Adaptive divergence in the thyroid hormone signaling pathway in the stickleback radiation

Abstract

During adaptive radiations, animals colonize diverse environments, which requires adaptation in multiple phenotypic traits. Because hormones mediate the dynamic regulation of suites of phenotypic traits, evolutionary changes in hormonal signaling pathways might contribute to adaptation to new environments. Here we report changes in the thyroid hormone signaling pathway in stream-resident ecotypes of threespine stickleback fish (Gasterosteus aculeatus), which have repeatedly evolved from ancestral marine ecotypes. Stream-resident fish exhibit a lower plasma concentration of thyroid hormone and a lower metabolic rate, which is likely adaptive for permanent residency in small streams. The thyroid-stimulating hormone-β2 (TSHβ2) gene exhibited significantly lower mRNA expression in pituitary glands of stream-resident sticklebacks relative to marine sticklebacks. Some of the difference in TSHβ2 transcript levels can be explained by cis-regulatory differences at the TSHβ2 gene locus. Consistent with these expression differences, a strong signature of divergent natural selection was found at the TSHβ2 genomic locus. By contrast, there were no differences between the marine and stream-resident ecotypes in mRNA levels or genomic sequence in the paralogous TSHβ1 gene. Our data indicate that evolutionary changes in hormonal signaling have played an important role in the postglacial adaptive radiation of sticklebacks.

Figure 1. Low plasma thyroid hormone concentration and metabolic rate in stream-resident stickleback

(A) Representative images of a marine ecotype (Duwamish River, Site 2 in Figure 1B) and a stream-resident ecotype of threespine stickleback (Big Soos Creek, Site 14 in Figure 1B). Skeletal structures are visualized with alizarin red staining. Scale bar = 10 mm. (B) Map of collection sites in the Pacific Northwest of North America (upper panel) and the Japanese Archipelago (lower panel). Red dots indicate the collection sites of marine fish, while blue dots indicate the collection sites of stream-resident fish. Numbers indicate the sampling sites: (1) Little Campbell River marine; (2) Duwamish River marine; (3) Clam Bay marine; (4) Seabeck Bay marine; (5) Quilcene Bay marine; (6) Akkeshi Pacific Ocean marine; (7) Akkeshi Japan Sea marine; (8) Little Campbell River stream; (9) Salmon River stream; (10) Evans Creek stream; (11) Forbes Creek stream; (12) Kelsey Creek stream; (13) May Creek stream; (14) Big Soos Creek stream; (15) Allen Creek stream; (16) Hiranoi Creek stream. (C) Plasma T4 concentration (mean ± SEM) of sticklebacks caught in early summer. Red bars indicate marine populations, while blue bars indicate stream-resident populations. Sample sizes for each population are shown above each bar. The number in parenthesis indicates the collection site shown in Figure 1B. (D) Plasma T4 concentration (mean ± SEM) of laboratory-raised sticklebacks sampled under a short photoperiod (left) or a long photoperiod (right). A grand mean (± SEM) of two independent families for each cross is shown in the graph. Data for each family are available in Figure S1A. Sample sizes for each group are shown above each bar. (E) Plasma T3 concentration (mean ± SEM) of laboratory-raised sticklebacks sampled under a short photoperiod (left) or wild fish (Little Campbell River marine and stream-resident ecotypes) caught in early summer (right). Sample sizes for each group are shown above each bar. (F) Effect of thyroid hormone inhibitor (thiourea) and thyroid hormone (thyroxine, T4) treatment on oxygen consumption rate. Oxygen consumption rate was divided by body weight (g) and shown as rate per hour. Mean ± SEM is shown. Sample sizes for each group are shown above each bar. Letters above the bars indicate the results of Waller-Duncan’s posthoc tests after ANOVA. (G) Expression levels of 74 genes involved in oxidative phosphorylation were compared between marine and stream-resident ecotypes by microarray. Red bars indicate the genes expressed more in marine fish than in stream-resident fish, while the blue bars indicate the genes expressed more in stream-resident fish than in marine fish. Fold change of transcript level (marine fish signal divided by the stream-resident fish signal) is shown as the logarithm to base 2, so 1 indicates that the signal intensity of the transcript is twice as high in marine fish than in stream-resident fish, while -1 indicates the signal intensity of the transcript is twice as high in stream-resident fish than in marine fish.

Figure 2. Divergent TSHβ2 transcript level and cis-regulatory sequence between marine and stream-resident sticklebacks

(A) Photoperiodic response of total pituitary TSHβι (upper panel) and TSHβ2 (lower panel) mRNA level. A grand mean (± SEM) of two independent families is shown in the graph. Sample sizes for each group are shown above each bar. Expression level was determined by comparing the amplification threshold cycle to that of a serially diluted standard cDNA sample, followed by multiplication of the amount of RNA isolated from each pituitary gland. (B) Three representative SNPs at the TSHβ1 locus did not show any significant segregation between marine and stream-resident ecotypes (left panel). Four representative SNPs at the TSHβ2 locus significantly differed in frequency between marine and stream-resident ecotypes (right panel). Results of genetic analyses are available in Table S1. In both panels, red squares indicate that an individual is homozygous for one SNP allele, blue squares indicate that an individual is homozygous for the alternative allele, and yellow squares indicate that an individual is heterozygous. Details of SNP information are shown in Figure S2. (C) Pyrosequencing of pituitary cDNA in F1 hybrid fish between a Little Campbell River marine female and a Little Campbell River-resident male. The left panel indicates a representative pyrogram showing a higher peak on C than on G at a SNP site (for the SNP, see Fig. S2G). The right panel indicates the ratio (mean ± SEM) of marine to stream-resident TSHβ2 allelic expression (n = 10).