Blogging on Peer-Reviewed Research

Fossils are cool, but some of us are interested in processes and structures that don’t fossilize well. For instance, if you want to know more about the evolution of mammalian reproduction, you’d best not pin your hopes on the discovery of a series of fossilized placentas, or fossilized mammary glands … and although a few fossilized invertebrate embryos have been discovered, their preservation relied on conditions not found inside the rotting gut cavity of dead pregnant mammals.

You’d think this would mean we’re right out of luck, but as it turns out, we have a place to turn to, a different kind of fossil. These are fossil genes, relics of our ancient past, and they are found by digging in the debris of our genomes. By comparing the sequences of genes of known function in different lineages, we can get a measure of divergence times … and in the case of some genes which have discrete functions, we can even plot the times of origin or loss of those particular functions in the organism’s history.

Here’s one example. We don’t have any fossilized placentas, but we know that there was an important transition in the mammalian lineage: we had to have shifted from producing eggs in which yolk was the primary source of embryonic nutrition to a state where the embryo acquired its nutrition from a direct interface with maternal circulation, the placenta. We modern mammals don’t need yolk at all … but could there be vestiges of yolk proteins still left buried in our genome? The answer, which you already know since I’m writing this, is yes.

First, a little background. It’s not that surprising to find traces of yolk proteins in our genomes, because we also have the evidence of embryology that shows that our embryos still make a yolk sac! Below is a series of diagrams of the human embryo over the last several weeks of the first month of pregnancy, and you can see the large sac hanging from the embryo; it’s a useless fluid filled space that contains no yolk at all, but is homologous to similar structures that form in birds and reptiles.

Sometimes people refuse to believe that we could have a yolk sac, and they don’t trust cartoons, so here’s a photo of a 28-somite stage embryo. The side view on the left nicely shows the branchial arches (they also don’t want to believe in those), but the one on the right is the same embryo rotated, so you can see the huge empty balloon of the yolk sac.


We retain the sac, but what about the contents? Where are the yolk proteins? The primary component of yolk is made from a protein called vitellogenin. Vitellogenin is a large (250-600kD) glycophospholipoprotein, which basically means that it has a protein core that is extensively modified by the addition of sugars, phosphates, and fatty acids — it’s a great greasy lump of protein, fat, and sugar, just the thing growing embryos need to eat. Animals with yolky eggs synthesize vitellogenin in their livers, and transport it the oviducts, the site of egg production, where it is deposited in the yolk sac, and also further broken down into the two major yolk proteins, phosvitin and lipovitellin. Mammals don’t make vitellogenin at all, although there are some interesting similarities between portions of vitellogenins and lipoproteins that we use to transport fats in our circulatory systems (the atherogenic lipoproteins that are the curse of our modern diets may be related to the lipoproteins our ancestors used to feed their embryos.)

We can follow the evolutionary history of the vitellogenin gene. Tetrapod ancestors, 350-400 million years ago, had two copies of the gene, called VIT1 and VITanc (multiple copies of a gene with high demand for its gene product, like yolk proteins, is advantageous for boosting output). Some time before the mammalian lineage diverged from the reptile/bird lineage, there was a duplication of VITanc to form VIT2 and VIT3 … so chickens have 3 vitellogenin genes, VIT1, VIT2, and VIT3.

How do we know that this duplication occured before the mammalian line split off? Because we also have VIT1, VIT2, and VIT3 in our genomes! They are irreparably broken and non-functional, and eroded by time, but Brawand et al. found them, and identified them by sequence similarity and by synteny, or the identity of the adjacent genes.

When non-functional genes, called pseudogenes, like this are found, one thing one can do is estimate the time of loss of function from the amount of decay. Natural selection is a force that maintains genes, and in its absence, they tend to slowly fall apart as they accumulate mutations. Browsing through the genome is like strolling through a run-down neighborhood. Houses that are still occupied will be maintained and kept up. Houses that have been recently abandoned might have an overgrown lawn and broken windows. Houses that have been neglected longer still might show signs of fire damage, or structural collapse, or might have been demolished right down to their foundations. By measuring the divergence of mammalian pseudogenes for vitellogenin from bird vitellogenin genes, for instance, we can estimate the time of loss.

Rather than counting broken windows, in genes we count the accumulation of stop codons (sequences that signal the end of transcription) and indels. An indel is a single insertion or deletion of a stretch of nucleotides in a gene, and in the lineages studied here they occur at a rate of slightly more than 1 x 10-10 per site per year, so it’s like a very slowly ticking clock that gradually scrambles the pseudogene.

The results are summarized in this diagram.

(Click for larger image)

VIT Gene Evolution in Tetrapods: The topology and divergence times of the tree are based on previous studies. Latin crosses indicate VIT inactivation events in eutherians and monotremes. Inactivation estimates (including approximated 95% prediction intervals) based on opossum VIT sequences are indicated by colored bars at the top (see also Figure 3). Duplications (?x2?) are indicated. VITanc is the likely ancestor of both the amphibian vtgA1/vtgA2 and VIT2/VIT3 genes in birds. Functional VIT genes in extant species are indicated in red. The inactivation time of VIT1* on the amphibian branch could not be estimated because of its absence in Xenopus tropicalis.

The loss of vitellogenin was not abrupt. VIT1 and VIT3 became nonfunctional about 150 million years ago (note, though, the wide range of possible times, caused by uncertainty in the methods), roughly corresponding to the evolution of eutherian ancestors and after viviparity. VIT2 hung in there until about 70 million years ago, suggesting that maybe those Cretaceous mammals were still pumping a little yolk protein into those yolk sacs, as a supplemental nutrition source.

The monotremes have also lost most of their vitellogenin genes, but still retain one, to no one’s surprise — they still lay eggs. Furthermore, VIT1 was only relatively recently lost, about 50 million years ago.

One other detail in the chart is of interest. It shows that nutritive lactation arose before placentation and loss of the vitellogenin genes. Again, no one has found fossil mammary glands; instead, they looked at genes important in milk production, in particular, the casein milk genes. Casein is a secreted calcium-binding phosphoprotein that is essential for transporting calcium to the embryo, and calcium is a critical growth-limiting mineral during embryogenesis. We have caseins, of course, and the platypus is found to have orthologous casein genes, which tells us that these genes arose before the monotreme and eutherian split.

Taken together, these data tell a story. Lactation evolved first, representing a gradual shift in parental investment from storage of yolk in eggs to later, post-hatching care. This reduced selective constraints on yolk production — three genes were overkill for the level of output needed — and was permissive in allowing the gradual decay of the VIT genes. Viviparity and placentation then made the yolk proteins more and more superfluous, as embryos became more and more reliant on simply tapping directly into the maternal blood supply. The process represents a pattern of change away from stockpiling massive quantities of nutritional supplies for future growth, to a more efficient just-in-time delivery system.

The story is all right there in your genes. You’re walking around carrying the crumbling record of hundreds of millions of years of history — all we need is the tools to extract it and read it.

Brawand D, Wahli W, Kaessmann H (2008) Loss of egg yolk genes in mammals and the origin of lactation and placentation. PLoS Biol 6(3):e63.

Sadler TW (2004) Langman’s Medical Embryology, 9th ed. Lippincott Williams and Wilkins, Baltimore.


  1. #1 Torbjörn Larsson, OM
    March 19, 2008

    So you are saying that I, as a mammal, can be egg-static over reproduction?

    Figures. But nice to know, so I’m joining the choir here.

  2. #2 David Marjanovi?, OM
    March 19, 2008

    The divergence dates of the tree are for the most part implausible. There’s a good fossil record of the Eutheria-Metatheria divergence… 125 million years ago, 130 perhaps, but never 180. 180 is where the Theria-Monotremata divergence should be put. Did you see how the tree implies that VIT2 was separately inactivated in the ancestry of primates and carnivores? The fossil record puts that divergence close to 65 million years ago, so VIT2 was probably inactivated just once, in the ancestry of Placentalia.

    Ironically, the 310-million-year date (actually 312) for the theropsid-sauropsid divergence is taken from the fossil record — but it’s the absolute minimum of a very badly constrained date! The maximum is somewhere around 335… People should really stop using this divergence as a calibration point.

    (And, as mentioned, monotremes are too mammals. And yes, stop codons signal the end of translation…)

    How do you get minerals to preserve their shape?

    Your question contains half of the answer. Fast decay can produce acids, which can precipitate minerals that happen to be around, et voilà.

    In really exceptional cases, namely the Tyrannosaurus blood vessels and not much else, decay is prevented altogether by… fast burial and dryness perhaps… that’s not well understood yet.

  3. #3 David Marjanovi?, OM
    March 19, 2008

    I understand why there’s a cross marking our Lutheran Ancestor, but what are the other crosses?

    When a gene goes extinct, it receives a Christian funeral…

    Clicking on the picture gives you a large, readable version.

  4. #4 David Marjanovi?, OM
    March 20, 2008

    One source said that multituberculates have pelvic anatomy that might indicate a marsupial type of reproduction with tiny young born after a brief gestation.

    Yes (the pelvic canal is really small), but Wikipedia probably has the phylogeny wrong and the multis are probably more closely related to us than the monotremes are.

    That’s not what is expected *at all*. Lactation occurs *after birth*. Yolk *and* placenta are *pre-birth* strategies. Lactation is a completely seperate issue having to do with the development stage of the animal *at birth*, not before.

    No: marsupials are born at a much earlier stage than monotremes hatch (and even these hatch quite early, like highly altricial birds AFAIK). That placentals spend the fetal stage in the womb instead of in a pouch is probably a secondary development.

    Keith Eaton, did you notice how self-defeating your point is? Fine, so the yolk sac is not useless — it still produces blood cells like it does in all other vertebrates! Yet another thing we have in common. Why should that be? Perhaps we all share a common ancestor? Hey, why don’t we produce our first blood cells in the placenta?

    Keep asking “why”. Science is about “why” questions. All those people who say science is only about “how” and philosophy and/or religion are about “why” don’t know what they’re talking about.

  5. #5 David Marjanovi?, OM
    March 20, 2008

    No: marsupials are born at a much earlier stage than monotremes hatch (and even these hatch quite early, like highly altricial birds AFAIK). That placentals spend the fetal stage in the womb instead of in a pouch is probably a secondary development.

    I should have explained what my point is: Normal amniotes spend the fetal stage in the egg and live off yolk. Marsupials, and probably live-bearing mammals generally, spend the fetal stage in the pouch and live off milk. Thus, milk is a yolk replacement, with monotremes representing the intermediate condition. That placentals spend the fetal stage in the womb and live off blood is a later development — a third strategy.

  6. #6 Mary
    August 15, 2008

    excellent! I work with Vitellogenin of tropical fish and I really enjoyed this article and the revision that make.. thanks!!