The hypothesis that evolution is truly irreversible, known as Dollo's Law, has become a question of increasing interest, as several recent studies have made claims that complex structures, such as an eye or insect wing, can be recovered after loss. For example, recent analysis of a family of cavefishes from the southeastern U.S. suggests that a surface-dwelling fish with functional eyes has evolved from a blind, cave-dwelling ancestor. The alternative is that an ancestor with functional eyes repeatedly gave rise to separate blind cavefish species. To test these hypotheses, this study examines genes involved in eye development. The alternative hypotheses make distinct predictions about similarities and differences between species with regard to molecular changes in eye-related genes in blind versus sighted lineages. However, the differences may be subtle and examining a multiple-gene dataset will provide the power needed to test the predictions of Dollo's Law versus reverse evolution. This study demonstrates a way forward when other analyses suggest, but cannot confirm, that reevolution of complex structures is likely.
This study will provide insight into the molecular evolution of several eye- and pigment-related genes in a vertebrate and might aid understanding of visual dysfunctions in humans. In addition, discoveries from this research will be incorporated into ongoing education and outreach efforts to local schools and organizations. Educational activities will confront common misconceptions about "use and disuse" in evolution, and promote understanding of how genes and environments jointly affect traits.
The repeated degeneration or loss of traits in many evolutionary lineages has long been of interest to biologists. Despite broad appreciation of the phenomenon of regressive evolution, the mechanisms underlying character loss remain poorly understood. Important questions include how character loss affects the evolution of genes formerly involved in its development and function, whether degeneration is driven by natural selection or random processes, and if lost characters can be reacquired by later descendants. Cave animals are popular examples of regressive evolution, as many lineages have independently lost their eyes and pigmentation. However, few studies have demonstrated that loss of eyes is associated with changes in vision-related genes at the molecular level. In particular, many studies have concluded that the visual photoreceptor gene rhodopsin, one of the best-studied retinal proteins, seems to have retained functionality in eyeless animals. Explanations for the apparent maintenance of function have focused on hypothetical roles for the protein in other, non-visual processes. In contrast, our study of molecular evolution of rhodopsin in amblyopsid cavefishes (see image) revealed repeated loss of functional constraint in this gene, with at least three cave lineages having independently accumulated unique loss-of-function mutations over the last 10 million years. Although several cave lineages still possess an ostensibly functional rhodopsin, they exhibit increased numbers of mutations that have greater effects on the structure and function of the protein compared to mutations in surface lineages (Figure 2). Existence of functional eye-related genes in some cave lineages may be explained simply because they are recently descended from sighted, surface-dwelling ancestors, and not enough time has passed for the random accumulation of loss-of-function mutations. Although our results are consistent with the neutral accumulation of mutations in rhodopsin, adaptive mechanisms for degeneration of vision cannot be ruled out, as changes in other genes upstream in the eye developmental pathway might be advantageous in cave populations. Another of our results regarding the evolutionary relationships of surface and cave-dwelling fishes raised the intriguing possibility that vision and use of surface habitats have re-evolved, in one instance, from a subterranean ancestor. A fundamental question in evolutionary biology is whether evolution in reversible. A theory dubbed "Dollo’s Law" posits that complex characters are unlikely to be reacquired once lost, yet the recent literature included several putative examples of re-evolution. When we took presence or absence of a functional eye as a single character and mapped it onto the phylogenetic tree of amblyopsids, our analysis supported re-evolution of eyes in a group called spring cavefishes. However, several other lines of evidence lead us to the alternative conclusion that spring cavefishes are descended from an unbroken line of sighted ancestors inhabiting the cave –surface interface, while eyeless cave specialists have repeatedly and independently branched off from that ancestral lineage. The evidence includes details of eye morphology (more nuanced than "presence or absence of a functional eye") and independent loss-of-function mutations in rhodopsin in different cave lineages. This work shows that ancestral reconstructions can occasionally yield strongly supported yet misleading results. Thorough analysis of molecular evolution of genes associated with a character of interest can provide important insights in studies of trait evolution and function. In addition to the basic research findings, this project created the opportunity for us to train undergraduate students and engage in formal and informal outreach/education activities with the Knoxville Zoo and National Speleological Society.
