November 2015 By guest authors Christopher A. Emerling, PhD, NSF Postdoctoral Fellow, UC Berkeley Museum of Vertebrate Zoology and Stephanie Keep, Editor of Reports of the National Center for Science Education, and science curriculum consultant for Keep Learning, LLC

Where's the evolution?

Snakes originated at least 100 million years ago. Some experts contend that snakes evolved on land and lost their limbs — first the forelimbs, then the hindlimbs — as they adapted to a burrowing lifestyle. Others prefer the hypothesis that they evolved from a group of aquatic reptiles, and lost their limbs as they adapted to the sea. Either way, snakes probably descended from a lineage of fully legged lizards based on three general lines of evidence. First, snakes are genetically nested deep within the lizard clade (Squamata). Second, the fossil record of snakes includes several species that retained hindlimbs (e.g., Eupodophis, Pachyrhachis), with a recently discovered putative (and controversial!) snake that possessed all four limbs (Tetrapodophis). Finally, some of the earlier branches of the snake phylogenetic tree, particularly boas and pythons, include species that possess embryonic hind limb buds that fail to develop into fully formed limbs.

In a previous post, we discussed how genes can be completely deleted from a genome or accumulate inactivating mutations, thereby rendering the gene nonfunctional. You might imagine that the genes encoding limbs were similarly inactivated or lost from the genomes of snakes. In reality, however, there are probably no true “limb genes.” Instead, limbs appear to develop based on the contributions of various developmental genes that have multiple functions. Such genes are called pleiotropic, and researchers do not typically expect pleiotropic genes to disappear or become “broken” during evolution since it could have widespread and deleterious effects on an organism’s phenotype.

If natural selection prevents animals from losing the genes that are involved in developing appendages, how is it that species like snakes have lost their limbs? One probable pathway is via the disruption of DNA regions known as enhancers. Enhancers are not genes, meaning they do not encode a protein, but they do regulate gene activity. They do this via proteins known as transcription factors, which bind to the enhancers, leading to the transcription of a (usually) nearby gene. Multiple enhancers can be associated with a single gene, and different transcription factors bind to distinct enhancers, controlling when and where the gene is turned on. The regulatory effects of transcription factors and enhancers helps to explain how an entire organism can develop from a single, undifferentiated fertilized egg. As sets of regulatory proteins interact with distinct enhancers, they direct different sets of genes to express themselves in various locations. This allows cells to specialize and differentiate, eventually forming tissues and organs.

If an enhancer were deleted, some functions of a gene may be lost, but enhancers associated with other sets of transcription factors — and therefore other gene functions — would be unaffected. Thus, the researchers were curious: Did snakes lose their limbs because limb-specific enhancers were disrupted during snake evolution? The team examined the genomes of a boa (Boa constrictor), Burmese python (Python bivittatus), and king cobra (Ophiophagus hannah) and compared them to four-legged reptilian relatives such as the American chameleon (Anolis carolinensis) and painted turtle (Chrysemys picta). Though they found some leg enhancers were missing in the genomes of snakes, many were retained, suggesting that they may have functions outside of limb development.

The researchers dove further into this question and discovered that much of the regulatory DNA associated with limb development, including enhancers, overlaps with the genital tubercle, the developmental predecessor to the phallus. They then examined the Tbx4 gene, a transcription factor expressed during formation of the mouse hindlimb and genital tubercle. They confirmed that Tbx4 is expressed in the embryonic hindlimb of the chameleon and the rudimentary hind limbs of the ball python (Python regius), as well as the phalluses of both species and the corn snake (Pantherophis guttatus). Next, they examined HLEB, an enhancer associated with Tbx4. When the researchers transmitted the HLEB enhancer from the chameleon to the mouse, it retained its activity in both the hindlimbs and the genital tubercle, whereas when they transmitted HLEB from the Burmese python and king cobra to the mouse, it lost all hindlimb activity! Together, these data suggest that snakes have retained limb-development genes and the limb-associated enhancers involved in phallus development, though these have evolved in such a way that their limb-development function has been lost.

One of the incredibly useful aspects about evolution is that multiple lineages can independently evolve the same phenotype, allowing for multiple tests of the same hypotheses. As such, understanding how snake genomes evolved to lose their limb-building toolkit can be useful in informing how other lineages lost their limbs. As we have discussed in a previous article, numerous other lineages of lizards have reduced their limbs or eliminated them entirely, including skinks, geckos, and many others. Do these legless lizards show a similar pattern of limb enhancer loss? Or, did they evolve leglessness by another mechanism altogether? Further research is necessary to test these hypotheses.

Among mammals, whales appear to have descended from small hoofed mammals, with the earliest whales having fully intact hind limbs. In most modern species, however, the hindlimbs have disappeared, though some retain a small remnant. Nonetheless, there are multiple recorded examples of individual dolphins with hind flippers, suggesting they may retain some of the genomic toolkit to develop these structures, perhaps including many of the hind limb specific enhancers. By contrast, almost all whales have retained their pelvis bones, though not as the “nonfunctional vestiges” that they are so often portrayed as being. Whale pelvic bones have an important function, and it’s one that broadens the hindlimb-phallus connection discovered in snakes: They are attachment sites for the muscles that control movement of the whale’s penis. In fact, recent research suggests that the size and shape of these whale pelvic bones may be evolving under sexual selection.

Finally, the caecilians represent a group of amphibians that no longer have legs, and also do not have true phalluses. Though they have a structure with a similar function, true phalluses probably appeared in the lineage that gave rise to mammals, reptiles and birds. In fact, a paper published just within the last week showed that embryos of a very basal lineage of reptiles, the tuatara, have the same phallus precursors as other reptiles, birds and mammals, despite not having a phallus as an adult. Since caecilians independently evolved a phallus and lack limbs, they may provide a better understanding of how the development of limbs and phalluses became intertwined.

So what can we learn about our limbs from the limbless? Quite a bit it turns out, especially in regards to their relation to the penis! By studying the genomes evolution of other lineages of limbless animals, certainly many more answers about limb development can be gleaned.