Parallel genetic origins of pelvic reduction in vertebrates

Abstract

Despite longstanding interest in parallel evolution, little is known about the genes that control similar traits in different lineages of vertebrates. Pelvic reduction in stickleback fish (family Gasterosteidae) provides a striking example of parallel evolution in a genetically tractable system. Previous studies suggest that cis-acting regulatory changes at the Pitx1 locus control pelvic reduction in a population of threespine sticklebacks (Gasterosteus aculeatus). In this study, progeny from intergeneric crosses between pelvic-reduced threespine and ninespine (Pungitius pungitius) sticklebacks also showed severe pelvic reduction, implicating a similar genetic origin for this trait in both genera. Comparative sequencing studies in complete and pelvic-reduced Pungitius revealed no differences in the Pitx1 coding sequences, but Pitx1 expression was absent from the prospective pelvic region of larvae from pelvic-reduced parents. A much more phylogenetically distant example of pelvic reduction, loss of hindlimbs in manatees, shows a similar left-right size bias that is a morphological signature of Pitx1-mediated pelvic reduction in both sticklebacks and mice. These multiple lines of evidence suggest that changes in Pitx1 may represent a key mechanism of morphological evolution in multiple populations, species, and genera of sticklebacks, as well as in distantly related vertebrate lineages.

Fig. 1.

Pelvic morphology of intergeneric hybrid progeny. Total bilateral pelvic scores range from 0 (absent pelvis) to 8 (complete pelvis with 4 elements on each side). (a) Parental and representative hybrid fish from Mud Lake control cross showing strong development of pelvic structures. Distribution of scores is skewed toward complete pelvises. (b) In contrast, hybrid progeny from pelvisless parents show very weak or no pelvic development. (c and d) In crosses with one complete-pelvis and one pelvisless parent, distributions of hybrid progeny pelvic scores are skewed toward strong pelvic development. ec, left ectocoracoid; ps, pelvic spine. (Scale bars, 2 mm.)

Fig. 2.

Genomic structure and amino acid sequence of Pitx1 in Pungitius and Gasterosteus. (a) The coding sequence comprises five exons; both genera have splice variants missing exon 3. Putative translation start sites vary between Pungitius (black triangles) and Gasterosteus (white triangles), and stop sites are conserved (black diamonds). Exon 1 is noncoding in Gasterosteus. (b) Predicted amino acid sequences of Pitx1 splice variants in Pungitius (Pp1, Pp2) and Gasterosteus (Ga1, Ga2). Arrows mark exon boundaries.

Fig. 3.

Pelvic expression of Pitx1 differs between control and Fox Holes Lakes Pungitius larvae. (a, c, and d) Whole-mount in situ hybridization of larvae from a complete-pelvis (control) population shows Pitx1 expression in the mouth and lower jaw (enlarged lateral view in c) and the prospective pelvic region (enlarged ventral view in d). (b, e, and f) Pitx1 expression is also detected in the mouth and lower jaw of Fox Holes Lakes larvae, but expression is absent from the prospective pelvic region. [Scale bars, 1 mm (a and b) and 0.5 mm (c–f).]

Fig. 4.

Asymmetry is a morphological signature of pelvic reduction in multiple, distantly related vertebrates. (a) In both an F₂ threespine stickleback cross (Upper) and a natural population of Florida manatees (Lower), pelvic remnants tend to be larger on the left side of the body than on the right. Each bar on the histograms represents a different individual; negative values indicate a larger right remnant. (b) Complementation and mapping crosses suggest that Pitx1 is involved repeatedly in the evolution of pelvic reduction in different populations of threespine (Gasterosteus) and ninespine (Pungitius) sticklebacks. Freshwater populations diverged from marine populations 10,000–20,000 years ago, whereas the two genera shared a complete-pelvis common ancestor at least 10 million years ago. Similar genetic mechanisms may underlie pelvic reduction in manatees, which diverged hundreds of millions of years ago from the lineage that includes sticklebacks and tens of millions of years ago from complete-pelvis relatives, such as elephants. Animal drawings in (b) are modified after refs. , , , .