PMC

Evolved differences in tooth number, area, and spacing appear late during development. Developmental time courses of laboratory-reared marine (red) and benthic (blue) fish of different total body lengths (x axis) for tooth number (A), tooth plate area (B), and tooth spacing (C). All three traits have diverged after 20-mm fish length.

Fine mapping of the chromosome 21 tooth number QTL centers around Bmp6. Genotype–phenotype association for genetic markers (circles) across chromosome 21 (x axis). The position of Bmp6 is marked with the arrow, and the dashed line is the significance threshold of α = 0.05 determined by permutation tests (LOD score of 2.2).

Heritable evolved tooth gain in derived benthic fish. (A) MicroCT images of wild adult stickleback ventral pharyngeal tooth plates of marine fish from Rabbit Slough, Alaska (Left) and benthic fish from Paxton Lake, Canada (Right). (Scale bar, 1 mm.) (B and C) Total ventral pharyngeal tooth number in wild (B) and laboratory-reared (C) adults shows benthic fish have significantly higher tooth counts (P = 8.8 × 10⁻⁷ and P = 4.9 × 10⁻⁷ in two-tailed t tests for wild and laboratory-reared, respectively). Error bars are SEM.

Genome-wide linkage mapping for tooth number, tooth plate area, and tooth spacing in a marine × benthic F2 genetic cross. Genome-wide QTL mapping results for tooth number (A), tooth plate area (B), and intertooth spacing (C). All significant QTL are highlighted in red, and all chromosomes with significant effects on at least one tooth phenotype are shaded gray. The largest-effect tooth QTL on chromosome 21 is highlighted in yellow. The dashed line is the significance threshold of α = 0.05 determined by permutation tests (LOD scores of 4.1 for all three traits).

cis-regulatory up-regulation of the benthic allele of Bmp6 in late, not early, stages of tooth development. Shown are the ratios of benthic to marine alleles measured by pyrosequencing assays from either genomic DNA (light gray) or tooth plate cDNA (dark gray) from benthic × marine F1 hybrids at three different developmental stages. No significant difference in Bmp6 expression was detected between marine and benthic alleles at the larval stage, but at the juvenile and adult stage the benthic allele was significantly up-regulated (sample sizes and P values by the Wilcoxon signed rank test for early, juvenile, and adult are n = 12, P = 0.27; n = 18, P = 0.0003; and n = 13, P = 0.0005, respectively). Error bars are SEM.

Bmp6 is expressed in developing stickleback teeth. Gene expression in developing benthic (A–F and H) and marine (G) stickleback teeth at 7.5 d postfertilization (dpf) (A–E) and 15 dpf (8 mm, F–H) revealed by in situ hybridization in whole-mount (A–C, G, and H) and 40-µm vibratome sections of comparably staged developing tooth germs (D–F and ). (A–F) Tooth markers Shh (A and D) and Pitx2 (B and E) are detected in the odontogenic epithelium, whereas Bmp6 is expressed dynamically in odontogenic epithelium early (C and H) and in odontogenic mesenchyme in newly ossifying teeth (F and H). (G and H) Bmp6 continues to be expressed in teeth later in development in both marine and benthic larvae. White arrowheads, odontogenic epithelium; asterisks, newly mineralized developing teeth; black arrowheads, odontogenic mesenchyme. (Scale bars, A–F = 50 μm, G and H = 100 µm.)