By PZ Myers — What are the key innovations that led to the evolution of multicellularity, and what were their precursors in the single-celled microbial life that existed before the metazoa? We can hypothesize at least two distinct kinds of features that had to have preceded true multicellularity.
- The obvious feature is that cells must stick together; specific adhesion molecules must be present that link cells together, that aren’t generically sticky and bind the organism to everything. So we need molecules that link cell to cell. Another feature of multicellular animals is that they secrete extracellular matrix, a feltwork of molecules outside the cells to which they can also adhere.
- A feature that distinguishes true multicellular animals from colonial organisms is division of labor — cells within the organism specialize and follow different functional roles. This requires cell signaling, in which information beyond simple stickiness is communicated to cells, and signal transduction mechanisms which translate the signals into different patterns of gene activity.
These are features that evolved over 600 million years ago, and we need to use a comparative approach to figure out how they arose. One strategy is to pursue breadth, cast the net wide, and examine divergent forms, for instance by comparing multicellular plants and animals. This approach leads to an understanding of universal properties, of how general programs of multicellular development work. Another is to go deep and examine closer relatives to find the step by step details of our specific lineage, and that’s exactly what is being done in a new analysis of the choanoflagellate genome.
So what is a choanoflagellate? They are members of a diverse and common group of single-celled eukaryotes that possess a flagellum for motility and a collar of slender processes called microvilli that it uses to capture bacterial prey. It’s a very successful lifestyle that has allowed them to flourish in both marine and freshwater environments.
Choanoflagellate cells bear a single apical flagellum (arrow, b) and an apical collar of actin-filled microvilli (bracket, c). d, An overlay of β-tubulin (green), polymerized actin (red) and DNA localization (blue) reveals the position of the flagellum within the collar of microvilli. Scale bar, 2 µm.
Here’s the connection to multicellular animals, the metazoa: choanoflagellates are markedly similar to the choanocytes of sponges. These are cells lining the interior channels of the sponge, which beat their flagella to propel water through the animal, and use the microvilli to filter out food particles. Right away, we can see an adaptive reason for the evolution of multicellularity: ancestral choanoflagellate-like organisms that teamed up could more efficiently filter water to extract food. The question here is the identity of the specific molecules they used to form early colonies.
King and others have sequence the entire genome of Monosiga brevicollis and compared what they found there to similar genes in other organisms. The genome itself is about 41.6 megabases, and contains approximately 9,200 genes (about half of what is present in humans). These genes were then compared to those of a suite of organisms to sort out what was held in common with the multicellular animals, the metazoa, and what was different from our distant cousins, the fungi and plants.
The close phylogenetic affinity between choanoflagellates and metazoans highlights the value of the M. brevicollis genome for investigations into metazoan origins, the biology of the last common ancestor of metazoans (filled circle) and the biology of the last common ancestor of choanoflagellates and metazoans (open circle). Genomes from species shown with their abbreviation were used for protein domain comparisons in this study: human (Homo sapiens, Hsap), ascidian (Ciona intestinalis, Cint), Drosophila (Drosophila melanogaster, Dmel), cnidarian (N. vectensis, Nvec), M. brevicollis (Mbre), zygomycete (Rhizopus oryzae, Rory), basidiomycete (Coprinus cinereus, Ccin), ascomycete (Neurospora crassa, Ncra), hemiascomycete (Saccharomyces cerevisiae, Scer), slime mould (Dictyosteliumdiscoideum, Ddis) and Arabidopsis (Arabidopsisthaliana, Atha).
So what did they find? Choanoflagellates have a surprisingly rich repertoire of cell adhesion molecules, with many members of families of genes that the metazoa also use. They have at least 23 cadherin genes; cadherins are calcium-dependent cell adhesion molecules that are not found in other multicellular organisms like fungi and plants, and are present in animals where they are used for essential processes in development like cell sorting and polarization, and in regulation of the morphogenetic movements of sheets of tissues … and there they are in the choanoflagellates as well. While some species of choanoflagellates will form clusters and at least transient colonies, M. brevicollis is not known to make such associations, so the function of these molecules in these particular organisms is a bit mysterious. They also contain integrin-α genes and genes with immunoglobulin domains — while you may be familiar with immunoglobulins as key proteins of the vertebrate immune system, the immunoglobulin motif is also a more general cell adhesion domain that is also found in many cells of the nervous system.
While these proteins that metazoans use to mediate interactions between cells are exciting to find in a choanoflagellate, and while their presence opens up new questions about their function, there’s another class of genes that are even more peculiar to find in a single-celled organism: genes for proteins that bind to the extracellular matrix. These are important in animals like us; we construct layers of extracellular matrix proteins during our development that are contained within our bodies, and cells bind to them and take advantage of them in embryogenesis. What do choanoflagellates use them for? They may be important in substrate attachment, or possibly these organisms secrete a more complex suite of molecules into their environment than is known.
So we see some remarkable homologies between choanoflagellates and metazoans in the genes that mediate cell adhesion and adhesions between cells and a matrix of molecules in the environment — our single-celled ancestors first built up a collection of tools to make them sticky, a cookbook of glue molecules that would later enable more sophisticated patterns of attachment to one another. The table below also shows that the choanoflagellates and their last common ancestor with the metazoa also evolved some common transcription factors, or gene regulators.
Note: sticky proteins and transcription factors are not unique to choanoflagellates and metazoans — bacteria, plants, fungi, etc. all also have them. What this work is showing is that the choanoflagellates and metazoa share an idiosyncratic, special set of sticky molecules and transcription factors.
M. brevicollis possesses diverse adhesion and ECM domains previously thought to be unique to metazoans (magenta). In contrast, many metazoan sequence-specific transcription factors are absent from the M. brevicollis gene catalogue. For adhesion and ECM domains, a filled box indicates a domain identified by both SMART and Pfam, a half-filled box indicates a domain identified by either SMART or Pfam, and an open box indicates a domain that is not encoded by the current set of gene models. The presence (filled box) or absence (empty box) of transcription factor families was determined by reciprocal BLAST and SMART/Pfam domain annotations. EC, extracellular domain; cyto, cytoplasmic domain; asterisk, collagen triple-helix-domains occur in the extended tandem arrays diagnostic of collagen proteins found only in metazoans and choanoflagellates.
At the beginning of this article I said that there were two properties essential to multicellularity: adhesion and signaling. Choanoflagellates have the molecular precursors needed for metazoan-style adhesivity, but they lack metazoan-specific signaling pathways. This makes sense. A general property like adhesion may well have utility to a single celled organism, but the specific pathways that would trigger region- and tissue-specific differentiation would be a later innovation.
Even in the case of these unrepresented elements of the metazoan genome, though, we see premonitions in the choanoflagellate. While complete, recognizable homologs of important signaling genes like hedgehog and Notch are not found, fragments of them are found scattered about. They didn’t appear out of nowhere — rather, there was a process of domain shuffling during metazoan evolution that built new signaling molecules by recombining elements present in the ancestral genome. So, while choanoflagellates reveal that the ancestor almost certainly did not have a true Notch gene, we can find 3 genes that contain pieces of Notch: one has the EGF domain, another the NL domain, and another the set of ankyrin repeats … and it’s easy to see that the Notch gene was not generated ex nihilo, but was assembled by splicing bits of pieces of extant genes into a novel protein.
Analysis of the draft gene set reveals that M. brevicollis possesses proteins containing domains characteristic of metazoan Notch (a, N1–N3) and hedgehog (b, H1 and H2). Some of these protein domains were previously thought to be unique to metazoans. The presence of these domains in separate M. brevicollis proteins implicates domain shuffling in the evolution of Notch and Hedgehog. Hh, hedgehog; N-hh, hedgehog N-terminal signalling domain; Hint, hedgehog/intein domain; TM, transmembrane domain; VWA, von Willebrand A domain.
Transitional fossils always get all the attention — and you’ve got to admit, a new collection of old bones is a sexy thing, an arresting attention grabber that has a lot of visual appeal. I think the more powerful modern evidence for evolution, though, are these examples of molecular transitions in which we can reconstruct the details of ancient changes. Not to belittle the fossil evidence, but changes within a single narrow lineage within a single phylum aren’t quite as dramatic or impressive as the kind of radical evolutionary event we see here — not just the reshaping of a femur, for instance, but the acquisition of whole new capabilities, the novel potential to build a femur in the first place. This is big stuff, a peek into core innovations that led to insects and jellyfish and grasshoppers and snails and cows, and that are held in common among all of us.
Yet despite the magnitude of the potential evolutionary consequences, we can also see revealed the mechanisms underlying them, and that they are small and simple changes, an expansion of capabilities present in miniscule, single-celled creatures. When evolving such fundamental and revolutionary features as multicellularity is such a patently feasible and explainable event, it does seem absurd that some people can still question relatively minor transformations, such as between varieties of ape.
by David Pescovitz
Biologist Nicole King studies tiny creatures that may be the closest living relatives to our single-celled ancestors.
Six-hundred million years ago, a pivotal turning point in the history of life occurred. In the ancient sea, multicellular organisms evolved that are now recognized as the world’s first animals. But what was the biology of the single-celled organism that made the transition? And how did it become the common progenitor of all animals? To answer these questions, UC Berkeley biologist Nicole King studies tiny creatures called choanoflagellates that may be the closest living relative to our single-celled ancestors.
“These early organisms are not preserved in the fossil record, so we don’t know very much about how multicellularity first evolved,” says King, a professor in the Departments of Integrative Biology and Molecular and Cell Biology. “But choanoflagellates might provide insight into that transition.”
Choanoflagellates are one-celled protozoans that live in fresh water and the ocean. Resembling sperm, the tiny organisms are approximately 10 microns across�nearly 100 would fit on the head of a pin. While choanoflagellates have long been suspected to be relatives of animals, studying their basic biology has historically been difficult. In recent years though, the genomics revolution has spawned techniques that are enabling King and her colleagues to look closely at the cellular secrets inside the organisms.
Propelled by their flagella, choanoflagellates move through water collecting bacteria on a collar of tentacles at the base of the cell body. (photo by Melissa Mott)
“When I first stumbled upon choanoflagellates, I was dumbfounded,” says King, who in September received a prestigious MacArthur Foundation “genius award.” “As a scientist interested in the cellular bases for animal development, I couldn’t believe what a goldmine these organisms might be. Once I realized that we could apply genomic tools to study them, this project really opened up.”
King’s earliest experiments helped confirm that choanoflagellates are indeed closely related to animals. Next, she and her colleagues surveyed the organism’s genes at a high level and quickly discovered that it contains genes that were previously thought to only exist in animals. The big surprise was that two of those genes are actually used by animals to express proteins for cell adhesion and cell communication. In other words, a single-celled animal is making proteins that are seemingly essential only to multicellular animals.
“It’s amazing.” King says. “We interpret that as evidence that some of the protein machinery for multicellularity actually evolved before the origin of animals, before multicellularity itself. The proteins predated their current function in animals.”
According to King, this “a classic example of co-option,” an evolutionary process in which an existing biological structure or system is adapted for a new function.
“Right now, we’re very interested in understanding how the proteins function in choanoflagellates and to use that as a tool in investigating what they might have been doing in the common ancestor,” she says.
Choanoflagellates were first considered to be close relatives of animals in the late nineteenth century. (courtesy the researchers)
As one of only a handful of laboratories around the world studying the choanoflagellates using methods from molecular and cell biology, King’s research group is developing most of their techniques from scratch. Meanwhile, they’re collaborating with scientists from the Department of Energy’s Joint Genome Institute, who are sequencing the whole genome of a choanaoflagellate. Once completed, the code will enable the King and her colleagues to reconstruct the minimal genome of the last common ancestor and seek out the hidden details of its evolutionary history.
“I was surprised to learn that so much of animal biology was in place before the origin of animals,” King says. “And I think that’s what motivates most scientists–not learning that you were right, but learning that you were wrong.”
Choanoflagellates represent an order of single-celled, transparent microbes that propel themselves with whiplike appendages. Photo courtesy University of Montreal.
GoogleNews.com, The New York Times, December 14, 2010, by Sean B. Carroll — RELATIVES Recent studies suggest that choanoflagellates are cousins to all animals in the same way that chimpanzees are cousins to humans. From left, a choanoflagellate colony, feeding cells of sponges that resemble choanoflagellates and a choanoflagellate with its long flagellum and collar of filaments. There can be millions of choanoflagellates in a gallon of sea water.
The Environmental Protection Agency is worried about a lot of things in our water — polychlorinated biphenyls, dibromochloropropane, Cryptosporidium parvum — to name just a few of the dozens of chemicals or organisms they monitor. However, in nearly every creek and lake, and throughout the oceans, there is one important group of multisyllabic microbes that the E.P.A. does not track, and until recently, most biologists heard and knew very little about — the choanoflagellates.
Before you spit out that glass of water or dunk your swimsuit in Clorox, relax. These tiny organisms are harmless. They are important for other reasons. They are part of the so-called nanoplankton and play critical roles in the ocean food chain. Choanoflagellates are voracious single-cell predators.
The beating of their long flagellum both propels them through the water and creates a current that helps them to collect bacteria and food particles in the collar of 30 to 40 tentaclelike filaments at one end of the cell.
There can be thousands to millions of choanoflagellates in a gallon of sea water, which may filter 10 to 25 percent of coastal surface water per day. Choanoflagellates in turn serve as food for planktonic animals like crustacean larvae, which are consumed by larger animals, and so on up the food chain.
Theirs is a humble existence compared with the larger, more charismatic residents of the oceans like lobsters, fish, squids and whales.
But recent studies suggest that these obscure organisms are among the closest living single-celled relatives of animals. In other words, choanoflagellates are cousins to all animals in the same way that chimpanzees are cousins to humans. Just as the study of great apes has been vital to understanding human evolution, biologists are now scrutinizing choanoflagellates for clues about one of the great transitions in history — the origin of the animal kingdom.
For most of the first 2.5 billion years of life on Earth, most species were microscopic, rarely exceeding one millimeter in size, and unicellular. Many different kinds of larger life forms, including fungi, animals and plants, subsequently evolved independently from separate single-celled ancestors.
The evolution of multicellularity was a critical step in the origin of each of these groups because it opened the way to the emergence of much more complex organisms in which different cells could take on different tasks. And the emergence of larger organisms drove profound changes in ecology that changed the face of the planet.
Scientists are eager to understand how transitions from a unicellular to multicellular lifestyle were accomplished. Reconstructing events that happened more than 600 million years ago, in the case of animals, is a great challenge. Ideally, one would have specimens from just before and immediately after the event. But the unicellular ancestor of animals and those first animals are long extinct. So information has to be gleaned from living sources.
This is where comparisons between choanoflagellates and animals come into play. The close kinship between choanoflagellates and animals means that there once lived a single-celled ancestor that gave rise to two lines of evolution — one leading to the living choanoflagellates and the other to animals. Choanoflagellates can tell us a lot about that ancestor because any characteristics that they share with animals must have been present in that ancestor and then inherited by both groups. By similar logic, whatever animals have but choanoflagellates lack probably arose during animal evolution.
There are striking physical resemblances between choanoflagellates and certain animal cells, specifically the feeding cells of sponges, called choanocytes. Sponge choanocytes also have a flagellum and possess a collar of filaments for trapping food. Similar collars have been seen on several kinds of animals cells. These similarities indicate that the unicellular ancestor of animals probably had a flagellum and a collar, and may have been much like a choanoflagellate.
But even more surprising and informative resemblances between choanoflagellates and animals have been revealed at the level of DNA. Recently, the genome sequence of one choanoflagellate species was analyzed by a team led by Nicole King and Daniel Rokhsar at the University of California, Berkeley. They identified many genetic features that were shared exclusively between choanoflagellates and animals. These included 78 pieces of proteins, many of which in animals are involved in making cells adhere to one another.
The presence of so many cell adhesion molecules in choanoflagellates was very surprising. The scientists are trying to figure out what all of those molecules are doing in a unicellular creature. One possibility is that the molecules are used in capturing prey.
Whatever the explanation, the presence of those genes in a unicellular organism indicates that much of the machinery for making multicellular animals was in place long before the origin of animals. It may be that rather than evolving new genes, animal ancestors simply used what they had to become multicellular. There may be selective advantages to forming colonies, like avoiding being eaten by other small predators. And in fact, some choanoflagellates do form multicellular colonies at stages of their life cycle.
Dr. King and her colleagues Stephen Fairclough and Mark Dayel investigated one such species to determine whether colony formation occurred by dividing cells staying together, the way animal embryos form, or by individual cells aggregating together, as some protists like slime molds do.
The scientists found that colonies formed exclusively by dividing cells staying together. They suggested that the ancient common ancestor of choanoflagellates and animals was capable of forming simple colonies and that this property may well have been a first step on the road to animal evolution.
The world is full of microbes, and we spend a lot of worry and effort trying to keep them off and out of our bodies. It is humbling to ponder that still swimming within that microscopic soup are our distant cousins.
Our Closest Single-Celled Cousin
Usually,when we want to understand the biology of humans and other complex animals, we use complex animal models–like primates, or canines, or fruitflies. A recent article I read pointed out that there are certain phenomena that are most easily understood with a much simpler model.
Recent work on how multicellularity evolved has focused in on a single-celled organism called a choanoflagellate. (I have shown two images here–one a highly magnified photograph, the other an artist’s rendering.) Choanoflagellates have, according to the article, “a distinctive form: a cell with an apical flagellum, a kind of propeller that can move it through the water or drive a flow of water over it, and a ring or collar of microvilli, thin projections that act like a net to capture bacteria for food.”
One interesting thing about choanoflagellates is that they have evolved cell-adhesion and signalling proteins to enable them to interact with other single-celled organisms. It is precisely these mechanisms, claims the article, that allowed for the development of multicellular life, because the same proteins that choanoflagellates use to interact with other cells are the proteins that more “advanced” forms of life use to negotiate between their own cells. Although the article does not make this point, this would seem to be a classic case of exaption–a mutation favorable to an earlier form for one reason (inter-organism interaction) being coopted by a later form for an entirely different purpose (integrity of a multicellular organism).
Two classes of proteins in particular are shared by choanoflagellates and multicellular animals: cadherins and integrins. Cadherins regulate cell adhesion though interaction with environmental calcium. And integrins help cells stick to the extracellular matrix. Without them, our cells would not be able to cohere into integral bodies.
In some senses, we may learn as much about complex animal life by studying these “simple” single-celled organisms as we do by looking at more obvious animal models.