Choanoflagellata

Choanoflagellata (collared flagellates) is a clade of ~360 described species of free-living, marine and freshwater heterotrophic flagellates that constitute the closest living unicellular/colonial sister group to the animal kingdom (Metazoa), uniting with animals to form the superclade Choanozoa within Holozoa (Opisthokonta). Ranging from 3 to 10 μm in cell body diameter, every choanoflagellate displays a diagnostic cell architecture first recognized by Henry James-Clark in 1866 as nearly identical to the choanocyte (collar cell) of sponges (Porifera): an ovoid or spherical cell body bearing a single apical flagellum encircled at its base by a funnel-shaped collar of 25 to 50 rigid, actin-filled microvilli held at uniform spacing by glycocalyx fibrils.

Comparative genomics of solitary species such as Monosiga brevicollis (King et al., 2008) and fakultatively colonial species such as Salpingoeca rosetta (Fairclough et al., 2013) and Choanoeca flexa (Brunet et al., 2019) has transformed evolutionary cell biology. Although choanoflagellates are not animals and do not undergo embryonic development, their genomes encode vast repertoires of protein domains once thought to be exclusively metazoan—including cadherin-like extracellular repeats (23–29 EC domains), C-type lectins, immunoglobulin-like domains, receptor and cytoplasmic tyrosine kinases (RTKs/Src), and core postsynaptic density and neurosecretory SNARE complexes. Furthermore, chromosome-scale assemblies of Salpingoeca rosetta preserve the unfused ancestral state of the 29 metazoan Ancestral Linkage Groups (ALGs), serving as the decisive outgroup for reconstructing animal synteny and the root of the animal tree of life.

Microvillar Feeding Current and Bacterial Phagocytosis

As an opisthokont flagellate, a choanoflagellate generates thrust via planar sinusoidal undulation of its single apical flagellum from base to tip. In accordance with low-Reynolds-number hydrodynamics (Re ≈ 10⁻⁴ to 10⁻³), base-to-tip flagellar waves propel free-swimming cells with the flagellum trailing posteriorly, or—when the cell attaches its basal pole to a substrate via a secreted pedicel or lorica—draw a toroidal water current upward past the cell body and through the inter-microvillar slits (~100 to 200 nm wide) of the actin collar. Bacterial prey (such as Algoriphagus, Vibrio, and Pseudomonas) suspended in the water column are trapped against the outer mucus mesh of the microvillar collar, transported toward the cell apex along microvillar surfaces, and engulfed by actin-driven phagocytosis into basal food vacuoles.

Taxonomically, Choanoflagellata is divided into two major orders based on extracellular coverings:

1. Craspedida (e.g., Monosiga brevicollis, Salpingoeca rosetta, Choanoeca flexa): Secrete purely organic coverings ranging from thin glycocalyx cups to flask-like thecae composed of sulfated polysaccharides and proteins.
2. Acanthoecida (e.g., Diaphanoeca grandis, Stephanoeca diplocostata): Construct intricate, basket-like extracellular cages (loricae) assembled from polymerized biogenic silica (SiO₂) costal strips that are synthesized intracellularly within silica deposition vesicles, exocytosed onto the collar, and glued into species-specific geodesic domes.

Bacterial Chemical Signaling and Multicellular Rosette Morphogenesis

In Salpingoeca rosetta, a single genome can transition facultatively among at least five distinct differentiated cell types and two multicellular states: solitary attached thecate cells, fast swimmers (dispersal cells with short collars), slow swimmers, linear chain colonies, and spherical rosette colonies (comprising 4 to 50 polarized cells arranged around a central extracellular matrix core, structurally reminiscent of a blastula). Crucially, rosette colonies do not form by cell-cell aggregation; instead, they arise via serial incomplete cytokinesis of a single founding slow swimmer in which sister cells remain linked by cytoplasmic bridges and a shared central ECM secreted into the basal core.

Remarkably, the switch from solitary proliferation to multicellular rosette development in Salpingoeca rosetta is triggered by inter-kingdom chemical signaling from its natural bacterial prey, the Bacteroidetes bacterium Algoriphagus machipongonensis:

• Rosette-Inducing Factor 1 (RIF-1) and Sulfonolipids: Algoriphagus outer membranes contain specific sulfonolipids (SLs)—notably RIF-1 and RIF-2—which activate rosette development at femtomolar-to-picomolar concentrations (10⁻¹⁵ to 10⁻¹² M).
• Synergistic Co-Activators and Inhibitors: A second Algoriphagus lipid class, lysosulfonolipids (Lyso-SLs), shows no activity alone but synergizes with RIF-1 to increase rosette maturation 10-fold, whereas closely related bacterial capnoids competitively inhibit RIF-1 signaling.
• Genetic Requirement for Rosetteless and Jumble: Forward genetic screens in S. rosetta demonstrate that rosette integrity requires the gene rosetteless (rtls), which encodes a secreted C-type lectin that localizes exclusively to the central ECM core of the rosette, together with chondroitin sulfate proteoglycans synthesized by glycosyltransferases such as Jumble.

Even more striking collective behavior was discovered in Choanoeca flexa (formerly Salpingoeca sp. from Curaçao splash pools): its cup-shaped monolayer sheet colonies of hundreds of cells undergo rapid, reversible inversion ("sheet-to-sphere" collective contractility). In light, bacterial rhodopsin-driven cyclic nucleotide signaling relaxes the apical actomyosin ring wrapping the base of each cell's microvillar collar, opening the colony into a concave feeding sheet with flagella pointing inward; in darkness, collar contraction bends the sheet inward so flagella point outward, converting the colony into a fast-swimming phototactic sphere—all without neurons or classical adherens junctions.

Choanoflagellate Cadherins (23–29 Genes) vs. Classical Metazoan Cadherins

Sequencing of Monosiga brevicollis and Salpingoeca rosetta overturned the long-standing view that cadherins evolved solely to weld animal cells into epithelia. M. brevicollis encodes 23 cadherin genes and S. rosetta encodes 29 cadherin genes—comparable in number to Drosophila (17) or amphioxus (~30). Structurally, choanoflagellate cadherins (such as MBCDH1, MBCDH2, and the giant 23-repeat cadherin arrays) contain canonical extracellular cadherin repeats (EC domains) with conserved DxNDN / DxD / LDRE Ca²⁺-binding hinge motifs laminated alongside epidermal growth factor (EGF), laminin G, and Hedgehog-like (Hint) domains.

However, choanoflagellate cadherins differ in two critical ways from metazoan classical cadherins:

1. Absence of the Cytoplasmic Catenin-Binding Domain (CBD): Although choanoflagellate genomes encode homologs of β-catenin (Armadillo) and α-catenin (vinculin-like), their cadherin cytoplasmic tails lack the conserved JMD/CBD motifs that link cadherins to β-catenin in animals. Instead, several choanoflagellate cadherins couple intracellularly to protein tyrosine phosphatase or SH2-binding signaling motifs.
2. Localization to the Feeding Collar: Immunofluorescence microscopy shows that M. brevicollis cadherins localize to the actin-filled microvillar collar, where they recognize and capture bacterial prey or mediate transient filopodial recognition during mating—demonstrating that cadherins originated as Ca²⁺-stiffened cell-surface receptors for environmental and prey sensing before being co-opted into animal adherens junctions via recruitment of a cytoplasmic catenin-binding tail.

Hyper-Diversification of Receptor Tyrosine Kinases (RTKs)

While fungi and plants completely lack receptor tyrosine kinases (RTKs), Monosiga brevicollis encodes 128 tyrosine kinases (including 88 RTKs, more than the ~58 RTKs in the human genome), along with 39 protein tyrosine phosphatases and 123 SH2 (Src Homology 2) phosphotyrosine-reader domains. Notably, choanoflagellates possess genuine orthologs of the non-receptor tyrosine kinase Src, its negative regulator CSK (C-terminal Src kinase), and Abl, establishing that the core phosphotyrosine signaling module (writer + reader + eraser) was fully operational in the unicellular Choanozoan ancestor before diversifying independently along the choanoflagellate and metazoan branches.

Are choanoflagellates primitive animals or sponges?

No. Choanoflagellates are not animals (Metazoa); they are the sister group to all animals within the clade Choanozoa. Unlike sponges, which are true multicellular animals possessing multiple permanently differentiated somatic cell types (choanocytes, pinacocytes, archaeocytes, sclerocytes), oogenesis/spermatogenesis, and embryonic cleavage, choanoflagellates are unicellular or facultatively colonial protists whose colonies form without gastrulation or germ layers.

Why do choanoflagellates have 23 to 29 cadherin genes if even solitary species never form colonies?

In solitary choanoflagellates such as Monosiga brevicollis, cadherins localize to the apical microvillar feeding collar and basal filopodia rather than cell-cell junctions. Their extracellular Ca²⁺-rigidified EC repeats function as tethered cell-surface sensors that bind bacterial surface glycans for phagocytosis and mediate gamete recognition. In the stem lineage leading to Metazoa, one family of these ancestral choanozoan cadherins acquired a cytoplasmic β-catenin-binding domain (CBD), coupling extracellular homophilic adhesion directly to the intracellular actomyosin cytoskeleton to create the animal adherens junction.

How did Salpingoeca rosetta help resolve the Ctenophora-sister vs. Porifera-sister debate?

Because amino-acid substitution models suffer from long-branch attraction over 600+ million years of evolution, resolving the deepest branch of the animal tree required an outgroup with an intact chromosome-level genome to polarize macrosynteny (chromosome fusion-with-mixing events). Chromosome-scale assembly of Salpingoeca rosetta (and the filasterean Capsaspora owczarzaki) revealed that the 29 metazoan Ancestral Linkage Groups (ALGs) existed in an unfused state in the unicellular outgroups and in Ctenophora, whereas sponges, cnidarians, and bilaterians share irreversible derived chromosome fusions (Schultz et al., 2023).

1. King, N., Westbrook, M. J., Young, S. L., Kuo, A., Abedin, M., Chapman, J., ... & Rokhsar, D. S. (2008). "The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans." Nature, 451(7180), 783–788. https://doi.org/10.1038/nature06617
2. Alegado, R. A., Brown, L. W., Cao, S., Dermenjian, R. K., Zuzow, R., Fairclough, S. R., ... & King, N. (2012). "A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals." eLife, 1, e00013. https://doi.org/10.7554/eLife.00013
3. Fairclough, S. R., Chen, Z., Kramer, E., Zeng, Q., Young, S., Robertson, H. M., ... & King, N. (2013). "Premetazoan genome evolution and the regulation of cell differentiation in the choanoflagellate Salpingoeca rosetta." Genome Biology, 14(2), R15. https://doi.org/10.1186/gb-2013-14-2-r15
4. Brunet, T., Larson, B. T., Linden, T. A., Vermeij, M. J. A., McDonald, K., & King, N. (2019). "Light-regulated collective contractility in a multicellular choanoflagellate." Science, 366(6463), 326–334. https://doi.org/10.1126/science.aay2346