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A. C. Hardy; On the Origin of the Metazoa. J Cell Sci 1 December 1953; s3-94 (28): 441–443. doi:

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A new view is expressed regarding the possible evolution of the Metazoa from unspecialized and relatively simple Metaphyta, instead of from colonial or partitioned Protozoa.

This is a short paper to present a very simple idea regarding the origin of the Metazoa, one which, as far as I am aware, has not been expressed before. It is in a sense a corollary to the views expressed by my old friend and colleague Dr. J. R. Baker in his article on ‘the Status of the Protozoa’ (1948); indeed, the idea owes its origin to that article for it occurred to me after reading it.

Baker, it will be remembered, defines a cell as ‘a mass of protoplasm, largely or completely bounded by a membrane, and containing within it a single nucleus formed by the telophase transformation of a haploid or diploid set of anaphase chromosomes’. He argues that while some Protozoa, such as the Ciliophora and certain Radiolaria, are not cells in the sense of this definition, there are many which can properly be regarded as unicellular organisms. He stresses that there is no fundamental difference between the cleavage of an egg and the multiplication of a unicellular protozoon by division. At the end of his article he discusses the evolution of Metazoa from Protozoa. It is well known that there are two schools of thought regarding this question. On the one hand, there are those who hold that the Metazoa were derived from a group of protozoan cells which had failed to separate after repeated division, i.e. from a colony of cells which have gained a new and corporate individuality; on the other hand, there are those who believe that the origin of the manycelled condition was due to the development of partitions within a multinucleate protozoon. Baker discusses both sides of the controversy and points out how it is more difficult for the zoologist to imagine the evolution of a metazoon from a protozoan colony than it is for the botanist to imagine a similar step in the evolution of multicellular plants. Since my suggestion springs directly from his argument of this point, I will, with his kind permission, quote him at some length. He writes as follows:

‘It may be remarked that while such forms as Volvox raise a storm of controversy among zoologists, they are not regarded as unusual by botanists, who are familiar with such colonial forms in groups other than the phytomonads. Botanists regard them as composed of cells, which are comparable on one hand with the individuals of what they call unicellular species, and on the other with the cells of higher but related forms, in which there is somatic differentiation (heterotrichous forms and other algal metaphytes). ‘If we seek the reason why zoologists are more divided in this controversy than botanists, we shall surely find it in a fundamental difference between plants and animals. In the lower plants each group tends to present both unicellular and metaphytic representatives, and botanists are repeatedly confronted with the intermediate forms. Metaphytes, in fact, have obviously originated independently many times, so that no one would, propose the word “Metaphyta” in any classification that was supposed to be based on evolutionary principles. With animals it is far different. Zoologists have not got intermediates again and again before their eyes. In the great group of Rhizopods, for example, there is no example of a multicellular form (the word “cell” being here used as defined above). It would not appear that anyone has discussed the reason for this striking difference between plants and animals. The following suggestion may be made. The unicellular plant absorbs nutriment from all sides equally, and when, in the course of ontogeny or phylogeny, it becomes a metaphyte, there is no fundamental change in this respect: a cell divides without separation and the two products continue to absorb nutriment over most of their surfaces. The passage from the unicellular form to the metaphyte is therefore easy. In the case of animals, however, there is an important change when a unicellular form becomes a metazoon: a new method of feeding must be adopted. We see this particularly clearly in the ontogeny of Dendrocoelum . The blastomeres feed at first saprozoically, but at last a profound change occurs: a set of digestive organs originates, and a new method of nutrition begins. Most animals overcome this difficulty in ontogeny by placing enough reserve food-stuffs within the single-celled stage to make feeding unnecessary. It is easy to see what evolutionary difficulties must be presented when a new system of nutrition must be acquired before advance can occur. The difficulties would be greatest when a protozoon had a localized mouth. If the products of division of such an animal were to adhere together and each were to acquire its own mouth, no advance would be made towards the evolution of a metazoan alimentary canal. This suggests that the Metazoa may have arisen from primitive Protozoa, unprovided with localized organs of assimilation. These considerations seem to make it clear why intermediates between unicellular animals and Metazoa are rare, while intermediates between unicellular plants and metaphytes are common. (The colonial phytomonads are, of course, only regarded as animals by zoologists on account of their motility: their nutrition, it need scarcely be said, is holophytic or saprophytic.)’

We are concerned here with the origin of the true Metazoa, leaving on one side the sponges, sometimes called the Parazoa, which with their more limited integration may well have been quite independently derived from aggregations of choanoflagellate-like protozoa. The simple idea I want to suggest is that the Metazoa have not been derived from the Protozoa at all, but from relatively simple metaphytes which, after they had evolved from protophytes began, perhaps as a result of a shortage of phosphates and nitrates, to capture and feed upon other small organisms as do the higher insectivorous plants. Among the Protista it is clear that animals have evolved from plants more than once; in several different groups of Protophyta, as Fritsch (1935) has so well shown, we can arrange parallel series passing from holophytic forms at one end to holozoic forms at the other. In addition to such changes among the flagellates of the class Euglenineae, Fritsch points out and compares these parallel evolutionary lines in four different classes of the simpler algae: Chlorophyceae, Xanthophyceae, Chrysophyceae, and Dinophyceae. If animals have evolved from plants several times at the unicellular level, is it not possible that animals might, at least once, have been so derived from plants at a not too specialized multicellular level ? This suggestion may perhaps be worth bearing in mind because it does get over the very real difficulty which Baker has pointed out, that of how a metazoon could have evolved from a colony of protozoa, each, as individuals, already adapted for the capture of prey.

It would be unprofitable to elaborate the idea in much detail. The many different devices evolved by the various carnivorous plants to enable them to secure their food suggest how such a metazoan organism may have been derived. It is not difficult to imagine a spherical volvox-like metaphyte developing a little pocket-like invagination in which small protozoa or protophyta might collect, die, and provide breakdown products to be absorbed by the cells lining the cavity. Such little pockets would be analogous with the bladders of Utricularia which capture water-fleas. How similar in general action, although different in physiological detail, are the ‘tentacles’ which capture flies on the leaf of the sundew Drosera to those which capture Crustacea on the polyps of a hydroid! The gradual transition from a simple metaphyte to a simple polyp-like metazoon—a bladder-like cavity with tentacles—seems no more difficult to conceive than the evolution of the higher animal-like insectivorous plants; we have only to imagine the process going so far as to cut out all photosynthesis, thus making the organism holozoic—as indeed has occurred repeatedly at the unicellular level of the flagellates and algae.

If the Metazoa should have evolved from Metaphyta then the findings of Gohar (1940) are of special interest; he claims to have shown that Alcyonarians of the family Xeniidae feed entirely upon the photosynthetic products of their symbiotic algae and never capture animals for food at all; by a circuitous route they appear now to be metazoa which are on their way back to becoming metaphytes and must, if the whole colony is to grow, be taking in nutritive salts from outside for the growth of their enclosed plant cells. (It is perhaps just possible, but most unlikely, that the zooxanthellae of the Anthozoa, which cannot be cultured outside their ‘host’, might be plant cells remaining as a legacy from the original metaphytic days and not separate symbiotic algae as in other such associations.)

I do not wish here to enter into the controversy between the former two opposing views as to the origin of the Metazoa; the sole purpose of this short communication is to state a third possible view for consideration. I am indebted to Dr. J. R. Baker not only for his article which gave rise to this idea, but for his interest in the suggestion and his discussion of it.

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Insights into the origin of metazoan multicellularity from predatory unicellular relatives of animals

  • Denis V. Tikhonenkov   ORCID: 1 , 2 ,
  • Elisabeth Hehenberger 3 ,
  • Anton S. Esaulov 4 ,
  • Olga I. Belyakova 4 ,
  • Yuri A. Mazei 5 ,
  • Alexander P. Mylnikov 1   na1 &
  • Patrick J. Keeling 2  

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The origin of animals from their unicellular ancestor was one of the most important events in evolutionary history, but the nature and the order of events leading up to the emergence of multicellular animals are still highly uncertain. The diversity and biology of unicellular relatives of animals have strongly informed our understanding of the transition from single-celled organisms to the multicellular Metazoa. Here, we analyze the cellular structures and complex life cycles of the novel unicellular holozoans Pigoraptor and Syssomonas (Opisthokonta), and their implications for the origin of animals.

Syssomonas and Pigoraptor are characterized by complex life cycles with a variety of cell types including flagellates, amoeboflagellates, amoeboid non-flagellar cells, and spherical cysts. The life cycles also include the formation of multicellular aggregations and syncytium-like structures, and an unusual diet for single-celled opisthokonts (partial cell fusion and joint sucking of a large eukaryotic prey), all of which provide new insights into the origin of multicellularity in Metazoa. Several existing models explaining the origin of multicellular animals have been put forward, but these data are interestingly consistent with one, the “synzoospore hypothesis.”


The feeding modes of the ancestral metazoan may have been more complex than previously thought, including not only bacterial prey, but also larger eukaryotic cells and organic structures. The ability to feed on large eukaryotic prey could have been a powerful trigger in the formation and development of both aggregative (e.g., joint feeding, which also implies signaling) and clonal (e.g., hypertrophic growth followed by palintomy) multicellular stages that played important roles in the emergence of multicellular animals.

The origin of animals (Metazoa) from their unicellular ancestors is one of the most important evolutionary transitions in the history of life. Questions about the mechanisms of this transformation arose about 200 years ago, but are still far from being resolved today. Most investigations on the origin of Metazoa have focused on determining the nature of the shared, multicellular ancestor of all contemporary animals [ 1 , 2 , 3 ]. However, even the branching order of early, non-bilaterian lineages of animals on phylogenetic trees is still debated: some consider either sponges (Porifera) [ 4 , 5 , 6 ] or Ctenophora [ 7 , 8 , 9 ] or Placozoa [ 10 , 11 ] to be the first branch of extant metazoans (although most data show the latter scenario to be the least realistic of these possibilities [ 2 , 12 ]).

While molecular clock-based studies and paleontological evidence indicate that multicellular animals arose more than 600 million years ago [ 13 , 14 ], we know very little about how animals arose. To establish the sequence of events in the origin of animals from unicellular ancestors, we also need to investigate their closest relatives, the unicellular opisthokont protists. Information on the diversity and biology of the unicellular relatives of animals, their placement within the phylogenetic tree of opisthokonts, and the identification of molecular and morphological traits thought to be specific for animals within their unicellular sister lineages has all strongly informed our understanding of the transition from single-celled organisms to the multicellular Metazoa [ 15 , 16 , 17 , 18 , 19 ].

Until recently, only three unicellular lineages, the choanoflagellates, filastereans, and ichthyosporeans, as well as Corallochytrium limacisporum , a mysterious marine osmotrophic protist described in association with corals, have been described as collectively being sisters to animals. Together with animals, they form the Holozoa within the Opisthokonta [ 19 , 20 , 21 ]. These unicellular organisms have extremely variable morphology and biology. Choanoflagellates represent a species-rich group of filter-feeding, bacterivorous, colony-forming protists, which possess a single flagellum surrounded by a collar of tentacles (microvilli). They are subdivided into two main groups—the predominantly marine Acanthoecida and the freshwater and marine Craspedida [ 22 ]. Filastereans are amoeboid protists producing pseudopodia. Until recently, they were represented by only two species: the endosymbiont of a freshwater snail, Capsaspora owczarzaki , and the free-living marine heterotroph, Ministeria vibrans [ 23 , 24 ], which was recently shown to also possess a single flagellum [ 19 , 25 ] . Ichthyosporeans are parasites or endocommensals of vertebrates and invertebrates characterized by a complex life cycle, reproduction through multinucleated coenocytic colonies, and flagellated and amoeboid dispersal stages [ 26 , 27 ]. Corallochytrium is a unicellular coccoid organism, which produces rough, raised colonies and amoeboid limax-like (slug-shaped) spores [ 28 ]. Additionally, molecular data predict a cryptic flagellated stage for Corallochytrium [ 19 ].

A large number of hypotheses about the origin of multicellular animals have been proposed. The most developed model for the origin of metazoan multicellularity is based on a common ancestor with choanoflagellates [ 16 , 29 , 30 , 31 , 32 , 33 ]. This idea was initially based on the observed similarity between choanoflagellates and specialized choanocyte cells in sponges. Molecular investigations also supported the idea by consistently indicating that choanoflagellates are the closest sister group to Metazoa. However, molecular phylogeny itself does not reveal the nature of ancestral states; it only provides a scaffolding on which they might be inferred from other data. The evolutionary positions of the other unicellular holozoans (filastereans, ichthyosporeans, and Corallochytrium ) are less clear and sometimes controversial (e.g., [ 19 , 24 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]).

As noted above, many molecular traits that were thought to be “animal-specific” are now known to be present in unicellular holozoans, while conversely, the loss of other traits has been shown to correlate with the origin of the animals. But gene content alone is not sufficient to provide a comprehensive understanding of the cell biology, life cycle, and regulation capabilities of the unicellular ancestor; it requires also analysis of the biology of the extant unicellular relatives of animals [ 41 ].

Recently, we described phylogenomic and transciptome analyses of three novel unicellular holozoans [ 37 ], which are very similar in morphology and life style but not closely related. Pigoraptor vietnamica and Pigoraptor chileana are distantly related to filastereans, and Syssomonas multiformis forms a new phylogenetic clade, “Pluriformea,” with Corallochytrium . The relationship of “Pluriformea” to other holozoans is controversial. Initial analysis indicated “Pluriformea” as a sister lineage to the clade uniting Metazoa, choanoflagellates, and filastereans (Fig.  1 a) [ 37 ]. Later, single-copy protein domain analysis recovered Pluriformea as sister lineage to Ichthyosporea (Fig.  1 b) with almost maximum statistical support [ 42 ], validating the Teretosporea hypothesis [ 19 ]. Both new genera of unicellular holozoans form the shortest and most slowly evolving branches on the tree, which improved support for many nodes in the phylogeny of unicellular holozoans. Also, comparison of gene content of the new taxa with the known unicellular holozoans revealed several new and interesting distribution patterns for genes related to multicellularity and adhesion [ 37 ].

figure 1

Schemes of the possible phylogenetic position and life cycles of Syssomonas and Pigoraptor . a Pluriformea as a sister lineage to the clade uniting animals, choanoflagellates, and filastereans (including Pigoraptor ) according to Hehenberger et al. [ 37 ]. b Pluriformea as a sister lineage to Ichthyosporea within Teretosporea according to López-Escardó et al. [ 42 ]. c Life cycle of Syssomonas multiformis . d Life cycle of Pigoraptor vietnamica and P. chileana

Here, we report the detailed morphological and ultrastructural analyses of these new species, as well as describing their life cycle in culture, which have important implications for understanding the origin of animals as are the genetic analyses. All three species are shown to be predatory flagellates that feed on large eukaryotic prey, which is very unusual for unicellular Holozoa. They also appear to exhibit complex life histories with several distinct stages, including interesting multicellular structures that might offer important clues to precursors of multicellularity. On the basis of these findings, we discuss the current hypotheses about the origin of multicellular animals from their unicellular ancestors.

Results and discussion

Three novel holozoan taxa were isolated from a freshwater pool ( Syssomonas multiformis ) and the silty sand on the littoral of a freshwater lake ( Pigoraptor vietnamica ) in tropical Vietnam, and from the sediment of a freshwater temporary water body in Tierra del Fuego ( Pigoraptor chileana ). The characteristics of the biotopes are specified in the “ Methods ” section. Samples were characterized by high species richness of heterotrophic flagellates including bodonids, chrysomonads ( Spumella spp., Paraphysomonas spp.), euglenids ( Petalomonas spp.), cercomonads, thaumatomonads, protaspids, and loricate bicosoecids. Predatory holozoans appeared to represent a minor fraction of the total abundance. Detailed morphological descriptions of their cells and aggregates are presented below. Note that the term “arrgeration(s)” and cognate words were always used to define a multicellular structure that formed from cells that came together as opposite to the term “clonal multicellularity,” which defines a multicellular structure that formed from a single founding cell that divided repeatedly. All stages of the life cycle (Fig.  1 c, d) were observed at 22 °C in the clonal cultures. The main life form in all three studied species is the swimming flagellate cell, which can turn into a cyst, especially in old (~ 1 month) cultures. The amoeboid and pseudopodial stages described below were apparent only after 2 years of cultivation and even then were extremely rare. The variation of temperature and pH, as well as variation of cultivation medium and agitation, did not result in the appearance of additional morphological forms or increase the frequency of occurrence of certain (e.g., amoeboid) life forms. However, increasing the temperature to 30–35 °C leads to suppression and immobilization of prey cells ( Parabodo caudatus ), which favored the feeding of opisthokont predators on slow-moving prey, which in turn lead to an increase in the number of cell aggregations arising from joint feeding.

Syssomonas multiformis morphology and life cycle

The organism is characterized by a large variety of life forms including flagellates, amoeboflagellates, amoeboid non-flagellar cells, and spherical cysts (Fig.  1 c). The most common stage in the life cycle, a swimming flagellate cell, resembles a typical opisthokont cell, reminiscent of sperm cells of most animals and zoospores of the chytrid fungi. Cells are round to oval and propel themselves with a single, long posterior flagellum (Fig.  2 a–c, x). The flagellum is smooth and emerges from the middle-lateral point of the cell, turns back, and always directs backward during swimming. The cell rotates during swimming (Video 1). Flagellar beating can be very fast, which can create the appearance of two flagella. Motile flagellates can suddenly stop and change the direction of movement. The flagellated cells measure 7–14 μm in diameter. The flagellum length is 10–24, rarely 35 μm. Cyst diameter is 5 μm (Fig.  2 d, y).

figure 2

External morphology and life forms of Syssomonas multiformis . a – c Swimming flagellated cells. d Cyst. e Attached flagellated cell. f – h Sucking of eukaryotic prey. i Simultaneous joint feeding of three cells of Syssomonas on one prey cell with attraction of other specimens to the feeding spot. j Amoeboflagellate. k , l Amoeboid cell. m Palintomic cell division inside the cyst: the number of observed daughter cells was always even, and up to 16 daughter cells have been seen. n Cell with inside vesicles. o Cyst with vesicles. p , q Cells of Syssomonas (arrows) with engulfed starch granules (bright field ( p ) and fluorescent microscopy, DAPI staining; arrows are pointing to Syssomonas cells). r Cells of Syssomonas with engulfed starch granules hiding into the starch crystals druse (circles and arrows show life cells of Syssomonas floating towards the starch crystals druse). s – u , w Cell aggregations of Syssomonas near the bottom of Petri dish. v Floating aggregation of flagellated cells. x General view of flagellated cell (scanning electron microscopy, SEM). y Cyst (SEM). ac, acroneme (pointed tip of flagellum); bc, bacterium; fl, flagellum; fp, filopodium; lb, lobopodium. Scale bars: a – p , s – w 10 μm, r 45 μm, x 3 μm, y 2 μm

Additional file 2 Video S1. Swimming of Syssomonas multiformis cell with rotation.

Solitary cells of Syssomonas can temporarily attach to the substrate by the anterior part of the cell body. They produce water flow by rapid flagellum beating posteriorly and in that state resemble cells of choanoflagellates or choanocytes from sponges (Fig.  2 e, Video 2). Floating flagellated cells can also move to the bottom and transform to amoeboflagellates (Fig.  2 j, Video 3) by producing both wide lobopodia and thin short filopodia. Flagellar beating becomes slower and then stops. Amoeboflagellates crawl along the surface using their anterior lobopodia and can take up clusters of bacteria. The organism can lose the flagellum via three different modes: the flagellum may be abruptly discarded from its proximal part of the cell; a stretched flagellum may be retracted into the cell; the flagellum may convolve under the cell body and then retract into the cell as a spiral (Video 4). As a result Syssomonas turns into an amoeba (Fig.  2 k, l, Video 4). Amoeboid cells produce thin, relatively short filopodia and sometimes have two contractile vacuoles. Amoeboid cells are weakly motile. The transformation of amoeboflagellates and amoebae back to flagellates was also observed.

Additional file 3 Video S2. Attached cell of Syssomonas multiformis and rapid flagellum beating.

Additional file 4 Video S3. Amoeboflagellate stage of Syssomonas multiformis. Cells of eukaryotic prey Parabodo caudatus are also visible.

Additional file 5 Video S4. Loss of flagellum in Syssomonas multiformis and transition to amoeba.

Amoeboid cells can also retract their filopodia, become roundish, and transform into a cyst (Fig.  2 d, Video 5). Palintomic divisions may occur inside the cyst, and up to 16 (2, 4, 8, or 16) flagellated cells are released as a result (Fig.  2 m, Video 6). Division into two cell structures was also observed in culture (Video 7), but it is hard to tell whether a simple binary longitudinal division of a Syssomonas cell with retracted flagellum has taken place, or the final stage of a division inside the cyst has been observed.

Additional file 6 Video S5. Transformation of amoeba into a cyst in Syssomonas multiformis .

Additional file 7 Video S6. Palintomic divisions inside the cyst of Syssomonas multiformis .

Additional file 8 Video S7. Division into two cell structures in Syssomonas multiformis .

Floating, flagellated cells containing vesicular structures were observed (Fig.  2 n, Additional file  1 : Fig. S1E, Video 8); however, the process of formation and the purpose of these vesicles are unknown. After some time, such cells lose their flagellum and transform into vesicular cysts with a thick cover (Fig.  2 o). Division inside vesicular cysts was not observed within 10 days of observation. Such structures could represent resting cysts or dying cells containing autophagic vacuoles (the partial destruction of one such cyst was observed after 4 days of observation, see Video 8).

Additional file 9 Video S8. Cell and cyst of Syssomonas multiformis with vesicular structures inside.

The organism is a predator; it takes up other flagellates (e.g., Parabodo caudatus and Spumella sp.) which can be smaller, about the same size, or larger than Syssomonas. But in contrast to many other eukaryotrophic protists, Syssomonas does not possess any extrusive organelles for prey hunting. After initial contact, Syssomonas attaches to the prey cell and sucks out their cytoplasm (without ingesting the cell membrane) (Fig.  2 f–h, Additional file  1 : Fig. S1A-C, Video 9). The organism feeds better on inactive, slow-moving, or dead cells and can also capture intact prey cells and cysts by means unobserved. After attaching to the prey, many other Syssomonas cells become attracted to the same prey cell (likely by chemical signaling) and try to attach to it. Joint feeding was observed: several cells of Syssomonas can suck out the cytoplasm of the same prey cell together (Fig.  2 i, Additional file  1 : Fig. S1D, Video 9).

Additional file 10 Video S9. Feeding of Syssomonas multiformis on eukaryotic prey.

In culture, Syssomonas can take up starch granules from rice grains; the granules can be the same size as the cells (Fig.  2 p, q). In the presence of Syssomonas cells, rice grains in Petri dishes crumble into small fragments and separate granules of starch (Additional file  1 : Fig. S2). Cells of Syssomonas with engulfed starch granules can hide within the starch crystals druses and lose the flagellum (Fig.  2 r, Additional file  1 : Fig. S1F). Numerous cysts integrated into the starch matrix were often observed in culture.

The organism can also feed on clusters of bacteria (Video 10) using short pseudopodia. After feeding, Syssomonas cells become 2–3 times bigger and a large food vacuole is formed at the posterior end of the cell body (Fig.  2 c). In the absence of eukaryotic prey (cultivation on bacteria and/or rice grain/starch only), Syssomonas either dies or forms resting cysts. Bacteria alone are not sufficient food for Syssomonas.

Additional file 11 Video S10. Feeding of Syssomonas multiformis on bacteria.

Solitary cells of Syssomonas can partially merge and form temporary cell aggregations. They are usually shapeless, observed near the bottom, and consist of about 3–10 flagellated or non-flagellated cells (Fig.  2 s–u, Video 11). Another type of aggregation is formed by only flagellated cells with outwards-directed flagella that can float in the water column and resemble the rosette-like colonies of choanoflagellates (Fig.  2 v, Video 12). Both types of aggregations break up easily, and it seems that the membranes of such aggregated cells are not fused.

Additional file 12 Video S11. Temporary cell aggregations of Syssomonas multiformis.

Additional file 13 Video S12. Floating rosette-like aggregation of Syssomonas multiformis.

However, in rich culture, solitary cells of Syssomonas can sometimes merge completely at the bottom of the Petri dish and form syncytium-like (or pseudoplasmodium) structures (it seems that the nuclei do not merge after cell fusion, Fig.  2 w). The budding of young flagellated daughter cells from such syncytia was observed (Video 13). Such syncytial structures with budding daughter cells have not been observed in other eukaryotes, to our knowledge, but multinucleated structures arising as a result of multiple cell aggregations or fusions of uninuclear cells are also known in Dictyostelia (Eumycetozoa) and Copromyxa (Tubulinea) in the Amoebozoa (sister group of Opisthokonta), as well as in other protists, such as Acrasidae in the Excavata, Sorogena in the Alveolata, Sorodiplophrys in the Stramenopiles, and Guttulinopsis in the Rhizaria [ 43 , 44 , 45 , 46 , 47 ]. Within the opisthokonts, aggregation of amoeboid cells is known in the sorocarpic species Fonticula alba (Holomycota) [ 48 ]. Transition from filopodial to aggregative stage was also observed in the filasterean Capsaspora owczarzaki [ 49 ].

Additional file 14 Video S13. Syncytium-like structures and budding of young flagellated daughter cells in Syssomonas multiformis.

We should also note that a syncytium is not an unusual cell structure in many fungi and animals; for example, most of the cytoplasm of glass sponges (Hexactinellida), the teguments of flatworms, and the skeletal muscles and the placenta of mammals [ 50 , 51 ] have a syncytial structure.

In Syssomonas , the processes of cells merging attract (again, likely by chemical signaling) many other cells of Syssomonas , which actively swim near aggregates or syncytium-like structures and try to attach to them. Some of these cells succeed to merge, and the aggregates grow.

All aggregations and syncytial-like structures appear to form by mergers of existing cells in the culture, as opposed to cell division (although, strictly speaking, all cells in the clonal culture are offspring of a single cell of Syssomonas ).

All of the above-described life forms and cellular changes do not represent well-defined phases of the life cycle of Syssomonas , but rather embody temporary transitions of cells in culture which are reversible.

Syssomonas grows at room temperature (22 °C) and can survive at temperatures from + 5 to 36 °C. At high temperature (30–35 °C), the prey cells (bodonids) in culture become immobile and roundish; Syssomonas actively feeds on these easily accessible cells, multiplies, and produces high biomass. In the absence of live eukaryotic prey, increasing the incubation temperature does not lead to an increase in cell numbers. The cells grow at pH values from 6 to 11. Agitation of culture does not lead to the formation of cell aggregates as was observed in the filasterean Capsaspora [ 49 ].

Syssomonas multiformis cell ultrastructure

The cell is naked and surrounded by the plasmalemma. The naked flagellum ends in a short, narrowed tip—the acroneme (Figs.  2 x and 3 d). A single spiral or other additional elements (e.g., a central filament typical for choanoflagellates) in the transition zone of the flagellum were not observed (Fig.  3 b, c). The flagellar axoneme (the central strand of flagellum) has an ordinary structure (9 + 2) in section (not shown). The flagellum can be retracted into the cell (Fig.  3 e). A cone-shaped rise at the cell surface around the flagellum base was observed (Fig.  3 b, c). The flagellar transition zone contains a transversal plate which is located above the cell surface (Fig.  3 b).

figure 3

General view and flagellar structure of Syssomonas multiformis (transmission electron microscopy, TEM). a General view of the cell section with flagellum and large anterior food vacuole (note that ruffled cell outline could be an artifact of fixation). b , c Arrangement of flagellum and basal bodies and their connection. d Twisting of the acronematic flagellum around the cell with nucleus and nucleolus. e Retracted flagellum axoneme inside the cell. f Basal body of the flagellum and nearest structures. ac, acroneme; ad, arc-like dense structure; an, flagellum axoneme; ds, dense spot; fb, fibril; fbb, flagellar basal body; fl, flagellum; fv, food vacuole; Ga, Golgi apparatus; mct, microtubule; mt, mitochondrion; n, nucleus; nfbb, non-flagellar basal body; nu, nucleolus; tp, transversal plate. Scale bars: a 2, b 0.5, c 0.5, d 2, e 0.5, f 0.5 μm

Two basal bodies, one flagellar and one non-flagellar (Fig.  3 a–c), lie approximately at a 45–90° angle to each other (Fig.  3 b, c). The flagellar root system consists of several elements. Arc-like dense material, representing satellites of the kinetosome, is connected with the flagellar basal body and initiates microtubules which run into the cell (Fig.  3 f). Radial fibrils originate from the flagellar basal body (Fig.  4 a–c, g) and resemble transitional fibers. At least two fibrils connect to the basal bodies (Fig.  3 b). It can be seen from serial sections that microtubules originate near both basal bodies (Fig.  4 a–f). Dense (osmiophilic) spots are situated near the basal bodies, and some of them initiate bundles of microtubules (Fig.  4 i, j, l). Microtubules originating from both basal bodies singly or in the form of a fan probably run into the cell (Fig.  3 b, Fig.  4 f–k). One group of contiguous microtubules begins from the dense spot (Fig.  4 l) and goes superficially close to the plasmalemma (Fig.  4 e, f, l).

figure 4

Arrangement of kinetosomes of Syssomonas multiformis . a – f Serial sections (50 nm thickness) of the kinetosomal area, non-flagellar and flagellar basal body with radial fibrils, and submembrane microtubules are visible. g – l Structures nearby the kinetosomes: microtubules run into the cell, submembrane microtubules begin from the dense spot ( l ) and go below plasmalemma. ds, dense spot; fb, fibril; fbb, flagellar basal body; mct, microtubule; nfbb, non-flagellar basal body; rf, radial fibrils; smmt, submembrane microtubules. Scale bars: a – j , l 0.5, k 1 μm

The nucleus is 2.6 μm in diameter, has a central nucleolus, and is situated closer to the posterior part of the cell (Fig.  3 a, d, Fig.  5 h). The Golgi apparatus is of usual structure and is positioned close to the nucleus (Fig.  3 b, Fig.  5 a). The cell contains several mitochondria with lamellar cristae (Fig.  5 b–d). Unusual reticulate or tubular crystal-like structures of unknown nature were observed inside the mitochondria (Fig.  5c, d ). A contractile vacuole is situated at the periphery of the cell and is usually surrounded by small vacuoles (Fig.  5 e).

figure 5

Cell structures and organelles of Syssomonas multiformis . a Golgi apparatus. b – d Mitochondria. e Contractile vacuole. f Food vacuole with remnants of eukaryotic prey ( Parabodo , flagella, and paraxial rods are seen) at the beginning of exocytosis. g Food vacuole containing cyst of Parabodo at the beginning of exocytosis. h Food vacuole containing starch granule. i Exocytosis of food vacuole. j Reserve substance and filopodia. k , l Cysts. ci, crystalloid inclusion; ct, cyst of the prey cell; cv, contractile vacuole; en, cyst envelope; fl, flagellum; fp, filopodia; fv, food vacuole; Ga, Golgi apparatus; mt, mitochondrion; n, nucleus; nu, nucleolus; rs, reserve substance; sg, starch granule. Scale bars: a 2, b 1, c 0.5, d 2, e 2, f 2, g 2, h 2, i 2, j 2, k 2, l 1 μm

A large food vacuole is usually located posteriorly or close to the cell center and contains either remnants of eukaryotic prey, e.g., cells (paraxial flagellar rods are seen) or cysts (fibrous cyst envelope is seen) of Parabodo caudatus , or starch granules (Fig.  3 a, Fig.  5 f–h). Exocytosis occurs on the posterior cell end (Fig.  5 i). Storage compounds are represented by roundish (presumably glycolipid) granules 0.8 μm in diameter (Fig.  5 a, d, j).

Thin filopodia are located on some parts of cell surface (Fig.  5 d, j).

A flagellum or flagellar axoneme, or two kinetosomes, as well as an eccentric nucleus, mitochondria with lamellate cristae and dense matrix, and a food vacuole with remnants of the prey cells, are all visible inside cysts containing dense cytoplasm (Fig.  5 k, l).

Extrusive organelles for prey hunting were not observed in any cell type.

Pigoraptor vietnamica cell ultrastructure

A single, naked flagellum with an acroneme originates from a small lateral groove and directs backward (Fig.  6 h, i). The cell is naked and surrounded by the plasmalemma. Two basal bodies, flagellar and non-flagellar, are located near the nucleus, lie approximately at a 90° angle to each other, and are not connected by visible fibrils (Fig.  7 a, b; Fig.  8 a, b; Fig.  9 a–f). The flagellum axoneme has an ordinary structure (9 + 2) in section (Fig.  7 d, e). A thin central filament, which connects the central pair of microtubules to the transversal plate, was observed (Fig.  7 c, f). The flagellum can be retracted into the cell (Fig.  10 c, see axoneme).The flagellar root system is reduced. Radial fibrils arise from the flagellar basal body (Fig.  8 c). Microtubules pass near the flagellar basal body (Fig.  8 b, Fig.  9 e, f). Serial sections show that the non-flagellar basal body does not initiate the formation of microtubules (Fig.  9 a, b).

figure 7

General view and flagellum structure of Pigoraptor vietnamica , TEM. a Longitudinal cell section. b , c Longitudinal section of flagellum. d – f Transverse flagellum sections in transitional area. cf, central filament; fbb, flagellar basal body; fl, flagellum; n, nucleus; rs, reserve substance. Scale bars: a 1, b 0.5, c 0.5, d – f 0.2 μm

figure 8

Nucleus and arrangement of basal bodies of Pigoraptor vietnamica . a Part of the cell containing nucleus and flagellar basal body. b Non-flagellar basal body. c Flagellar basal body and surrounding structures. fbb, flagellar basal body; mct, microtubule; n, nucleus; nu, nucleolus; rf, radial fibrils; rs, reserve substance. Scale bars: a 0.5, b 0.5, c 0.2 μm

figure 9

Arrangement of two basal bodies of Pigoraptor vietnamica relative to one another. a – f Serial sections of basal bodies. fbb, flagellar basal body; mct, microtubule; n, nucleus; nfbb, non-flagellar basal body. Scale bars: a – f 0.5 μm

figure 10

Sections of nucleus and other cell structures of Pigoraptor vietnamica . a Nucleus. b Golgi apparatus. c Mitochondrion. d , e Nucleus and filopodia. f Food vacuole. g Exocytosis. h Reserve substance. i Dividing symbiotic bacteria. an, flagellar axoneme; bc, bacterium; ec, ectoproct; fp, filopodium; fv, food vacuole; Ga, Golgi apparatus; mt, mitochondrion; n, nucleus; nu, nucleolus; rs, reserve substance; sb, symbiotic bacteria. Scale bars: a 1, b 0.5, c 0.5, d 0.5, e 0.2, f 1, g 1, h 1, i 0.5 μm

The roundish nucleus is about 1.5 μm in diameter, contains a prominent nucleolus (Fig.  7 a, b; Fig.  8 a; Fig.  10 a, d), and is situated closer to the posterior end of the cell. Chromatin granules (clumps) are scattered within the nucleoplasm. The Golgi apparatus is adjacent to the nucleus (Fig.  10 b). Cells contain several mitochondria that possess lamellar cristae (Fig.  10 a, c). Rare thin filopodia have been observed on the cell surface (Fig.  10 d, e). Cells usually contain one large food vacuole (Fig.  10 f), which contains remnants of eukaryotic prey and bacteria. Exocytosis takes place on the anterior cell end (Fig.  10 g). Storage compounds are represented by roundish (presumably glycolipid) granules 0.3–0.4 μm in diameter (Fig.  7 a, Fig.  8 a, Fig.  10 c, h). Some cells contain symbiotic bacteria, which are able to divide in the host cytoplasm (Fig.  10 h, i). A single contractile vacuole is situated close to the cell surface (not shown on cell sections but visualized by SEM, Fig.  6 h).

Pigoraptor chileana cell ultrastructure

The cell is naked and surrounded by the plasmalemma. The nucleus is positioned close to the posterior cell end (Fig.  12 a). The flagellum is naked, and the flagellar axoneme has an ordinary structure (9 + 2) in section (Fig.  12 b–d). The flagellum can be retracted into the cell which is visible in some sections (Fig.  12 a, c). Flagellar and non-flagellar basal bodies are located near the nucleus (Fig.  12 a) and lie approximately at a 60–90° angle to each other (Fig.  13 a–f). The flagellar basal body contains a wheel-shaped structure in the proximal part (Fig.  12 e, f). Single microtubules and microtubule bundles are situated near this basal body (Fig.  12 e–h). Some microtubules arise from dense spots close to the basal body (Fig.  12 h).

figure 12

General view, flagellum, and flagella root system of Pigoraptor chileana . a General view of the cell section. b – d Flagellum. e – h Flagellar basal body and surrounding structures. an, axoneme; ds, dense spot; fbb, flagellar basal body; fv, food vacuole; mct, microtubule; n, nucleus; nfbb, non-flagellar basal body; nu, nucleolus. Scale bars: a 0.5, b 0.2, c 0.2, d 0.5, e 0.5, f 0.5, g 0.5, h 0.5 μm

figure 13

Arrangement of basal bodies and structure of filopodia of Pigoraptor chileana . a – f Serial sections of basal bodies. g – i Filipodia. fbb, flagellar basal body; fl, flagellum; fp, filopodium; mct, microtubule; n, nucleus; nfbb, non-flagellar basal body. Scale bars: a – f 0.2, g – i 0.5 μm

Rare, thin, sometimes branching filopodia may contain superficially microtubule-like profiles (Fig.  13 g–i). The roundish nucleus is about 1.5 μm in diameter and has a central nucleolus (Fig.  12 a). Chromatin granules are scattered within the nucleoplasm . The Golgi apparatus was not observed. The mitochondria contain lamellar cristae and empty space inside (Fig.  14 a, b). Cells usually contain one large food vacuole (Fig.  12 a, Fig.  14 c), which contains remnants of eukaryotic prey and bacteria. Storage compounds are represented by roundish (presumably glycolipid) granules 0.2–0.4 μm in diameter (Fig.  14 c). The single ultrathin section of the dividing cell (possible open orthomitosis) was obtained in metaphase stage (Fig.  14 d).

figure 14

Mitochondria, food vacuole, and nucleus division of Pigoraptor chileana . a , b Mitochondria. c Food vacuole and exocytose. d Nucleus division in metaphase stage. chr, chromosomes; fv, food vacuole; mct, microtubule; mt, mitochondrion; rs, reserve substance. Scale bars: a 0.5, b 0.5, c 0.5, d 1 μm

Key features of novel unicellular opisthokonts and origin of multicellularity in Metazoa

Our understanding of the origin and early evolution of animals has transformed as a result of the study of their most closely related sister groups of unicellular organisms: choanoflagellates, filastereans, and ichthyosporeans [ 16 , 17 , 18 ]. Our discovery of previously unknown unicellular Holozoa from freshwater bottom sediments in Vietnam and Chile provides new material for analysis.


A distinctive feature of all three new species is their feeding on eukaryotic prey of similar or larger size, which is unusual (if not unique) for known unicellular Holozoa. While they consume entire prey cells or only the cytoplasmic contents of eukaryotic cells, which resembles the phagocytotic uptake of the contents of Schistosoma mansoni sporocysts by the filasterean Capsaspora owczarzaki in laboratory conditions [ 52 ], they can also feed on clusters of bacteria, resembling the phagocytic uptake of bacteria by choanoflagellates [ 53 ]. It is particularly noteworthy that the organisms we discovered do not possess extrusive organelles for paralyzing and immobilizing the prey, which is typical for eukaryovorous protists. Our observations show that prior to absorption, they somehow adhere to the surface of the prey cell. Studies on the choanoflagellate Monosiga brevicollis have shown that cadherins, which function as cell-cell adhesion proteins in animals, are located on the microvilli of the feeding collar and colocalize with the actin cytoskeleton [ 54 ]. M. brevicollis is non-colonial, thus suggesting that cadherins participate in prey capture, not colony formation. In addition, studies of the colonial choanoflagellate Salpingoeca rosetta and ichthyosporean Sphaeroforma arctica did not indicate a role of the cadherins in colony or “multicellular” epithelial-like structure formation [ 26 , 55 ], further supporting the notion that cadherins do not play a role in cell-cell adhesion in choanoflagellates and ichthyosporeans, and perhaps also did not in the unicellular ancestor of animals [ 41 ]. Also, non-cadherin-based epithelial structure is present in slime molds [ 56 ].

In the case of the unicellular predators Syssomonas and Pigoraptor , adherence to a large and actively moving prey seems to be crucial for feeding and important for survival. Interestingly, the Syssomonas transcriptome does not include cadherin genes, but it does express C-type lectins (carbohydrate-binding proteins performing various functions in animals, including intercellular interaction, immune response, and apoptosis). The opposite is seen in Pigoraptor , where cadherin domain-containing transcripts were found but no C-type lectins [ 37 ].

The presence of eukaryotrophy as a type of feeding within both filastereans and Pluriformea (although the second representative, Corallochytrium , is an osmotroph) suggests that predation could be or have once been widespread among unicellular relatives of animals, and perhaps that the ancestor of Metazoa was able to feed on prey much larger than bacteria. The “joint feeding” we observed many times in cultures of Syssomonas or Pigoraptor , including the behavior where cells are attracted to the large prey by other predators already feeding on it, is probably mediated by chemical signaling of the initially attached predator cell. The newly arriving cells also adhere to the plasmalemma of the prey, partially merging with each other and sucking out the contents of the large prey cell together (Fig.  2 i, Video 9, 14, 15). The merging of predator cells during feeding is quite unusual and may represent a new factor to consider in the emergence of aggregated multicellularity. In addition, putative chemical signaling to attract other cells of its species is observed during the formation of syncytial structures in these species. In this context, alpha- and beta-integrins and other components of the so-called integrin adhesome, which are responsible for interaction with the extracellular matrix and the transmission of various intercellular signals, were found in the transcriptomes of all three studied species [ 37 ].

Starch breakdown by Syssomonas

An interesting phenomenon was observed in clonal cultures of Syssomonas , where the predator can completely engulf starch granules of the same size as the cell, also mediating the rapid destruction of rice grains into smaller fragments and individual starch crystals (Additional file  1 : Fig. S2). It is possible that Syssomonas secretes hydrolytic enzymes that provide near-membrane extracellular digestion. The appearance of extracellular digestion is considered to be a major step in animal evolution [ 57 ], since it is central to the breakdown of many organic molecules when combined with the direct absorption of nutrient monomers by the gut epithelia using transmembrane transport in animals [ 58 ].

This ability of Syssomonas to feed on starch is likely promoted by the expression of numerous enzymes that are putatively involved in starch breakdown (several putative α-amylases and α-glucosidases, a glycogen debranching enzyme, and a glycogen phosphorylase) (Table  1 ). For example, Syssomonas has five distinct putative α-amylases, one of which was not found in any other Holozoa present in our database (Table  1 ). Similarly, one of the four putative α-glucosidases in S. multiformis seems to be specific to this lineage, and possibly the Filasterea, within the Holozoa. While α-amylases and α-glucosidases are able to hydrolyze α-1,4-linked glycosidic linkages, mobilization of the starch molecule at the α-1,6 glycosidic bonds at branch points requires the activity of debranching enzymes. A possible candidate for the catalysis of this reaction is a conserved glycogen debranching enzyme in S. multiformis , orthologous to the human AGL gene (Table  1 ). Additionally, we identified a transcript for a glycogen phosphorylase (orthologous to the human PYGB , PYGL , and PYGM genes), an enzyme involved in the degradation of large branched glycan polymers.

Our observations also show that in the presence of starch in culture, Syssomonas can form resting stages of unidentified genesis, which tend to adhere to each other and to starch grains.

Structural features

Syssomonas and Pigoraptor both display a broad morphological plasticity: all three species have a flagellar stage, form pseudopodia and cysts, and can form aggregations of several cells. Syssomonas multiformis also has an amoeboid non-flagellar stage. The dominant life form of all three species in culture is the uniflagellar swimming cell. Interestingly, amoeboid and pseudopodial life forms were detected in cultures only after 2 years of cultivation and observation, suggesting they may be extremely rare in nature. Overall, the morphological differences between cells of the same type of the two genera, Syssomonas and Pigoraptor , are few and subtle. Given these genera are distantly related within the tree of Holozoa in all current phylogenomic reconstructions, it is interesting to speculate that they may be the result of morphostasis and by extension retain features resembling those of an ancestral state of extant holozoan lineages. Although other unknown lineages of unicellular holozoans undoubtly exist and their morphology remains to be investigated, we propose such lineages will likely also possess a similar morphological plasticity, with flagellated and pseudopodial stages, and characteristics overall similar to Syssomonas and Pigoraptor . It has been established that single cells of Syssomonas and Pigoraptor can temporarily attach themselves to the substrate (Fig.  1 c, d) and, by beating their flagellum, can create water currents to putatively attract food particles, similar to choanoflagellates and sponge choanocytes (Fig.  2 e, Video 2), although such behavior could be analogous. Choanocytes and choanoflagellates possess, in addition to the flagellum, a collar consisting of cytoplasmic outgrowths reinforced with actin filaments (microvilli) that serve to capture bacterial prey. The thin filopodia that are observed on the cell surface of all three Syssomonas and Pigoraptor species may thus be homologous to collar microvilli. But this will require further evidence in form of homologous proteins in these structures or evidence of their function in Syssomonas and Pigoraptor . While the filopodia of Syssomonas have no obvious structural contents, the outgrowths of Pigoraptor sometimes contain microtubular-like profiles. Cross-sections of these structures were not obtained, but they may represent parallel microfilaments such as recently found in the filopodial arms of Ministeria vibrans [ 25 ]. The organization of the Ministeria filopodial arms resembles the microvilli of choanoflagellates, which have stable bundles of microfilaments at their base. It has been proposed previously that the ancestor of Filozoa (Filasterea + Choanoflagellida + Metazoa) probably had already developed filose tentacles, which have aggregated into a collar in the common ancestor of choanoflagellates/sponges [ 24 ], and that microvilli were present in the common ancestor of Filozoa [ 25 ].

A single, posterior flagellum is the defining characteristic of opisthokonts [ 34 ]. However, the flagellum has not yet been found in all known Opisthokonta lineages. Torruella et al. [ 19 ] have found several proteins corresponding to key components of the flagellum in Corallochytrium and the filose amoeba Ministeria vibrans , which have been considered to lack flagella. These authors have shown that the stalk used by Ministeria to attach to the substrate is a modified flagellum. Recently, morphological observations on another strain of Ministeria vibrans (strain L27 [ 25 ]) revealed that this strain lacks the stalk for substrate attachment, but possesses a typical flagellum that projects forward and beats at attached to the substrate cells (see Fig.  2 h and Video S1 in [ 25 ]). The authors concluded that the filasterean ancestor possessed a flagellum, which was subsequently lost in Capsaspora owczarzaki . In the case of Corallochytrium limacisporum , it was suggested that it has a cryptic flagellate stage in its life cycle [ 19 ], as has been proposed for other eukaryotes ( Aureococcus and Ostreococcus , for instance) based on their genome sequences [ 59 ]. Therefore, the flagellate stage could have been the one morphological trait uniting Corallochytrium and Syssomonas within “Pluriformea.” Interestingly, the ancestor of ichthyosporeans probably also had a flagellum, which is preserved in the Dermocystida (at the stage of zoospores), but was again lost in the Ichthyophonida. However, some membrane-decorated vesicles with short flagellum-like strands were visualized by TEM on cell sections in ichtyophonid Sphaeroforma sirkka and S. napiecek [ 60 ].

The central filament of the flagellum, which connects the central pair of microtubules with the transversal plate in Pigoraptor (Fig.  7 c, f), is also noteworthy, since this character was previously known only in the choanoflagellates [ 61 ] and was considered a unique feature for this lineage. The cone-shaped elevation of the surface membrane around the base of the flagellum in Syssomonas (Fig.  3 b, c) is also typical for choanoflagellates (see Fig.  5 a, b in [ 61 ]).

One interesting ultrastructural peculiarity is the unusual reticulate or tubular structures in Syssomonas mitochondria which were not observed in other eukaryotes we are aware of.

Another noteworthy feature is the presence of symbiotic bacteria in the cells of Pigoraptor vietnamica . Symbiotic bacteria are common in both vertebrate and invertebrate animals including placozoan Trichoplax [ 62 ] and fungi [ 63 ]. Bacterial endosymbionts are also known in unicellular protists from all eukaryotic supergroups and perform a multitude of new biochemical functions in the host [ 64 , 65 ]. But as far as we know, there are only two documented cases of opisthokont protists with prokaryotic symbionts: nucleariid amoebae (sister lineage of fungi) with several groups of Proteobacteria and the choanoflagellate Codosiga balthica , which harbored two different endosymbiotic bacteria inside the cytoplasm [ 66 , 67 ]. Future investigations of the intracellular bacteria in Pigoraptor will be essential to understanding the role of prokaryotic symbionts in the biology and cell functions of unicellular relatives of animals.

Availability of data and materials

Videos of morphology, life cycle, and feeding of novel predatory unicellular relatives of animals are available on figshare [ 105 ].

The raw tree files in newick, colored trees with taxon information (and accession numbers where available) in pdf format, and trimmed alignments have been deposited to the same figshare deposition [ 105 ] doi: .

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We thank Kristina I. Prokina for sample collection in Chile, Dr. Hoan Q. Tran and Tran Duc Dien for assistance with sample collection and trip management in Vietnam, Sergei A. Karpov for help with interpretation of transmission electron microscopy images, Jürgen F.H. Strassert for help with DAPI staining, and Vladimir V. Aleshin and Kirill V. Mikhailov for fruitful discussion on different aspects of the origin of Metazoa. Field work in Vietnam is part of the project “Ecolan 3.2” of the Russian-Vietnam Tropical Centre.

This work was supported by the Russian Science Foundation grant no. 18-14-00239 (cell isolation and culturing, light and electron microscopy, and analyses) and by the Natural Sciences and Engineering Research Council of Canada (grant no. 227301).

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Alexander P. Mylnikov is deceased.

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Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Russia, 152742

Denis V. Tikhonenkov & Alexander P. Mylnikov

Department of Botany, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada

Denis V. Tikhonenkov & Patrick J. Keeling

Ocean EcoSystems Biology Unit, RD3, GEOMAR Helmholtz Centre for Ocean Research Kiel, Duesternbrookerweg 20, 24105, Kiel, Germany

Elisabeth Hehenberger

Penza State University, Penza, Russia, 440026

Anton S. Esaulov & Olga I. Belyakova

Moscow State University, Moscow, Russia, 119991

Yuri A. Mazei

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DVT, APM, EH, and PJK designed the study. DVT and APM isolated and cultured the cells and performed the electron microscopy analysis. DVT, ASE, OIB, and YAM performed the light microscopy analysis. EH performed the starch-degrading enzyme analysis. DVT, EH, and PJK wrote the manuscript with input from all authors. All authors read and approved the final manuscript.

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Correspondence to Denis V. Tikhonenkov or Patrick J. Keeling .

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Additional file 1 : fig. s1..

A-C – Syssomonas multiformis sucks out the cytoplasm of the prey; D – three cells of Syssomonas ( s ) suck out the cytoplasm of the same prey cell together, other Syssomonas cells (arrows) become attracted and swim to the same prey cell; E – unusual flagellated cell of Syssomonas containing vesicular structures; F – cells of Syssomonas with engulfed starch granules swim to the starch crystals druse and hide within the starch crystals. Fig. S2. Rice grain destruction in Petri dish with Pratt medium and presence of the cells of Parabodo caudatus (prey) only (A) and Syssomonas multiformis (B) after 9 days of incubation.

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Tikhonenkov, D.V., Hehenberger, E., Esaulov, A.S. et al. Insights into the origin of metazoan multicellularity from predatory unicellular relatives of animals. BMC Biol 18 , 39 (2020).

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The origin of Metazoa: a transition from temporal to spatial cell differentiation

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The evolution of metazoan extracellular matrix

The modular domain structure of extracellular matrix (ECM) proteins and their genes has allowed extensive exon/domain shuffling during evolution to generate hundreds of ECM proteins. Many of these arose early during metazoan evolution and have been highly conserved ever since. Others have undergone duplication and divergence during evolution, and novel combinations of domains have evolved to generate new ECM proteins, particularly in the vertebrate lineage. The recent sequencing of several genomes has revealed many details of this conservation and evolution of ECM proteins to serve diverse functions in metazoa.

ECM proteins are typically composed of multiple protein domains, and their gene structures were some of the first recognized to have arisen by exon shuffling ( Engel, 1996 ; Patthy, 1999 ; Hohenester and Engel, 2002 ). Biochemical analyses of ECM proteins began in vertebrates. However, as cDNA and genomic sequences became available, it became increasingly evident that many ECM genes such as collagens and laminins are very ancient, and, in the last decade, as genomic sequences were determined for many metazoa, it was recognized that many ECM-encoding genes originated early in metazoan evolution. In particular, genomes of diverse bilaterian organisms (mammals, flies, worms, sea urchins, and ascidians) revealed a common set of ECM proteins shared by all bilateria ( Hynes and Zhao, 2000 ; Whittaker et al., 2006 ; Huxley-Jones et al., 2007 ), which is consistent with the presence of common ECM structures such as basement membranes in all these organisms. Most recently, genomes of nonbilaterian eumetazoa and basal metazoa (see Box 1 and Fig. 1 for a summary of metazoan phylogeny), as well as unicellular relatives of metazoa, have allowed investigation of the origins of this common set of ECM proteins. Furthermore, the increasing amount of genomic information has allowed investigation of the elaboration, diversification, and specialization of ECM proteins in different evolutionary lineages to subserve differing functional roles. In this brief review, I will summarize our current understanding of the diversity and evolution of ECM proteins and attempt to relate them to the evolution of multicellularity and the subsequent evolution of metazoa.

Box 1. Outline of metazoan phylogeny

Any phylogenetic group, such as a phylum, class, genus, or species.

A group of organisms that all share a common ancestor. Also applied to groups of proteins that are related by evolution and divergence.

Multicellular animals.

All metazoan animals apart from Porifera (sponges), Placozoa, and a few other obscure taxa. Within the eumetazoa there are two well defined clades of bilaterally symmetric animals; protostomes and deuterostomes, which are grouped together as Bilateria. Protostomes have two subdivisions: ecdysozoa, which include arthropods and nematodes; and lophotrochozoa, which include mollusks, annelids, flatworms, and others. Deuterostomes include echinoderms, hemichordates, protochordates, and chordates. Eumetazoa also include two additional clades: ctenophores (comb jellies) and cnidaria ( Hydrazoa , sea anemones, jelly fish, and the like), which are traditionally viewed as radially symmetrical (but see Martindale et al., 2002 ; Ball et al., 2004 ). They are sometimes classed together as Radiata or Coelenterata; however, the phylogenetic relationships between ctenophores and cnidaria are not certain and there are, as yet, no complete genomic sequences available for ctenophores. The major eumetazoan phylogenetic divisions are outlined in Fig. 1 ; they all arose before the Cambrian era, >540 million years ago.

All eumetazoa have epithelial layers showing apical-basal polarity and underlain by basement membranes. Bilateria have three germ layers (ectoderm, endoderm, and mesoderm), whereas Radiata have two epithelial layers with limited interstitial cells among and between them. Two metazoan phyla (sometimes grouped as Parazoa) are basal to the eumetazoa and lack any obvious axes of symmetry: the Placozoa, which are flat, bilayered organisms with a very limited number of cell types (approximately four) and no obvious basement membranes or ECM; and Porifera, or sponges, in which most cells lack epithelial organization. Most sponges lack basement membranes, but interstitial ECM is present. The exact evolutionary relationships between the Parazoa and Eumetazoa remain incompletely defined. Most phylogenetic analyses place Placozoa closer than Porifera to the Eumetazoa, as shown in Fig. 1 (but see Schierwater et al., 2009 ), and, as we will see, analyses of the complement of ECM proteins conform with this conclusion.

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Object name is JCB_201109041_Fig1.jpg

Eukaryotic phylogeny and the appearance of ECM proteins. The figure diagrams the major taxonomic divisions of eukaryotes ( Box 1 ), and is based on published phylogenetic analyses (e.g., Philippe et al., 2005 , 2009 ; Shalchian-Tabrizi et al., 2008 ; Pick et al., 2010 ) as well as on analyses of whole genome sequences (see main text). Taxa for which whole genome sequences are available are marked with asterisks. The diagram represents topologies of relationships, and the branch lengths are not intended to reflect accurate evolutionary distances. The relationships of apusomonads and placozoa are not well defined; their placement in the diagram is influenced in part by evidence from analyses of ECM-related genes (see main text). Taxa with the complement of ECM proteins typical of all bilateria are within the light blue hexagon. Taxa highlighted with green or blue text have some homologues of bilaterian ECM proteins or their receptors (see main text), but lack a complete set. The complexity of the known ECM protein sets in these organisms increases from left to right. In contrast, the taxa within the gray trapezoid show no evidence of any credible examples of metazoan ECM proteins. First known appearances of relevant genes/proteins or domains are marked in red. Note that taxa appearing to the right of (i.e., subsequent to) the origin of a given feature may have lost it; examples would be the absence of integrins in choanoflagellates sequenced to date and the absence of fibrillar collagens in Drosophila (in both cases, presumably by gene loss).

Major characteristics and categories of ECM proteins in metazoa

ECMs are, by definition, relatively or completely insoluble assemblies of proteins that form structures such as basement membranes, interstitial matrices, tendons, cartilage, bones, and teeth. The proteins that comprise these various ECMs are frequently large, with multiple characteristic domains specialized for protein interactions necessary for ECM assembly or for the recruitment of cells or other proteins (such as growth factors or cytokines) to the ECM ( Hynes, 2009 ; Hynes and Naba, 2011 ; see Figs. 2 and ​ and3 3 for illustration of domain structures). ECM proteins are frequently cross-linked by enzymatic and nonenzymatic reactions, further contributing to their insolubility. The large size, complexity, and insolubility of ECM proteins has made their analysis challenging, but the availability of complete genome sequences and their inferred complement of encoded proteins has made available reasonably reliable inventories of ECM proteins and allowed comparative analyses among species. These analyses have made clear that all bilaterian taxa share a common set of ECM proteins, with occasional examples of gene loss in certain lineages and many examples of taxon-specific elaborations based on the common set.

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Object name is JCB_201109041_Fig2.jpg

The basement membrane toolkit. The figure shows the domain structures of the core proteins of metazoan basement membranes, based on domain predictions largely using SMART and Pfam domain definitions. All bilaterian clades encode this set of nine proteins in their genomes. There are typically two distinct laminin α subunits and one each of the laminin β and γ subunits. Each subunit has a characteristic domain organization. A laminin protomer is an αβγ trimer (shown at the top left) associated through coiled-coil domains in each subunit (red) and disulfide bonds (not depicted). Type IV collagen is a trimer of two homologous subunits, α1 and α2, usually adjacent in the genome in a head-to-head arrangement with a single promoter between the two genes. Signature pairs of C4 domains lie at the C termini of all type IV collagens, and the collagen segment (fuschia) is interrupted, allowing flexibility. Type IV collagen protomers associate through their N and C termini and through disulfide bonding to form a “chicken wire” network that provides structural strength to the basement membrane. Laminins bind to the collagen network and to nidogen. Perlecan, which is a complex heparan sulfate proteoglycan, is also incorporated into the basement membrane. Two other collagens, types XV and XVIII, are also associated with vertebrate basement membranes, and an orthologue is present in all bilaterian clades. The high degree of conservation of this “toolkit” over more than half a billion years testifies to the essentiality of basement membranes.

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Object name is JCB_201109041_Fig3.jpg

Examples of ECM proteins. (A) Proteins common to all bilaterian clades: agrin, slits, and thrombospondins. The figure shows domain maps of vertebrate proteins. Agrin is involved in synapse organization and slit family members in axonal guidance, although both are also involved in other processes as well. Proteins with similar arrangements of domains are found in all bilaterian clades, although occasionally missing domains, at least according to current gene predictions. Thrombospondins are characterized by the C-terminal set of domains (bracketed), which can be associated with a variety of additional N-terminal domains ( Bentley and Adams, 2010 ; Adams and Lawler, 2011 ). The particular set of domains shown (including TSPN, VWC, and TSP1 repeats) is that of so-called type A thrombospondins. Type A thrombospondins are found only in deuterostomes, but all bilateria encode examples of type B thrombospondins, which lack the TSPN, VWC, and TSP1 domains but often have EGF repeats and sometimes others. (B) Proteins found only in the chordate lineage: tenascin, fibronectin, and VWF. These proteins evolved in the deuterostome lineage (see main text) and exemplify different sorts of domain shuffling. Tenascins are built from ancient domains (EGF, FN3, and FBG) found in many proteins throughout metazoa (and even in lower organisms). However, the particular set of domains in tenascins appears first in Branchiostoma (amphioxus), and the family is expanded in vertebrates. Fibronectins are built from a mixture of ancient domains (FN3), more recent ones (FN2), and chordate-specific domains (FN1). The only true fibronectins are vertebrate-specific and, within that subphylum, highly conserved and essential. VWF is based on an ancient gene structure (mucins) altered by insertion of three VWA domains (bracketed), which incorporate many of the key functions of VWF in hemostasis in vertebrates. All three of these characteristic vertebrate ECM proteins contain RGD motifs (asterisks), which are sites for binding of integrins.

Basement membranes are a characteristic feature of most metazoa, arguably an essential feature of tissue and epithelial organization, providing a locus for adhesion of epithelial cell layers and definition of basal-apical polarity of the cells ( Fahey and Degnan, 2010 ). Studies, initially in vertebrates but more recently in invertebrates, have defined the major protein components of basement membranes ( Fig. 2 ). All basement membranes are composed of a common set of interacting proteins ( Yurchenco, 2011 ): a core network of cross-linked type IV collagen is associated with laminin (a trimer of related α, β, and γ subunits); nidogen, a laminin-binding glycoprotein; and perlecan, a very large and complex heparan sulfate proteoglycan. Strikingly, genes encoding this characteristic set of proteins, long-defined in vertebrates, were found in the genomes of two model protostomes, Caenorhabditis elegans ( Hutter et al., 2000 ) and Drosophila melanogaster ( Hynes and Zhao, 2000 ), when they were sequenced a little over a decade ago. The two homologous minor collagens XV and XVIII had also been observed to be associated with vertebrate basement membranes, although their functions were, and are, less clear. Genes encoding a collagen XV/XVIII orthologue were also found in both the fly and worm genomes. This set of 9–10 genes (2 laminin α, 1 laminin β, 1 laminin γ, 2 type IV collagen subunits, nidogen, perlecan, and 1–2 collagen XV/XVIII homologues; Fig. 2 ) has subsequently shown up in essentially every bilaterian genome sequenced, and we have called it the “basement membrane toolkit” ( Whittaker et al., 2006 ). As is typical of most ECM proteins, the core constituents of basement membrane proteins are built from a set of well-defined protein domains ( Fig. 2 ; Engel, 1996 ; Hohenester and Engel, 2002 ). This highly conserved set of genes has persisted in bilaterian genomes for well over half a billion years. This conservation indicates the essential nature of both this toolkit and the individual domains of its constituent proteins.

The most prevalent and earliest described collagens of vertebrates are those with long uninterrupted series of collagen repeats, typically ∼1,000 amino acids long. They comprise multiple repeats of the tripeptide unit Gly-X-Y, where X is frequently proline and Y is often hydroxyproline. This repeating amino acid structure allows collagen subunits to assemble into triple-helical protomers. A primordial exon (54 bp) encoding exactly six repeating Gly-X-Y tripeptides underwent duplications and modifications (such as deletions and fusions), always retaining the same phasing of introns, so that exons encoding collagen repeat units can be assembled in varying numbers and with other domains. In vertebrates, there are >40 collagen genes encoding diverse collagens ( Ricard-Blum, 2011 ). Mammalian fibrillar collagens (11 genes) have collagen repeats flanked by characteristic noncollagenous domains at the N terminus and COLFI domains at the C terminus. In contrast, type IV collagen genes encode interrupted collagen repeats and a characteristic pair of C-terminal C4 domains ( Fig. 2 ). Other vertebrate collagens have variations on these themes, with diverse arrays of collagen repeats with and without interruptions, interspersed with other ECM domains, such as FN3 and VWA domains ( Ricard-Blum, 2011 ). We will discuss taxon-specific expansions of the collagen family later (see “Taxon-specific elaborations”).

As mentioned previously, type IV collagens have a pre-Cambrian origin. The same is true for fibrillar collagens. The fibrillar collagens assemble into the characteristic striated collagen fibrils of interstitial connective tissue matrices and provide structural strength to those ECMs. As such, they play crucial roles in the integrity of multicellular organisms. Fibrillar collagens are found in sponges, the most primitive metazoan phylum ( Box 1 and Fig. 1 ). Three fibrillar collagen subclades (A, B, and C) arose before the eumetazoan radiation and are widespread, although not universal, in bilateria ( Exposito et al., 2008 , 2010 ; Heino et al., 2009 ). For example, Drosophila lacks any fibrillar collagens, which indicates the loss of the relevant genes in that lineage.

In addition to perlecan, vertebrate genomes encode many other proteoglycans, around three dozen in mammals. Many of these fall into two families ( Merline et al., 2009 ; Schaefer and Schaefer 2010 ): one built of LRR domains and one, known as hyalectans, containing N-terminal IgV and LINK domains and C-terminal EGF-CLEC-CCP domain units, flanking a central section bearing attached glycosaminoglycans. In addition, a small family of proteins named SPOCKs or testicans are related to the ECM glycoprotein SPARC/osteonectin. The testicans, LRR repeat proteoglycans, and hyalectans have been reported only in chordates, and will be discussed later. Two membrane-bound families of proteoglycans—syndecans and glypicans ( Couchman, 2010 )—like perlecan, are found throughout bilateria ( Ozbek et al., 2010 ).

Mammalian genomes encode around 200 further ECM glycoproteins distinct from collagens and proteoglycans ( Hynes and Naba, 2011 ; Naba et al., 2011 ). These ECM glycoproteins are also built from characteristic arrays of domains of >50 different types ( Figs. 2 and ​ and3). 3 ). Like the collagen repeats, these domains are typically encoded by single exons or groups of exons that have allowed shuffling during evolution of the exonic units encoding these domains to build a large variety of ECM proteins. Although the same domains can occur in many different proteins, including both ECM and non-ECM proteins, the domain composition, order, and number are characteristic of individual ECM proteins; that is, they are defined by their domain architectures. This is illustrated in Fig. 2 , where the laminin subunits are clearly related to each other and share domains with nidogen and perlecan. Many of the mammalian and vertebrate ECM proteins are restricted to later-evolving taxa, as we will discuss. However, some of them are widespread in bilateria and a few more examples are shown in Fig. 3 A . These ancient ECM glycoproteins, like those of the basement membrane toolkit ( Fig. 2 ), have been subject to strong selection since the divergence of bilateria >600 million years ago and must have fundamental functions.

Analyses of the evolution of ECM proteins present some challenges. As discussed, ECM proteins are large and complex, with multiple domains, which they share both among themselves and with many other proteins. Domains such as EGF, LRR, FN3, and Ig are widespread in many proteins encoded by metazoan genomes and do not themselves define ECM proteins. Therefore, simple Basic Local Alignment Search Tool (BLAST) or domain searches yield multiple partial homologues for most ECM proteins and can be misleading if not supplemented by analyses of domain composition. It is the patterns or arrangements of domains that are diagnostic of specific ECM proteins. However, because the genes are large, with many exons, they are frequently incomplete or interrupted in current databases of genomes, ESTs, cDNAs, and inferred proteins. Therefore, gene predictions for ECM proteins are significantly harder than for many other genes. Thorough analyses require high-quality genomic or cDNA sequences and, often, further annotation to yield complete and reliable ECM protein predictions. This has only become possible fairly recently for many taxa, but there has been an explosion of genomic information in recent years that has shed light on the origins of ECM proteins and, indeed, of ECM itself. These data have allowed extension of the comparative genomics of ECM beyond bilateria.

The genomes of Nematostella vectensis (starlet sea anemone; Putnam et al., 2007 ) and Hydra magnipapillata ( Chapman et al., 2010 ) reveal that cnidaria share many but not the entire core set of ECM proteins found in bilateria. Some of these proteins had been described previously, based on cDNA cloning, but the completed genomes allow conclusions about what is absent as well as what is present (subject to the qualifications mentioned in the previous paragraph). The comparative analyses by Fahey and Degnan (2010) are particularly informative. They show clearly that N. vectensis encodes good homologues of most of the basement membrane toolkit: laminin (1α, 1β, and 1γ), nidogen, perlecan, and collagens (IV and XV/XVIII). H. magnipapillata also encodes these proteins, and cnidaria encode examples of all three fibrillar collagen clades. They also encode homologues of fibrillins and thrombospondins ( Fig. 3 A ), as well as some other ECM proteins. Also conserved across eumetazoa are cellular receptors for ECM proteins: integrins, which bind many ECM proteins; dystroglycan, which binds laminin and agrin; and CD36, which binds thrombospondins, as well as the membrane proteoglycans, syndecan, and glypican ( Hynes and Zhao, 2000 ; Huhtala et al., 2005 ; Ewan et al., 2005 ; Whittaker et al., 2006 ; Knack et al., 2008 ; Ozbek et al., 2010 ). Therefore, it appears that all eumetazoan genomes encode a common set of ECM proteins, although data for ctenophores are sparse. Individual taxa may lack some of this set but it is clear that the common ancestor of eumetazoa had a reasonably complex repertoire of ECM proteins that has been largely conserved throughout subsequent evolution.

Given this strong conservation of a core set of ECM proteins in all eumetazoa, it is of obvious interest to ask when the genes encoding these proteins arose during evolution and to attempt to correlate their emergence with the acquisition of novel morphological and developmental features. The taxa closest to Metazoa are the Placozoa and the Porifera (sponges). Genomes from these two phyla have recently been completed: the placozoan Trichoplax adhaerens ( Srivastava et al., 2008 ) and the demosponge Amphimedon queenslandica ( Srivastava et al., 2010 ). These genomes have proven quite informative concerning the origins of ECM proteins (see also Fahey and Degnan, 2010 and Ozbek et al., 2010 ). As mentioned earlier, neither organism has any true basement membranes. However, the T. adhaerens genome encodes reasonably good orthologues of type IV collagen (two subunits); laminin α, β and γ subunits; and nidogen and perlecan—essentially the entire basement membrane toolkit apart from type XV/XVIII collagen. This is a surprising result given the reported absence of basement membranes in T. adhaerens and it suggests that T. adhaerens has the ingredients to make a basement membrane. Perhaps there are stages in the T. adhaerens life cycle where basement membranes are assembled or perhaps some other protein is needed for coassembly or as a cell-surface receptor. T. adhaerens does encode potential laminin receptors, including dystroglycan as well as an integrin, although the homology of the latter with subclasses of bilaterian integrins has not yet been explored. It will be of interest to determine the biosynthetic patterns and distributions of the basement membrane proteins and these potential receptors in T. adhaerens.

In contrast, the A. queenslandica genome encodes homologues of all three laminin subunits, albeit with imperfect matches in domain composition ( Fahey and Degnan, 2010 ), but does not encode any of the other proteins of the basement membrane toolkit, which is consistent with the absence of basement membranes in demosponges. The more complete set of basement membrane proteins encoded by T. adhaerens as compared with A. queenslandica is consistent with a closer evolutionary relationship of Placozoa with eumetazoa, as shown in Fig. 1 . However, it should be noted that sponges are diverse, with four distinguishable clades ( Gazave et al., 2010 ), one of which, homoscleromorphs, has been reported to have basement membranes. Indeed, type IV collagen cDNA has been isolated from Pseudocorticium jarrei , a homoscleromorph sponge ( Boute et al., 1996 ). Thus, it remains plausible that some sponges may express the basement membrane toolkit and assemble basement membranes, an obvious topic for future investigations.

The T. adhaerens genome also encodes many other candidate ECM glycoproteins, including a homologue of B-type thrombospondins (although in the current genome assembly, the gene may be fused with another) and a partial match with agrin. The genome includes many genes with known ECM domains in unusual combinations not seen in eumetazoa. Some of these inferred proteins include predicted transmembrane domains and may, in fact, be surface glycoproteins rather than true ECM proteins. In contrast with sponges, there is little evidence for collagens other than type IV in T. adhaerens. However, it is clear that this simple organism with only four known cell types has elaborated large numbers of genes encoding multiple ECM domains. The elaboration of ECM proteins appears further developed in Placozoa than in the sponge species analyzed to date. Further comparative analyses of the T. adhaerens genome and those of sponges should shed further light on the evolution of diverse combinations of extracellular domains in these simple metazoan animals.

There is widespread agreement that choanoflagellates are the closest unicellular relatives of metazoa ( King et al., 2003 , 2008 ). Their characteristic cellular organization, with a collar of actin-based filopodia surrounding a single apical flagellum, is similar to that of choanocytes, the feeding cells of sponges. The complete genome of Monosiga brevicollis ( King et al., 2008 ) and the partial one of Salpingoeca rosetta (Broad Institute Origins of Multicellularity Initiative; ) have revealed that these two choanoflagellates encode several proteins previously considered to be specific to metazoa. These include homologues of the cell–cell adhesion receptor cadherins. The presence of some integrin domains in choanoflagellates might also suggest a role in ECM-mediated adhesion, but there are no true integrins. There are a few genes encoding α integrin repeats, but none of them looks like a fully developed integrin subunit, and there is no evidence for any β subunits ( King et al., 2008 ). Furthermore, neither genome encodes any of the proteins of the basement membrane toolkit. Although there are several proteins that include one or more laminin domains, only one approaches eumetazoan (or Placozoan or sponge) laminin subunits in the complexity of domain organization. However, it lacks some domains and is not a true orthologue, and there is no evidence for laminin αβγ heterotrimers. Also, collagen IV, nidogen, and perlecan all appear to be absent ( King et al., 2008 ; unpublished data). Both choanoflagellate species encode several proteins with collagen repeats and others with COLFI domains, but so far never in the same protein, which indicates that they lack true fibrillar collagens. Both choanoflagellate species do encode a protein with multiple collagen repeats and VWA domains. This is superficially reminiscent of certain vertebrate collagens, but the matches in domain architecture are not at all good (unpublished data).

Thus it appears that choanoflagellates do encode several characteristic ECM domains, but, to date, no true matches with bilaterian ECM proteins have been found ( King et al., 2008 ; Ozbek et al., 2010 ; unpublished data). The unusual VWA collagen may represent an early ECM protein, and it has been suggested that there is a putative fibrillin-like protein encoded in each genome ( Ozbek et al., 2010 ). However, these proposed fibrillin-like proteins consist solely of EGF repeats, lack the TGF-β–binding TB domains of fibrillins, and have transmembrane domains, so their homology with fibrillins is not at all close (unpublished data). Fibrillins and the homologous latent transforming growth factor β-binding proteins (LTBPs) are involved in binding and regulating TGF-β family members but, to date, appear to be eumetazoan in origin ( Robertson et al., 2011 ); placozoa, sponges, and choanoflagellates do not have the TB domain. In fact, M. brevicollis does not actually encode very many ECM-type proteins, and many known ECM domains, which play important roles in conserved bilaterian ECM proteins (compare Figs. 2 and ​ and3), 3 ), appear to be absent from the genome. There are also very few Ig family domains and only one or two copies of several other ECM domains, all of which are, in contrast, extremely prevalent in the T. adhaerens genome (unpublished data).

In conclusion, at this point it is clear that choanoflagellate genomes contain some domains typical of ECM proteins (LamNT, LamG, FN3, VWA, EGF, COLFI, and collagen repeats) but do not appear to have assembled them into the characteristic arrangements of domains seen in metazoan ECM proteins. They also lack many other ECM domains. Most choanoflagellates are unicellular, although S. rosetta does have a colonial phase. The transition to multicellularity therefore seems to have involved both considerable shuffling of preexisting domains ( King et al., 2008 ) as well as evolution of many new ones.

The taxon that contains metazoa and choanoflagellates as well as fungi and several other unicellular relatives is called the opisthokonts. Although fungi contain no credible homologues of ECM proteins (or integrins), several of the other opisthokonts do encode some integrin subunits ( Shalchian-Tabrizi et al., 2008 ; Sebé-Pedrós et al., 2010 ), but so far there have been no reports of ECM proteins. One additional unicellular organism that encodes an integrin β subunit but, so far, no α subunits ( Thecamonas trahens formerly known as Amastigomonas sp .), is an apusomonad ( Sebé-Pedrós et al., 2010 ). This group is of uncertain phylogenetic position, but the shared integrin subunit suggests a relationship with the other unicellular organisms discussed here (compare Fig. 1 ). The presence of integrin homologues of unknown function in these unicellular opisthokonts suggests that integrins may have been lost in the choanoflagellate lineage. Why these unicellular organisms encode integrins is unclear. One possibility is that the integrins function in phagocytosis, as has been suggested for the cadherins in choanoflagellates ( King et al., 2008 ). It will be of considerable interest to see the entire genomes of representatives of these unicellular taxa and to investigate the expression and functions of their integrins and whether or not there are any ECM ligands.

As for most other categories of genes and proteins, there is a steady increase in the complexity of the “matrisome,” the set of proteins contributing to the ECM, as one ascends the tree of life. This increase comprises several different processes. There are notable examples of taxon-specific elaborations of the matrisome, both by duplication and divergence of existing genes as well as by the addition of new domains, including domains not observed at all in the genomes of earlier taxa. In this section, we will consider some examples to illustrate these processes.

Basement membranes.

As discussed earlier, essentially all eumetazoan genomes studied to date encode a set of proteins that make up basement membranes ( Fig. 2 ). This core basement membrane toolkit is found in placozoa, cnidaria, protostomes, and invertebrate deuterostomes with very little change, and appears sufficient for assembly of all the basement membranes of all these organisms. However, vertebrates encode multiple paralogs of most of these proteins; only perlecan remains a unique gene/protein in vertebrate genomes. Mammals have multiple laminin subunits: three pairs of type IV collagen subunits, both collagen XV and collagen XVIII, and two nidogens. This expansion is consistent with the two whole genome duplications that have occurred during the evolution of vertebrates. These paralogs have undergone divergence, both in structure and in patterns of expression. For example, among the duplicated laminin subunits (6α, 3β, and 3γ), some have altered patterns of domains and assemble into trimeric laminin protomers with different shapes ( Yurchenco, 2011 ), and the three type IV collagen gene pairs are differentially expressed during development and in different tissues. Thus, the basement membranes of vertebrate tissues differ from one another and, although we do not yet understand the full implications of this divergence, it is clear that it contributes to the increased complexity of vertebrates.

The collagen gene family offers many examples of taxon-specific divergence to suit particular purposes. Although the three clades of fibrillar collagens have an ancient origin before the divergence of eumetazoa ( Exposito et al., 2008 , 2010 ; Heino et al., 2009 ), individual lineages have expanded the set in different ways. Again, vertebrates provide some prime examples. Each of the three clades has expanded (to give a total of 11 fibrillar collagen genes), and individual members of each clade have become specialized for different functions; one from each clade of collagens is expressed selectively in notochord, cartilage, and bone ( Wada et al., 2006 ). Vertebrate genomes also encode complex collagens with additional ECM domains, such as VWA and FN3. These are not newly developed domains; both are widespread and found in many other genes ( Fig. 3 ; Whittaker and Hynes, 2002 ), and VWA domains do occur in collagen genes of unknown function in H. magnipapillata ( Zhang et al., 2007 ) and, as mentioned, in choanoflagellates. There are several specialized vertebrate collagens incorporating VWA and FN3 domains. These include FACIT collagens, which form side branches on collagen fibrils; and collagens VI and VII, which assemble into short fibrils connecting basement membranes to underlying interstitial ECM in locations such as the skin (for review see Ricard-Blum, 2011 ). The inclusion of these extra domains confers additional interaction capabilities on these collagens, allowing assembly of higher-order structures important for the organisms.

Another example comes from sponges. They encode a family of short-chain collagens (∼120 Gly-X-Y repeats) called spongins, which form exoskeletons (familiar in the form of bath sponges). Spongins have a C-terminal domain distantly related to that of type IV collagens, and appear to have diverged from those basement membrane collagens before the parazoa/metazoa split ( Aouacheria et al., 2006 ). Relatives of spongins are found in other invertebrates, although not in ecdysozoa or vertebrates, the spongin genes presumably having been lost in those lineages. The nematode, C. elegans , is one such ecdysozoan. The genome of this worm instead encodes a large number (>160) of collagen genes ( Hutter et al., 2000 ; Myllyharju and Kivirikko, 2004 ). These encode short collagen chains (∼50 collagen Gly-X-Y repeats), which form the cuticle of the worm, a structure that undergoes remodeling at each larval molt. Different sets of cuticle collagen genes are expressed at different times. This is therefore a nematode-specific expansion of this family of specialized collagens for a taxon-specific ECM function, the cuticle. In contrast, flies (also ecdysozoans), which have a chitin-based exoskeleton, have entirely dispensed with fibrillar collagens and have lost those genes as well.

Deuterostomes and vertebrates.

The structure of collagen genes, built of multiple exons with common codon phasing, allows exon shuffling to generate the diverse collagens discussed above. Similarly, most ECM domains are encoded as exonic units, and that has allowed exon shuffling to develop new genes encoding ECM proteins with novel domain architectures. Examples of the evolution of novel ECM gene and protein architectures are particularly prevalent in the deuterostome lineage leading to vertebrates ( Fig. 4 ). Whereas invertebrates of the protostome and deuterostome clades have similar sets of ECM proteins (aside from occasional taxon-specific expansions as discussed for collagens), vertebrates have a significantly expanded set of ECM proteins encoding diverse and novel ECM proteins. Thus, although deuterostome sea urchins share most of their ECM proteins with the protostome taxa of flies and nematodes, they lack many ECM genes found in vertebrates (see Whittaker et al., 2006 ; Huxley-Jones et al., 2007 ; and Ozbek et al., 2010 for more complete lists). We have already mentioned the large increase in number of collagen genes, both by duplication and divergence (e.g., fibrillar collagens) and by the development of novel domain architectures. Vertebrates also encode several families of proteoglycans (LRR-repeat PGs, hyalectans, and testicans), all of which are absent from the sea urchin genome and from protostomes and cnidaria. The hyalectans include the novel LINK domain, which is not found in protostomes or cnidaria and only twice in sea urchins (and then not in a context like that in hyalectans). This domain binds to hyaluronic acid, a high molecular weight glycosaminoglycan, and allows proteoglycans to assemble into multiprotein aggregates, which is important for the structure of cartilage but also for other ECMs. Many other vertebrate-specific ECM proteins are also probably involved in assembly and function of the major structural ECMs that define vertebrates. However, there are also many novel vertebrate ECM proteins whose functions do not appear obviously linked to cartilage, bones, or teeth.

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Object name is JCB_201109041_Fig4.jpg

Deuterostome phylogeny and elaboration of ECM proteins. The figure diagrams deuterostome evolution according to the same principles outlined in Fig. 1 . The core set of ECM proteins encoded in the genomes of all bilateria is boxed in black. Many of these proteins are also found in cnidaria (see main text). The main taxa of the deuterostome lineage and their relationships as currently understood are indicated with representative animals noted. The first reported appearances of particular proteins are marked in red.

Among the ECM proteins missing from sea urchins as well as other invertebrates are tenascin, fibronectin, and von Willebrand factor (VWF; Whittaker et al., 2006 ). All three proteins comprise novel assemblages of domains in combinations not found in other ECM proteins ( Fig. 3 B ), and they serve to illustrate some issues common to the many other vertebrate-specific ECM proteins. Tenascins include multiple EGF and FN3 domains and a single C-terminal FBG domain. All of these domains are ancient in origin, but the combination is only found in deuterostomes. The sea urchin genome does not encode a tenascin, but those of Branchiostoma floridae (amphioxus, lancelet, cephalochordate; Putnam et al., 2008 ), Ciona intestinalis , and Ciona savignyi (sea squirts, ascidians, tunicates, urochordates; Dehal et al., 2002 ) all do, and all vertebrates encode multiple tenascins ( Tucker and Chiquet-Ehrismann, 2009 ; Chiquet-Ehrismann and Tucker, 2011 ). The different vertebrate tenascins are differentially expressed in various ECMs, including those in the central nervous system (CNS) and during inflammatory and carcinogenic processes, and, given their association with disease states, clearly play important roles in vertebrates ( Chiquet-Ehrismann and Tucker, 2011 ). Fibronectin appeared even later in the deuterostome lineage. In contrast with tenascins, fibronectin does include novel domains; although FN3 domains are ancient in origin, FN2 and FN1 domains are much more recent developments largely confined to chordates. The structure of vertebrate fibronectin is highly conserved in the entire vertebrate subphylum—once assembled, this gene appears to have been under strong selection—and it is essential for life in every species tested. Ascidians do encode a fibronectin-related gene ( Tucker and Chiquet-Ehrismann, 2009 ) with all three fibronectin domains (FN1, -2, and -3), but it lacks key features (domains and motifs) of fibronectin structure and function, has additional domains not found in vertebrate fibronectins, and is best viewed as a proto-fibronectin (unpublished data). VWF is the final vertebrate ECM protein we will discuss in this context. This gene appears conserved in mammals, birds, amphibians, and fish (and presumably other vertebrates). As for fibronectin, there appears to be a proto-VWF in ascidians with similar domains but differentially arranged and including additional domains (unpublished data). VWF is a key protein in hemostasis, being responsible for platelet adhesion under high-shear conditions such as those in arterioles ( Sadler, 2009 ; Bergmeier and Hynes, 2012 ). So, its function would appear to be necessary only in vertebrates. Its domain structure reveals that it is related to mucins, which are found in many invertebrates; the key innovation is the inclusion of a set of three VWA domains that are involved in binding to collagen (as in certain integrins) and to the cell-surface receptor, GPIb/V/IX, on platelets.

These three proteins, as well as the collagens, exemplify the role of domain shuffling and the addition of novel domains to ECM proteins to confer novel functions. For VWF, it is plausible to infer the novel functions from our knowledge of its hemostatic role in mammals. What could be the novel functions that selected for the evolution of tenascins and fibronectin in vertebrates? It could be that they are necessary for the development of vertebrate-specific structural ECMs like cartilage (as for some collagens and proteoglycans), but tenascin and fibronectin do not have obvious roles in such ECMs. Another possibility is neural crest migration, a key feature of vertebrate development; both tenascin C and fibronectin are strongly expressed in neural crest, and fibronectin has been functionally implicated in this migration as well as in condensation of somites ( Hynes 1990 ), another vertebrate synapomorphy. Development and function of an endothelium-lined vasculature and high-pressure circulation is also a specialization of vertebrates. Fibronectin clearly plays a role there, and tenascins are expressed in the vertebrate CNS ( Chiquet-Ehrismann and Tucker, 2011 ), as are many other ECM proteins, including both the pan-eumetazoan proteins, laminins, netrins, slits, and agrin, as well as later-evolving proteins (e.g., reelin and thrombospondin-1) and vertebrate-specific proteins such as proteoglycans and SCO-spondin ( Barros et al., 2011 ).

The recent completion of genome sequences for cnidaria has shown that the common set of ECM proteins already known to be shared by all bilaterian taxa originated before the eumetazoan radiation >600 million years ago, and that many of these proteins have been conserved ever since, which indicates the importance of ECM for metazoan life. Furthermore, genome sequences of basal metazoa have shown that placozoans have many of the same proteins, most notably including the basement membrane toolkit. Sponges have a somewhat simpler repertoire of ECM proteins, and the demosponge for which genomic information is available lacks the basement membrane toolkit. Thus, it appears that, with respect to ECM content, placozoa are closer to eumetazoa than are demosponges, although information on other sponge clades will be of future interest. With the core of eumetazoan ECM proteins as a point of reference, one can ask when these proteins arose in premetazoan organisms and how the repertoire has been expanded in higher-order taxa.

Genomes of the closest unicellular relatives of metazoa—choanoflagellates—encode some domains characteristic of ECM proteins but appear not to have organized them in the combinations and patterns typical of metazoan ECM proteins. Choanoflagellates also lack ECM receptors such as integrins. However, some other unicellular opisthokonts do encode integrins, although no metazoan-type ECM proteins have yet been detected. Therefore, assembly of complex domain structures in ECM proteins seem to have accompanied the acquisition of multicellularity, with placozoa showing extensive elaboration of novel ECM proteins with domain combinations not reported elsewhere. The core set of ECM proteins has shown multiple taxon-specific expansions to meet particular needs. This is particularly evident in the deuterostome lineage leading to chordates and vertebrates. These taxa have greatly expanded the repertoire of ECM proteins both by gene duplication and divergence, and by the evolution of novel ECM proteins incorporating novel arrangements of old domains as well as the occasional addition of new ones. Evolution of this diverse set of ECM proteins has been enabled by their modular protein structures, with individual domains encoded as exonic units allowing shuffling during evolution.


I would like to thank Charlie Whittaker and Sebastian Hoersch of the Swanson Biotechnology Bioinformatics Facility in the Koch Institute and Alexandra Naba of my own laboratory for their collaborations on annotation of ECM proteins.

I would like to thank the Howard Hughes Medical Institute for financial support.

Abbreviations used in this paper:

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  • Published: 27 April 2020

The origin of metazoan larvae

  • Konstantin Khalturin   ORCID: 1  

Nature Ecology & Evolution volume  4 ,  pages 674–675 ( 2020 ) Cite this article

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A comparative analysis of developmental transcriptomes across Metazoa provides a quantitative approach to test scenarios of life-cycle evolution and supports an ancestral adult form with later intercalation of larval stages.

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IMF Working Papers

The riskiness of credit origins and downside risks to economic activity.


Claudio Raddatz ; Dulani Seneviratne ; Jerome Vandenbussche ; Peichu Xie ; Yizhi Xu

Publication Date:

March 29, 2024

Electronic Access:

Free Download . Use the free Adobe Acrobat Reader to view this PDF file

Disclaimer: IMF Working Papers describe research in progress by the author(s) and are published to elicit comments and to encourage debate. The views expressed in IMF Working Papers are those of the author(s) and do not necessarily represent the views of the IMF, its Executive Board, or IMF management.

We construct a country-level indicator capturing the extent to which aggregate bank credit growth originates from banks with a relatively riskier profile, which we label the Riskiness of Credit Origins (RCO). Using bank-level data from 42 countries over more than two decades, we document that RCO variations over time are a feature of the credit cycle. RCO also robustly predicts downside risks to GDP growth even after controlling for aggregate bank credit growth and financial conditions, among other determinants. RCO’s explanatory power comes from its relationship with asset quality, investor and banking sector sentiment, as well as future banking sector resilience. Our findings underscore the importance of bank heterogeneity for theories of the credit cycle and financial stability policy.

Working Paper No. 2024/072



Please address any questions about this title to [email protected]


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