A large colonial choanoflagellate from Mono Lake harbors live bacteria.

As the closest living relatives of animals, choanoflagellates offer insights into the ancestry of animal cell physiology. Here, we report the isolation and characterization of a colonial choanoflagellate from Mono Lake, California. The choanoflagellate forms large spherical colonies that are an order of magnitude larger than those formed by the closely related choanoflagellate Salpingoeca rosetta. In cultures maintained in the laboratory, the lumen of the spherical colony is filled with a branched network of extracellular matrix and colonized by bacteria, including diverse Gammaproteobacteria and Alphaproteobacteria. We propose to erect Barroeca monosierra gen. nov., sp. nov. Hake, Burkhardt, Richter, and King to accommodate this extremophile choanoflagellate. The physical association between bacteria and B. monosierra in culture presents a new experimental model for investigating interactions among bacteria and eukaryotes. Future work will investigate the nature of these interactions in wild populations and the mechanisms underpinning the colonization of B. monosierra spheres by bacteria. IMPORTANCE: The diversity of organisms that live in the extreme environment of Mono Lake (California, USA) is limited. We sought to investigate whether the closest living relatives of animals, the choanoflagellates, exist in Mono Lake, a hypersaline, alkaline, arsenic-rich environment. We repeatedly isolated members of a new species of choanoflagellate, which we have named Barroeca monosierra. Characterization of B. monosierra revealed that it forms large spherical colonies containing diverse co-isolated bacteria, providing an opportunity to investigate mechanisms underlying physical associations between eukaryotes and bacteria.

Choanoflagellates alongside diverse uncultured predatory protists consume the abundant open-ocean cyanobacterium Prochlorococcus.

Prochlorococcus is a key member of open-ocean primary producer communities. Despite its importance, little is known about the predators that consume this cyanobacterium and make its biomass available to higher trophic levels. We identify potential predators along a gradient wherein Prochlorococcus abundance increased from near detection limits (coastal California) to >200,000 cells mL-1 (subtropical North Pacific Gyre). A replicated RNA-Stable Isotope Probing experiment involving the in situ community, and labeled Prochlorococcus as prey, revealed choanoflagellates as the most active predators of Prochlorococcus, alongside a radiolarian, chrysophytes, dictyochophytes, and specific MAST lineages. These predators were not appropriately highlighted in multiyear conventional 18S rRNA gene amplicon surveys where dinoflagellates and other taxa had highest relative amplicon abundances across the gradient. In identifying direct consumers of Prochlorococcus, we reveal food-web linkages of individual protistan taxa and resolve routes of carbon transfer from the base of marine food webs.

What is it like to be a choanoflagellate? Sensation, processing and behavior in the closest unicellular relatives of animals.

All animals evolved from a single lineage of unicellular precursors more than 600 million years ago. Thus, the biological and genetic foundations for animal sensation, cognition and behavior must necessarily have arisen by modifications of pre-existing features in their unicellular ancestors. Given that the single-celled ancestors of the animal kingdom are extinct, the only way to reconstruct how these features evolved is by comparing the biology and genomic content of extant animals to their closest living relatives. Here, we reconstruct the Umwelt (the subjective, perceptive world) inhabited by choanoflagellates, a group of unicellular (or facultatively multicellular) aquatic microeukaryotes that are the closest living relatives of animals. Although behavioral research on choanoflagellates remains patchy, existing evidence shows that they are capable of chemosensation, photosensation and mechanosensation. These processes often involve specialized sensorimotor cellular appendages (cilia, microvilli, and/or filopodia) that resemble those that underlie perception in most animal sensory cells. Furthermore, comparative genomics predicts an extensive "sensory molecular toolkit" in choanoflagellates, which both provides a potential basis for known behaviors and suggests the existence of a largely undescribed behavioral complexity that presents exciting avenues for future research. Finally, we discuss how facultative multicellularity in choanoflagellates might help us understand how evolution displaced the locus of decision-making from a single cell to a collective, and how a new space of behavioral complexity might have become accessible in the process.

The premetazoan ancestry of the synaptic toolkit and appearance of first neurons.

Neurons, especially when coupled with muscles, allow animals to interact with and navigate through their environment in ways unique to life on earth. Found in all major animal lineages except sponges and placozoans, nervous systems range widely in organization and complexity, with neurons possibly representing the most diverse cell-type. This diversity has led to much debate over the evolutionary origin of neurons as well as synapses, which allow for the directed transmission of information. The broad phylogenetic distribution of neurons and presence of many of the defining components outside of animals suggests an early origin of this cell type, potentially in the time between the first animal and the last common ancestor of extant animals. Here, we highlight the occurrence and function of key aspects of neurons outside of animals as well as recent findings from non-bilaterian animals in order to make predictions about when and how the first neuron(s) arose during animal evolution and their relationship to those found in extant lineages. With advancing technologies in single cell transcriptomics and proteomics as well as expanding functional techniques in non-bilaterian animals and the close relatives of animals, it is an exciting time to begin unraveling the complex evolutionary history of this fascinating animal cell type.

Flow cytometric sorting of loricate choanoflagellates from the oligotrophic ocean.

It is challenging to study protists with extensive, loosely-associated extracellular structures because of the problems with keeping specimens intact. Here we have tested the suitability of high-speed flow cytometric sorting as a tool for studying such protists using oceanic loricate choanoflagellates as a model. We chose choanoflagellates because their lorica-to-cell volume ratio is > 10 and the voluminous loricae, i.e., the siliceous cell baskets essential for taxonomic identification, only loosely enclose the cells. Besides, owing to low concentrations, choanoflagellates are grossly under-sampled in the oligotrophic ocean. On four research cruises the small heterotrophic protists from samples collected in the photic layer of the South Atlantic and South Pacific oligotrophic (sub)tropical gyres and adjacent mesotrophic waters were flow sorted at sea for electron microscopy ashore. Among the flow-sorted protozoa we were able to select loricate choanoflagellates to assess their species diversity and concentrations. The well-preserved loricae of flow-sorted choanoflagellates made identification of 29 species from 14 genera possible. In the oligotrophic waters, we found neither endemic species nor evident morphological adaptations other than a tendency for lighter silicification of loricae. Common sightings of specimens storing extra costae in preparation for division, indicate choanoflagellates thriving in oligotrophic waters rather than enduring them. Thus, this case study demonstrates that high-speed flow sorting can assist in studying protists with extracellular structures 16-78× bigger than the enclosed cell.

Notable predominant morphology of the smallest most abundant protozoa of the open ocean revealed by electron microscopy.

In the microbe-driven ecosystems of the open ocean, the small heterotrophic flagellates (sHF) are the chief microbial predators and recyclers of essential nutrients to phototrophic microbes. Even with intensive molecular phylogenetic studies of the sHF, the origins of their feeding success remain obscure because of limited understanding of their morphological adaptations to feeding. Here, we examined the sHF morphologies in the largest, most oligotrophic South Pacific and Atlantic (sub)tropical gyres and adjacent mesotrophic waters. On four research cruises, the sHF cells were flow cytometrically sorted from bacterioplankton and phytoplankton for electron microscopy. The sorted sHF comprised chiefly heterokont (HK) biflagellates and unikont choanoflagellates numerically at around 10-to-1 ratio. Of the four differentiated morphological types of HK omnipresent in the open ocean, the short-tinsel heterokont (stHK), whose tinsel flagellum is too short to propagate a complete wave, is predominant and a likely candidate to be the most abundant predator on Earth. Modeling shows that the described stHK propulsion is effective in feeding on bacterioplankton cells at low concentrations; however, owing to general prey scarcity in the oligotrophic ocean, selective feeding is unsustainable and omnivory is equally obligatory for the seven examined sHF types irrespective of their mode of propulsion.

Nitric oxide signaling controls collective contractions in a colonial choanoflagellate.

Although signaling by the gaseous molecule nitric oxide (NO) regulates key physiological processes in animals, including contractility,1-3 immunity,4,5 development,6-9 and locomotion,10,11 the early evolution of animal NO signaling remains unclear. To reconstruct the role of NO in the animal stem lineage, we set out to study NO signaling in choanoflagellates, the closest living relatives of animals.12 In animals, NO produced by the nitric oxide synthase (NOS) canonically signals through cGMP by activating soluble guanylate cyclases (sGCs).13,14 We surveyed the distribution of the NO signaling pathway components across the diversity of choanoflagellates and found three species that express NOS (of either bacterial or eukaryotic origin), sGCs, and downstream genes previously shown to be involved in the NO/cGMP pathway. One of the species coexpressing sGCs and a bacterial-type NOS, Choanoeca flexa, forms multicellular sheets that undergo collective contractions controlled by cGMP.15 We found that treatment with NO induces cGMP synthesis and contraction in C. flexa. Biochemical assays show that NO directly binds C. flexa sGC1 and stimulates its cyclase activity. The NO/cGMP pathway acts independently from other inducers of C. flexa contraction, including mechanical stimuli and heat, but sGC activity is required for contractions induced by light-to-dark transitions. The output of NO signaling in C. flexa-contractions resulting in a switch from feeding to swimming-resembles the effect of NO in sponges1-3 and cnidarians,11,16,17 where it interrupts feeding and activates contractility. These data provide insights into the biology of the first animals and the evolution of NO signaling.

The history of Salpingoeca rosetta as a model for reconstructing animal origins.

Choanoflagellates, the closest living relatives of animals, have the potential to reveal the genetic and cell biological foundations of complex multicellular development in animals. Here we describe the history of research on the choanoflagellate Salpingoeca rosetta. From its original isolation in 2000 to the establishment of CRISPR-mediated genome editing in 2020, S. rosetta provides an instructive case study in the establishment of a new model organism.

Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates.

Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.

STING mediates immune responses in the closest living relatives of animals.

Animals have evolved unique repertoires of innate immune genes and pathways that provide their first line of defense against pathogens. To reconstruct the ancestry of animal innate immunity, we have developed the choanoflagellate Monosiga brevicollis, one of the closest living relatives of animals, as a model for studying mechanisms underlying pathogen recognition and immune response. We found that M. brevicollis is killed by exposure to Pseudomonas aeruginosa bacteria. Moreover, M. brevicollis expresses STING, which, in animals, activates innate immune pathways in response to cyclic dinucleotides during pathogen sensing. M. brevicollis STING increases the susceptibility of M. brevicollis to P. aeruginosa-induced cell death and is required for responding to the cyclic dinucleotide 2'3' cGAMP. Furthermore, similar to animals, autophagic signaling in M. brevicollis is induced by 2'3' cGAMP in a STING-dependent manner. This study provides evidence for a pre-animal role for STING in antibacterial immunity and establishes M. brevicollis as a model system for the study of immune responses.

Domain Analysis and Motif Matcher (DAMM): A Program to Predict Selectivity Determinants in Monosiga brevicollis PDZ Domains Using Human PDZ Data.

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 µM affinity, a value commonly considered the threshold for cellular PDZ-peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.

Loricate choanoflagellates (Acanthoecida) from warm water seas. IX. Coronoeca gen. nov., Polyfibula Manton and spiny forms of Parvicorbicula Deflandre.

The ambition to generate an overview of warm water loricate choanoflagellate biodiversity, based on a classic morphometric approach, is here completed by analyses of a range of tiny forms with anterior spines or projections and in most cases also a posterior pedicel. The warm water study complements previously obtained results from the more extensively studied temperate and polar regions of the world's oceans. It thus contributes to a significantly more balanced approach to global diversity patterns for these organisms. The current survey includes taxa such as Polyfibula elatensis, Parvicorbicula pedicellata, as well as a range of primarily undescribed and taxonomically challenging species, that are in an interim approach allocated to Coronoeca gen. nov. (C. kosmaniae sp. nov., C. conicella sp. nov., C. superpositus (Booth) comb. nov., C. marchantii sp. nov., C. tongiae sp. nov., and C. patongiensis sp. nov.). The analysis of warm water acanthoecid biodiversity has revealed in total 80 species from the six geographic regions sampled, corresponding to approximately 50% of all loricate species described. Nineteen species are previously undescribed forms. The Andaman Sea, Thailand, and West Australia are in a global context the most species-rich regions with 62 and 64 species respectively.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VIII. Crinolina Thomsen and Diaphanoeca Ellis.

The loricate choanoflagellate genera Diaphanoeca Ellis and Crinolina Thomsen encompass a total of ten species. The majority of these are recorded from the warm water regions reported on here. A distinct morphological dichotomy characterizes the genus Diaphanoeca as currently circumscribed. The species distribute themselves within a 'D. grandis subgroup' and a 'D. pedicellata subgroup' distinguished on e.g., the position of the protoplast inside the lorica chamber and the elaboration of the anterior projections. We are, while awaiting in particular further molecular evidence, taking a conservative approach and abstain from dealing with the subgroup issue at the generic level. The examination of material from the warm water regions of the world's oceans has resulted in the description of D. sargassoensis sp.n., D. pseudoundulata sp.n., and D. throndsenii sp.n., and a thorough re-examination of D. undulata. Species of Crinolina share multiple features with in particular the D. grandis species subgroup. It is yet relevant, both in a morphological and molecular perspective, to retain the genus Crinolina which remains unambiguously defined based on the posteriorly open lorica. A high level of agreement is found when contrasting morphological and molecular based phylogenetic schemes.

Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa.

Division of labor and establishment of the spatial pattern of different cell types of multicellular organisms require cell type-specific transcription factor modules that control cellular phenotypes and proteins that mediate the interactions of cells with other cells. Recent studies indicate that, although constituent protein domains of numerous components of the genetic toolkit of the multicellular body plan of Metazoa were present in the unicellular ancestor of animals, the repertoire of multidomain proteins that are indispensable for the arrangement of distinct body parts in a reproducible manner evolved only in Metazoa. We have shown that the majority of the multidomain proteins involved in cell-cell and cell-matrix interactions of Metazoa have been assembled by exon shuffling, but there is no evidence for a similar role of exon shuffling in the evolution of proteins of metazoan transcription factor modules. A possible explanation for this difference in the intracellular and intercellular toolkits is that evolution of the transcription factor modules preceded the burst of exon shuffling that led to the creation of the proteins controlling spatial patterning in Metazoa. This explanation is in harmony with the temporal-to-spatial transition hypothesis of multicellularity that proposes that cell differentiation may have predated spatial segregation of cell types in animal ancestors.

Evolution and Diversity of Semaphorins and Plexins in Choanoflagellates.

Semaphorins and plexins are cell surface ligand/receptor proteins that affect cytoskeletal dynamics in metazoan cells. Interestingly, they are also present in Choanoflagellata, a class of unicellular heterotrophic flagellates that forms the phylogenetic sister group to Metazoa. Several members of choanoflagellates are capable of forming transient colonies, whereas others reside solitary inside exoskeletons; their molecular diversity is only beginning to emerge. Here, we surveyed genomics data from 22 choanoflagellate species and detected semaphorin/plexin pairs in 16 species. Choanoflagellate semaphorins (Sema-FN1) contain several domain features distinct from metazoan semaphorins, including an N-terminal Reeler domain that may facilitate dimer stabilization, an array of fibronectin type III domains, a variable serine/threonine-rich domain that is a potential site for O-linked glycosylation, and a SEA domain that can undergo autoproteolysis. In contrast, choanoflagellate plexins (Plexin-1) harbor a domain arrangement that is largely identical to metazoan plexins. Both Sema-FN1 and Plexin-1 also contain a short homologous motif near the C-terminus, likely associated with a shared function. Three-dimensional molecular models revealed a highly conserved structural architecture of choanoflagellate Plexin-1 as compared to metazoan plexins, including similar predicted conformational changes in a segment that is involved in the activation of the intracellular Ras-GAP domain. The absence of semaphorins and plexins in several choanoflagellate species did not appear to correlate with unicellular versus colonial lifestyle or ecological factors such as fresh versus salt water environment. Together, our findings support a conserved mechanism of semaphorin/plexin proteins in regulating cytoskeletal dynamics in unicellular and multicellular organisms.

A flagellate-to-amoeboid switch in the closest living relatives of animals.

Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.

Towards understanding the origin of animal development.

Almost all animals undergo embryonic development, going from a single-celled zygote to a complex multicellular adult. We know that the patterning and morphogenetic processes involved in development are deeply conserved within the animal kingdom. However, the origins of these developmental processes are just beginning to be unveiled. Here, we focus on how the protist lineages sister to animals are reshaping our view of animal development. Most intriguingly, many of these protistan lineages display transient multicellular structures, which are governed by similar morphogenetic and gene regulatory processes as animal development. We discuss here two potential alternative scenarios to explain the origin of animal embryonic development: either it originated concomitantly at the onset of animals or it evolved from morphogenetic processes already present in their unicellular ancestors. We propose that an integrative study of several unicellular taxa closely related to animals will allow a more refined picture of how the last common ancestor of animals underwent embryonic development.

Structural characterization and computational analysis of PDZ domains in Monosiga brevicollis.

Identification of the molecular networks that facilitated the evolution of multicellular animals from their unicellular ancestors is a fundamental problem in evolutionary cellular biology. Choanoflagellates are recognized as the closest extant nonmetazoan ancestors to animals. These unicellular eukaryotes can adopt a multicellular-like "rosette" state. Therefore, they are compelling models for the study of early multicellularity. Comparative studies revealed that a number of putative human orthologs are present in choanoflagellate genomes, suggesting that a subset of these genes were necessary for the emergence of multicellularity. However, previous work is largely based on sequence alignments alone, which does not confirm structural nor functional similarity. Here, we focus on the PDZ domain, a peptide-binding domain which plays critical roles in myriad cellular signaling networks and which underwent a gene family expansion in metazoan lineages. Using a customized sequence similarity search algorithm, we identified 178 PDZ domains in the Monosiga brevicollis proteome. This includes 11 previously unidentified sequences, which we analyzed using Rosetta and homology modeling. To assess conservation of protein structure, we solved high-resolution crystal structures of representative M. brevicollis PDZ domains that are homologous to human Dlg1 PDZ2, Dlg1 PDZ3, GIPC, and SHANK1 PDZ domains. To assess functional conservation, we calculated binding affinities for mbGIPC, mbSHANK1, mbSNX27, and mbDLG-3 PDZ domains from M. brevicollis. Overall, we find that peptide selectivity is generally conserved between these two disparate organisms, with one possible exception, mbDLG-3. Overall, our results provide novel insight into signaling pathways in a choanoflagellate model of primitive multicellularity.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VII. Calotheca Thomsen and Moestrup, Stephanacantha Thomsen and Syndetophyllum Thomsen and Moestrup.

The tectiform loricate choanoflagellate genera Calotheca, Stephanacantha and Syndetophyllum have all been first described from warm water habitats and share the presence of flattened and often elaborate costal strips in the lorica. The current reinvestigation does confirm both the widespread occurrence of these taxa within the global warm water belt, and largely corroborates the established genus and species matrix. We describe here Stephanacantha oceanica sp. nov. which closely resembles S. campaniformis, and transfer Parvicorbicula zigzag to the genus Stephanacantha, despite differences in costal strip morphology, but based on a complete agreement in lorica constructional details.

Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta.

In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.