflagellates is common among several algal classes and may serve various nutritional needs, including the need for organic b Light carbon for energy and growth (as in some Ochromonas spp.), the need to eliminate competing algae (as in Prymnesium parvum) or the need to supplement photosynthetic growth Prymnesium parvum Dinobryon spp. Ochromonas spp. with as-yet poorly defined growth factors (as in some Dinobryon spp.). These protists span a range of nutritional modes from nearly completely phototrophic (left) to nearly completely heterotrophic (right). These differing strategies Trace elements, for prey ingestion are reflected in the gene expression 106 vitamins and other Carbon, energy profiles of these species in response to light and prey . Macronutrients growth factors and macronutrients c | The retention of functional chloroplasts (kleptoplastidy) contained in prey contained in prey contained in prey from ingested algal prey (shown by arrows in the direction Phagotrophic of acquisition) is carried out by numerous heterotrophic Phototrophic dinoflagellates and ciliates, and by a few foraminifera. In one case, cryptophyte chloroplasts retained by the ciliate Mesodinium rubrum are also retained by the predators of c Primary kleptoplastidy Secondary kleptoplastidy the ciliate, dinoflagellates within the genus Dinophysis. Another example is the retention of diatom chloroplasts by a benthic foraminiferan (for example, Elphidium excavatum).

stramenopile Nannochloropsis oceanica, in which >7,600 genes (representing >60% of the genome) were shown to cycle in response to the light–dark period65. Cryptophyte Planktonic ciliate Planktonic prey (Mesodinium rubrum) dinoflagellate Diatoms, as major contributors to primary production (Dinophysis spp.) in the ocean, have become a focus of comparative genomic and transcriptomic analyses to identify genes associated with the metabolic processes that might explain the eco- logical dominance of these species. Analyses of available diatom genomes, coupled with experiments enabled by Diatom prey Benthic foraminiferan transcriptomics data, have highlighted unique aspects of (Elphidium excavatum) nitrogen metabolism in these species, such as a complete ornithine–urea cycle, which is an important link between Nature Reviews | Microbiology nitrogen and inorganic-carbon metabolism66–68. C. reinhardtii have also demonstrated sweeping shifts The widely distributed species Thalassiosira in gene expression (80% of the transcriptome) over a pseudonana­ has become a model for linking the genetic diurnal cycle62. and physiological responses of diatoms to environmen- Transcriptomic studies, aided by the availability of tal cues, in part because of the availability of a sequenced sequenced genomes, have also yielded unique insights genome68. Whole-genome expression profiling of this spe- into the cellular responses of important marine green cies has revealed a set of 75 genes that is induced during algae. One such study compared the transcriptomes of silicon limitation but not other types of stress69. Most of the prasinophyte M. pusilla grown under phosphorus- these genes were predicted to have roles in signalling or replete and phosphorus-deficient conditions63, finding transport, which might explain the rapid response of dia- that genes that are involved in the acquisition, trans- toms to the changing availability of the element in nature. port and cellular reallocation of phosphorus are upreg- Furthermore, another analysis used proteomic and tran- Prasinophyte ulated during phosphorus deficiency. Transcriptomic scriptomic data to demonstrate that nitrogen starvation A type of green alga (phylum studies of the prasinophyte Ostreococcus tauri revealed upregulates components of the tricarboxylic acid (TCA) Chlorophyta, supergroup extensive rhythmic changes in the gene expression of cycle and glycolysis in T. pseudonana70. The authors of Archaeplastida). Prasinophytes are important primary cultures grown in a light–dark cycle, indicating that the this study speculated that this response might provide 64 producers in freshwater and photoperiod exhibits strong transcriptional control . A carbon and energy for nitrogen assimilation, which would marine ecosystems. similar result was observed in the sequenced marine be a significant adaptation in the ocean, in which rapidly

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changing nitrogen availability plays a fundamental part exemplifying the power of exploiting gene expression in controlling primary productivity. Physiological ver- data to interpret the behaviour and nutritional status of satility and responsiveness may also explain the general natural populations78. cellular reorganization that takes place in another diatom, Other HAB species use various compounds to slow Phaeodactylum tricornutum, in response to iron starva- the growth of competing microalgae (allelopathy)79, to tion; this reorganization includes the downregulation of deter grazers80 or to subdue prey81. They include such iron‑rich processes71. notable compounds as the prymnesins, which are used Studies of gene expression in diatoms have revealed by the toxic haptophyte Prymnesium parvum to subdue commonalities that could eventually explain the over- and kill prey82, and dimethylsulfoniopropionate, which all competitive strategy of these protists within natural is used by some algae as a deterrent to zooplankton graz- phytoplankton assemblages, whereas comparative stud- ing83. Transcriptomics and genomics are improving our ies among diatom taxa have uncovered many species-­ knowledge both of the sources and biochemical pathways specific genes that underlie their unique autecologies. that generate these compounds, and of the environmen- A comparative transcriptomic study of three diatoms tal conditions that induce their production. For exam- (T. pseudonana, Fragilariopsis cylindrus and Pseudo- ple, powerful neurotoxins called saxitoxins, which affect nitzschia multiseries) demonstrated that less than 5% sodium channel function, were thought to be encoded of the >5,500 orthologous genes in these species exhibit and produced by bacteria that are associated with dino- the same transcriptional response to nitrate limitation, flagellates in the genus Alexandrium, but recent work has despite some overall similarities in the cellular responses, revealed that genes that regulate saxitoxin production are such as reduced carbon fixation72. present in the nuclear genomes of several Alexandrium Whole-genome expression profiling has now spp., which indicates that these toxins might be produced expanded well beyond several green algae and dia- by the protists, either exclusively or in combination toms, providing valuable datasets for the interpre- with bacterial production84. Furthermore, studies of tation of transcriptomic data from other important P. parvum have hinted at the possible biosynthetic path- taxa. Dinoflagellates have been highly desirous targets ways that lead to toxin production in that species85,86. for these studies because they exhibit an especially Moving forward, the transcriptome assemblies generated wide range of nutritional modes and endosymbiotic for HAB species will provide a targeted reference data- relationships, ranging from pure heterotrophy, to base for identifying transcriptional patterns, examining obligate endosym­biosis that provides photosynthetic genetic variation and contextualizing those signals among benefits to the heterotrophic host, through to fully populations in nature87. integrated and functional chloroplasts73. In addition, many species are harmful algal bloom-forming (HAB) Metatranscriptomics of natural assemblages of phyto­ species. The very large genome sizes of dinoflagellates plankton. Large genome sizes, high species richness, have made transcriptome sequencing particularly present sequencing depth and cost of sequencing have useful. Recent studies of these species have reported limited the number of available metatranscriptomic and complex transcriptional responses to nutrient limita- metagenomic datasets of natural assemblages of eukar- tion or the presence of algicides74,75. These complex yotes. These approaches are now becoming tractable, in responses are not unanticipated, given the results part owing to improvements in the reference datasets obtained with other well-studied algal groups. Moreover, for gene annotation and in part through focusing on they demonstrate the potential for gaining insight into communities with limited species richness7,88,89. Autecologies the cellular processes in eco­logically important species Metatranscriptomic studies of natural phyto­ The ecologies of individual with genomes that are too large for modest sequencing plankton communities are providing early insights into species and their interactions projects. why diatoms are so successful. A study that investigated with the surrounding Several non-dinoflagellate HAB species have been the response of diatoms to iron enrichment noted the environment and co‑occurring species. investigated to understand their competitive superiority upregulation of large suites of genes that are associated in nature. The pelagophyte Aureococcus anophagefferens, with nitrate assimilation and synthesis of carbon storage Allelopathy another recently sequenced species, has been screened compounds, which indicates that diatoms respond to The production of for differential responses of metabolic pathways under iron with stimulated nitrogen acquisition and renewed growth-inhibiting chemicals three different culture conditions76. Genes and pathways photo­synthesis and growth90. These findings are con- by one species to target competing species. The term that are associ­ated with the uptake and transforma- sistent with the observation that diatoms are typically is commonly used in reference tion of nitrogen, the hydrolysis of organic phosphorus the dominant phytoplankton group appearing during to chemical warfare among compounds and the catabolism of organic compounds blooms in response to iron enrichment experiments co‑occurring phytoplankton. were upregulated under conditions of low nitrogen, in high-nitrate low-chlorophyll regions (HNLC regions)91.

High-nitrate low-chorophyll phosphorus or light levels, respectively. The results Similarly, comparative transcriptomics of diatom regions explain field and laboratory studies indicating that the assemblages from three locations off Antarctica revealed (HNLC regions). Regions of the alga has a considerable capacity for supplementing its species-­specific responses to nutrient status, tempera- global ocean in which inorganic nutrition through the uptake of organic compounds77. ture and light that indicated the unique physiological nitrogen does not limit More recently, it was demonstrated that a phosphorus strategies of the different diatom species that dominated phytoplankton growth, and 92 other elements (most notably transporter is highly expressed under phosphorus- at each of the sites . Furthermore, quantitative trans­ iron) limit the rate and amount limiting conditions both in laboratory culture and criptomic studies conducted in coastal Southern Ocean of primary production. during a natural bloom of A. anophagefferens, thus waters have resulted in new insights into the molecular

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underpinnings of cobalamin and iron limitation and taxon enables physiological responsiveness to changing have provided evidence that this limitation is driven by environmental conditions, in part explaining the broad several phytoplankton–bacterium interactions93. geographical distribution of the species. Bioinformatic analyses of natural assemblages of Sinking-particle flux at station ALOHA during the phytoplankton have leveraged available microalgal summer shows a reproducible annual maximum in genomes and expressed sequence tag (EST) datasets to the export of material from surface waters, which is map sequence data89,94, but gene annotation has been potentially associated with diatom blooms100. Nutrient challenging owing to the lack of extensive, well-curated­ supply from the mixing of deep, nutrient-rich water into datasets for free-living protists. One recent study the euphotic zone (for example, through eddy-driven showed that approximately 40% of the transcriptome upwelling)101 is likely to be important for bloom for- of T. pseudonana changed during the photoperiod95. mation. A simulated mixing event (an experiment in Quantitative metabolic However, gene annotation must be improved sub- which deep water was added to surface water) changed fingerprints stantially if we are to gain a better mechanistic under- the QMF of the major functional groups of phytoplank- (QMFs). Comparisons of the relative expression levels of standing of the cellular pathways and processes that are ton, including expression changes for genes that are genes or gene families for a affected by these changes. More than half of the proteins involved in carbohydrate, protein and sulfur metabo- species under different that are predicted in the iron limitation study discussed lism, nutrient transport, and photosynthesis97 (FIG. 4c). environmental conditions or above69 had no homology to known proteins. Analysis indicated that diatoms have the metabolic plas- for different species under the same conditions. The The application of bioinformatic pipelines using new ticity to capitalize on such nutrient pulses, as evidenced fingerprints can indicate and more extensive datasets for environmental samples by the upregulation of pathways that are associated with specific pathways that are will lead to improved metatranscriptomic analyses. For photosynthesis and carbon fixation. It is still rare to see upregulated or downregulated, example, a metatranscriptomic analysis of two dominant the application of transcriptomic data to help explain and can thus provide insight diatom species, Skeletonema costatum and Thalassiosira shifts in primary production and in the taxonomic into the metabolic responses of the organism. rotula, showed that both species express core metabolic composition of phytoplankton in response to changing pathways for nitrogen and phosphorus metabolism, but environmental conditions, but this approach constitutes Kleptoplastic quantitative metabolic fingerprints (QMFs) of the two spe- a new and powerful tool in biological oceanography. Able to ingest and partially cies showed opposite transcriptional responses of many digest photosynthetic prey, 96 but retain the chloroplasts genes that are involved in these pathways . Both pro- Mixotrophic behaviour and nutrition. One of the from the prey in a functional tists were shown to have homologues that encode nitrate, least appreciated but common behaviours of many state, usually for periods of ammonium and amino acid transporters, but expression protists is their ability to combine phototrophic and days to a few weeks. of these genes was temporally shifted between the two heterotrophic nutrition. Mixotrophic protists include species. These different capabilities for transporting and phototrophic species that also consume prey, and Ciliates Members of the phylum transforming phosphorus and nitrogen imply that there hetero­trophic species that retain functional chloroplasts Ciliophora (supergroup is a differential utilization of nutrients (and differential from their prey (kleptoplastidic ciliates, dinoflagellates Alveolata), a monophyletic timing of that utilization) between these species, and and some rhizaria). group of heterotrophic and this example of resource partitioning might explain their Heterotrophic nutrition in chloroplast-bearing pro- mixotrophic (kleptoplastidic) phagotrophy protists characterized by the co‑occurrence. tists generally involves . Alternatively, or in presence of cilia that are used Using samples that were collected as a part of the addition to this, many phototrophic protists are capable for motility and feeding. The Hawaii Ocean Time series (station ALOHA), the expres- of osmotrophy at very high concentrations of labile organic ciliates are major consumers of sion patterns of three functional groups of phyto­plankton compounds102, and osmotrophy may provide planktonic phytoplankton, bacteria and (diatoms, dinoflagellates and haptophytes) were investi- algae with specific organic substances required in trace other microorganisms. gated; this analysis indicated group-specific differences in amounts (for example, vitamins)103. However, there is Phagotrophy metabolic responsiveness to environmental change (FIG. 4), no convincing evidence that osmotrophy alone can sup- A mode of nutrition that is a finding that was supported and refined by the MMETSP port measureable population growth of algae in marine characterized by the dataset97. A comparison of the results for two environmen- pelagic ecosystems in which concentrations of organic engulfment and digestion of particulate material, usually tal samples yielded a roughly 5‑fold increase in sequence compounds are exceedingly low. microbial prey. read identification using the MMETSP dataset relative Phagotrophy is widespread among the major to a dataset containing only the available whole-genome taxonomic groups of phytoplankton, including the Osmotrophy reference data for free-living phytoplankton97 (FIG. 4b). Use chrysophytes, cryptophytes, dinoflagellates, haptophytes A mode of nutrition that of the MMETSP dataset revealed the dominance of dino- and green algae. These mixotrophic groups can use pre- is characterized by the absorption and utilization of flagellate-derived reads in these two samples. However, dation to satisfy various nutritional requirements (the dissolved organic compounds. this dominance was not evident in the earlier analyses, need for carbon, energy, major nutrients, trace metals or indicating that expansion of the available transcriptomic vitamins), and they have very variable phototrophic and Chrysophytes and genomic information on under-represented­ pro- heterotrophic capabilities (FIG. 3b). Experimental studies Small, flagellated algal protists of the class Chrysophyceae tists will improve our understanding of phytoplankton have attempted to resolve the ecological strategies and (supergroup Stramenopiles), ecology. The QMF of the dinoflagellates was stable over resulting competitive advantages of mixotrophy in these which contains photosynthetic, time, whereas haptophytes showed a variable QMF97. The species and have tried to incorporate those properties heterotrophic and mixotrophic cosmopolitan haptophyte Emiliania huxleyi was highly into food web models104,105, but we currently have a poor species. Chloroplast-bearing represented in the dataset. This species has a set of core understanding of this behaviour. species were previously referred to as golden-brown genes observed in specimens collected globally (a pange- Comparative transcriptomic studies have begun 98 algae, an imprecise term no nome) , but also large genetic variability among cultured to shed light on the physiological abilities, and longer used by specialists. isolates99. It is likely that the genetic variability in this thereby ecological strategies, of mixotrophic algae.

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Field sampling Extraction of nucleic acids Metatranscriptomic sequencing

b c S1 S2 DW Diatom Dinoflagellate Haptophyte S1 S2 DW S1 S2 DW S1 S2 DW mapping Genome KEGG module mapping MMETSP

Dinoflagellates Other ochrophytes Green algae Diatoms Haptophytes Other

10–5 10–4 10–3 10–2 10–1 Percent annotated reads Figure 4 | Improved gene identification and understanding of substantial increase in the ability to assign genes to specific phytoplankton phytoplankton functional groups. a,b | Metatranscriptomes from a groups97. c | A heat map was generated ofNature the quantitative Reviews | Microbiology metabolic natural phytoplankton assemblage (part a) were mapped to available fingerprints (QMFs) of the major eukaryotic phytoplankton functional genomes of free-living algae (part b; top row) and to a custom-built Marine groups (diatoms, dinoflagellates and haptophytes) in the nanoplankton Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP)-based to microplankton size class (>5 μm). Gene identity was based on mapping to transcriptome dataset (part b; bottom row); mapping was carried out for MMETSP KEGG orthology and revealed group-specific responses to the two near-surface in situ assemblage samples, S1 and S2, and also for a addition of nutrient-rich deep water to surface-water communities. sample that was cultured with deep water added (DW). In comparison to Parts b and c adapted with permission from REF. 97, National Academy the use of the available whole genomes, the MMETSP dataset enabled a of Sciences.

A comparative study of two species of chrysophyte revealing a strong dependence on phototrophy even (Dinobryon sp. and Ochromonas sp.) and a hapto- though this species also readily consumes prey. The phyte (P. p ar v um ) showed that these species possess comparative transcriptomic approach is an impor- different phototrophic and heterotrophic abilities tant step towards understanding the ecology of these (FIG. 3b) and exhibit corresponding differences in gene mixotrophic algae in nature. expression106. Ochromonas sp., a predominantly het- On the other end of the trophic spectrum, many erotrophic species107, grew rapidly in the presence heterotrophic dinoflagellates and ciliates, as well as a of bacteria regardless of light conditions, whereas few foraminifera, supplement heterotrophic nutrition its growth in the absence of prey was a fraction of its through organic-carbon and energy acquisition by the heterotrophic growth potential. Expression levels of retention of functional chloroplasts from ingested pho- the rRNA genes of the alga were relatively unchanged tosynthetic prey108,109. Genetic studies of these kleptoplas- between light and dark, as long as bacterial prey was tidic mixotrophs (FIG. 3c) are benefiting from the growth present, which indicates the preference of this species of public transcriptomic reference databases to aid gene for heterotrophy. By contrast, P. p ar v um readily immo- annotation. It is now clear that the ciliate Mesodinium bilized and killed ciliate prey, but the expression of rRNA rubrum (Myrionecta rubra) can maintain transcription- genes was strongly upregulated in the light compared ally active chloroplasts that are acquired from its microal- with levels in the dark, consistent with a physiology that gal prey110. Moreover, a comparison of gene expression is more dependent on phototrophy than on heterotro- in the photosynthetic prey in the free-living state and in phy. The upregulation of rRNA genes by Dinobryon sp. the host-sequestered state indicate that specific meta- in the light was similar to the response of P. p ar v um , bolic pathways are maintained in the sequestered state

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(notably, photosynthesis and oxidative-stress reduc- Beyond these meagre insights concerning marine tion)111. Future efforts to decipher the molecular com- heterotrophic protists, there has been little examination munication between host and prey organelles will rely of gene expression in heterotrophic species. The hetero­ heavily on investigations of gene expression. trophic dinoflagellate Oxyrrhis marina has recently emerged as a model system for studying the genetics Heterotrophic protists: a neglected majority. Predator– of free-living heterotrophic protists, but transcriptomic prey and parasite–host relationships that involve analyses of this species have focused not on processes protists are crucial for the food webs of aquatic and such as phagotrophy but rather on the evolutionary soil ecosystems (FIG. 1). The Tara Oceans expedition origin of dinoflagellate features117 and on rhodopsin collected thousands of plankton samples for analysis expression in response to starvation118. and thus generated a vast amount of data concerning marine protists, network analysis of which has demon- Protist–bacterium interactions. Whereas work on strated the tremendous diversity and trophic connec- predator–prey, host–symbiont and host–parasitoid rela- tivity of these organisms112. Findings that reveal the tionships is only just beginning, transcriptomic inves- high diversity and connectivity of eukaryotic parasitic tigations of phototrophic protists and their associ­ated taxa, in particular, are changing our view of the com- bacterial assemblages have rapidly advanced our under- plexity of food web structures within these commu- standing of protist–bacterium interactions119 (FIG. 5b–d). nities. Nevertheless, there have been very few genetic The phycosphere — the chemical and physical micro­ studies of parasitic or free-living heterotrophic protists environment that surrounds phytoplankton cells — has aimed at understanding these food web interactions, been a topic of research for many years120, but studies owing mostly to the lack of well-curated culture col- of the diversity and gene expression of protistan hosts lections for heterotrophs. Consequently, knowledge and their co‑existing bacteria are now revealing the of the metabolic processes that are involved in hetero- true nature of these associations (FIG. 5b). Algae impose trophic nutrition has been gleaned mostly from stud- strong selection on the bacterial community121,122, nur- ies of non-marine model systems or from studies with turing the growth of specific bacterial taxa that in turn other motivations, such as understanding evolution or provide growth substances required by the protist, pathogenicity. resulting in mutualistic metabolic relationships88,123. A few transcriptomic studies focused on identifying The ecological roles of these interactions for the protist the genes involved in phagocytosis and examining how range from nitrogen acquisition, such as in associations metabolism is affected by this process, and these studies between nitrogen-fixing cyanobacteria and algae124, to have begun to provide some insight into the response vitamin acquisition in many microalgae125. of protists to prey (FIG. 5a). Nearly 75% of genes in slime The ability of bacteria to directly affect gene expres- moulds of the genus Dictyostelium were found to be sion in some diatoms and dinoflagellates has also been differentially expressed in the presence or absence of demonstrated126,127. For example, one investigation prey113, which indicates that these species have a com- showed that in the toxic dinoflagellate Alexandrium plex response to phagotrophic nutrition, including tamarense, S‑adenosylmethionine synthetase (SAMS)- changes in the expression of genes that are responsi- encoding genes are downregulated in the presence of ble for prey recognition at the membrane, adhesion, bacteria127. The authors noted the similarity between transcription regulation and sterol metabolism (the the response of the dinoflagellate to bacteria and the latter seems to contribute to phagosomal membrane relationship reported between Amoeba proteus and its formation). In the phagotrophic haptophyte P. p ar v um , endosymbiotic bacterium, in which the presence of the predation by the alga resulted in upregulation of genes endosymbiont downregulates host genes that encode that are involved in fatty acid oxidation and the TCA SAMS128. This activity seems to make the host depend- cycle114, whereas genes that are related to inorganic-ni- ent on SAMS produced by the bacterium, and this pos- trogen uptake and transformation and iron uptake sibly solidifies the symbiotic relationship by linking the were substantially downregulated when prey was pres- survival and growth of the host to its bacterial associate. ent, implying that nitrogen and iron are obtained from Another study noted the production of algicidal ingested prey. compounds by a bacterial associate of the haptophyte Genomic and transcriptomic investigations of pro- E. huxleyi and, furthermore, determined that this was tistan human parasites have uncovered factors that are elicited by compounds that are produced by senescent involved in host invasion, cytotoxicity, metabolism, viru­ algal cells129 (FIG. 5c). The investigators proposed a model lence and immunosuppression or immunoevasion115. whereby the production of algicidal compounds con- By comparison, little is known about gene expres- verted a normally mutualistic relationship into a para­ sion in marine parasitoids, but one recent study of an sitic one. There is a high probability that many such Amoebophrya sp. (the genus is a major parasitoid group, complex and specific interactions between bacteria and members of which infect and kill numerous phytoplank- marine protists await discovery. ton) has indicated the probable role of lectins in attach- Colony formation in the choanoflagellate Salpingoeca ment to the host116. Interestingly, in another study46 rosetta has been shown to be stimulated (or inhibited) lectins seemed to be involved in the establishment of by specific lipids produced by co‑occurring bacteria130 endosymbiotic associations between large rhizaria and (FIG. 5d), a finding that may have implications for the photosynthetic dinoflagellates. development of multicellularity in the supergroup

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a Phagocytosis and b Chemical selection and c Release of algicidal d Induction of colony digestion of prey stimulation of bacterial compounds by bacteria formation by bacterial community by algal exudates exudates Food vacuole

Chemical induction of phagocytosis Chemical signal from Exchange of bacteria chemical signals between algae and bacteria Cell death

Figure 5 | Interactions between protists and other microorganisms. A myriad of interactions between protistan species and other organisms await discovery and characterization. Transcriptomic studies areNature now Reviews beginning | Microbiology to reveal changes in gene expression and the metabolic pathways that are associated with some of these interactions. a | Phagotrophy of a prey organism is induced by chemicals that are contained in the prey114 and can lead to changes in gene expression, such as upregulation of receptor genes and downregulation of genes involved in the uptake of nutrients that can be obtained from the prey (instead of by direct uptake from the environment). b | Chemical communication between bacteria and algae can give rise to species-specific mutualistic associations121. c | Chemical warfare between algae and some co‑occurring bacteria can result in algal death129. d | Chemical signalling can alter the life cycle of some protists. In one example, the formation of a colony by protists is induced (or inhibited) in response to lipids that are produced by co-existing bacteria130.

Opisthokonta, which also contains the animals and are also providing a much-needed reference dataset fungi. Interestingly, a colony-inducing sulfonolipid for the interpretation of genetic data obtained from produced by the bacterium Algoriphagus machipon- the massive field studies that have recently been com- gonensis resembles molecules that are produced by pleted4,18. What is more, the promise of this approach plants, animals and fungi and that have roles in signal is stimulating genomic and transcriptomic projects transmission. on species from lineages not yet represented among Many interactions undoubtedly also exist between sequenced (or even cultured) eukaryotes. marine protists and larger, multicellular organisms, and Recent efforts to obtain extensive genetic informa- the molecular interactions that control competition, tion about free-living marine protists have generated a predation, parasitism, mutualism and commensalism much-expanded database of protistan transcriptomes, are just beginning to be characterized. Likewise, the facilitating genetic studies on a broad diversity of these interplay between protists and viruses is particularly species. In addition, the transcriptomes of a few fresh- poorly understood. Although viruses of eukaryotic water protists (for example, glaucophytes) were com- algae have been known for many years131, and more pleted among the 396 strains that were sequenced by recently viruses of heterotrophic protists have also been the MMETSP project, because many protistan lineages characterized132, the factors that control protist suscep- contain species from marine, freshwater and terrestrial tibility and resistance, and the overall ecological role of environments. Therefore, the discoveries that are made these interactions, are poorly understood. possible by new datasets will have a substantial impact on investigations into a broad spectrum of free-living Outlook and conclusions protists. These transcriptomes also provide a starting Studies of protistan biology using traditional approaches point for comparative studies that aim to understand the have broadly framed the contributions of these species genetic foundations of adaptation to marine, freshwater to important biogeochemical processes. However, phys- and terrestrial existence. iological details about species and functional groups of many eukaryotes have thus far proved insufficient to The need for functional genomics. Eukaryotic tran- produce global models with strong predictive power. scriptomics (and, to some degree, metatranscriptomics) The next great challenge is the integration of new infor- have expanded rapidly in recent years. RNA deple- mation regarding various aspects of protistan biology, tion and poly‑A selection (the latter approach misses from the genetic potential of these organisms to the non-coding RNA and non-poly‑A RNA transcripts) behaviours of species, populations and ecosystems as have been used effectively to lower sequencing costs and a whole. In‑depth genetic investigations, enabled by improve sequencing depth. However, transcriptomes large-scale genomic and transcriptomic datasets, are are not genomes. Transcriptomes recover only the genes beginning to meet that challenge. These approaches that are expressed at the time of sampling and so do not provide a fundamental mechanistic understanding of define all the genetic potential of a species. This issue the metabolic potential of protists and their responses can be addressed to some extent by combining multi- to specific conditions and interactions. Transcriptomes ple transcriptomes from a strain that is cultured under

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different environmental conditions, yielding assemblies little is known about the distribution of many unique, that provide a more complete picture of the genetic uncultured taxa135. This technology can also be used to potential of the taxon86. Even so, gene expression does characterize the viral, bacterial and archaeal assemblages not necessarily imply protein function, owing to the that are associated with particular protistan strains and potential for post-transcriptional and post-translational species136,137, facilitating experimental investigation of the processing. nature of these ecological interactions. In addition, most protistan species have not yet been Although transcriptomics provides a robust starting cultured and are therefore unavailable for transcriptome point for gaining a better understanding of free-living sequencing. Metatranscriptomes are a partial solution marine protists, there are limitations to the approach, as to this problem, but as well as sequencing costs, gene noted above. Moreover, genomic features that regulate annotation remains a problem (for example, up to 75% transcription, such as promoters and methylation, are of genes cannot yet be ascribed a function47,96), and many not well understood138, nor are post-translational modi- genes cannot even be assigned to a particular taxon. fications. In addition, the total amount of RNA per cell is Sequenced genomes, and the reference databases that often assumed to be fairly constant, but this is not neces- they provide, will therefore be exceptionally important sarily true. The existence of different levels of total RNA in the future. They are fundamental for the alignment in any two cells to be compared (transcriptional ampli- and annotation of datasets derived from transcriptomes fication) can lead to erroneous conclusions regarding and metatranscriptomes, and they enable analyses using relative gene expression levels139. Some normalization in‑depth functional genomics methods. strategies being developed include, but are not limited to, Genomic studies are becoming increasingly feasible the use of exogenous standards and cell counts, or nor- with present-day sequencing power, even for species with malization to stable reference genes. For these and other large genomes, such as dinoflagellates42. Consequently, reasons, it will be necessary to combine transcriptomic, transcriptomic analyses for species with sequenced proteomic, metabolomic and gene manipulation studies genomes are appearing for species other than green algae of marine protists to validate any inferences made from and diatoms133,134. Beyond individual genomes, eukary- studies of gene expression140,141. otic metagenomics and the reconstruction of protistan Collectively, the insights gained from this research genomes from these datasets are currently feasible for on the evolution, biochemical capabilities and activi- only a limited number of species with small genomes, for ties of ecologically important protistan species will be samples from environments with very low species rich- widely felt. For example, genomic sequencing of the ness (for example, some extreme environments) or in tsetse fly and its associated protist, which causes tryp- only exceptionally well-funded projects. Even when these anosomiasis142, enhanced our knowledge of the inter- approaches are feasible, the interpretation of their results play between these two species and presented possible will be limited by the lack of suitable reference databases research avenues for the prevention of human disease. for assigning taxonomy and for gene annotation. These Similarly, the study of free-living protists will advance studies will continue to lag behind similar studies of our understanding of how environmental change might prokaryotes. lead to shifts in food web structure and ecosystem func- tion. In turn, this knowledge will guide the design of Future directions. Future applications of transcriptom- biogeochemical models to better predict these out- ics to the study of protistan biology will most probably comes and to help determine ecosystem-level manage- integrate other technologies that are now being used for ment efforts on local, regional and global scales143,144. In prokaryotes. Most notably, single-­cell sorting techniques recognition of these possibilities, it is true to say that and similar methods will be essential for isolating, ana- molecular ecological studies of marine protists have lysing and culturing protists from environments in which finally come of age.

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