Assembling Vertebrate Body Plans

In our last post, we introduced the distinction between “stem group” and “crown group” organisms, and discussed how the “arthropod body plan” – i.e. the body plan of crown group arthropods – arose through a gradual process. In this post, we’ll explore the origins of vertebrates, the group that would ultimately give rise to mammals such as our own species. Like arthropods, vertebrates make their first appearance in the fossil record in the Cambrian; and, as we have seen for arthropods, the vertebrate “body plan” shows signs of having been assembled over a lengthy period for exactly the same reasons – we see “stem group” organisms with only a subset of the traits characteristic of crown-group organisms. Before we discuss the origin and diversification of the vertebrates, however, let’s expand on a point we touched on briefly in the last post – the expectation that stem group species are not the direct ancestors of crown group species, but nonetheless are informative about the evolutionary lineage leading to the crown group.

As a scientist, reading popular news reports about biological discoveries is often a painful experience. Most news stories about fossil discoveries, for instance, are plagued with misconceptions. One very common misconception is that paleontology is the search for the direct ancestors of present-day organisms. While paleontologists are certainly interested in what the ancestors of present-day organisms looked like, paleontology is not well suited for finding direct ancestors. Counter-intuitively, however, this does not prevent scientists from learning a great deal about lineages leading to a present-day crown group. Let’s use human family trees as an analogy to explain why this is the case.

With “Venema” as a last name, it won’t be a surprise that my ancestry is rooted in the Netherlands. If I were to travel to the Netherlands and visit a medieval graveyard, the chances that any particular grave would hold the remains of one of my direct ancestors would be tiny. On the other hand, studying the remains of anyone in the graveyard would be highly informative about my ancestry, because nearly all individuals found there would be (fairly) close relatives of mine. In other words, I would share an ancestor in common with them – though some number of generations back. If I were to call my own immediate family a “crown group,” then these relatives that branch off my direct lineage a few generations back would be analogous to “stem groups” – and studying the characteristics of these “stem group” relatives would be an excellent way for me to learn about my own lineage, even if I knew nothing about it directly, since I would be studying close relatives of my direct ancestors.

So too with fossil species: the probability that any given fossil species is a direct ancestor of a modern-day species is vanishingly small. Fossilization is a highly infrequent event – fossils spaced 10,000 or even 100,000 years apart would be considered to be nearly simultaneous in their timing from a geological perspective – and the chances of such an infrequent event preserving a direct ancestor is highly unlikely. On the other hand, the probability that the fossil record preserves relatives of modern-day species is quite good, though these relatives might be fairly distant ones (certainly much more distant than the relatives I might find in a Dutch cemetery).

Let’s briefly return to the diversification of arthropods to illustrate what we mean. In the phylogeny below, the direct lineage of crown-group arthropods is outlined in blue, and various stem-groups branch off this lineage along the way. Studying these stem-group species allows us to infer what characteristics were present at different time points along the direct crown-group lineage, and the order in which the characteristics were acquired.

For example, because we see stem-group arthropods with specialized appendages and body segments (but without a hardened exoskeleton) we can infer that these characteristics arose first on the lineage leading to the crown-group. Note well – these stem-group species (X1, X2 and X3 on the diagram above) are not ancestors of crown-group arthropods, but relatives. Put another way, they are not transitional forms leading to the crown group. They are, however, species that give us information about the actual transitional forms on the direct crown-group lineage. In this way, the stem-group species display transitional characteristics – the stepwise accumulation of traits that we use to define the crown group.

Another point worth mentioning here is that because stem-group species are not ancestors of the crown group, there is no expectation that they will be older than the last common ancestral species that ultimately gives rise to the crown group. For example, the stem-group species in the diagram above (X1, X2 and X3) are all species found in the fossil record alongside crown-group species. At the time point marked by the dashed red line, for example, it would be no surprise to find additional species that were stem-group species with one, two or three crown-group characteristics. Once lineages separate, they are independent of each other.

(As an aside, it is fairly common to see misunderstandings of these concepts in the popular press and in Christian antievolutionary writings. Often, the above phylogeny would be interpreted as species X3 being the direct ancestor of species X2, which is in turn the direct ancestor of species X1, which in turn is the direct ancestor of the modern-day species. At times this (erroneous) expectation is even specifically derided as impossible, since (for example) species X2 appears in the fossil record later than species X1, even though species X2 is “supposed to be the more primitive species.” The misunderstanding arises from (a) the expectation that evolution is a ladder-like progression directly towards present-day species rather than a branching tree of related species and (b) the expectation that the fossil record shows us the preconceived direct progression of transitional forms rather than an infrequent sampling from various parts of the branching tree.)

The origins of vertebrates

With these concepts in place, then, we are, at last, ready to delve into the Cambrian origins of our own group – the vertebrates. As you now understand, seeking to understand the origins of the defining characteristics of vertebrates is to look for stem groups on the vertebrate lineage and examine their characteristics.

Vertebrates are a monophyletic group nested within a larger group known as chordates, which means that understanding vertebrate evolution requires us to examine chordate evolution first. Chordates are defined as organisms that have (1) a hollow dorsal nerve cord, (2) a rod-like, flexible structure called the notochord, (3) a pharynx (with pharyngeal openings, sometimes referred to as “gill slits”) and (4) a tail that extends past the anal opening (a “post-anal tail”). Vertebrates have all of the features of chordates, but add others, such as a (5) brain encased within a skull, and (6) a backbone that replaces the embryonic notochord later in development. As you might expect, there are several Cambrian stem-group species on the vertebrate lineage that allow us to see how the defining vertebrate characteristics were assembled over time – a topic we will explore next.

So far, we have introduced the defining features of chordates:

1. a hollow dorsal nerve cord
2. a rod-like, flexible structure called the notochord
3. a pharynx (with pharyngeal openings, or “gill slits”)
4. a tail that extends past the anal opening (a “post-anal tail”)

We further noted that vertebrates are chordates – since they have all of the above features – but also have a brain encased within a skull, and a backbone. Non-vertebrate chordates include two groups with present-day representatives – cephalochordates (lancelets) and tunicates (sea squirts). Together, these three monophyletic groups form the crown group chordates (i.e. all living chordates, their last common ancestral population, and all of its descendant species, whether living or not). Crown group vertebrates, in turn, are nested within chordates, and chordates themselves are nested within a larger group known as deuterostomes:

Phylogenetic relationships within deuterostomes, showing the nested relationships of vertebrates (blue box) within chordates (red box), and chordates within deuterostomes (green box). All vertebrates are thus chordates, but not all chordates are vertebrates, and so on. The arrow indicates that the position of tunicates and cephalochordates may in fact be reversed. Not to scale.

Building the vertebrate body plan: stem-group deuterostomes

Having located vertebrates in their broader phylogenetic context, we can now trace some of the key innovations that ultimately led to the vertebrate body plan. As we discussed in the last post, this tracing process is not based on finding direct ancestors of crown-group vertebrates, but rather by looking in the fossil record for organisms that branch off the vertebrate lineage at various time points, carrying with them their up-to-that-point-in-time set of characteristics. In other words, we can infer a great deal about the true vertebrate lineage by examining stem-group species in the fossil record.

One challenge for reconstructing the vertebrate family tree is the fact that stem-group deuterostomes (and for that matter, stem-group chordates) were soft-bodied animals, and thus did not fossilize readily. Indeed, what we know about these ancient, soft-bodied species comes from a handful of key fossil sites, where fortuitous circumstances led to their preservation – sites such as the Burgess Shale in Canada, and the Chengjiang region of China. At these sites, we see brief geological “snapshots” of life in the Cambrian period. The Chengjiang fossils capture a window at 525-520 million years ago, and the Burgess Shale captures a slightly later period of the Cambrian at 505 million years ago. Moreover, the flourishing life we see at these sites (and times) of exceptional preservation indicates that the “regular” fossil record of this period, where circumstances did not favor the preservation of soft-bodied organisms, is woefully sparse.

One group of organisms that sheds light on the early origins of deuterostomes is the vetulicolians – a group of Cambrian organisms that remained somewhat of a mystery until recent work, based on new fossils found in Chengjiang, places them as a likely stem-group deuterostome lineage.

The evidence for Vetulicolians as stem-group deuterostomes is based on what appear to be a pharynx and pharyngeal openings. These structures presumably allowed them to expel swallowed seawater and feed as suspension feeders. The fact that a group of species has these traits – without other defining characteristics seen in crown-group deuterostomes – strongly suggests that an early step towards the “vertebrate body plan” was the development of pharyngeal openings:

Building the vertebrate body plan: stem-group chordates

The Cambrian also shows evidence of stem-group chordates in addition to stem-group deuterostomes. One such organism, known from the Burgess Shale, is Pikaia gracilens – a species immediately noted for its resemblance to cephalochordates upon its discovery in the early 1900s. A recent large-scale re-evaluation of Pikaia places it as a stem-group chordate. In support of this placement, Pikaia exhibits a pharynx with pharyngeal openings, a notochord, and a dorsal hollow nerve cord – but appears to lack other defining chordate features (such as a post-anal tail). Accordingly, this evidence supports the hypothesis that the next “step” towards crown-group vertebrates was the development of the dorsal nerve cord and notochord:

Accordingly, the next characteristic to be a acquired would be a post-anal tail, to finally arrive at the defining features of what we now recognize as a hugely successful monophyletic group: the crown-group chordates.

Building the vertebrate body plan: Cambrian crown-group vertebrates

The Chengjiang area is perhaps most famous for the discovery of Cambrian species that are thought to be true vertebrates, including Myllokunmingia and Haikouichthys. These species are thought to be the earliest-known examples of jawless fish, with skulls and backbones made of cartilage. If indeed these species are true vertebrates, they would be part of the vertebrate crown group (though as we shall see in later posts, these species also sit as a stem group to later sub-categories of vertebrates as additional characteristics, such as jaws, are added):

Summing up – from deuterostome to vertebrate

Taken together, what we have seen is that though the vertebrate body plan first appears in the fossil record in the Cambrian period (due to its fortuitous preservation at Chengjiang), there are numerous species in the Cambrian that are identifiable as stem groups on the vertebrate lineage. These stem groups show us that the vertebrate body plan was assembled over time in a stepwise fashion, and that its “sudden appearance” in the Cambrian record is in fact not sudden at all, but rather the end result of a process that extends much deeper into the past.

Previously, we examined the origins of vertebrates (chordates with a skull and a backbone). From their origin as jawless fish, vertebrates have diversified into a number of distinct body plans – body plans as diverse as jawed fish, whales, bats, snakes, and birds. The latter four groups are tetrapods – a remarkably successful monophyletic group of vertebrates that have four limbs as one of their defining characteristics (yes, snakes are tetrapods – we will discuss the loss of limbs in the snake lineage in a future post). The closest-living relatives of tetrapods are lungfish and coelacanths, both of which are groups of lobe-finned fish. Lobe-finned fish, or Sarcopterygii bear their fins on fleshy appendages (from the Greek sarx (flesh) and pteryx (fin)). Numerous lines of evidence support the inclusion of tetrapods within Sarcopterygii, not least of which is the recent whole-genome sequence analysis of coelacanths, and extensive genome comparisons between tetrapods, coelacanths, and lungfish. These lines of evidence place lungfish as the closest living relatives of crown-group tetrapods:

A phylogeny of crown-group Sarcopterygii: modern-day tetrapods, lungfish, and coelacanths, their last common ancestral population, and all of its extinct descendant species. Stem-group tetrapods (marked with a “?”) fill in the “space” between the lungfish and tetrapod crown groups.

Stem tetrapods: the long road from fish to amphibian

Several years ago my family had the opportunity to visit a traveling fossil exhibit at a local science center. I recall the day well, as I was dragged from one spectacular dinosaur to another by a wide-eyed toddler who was experiencing them in person for the first time. Tucked in between the impressive giants my son was bent on seeing, however, was an odd little fish that suddenly I was very excited about – much to the amusement of everyone nearby at the time, who barely gave it a second glance. The object of my excitement was a member of the genus Eusthenopteron – a stem tetrapod close to the last common ancestral population of lungfish and tetrapods.

As we have seen in the last few posts, the key to understanding the transitions that a lineage makes over time is to look for stem groups that branch off the lineage leading to the crown group. While stem group species are not likely to be direct ancestors of the crown group it is possible to find ever-closer relatives of the crown group. These species – though not the direct, ancestral transitional forms leading to the crown group – nonetheless preserve the transitional features that the crown-group lineage passed through.

Perhaps those around me who were passing by the Eusthenopteron exhibit can be excused for their lack of excitement: after all, Eusthenopteron looks like a small, unimpressive fish. Its skeletal structure, however, is anything but dull to a paleontologist. Eusthenopteron had articulated bones in its front lobe fins – bones we recognize in modern-day tetrapods as the humerus, radius, and ulna. These are the long bones that make up the forelimb in crown-group tetrapods – but in Eusthenopteron, these bones are short, and serve as the support for fins, not limbs (in the tetrapod sense). The articulation of these three bones in the forelimb is a trait that Eustenopteron shares with lungfish, but not coelacanths, indicating that this characteristic was present in their last common ancestral population:

The various species of Eustenopteron lived around 385 million years ago, in the Devonian period – a period of earth’s history that was seminal for tetrapod origins, and a fruitful period to search for additional stem tetrapod species.

When you’re going out on a limb, it’s all in the wrist

Once researchers narrow down a timeframe during which a specific set of transitions took place, a concentrated effort to find numerous stem group species in that timeframe is possible. Early amphibian-like (stem-group) tetrapods such as Acanthostega and Ichthyostega appear in the fossil record in the mid-Devonian at about 365 million years ago. This placed the transition from lobe fins to tetrapod limbs within a range – from the branch point of the tetrapod lineage with Eustenopteron (perhaps around 400 – 390 million years ago) to the appearance of these amphibious tetrapods. Accordingly, in the last few decades, much effort has been expended to search for stem-group tetrapods from this time period to illuminate the transition to the crown-group tetrapod state.

One such find that generated significant interest when it was announced was Tiktaalik roseae, a stem-group tetrapod first discovered in the Canadian Arctic in 2004. Tiktaalik is a remarkable fossil in that it is a lobe-finned fish with strikingly tetrapod-like features, such as elbows, flexible wrists (including wrist bones that correspond to the wrist bones of crown-group tetrapods), a flexible neck, and forelimbs (forefins?) capable of supporting its weight, leading to speculation that it may have propped itself up out of its shallow water habitat from time to time.

These discoveries (and many others – feathered dinosaurs don’t even seem to make the popular press anymore, since they are now so common) thus “fill in the space” between non-avian theropods and birds – to the point where the boundary is so blurred that some new discoveries are debated as being “true birds” or merely very close non-avian relatives.

The use of “half a wing”

So, despite decades of protests from antievolutionists that no possible intermediate forms for birds could exist, we see a group of fossil species that meets the criteria handily – and demonstrates that yes, there was a use for “partially-formed feathers and wings.” Long before birds took to the air, feathers were a common feature of terrestrial, non-flying dinosaurs with a body plan markedly similar to birds. Indeed, at this time and place in biological prehistory, the body plan of “non-avian, feathered theropod” (and later, “quasi-avian, feathered theropod”) seems to have been a remarkably successful one, as evidenced by the number of species we observe in the fossil record that fit this general description. Feathers and wings are thus examples of exaptation– the repurposing of parts through evolution. Feathers were originally selected for a non-flight function (insulation, for example) and later were co-opted for another function (flight). Likewise, the wing is a repurposed (i.e. exapted) tetrapod forelimb. Had Darwin lived to see their discovery, no doubt he would have seen feathered theropods as another “grand case” for his theory.

Next, we’ll return to the tetrapod lineage leading to our own species – that of mammals.

Stem groups as “transitional forms”

Though we’ve repeatedly emphasized that stem-group species are not the direct ancestors of a crown group, it is by now hopefully clear that stem groups are in fact the “transitional forms” that the fossil record can be reasonably expected to show, given the infrequent nature of the fossilization process. The fact that a species like Tiktaalik is highly unlikely to be the direct ancestor of modern tetrapods is no objection to appreciating the combination of characteristics it possesses, and how (almost perfectly) intermediate it is in form between lobe-finned fish and tetrapods. To object that stem-group species are not direct ancestors – and thus not informative about modern-day species – is to miss the pattern that the fossil record presents to us: groups of species in nested sets, but sets whose boundaries are blurred wherever we have sufficient data to see it.

Next, we’ll trace the tetrapod lineage further into the future — and examine the diversification of birds and mammals.

As a brief recap, we have traced the origins and diversification of vertebrates from their (pre) Cambrian origins, through jawless and jawed fishes, and on to the origins and diversification of stem-group tetrapods (“fishapods”?) in the late Devonian.

From this amphibian-like origin, crown-group tetrapods continued to diversify and acquire new characteristics that we can mention only briefly – such as the key transition to laying eggs on land with a membrane separating the egg from the terrestrial environment (i.e. an amniotic egg), and the subsequent diversification of amniotes into reptiles, birds, and mammals (as well as a number of extinct lineages that are known only from the fossil record).

Just winging it

With all of this tetrapod diversity to explore, we can only hope to visit a few points of special interest along the way – transitions that will also serve to illustrate other features of evolution that we have not yet discussed in great detail.

One transition that has long fascinated scientists is the origin of birds, with their striking adaptations of feathers and powered flight. These adaptations place modern birds at a sizeable distance from other present-day tetrapods – a fact that was troublesome to Darwin. Only a few years after the publication of his On the Origin of Species, however, a stunning stem-group bird was discovered in Germany – Archaeopteryx lithographica.

Even to a casual observer, Archaeopteryx displays a mix of “reptilian” and “avian” characteristics. Like reptiles, Archaeopteryx had a long bony tail, teeth, and forelimbs with clawed digits – but combined these features with a trait that until then had been thought to be the sole hallmark of birds – feathered wings. For Darwin, Archaeopteryx was simultaneously support for his theory as well as a reminder of the paucity of the fossil record. He would express these thoughts in a letter to a colleague in 1863:

The fossil Bird with the long tail & fingers to its wings (I hear from Falconer that Owen has not done the work well) is by far the greatest prodigy of recent times. It is a grand case for me; as no group was so isolated as Birds; & it shows how little we know what lived during former times.

Later, he would include a discussion of Archaeopteryx in a revised edition of On the Origin, as well as note that it had “affinities” with a then-known theropod dinosaur, Compsognathus:

Let us now look to the mutual affinities of extinct and living species. They all fall into a few grand classes; and this fact is at once explained on the principle of descent. The more ancient any form is, the more, as a general rule, it differs from living forms. But, as Buckland long ago remarked, all extinct species can be classed either in still existing groups, or between them. That the extinct forms of life help to fill up the intervals between existing genera, families, and orders, cannot be disputed. For if we confine our attention either to the living or to the extinct alone, the series is far less perfect than if we combine both into one general system. With respect to the vertebrata, whole pages could be filled with illustrations from Owen, showing how extinct animals fall in between existing groups… Another distinguished palæontologist, M. Gaudry, shows that very many of the fossil mammals discovered by him in Attica connect in the plainest manner existing genera. Even the wide interval between birds and reptiles has been shown by Professor Huxley to be partially bridged over in the most unexpected manner, by, on the one hand, the ostrich and extinct Archeopteryx (sic), and on the other hand, the Compsognathus, one of the Dinosaurians—that group which includes the most gigantic of all terrestrial reptiles.

So, even in Darwin’s time, the evidence supported the hypothesis that Archaeopteryx was a transitional form in the sense that we have been discussing – as a stem group on the lineage leading to modern birds – with that stem rooted within theropod dinosaurs.

Theropods of a feather, group together

Despite the early discovery of Archaeopteryx, other fossil species that “fill up the interval” between crown-group birds and extinct theropods were unknown until over 100 years later. In the mid 1990s, however, the first of what would be a number of significant discoveries was made in deposits of the Yixian Formation in China – namely, a feathered, non-avian theropod dinosaur named Sinosauropteryx prima. This fossil was noteworthy not merely because it was feathered and related to Compsognathus, but also because of the nature of its feathers – this theropod possessed only relatively simple “protofeathers” – unbranched filaments that could serve as insulation. Sinosauropteryx seems to have branched off the avian lineage at a time when feathers were not (yet) even branched, let alone adapted for flight.

Not long after the discovery of Sinosauropteryx, an additional “transitional form” – i.e. stem group on the avian lineage displaying transitional characteristics – was found in China that revealed a progression in feather evolution towards the feathers found in modern birds. Sinornithosaurus is not only a feathered, non-avian theropod, but one with tufted and branched feathers, which up until its discovery, had only been observed in birds:

These discoveries (and many others – feathered dinosaurs don’t even seem to make the popular press anymore, since they are now so common) thus “fill in the space” between non-avian theropods and birds – to the point where the boundary is so blurred that some new discoveries are debated as being “true birds” or merely very close non-avian relatives.

The use of “half a wing”

So, despite decades of protests from antievolutionists that no possible intermediate forms for birds could exist, we see a group of fossil species that meets the criteria handily – and demonstrates that yes, there was a use for “partially-formed feathers and wings.” Long before birds took to the air, feathers were a common feature of terrestrial, non-flying dinosaurs with a body plan markedly similar to birds. Indeed, at this time and place in biological prehistory, the body plan of “non-avian, feathered theropod” (and later, “quasi-avian, feathered theropod”) seems to have been a remarkably successful one, as evidenced by the number of species we observe in the fossil record that fit this general description. Feathers and wings are thus examples of exaptation– the repurposing of parts through evolution. Feathers were originally selected for a non-flight function (insulation, for example) and later were co-opted for another function (flight). Likewise, the wing is a repurposed (i.e. exapted) tetrapod forelimb. Had Darwin lived to see their discovery, no doubt he would have seen feathered theropods as another “grand case” for his theory.

Next, we’ll return to the tetrapod lineage leading to our own species – that of mammals.

Stem-group mammals: the synapsids

Mammals are the only living representatives of a group called synapsids, a group that parted ways with the dinosaur/avian lineage (the sauropsids) and went on to diversify beginning in the late Carboniferous period, around 325 million years ago. The numerous synapsids known from the fossil record are stem-group mammals – organisms related to mammals that branch off from the lineage leading to crown-group mammals (the last common ancestral population for all living mammals, and all of that population’s descendant species). The fact that some of these stem-group, extinct synapsids were once known as “mammal-like reptiles” and others as “reptile-like mammals” reveals the transitional nature of their features – they blur the distinction between “reptiles” and a “mammals” in the way we are now familiar with: through the gradual accumulation of traits characteristic of crown-group mammals, and in a branching pattern that indicates the order in which those characteristics were acquired. Examples of such acquired traits include jaw morphology (including the co-option and repurposing – i.e. exaptation – of jaw bones for a specialized hearing function in the inner ear), the development of hair, and lactation (the secretion of milk for feeding young).

From egg to placenta

Sometimes I encounter non-biologists (and even some biologists) who are surprised to learn that “live birth” is not a defining characteristic of mammals (or, more precisely, ofcrown-group mammals). The reason for this is because a lineage of egg-laying mammals, the monotremes, has living representatives. The fact that egg-laying mammals exist in the present day means that they are part of the crown group (by definition), and as such any characteristic that they lack cannot be a defining feature of the crown group:

Crown-group mammals include egg-laying mammals (monotremes) as well as non-egg-laying mammals (marsupials and placental mammals). Of the features shown on this phylogeny, only lactation is a characteristic common to the entire crown group.

In egg-laying mammals, such as the platypus and the various species of echidna, young hatch from the egg and then are nourished by their mother’s milk, which is secreted from a patch on the skin, and lapped by the young. In marsupials, pregnancy is much shorter than in placental mammals, and after birth the (still very much embryonic) young crawl to a protected pouch where they nurse at a teat in order to complete their development. In marsupials a brief connection is formed in utero between the embryo and the mother through the yolk sac membrane to form a “yolk sac” placenta. Yes, it is somewhat confusing that both marsupials and “placental” mammals – i.e. eutherians – both have a placenta. The difference is that placental mammals form their placenta from a different membrane – the chorioallantoic membrane.

While monotremes and marsupials are not stem-group mammals (since their lineages persist to the present day) we can appreciate their features in exactly the same way that we have done for stem groups. (Put another way, if the monotreme and marsupial lineages had in fact gone extinct, we would call eutherians “mammals” and monotremes and marsupials would be stem groups on the eutherian lineage.) Similarly to what we have seen with stem groups, monotremes and marsupials demonstrate that the eutherian state was arrived at over time, and through a series of gradual steps. Though in reality this process was a gradient, we can arbitrarily denote some “stages” along the way:

• Egg laying with after-hatching lactation (monotreme state)
• Short gestation with a yolk-sac placenta with post-birth lactation (marsupial state)
• Gestation with a combination of yolk-sac and chorioallantoic placentas (that extended gestation time) with post-birth lactation
• Reduction of the yolk-sac placenta in favor of the chorioallantoic placenta (with further extended gestation time) with post-birth lactation
• Long gestation with a chorioallantoic placenta with post-birth lactation (the eutherian condition).

Along the way, metatherians and eutherians would shift away from yolk-based nutrition for their embryos in utero, and shift towards nutrition based on their placentas. In doing so, the biochemical machinery for yolk-based nutrition would be predicted to become less and less important – and eventually, useless altogether. At the genetic level, genes required for yolk production would eventually no longer contribute to the survival or reproduction of the organism – meaning that they were no longer under selection, and now free to accumulate mutations without consequence to the organism.

Seeking the dead among the living

In practical terms, when a gene is no longer under natural selection, it is then maintained only by the overall precision of DNA replication during cell division. While quite accurate, DNA replication is not perfect. For sequences under natural selection, mutations are removed from the population if the individuals carrying those variants cannot reproduce at the same frequency as their non-mutated relatives. For genes no longer subject to natural selection, mutations will accumulate slowly over time.

For marsupial and placental mammals, one such gene, named vitellogenin, is one that would be expected to be released from selection after the establishment of a placenta. Vitellogenin is vitally important to the formation of egg yolk, since it acts as a major carrier of nutrients from the liver to the forming egg yolk in egg-laying organisms. In 2008, a research group went looking for the remains of vitellogenin sequences in placental, marsupial, and monotreme mammals. Monotremes, as you would expect, have a functional vitellogenin gene sequence, since they are egg-laying mammals. While marsupials and placental mammals do not have functional vitellogenin gene sequences, they do have the (heavily) mutated remains of vitellogenin sequences, indicating that these lineages once did have functional biochemical machinery to transfer nutrients in bulk to egg yolk. This observation makes perfect sense in light of a phylogenetic prediction that placentals and marsupials share a common ancestral population with monotremes (and of course other tetrapods) – with egg-laying as the ancestral state that was subsequently lost in the marsupial – placental common ancestral population: