A broad survey of choanoflagellates revises the evolutionary history of the Shaker family of voltage-gated K+ channels in animals.

The Shaker family of voltage-gated K+ channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K+ currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K+ channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K+ channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates.

What choanoflagellates can teach us about symbiosis.

Environmental bacteria influence many facets of choanoflagellate biology, yet surprisingly few examples of symbioses exist. We need to find out why, as choanoflagellates can help us to understand how symbiosis may have shaped the early evolution of animals.

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.

Morphological and molecular reinvestigation of acanthoecid species III. - Kalathoeca cupula (Leadbeater, 1972) gen. et comb. nov. (=Stephanoeca cupula (Leadbeater) Thomsen, 1988).

Based on a further re-examination of loricate choanoflagellate species with detailed morphological (SEM/TEM) and molecular data of the SSU and LSU rRNA, the present study aims to give new insights for Stephanoeca cupula. In contrast to the original allocation within the family of tectiform reproducing species, morphological and molecular data of S. cupula sensu Leadbeater, 1972 points towards an affiliation within the nudiform reproducing family. Based on these new data, we here erect the nudiform genus Kalathoeca with its type species K. cupula gen. et comb. nov. Our data challenges morphological species assignments, as K. cupula shares its morphological lorica characteristics with tectiform reproducing species of Stephanoeca sensu stricto. Kalathoeca cupula is an interesting candidate for further investigating and understanding the evolutionary relationship of tectiform and nudiform reproducing species. Stephanoeca cupula sensu Thomsen, 1988 has been morphologically re-examined based on the renewed understanding of the morphological variability associated with S. cupula sensu Leadbeater, 1972 (=K. cupula), allowing us now to distribute the different morphological forms investigated within K. cupula and Pseudostephanoeca quasicupula.

First putative occurrence in the fossil record of choanoflagellates, the sister group of Metazoa.

Choanoflagellates are microeukaryotes that inhabit freshwater and marine environments and have long been regarded as the closest living relatives of Metazoa. Knowledge on the evolution of choanoflagellates is key for the understanding of the ancestry of animals, and although molecular clock evidence suggests the appearance of choanoflagellates by late Neoproterozoic, no specimens of choanoflagellates are known to occur in the fossil record. Here the first putative occurrence of choanoflagellates in sediments from the Cretaceous (Cenomanian-Turonian) is described by means of several cutting-edge petrographic techniques, and a discussion of its paleoenvironmental significance is performed. Furthermore, their placement in the organic matter classification systems is argued, with a placement in the Zoomorph Subgroup (Palynomorph Group) of the dispersed organic matter classification system being proposed. Regarding the ICCP System 1994, incorporation of choanoflagellates is, at a first glance, straightforward within the liptinite group, but the definition of a new maceral may be necessary to accommodate the genetic origin of these organisms. While modern choanoflagellates may bring light to the cellular foundations of animal origins, this discovery may provide an older term of comparison to their extant specimens and provide guidelines for possible identification of these organic components in other locations and ages throughout the geological record.

Re-evaluating Loricate Choanoflagellate Phylogenetics: Molecular Evidence Points to the Paraphyly of Tectiform Species.

Lorica-bearing choanoflagellates belong to the order Acanthoecida, a taxon which has been consistently recovered as monophyletic in molecular phylogenies. Based upon differences in lorica development and morphology, as well as the presence or absence of a motile dispersal stage, species are labelled as either nudiform or tectiform. Whilst Acanthoecida is robustly resolved in molecular phylogenies, the placement of the root of the clade is less certain with two different positions identified in past studies. One recovered root has been placed between the nudiform family Acanthoecidae and the tectiform family Stephanoecidae. An alternative root placement falls within the tectiform species, recovering the monophyletic Acanthoecidae nested within a paraphyletic Stephanoecidae. Presented here is a 14-gene phylogeny, based upon nucleotide and amino acid sequences, which strongly supports tectiform paraphyly. The horizontal transfer of a ribosomal protein gene, from a possible SAR donor, into a subset of acanthoecid species provides further, independent, support for this root placement. Differing patterns of codon usage bias across the choanoflagellates are proposed as the cause of artefactual phylogenetic signals that lead to the recovery of tectiform monophyly.

Premetazoan Origin of Neuropeptide Signaling.

Neuropeptides are a diverse class of signaling molecules in metazoans. They occur in all animals with a nervous system and also in neuron-less placozoans. However, their origin has remained unclear because no neuropeptide shows deep homology across lineages, and none have been found in sponges. Here, we identify two neuropeptide precursors, phoenixin (PNX) and nesfatin, with broad evolutionary conservation. By database searches, sequence alignments, and gene-structure comparisons, we show that both precursors are present in bilaterians, cnidarians, ctenophores, and sponges. We also found PNX and a secreted nesfatin precursor homolog in the choanoflagellate Salpingoeca rosetta. PNX, in particular, is highly conserved, including its cleavage sites, suggesting that prohormone processing occurs also in choanoflagellates. In addition, based on phyletic patterns and negative pharmacological assays, we question the originally proposed GPR-173 (SREB3) as a PNX receptor. Our findings revealed that secreted neuropeptide homologs derived from longer precursors have premetazoan origins and thus evolved before neurons.

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.

Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor.

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

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.

Extended divergence estimates and species descriptions of new craspedid choanoflagellates from the Atacama Desert, Northern Chile.

In contrast to previous perspectives, hypersaline environments have been proven to harbour a variety of potentially highly adapted microorganisms, in particular unicellular eukaryotes. The isolated, hypersaline waterbodies in the Atacama Desert, Northern Chile are exposed to high UV radiation and deposition of toxic heavy metals, making them of great interest regarding studies on speciation and evolutionary processes. In the past two years, among a variety of other protist species, five new species of heterotrophic choanoflagellates were described and analysed from this area, showing an adaptation to a broad range of salinities. Morphological data alone does not allow for species delineation within craspedid species, additional molecular data is essential for modern taxonomy. In addition, molecular clock analyses pointed towards a strong selection force of the extreme environmental conditions. Within this study, we describe three additional craspedid choanoflagellate species, isolated from different aquatic environments. Phylogenetic analyses show two distinct clades of choanoflagellates from the Atacama, suggesting two independent invasions of at least two ancestral marine species, and, as indicated by our new data, a possible dispersal by Andean aquifers. The extended molecular clock analysis based on transcriptomic data of choanoflagellate strains from the Salar de Llamará, a hypersaline basin within the Central Depression of the Atacama Desert, reflects colonisation and divergence events which correspond to geological data of the paleohydrology.

Detection of horizontal gene transfer in the genome of the choanoflagellate Salpingoeca rosetta.

Horizontal gene transfer (HGT), the movement of heritable materials between distantly related organisms, is crucial in eukaryotic evolution. However, the scale of HGT in choanoflagellates, the closest unicellular relatives of metazoans, and its possible roles in the evolution of animal multicellularity remains unexplored. We identified at least 175 candidate HGTs in the genome of the colonial choanoflagellate Salpingoeca rosetta using sequence-based tests. The majority of these were orthologous to genes in bacterial and microalgal lineages, yet displayed genomic features consistent with the rest of the S. rosetta genome-evidence of ancient acquisition events. Putative functions include enzymes involved in amino acid and carbohydrate metabolism, cell signaling, and the synthesis of extracellular matrix components. Functions of candidate HGTs may have contributed to the ability of choanoflagellates to assimilate novel metabolites, thereby supporting adaptation, survival in diverse ecological niches, and response to external cues that are possibly critical in the evolution of multicellularity in choanoflagellates.

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 Needle in the Haystack - Mapping Sequences to Morphology Exemplified by the Loricate Choanoflagellate Enibas thessalia sp. nov. (Acanthoecida, Acanthoecidae).

Environmental sequencing surveys unveil an unexpected magnitude of protist biodiversity and help to understand environmental community structure as well as biogeographical patterns. The interpretation of these data is still hindered by the lack of a verified and reliable reference database, which is the important basis for all analyses. References should rely on detailed and valid taxonomical descriptions including both morphology and autecological properties. In fact, obtaining such data is still a major challenge as cultivation-based approaches are very selective. In the present study, we highlight the potential to resample habitats which showed phylogenetically interesting sequences from environmental molecular surveys. We have been able to reveal a choanoflagellate species with the use of a single cell isolation approach in order to achieve a morphological description to the target sequence. This new species, Enibas thessalia sp. nov. now extends a recently described monospecific genus. In addition, we illustrate a nudiform lorica reproduction of the genus Enibas by observation of living cells. The genus belongs to the family of Acanthoecidae, which comprises five genera. The morphology of the genus Enibas shows a striking resemblance to the genus Stephanoeca, which belongs to the other family of loricate choanoflagellates, the Stephanoecidae, indicating that morphology alone might not reflect phylogenetic relations. We demonstrate that mapping sequences to a taxonomical description of species is a valuable tool to verify the organism behind an environmental amplicon. We emphasize the urgent need of integrative taxonomy matching molecular data with morphological features to verify the outcome of phylogenetic analyses.

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 insights into the mechanism of rhodopsin phosphodiesterase.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

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.

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.

Gene Similarity Networks Unveil a Potential Novel Unicellular Group Closely Related to Animals from the Tara Oceans Expedition.

The Holozoa clade comprises animals and several unicellular lineages (choanoflagellates, filastereans, and teretosporeans). Understanding their full diversity is essential to address the origins of animals and other evolutionary questions. However, they are poorly known. To provide more insights into the real diversity of holozoans and check for undiscovered diversity, we here analyzed 18S rDNA metabarcoding data from the global Tara Oceans expedition. To overcome the low phylogenetic information contained in the metabarcoding data set (composed of sequences from the short V9 region of the gene), we used similarity networks by combining two data sets: unknown environmental sequences from Tara Oceans and known reference sequences from GenBank. We then calculated network metrics to compare environmental sequences with reference sequences. These metrics reflected the divergence between both types of sequences and provided an effective way to search for evolutionary relevant diversity, further validated by phylogenetic placements. Our results showed that the percentage of unicellular holozoan diversity remains hidden. We found novelties in several lineages, especially in Acanthoecida choanoflagellates. We also identified a potential new holozoan group that could not be assigned to any of the described extant clades. Data on geographical distribution showed that, although ubiquitous, each unicellular holozoan lineage exhibits a different distribution pattern. We also identified a positive association between new animal hosts and the ichthyosporean symbiont Creolimax fragrantissima, as well as for other holozoans previously reported as free-living. Overall, our analyses provide a fresh perspective into the diversity and ecology of unicellular holozoans, highlighting the amount of undescribed diversity.