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What did the evolution of multicellular animals look like?
Aspects of this question include:
(1) Are there any living organisms that might be helpful in visualizing "transitional forms" between unicellular organisms and multicellular animals? E.g. I have heard of slime molds as one example. Would love to hear of as many good examples as you can come up with. Not looking for strict hereditary relationships here, and I am fine with plausible but unproven theories. E.g., mudskippers might help a person visualize the evolution of amphibians, even if they aren't in fact closely related to amphibian ancestors.
(2) Do we think that the first animals evolved into sponges, or cnidarians/ctenophoroans, or something else? Did sponges and cnidarians/ctenophoroans evolve multicellularity separately? As this is probably a big topic, feel free to just post a link or two.
(3) For any examples that you can think of for #1, would love to hear of any good citations of articles or books for me to read, but keep in mind that I am not a scientist and not looking for anything extremely technical.
Evolutionary biologists have frequently cited volvocine green algae, which include both unicellular and multicellular members, as a useful model system (your point #1), e.g. see this article
The life cycle of myxobacteria (e.g., Myxococcus coralloides, Myxococcus disciformis, Myxococcus flavescens, Myxococcus fulvus, Myxococcus macrosporus, Myxococcus stipitatus, Myxococcus virescens, Myxococcus xanthus---for references to the original articles describing these species, see Bacteria I. Taxonomy: Genera and Species) includes the formation of fruiting bodies, which is an obligate multicellular process requiring cooperativity between individual bacterial cells (see, e.g., Cao et al., 2015).
What did the evolution of multicellular animals look like?
There is no agreed scenario of evolution of animals. Molecular evidence is ambiguous, early fossils are puzzling in form, and even more in relations.
Molecular phylogenies agree that closest unicellular relatives of animals are choanoflagellates. Most (nearly all) researchers interpret it that our distant ancestor was choanoflagellate-like organism. However there are people that think that choanoflagellates are sponges that "returned" to unicellular life.
Do we think that the first animals evolved into sponges, or cnidarians/ctenophoroans, or something else?
Molecular phylogenies of animals are not clear. I write below few examples of most basal taxa from different articles, all based on molecular evidence. Most basal is on the left.
- ctenophora, porifera, placozoa, cnidaria, bilateria ref
- ctenophora, demospongia, placozoa, homoscleromorpha, cnidaria, bilateria ref
- porifera, ctenophora, placozoa, cnidaria, bilateria ref and ref
- porifera, placozoa, ctenophora, cnidaria, bilateria ref
- porifera*, placozoa, ctenophora, cnidaria, bilateria ref
Porifera are sponges, I marked with porifera* paraphyletic reconstruction, and with porifera monophyletic. Groups homoscleromorpha, and demospongia are subgroups of sponges. As we see the most basal are either sponges or ctenophora (comb jellies), after them usually placozoa are second. Cnidaria with bilateria are least basal.
Most animals except most sponges and placozoa have basement membrane. It is a non cellular collagen matrix under virtually all epithelial tissues (tissue organized as 2d sheet of cells). In sponges only one group, called homoscleromorpha in larval stage was reported to have this membrane. This would suggest that all other animals derive from that group. However other source contradicts that finding: https://www.elsevier.com/books/book-series/advances-in-marine-biology vol 61.
Fossil evidence of most basal animal is even more problematic. There are plenty of early sponge fossils, some of them going even before Marinoan glaciation (about 90My before Cambrian), check this one, however none of the Precambrian fossils are uncontested. This is especially significant as sponge fossils with their robust spicula, and macroscopic sizes, are usually quite well-preserved fossils. Moreover, there are great richness of soft-bodied animals preserved from Ediacaran, but sponges are missing. This suggests that sponges before Cambrian would have to be very small, and without spicula, what is possible, however not very likely.
If precambrian sponges are rejected then sponge-like animals could not be basal animals. Scenario consistent with sponges being most basal is that they either evolved multicellularity independently in Cambrian, or that they evolved in Cambrian from animals of different form that separated from our lineage in Precambrian.
If we reject sponges, then most basal living animals are either placozoa or ctenophora. Placozoa taxon contains just one genus Trichoplax with few species of a very weird animal. Ctenophora (comb-jellies) is a small group with about 100 species of slowly moving marine predators.
Earliest unquestionably animal fossils are from Avalon assemblage from Ediacaran period (about 30My before Cambrian). Figure 1 from here is quite telling. Those earliest fossils seem to be sessile growing on surface of bacterial mat, bound to the ground with gelatin-like substance produced by bacteria. They were likely filter-feeders, possibly with symbiotic algae, just like modern sponges. However their form is very different from sponges; in shape they are more similar to plants or fractals. You can read about those sessile forms here, and here. Later mobile animals evolved, most iconic of them Dickinsonia. Here is an album of many of Ediacaran forms, that could be a good starting point. First mobile animals like Dickinsonia, or Yorgia were probably feeding on biomats. They didn't have mouth, probably digesting bacteria externally under bottom of their body. Only extant animal that feeds that way is placozoan Trichoplax. There is possibility that they are related.
There are plenty of sources to learn about Ediacaran biota, a lot is known about them. However question which one of them, if any, was the most basal animal was not answered till now. In other words you can read a lot about those fossils, but still you will not learn how the first animal looked.
For any examples that you can think of for living organisms that might be helpful in visualizing "transitional forms"
Though probably not the most basal animal, it will be helpful for you to read about Trichoplax. I suggest this, especially figure 7 is nice ilustration.
Choanoflagelates are single-celled eukaryotes that are almost identical to the flagellated cell type in sponges, and are thought to be the sister taxon of Metazoa (Multicellular animals) http://www.ucmp.berkeley.edu/protista/choanos.html
Choanoflagellates and the Origin of Animal Multicellularity
00:00:07.27 So, animals are incredible!
00:00:10.06 Some of them can fly through the air,
00:00:12.09 some of them can swim.
00:00:14.06 Animals have incredibly diverse body plans,
00:00:16.29 for instance this nudibranch.
00:00:19.13 Some of them can pattern their coloration
00:00:22.02 in different ways,
00:00:23.19 like this moth,
00:00:25.09 and even what we might consider simple organisms,
00:00:27.20 like the jellyfish that we see here
00:00:30.06 or a sponge.
00:00:32.22 these are incredibly interesting organisms as well,
00:00:35.11 and all of these animals share in common
00:00:37.16 something important,
00:00:39.02 which is they are composed of thousands and millions of cells
00:00:41.16 and these cells are working together
00:00:43.19 to make the organism work properly.
00:00:46.11 How did this all come to be?
00:00:48.16 Well, that's the focus of the talk
00:00:50.21 that I'm going to give you today.
00:00:52.12 The work in my laboratory has to do
00:00:54.03 with the origin of multicellularity.
00:00:56.12 My name is Nicole King.
00:00:58.01 I'm an investigator with the Howard Hughes Medical Institute
00:01:00.04 and a professor at the University of California at Berkeley,
00:01:02.23 and I'm excited to be here today
00:01:04.15 to tell you about my research.
00:01:06.18 Now, in the closing line of
00:01:09.25 Darwin's Origin of Species,
00:01:11.17 he remarked on endless forms most beautiful,
00:01:13.19 and he was referring to
00:01:16.28 the incredible diversity of body plans that we can see here,
00:01:19.10 and much of his research and thinking
00:01:22.14 had to do with trying to understand,
00:01:24.22 how do we get this diversity of organisms?
00:01:27.00 And there's been a great deal of progress in this regard,
00:01:29.24 largely from the work of embryologists
00:01:33.07 and evolutionary biologists
00:01:34.22 and geneticists working together
00:01:36.13 to try to understand what are the molecular
00:01:38.20 and mechanistic underpinnings
00:01:40.12 of the diversification of animal body plans.
00:01:43.07 But, in fact, there's something else important
00:01:45.14 that we need to keep in mind,
00:01:46.26 and that is that animals are united
00:01:48.21 by their shared ancestry.
00:01:50.08 They all share a common ancestor
00:01:51.27 that you can see here, indicated by this red dot.
00:01:55.07 And, in fact, we know relatively little
00:01:57.03 about the nature of that organism.
00:01:59.10 We don't know much about what its biology was like
00:02:01.21 or what its genome contained,
00:02:05.00 and we know even less
00:02:06.25 about the organisms from which it evolved,
00:02:09.05 but we can make some reasonable inferences
00:02:12.17 about the prehistory,
00:02:14.07 the pre-metazoan history of animals.
00:02:16.15 What we can reasonably infer
00:02:19.01 is that there some important evolutionary processes
00:02:22.05 that predate animal origins,
00:02:24.16 and these have to do with the origin of multicellularity,
00:02:27.22 the transition from a single-celled lifestyle
00:02:30.09 to one with organisms that were capable
00:02:33.07 of being multicellular
00:02:35.15 and coordinating the activities
00:02:37.07 of their different cells.
00:02:38.28 So, what I'd like to talk to you about today,
00:02:40.23 in this first part of my talk,
00:02:42.29 is what are the big questions that we want to ask
00:02:45.23 when we want to think about reconstructing animal origins,
00:02:49.25 and I think there are some discrete questions
00:02:51.27 that we can start to address.
00:02:53.26 The first is:
00:02:55.24 how did genome evolution contribute to animal origins?
00:02:59.07 It's clearly the case
00:03:01.21 that different groups of organisms on the tree of life
00:03:04.21 have different types of genes in their genomes,
00:03:07.03 and what we're interested in in my lab
00:03:09.10 is trying to understand how changes in gene sequences
00:03:12.17 and the composition of genomes
00:03:15.10 might have contributed to animal origins.
00:03:17.06 In addition, we're interested in understanding
00:03:19.21 how genes that are required for animal development
00:03:22.09 might have functioned before animals first evolved.
00:03:26.13 One of the special things about animals
00:03:28.08 is they have different cell types
00:03:30.15 that are not found in other groups of organisms.
00:03:32.23 These might include neurons
00:03:34.20 or the epithelial cells that make up your skin
00:03:37.01 and the lining of your gut.
00:03:38.28 How did those specialized cell types first evolve?
00:03:42.25 And then, in a topic that
00:03:45.29 we didn't expect to be studying,
00:03:47.20 we find that we're becomingly increasingly interested
00:03:49.24 in how interactions with bacteria
00:03:51.21 might have influenced animal origins,
00:03:53.19 and I'm gonna come back to that topic in part two.
00:03:56.27 And, of course, in the background of all of this
00:04:01.01 we're interested in understanding
00:04:03.23 the evolutionary implications of multicellularity,
00:04:05.21 and this is a topic of research that is ongoing.
00:04:12.00 Now, historically,
00:04:14.12 we've been very interested.
00:04:16.15 evolutionary biologists
00:04:18.29 have approached the evolution of animals
00:04:21.00 and the diversification of body plans
00:04:23.01 by really focusing on the fossil record,
00:04:25.12 and fossils have been great.
00:04:26.26 They tell us about the age of certain animal groups
00:04:29.03 and they can tell us about the shapes
00:04:31.07 of some of their body parts.
00:04:33.24 So, for instance, these beautiful star-shaped objects
00:04:36.21 are actually spicules from an ancient sponge,
00:04:39.27 this is a hypothesized embryo
00:04:43.08 that has recently been recovered,
00:04:45.20 and here we have a fossil of a coral,
00:04:47.17 and so we can see the fossil remnants of animals,
00:04:50.24 but it really doesn't tell us the whole story.
00:04:52.20 It doesn't tell us how animals came to be
00:04:55.02 and it doesn't tell us how cells
00:04:57.23 in those ancient organisms actually interacted.
00:05:01.18 To really understand animal origins,
00:05:03.15 I think we need to be focusing
00:05:05.20 on comparing living organisms,
00:05:07.13 and so what I'm going to tell you in this first part
00:05:09.22 of my iBio seminar
00:05:11.17 is about an unusual group of organisms
00:05:13.15 called the choanoflagellates
00:05:14.29 and how they can give us special insight into animal origins.
00:05:18.21 And then I'm going to tell you about
00:05:20.23 how the study of choanoflagellates,
00:05:22.06 and comparisons with animals,
00:05:24.10 have started to reveal the genome composition
00:05:26.11 and biology of the first animals,
00:05:28.15 organisms that lived and died
00:05:31.04 almost a billion years ago,
00:05:32.27 and yet by studying living organisms
00:05:34.12 we can learn about how they functioned.
00:05:37.01 In Part II, which I will come to later,
00:05:39.08 I will tell you that some choanoflagellates
00:05:41.22 can transition between being single-celled
00:05:43.26 and multi-celled,
00:05:45.16 and I'll tell you about how that happens,
00:05:47.22 and in addition I will tell you
00:05:50.03 about how that's regulated.
00:05:51.26 There are intrinsic and extrinsic influences on this process.
00:05:54.21 But, let me get back to this big question:
00:05:57.21 how did animals first evolve?
00:06:00.01 And in particular, can we focus on multicellularity?
00:06:03.14 So, let me remind you that
00:06:06.05 animals are not the only multicellular organisms out there.
00:06:08.29 We are only one of many
00:06:11.23 diverse multicellular forms out there.
00:06:13.09 So, of course, we have representative animals,
00:06:15.21 but plants are a remarkable example of multicellularity.
00:06:18.28 There are also green algae,
00:06:20.28 the fungi,
00:06:22.11 and, on the far side of the slide,
00:06:24.12 the slime molds,
00:06:25.23 and there are, you know,
00:06:27.13 probably 20 different lineages that are multicellular,
00:06:30.01 and so each of these lineages
00:06:34.01 has an interesting history in terms of multicellularity
00:06:37.04 and you might think that we could compare
00:06:39.01 among all of these lineages
00:06:40.17 and learn something about the origins of multicellularity,
00:06:43.21 but it turns out that that's not possible,
00:06:46.00 and that's not possible for a few reasons.
00:06:48.05 One is that if we look at the cell biology
00:06:50.14 of each of these different multicellular lineages,
00:06:53.01 we see that their multicellularity
00:06:55.08 is set up differently.
00:06:56.24 So, some organisms like plants and green algae,
00:06:59.19 they have stiff cell walls
00:07:02.20 that mean that a cell is born where it's going to die,
00:07:06.18 they're not able to move around relative to each other,
00:07:08.29 whereas animals and the slime mold
00:07:12.12 don't have a cell wall and the cells are able to move around
00:07:15.09 and resculpt,
00:07:17.05 and that changes their ability to form complex structures
00:07:20.02 and interact with their environment.
00:07:22.17 So, these differences as the cell biological level
00:07:24.23 also help us to understand
00:07:27.03 something that we see at the level of genomes.
00:07:29.15 Now, you might imagine that you could
00:07:32.20 compare the genomes of different multicellular organisms,
00:07:34.26 and the genes they share in common,
00:07:36.22 which are indicated here at the intersection,
00:07:38.17 that these would be the ones involved in multicellularity,
00:07:40.20 but in fact that is not the case.
00:07:42.20 The genes found at the intersection
00:07:44.13 of comparing the genomes
00:07:46.09 of these different multicellular lineages
00:07:48.23 are the genes that are involved
00:07:51.18 in basic housekeeping functions in the cell:
00:07:53.26 DNA replication, translation, repair, etc.
00:07:58.09 The genes that are involved
00:07:59.05 in mediating interactions between cells
00:08:02.04 are actually the genes that are unique
00:08:04.18 within each of these genomes.
00:08:06.11 Why? Why is that the case?
00:08:08.22 Well, to explain why the genes for multicellularity
00:08:12.14 are different in each of these lineages,
00:08:14.07 I need to introduce you to a simple tree.
00:08:17.02 So, what I'm showing you here is
00:08:20.25 a very simple tree depicting the relationships
00:08:23.15 between three different major multicellular lineages
00:08:25.24 -- the animals,
00:08:27.11 which are also called the metazoa,
00:08:29.03 the fungi, which include the mushrooms,
00:08:31.23 and the plants --
00:08:34.04 and what I hope you can see is that
00:08:36.03 there are a few surprises in looking at this tree.
00:08:38.16 First of all, it's only recently been appreciated
00:08:40.27 that the closest living multicellular relatives of animals
00:08:44.23 are the fungi,
00:08:46.15 but the other thing I need to tell you
00:08:49.01 is that, by looking at diverse organisms,
00:08:51.28 it has now become clear that multicellularity
00:08:54.14 evolved independently in each of these lineages,
00:08:57.12 and that's depicted by these yellow bars.
00:08:59.22 So we think, actually,
00:09:01.15 that the last common ancestor,
00:09:03.13 for instance, of the animals and the fungi,
00:09:05.16 was not multicellular.
00:09:07.09 In fact, it was unicellular.
00:09:09.20 So, we have a rich history
00:09:11.19 of unicellular life
00:09:14.00 before the origin of these different multicellular lineages,
00:09:16.19 and then these lineages evolved multicellularity
00:09:21.06 Well, what are we going to do?
00:09:22.28 How do we operate within this framework
00:09:24.24 to learn anything about the nature
00:09:27.06 of the organisms from which animals first evolved?
00:09:30.05 Well, the way we do that
00:09:31.24 is to try to find lineages
00:09:34.00 between this long-extinct unicellular ancestor
00:09:38.06 and the origin of multicellularity, here,
00:09:40.11 in the animals.
00:09:42.04 And we do that using a group of organisms
00:09:44.12 that sits in this sweet spot on the phylogenetic tree,
00:09:47.10 and these are the choanoflagellates.
00:09:49.22 So, choanoflagellates were discovered long ago
00:09:53.03 and I'm going to tell you
00:09:54.09 quite a bit about them in the next few slides,
00:09:56.04 but I want to say that the evidence for them sitting
00:09:59.19 on this spot on the tree, as the sister group of animals, or metazoa,
00:10:03.13 is that they have shared cell biological features with animals
00:10:07.02 that are not seen anywhere else in diversity.
00:10:09.21 Phylogenetic analyses of diverse genes
00:10:12.12 have indicated that choanoflagellates
00:10:14.20 are the closest living relatives of animals,
00:10:16.18 and then I'm going to tell you, very excitingly,
00:10:18.21 that we've sequenced the genomes
00:10:20.25 of diverse choanoflagellates,
00:10:23.18 and when we compare the composition
00:10:26.04 of choanoflagellate genomes to those of animals
00:10:28.15 it's very clear that they share a very close relationship
00:10:32.24 to animals.
00:10:34.29 Let me tell you about these organisms
00:10:36.15 because you may never have heard about them before.
00:10:39.02 Choanoflagellates are single-celled microbial eukaryotes.
00:10:43.11 They're about the size of a yeast cell,
00:10:45.18 and they have some diagnostic features
00:10:49.04 that tell you that you're looking at a choanoflagellate.
00:10:51.24 They have a spherical or ovoid cell body.
00:10:54.10 At the top of the cell,
00:10:56.18 which we call the apical surface of the cell,
00:10:58.12 they have, as you can see in red here,
00:11:00.23 something that's called a collar,
00:11:02.25 and this is actually the source of the name choanoflagellate.
00:11:07.24 The phrase choano- refers to the collar,
00:11:09.29 and the choanoflagellates
00:11:12.19 also have a long flagellum,
00:11:14.06 and you can reasonably think of these cells
00:11:16.08 as resembling sperm cells,
00:11:18.16 with the addition of this collar.
00:11:20.23 Now, choanoflagellates are actually quite diverse.
00:11:23.18 They can come in many different shapes and forms.
00:11:26.19 So, almost all choanoflagellates
00:11:29.08 have a single-celled phase to their life history
00:11:31.23 as you can see here.
00:11:33.28 And, as I said, all choanoflagellates
00:11:36.10 have a flagellum and collar,
00:11:38.03 but some of them can form beautiful colonial structures,
00:11:41.06 such as you can see here.
00:11:42.26 This species can actually
00:11:45.02 fluctuate between colonial and single-celled,
00:11:47.25 and some of them form very ornate extracellular structures,
00:11:52.12 such as this beautiful organism,
00:11:54.24 which can actually biomineralized silica
00:11:57.00 to form a rigid structure that protects the cell
00:11:59.23 and mediates its interactions with other organisms
00:12:02.16 in the open ocean.
00:12:05.26 Why do choanoflagellates
00:12:08.07 have this combination of the flagellum and the collar?
00:12:11.16 What does that do for the choanoflagellate?
00:12:14.07 Well, let me show you.
00:12:16.00 What you're going to see, this is a movie,
00:12:18.04 and the flagellum is undulating back and forth,
00:12:21.15 and what this does is it actually creates fluid flow,
00:12:24.20 indicated by the arrows, that pulls water
00:12:28.14 along the surface of the collar,
00:12:30.22 and the flagellum pushes water out
00:12:33.25 behind the cell,
00:12:35.20 and so this has two consequences.
00:12:37.25 If the choanoflagellate cell is not attached to anything,
00:12:40.28 the movement of flagellum allows it
00:12:43.25 to swim along through the water column,
00:12:46.23 but that fluid flow also has a second important function,
00:12:49.17 and that is it allows the choanoflagellate
00:12:52.01 to pull bacteria up against the surface of the collar,
00:12:55.01 and so you can see in this picture right here
00:12:58.07 a bacterial cell that's been trapped
00:13:00.18 up against the side of the collar,
00:13:02.12 and so choanoflagellates actually have an important
00:13:04.25 and intimate interaction with choanoflagellates that.
00:13:08.16 errr, sorry, with bacteria.
00:13:10.14 that is essential for their viability.
00:13:13.03 Now, choanoflagellates were actually,
00:13:14.28 although they are not widely known,
00:13:17.04 choanoflagellates were actually first discovered
00:13:19.21 a long time ago, in the mid to late 1800s,
00:13:23.21 and people like Ernst Haeckel and William Saville-Kent
00:13:26.18 were obsessed with choanoflagellates.
00:13:28.29 Saville-Kent actually wrote a large monograph
00:13:32.24 called the Manual of Infusoria,
00:13:34.27 and there are many, many plates dedicated to the choanoflagellates,
00:13:38.23 showing their incredible diversity.
00:13:41.07 And, one of the things that excited Saville-Kent
00:13:44.00 about choanoflagellates
00:13:46.03 was that, to his eye,
00:13:48.15 they were completely indistinguishable
00:13:50.25 from another group of cells that he saw
00:13:53.01 in the natural world, and that was in sponges.
00:13:56.03 So, he noticed this similarity
00:13:58.07 between the morphology of choanoflagellates
00:14:00.09 and the morphology of sponges,
00:14:02.23 and from that he made the argument that
00:14:05.04 choanoflagellates and sponges might be closely related,
00:14:07.28 and you can see that similarity, I think,
00:14:10.09 even more clearly in this electron micrograph,
00:14:16.01 in which you can see, again, a choanoflagellate cell
00:14:18.22 with its cell body, its collar, and its flagellum,
00:14:22.06 and here you can see, in SEM,
00:14:25.15 a group of choanocytes,
00:14:27.25 that's the name for the collar cells in sponges,
00:14:30.21 arranged in a circle, and they're doing the same thing.
00:14:33.29 They're actually creating fluid flow to capture bacteria.
00:14:37.26 And, I think the power.
00:14:41.07 or the organization of these choanoflagellates,
00:14:44.00 or sorry choanocytes,
00:14:46.08 into this choanocyte chamber
00:14:48.14 is actually a very nice demonstration
00:14:50.22 of what happens when an organism becomes multicellular.
00:14:54.23 And so, an example of this,
00:14:56.18 I'm going to just show you in this movie,
00:14:59.01 is that the coordinated action of collar cells in sponges
00:15:03.14 allows for tremendous fluid flow.
00:15:06.19 And so, what you're going to see in this movie,
00:15:09.11 taken by PBS,
00:15:12.26 is that a diver comes in
00:15:15.13 and releases a cloud of fluorescent water
00:15:19.17 just near a sponge,
00:15:21.28 and now watch what the sponge can do with this,
00:15:24.04 just through the movement and activity of choanocytes.
00:15:28.06 So, the diver comes in,
00:15:30.12 this fluorescent dye is released near the sponge,
00:15:33.00 and now as the camera pan back you see that the sponge,
00:15:35.22 which we think of as a very simple organism,
00:15:38.17 is creating coordinated fluid flow
00:15:41.19 and sponges, through this action, are able to
00:15:44.10 capture enormous amounts of bacteria out of the water column.
00:15:50.25 So, choanoflagellates and sponges
00:15:53.20 are using an indistinguishable cell type
00:15:56.13 to capture bacteria out of the water column,
00:15:59.12 and it turns out that cells that resemble
00:16:02.12 choanocytes and choanoflagellates
00:16:04.12 are actually also found in other groups of organisms,
00:16:06.20 including in the form of epithelia and sperm.
00:16:10.04 When we map the distribution
00:16:12.14 of these types of cells, the collar cells,
00:16:14.20 onto a phylogenetic tree,
00:16:16.21 we can infer that because collar cells
00:16:19.27 are widespread within animals
00:16:22.01 and they're also found in all choanoflagellates,
00:16:24.17 then we can reasonably make an inference
00:16:26.24 that choanocytes and collar cells
00:16:29.03 were also present in their last common ancestor.
00:16:31.21 And we can also compare other features
00:16:33.21 of the biology of choanoflagellates and animals
00:16:36.11 within the context of a phylogenetic tree
00:16:38.21 and that brings us to a very exciting point,
00:16:41.00 which is that we can start to make
00:16:43.06 specific inferences about the cell biology
00:16:45.10 and life history of the first animals.
00:16:48.01 So, in this schematic,
00:16:49.21 what I'm showing you is what we now infer
00:16:53.01 to have been the case for the biology of the first animals.
00:16:56.18 We think that it had a simple epithelium,
00:17:00.08 this planar sheet of cells.
00:17:02.24 We think those cells were adhering tightly to each other.
00:17:06.21 We think that some of those cells, at least,
00:17:09.03 were capable of differentiating into collar cells
00:17:11.22 and, importantly, that those cells
00:17:14.02 were actually eating bacteria.
00:17:16.08 So, the first animals were bacterivorous.
00:17:19.13 We think that the first animal
00:17:22.00 also was capable of undergoing apoptosis,
00:17:24.01 or programmed cell death,
00:17:25.29 and that there were different cell types in the first animal,
00:17:28.18 indicative of cell differentiation within the soma.
00:17:33.01 Moreover, it's become clear,
00:17:35.13 by looking at the distribution
00:17:39.16 of different modes of sexual reproduction,
00:17:41.14 sperm and egg in animals,
00:17:44.02 it's become clear that the first animal
00:17:46.26 from which all living animals evolved
00:17:48.26 was capable of undergoing gametogenesis,
00:17:52.05 and that it produced differentiated eggs and sperm
00:17:55.21 and that these merged, in a process of fertilization,
00:17:58.24 to produce a zygote,
00:18:00.29 and then that zygote underwent multiple rounds of cell division
00:18:03.29 and cell differentiation
00:18:05.28 to produce this adult form that I just told you about.
00:18:08.11 So, I think this is an exciting time in which we're starting
00:18:11.19 to see the power of comparative biology,
00:18:13.27 and we can compare the cell biology of choanoflagellates
00:18:16.22 to animals
00:18:18.19 and start to really make specific inferences
00:18:20.16 about the biology of their last common ancestor.
00:18:24.02 Moreover, with the advent of genomic approaches,
00:18:28.02 we can start to learn something
00:18:30.11 about the genome of this organism.
00:18:34.00 Now, choanoflagellates
00:18:36.11 have really been relatively poorly studied
00:18:38.24 by molecular biologists.
00:18:40.18 There was this flurry in the mid-1800s
00:18:43.01 in which people were spending a lot of time
00:18:45.10 looking at and thinking about choanoflagellates
00:18:47.23 and then they were relatively forgotten
00:18:49.23 within the world of molecular biology,
00:18:52.22 and during the molecular biology revolution.
00:18:56.01 And so, one of the first things I did
00:18:58.13 when I started studying choanoflagellates
00:19:00.27 was to collaborate with the Joint Genome Institute
00:19:03.00 and the Broad Institute
00:19:04.17 to sequence the genomes of two different choanoflagellates,
00:19:06.27 Monosiga brevicollis,
00:19:08.23 which so far we have only seen in unicellular form,
00:19:11.14 and S. rosetta, which can be single-celled or colonial.
00:19:15.12 These genomes have a modest number of genes,
00:19:19.05 between 9-12000 genes in their genomes,
00:19:22.03 and we can compare the composition
00:19:24.08 of those genomes with animal genomes
00:19:26.14 to make inferences about the genome of their last common ancestor.
00:19:29.22 In addition, we've recently started sequencing
00:19:34.16 the transcribed and translated genes
00:19:38.23 in the genomes of twenty other
00:19:42.10 additional choanoflagellates that are in culture,
00:19:45.07 and I just want to make the point that
00:19:47.15 there's a lot of diversity in choanoflagellates,
00:19:49.19 and by surveying the genomes
00:19:52.05 of many, many different choanoflagellates
00:19:53.28 we're starting to get an increasingly complete
00:19:56.01 and complex picture
00:19:58.07 of what the genomic landscape of animal origins
00:20:00.18 might have been.
00:20:02.06 Now, I'm not going to tell you about
00:20:04.03 all of the different genes that are found in that ancestral genome,
00:20:06.14 but I do want to summarize some of the exciting findings.
00:20:10.03 When we analyzed these genomes,
00:20:13.03 we particularly focused on genes
00:20:16.06 whose functions are required for
00:20:19.26 animal multicellularity and animal development,
00:20:22.03 and in particular we focused on genes that are required
00:20:24.18 for cells to adhere to each other,
00:20:26.19 genes that are involved in cell signaling,
00:20:28.13 that is, allowing cells to talk to each other
00:20:30.08 and coordinate their functions,
00:20:32.16 genes that are required for gene regulation,
00:20:34.25 which allows one cell to differentiate
00:20:36.19 its function from the other,
00:20:38.25 and genes that are involved in interactions
00:20:41.07 with what's called the extracellular matrix, the ECM,
00:20:44.08 and these are the genes and proteins
00:20:46.16 whose functions allow cells to create this matrix,
00:20:50.27 this structure that provides a landing spot
00:20:54.27 and scaffold for cell-cell interactions.
00:20:57.21 So, we can think about these as being essential functions
00:21:00.00 for animal multicellularity.
00:21:02.17 Many of the genes that are required for these processes
00:21:04.17 in animals
00:21:06.19 had not previously been found in a non-animal before,
00:21:09.14 and now we can ask, if we look at choanoflagellates,
00:21:12.06 what does that tell us about the ancestry of these genes?
00:21:15.15 Are they really animal-specific?
00:21:17.10 Or, might some of these genes
00:21:19.06 have evolved earlier to serve other functions?
00:21:21.24 Now, remember,
00:21:23.04 we have to do this within a phylogenetic framework,
00:21:25.06 and so we're going to ask two different questions.
00:21:29.00 If we are focused on these classes of genes,
00:21:31.14 what fraction of them seem to be restricted to animals?
00:21:35.04 And, what fraction of them
00:21:37.05 are also in choanoflagellates
00:21:38.22 and therefore, we infer,
00:21:40.15 present in their last common ancestor with animals?
00:21:42.25 Some of these genes might have evolved
00:21:45.02 much earlier in the colonial and unicellular
00:21:48.02 progenitors of animals.
00:21:50.11 So, when we do these types of comparisons,
00:21:53.02 and when we did them, it was really quite exciting.
00:21:56.08 I think it helped to motivate
00:21:58.08 a lot of the future study for choanoflagellates,
00:22:00.15 and that's because choanoflagellates
00:22:03.17 turned out to express many different components of the.
00:22:07.29 or, many different genes that are required
00:22:11.12 for the functions that I was just discussing.
00:22:13.29 So, we can find genes that are required
00:22:16.12 for cell signaling in animals,
00:22:18.08 including things like.
00:22:19.27 it's a bit of a chicken soup,
00:22:21.22 but the GPCRs, these are protein coupled receptors,
00:22:24.02 the receptor tyrosine kinases,
00:22:26.09 proto-oncogenes like Src and Csk.
00:22:29.10 We can also find genes whose functions
00:22:32.07 are both necessary and sufficient for allowing cells
00:22:34.11 to stick together.
00:22:35.28 These include the cadherins and C-type lectins.
00:22:38.01 We can find representatives of various transcription factors
00:22:41.18 that are involved in gene regulation,
00:22:43.03 Myc, p53, and Forkhead,
00:22:45.12 and we even find genes that are involved
00:22:48.13 in forming and coordinating the interactions
00:22:52.24 of animals cells with an extracellular matrix.
00:22:55.13 But, remember,
00:22:57.00 we're finding representatives of these genes
00:22:58.21 in non-animals, the choanoflagellates,
00:23:00.27 and so I think an exciting future area of research
00:23:03.08 is to try to figure out
00:23:05.15 how these genes function in choanoflagellates,
00:23:07.25 and try to make inferences
00:23:10.13 about how they might have functioned
00:23:12.07 in our long-ancient progenitors.
00:23:14.17 Now, it was very exciting to find all these animal genes
00:23:17.06 in choanoflagellates,
00:23:18.29 but I think we all need to agree that choanoflagellates
00:23:21.03 are not animals.
00:23:22.21 So, what makes animals different?
00:23:24.20 And, what is exciting is that these genomic interactions.
00:23:28.22 or, sorry, these genomic comparisons,
00:23:30.24 allow us to learn about
00:23:33.27 what types of genes and genomic innovations
00:23:36.12 might have actually contributed to animal origins.
00:23:38.20 And so, when we look at the gene complement of animals
00:23:42.20 and compare it to choanoflagellates
00:23:44.20 we find that there are some genes
00:23:47.02 that thus far have never been found
00:23:49.13 in a non-animal.
00:23:51.06 And so, these are representatives
00:23:53.11 from each of these different
00:23:56.13 groups of processes as well,
00:23:58.17 and they include important genes involved
00:24:00.17 in developmental signaling,
00:24:02.27 one special class of cadherins,
00:24:05.07 the classical cadherins,
00:24:07.06 that are essential for allowing epithelial cells to interact,
00:24:10.19 important and famous developmental patterning genes
00:24:13.21 like the Hox genes,
00:24:15.17 so far have never been found in a non-animal,
00:24:17.19 and very specialized forms of extracellular matrix components,
00:24:21.00 including the Type IV collagens.
00:24:23.19 So, having genome sequences
00:24:26.22 from living organisms
00:24:28.25 has now allowed us to reconstruct,
00:24:30.28 in increasing detail,
00:24:32.06 the genomic landscape of animal origins.
00:24:36.03 So, what I want to say, then,
00:24:39.17 and what I've tried to say in Part I,
00:24:41.26 is that by studying
00:24:45.07 these previously enigmatic organisms,
00:24:47.27 that had been poorly studied,
00:24:50.16 we're starting to grow and develop
00:24:53.01 a new model for animal origins,
00:24:55.12 and we can study these organisms, now,
00:24:58.15 in a modern context to start to learn
00:25:01.07 about animal origins and details.
00:25:03.14 So, what I've told you in this first section
00:25:06.00 is that choanoflagellates, the study of choanoflagellates,
00:25:08.10 has illuminated the cell biology and genome
00:25:11.14 of the progenitors of animals,
00:25:13.19 and told us that those first animals
00:25:16.09 probably ate bacteria and they had collar cells.
00:25:19.02 And, the second important thing that we've learned
00:25:21.05 by studying choanoflagellates
00:25:23.18 is that a remarkable number of genes
00:25:25.10 required for multicellularity in animals
00:25:27.18 actually evolved before the origin of multicellularity,
00:25:31.19 and an exciting future area of research
00:25:33.25 will be to figure out what those genes were doing
00:25:36.23 before they were required for mediating cell-cell interactions.
00:25:41.23 So, that is the completion of Part I,
00:25:44.20 and in Part II
00:25:47.10 I will tell you about a transition to multicellularity
00:25:49.27 that didn't happen hundreds of millions of years ago,
00:25:52.25 but actually happens every day
00:25:55.25 in one particular choanoflagellate,
00:25:58.00 and I'm going to tell you about how that's regulated.
00:26:01.21 Finally, this work wouldn't have been possible
00:26:04.10 without the help of my past and current lab members,
00:26:07.27 and I'm also very grateful to all the collaborators
00:26:10.20 that made all this work possible.
00:26:13.10 Finally, I'm very grateful
00:26:16.07 for the generous support that's come
00:26:18.13 from the National Institutes of Health,
00:26:20.08 the Gordon and Betty Moore Foundation,
00:26:22.02 the Canadian Institute for Advanced Research,
00:26:24.07 and most recently the Howard Hughes Medical Institute.
00:26:26.10 Thank you very much.
How Did Multicellular Life Evolve?
Scientists are discovering ways in which single cells might have evolved traits that entrenched them into group behavior, paving the way for multicellular life. These discoveries could shed light on how complex extraterrestrial life might evolve on alien worlds.
Researchers detailed these findings in the Oct. 24 issue of the journal Science.
The first known single-celled organisms appeared on Earth about 3.5 billion years ago, roughly a billion years after Earth formed. More complex forms of life took longer to evolve, with the first multicellular animals not appearing until about 600 million years ago.
The evolution of multicellular life from simpler, unicellular microbes was a pivotal moment in the history of biology on Earth and has drastically reshaped the planet’s ecology. However, one mystery about multicellular organisms is why cells did not return back to single-celled life.
“Unicellularity is clearly successful — unicellular organisms are much more abundant than multicellular organisms, and have been around for at least an additional 2 billion years,” said lead study author Eric Libby, a mathematical biologist at the Santa Fe Institute in New Mexico. “So what is the advantage to being multicellular and staying that way?”
The answer to this question is usually cooperation, as cells benefitted more from working together than they would from living alone. However, in scenarios of cooperation, there are constantly tempting opportunities “for cells to shirk their duties — that is, cheat,” Libby said.
When social amoeba Dictyostelium discoideum starves, it forms a multicellular body. Credit: Scott Solomon
“As an example, consider an ant colony where only the queen is laying eggs and the workers, who cannot reproduce, must sacrifice themselves for the colony,” Libby said. “What prevents the ant worker from leaving the colony and forming a new colony? Well, obviously the ant worker cannot reproduce, so it cannot start its own colony. But if it got a mutation that enabled it to do that, then this would be a real problem for the colony. This kind of struggle is prevalent in the evolution of multicellularity because the first multicellular organisms were only a mutation away from being strictly unicellular.”
Experiments have shown that a group of microbes that secretes useful molecules that all members of the group can benefit from can grow faster than groups that do not. But within that group, freeloaders that do not expend resources or energy to secrete these molecules grow fastest of all. Another example of cells that grow in a way that harms other members of their groups are cancer cells, which are a potential problem for all multicellular organisms.
Indeed, many primitive multicellular organisms probably experienced both unicellular and multicellular states, providing opportunities to forego a group lifestyle. For example, the bacterium Pseudomonas fluorescens rapidly evolves to generate multicellular mats on surfaces to gain better access to oxygen. However, once a mat has formed, unicellular cheats have an incentive to not produce the glue responsible for mat formation, ultimately leading to the mat’s destruction.
Groups of yeast cells. If key cells die a programmed death, these groups can separate. Credit: E. Libby et al., PLOS Computational Biology
To solve the mystery of how multicellular life persisted, scientists are suggesting what they call “ratcheting mechanisms.” Ratchets are devices that permit motion in just one direction. By analogy, ratcheting mechanisms are traits that provide benefits in a group context but are detrimental to loners, ultimately preventing a reversion to a single-celled state, said Libby and study co-author William Ratcliff at the Georgia Institute of Technology in Atlanta.
In general, the more a trait makes cells in a group mutually reliant, the more it serves as a ratchet. For instance, groups of cells may divide labor so that some cells grow one vital molecule while other cells grow a different essential compound, so these cells do better together than apart, an idea supported by recent experiments with bacteria.
Ratcheting can also explain the symbiosis between ancient microbes that led to symbionts living inside cells, such as the mitochondria and chloroplasts that respectively help their hosts make use of oxygen and sunlight. The single-celled organisms known as Paramecia do poorly when experimentally derived of photosynthetic symbionts, and in turn symbionts typically lose genes that are required for life outside their hosts.
These ratcheting mechanisms can lead to seemingly nonsensical results. For instance, apoptosis, or programmed cell death, is a process by which a cell essentially undergoes suicide. However, experiments show that higher rates of apoptosis can actually have benefits. In large clusters of yeast cells, apoptotic cells act like weak links whose death allows small clumps of yeast cells to break free and go on to spread elsewhere where they might have more room and nutrients to grow.
A fossil of a 600 million-year-old multicellular organism displays unexpected evidence of complexity. Credit: Virginia Tech
“This advantage does not work for single cells, which meant that any cell that abandoned the group would suffer a disadvantage,” Libby said. “This work shows that a cell living in a group can experience a fundamentally different environment than a cell living on its own. The environment can be so different that traits disastrous for a solitary organism, like increased rates of death, can become advantageous for cells in a group.”
When it comes to what these findings mean in the search for alien life, Libby said this research suggests that extraterrestrial behavior might appear odd until one better understands that an organism may be a member of a group.
“Organisms in communities can adopt behaviors that would appear bizarre or counterintuitive without proper consideration of their communal context,” Libby said. “It is essentially a reminder that a puzzle piece is a puzzle until you know how it fits into a larger context.”
Libby and his colleagues plan to identify other ratcheting mechanisms.
“We also have some experiments in the works to calculate the stability provided by some possible ratcheting traits,” Libby said.
The origin of animals: an ancestral reconstruction of the unicellular-to-multicellular transition
How animals evolved from a single-celled ancestor, transitioning from a unicellular lifestyle to a coordinated multicellular entity, remains a fascinating question. Key events in this transition involved the emergence of processes related to cell adhesion, cell–cell communication and gene regulation. To understand how these capacities evolved, we need to reconstruct the features of both the last common multicellular ancestor of animals and the last unicellular ancestor of animals. In this review, we summarize recent advances in the characterization of these ancestors, inferred by comparative genomic analyses between the earliest branching animals and those radiating later, and between animals and their closest unicellular relatives. We also provide an updated hypothesis regarding the transition to animal multicellularity, which was likely gradual and involved the use of gene regulatory mechanisms in the emergence of early developmental and morphogenetic plans. Finally, we discuss some new avenues of research that will complement these studies in the coming years.
1. An overview of animal origins
Animals (Metazoa) are among the major groups of complex multicellular organisms. They rely on a wide variety of differentiated cell types that are spatially organized within physiological systems. At the same time, animal cells perform specialized functions, and thus evolved the capacity to integrate and coordinate them using tightly regulated developmental programmes. However, we still do not know which genetic and mechanistic factors underpinned the origin and evolution of animal multicellularity.
All extant animals living today diversified from a common multicellular ancestor, also known as the last common ancestor (LCA) of animals or the animal LCA (box 1). The animal LCA evolved from a single-celled ancestor more than 600 million years ago (Ma), transitioning from a unicellular ancestral state to complex multicellularity (box 1, figure 1a). By comparing the nature of these two ancestral states—the last unicellular ancestor and the animal LCA—we can uncover the major changes that drove the transition to animal multicellularity and create new, testable hypotheses about the origin of animals. The questions are, then: What were these two animal ancestors like? Was the last unicellular ancestor very simple, or was it quite complex, establishing the foundations for cell differentiation and multicellularity? And what was the animal LCA like? Was it simple, gradually acquiring new developmental capabilities while diversifying into different body plans, or was it already complex, creating the genetic conditions for a successful animal diversification?
Figure 1. Phylogenetic classification of animals and their unicellular relatives. (a) A timeline of different events during early animal evolution. The transition to animal multicellularity, and therefore the origin of the first animals, occurred sometime at the end of the Tonian period, according to molecular clock estimates. The oldest fossil or geological evidence of recognizable animals dates back to the Ediacaran period, with molecular clocks extending the emergence of different animal phyla back to the Cryogenian [15–17]. Time units are million years ago (Ma). (b) Cladogram representing the major clades of the tree of animals and the major groups of unicellular relatives of animals: choanoflagellates, filastereans, ichthyosporeans and corallochytreans/pluriformeans. Coloured nodes indicate different ancestors that we can reconstruct and that are important to understand the transition to animal multicellularity the highlighted internal branch (from the Urchoanozoan to the animal LCA) indicates the animal stem (see box 1 LCA = last common ancestor). Uncertain positions within the animal tree [18–23] and within Holozoa [24–26] are represented with polytomies.
Box 1. Terminology used in this review.
Last common ancestor of animals (animal LCA): The ancestral stage from which all animal phyla living today radiated. Reconstructed from features present in, and shared by, extant animals. Undoubtedly presenting all the features shared by all animals, including complex, coordinated multicellularity. Therefore, it can be classified as an animal.
Last unicellular ancestor of animals: The single-celled ancestor immediately preceding the emergence of the first animal.
Complex multicellularity: An assembly of cells displaying a three-dimensional organization and complex body plans arising from a centralized developmental programme.
Simple multicellularity: An assembly of cells, including filaments, clusters, balls, sheets or mats, that arise via mitotic cell division from a single progenitor or by aggregation of independent cells. Simple multicellularity can be found in prokaryotes and eukaryotes.
First animal: First multicellular ancestor of all extant animals. Partly reconstructed from features shared between early diverging animal lineages (i.e. sponges, ctenophores, placozoans and cnidarians), even if these features are absent from bilaterians. This ancestor lived subsequent to changes that led to the foundations of complex multicellularity in animals and is unlikely to be the same as the animal LCA.
Animal stem: The evolutionary lineage leading to all animals, from the common ancestor of animals and choanoflagellates (Urchoanozoan) to the animal LCA. The subsequent transition from unicellularity to multicellularity occurred along the animal stem lineage.
Urmetazoa: A term used in the literature, that is variously defined as the first animal, the animal LCA, or as an amalgam of the two. To avoid confusion, we do not use this term in this review
Urchoanozoan: The last common ancestor of animals and choanoflagellates. It may or may not be the same as the last unicellular ancestor of animals.
Holozoa: Eukaryotic group comprising animals, choanoflagellates, filastereans, ichthyosporeans and corallochytreans/pluriformeans. The largest clade including Homo sapiens but not Neurospora crassa .
Last common ancestor of Holozoa (Holozoa LCA): The ancestor shared by Metazoa, Choanoflagellatea, Filasterea, Ichthyosporea and Corallochytrea/Pluriformea.
Metacell: In single-cell genomics, a subgroup of homogeneous scRNASeq profiles with only local variance relative to the total dataset, useful for clustering and quantitative gene expression analyses . Ultimately, it can be related to certain cell types, but only upon experimental validation.
Cell type: In its simplest definition, a cell type was defined as a unit of classification to distinguish forms of cells according to different morphologies or phenotypes. Cell types are often related to different germ layers during the formation of the embryo, with nerve and epithelial cells coming from the ectoderm, muscle and blood cells from the mesoderm, and gut cells from the endoderm [3–5]. Whereas vertebrate cell types are often defined by their committed fate and being unable to de-differentiate, cells from early branching animals are known to transdifferentiate and change their cell types . This has led to numerous revisions of the concept at the functional, developmental, and even molecular (gene expression) level. Here, we use the term ‘cell type’ as ‘a classification unit based on the combined observations of a cell morphology and gene expression profile, which is driven by a gene regulatory network and can be repeatedly found within the context of a species'. These cell types can be part of either a spatially or a temporally integrated life cycle.
Aggregative multicellularity: One of the two known mechanisms for evolving multicellularity. Aggregative multicellularity is the result of two or more independent and genetically distinct cells binding to or aggregating with each other. The resulting multicellular structure consists of a heterogeneous population of cells, and it is often formed for the purpose of reproduction and dispersion [7–9]. It has evolved repeatedly across different eukaryotic lineages [10–14].
Clonal multicellularity: One of the two known mechanisms for evolving multicellularity. Clonal multicellularity arises through successive rounds of cell division from a single founder cell (spore or zygote) with incomplete cytokinesis (i.e. division of the cytoplasm of the parental cell into two daughter cells). It has appeared on fewer occasions and is responsible for the best-known radiations of complex multicellular life forms in the tree of life: land plants, fungi and animals.
Recent data from a broad representation of animal species, especially from non-bilaterian animals (sponges, ctenophores, placozoans and cnidarians), and also from unicellular species related to animals, have enabled us to better answer these questions. Their genome content, gene regulatory capabilities and biological features can be compared to reconstruct the cellular foundations of animal evolution and infer the minimal genomic complexity of both the last unicellular ancestor of animals and the animal LCA. Moreover, the advent of sequencing technologies, such as single-cell omics, and the development of genetic tools among unicellular relatives of animals are opening new avenues of research for gene function studies, pointing to an ever-expanding breadth of exciting questions that will complement these inferences from a functional and biological perspective.
In this review, we provide an updated reconstruction of these two evolutionary stages that are key to better understanding the transition to animal multicellularity: 1) the last unicellular ancestor of animals and 2) the animal LCA. We summarize current knowledge on the genetic toolkit, cell-type diversity and ecological context of these ancestors, inferred by comparative genomic analyses between animals with their closest unicellular relatives and between the earliest branching animals and those radiating later. On this basis, we propose an updated hypothesis to explain the transition to animal multicellularity, stressing that animal foundations were laid before the origin of animals and that the gradual complexification of genetic regulatory mechanisms was key to the progressive acquisition of animal axial cell patterning and cell-type identity. Finally, we discuss some of the research areas that we predict will be key to studying animal origins in the coming years.
1.1. Phylogenetic framework of animals and their unicellular relatives
The reconstruction of any evolutionary event relies on a well-supported phylogenetic framework. Thus, to infer the genomic and biological features of the last unicellular ancestor of animals and the animal LCA, the first step is to define the evolutionary relationships between animals and between animals and their closest relatives. The animal tree of life has been deeply studied [18,27–31] (see  for a review), yet a consistent, well-supported phylogeny remains elusive. Some areas of uncertainty remain, especially around the root of Metazoa, due largely to choices made in different phylogenomic analyses, such as the genes selected, taxon sampling used, the assembly of the phylogenomic data matrix or the model of sequence evolution [18,31–33]. The latter can contribute to violations of model assumptions, known as systematic errors (e.g. long-branch attraction artefacts) these may also impact animal tree reconstruction . This lack of consensus on relationships between the earliest branching Metazoa [18,19,31,33,34] has hindered the reconstruction of certain metazoan traits [33,35]. For instance, uncertainty regarding the position of Ctenophora or Porifera as the sister group of all other animals has led to continued debate regarding the origin and evolution of the nervous system [18–23,33,36–40]. Nonetheless, the robustness of other positions in the animal phylogeny allow us to infer many other features of the animal LCA .
Until recently, we knew very little about the tree of life surrounding animals, especially because a well-supported phylogeny relies on the availability of well-annotated genome-scale data and the placement of key taxa. In the last decade, the genome sequencing of several unicellular species has improved the phylogenetic framework of animals and their unicellular relatives [24,25,41–45]. Now we know that animals are closely related to a heterogeneous assembly of unicellular lineages known as unicellular holozoans, which together comprise the Holozoa clade within the eukaryotic group Opisthokonta (figures 1b and 3 box 1) [25,46–51]. The closest unicellular lineage to animals is Choanoflagellatea, a group of more than 250 species of spherical/ovoid heterotrophic flagellates (figure 1b) . Their representatives, the choanoflagellates, have been linked to animals for over a century because of their morphological resemblance to choanocytes, a specific cell type of sponges . This similarity, together with the confirmation from molecular phylogenies of their position as a sister group of animals (figures 1b and 3a,b) [47,48,52,54–59], has historically given rise to hypotheses of animals evolving from a choanoflagellate-like ancestor [60–63]. Molecular phylogenies have confirmed two additional independent lineages within Holozoa: Filasterea and Ichthyosporea (figure 1b). Filasterea is the sister group of Choanoflagellatea and Metazoa, and is so far known to include only five amoeboid and amoeboflagellate species (figures 1b and 3c,d) [25,26,48–50,55,64–71]. Ichthyosporea is the sister group to the rest of Holozoa and is a diverse group of around 40 osmotrophic and saprotrophic protists (figure 1b and 3e,f) [72–82]. Nevertheless, the addition of new species has left some uncertainties in the holozoan phylogeny, which appears to be highly sensitive to taxonomic sampling.
One open question concerns the position of the free-living osmotroph Corallochytrium limacisporum (figures 1b and 3g) . Corallochytrium was previously classified as the sister group to Ichthyosporea, forming a monophyletic group named Teretosporea [24,25]. However, recent analyses including the newly described predatory flagellate Syssomonas multiformis (figure 3h) [26,70] grouped Corallochytrium and Syssomonas together in a new independent clade named Pluriformea, which branches between Filasterea and Ichthyosporea (figure 1b) . A similar case concerns the unresolved position of the recently discovered Tunicaraptor unikontum, another predatory flagellate closely related to animals . Depending on the taxon sampling used, T. unikontum may be sister to filastereans, Filozoa (which includes the filasterean–choanoflagellate–animal group), or it may be the earliest branching holozoan lineage . Environmental surveys have also identified other putative new species falling within or related to different unicellular holozoan clades and even a potential novel lineage [85–93]. This indicates that there is still a substantial hidden diversity within the Holozoa clade, which may affect our reconstruction of the evolution of certain traits along the Holozoa stem. We expect future studies will improve our understanding of unicellular holozoan diversity and clarify the evolutionary relationships of the tree surrounding animals. Nevertheless, despite the previously mentioned conundrums in the Holozoa phylogeny, we can still make inferences based on the current data that we review in the following sections.
2. Reconstruction of the last unicellular ancestor of animals and the last common ancestor of animals
Under the Holozoa phylogenetic framework we can compare the genomic and biological features between unicellular holozoans and animals and reconstruct the two key evolutionary stages from which animals originated: the last unicellular ancestor of animals and the animal LCA (see box 2 for clarification).
Box 2. Was the first animal similar to the animal LCA?
The shared common multicellular ancestor from which all extant animals diversified (the animal LCA) may have not been the same as the first animal (box 1). The first animal was the first multicellular ancestor of all extant animals, and likely gave rise to other lineages that subsequently became extinct prior to the divergence of all modern animal lineages from the animal LCA. Despite research being so far limited to the reconstruction of the animal LCA (and the different unicellular ancestors of animals), we can partly reconstruct the first animal based on our current knowledge of the animal LCA and also from features shared between early diverging animals. For instance, we can infer that the genetic toolkit of the first animal was very rich in genes related to metazoan innovations, ranging from the cellular foundations of epithelial-like layers to neuron-like signalling cells and occurrence of muscle-like contractile cells. Many animal-specific pathways and mechanisms were thus largely complete in the animal LCA (similar to the observations about the cnidarian–bilaterian LCA by Putnam et al. ), suggesting that they were also present in previous ancestral states, possibly even in the first animals (figure 1, box 1). Similarly, based on our inferences of cell-type diversity in the animal LCA, those ancestors prior to the animal LCA likely had the ability to regulate cell differentiation by means of hierarchical TF networks and distal regulation in different cells within the multicellular collective, which translates to a certain degree of spatial cell differentiation possibly present in the first animals. Rather than a drastic bloom of innovations, it is likely that gene expansion, co-option, increased regulatory sophistication and a transition from temporal to spatial gene regulation had a crucial impact on the gradually increasing complexity of the first animals (, and references within).
Currently, phylogenomic studies and analysis for the reconstruction of animal ancestors are limited by the data available for such comparisons. For instance, genomic data on early branching animals is limited to a handful of species, which may or may not be good representatives due to gene loss and rapid evolution. Likewise, our findings would be biased towards the assumption of numerous innovations in the animal lineage unless we include other lineages in our comparisons. For these reasons, studying the origin and evolution of animals requires us to sequence more early branching animal genomes, and just as importantly, to expand our focus to other lineages outside Metazoa.
2.1. Reconstruction of the genomic features of the last unicellular ancestor of animals and the last common ancestor of animals
2.1.1. The genetic toolkit of the last unicellular ancestor of animals
The nature of the last unicellular ancestor of animals can only be reconstructed through comparative studies between animals and their closest extant unicellular relatives, the unicellular holozoans. In the last decade, multiple omics-scale datasets have been generated from a broad representation of unicellular holozoan species. We currently have 11 complete genomes at our disposal [24,25,41–45] and around 30 transcriptomes and proteomes of several species, including representatives of each unicellular holozoan lineage [24–26,42,45,51,84,96–101]. These datasets have allowed us to identify the genomic features that are shared between extant unicellular holozoans and animals, which are thus inferred to be present in their last unicellular common ancestor.
Strikingly, the genomes of extant unicellular holozoans indeed encode a large repertoire of genes that are homologous to genes critical for multicellularity-related functions in animals [24–26,41,42,44,45,97,98,100–104]. These include genes related to cell adhesion, signalling pathways and transcriptional regulation (figure 2a) [95,122,123]. For instance, a rich repertoire of genes related to cell adhesion in animals is found in the genomes of several unicellular holozoans. These include key genes mediating animal cell–cell adhesion, such as cadherin domain-containing proteins or C-type lectins, which are present in choanoflagellates and have a patchy distribution in other holozoans [84,97,105,124,125]. Integrins and associated scaffolding proteins, which mediate animal cell–extracellular matrix adhesion, are present in filastereans, ichthyosporeans, C. limacisporum, S. multiformis and T. unikontum [26,84,97,98,103,126]. Some choanoflagellate species also possess a small subset of the integrin adhesome system [97,98,103]. Moreover, other structural remodelling proteins, such as fascin or Ezrin–Radixin–Moesin and some basal lamina elements (i.e. collagen, laminin and fibronectin), are present in a few unicellular holozoan species [84,98,106]. Choanoflagellates and T. unikontum also encode several domains with affinity to the animal Ig-like domain families [41,84,97]. Altogether, this indicates that several genes from the animal cell adhesion machinery were already present in the last unicellular ancestor of animals (figure 2a).
Figure 2. An inferred gene repertoire of the last unicellular ancestor and the last common ancestor of animals. (a) The reconstruction of the last unicellular ancestor of animals is based on the presence of key metazoan genes in the genomes of unicellular relatives of animals. (b) Inferred gains present in the last common ancestor (LCA) of animals. Yellow indicates genes that originated prior to the emergence of the Holozoa LCA (pre-holozoan origins) green, genes that originated in Holozoa prior to the animal LCA (Holozoa origins) red, animal-specific genes that originated at the root of animals (animal origins). bHLH, basic helix–loop–helix transcription factors BRA, Brachyury CSK, C-terminal Src kinase DRFs, diaphanous-related formins EPS8, epidermal growth factor receptor kinase substrate 8 ERM, Ezrin–Radixin–Moesin proteins GPCRs, G protein-coupled receptors GSK3, glycogen synthase kinase 3 HD, homeodomain MAGUKs, membrane-associated guanylate kinases MAPKs, mitogen-activated protein kinases MEF2, myocyte-specific enhancer factor 2 NF-κB, nuclear factor-κB PI3 K, phosphatidylinositol 3-kinase RFX, regulatory factor X transcription factors RTKs, receptor tyrosine kinases STAT, signal transducer and activator of transcription TALEs, three amino acid loop extensions TFs, transcription factors TGFß, transforming growth factor beta. Data from [24,26,44,45,97,102,105–121].
The genomes of unicellular holozoans also encode homologues of key metazoan intracellular signalling components related to cell–cell communication, immunity and environmental signal/response pathways. These include Notch, Delta, receptor tyrosine kinases and homologues of the animal Toll-like receptor genes (figure 2a) [97,107,125,127–131]. By contrast, several upstream receptors and ligands, such as the spatial signalling genes Hedgehog, Wnt, TGF-β and JAK from the JAK-STAT network, are absent in unicellular holozoans and were likely absent from the last unicellular ancestor of animals (figure 2a) . A similar pattern is observed among some members of the Myc–Max network  and the Hippo signalling pathway . For example, in the latter case, some intracellular components are present in Capsaspora owczarzaki, whereas their metazoan upstream receptors Crumbs and Fat are animal specific [95,108]. Thus, despite several upstream receptors and ligands evolving after the transition to animal multicellularity, the last unicellular ancestor of animals already encoded several components of key metazoan signalling pathways (figure 2a).
A number of transcription factors (TFs) formerly thought to be animal specific are also present in unicellular holozoans. For example, several transcriptional activators of the previously mentioned Hippo signalling pathway and the Myc–Max network are present in some unicellular holozoans [100,108]. A few choanoflagellates and ichthyosporeans, as well as Capsaspora and Corallochytrium, encode LIM Homeobox TFs [24,104]. Several unicellular holozoans also encode homologues of key animal developmental TFs, such as nuclear factor-κB, the p53/63/73 family, RUNX and T-box TFs, such as Brachyury [84,95,102,109,133]. Interestingly, some of these TFs already display the potential to participate in gene regulatory networks (GRNs) well established in Metazoa, such as Brachyury and Myc . This indicates that the last unicellular ancestor of animals already possessed a diverse repertoire of TFs and some of them could potentially have had similar regulatory roles to those found in animals (figure 2a).
Finally, a few unicellular holozoans also exhibit some of the mechanisms that animals use to regulate TF recruitment and gene expression. For example, some species encode genes involved in the control of chromatin accessibility, such as the histone acetyltransferase p300/CBP or many histone post-translational modifiers [24,100]. In Capsaspora, life-stage transitions are associated with changes of chromatin accessibility in only the proximal cis-regulatory regions . In addition, its regulatory genome lacks animal promoter types and signatures of animal enhancers, indicating that Capsaspora cis-regulatory regions are small and proximal . Moreover, the first evidence for post-transcriptional regulation of mRNA via miRNAs has been reported in ichthyosporeans, as some species encode several miRNA genes and homologues of the animal miRNA biogenesis machinery (including Drosha and Pasha) . This indicates a unicellular origin of animal miRNAs and the associated microprocessor complex . Altogether, this suggests that the last unicellular ancestor of animals likely followed a primarily proximal gene regulatory strategy and used few epigenomic mechanisms to control chromatin accessibility, which potentially could also regulate transitions between different life stages.
Thus, these findings indicate that the last unicellular ancestor of animals had a gene-rich and regulatorily complex genome. Some of the genes that were already present in the last unicellular ancestor are important for animal multicellularity-related functions, especially those involved in differential gene regulation (e.g. TFs and signalling pathways), cell adhesion (e.g. cadherins and integrins), cell-type specification, cell cycle and immunity (figure 2a) [34,97,122]. Nevertheless, these inferences are based on a still limited number of currently available genomes, the gene content of which varies considerably between unicellular holozoan species and lineages [41,42,97]. We expect to continue elucidating the genetic toolkit of the last unicellular ancestor of animals as more genomic data are available for more unicellular holozoans in the coming years.
2.1.2. The genetic toolkit of the last common ancestor of animals
The genetic toolkit of the animal LCA can be reconstructed by comparing the genomes of extant animals. However, comparisons between extant animals and unicellular holozoans can also yield valuable insights into reconstructing the genomic features of the animal LCA [33,34,95]. Specifically, those features that are shared between unicellular holozoans and animals, which are traced back to the last unicellular ancestor of animals (see §2.1.1), can also be inferred to be present in the animal LCA (figure 2). For example, cadherins (molecules mediating cell–cell interactions), integrins (mediating cell–extracellular matrix interactions) and some basal lamina elements are shared between unicellular holozoans and most animals and are thus inferred to be present both in the last unicellular ancestor of animals and the animal LCA (figure 2) [20,22,94,135–137]. The same happens with several of the aforementioned components related to key intracellular signalling pathways and TFs (figure 2) [24–26,41,42,44,100,102]. Thus, the animal LCA also possessed key genes related to cell adhesion, signal transduction and transcriptional regulation that evolved in a unicellular context (see §2.1.1, figure 2).
Other features that are well conserved between unicellular holozoans and some animal lineages but absent in some early branching animals can also be traced back to the animal LCA [33,123]. For example, the hedgling cadherin family is inferred to have been present in the last unicellular ancestor of animals, as it is present in the genomes of some choanoflagellates, sponges and cnidarians (figure 2a) [33,41,42,138,139] but is absent in ctenophores, placozoans and bilaterians [33,105,138,139]. Similarly, Toll-like receptors are found in several choanoflagellate species and in nearly all bilaterians and cnidarians but are absent in placozoans and ctenophores and incomplete (i.e. partial domain architectures) in sponges [97,140,141].
Lastly, those features exclusively shared between bilaterian and non-bilaterian animals but absent from unicellular holozoans can be inferred to be present in the animal LCA. These features can be considered key animal innovations and can help identify the set of genes and mechanisms that evolved to support the fundamentals of animal multicellularity. Strikingly, most of these genes are enriched in functions of DNA binding, signalling pathways and innate immunity, as well as cell adhesion and cytoskeletal regulation [34,97,110]. For example, a key animal innovation includes the emergence of several new classes of TFs [102,110,133]. Some of these new TF classes include ETS, SMAD, nuclear receptor, Doublesex and interferon-regulatory factor TFs [110,133]. As importantly, other TF families which expanded along the animal stem (see box 1 for a definition) greatly enhanced the regulatory capabilities of the first animals. These include members of the homeobox TF family, such as Pax, Sox, basic helix–loop–helix and zinc-finger TF families [110,133]. Thus, the foundations of the animal TF toolkit were already integrated in the animal LCA (figure 2b).
Components of key signalling pathways also originated along the animal stem and are inferred to be present in the animal LCA. The first example includes the Wnt signalling pathway, which orchestrates cell–cell communication-mediated cooperation, specialization and polarity during animal development. For instance, frizzled, dishevelled and β- and δ-catenins are inferred to have been present in the animal LCA. Some of these members are indeed expressed among early branching animals, such as in sponge larvae, during cnidarian development, and in several structures of both adult sponges and adult ctenophores [136,141–145]. Others are present only in a few highly derived taxa [146,147]. Another key signalling pathway that evolved at the root of Metazoa includes the developmental TGF-β signalling pathway. Although its core components show a more scattered distribution between lineages and species across the animal tree, it is also inferred to be present in the animal LCA [20,22,141]. Similarly, many other animal signalling pathways which expanded along the animal stem (including those responsible for patterning in bilaterians and innate immunity) are present in early branching animal lineages, despite also being patchily distributed and incomplete in some species [34,141,148]. For instance, there is abundant evidence of innate immunity components occurring in different animal lineages, from Toll-like and Ig receptors to TFs and complement system in sponges and cnidarians [140,141,149–151]. Thus, the animal LCA already contained a rich repertoire of genes related to key animal signalling pathways. These key animal-specific acquisitions, especially related to members of the Wnt and TGF-β signalling pathways, are considered hallmarks of animal development and the acquisition of stable multicellularity [34,97,143,145,152].
Several genes related to cell–cell adhesion and cytoskeletal regulation also emerged at the onset of Metazoa and are inferred to be present in the animal LCA. These include, for example, Dystroglycan, Hemicentin, Fermitin  and the multifunctional Espin gene (figure 2b) [153,154]. Other components related to adherens junctions and cell polarity functions are fairly well conserved in sponges [105,136,155] with some homologues missing in ctenophores .
Finally, those features absent from unicellular holozoans and most non-bilaterian animals are more difficult to infer as present in the animal LCA [33,35]. An example includes the reconstruction of genes critical to the development and physiology of the nervous system [37,39,40,94]. Interestingly, some relevant genes are present in sponges, despite the apparent absence of a nervous system in this group [136,141]. By contrast, ctenophores lack neurotransmitters of the canonical nervous system toolkit present in other animals , leading some authors to hypothesize a parallel evolution of the nervous system in this lineage [39,40]. Nevertheless, some observations indicate that early branching animals could use this ‘simpler’ nervous system to communicate information about their microbiomes [157,158], sharing a common origin of the foundations of the neural and the immune systems at the functional level. A similar scattering pattern is observed with genes related to the development of germ layers. Ctenophores possess an independently derived mesodermal tissue, despite their lack of key bilaterian mesoderm specification genes [20,22,159]. This suggests that the regulatory mechanisms necessary for establishing early fates in layers of cells (such as the muscle cells in the ctenophore-specific mesoderm) were present before the emergence of bilaterians. If we consider ctenophores as the earliest branching animal lineage, then these mechanisms would likely have been present in the animal LCA. Thus, although the origins of the nervous system and of developmental processes remain elusive, the relevant toolkit may have existed in a simpler form in the animal LCA and later evolved into more specialized and complex systems in different lineages during animal diversification.
Overall, the emergence and expansion of key TFs and members of several signalling pathways (such as Wnt and TGF-β), as well as the evolution of elements involved in innate immunity, development and cell adhesion, were critical acquisitions that originated in the animal LCA. These systems may have helped establish the foundations of axial patterning and the acquisition of stable multicellularity in animals.
2.1.3. Major forces shaping the evolution of animal genomes
Which major evolutionary mechanisms shaped the evolution of animal genomes during the transition from unicellularity to multicellularity? Previously, the innovation of some genes key to animal multicellularity was considered the most important driving force for the origin of animals. And indeed, a relatively large number of novel gene families (around 2000), which take part in processes that differentiate animals from other lineages, originated in the animal stem lineage [34,42,44,97,160]. However, only around 2% of these gene families are conserved across animal phyla, indicating that most genes originating in the animal LCA were secondarily lost in extant phyla [34,97]. Some studies estimate that the rate of gene innovation in or immediately prior to the animal LCA was larger than at other points of the animal stem. This suggests a high gene birth rate at the onset of animals which progressively decreased as animals diversified into clades [34,161]. Other studies estimate approximately equal numbers of gains and losses, finding evidence for a burst of gene family expansions in the last unicellular ancestor of animals stem (box 1), and an accelerated churn (i.e. both gains and losses, rather than only gains) of gene families that later evolved along the Metazoa stem [97,162]. In fact, a similar number of gene losses and gains are detected in animals compared to their unicellular relatives, mostly affecting pathways such as amino acid biosynthesis and osmosensing [34,97]. This points to a high turnover of genes and the potential for increased genomic plasticity during the diversification of animals, implying that a remarkable amount of gene losses and gene innovation contributed to shaping the genome composition of animals [34,97,161,163–165].
As discussed in previous sections, analyses of the genomes of extant unicellular holozoans have revealed that they indeed share an unexpectedly large repertoire of multicellularity-related genes with animals these genes are therefore inferred to have been present both in the last unicellular ancestor of animals and in the animal LCA (figure 2) [24–26,41,42,44,45,97,98,100–104]. For instance, approximately one-quarter of the genes shared between animals and their unicellular relatives were already present in the LCA of Opisthokonta or gained at the root of Holozoa (figure 1a and box 1). This suggests that gene co-option of these pre-existing ancestral genes to perform new or specialized functions was an important driving force for animal origins [24,25,41,42,44,45,97,102,125,166].
The changes in gene content mentioned above were facilitated in part by two major genome expansions that contributed to gene family expansion and diversification in animals . Gene family expansion and diversification specifically led to changes in the regulatory capacities of animals [34,97,110,133]. For instance, several classes of TFs also expanded to give rise to new families at the onset of Metazoa (see §2.1.2) [102,110,133]. This expansion of TFs in terms of classes and families triggered the rewiring and integration of some pre-existing core regulatory networks into more complex regulatory programmes during animal evolution [100,133]. In parallel, the evolution of non-coding genes and novel epigenetic mechanisms, such as the appearance of developmental promoters and distal enhancer elements, also increased cis-regulatory complexity in the animal stem lineage . Finally, an additional level of acquired transcriptomic regulatory complexity, including alternative splicing events by exon shuffling, exon skipping or intron retention [24,167], also contributed to novel sources of transcriptomic innovation [24,168–171].
Overall, the evolution of animal genomes from a unicellular ancestor was made possible through a combination of ancient gene families with newly evolved genes in the animal stem lineage, shaped by an unbalanced distribution of gene gain and duplications, rampant gene family losses, gene co-option, gene family expansion and subfunctionalization (especially of several key TFs). The emergence of novel GRNs (especially distal regulatory elements such as enhancers and chromatin-structural modifications) was then a key mechanism for the evolution of animal genomes from a unicellular ancestor [24,25,34,41,42,44,45,97,100,102,110,125,136,161,166,172–174].
2.2. Reconstruction of the biological features of the last unicellular ancestor of animals and last common ancestor of animals
2.2.1. Potential lifestyles of the last unicellular ancestor of animals
Besides analyses of their genomes, comparisons of unicellular holozoans' biological traits can also provide a comprehensive reconstruction of the cellular foundations of the last unicellular ancestor of animals. In recent years, the lifestyles and cell biology of several unicellular holozoan species have been characterized at the transcriptomic and morphological level [24–26,42,45,51,70,84,96–99,175–180]. Strikingly, each unicellular holozoan lineage features unique and distinctive traits that have changed our understanding of the biological nature of the last unicellular ancestor of animals.
For example, choanoflagellates are widely distributed worldwide in a range of primarily aquatic environments [89,181–186]. Despite being mostly unicellular flagellates, some species, such as Salpingoeca rosetta, are able to form simple multicellular structures of stably adherent cells as a result of oriented cell divisions from a single founder cell (figure 3a, box 1) [61,187]. Under certain conditions, S. rosetta flagellate cells are also able to transdifferentiate into amoeboid cells . Other species, such as the recently described Choanoeca flexa, are able to form enormous cup-shaped colonies (figure 3b) . Notably, these colonies reversibly invert their curvature in response to light through a rhodopsin-cGMP pathway, representing a similar behaviour to concerted movement and morphogenesis in animals .
Filastereans are found in freshwater, marine and animal-associated environments [25,26,50,55,64–68,70,71]. Like choanoflagellates, some filasterean species are able to form simple multicellular structures. But, in contrast to the clonal colonies found in choanoflagellates, these are formed through the active aggregation of independent cells (figure 3c,d, box 1) [26,67,98]. The best-described species, Capsaspora owczarzaki, has three different life stages, including an aggregative stage these stages are differentially regulated at the transcriptomic, proteomic and phosphoproteomic levels (figure 3c) [65,98,100,101]. Others, such as Pigoraptor spp., are morphologically very plastic and are able to transition from amoeba and amoeboflagellate stages to cysts and aggregates of cells (figure 3d) [26,70].
Figure 3. Temporally alternating life cycles of unicellular holozoans. Each panel shows life stage transitions of two unicellular holozoan species representing each clade. Arrows indicate directionality of the transition. Loop arrows indicate cell division. Dotted arrows with question marks between stages indicate potential (unconfirmed) life-stage transitions. (a) Life stages of the colonial choanoflagellate Salpingoeca rosetta [176,187]. The asexual life cycle (on the right) includes a single-celled sessile thecate stage (adhered to the substrate), slow and fast swimming single-celled stages, and two types of clonal colonial stages (chain and rosette colonies), in which neighbouring cells are linked by intercellular bridges [188–190]. Starvation triggers the S. rosetta sexual cycle (on the left), in which diploid cells (slow swimmers) undergo meiosis and recombination, and the resulting haploid cells (which can also divide asexually) mate anisogamously [176,178]. (b) Life stages of the colonial choanoflagellate Choanoeca flexa . Light-to-dark transitions induce C. flexa colonies to rapidly and reversibly invert their curvature while maintaining contacts among neighbouring cells between their collar microvilli, alternating between two colony conformations. In response to light, colonies exhibit a relaxed (flagella-in) feeding form. In the absence of light, colonies transition to an inverted (flagella-out) swimming form. (c) Life stages of the filasterean Capsaspora owczarzaki [64,65,98]. In the trophic proliferative (filopodial) stage, cells are amoebae adhered to the substrate, extending several long, thin actin-based filopodia. These amoebas can detach from the substrate and actively aggregate in the aggregative or ‘multicellular’ stage, producing an extracellular matrix that presumably binds them together. In response to crowding or stress, cells from both the amoeba and the aggregative stages can encyst by retracting the filopodia into a cystic or resistance stage. (d) Putative life stages of the filasterean Pigoraptor vietnamica [26,70]. Swimming flagellated cells can form long, thin, sometimes branching filopodia that can attach to the substrate. Flagellated cells can sometimes present wide lobopodia. Flagellated cells can retract the flagellum and become roundish, to either divide into two daughter flagellated cells or transition to a cystic stage. This can, in turn, produce two flagellated daughter cells. Cells can also form easily disintegrating aggregations of cells and feed jointly. The life stages of Pigoraptor chileana are very similar to the ones of P. vietnamica, but P. chileana shows a much reduced capability to produce filopodia and lobopodia (both stages are extremely rare in P. chileana). (e) Life stages of the ichthyosporean Creolimax fragrantissima [45,77]. Single-nucleated amoebae disperse until they settle and encyst. The rounded cell undergoes multiple rounds of synchronous nuclear division (coenocytic division) without cytoplasmic division. Nuclei are later arranged at the periphery of the cell as a large central vacuole grows. Finally, the coenocyte cellularizes and new amoebas are released to start the cycle over again. (f) Life stages of the ichthyosporean Sphaeroforma arctica [99,180]. Single-nucleated cells undergo multiple rounds of synchronous nuclear division (coenocytic division) without cytoplasmic division. Nuclei are later arranged at the periphery of the cell. Finally, the coenocyte cellularizes, releasing a number of daughter cells to start the cycle over again. (g) Life stages of the corallochytrean Corallochytrium limacisporum [22,83,191]. Reproduction in C. limacisporum occurs mainly through binary fission (99% of the cases), during which a binucleated cell divides into two, symmetrical, uninucleate cells. Binucleate cells can form two lobes that can lead to cellular division (forming two monoucleate cells), or can reverse towards spherical cells. At this point (*), cells can transition to coenocytic growth (1% of the cases) and continue dividing their nuclei further forming quadrinucleated cells. Quadrinucleated cells can often form a clover-like shape (similar to bilobed cell), that generates either four mononucleate cells or returns to spherical shape and further divides to an eight, 12 and up to 32 nuclei coenocyte. Coenocytes can release dispersive amoebas to start the cycle over again. (h) Putative life stages of the pluriformean Syssomonas multiformis [26,70]. A swimming flagellated cell can temporarily attach to the substrate through the anterior part of the cell body or move to the bottom and transform to an amoeboflagellate form by producing both wide lobopodia and thin short filopodia. Flagellated cells can lose the flagellum via different modes and transition into an amoeba stage, which produces thin, relatively short filopodia. Both amoeboflagellate and amoeba stages can transition back to the flagellate stage. Amoeboid cells can also encyst by retracting their filopodia and rounding the cell body. Palintomic divisions may occur in the cystic stage to release several flagellated daughter cells. Flagellated cells can partially merge and form temporary shapeless cell aggregates of both flagellated or non-flagellated cells and rosette-like colonies composed by only flagellated cells (showing outwards-directed flagella). In rich medium, solitary flagellated cells can sometimes actively merge and form a syncytium-like structure, which undergoes budding and releases flagellated daughter cells.
Ichthyosporeans are found in commensal, mutualistic or parasitic relationships with aquatic (both freshwater and marine) and terrestrial animals. Most of them have been directly isolated from different animal tissues, especially guts of molluscs and arthropods [73,76–79]. Some species exhibit distinct phenotypes, such as motile pseudopodia, hyphal or plasmodial structures . Ichthyosporeans also present a broadly conserved developmental mode consisting of large, multinucleated spherical or ovoid coenocytes that sometimes release multiple spherical propagules or motile limax-shaped amoebas by cellularization of the internal nuclei (figure 3e,f) [76–78,99,180,193]. Intriguingly, at least one of these species appears to generate a self-organized polarized layer of cells in the course of cellularization (figure 3f) .
Members of the Corallochytrea/Pluriformea group and T. unikontum also exhibit complex behaviours and developmental modes, sometimes resembling those observed in ichthyosporeans and filastereans. For example, C. limacisporum, is a small spherical free-living osmotroph originally isolated from marine coral reefs with a still unresolved complex developmental mode (figure 3g) [25,83]. Usually, cells undergo binary cell division but occasionally cell division occurs by coenocytic development followed by the release of propagules or limax-shaped amoebas, similar to ichthyosporeans (figure 3g) [83,191]. Syssomonas multiformis is a freshwater-dwelling predatory flagellate that feeds on large eukaryotic prey [26,70]. Similar to the filasterean Pigoraptor sp., it also has a complex developmental mode that includes amoeboflagellate, amoeboid cells, motile swimming cells, spherical cysts and sometimes clusters of multiple cells (figure 3h) [26,70]. Finally, T. unikontum is a marine free-living predatory flagellate that also feeds on eukaryotic prey . Besides its flagellate form, solitary cells temporarily aggregate into flagellated or non-flagellated cell clumps as observed in S. multiformis or the filasterean Pigoraptors spp. .
This diversity of phenotypes observed in each unicellular holozoan lineage, and the evidence of temporarily regulated life-stage transitions among some of their representatives [42,45,98,100], indicate that the last ancestral unicellular state was probably relatively plastic, rather than a simple unicellular entity (figure 4a) [95,123]. The last unicellular ancestor of animals could probably sense environmental stimuli and respond by transitioning to different cell stages (figure 4a,b). Its life cycle could have included a differentiated sedentary filter-feeding or heterotrophic life stage (most likely bacterivorous), and a proliferative stage, possibly including dispersive forms. It could also have included cysts or resistance forms and at least one multicellular stage. These distinct cell stages could have been regulated via temporal gene regulatory programmes, which in turn controlled life-stage transitions. Thus, the data gathered among unicellular relatives of animals suggest that the last unicellular ancestor of animals likely presented a complex life cycle integrating distinct transient cell identities, or states, and likely included a multicellular state exhibiting the spatial coexistence of different labile cell types. Future studies will provide deeper insights into whether the temporal regulation of these distinct labile cell types or stages in the last unicellular ancestor could have gradually evolved into spatio-temporal differentiation of cell types in the animal stem lineage. In fact, recent and ongoing efforts are investigating whether the multicellular structures exhibited in various unicellular holozoan species are formed by distinct cells coexisting in those multicellular stages (at the morphologic and genetic level) ([188,189,191] S. R. Najle 2021, personal communication). If this is indeed the case, then it would suggest that spatio-temporal differentiated cell types might have been present in the last unicellular ancestor of animals.
Figure 4. Our current perspective on important changes in the origin of animals. (a) The last unicellular ancestor of animals likely possessed a life cycle comprising different temporally regulated stages, including a sexually reproductive stage and at least one multicellular stage. (b) Cells within this multicellular structure were able to respond to different environmental stimuli thanks to a complex repertoire of signalling molecules and gene regulatory networks (GRNs), transitioning to labile cell stages. (c) This multicellular entity might have had a certain ability to integrate positional information from within the structure but lacked any axial/positional patterning. (d) The transition to animal origins likely involved some changes in this life cycle, already present by the time of the last common ancestor (LCA) of animals. (e) Cells within the multicellular structure acquired the ability to integrate spatial information from within the organism by making use of morphogenetic tools (such as ligands, receptors, and GRNs) (d′), which allowed the spatial organization of cell types (d″). Concomitantly, this developmental programme was conjoined with the sexual reproduction programme, by which gamete fusion was able to trigger the formation of a multicellular structure through serial division. (f) A greater ability to establish different cell types independently of the environment translates into the emergence of rudimentary morphogenetic plans, consisting of simple positional patterns (such as a primary axis) where different cell types localize to different regions of the organism (axial/positional patterning). It is worth emphasizing that the visual depictions presented here are mere representations of general concepts, and that we are by no means taking positions regarding specific details, such as the real structure of the life cycles, the number of cells, genes, molecules and GRNs implicated, the axial patterning or the morphological details of these organisms.
2.2.2. Potential lifestyles of the last common ancestor of animals
Comparative analyses between unicellular holozoans and animals also allow us to reconstruct the biological and ecological features of the animal LCA. In this case, those features inferred to be present in the animal LCA include traits predicted to have evolved along the animal stem. For example, the animal LCA was likely aquatic and featured obligate, clonal multicellularity [122,123]. Importantly, the animal LCA likely presented cell–cell communication-mediated cooperation, specialization and polarity, allowing the spatial distribution of labour between distinct coexisting cells. Each cell type (box 1) was specialized to perform a different role within the whole organism, with molecular features resembling those seen in the main cell types of extant animals . For instance, each cell type would also have their own sets of expressed genes used in different processes (e.g. contraction, secretion, signalling and reception), regulated by well-defined genetic programmes (a set of TFs and other specific regulatory mechanisms). This implies that some genes would be expressed by certain cell types but not others (i.e. each cell type expresses a limited number of genes encoded in the genome). The genome partitioning into functional modules accessed by different cell types reflects an increase in regulatory mechanisms to determine diverse cell fates .
From our previous ancestral gene content reconstruction, we can also predict that the animal LCA featured cell–cell adhesion using cadherins, cell–ECM adhesion through integrin-related proteins, and orchestrated collective movement by cell contractility . It also had the capacity to sense the environment, communicate between cells via synapse-like pathways and employ an epithelium-like cell layer used in part to capture bacterial or eukaryotic prey as a food source [122,123]. Moreover, it probably reproduced sexually using sperm and eggs, thus differentiating distinct gametes through spermatogenesis and oogenesis (i.e. oogamy) [122,123]. Finally, the animal LCA likely presented a form of developmental processes through mechanisms of cell division, cell differentiation and invagination present in all animals [122,123]. Such diversity of cell types and complex organization was in turn regulated by a diverse set of TFs and epigenomic machinery involving distal regulation, and the initial steps of development likely involved coordinated signalling through members of the Wnt and TGF-β pathways, paving the path to spatial distribution of labour among coexisting cells. Thus, we can conclude that the animal LCA was already rich in cell types which share some of their cellular foundations with those found in extant species.
3. Our current perspective on the origin of animals
The updated reconstruction of the genomic and biological features of both the last unicellular ancestor of animals and the animal LCA have allowed us to identify key features and major forces shaping animal evolution. In past, this identification was restricted by the limited information on the evolutionary relationships of animals and other eukaryotes. For instance, classical studies compared animals with unicellular organisms like yeast and designated features absent from yeast as potentially key to the origin of animals [194,195]. Now we know that such an approach was far from ideal due to the long evolutionary distances separating these lineages. In recent years, we have seen this perspective gradually changing with the study of animals’ closest unicellular relatives and their comparison to early branching animals, as discussed in previous sections. In addition, numerous studies have increased our knowledge of the environment in which animals originated and diversified. These studies have allowed us to rethink the context and major forces that drove the transition to animal multicellularity.
3.1. The ecological context of the transition
External factors and ecological triggers were possibly as important as genomic changes during animal evolution . One example is the biogeochemical context in which animals originated and diversified. Some of the potential ecological triggers include changes in ocean chemistry, such as the availability of iron and copper [196–201] or the great oxygenation event that occurred around 700 Ma  (although some authors argue the latter was not as critical: [203,204]). As multicellular organisms, the origin of animals could also have been influenced by all the advantages derived from being multicellular. For example, the emergence of new ecological niches  and selection for multicellularity as an escape from predation were also potential driving forces for the origin of animals [206,207] (but see also ).
The ecological context might have also had an impact on animal evolution, such as in shaping animal feeding modes and morphological features . For instance, animals evolved in an environment teeming with bacteria and other eukaryotes, and have lived in close association with these organisms throughout their subsequent evolutionary history. Indeed, host-associated microbiota can actually regulate development and gut morphogenesis in animals . In this context, being in a close relationship with bacteria could have impacted animal evolution by requiring a system of cell communication to harbour bacterial symbionts and commensals, and a defence system to deal with bacterial pathogens. Interestingly, bacterial interactions are also observed among the closest unicellular relatives of animals, especially among choanoflagellates. For instance, rosette development in the choanoflagellate S. rosetta is known to be triggered and enhanced by a bacterial sulfonolipid [42,61,177,187,210]. Bacterial lipids also regulate developmental switches both activating and inhibiting rosette formation in S. rosetta . This is not the sole example of environmental bacteria playing a key role during its life stages transitions, as S. rosetta is also capable of sexually reproducing upon induction by a bacterial chondroitinase [176–178]. Interestingly, the S. rosetta sexual cycle is induced by a bacterial species that also regulates light-organ development in a squid . Numerous studies in other choanoflagellates highlight the role of bacterial interactions [179,212]. An example is Salpingoeca monosierra, a new choanoflagellate species harbouring the first known choanoflagellate microbiome . Salpingoeca monosierra forms large colonies of more than 100 µm in diameter (more than an order of magnitude larger than those formed by S. rosetta) and harbour around 10 bacterial symbionts within a single colony . Overall, the ecological context during animal evolution was also key for the transition to multicellularity. Living in an environment teeming with bacteria likely provided the foundations of animal-associated microbiomes and the origin of animal interactions with microorganisms.
3.2. The origin of animals
Besides the ecological context, former biological definitions of animals involved the capacity for cell coordination at the multicellular level, the presence of spatial cell differentiation and a coordinated developmental plan starting from a single cell. Thus, theories explaining the origins of animals involve the acquisition of mechanisms necessary to generate epithelium-like multicellular structures. Further studies and comparisons revealed that the mechanisms underpinning these features likely developed in the stem lineage of animals, building upon pathways and features present in their unicellular ancestors [24,25,45,95,98,100,122,123]. Thus, some revised theories proposed the acquisition of spatial regulation as one of the main drivers of the origin of animals, in contrast to the temporal regulation of cell types exhibited by their unicellular relatives [214,215].
We here propose an updated review of which changes might have been key to the emergence of animals (figure 4). To start with, in our view, multicellular structures with different labile cell types coexisting were likely present prior to the origin of animals. We envision an initial scenario of an ancestral organism with a complex ontogeny and temporal regulation of different transient life stages, as proposed in Zakhvatkin  and revised in Mikhailov  (figure 4a–c). Each stage consisted of different cell types using distinct pathways to perform specific roles, such as substrate attachment, feeding, swimming and mating. One of those stages was a multicellular structure likely originating through clonal division, displaying spatial coexistence of different, non-committed cell identities driven by unique genetic programmes of transdifferentiation (figure 4b,c). In this temporal multicellular stage, different functions (feeding, motion and secretion) occurred simultaneously as they were performed by different cells. Thus, we propose that spatial regulation itself was present in the last unicellular ancestor of Metazoa.
Below, we speculate about some aspects that may have played a key role in the origin of animals, in relation to some of their features and in no particular order, and always in the context of incremental complexity discussed in this review.
3.2.1. Increased genomic innovation and co-option of pre-existing elements
The origin of animals was accompanied by increased genomic innovation, including many new, rapidly evolving and subsequently widely conserved genes. These genes encoded proteins known to have regulatory functions in animal multicellularity: gene regulation, signalling, cell adhesion and cell-cycle regulation. Nevertheless, co-option of and regulatory changes in pre-existing elements present among unicellular holozoans set the foundations for further gene family expansions and diversifications. This in turn contributed to an increased layer of regulation for cell-type specification in the animal stem lineage, and likely played a major role in the events discussed below.
3.2.2. Progressive acquisition of axial patterning and cell-type identity
As previously proposed, the last unicellular ancestor of animals had a mixture of labile cell types coexisting in the same entity (figure 4b,c) . However, analyses have so far not yet shown conclusive evidence that unicellular relatives of animals have specific arrangements of differentiated cell types when forming a multicellular structure. The last unicellular ancestor of animals was likely able to respond to external cues in a changing environment thanks to the signalling and genome regulatory mechanisms discussed above (figure 4b,c). Co-option of such genes for spatial cell signalling between neighbouring cells might have led to the ability to integrate positional information from within the organism. The pathways in question would involve the triggering of adjustable, non-binary responses, as in animal morphogens, and at least one mechanism of genome regulation determining different phenotypes. One potential candidate could be the Wnt/β-catenin signalling pathway, known to regulate the anteroposterior axis of the body plan even in early branching animals [142,144]. A primary axis likely arose as a result of spatial separation between different groups of cells. These primary axes could have provided a nucleating architecture for the different cell types to arrange and may have led to the formation of simple morphogenetic plans . With this, spatial coordination of cells came to be equally important to define different functions in the organism, rather than just individual coexisting cells.
The integration of temporally regulated and spatially coexisting cell types could have contributed to a gradual regionalization of functions that in turn fostered the emergence of morphogenetic programmes (figure 4d–f) . Flexible cell identity (and in turn GRNs) became less dependent on external factors, leading to a certain commitment of cell fate (figure 4e). This might have occurred through GRNs becoming more linked or dependent on signals within the organism, thereby overriding the freedom of the cell to respond to its environment by transdifferentiation. The emergence of cell types would allow selection to operate at the level of individual cells in terms of collective fitness, constituting a fine-tuning of within-group selection . Inherently, the emergence of multicellular structures might have enhanced the differences between cells in different regions of this multicellular entity . Thus, the transition to animal origins likely involved the progressive integration of GRNs and a gradual regionalization of functions, allowing the establishment of different spatially coexisting cell types.
3.2.3. Emergence of a conjoined gene regulatory programme of fertilization and multicellular development
Animals produce very distinct kinds of gametes. Gamete fusion determines initial polarity and triggers the developmental programme in animal eggs [218,219], meaning that in earlier stages of animal evolution it could have served as an early trigger for asymmetric cell division, generation of a rudimentary axis and establishment of cell fates. During development and throughout the animal's life, animal cells are able to proliferate in response to signals from within the organism by controlling entry into the cell cycle. The set of Capsaspora cell cycle regulators shares some traits with those of animals, with some conserved TFs related to proliferation as well as the timing of expression of cell cycle checkpoint genes [100,220]. However, unicellular holozoans lack the genes required to trigger cell cycle progression in response to extracellular signalling in animals [220–222]. So far, we do not know of any unicellular holozoan where the formation of the multicellular stage is linked to the fusion of gametes. At some point along the stem lineage leading to animals, an ancestor with the ability to both generate a multicellular morphogenetic plan through axial patterning and perform sexual reproduction likely integrated these two programmes in a single developmental plan (figure 4).
3.2.4. Relegation of unicellular stages in favour of a multicellular stage
The origin of animals likely involved a long, gradual evolutionary process rather than a single evolutionary leap, paving the way to animal multicellularity by coupling complex development, sperm–egg fusion and serial cell division in parallel with the integration of spatial cell differentiation [95,123]. The multicellular stage could have prevailed over the unicellular stage by favouring escape from predators, enhanced resource exploitation and relaxation of ecological constraints due to increases in the availability of some nutrients. The relegated unicellular stages could have later become simple forms for dispersion, or gametes, as the emerging properties concomitant with multicellularity, like the division of labour, could have led the multicellular stage to thrive as a proliferative stage .
4. New avenues of research into animal origins
The improved phylogenetic framework of animals and their unicellular relatives along with the sequencing of various omics-scale datasets has allowed an updated reconstruction of the genomic and biological features of both the last unicellular ancestor of animals and the animal LCA. These comparative studies have also highlighted various evolutionary mechanisms as important driving forces for the origin of animals. For instance, we now know that co-option of ancestral genes into new functions expansion of pre-existing GRNs combined with the emergence of novel genomic regulatory strategies and the progressive acquisition of spatio-temporal cell-type identities, were probably key for animal evolution. Nevertheless, many questions are still left unanswered, and further studies are needed to fully understand how those mechanisms might have impacted the transition to animal multicellularity.
For example, many genes critical for animal multicellularity-related functions have homologues in unicellular holozoans, but we still do not understand the function of these homologues in non-metazoans. In addition, some genes underwent duplications along the animal stem lineage, and their functions prior to duplication (and sub- or neofunctionalization) are not known. The functions of these genes in extant unicellular holozoans are not necessarily identical to those in the unicellular ancestors of animals nevertheless, understanding their function in a unicellular context is essential to fully address the role of co-option during the unicellular-to-multicellular transition. In this regard, the development of genetic tools among unicellular holozoans is crucial to fully understand the function of these genes of interest and assess to which extent the unicellular holozoan orthologues perform similar or different functions in a unicellular context . In recent years, our joint efforts have successfully developed transfection in several unicellular species representing all major unicellular Holozoa clades [191,193,224–227]. This tool has already provided some insights into the cell biology of several unicellular holozoans. For instance, transfection in the choanoflagellate S. rosetta allowed the first in vivo characterization of septins, a major class of cytoskeletal proteins . Interestingly, the S. rosetta septin orthologue localized to the basal poles of the cells, resembling the localization of septins in animal epithelia . Transient transfection in the filasterean C. owczarzaki revealed the three-dimensional organization of filopodia and actin bundles in live cells . In the ichthyosporean Creolimax fragrantissima, transient transfection allowed tracing of nuclear divisions in a growing cell in vivo, and revealed that these divisions were strictly synchronized . Moreover, two gene silencing strategies using RNA interference by small interfering RNAs (siRNA) and morpholinos have also been developed in C. fragrantissima . This tool has been used to analyse the function of c-Src kinase animal homologue throughout its life cycle, and revealed that an existing tyrosine-specific phosphatase was potentially co-opted for the role of Src regulation in the highly reduced kinome of C. fragrantissima [131,193]. Finally, transfection has also been recently developed for two additional unicellular holozoan species: the ichthyosporean Abeoforma whisleri  and the corallochytrean C. limacisporum [191,228]. Both species can be transiently transfected with fluorescently tagged reporter cassettes containing endogenous genes, using the same approach developed in S. rosetta [191,225,227]. Indeed, C. limacisporum transfectants can also be stably maintained using antibiotic-based selection, a strategy that has allowed the reconstruction of the life cycle of C. limacisporum with an unprecedented level of detail . More recently, CRISPR/Cas9-mediated genome editing tool has been developed for S. rosetta, opening new avenues of research for gene function studies using reverse genetics . Under this scenario, we expect future efforts to be invested in two main directions. First, towards taking advantage of the tools developed to investigate the function of key animal ‘multicellularity-related’ genes, such as those involved in animal cell adhesion, cell communication or transcriptional regulation, in the aforementioned unicellular holozoan species. And second, towards developing genetic tools in a broader representation of unicellular holozoan species to continue expanding the functional platform of experimentally tractable systems to address animal origins.
Another important pending question concerns genome regulation in a wider representation of unicellular holozoan species. Until now, our inferences have been based on the analysis of the regulatory genome of only one single species, the filasterean C. owczarzaki . Based on this study, we inferred that the last unicellular ancestor of animals probably followed a primarily proximal gene regulatory strategy, lacking some animal promoter types and signatures of animal enhancers [95,100]. However, we still need to characterize the genomic regulatory landscape of other unicellular holozoan species to accurately infer the regulatory capability of the last unicellular ancestor and fully understand how genome regulation evolved during the origin of animals. Thus, we expect future research to be directed to comparatively investigate the epigenome (including chromatin accessibility and regulatory dynamics, and transcription factor networks) of additional species representing other unicellular holozoan clades (i.e. choanoflagellates, ichthyosporeans and corallochytreans). This will allow for a more comprehensive reconstruction of the regulatory capabilities of the last unicellular ancestor of animals address whether metazoan-like distal regulation was or was not an animal innovation and also provide mechanistic insights into the evolution of genome regulation during the unicellular-to-multicellular transition.
We also still do not know how animal cell types appeared nor whether spatial cell differentiation was already established in a unicellular context. Although analyses in the filasterean C. owczarzaki revealed that some of the mechanisms required for animal spatial cell differentiation were already present in the last unicellular ancestor of animals , it has been assumed that spatial cell differentiation per se evolved at the Metazoa stem. However, we still have not investigated whether the multicellular structures exhibited by unicellular holozoans are indeed composed of morphologically and genetically identical cells or, on the contrary, they are composed of distinct cell types. Recently, the tridimensional reconstruction of rosette colonies in the choanoflagellate S. rosetta has unexpectedly revealed that cells within rosette colonies exhibit spatial cell disparity, varying significantly in cell size, shape and nuclear and mitochondrial content [188,189]. In parallel, microscope observations in other unicellular holozoan species, such as in the filasterean C. owczarzaki, have also pointed to at least different cell morphologies within the same multicellular structure (S. R. Najle 2021, personal communication). This indicates that unicellular holozoan colonies may not be just formed from the assemblage of identical single cells, but they may subsequently differentiate into distinct cell types displaying morphological modifications and, potentially, genetic modifications. Thus, we expect future studies to be directed towards analysing cell-type diversity at the genetic and morphologic level across the multicellular structures of several unicellular holozoan species representing major unicellular Holozoan clades. The integration of newly developed single-cell techniques will indeed provide a unique opportunity into these studies as they can allow to detect novel, undiscovered cell types and signatures of cell-type specific gene expression profiles [2,229–233]. Moreover, molecular data at a single-cell resolution from several animal taxa, especially among non-bilaterian animals (i.e. sponges, comb jellies and placozoans) [229–232], will also complement these studies from a comparative perspective to address animal cell-type evolution.
Finally, we also predict future research to be directed towards isolating and characterizing under-studied unicellular holozoan species. Particularly, those species falling within or related to different known unicellular holozoan clades identified from molecular environmental data, and those related to potential novel unicellular holozoan clades . First, because the discovery of new unicellular holozoan species will clarify the evolutionary relationships of the tree surrounding animals. And second, because their huge diversity of morphologies, lifestyles and genetic repertoires will help us continue refining the genome content and biological features of both the last unicellular ancestor of animals and the animal LCA.
In the coming years, the development of emerging model systems among unicellular holozoans combined with the use of modern research tools will allow us to fully address these new outstanding questions with an unprecedented level of detail. We look forward to seeing advances in this field as we are now entering an exciting era in the study of the origin of animals.
5. Concluding remarks
In recent years, a vast body of knowledge from molecular omics has provided not only a better phylogenetic framework of animals and their closest unicellular relatives but also a better understanding of the evolutionary history of genes key to animal multicellularity. To further expand this knowledge, we must aim to improve our understanding of the closest unicellular relatives of animals from different perspectives. For instance, more genome sequences are needed to better pinpoint the origin of some genes key to animal multicellularity. Moreover, functional studies of some proteins would allow us to understand how they could have been co-opted. Efforts at the taxonomic level should also allow the identification and isolation of more unicellular holozoan species. Likewise, studying their biology through cell biological and developmental approaches might help to uncover additional aspects of their temporal multicellular stages and their potential homology to similar structures in animals. Finally, the recent establishment of genetic tools in those taxa also promises to contribute to this end. Overall, we believe the years ahead of us will be crucial to better understand this transition and we find ourselves excited about, but most importantly eager to begin, unravelling the origins of animals.
In Search of the First Animals
All animals–from corgis to Greenland sharks, from dog ticks to toucans to you–descend from a common ancestor. The fossil record of animals, which runs back over 600 million years, can help us travel back some of the way through animal evolution towards the origin of the kingdom. But those early rocks contain precious few remains of animals, and so fossils alone can’t tell us what our common animal ancestor looked like.
Scientists can add to their supply of clues by studying living animals. And it now looks as if some of the most important clues to how animals got their start come from a beautiful creature called the comb jelly. This video from the Monterey Bay Aquarium is a good introduction to their luminescent loveliness.
In the past, a lot of scientists would not have put so much importance on comb jellies. If you asked them (as I did) how animals evolved, they’ve sketch out a version of events that runs like this:
1. Before multicellular animals evolved, their ancestors were single-celled protozoans that may have formed colonies. Our DNA shows that our closest non-animal relatives are critters called choanoflagellates. I wrote about our single-celled cousins in the New York Times.
I wrote about our single-celled cousins in the New York Times.
I wrote about our single-celled cousins in the New York Times.
2. Our ancestors then crossed the line from colonial life to life as multicellular creatures. They became the first animals.
3. The animal lineage then started to split into new branches. Many branches are now extinct. Among living animals, the first split divided the ancestors of today’s sponges from all other species.
4. That could tell you something important about what the earliest animals were like. Sponges have no nervous system and no muscles, for example. All other animals, from comb jellies to starfish to clams to us, do. So the early animals didn’t have muscles and neurons yet, and didn’t evolve them until after sponges split off on their own. You might even go so far as to say that our own direct ancestors were sponges–animals anchored to the sea floor, filtering food through small pores.
5. Later, the common ancestor of the non-sponge animals evolved muscles and neurons. These animals began to move around–as pulsating jellyfish, crawling worms, and, eventually, swimming fish.
6. Later still, the muscle-and-neuron-carrying animals split apart into two main lineages. Jellyfish belong to a lineage called cnidarians. The other branch is known as bilaterians. It includes all the animals with heads, brains, and tails, from insects to mammals.
Comb jellies seemed to many researchers to simply be a cousin lineage to cnidarians. And that meant they weren’t important to understanding how animals first evolved.
But a few years ago something weird started to happen. When scientists compared the DNA from more and more species, some of them would end up with animal trees in which the comb jellies split off first, not the sponges. (Here’s a piece I wrote about that work for the Boston Globe in 2008.)
A lot of debate ensued. Drawing evolutionary trees is no simple task, especially when you’re looking at branches that split off from each other hundreds of millions of years ago. Teasing apart the order of the branches can be as tricky as teasing apart the stars in a distant corner of the galaxy. So evolutionary biologists built themselves a better telescope.
A new paper today in ScienceA new paper today in Science is that telescope. In the past, scientists have compared limited segments of DNA from different species to work out the animal tree. A team of researchers at the National Institutes of Health and elsewhere decided that a more powerful view of the tree might come from looking at the entire genomes of many animals. And it just so happened that of all the major groups of animal species (known as phyla), only the comb jellies were without a sequenced genome.
The scientists rectified the matter, sequencing the first comb jelly genome, belonging to a species called Mnemiopsis leidyi. They then compared the genomes of 13 species, and then did a second comparison of smaller pieces of DNA from 58 species. The studies all pointed in the same direction: comb jellies, not sponges, split first. And the statistical support for that split was very strong.
This diagram, published in Science, sums up the findings nicely:
Having sequenced a comb jelly genome, the scientists could also march through its catalog of genes to see how many genes for different functions it shares in common with us. Despite the fact that comb jellies have muscles, they lack many of the genes essential for muscles in other animals. This would suggest that muscles evolved twice in the animal kingdom.
On the other hand, comb jellies have a lot of nervous system-related genes in common with bilaterians and cnidarians. It’s therefore possible that the common ancestor of all living animals had a simple nervous system. Sponges lost their nervous system and muscles entirely as they adapted to a quiet existence as filter feeders. (The placozoans shown on this tree are an obscure group of weird animals that are just tiny sheets of cells that creep across the sea floor. If the new study is right, they lost their nervous system and muscles too.)
I asked Antonin Rokas, an expert on animal relationships at Vanderbilt University, what he thought of the research. ” I think it’s a step in the right direction,” he told me, “but I doubt that it will silence those who have championed sponges as the earliest branching animal phylum.” The idea of comb jellies as belong to such an ancient lineage runs counter to a lot of thinking in zoology for over a century. It may take studies of more genomes to convince the tougher skeptics.
Aside from basic curiosity, there are other good reasons to settle the debate–and they help explain why the National Institutes of Health spearheaded this study. The NIH sponsors research on animals as models for human diseases. That’s because we share a lot of genes in common with them. Of particular interest to health researchers are so-called “disease genes”–human genes that are associated with diseases if they acquire mutations.
It turns out that over half of known disease genes are present in comb jellies–including many that are missing from species such as fruit flies that scientists use a lot to study human diseases. A lot of our diseases may arise from damage to the fundamental system for building an animal that evolved some 700 million years ago.
(For more on comb jellies, read this feature from earlier this year by Amy Maxmen in Science News.)
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Our research is aimed at uncovering the genetic and genomic basis of morphological complexity in animals, from both developmental and evolutionary perspectives. From the developmental perspective, we are interested in the early stages of development, including symmetry breaking during formation of the embryo and then during growth of the organisms. From the evolutionary perspective, we are interested in both macroevolutionary transitions (from colonial protists to the first animals, and from simple to complex animals), as well as in microevolutionary transitions (body form variations among related species). We are using marine sponges as primary model species, and a variety of animals (especially cnidarians) and their relatives in comparative analyses. Our work combines a bit of field sampling with lots of next-generation sequencing, gene and protein expression analyses and experimental manipulations of sponges, including studies of regeneration.
- Cuneyt Caglar
- Di Pan
- Xinran Cui
- Wenbo Yue
Animal embryos evolved before animals
Computer models based on X-ray tomographic microscopy of the fossils, showing the successive stages of development. Credit: Philip Donoghue and Zongjun Yin
Animals evolved from single-celled ancestors, before diversifying into 30 or 40 distinct anatomical designs. When and how animal ancestors made the transition from single-celled microbes to complex multicellular organisms has been the focus of intense debate.
Until now, this question could only be addressed by studying living animals and their relatives, but now the research team has found evidence that a key step in this major evolutionary transition occurred long before complex animals appear in the fossil record, in the fossilised embryos that resemble multicellular stages in the life cycle of single-celled relatives of animals.
The team discovered the fossils named Caveasphaera in 609 million-year old rocks in the Guizhou Province of South China. Individual Caveasphaera fossils are only about half a millimeter in diameter, but X-ray microscopy revealed that they were preserved all the way down to their component cells.
Kelly Vargas, from the University of Bristol's School of Earth Sciences, said: "X-Ray tomographic microscopy works like a medical CT scanner, but allows us to see features that are less than a thousandth of a millimeter in size. We were able to sort the fossils into growth stages, reconstructing the embryology of Caveasphaera."
Co-author Zongjun Yin, from Nanjing Institute of Geology and Palaeontology in China, added: "Our results show that Caveasphaera sorted its cells during embryo development, in just the same way as living animals, including humans, but we have no evidence that these embryos developed into more complex organisms."An embryo of Caveasphaera showing its cellular structure and the growing tips where cells are increasing in number through division. This image was obtained using Scanning Electron Microscopy. The fossil specimen is less than half a millimetre in diameter. Credit: Philip Donoghue and Zongjun Yin
Co-author Dr. John Cunningham, also from University of Bristol, said: "Caveasphaera had a life cycle like the close living relatives of animals, which alternate between single-celled and multicellular stages. However, Caveasphaera goes one step further, reorganising those cells during embryology."
Co-author Stefan Bengtson, from the Swedish Museum of Natural History, said "Caveasphaera is the earliest evidence of this most important step in the evolution of animals, which allowed them to develop distinct tissue layers and organs".
Co-author Maoyan Zhu, also from Nanjing Institute of Geology and Palaeontology, said he is not totally convinced that Caveasphaera is an animal. He added: "Caveasphaera looks a lot like the embryos of some starfish and corals—we don't find the adult stages simply because they are harder to fossilise
Co-author Dr. Federica Marone from the Paul Scherrer Institute in Switzerland said "this study shows the amazing detail that can be preserved in the fossil record but also the power of X-ray microscopes in uncovering secrets preserved in stone without destroying the fossils."
Co-author Professor Philip Donoghue, also from the University of Bristol's School of Earth Sciences, said "Caveasphaera shows features that look both like microbial relatives of animals and early embryo stages of primitive animals. We're still searching for more fossils that may help us to decide.
"Either way, fossils of Caveasphaera tell us that animal-like embryonic development evolved long before the oldest definitive animals appear in the fossil record."
Model of multicellular evolution overturns classic theory
Cells can evolve specialized functions under a much broader range of conditions than previously thought, according to a study published today in eLife.
The findings, originally posted on bioRxiv, provide new insight about natural selection, and help us understand how and why common multicellular life has evolved so many times on Earth.
Life on Earth has been transformed by the evolution of multicellular life forms. Multicellularity allowed organisms to develop specialized cells to carry out certain functions, such as being nerve cells, skin cells or muscle cells. It has long been assumed that this specialization of cells will only occur when there are benefits. For example, if by specializing, cells can invest in two products A and B, then evolution will only favor specialization if the total output of both A and B is greater than that produced by a generalist cell. However, to date, there is little evidence to support this concept.
"Rather than each cell producing what it needs, specialized cells need to be able to trade with each other. Previous work suggests that this only happens as long as the overall group's productivity keeps increasing," explains lead author David Yanni, Ph.D. student at Georgia Institute of Technology, Atlanta, US. "Understanding the evolution of cell-to-cell trade requires us to know the extent of social interactions between cells, and this is dictated by the structure of the networks between them."
To study this further, the team used network theory to develop a mathematical model that allowed them to explore how different cell network characteristics affect the evolution of specialization. They separated out two key measurements of cell group fitness—viability (the cells' ability to survive) and fecundity (the cells' ability to reproduce). This is similar to how multicellular organisms divide labor in real life—germ cells carry out reproduction and somatic cells work to ensure the organism survives.
In the model, cells can share some of the outputs of their investment in viability with other cells, but they cannot share outputs of efforts in reproduction. So, within a multicellular group, each cell's viability is the return on its own investment and that of others in the group, and gives an indication of the group's fitness.
By studying how the different network structures affected the group fitness, the team came to a surprising conclusion: they found that cell specialization can be favored even if this reduces the group's total productivity. In order to specialize, cells in the network must be sparsely connected, and they cannot share all the products of their labor equally. These match the conditions that are common in the early evolution of multicellular organisms—where cells naturally share viability and reproduction tasks differently, often to the detriment of other cells in the group.
"Our results suggest that the evolution of complex multicellularity, indicated by the evolution of specialized cells, is simpler than previously thought, but only if a few certain criteria are met," concludes senior author Peter Yunker, Assistant Professor at Georgia Institute of Technology, Atlanta, US. "This contrasts directly to the prevailing view that increasing returns are required for natural selection to favor increased specialization."
The event that transformed Earth
If you could build a time machine and go back to Earth's distant past, you'd get a nasty surprise. You wouldn't be able to breathe the air. Unless you had some breathing apparatus, you would asphyxiate within minutes.
For the first half of our planet's history, there was no oxygen in the atmosphere. This life-giving gas only started to appear about 2.4 billion years ago.
This "Great Oxidation Event" was one of the most important things to ever happen on this planet. Without it, there could never have been any animals that breathe oxygen: no insects, no fish, and certainly no humans.
For decades, scientists have worked to understand how and why the first oxygen was pumped into the air. They have long suspected that life itself was responsible for creating the air that we breathe.
But not just any life. If the latest findings are to be believed, life itself was undergoing a tremendous transformation just before the Great Oxidation Event. This evolutionary leap forward may be the key to understanding what happened.
Earth was already 2 billion years old at the time of the Great Oxidation Event, having formed 4.5 billion years ago. It was inhabited, but only by single-celled organisms.
They evolved a way to take energy from sunlight
It's not clear exactly when life began, but the oldest known fossils of these microorganisms date back 3.5 billion years, so it must have been before that. That means life had been around for at least a billion years before the Great Oxidation Event.
Those simple life-forms are the prime suspects for the Great Oxidation Event. One group in particular stands out: cyanobacteria. Today, these microscopic organisms sometimes form bright blue-green layers on ponds and oceans.
Their ancestors invented a trick that has since spread like wildlife. They evolved a way to take energy from sunlight, and use it to make sugars out of water and carbon dioxide.
This is called photosynthesis, and today it's how all green plants get their food. That tree down your street is pretty much using the same chemical process that the first cyanobacteria used billions of years ago.
It was the cyanobacteria, pumping out unwanted oxygen, that transformed Earth's atmosphere
From the bacteria's point of view, photosynthesis has one irritating downside. It produces oxygen as a waste product. Oxygen is of no use to them, so they release it into the air.
So there's a simple explanation for the Great Oxidation Event. It was the cyanobacteria, pumping out unwanted oxygen, that transformed Earth's atmosphere.
But while this explains how it happened, it doesn't explain why, and it certainly doesn't explain when it happened.
The problem is that cyanobacteria seem to have been around long before the Great Oxidation Event. "They're probably among the first organisms we have on this planet," says Bettina Schirrmeister of the University of Bristol in the UK.
Maybe the cyanobacteria changed
We can be confident that there were cyanobacteria by 2.9 billion years ago, because there is evidence of isolated "oxygen oases" at that time. They might date as far back as 3.5 billion years, but it's hard to tell because the fossil record is so patchy.
That means the cyanobacteria were busy pumping out oxygen for at least half a billion years before oxygen started appearing in the air. That doesn't make a lot of sense.
One explanation is that there were a lot of chemicals around &ndash perhaps volcanic gases &ndash that reacted with the oxygen, effectively "mopping it up".
But there's another possibility, says Schirrmeister. Maybe the cyanobacteria changed. "Some evolutionary innovation in cyanobacteria helped them to become more successful and more important," she says.
Some modern cyanobacteria have done something that, by bacterial standards, is remarkable. While the vast majority of bacteria are single cells, they are multicellular.
Multicellularity could have been a game-changer for Earth's early cyanobacteria
The individual cyanobacterial cells have joined up into stringy filaments, like the carriages of a train. That in itself is unusual for bacteria, but some have gone further.
"Many cyanobacteria are able to produce specialised cells that lose their ability to divide," says Schirrmeister. "This is the first form of specialisation we see." It's a simple version of the many specialised cells that animals have, such as muscle, nerve and blood cells.
Schirrmeister thinks multicellularity could have been a game-changer for Earth's early cyanobacteria. It offers several possible advantages.
On the early Earth, single-celled organisms often lived together in flat layers of gunk called "mats". Within each mat there would have been many different species of cyanobacteria, and a host of other things to boot.
The Earth was being bombarded with harmful ultraviolet radiation from the Sun
A multicellular cyanobacterium would have one clear advantage compared to its single-celled rivals. It would find it easier to spread, because its larger surface area would mean it was better at attaching itself to slippery rocks. Such an organism would be "less likely to wash away in the current", says Schirrmeister.
Many modern multicellular cyanobacteria can move around within their mats. "They're not extremely fast but they can move," says Schirrmeister. That suggests the primordial ones could as well.
Moving could have helped them survive. At the time the Earth was being bombarded with harmful ultraviolet radiation from the Sun, and there was no ozone layer to keep it out.
"In modern mats, cyanobacteria will turn around and appear vertical instead of horizontal to protect themselves from excess sunlight," says Schirrmeister. "You have also movement between layers. It might be these multicellular cyanobacteria had the ability to position themselves optimally within the mat."
It's a neat idea. But for it to be true, cyanobacteria must have evolved multicellularity before the Great Oxidation Event.
Schirrmeister has spent the last few years trying to figure out when cyanobacteria first evolved multicellularity.
The clues lie in their genes. By examining genes that all cyanobacteria share, and identifying tiny differences between them, Schirrmeister could figure out how they are all related &ndash essentially drawing up a family tree of cyanobacteria.
With that tree in place, Schirrmeister could then home in on the multicellular cyanobacteria, and estimate roughly when they first became multicellular.
Her first attempt, published in 2011, suggested that most modern cyanobacteria are descended from multicellular ancestors. That suggested multicellularity was ancient, but it was difficult to put a firm date on it.
Her family tree was only based on one gene
Schirrmeister refined her methods for a second paper, published in 2013. This suggested that multicellularity evolved not long before the Great Oxidation Event, at a time when cyanobacteria were diversifying rapidly.
But that didn't clinch the argument. Her family tree was only based on one gene, albeit a gene shared by every single species of cyanobacterium. That meant the tree was suspect.
So Schirrmeister has now gone one better.
"This time I worked with 756 genes," says Schirrmeister. "The genes I took are present in all cyanobacteria."
We have multicellularity evolving before the Great Oxidation Event
Her estimate of the origin of multicellularity is still rough, but it seems to be around 2.5 billion years ago &ndash before the Great Oxidation Event.
There are several different ways to calculate these family trees, and they all gave the same answer. "No matter how we calibrate our phylogeny, it seems more likely we have multicellularity evolving before the Great Oxidation Event," says Schirrmeister.
This may not be the end of the story. Even if Schirrmeister's results are confirmed, and cyanobacteria did become multicellular just before the Great Oxidation Event, there are two big questions.
It is one of the most important things to ever happen on this planet
The first is, did multicellularity really offer them the advantages she thinks it did? We don't know, but we could find out: by testing how modern single-celled and multicellular cyanobacteria cope with different situations.
The second question is harder: why did it take so long for cyanobacteria to become multicellular? If it is so advantageous, why did they not evolve it sooner, and trigger an earlier Great Oxidation Event?
"The next step is to find out which genes are responsible for multicellularity in cyanobacteria," says Schirrmeister. "Then I could say why did it take that long, why didn't it evolve earlier." If lots of new genes were required, it becomes understandable that it took the cyanobacteria a long time to evolve it.
Whatever caused the Great Oxidation Event, it's clear that it is one of the most important things to ever happen on this planet.
In the short term, it was probably rather bad news for life.
"Oxygen would have been lethal for many bacteria," says Schirrmeister. "It's hard to prove, because from the fossil record we don't have a lot of deposits from that time&hellip [but] we can assume we had a lot of bacteria dying at that point."
Those first multicellular cyanobacteria triggered the evolution of complex life
But in the longer term, it allowed a whole new kind of life to evolve. Oxygen is a reactive gas &ndash that's why it starts fires &ndash so when some organisms figured out how to harness it, they suddenly had access to a major new source of energy.
By breathing oxygen, organisms could become much more active, and much larger. Moving beyond the simple multicellularity developed by cyanobacteria, some organisms became far more intricate. They became plants and animals, from sponges and worms to fish and, ultimately, humans.
If Schirrmeister is right, those first multicellular cyanobacteria triggered the evolution of complex life, including us, by producing oxygen on a global scale. "It made complex life possible," she says.
Multicellular Life: Setting the Stage
Nearly 80% of Earth&rsquos history passed before multicellular life evolved. Up until then, all organisms existed as single cells. Why did multicellular organisms evolve? What led up to this major step in the evolution of life? To put the evolution of multicellularity in context, let&rsquos return to what was happening on planet Earth during this part of its history.
The Late Precambrian
The late Precambrian is the time from about 2 billion to half a billion years ago. During this long span of time, Earth experienced many dramatic geologic and climatic changes.
- Continents drifted. They collided to form a gigantic supercontinent and then broke up again and moved apart. Continental drift changed climates worldwide and caused intense volcanic activity. To see an animation of continental drift, go to this link:http://www.ucmp.berkeley.edu/geology/anim1.html.
- Carbon dioxide levels in the atmosphere rose and fell. This was due to volcanic activity and other factors. When the levels were high, they created a greenhouse effect. More heat was trapped on Earth&rsquos surface, and the climate became warmer. When the levels were low, less heat was trapped and the planet cooled. Several times, cooling was severe enough to plunge Earth into an ice age. One ice age was so cold that snow and ice completely covered the planet. Earth during this ice age has been called snowball Earth(see Figurebelow).
Snowball Earth. During the late Precambrian, Earth grew so cold that it was covered with snow and ice. Earth during this ice age has been called snowball Earth.
Life During the Late Precambrian
The dramatic changes of the late Precambrian had a major impact on Earth&rsquos life forms. Living things that could not adapt died out. They were replaced by organisms that evolved new adaptations. These adaptations included sexual reproduction, specialization of cells, and multicellularity.
- Sexual reproduction created much more variety among offspring. This increased the chances that at least some of them would survive when the environment changed. It also increased the speed at which evolution could occur.
- Some cells started to live together in colonies. In some colonies, cells started to specialize in doing different jobs. This made the cells more efficient as a colony than as individual cells.
- By 1 billion years ago, the first multicellular organisms had evolved. They may have developed from colonies of specialized cells. Their cells were so specialized they could no longer survive independently. However, together they were mighty. They formed an organism that was bigger, more efficient, and able to do much more than any single-celled organism ever could.
The Precambrian Extinction
At the close of the Precambrian 544 million years ago, a mass extinction occurred. In a mass extinction, many or even most species abruptly disappear from Earth. There have been fivemass extinctions in Earth&rsquos history. Many scientists think we are currently going through a sixth mass extinction. What caused the Precambrian mass extinction? A combination of climatic and geologic events was probably responsible. No matter what the cause, the extinction paved the way for a burst of new life, called the Cambrian explosion, during the following Paleozoic Era.