Accelerated regulatory gene evolution in an adaptive radiation

Abstract

The disparity between rates of morphological and molecular evolution remains a key paradox in evolutionary genetics. A proposed resolution to this paradox has been the conjecture that morphological evolution proceeds via diversification in regulatory loci, and that phenotypic evolution may correlate better with regulatory gene divergence. This conjecture can be tested by examining rates of regulatory gene evolution in species that display rapid morphological diversification within adaptive radiations. We have isolated homologues to the Arabidopsis APETALA3 (ASAP3/TM6) and APETALA1 (ASAP1) floral regulatory genes and the CHLOROPHYLL A/B BINDING PROTEIN9 (ASCAB9) photosynthetic structural gene from species in the Hawaiian silversword alliance, a premier example of plant adaptive radiation. We have compared rates of regulatory and structural gene evolution in the Hawaiian species to those in related species of North American tarweeds. Molecular evolutionary analyses indicate significant increases in nonsynonymous relative to synonymous nucleotide substitution rates in the ASAP3/TM6 and ASAP1 regulatory genes in the rapidly evolving Hawaiian species. By contrast, no general increase is evident in neutral mutation rates for these loci in the Hawaiian species. An increase in nonsynonymous relative to synonymous nucleotide substitution rate is also evident in the ASCAB9 structural gene in the Hawaiian species, but not to the extent displayed in the regulatory loci. The significantly accelerated rates of regulatory gene evolution in the Hawaiian species may reflect the influence of allopolyploidy or of selection and adaptive divergence. The analyses suggest that accelerated rates of regulatory gene evolution may accompany rapid morphological diversification in adaptive radiations.

Figure 1

(A) Gene maps of ASAP3/TM6 and ASAP1 loci. Exons are shown as numbered boxes. The gene maps depicted are for the A copies of the loci. Upright and inverted triangles represent major deletions and insertions, respectively, that characterize the B duplicate copies. Numbers above the triangles provide the sizes of the indels. Arrows show positions of PCR primers used to isolate genomic sequences. Circles indicate approximate location of copy-specific RT-PCR primers used in gene expression assays. A 100-bp scale bar is provided. Relative sizes of exons and introns in the amplified regions were derived from comparison of genomic and cDNA sequences. Exon sizes outside the amplified regions are estimates based on comparison with data from A. thaliana orthologues. (B) Expression of the ASAP3/TM6 and ASAP1 floral regulatory genes in developing inflorescences of D. arborea. Expression was assayed with gene-specific primers (for the duplicate A and B copies) that amplified ≈300 nucleotides of cDNA in RT-PCR reactions. Control reactions using cloned A and B gene copies indicate the primers are copy-specific. Identity of amplified products was confirmed by sequencing.

Figure 2

Distribution of Ka/Ks values for the ASAP3/TM6, ASAP1, and ASCAB9 genes in the North American tarweeds and Hawaiian silversword alliance. Mean values and standard deviations are indicated. Pairwise comparisons that had no synonymous substitutions are not shown in the histograms and were not included in the analyses. Individual pairwise Ka and Ks estimates are shown in Tables 2–11, which are published as supplemental data on the PNAS web site.

Figure 3

Phylogenies of the (A) ASAP3/TM6 and (B) ASAP1 genes in the North American tarweeds (green) and Hawaiian silversword alliance (blue). The phylogenies were reconstructed by using coding region and noncoding region sequences. The numbers of nonsynonymous (N) and synonymous (S) nucleotide substitutions in the coding regions inferred along each branch by maximum likelihood ancestral state reconstructions are given as a ratio (N/S). Levels of bootstrap support are shown next to the nodes. Nodes with less than 70% bootstrap support, and with no coding region nucleotide substitutions inferred along the subtending branch, are collapsed. For the North American species, the generic abbreviations are: A, Anisocarpus; Ad, Adenothamnus; C, Carlquistia; Ca, Calycadenia; Ce, Centromadia; D, Deinandra; H, Harmonia; K, Kyhosia; M, Madia; O, Osmadenia; and R, Raillardella. For the Hawaiian species, the generic abbreviations are: A, Argyroxiphium; D, Dubautia; and W, Wilkesia.