Cryptic variation in morphological evolution: HSP90 as a capacitor for loss of eyes in cavefish

Abstract

In the process of morphological evolution, the extent to which cryptic, preexisting variation provides a substrate for natural selection has been controversial. We provide evidence that heat shock protein 90 (HSP90) phenotypically masks standing eye-size variation in surface populations of the cavefish Astyanax mexicanus. This variation is exposed by HSP90 inhibition and can be selected for, ultimately yielding a reduced-eye phenotype even in the presence of full HSP90 activity. Raising surface fish under conditions found in caves taxes the HSP90 system, unmasking the same phenotypic variation as does direct inhibition of HSP90. These results suggest that cryptic variation played a role in the evolution of eye loss in cavefish and provide the first evidence for HSP90 as a capacitor for morphological evolution in a natural setting.

Fig. 1

Reduction of HSP90 levels in Astyanax mexicanus using the chemical inhibitor Radicicol. (A) Inhibition of HSP90 using 500nM Radicicol leads to activation of BAG3 and HSPB1 (two-tailed t-test: **= p<0.005, ***= p<0.0005). Time scale refers to hours of treatment. (B) Variable eye sizes in surface A. mexicanus larvae after treatment. (C) Quantification of eye size in adult F2 hybrids after larval treatment of Radicicol reveals a significant increase in standard deviation of eye size while average eye size is not affected (two sided F-test: p=0.0004; Bartlett’s test: p=0.001; Levene’s test: p=0.03). Note that raising the fish in the dark alone does not affect eye size. Values were corrected for body size using standard length of the fish. (D) Examples of eye size variation in F2 population of hybrid A. mexicanus.

Fig. 2

HSP90 inhibition in natural populations of Astyanax mexicanus. (A,B) HSP90 inhibition in natural populations of surface Astyanax mexicanus leads to an increase of variation in eye size (A) and orbit size (B). Eye size SD+ 83% - two sided F-test: p=8.1E-6; Bartlett’s test: p<0.001, Levene’s test: p<0.001; Orbit size SD + 108% - two sided F-test: p=3.4E-6; Bartlett’s test: p<0.001, Levene’s test p<0.001. (C) Orbit size decreases after Radicicol treatment in Tinaja cave populations (t-test: p=0.02), while change in variation is observed (SD -12%: two sided F-test: p=0.239). Control is DMSO. Asterisks in (A) and (B) compare standard deviations, while (C) compares averages.

Fig. 3

Genetic assimilation. (A–B) Selection for small eye size in surface fish generated by Radicicol treatment resulted in offspring with significantly smaller orbit size (A) and eye size (B) in the absence of treatment (two-tailed t-test: p=6.5E-13 for orbit size and p=7.2E-16 for eye size). The resultant range exceeded the range seen in any cross of untreated surface fish.

Fig. 4

Low conductivity conditions in the cave natural habitat have a similar effect to Radicicol treatment on surface populations. (A) qRT-PCR of BAG3 and HSPB1 for surface fish reared under low conducitivity (230 μS) conditions compared to control conductivity conditions (two-tailed t-test: *= p<0.05, **= p<0.005, ***= p<0.0005). Time scale refers to hours of treatment. (B–C) Lower conductivity conditions reveal an increase in variation of orbit size (B) and eye size (C) (Eye size SD +50% - two sided F-test: p=0.0018; Bartlett’s test: p=0.006, Levene’s test: p=0.005; Orbit size SD +58% - two sided F-test: p=5.9E-4; Bartlett’s test: p=0.001, Levene’s test p=0.01).