Cave fishes differ from surface fishes in numerous ways because of their evolutionary adaptations to life in the dark. Most noticeably, they have smaller eyes and less pigmentation, but they also have better senses of smell and taste, and are more efficient metabolically. All of these differences are under genetic control; the gene copies in the cave populations affecting important developmental systems differ from those in the surface populations. The Mexican Tetra is unique in that the species has both cave and surface forms. Because they can hybridize, they afford the opportunity to investigate the differences through genetic analysis. In this collaborative research project, the genes responsible for these differences will be identified by genetic mapping and functional analyses of selected candidate genes. The results will lead to a better understanding of the genetic pathways that control the development and maintenance of: (1) the visual system; (2) pigmentation; (3) metabolic efficiency; and (4) various other physiological and anatomical systems. In terms of Broader Impacts, the work on cave fishes has been well covered by the media, and the extraordinary ability of this system to engage people's interest and attention will have broad positive impacts on public education in biology, genetics and evolution. The research will also impact the training of undergraduate and graduate students and postdoctoral fellows, particularly in novel concepts of evolution and development (evodevo). In terms of societal impacts, the results may also contribute to a general understanding of the causes of eye degeneration and metabolic disorders and could lead to a better insight into how environmental changes (i.e., the shift from surface to cave environments) can modulate the direction of evolutionary change.
The broad goal of this investigation is to learn more about the genetic changes responsible for differences in morphology, physiology and behavior between closely related populations or species. We study the Mexican tetra, a freshwater fish with some populations inhabiting surface rivers and others inhabiting caves. The cave fish populations are derived from surface ancestors that colonized the cave environment and adapted through evolution to life in perpetual darkness and limited access to food. Compared to their surface relatives, the cave forms have greatly reduced eyes and pigmentation (Image1), more efficient metabolisms, and altered patterns of behaviors. Study of these trait differences is important because they all have genetic bases; knowledge of the genes responsible for the differences will tell us much about the genetics of normal (and abnormal) eye development, physiology and behavior. What we learn will be as relevant to humans as to fish. During the course of the investigation we constructed high density genetic linkage maps which gave the positions of genes and other genetic markers (called "SNPs") along the chromosomes. Using these SNP maps we were able to find previously undetected regions in the genome where there were genetic differences (called "QTLs") between cave and surface fish that were responsible for the phenotypic differences. Thus, the QTLs mark the positions in the genome where important evolutionary changes occurred. By analyzing variation in SNP frequency among surface and cave populations, we identified a subset of SNPs whose variation bore hallmarks of natural selection. Many of these "selected SNPs" mapped exactly to the locations of QTL. This established that natural selection played important roles in the evolutionary adaptation of fish to the cave environment. We identified QTL for cave adapted traits in independently evolved cave populations and established that the different populations often converged on the same phenotypes (e.g., eye and pigment loss) because of changes in different genes. We also showed that many of the genes selected for in the cave populations were not new mutations that occurred after the fish had colonized the caves, but were pre-existing at low frequencies in the ancestral surface populations. Thus, adaptation to the cave environment by selection appears to have begun immediately upon colonization and did not depend upon the slow acquisition of new mutations. We detected numerous new QTL for cave-adapted feeding and schooling behaviors. We established that many of the "selected SNPs" fall within or very near to genes that are likely candidates for the traits detected by QTL analysis. These genes are now being sequenced and will be tested for the effect of their variation on the relevant trait. The cave fish scientific community received a big boost from the new availability of a sequenced genome of the cave form (sequenced at Washington University). We were instrumental in advancing the genome sequencing effort, supplying the Beijing Genome Institute (BGI) with DNA from the surface form and the rationale for its sequencing. BGI has sequenced and assembled the surface genome and is in the process of annotation. When this becomes available, researchers will be able to compare the two genomes directly, facilitating discovery of important genetic differences. An unanticipated, but important outcome of this research was our discovery that cave fish sleep much less than related surface fishes. Surface fish average about 12 hours of sleep per day, whereas cave fish sleep only two or three hours (Image 2). Patterns of sleep can be analyzed in terms of numbers and durations of sleep bouts. Numbers of sleep bouts per night do not differ between cave and surface fish, but the lengths of sleep bouts in cave fish are much shorter. That is, cave fish fall asleep as often as surface fish, but they don’t stay asleep for nearly as long (Image 3). This pattern of sleeplessness closely resembles that found in certain types of insomnia in humans, where sufferers fall asleep but soon awaken and remain awake for extended periods. We discovered that treating cave fish with propranolol, a blocker of beta adrenergic signaling, restored "insomniac" cave fish to the normal sleeping pattern. This suggests that further investigations of the underlying genetics of this cave fish trait could aid in the treatment of some human sleep disorders. We successfully performed QTL analysis of sleep differences and identified six regions in the genome which might contain genes influencing sleep patterns. Significantly, two of the "selected SNPs" exactly correspond with two of these QTL, and more significantly, one of them sits within a gene for a beta adrenergic signaling protein. This is work in progress. In summary, the work conducted during the course of this grant has identified genes and numerous new candidates affecting eye and pigmentation development as well as behavior, and has opened a new line of investigation that might result in ameliorating poor sleep patterns in humans.
