Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer.

The molecular mechanisms underlying major phenotypic changes that have evolved repeatedly in nature are generally unknown. Pelvic loss in different natural populations of threespine stickleback fish has occurred through regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The high prevalence of deletion mutations at Pitx1 may be influenced by inherent structural features of the locus. Although Pitx1 null mutations are lethal in laboratory animals, Pitx1 regulatory mutations show molecular signatures of positive selection in pelvic-reduced populations. These studies illustrate how major expression and morphological changes can arise from single mutational leaps in natural populations, producing new adaptive alleles via recurrent regulatory alterations in a key developmental control gene. E volutionary biology has been animated by long-standing debates about the number and type of genetic alterations that underlie evolutionary change. Questions about the roles of genetic changes of infinitesimally small versus large effects, the origin of traits by either natural selection or genetic drift, and the relative importance of coding and regulatory changes in evolution are currently being actively investigated (1-4). One of the classic examples of major evolutionary change in vertebrates is the extensive modification of paired appendages seen in different species (5). Although essential for many forms of locomotion, paired appendages have also been repeatedly lost in some fish, amphibian, reptile, and mammalian lineages, probably via selection for streamlined body forms (6). Threespine stickleback fish (Gasterosteus aculeatus) make it possible to analyze the evolution, genetics, and development of major skeletal changes in natural populations (7). The pelvic apparatus of marine sticklebacks consists of prominent serrated spines that articulate with an underlying pelvic girdle that extends along the ventral and lateral sides of the fish (inspiring the scientific name Gasterosteus aculeatus, or bony stomach with spines). Although most sticklebacks develop a robust pelvic apparatus, over two dozen widely distributed and probably independent freshwater stickleback populations show partial or complete loss of pelvic structures (8). Several factors may contribute to repeated evolution of pelvic reduction, including the absence of gape-limited predatory fish, limited calcium availability, and predation by grasping insects (9-12). Genome-wide linkage mapping has identified a single chromosome region that explains more than two thirds of the variance in pelvic size in crosses with pelvic-reduced sticklebacks (13)(14)(15). This region contains Pituitary homeobox 1 (Pitx1), a gene expressed in hindlimbs but not forelimbs of many different vertebrates and required for normal hindlimb development (13). Although the Pitx1 gene of pelvic-reduced sticklebacks shows no protein-coding changes as compared with that of ancestral marine fish, its expression in the developing pelvic region is almost completely lost (13, 16). On the basis of the map location, changes in expression, and directional asymmetry shared in both Pitx1-null mice and pelvic-reduced sticklebacks, cis-regulatory mutations at the Pitx1 locus have been proposed as the basis of stickleback pelvic reduction (13). However, regulatory mutations are difficult to identify, and the actual sequences controlling pelvic reduction have remained hypothetical (2). cis-regulatory changes at Pitx1 locus. Although Pitx1 represents a strong candidate gene for pelvic reduction, other genes in the larger chromosome region could be the real cause of pelvic loss, leading to secondary or trans-acting reduction of Pitx1 expression (2). To test this possibility, we generated F1 hybrids between pelvic-complete [Friant Low (FRIL) and pelvic-reduced (Paxton Lake Benthic (PAXB)] sticklebacks [see table S1 for geographic location of all populations used in this stud