Q: What unique organ is found only in mammals, but not in fish, amphibians, reptiles, or birds?

The title and that little picture to the left ought to be hint enough, but if not, read on.

A: The vagina. Aren't we lucky?

There's an old joke going around about poor design: what kind of designer would route the sewer pipes right through the center of the entertainment center? It's a good point. It doesn't make sense from a design standpoint to have our reproductive and excretory systems so intimately intermingled, but it does make a heck of a lot of sense from a purely historical point of view. In a sense, reproduction is an excretory function—we are shedding gametes produced internally, and we already have a perfectly good set of pipes running from our insides to the outside, so why not use them? It's just that in our lineage, which has specialized in giving great care to our gametes and zygotes, that plumbing has become increasingly elaborate, and that part of the system that was once just a convenient throughway has become a destination and a long-term residence in its own right.

Development tells us part of the story. The reproductive and urinary tracts are all tangled together in early development, arising together from two pairs of ducts, the Müllerian and Wolffian ducts, which are modified in complex ways to form a series of kidneys (we keep only the last one, the metanephros), one set of pathways for the male testes, and yet another set for the female ovaries.

In non-therian mammals, all of these complicated pipes have one common destination, a single outlet to the external world: the cloaca. "Cloaca" is Latin for sewer, and it is appropriately named. The terminus of the large intestine is here, as well as the ends of the ureters from the kidneys and the ducts from the ovaries or testes. Everything gets dumped in to the cavity of the cloaca, making a nice stew of feces, urine, and sperm or eggs. Mmm-mmm. The cloaca is the grey cylinder at the bottom of figure A, below, in the first three organisms, amphibians, birds/reptiles, and monotremes (my apologies for the murkiness of the image; it's the best copy I have).

Evolution of the tetrapod reproductive system. (A) Female urogenital system from major tetrapod lineages. Inf, infundibulum; Ov, ovary; Ovd, oviduct; Ut, uterus (or shell-producing region in non-therian animals); Vg, vagina; Kd, kidney; Ud, urinary duct; Rc, rectum; Ub, urinary bladder; Cl, cloaca. (B) Tetrapod egg. *, the shell coat of birds and some reptiles is highly calcified. MPS, marsupial-specific mucopolysaccharide layer. (C) Tetrapod phylogeny showing major transitions in mammalian reproduction. Divergence of amphibians and amniotes (A). Divergence of birds/reptiles and mammals (B). Divergence of monotremes and therians (C). Divergence of marsupials and placentals (D).

The fundamental organization of the reproductive part of the vertebrate urogenital tract is straightforward: it's a tube with a funnel at one end that captures eggs released by the ovary, and conducts them to an external orifice. Along the way, cells lining the tube secrete useful products like albumin and yolk, and deposit a shell, and may act to temporarily store the egg before its final release.

Marsupial and placental mammals have dispensed with most of those functions, and expanded on others. One part of the oviduct has acquired a richly vascularized epithelium and specializations for investing and nurturing a resident embryo, becoming a uterus. That's an amazing and innovative function in itself, but in addition, it has formed a new, separate channel, the vagina. The vagina is an entirely new structure, which has no homolog in amphibians or reptiles.

That is an interesting observation. It's a wholly original structure that arose sometime after the monotreme-marsupial split, an evolutionary novelty. How did that happen? How can we study a unique event that occurred over 150 million years ago?

Evolutionary tree showing placement of the three groups of living mammals (colored boxes and icons at top) with respect to selected Mesozoic taxa. Branching times for the black tree are based on the earliest known fossil occurrences of taxa (black dots).The red tree is based on molecular divergence times for monotremes-therians and eutherians- metatherians.

Wagner and Lynch have a proposal to answer both questions. The general mechanism for generating novel structures is evo-devo orthodoxy:

1. An epigenetic side effect of other evolutionary changes in the body leading to a novel physical structure in the organisms.
2. The genetic consolidation and individuation of the novel structure.

(Note that this proposes phenotype before genotype, which is somewhat heretical for neodarwinism. It shouldn't trouble the evo-devo gang in the slightest, of course.)

How to study such a process from the past?

The basic assumption of a molecular evolutionary approach to the study of evolutionary novelties is that changes in developmental regulation have left traces in the molecular structure of the genome and a comparative study of genomic structures should be able to identify genetic changes coincidental with a phenotypic novelty. (emphasis mine)

That process of consolidation and individuation would have left detectable scars in the genome—the genes involved would have acquired changes necessary to fix the phenotype in the population. Again, as we'd expect from the evo-devo perspective, those changes would have been made to the regulatory genes that control tissue-specific gene expression. What genes should we examine? Let's look at the therian organs of interest, and here are some likely candidates: the HoxA genes that have region-specific domains in the female reproductive tract.

Hox gene expression pattern and the evolution of the female reproductive tract. (a) HoxA-13 to HoxA-9 are located at the 5' end of the HoxA cluster and are expressed in the same regions in the adult as in the embryo: HoxA-13 (green), HoxA-11 (yellow), HoxA-10 (orange) and HoxA-9 (blue). (b) Tetrapod phylogeny showing representative female reproductive systems from each group (amphibian ovaries shown only on the left).

Phylogenetic relationships among a small set of vertebrate species including representatives of the major mammalian clades: monotremes (platypus), marsupials (opposum) and placentals (Hyrax and human). Above some branches the estimated number of non-synonymous and synonymous substitutions of HoxA-11. The estimates are obtained from a maximum likelihood codon model as implemented in PAML. Note that in the stem lineages of therians (i.e., the lineage leading to the most recent common ancestor of opossum and the placentals), there are five to six non-synonymous substitutions but no synonymous substitution. This indicates a very strong selection coincidental with the evolution of the internal developmental mode of mammals.

The HoxA-9 through HoxA-13 genes are expressed in order along the length of the embryonic Müllerian duct, and also continue to be expressed in adulthood; so the cells of the vagina are all expressing HoxA-13, while the cells of the cervix all have HoxA-11 turned on (for some reason, I find that to be a wonderful piece of knowledge, and I just have to say…Hooray for HoxA-13! It has just become my favorite Hox gene.)

So the question is whether there is any evidence that these particular Hox genes have signs of any set of changes that are associated with particular transitions in vertebrate evolution—in particular, are there differences that can be traced to the transition between monotremes and the theria, and between the placentals and marsupials. The answer seems to be yes: the diagram to the right is a measure of the number of synonymous to nonsynonymous changes in HoxA-11, which is an indicator of the selective pressures that have shaped the gene.

Furthermore, they've identified where these changes have occurred, and they are not in the homeodomain (the part of the protein that binds to specific sequences in the DNA, but in the amino terminal end.

Approximate positions of the amino acids positions of HoxA-11 which are under directional selection between the most recent common ancestor of all extant mammals and the most recent common ancestor of placentals. Note that all of these substitutions are N-terminal of the homeodomain and affect small clusters of amino acids.

The 3-D models below show where the relevant amino acids (in yellow) end up in the folded protein. The interesting thing here is that regulatory proteins don't just interact with each other, but also with other regulatory proteins that are simultaneously binding. It's a whole chain of interactions—regulatory proteins binding to the DNA, and also binding between each other in a complex called the enhancersome—that determines the level of expression of a particular gene.

HoxA-11 protein structure. This three-dimensional protein model was calculated by comparative modeling as part of the MODBASE project. (A) Model shown as ribbons. (B) Model rendered with a molecular surface. The DNA-binding homeodomain is shown in red. The carboxy-terminal region of exon 2 is shown in blue. Residues identified as being under directional (positive) selection in the stem lineage of eutherians are shown in yellow. Residues replaced in the stem lineage of therians but not identified under selection are shown in green. Note that all of these amino acid sites affect amino acids that are predicted to be placed on the surface of the molecule as expected if selection is driven by novel protein-protein interactions.

There is a great deal left to be done. Hox genes are rather high up the chain of regulatory genes, so there are many more genes downstream that have to be puzzled out. We also are a long ways from figuring out how these patterns of gene expression define the morphogenetic processes that create this lovely novel structure, the vagina. The important thing, though, is that there are these questions waiting to be answered—the investigators have a research program.

We propose that a research program to explain evolutionary novelties has to focus on the question of whether novel characters arise through the evolution of novel regulatory links among developmental genes. We further propose that a detailed analysis of the evolution of developmental genes involved in the development of a derived, novel character can reveal molecular changes that could be causally involved in the origin of evolutionary novelties. The case study presented here suggests that the statistical methods of molecular evolution are strong enough to provide specific hypothesis for experimental test. The success of this research program will depend on the ability to connect the patterns of molecular evolution with the functional role of these molecular changes.

That's the cool thing about evolutionary biology: exciting questions, titillating ancestors, and the promise of tools to answer more.

Lynch VJ, Roth JJ, Takahashi K, Dunn CW, Nonaka DF, Stopper GF, Wagner GP (2004) Adaptive evolution of HoxA-11 and HoxA-13 at the origin of the uterus in mammals. Proc Biol Sci. 271(1554):2201-7.

Wagner GP, Lynch VJ (2005) Molecular evolution of evolutionary novelties: the vagina and uterus of therian mammals. J Exp Zoolog B Mol Dev Evol. [Epub ahead of print]

Cifelli RL, Davis BM (2003) Marsupial Origins. Science 302:1899-1900.