Complex Evolutionary and Genetic Patterns Characterize the Loss of Scleral Ossification in the Blind Cavefish Astyanax mexicanus

Abstract

The sclera is the tough outer covering of the eye that provides structural support and helps maintain intraocular pressure. In some fishes, reptiles, and birds, the sclera is reinforced with an additional ring of hyaline cartilage or bone that forms from scleral ossicles. Currently, the evolutionary and genetic basis of scleral ossification is poorly understood, especially in teleost fishes. We assessed scleral ossification among several groups of the Mexican tetra (Astyanax mexicanus), which exhibit both an eyed and eyeless morph. Although eyed Astyanax surface fish have bony sclera similar to other teleosts, the ossicles of blind Astyanax cavefish generally do not form. We first sampled cavefish from multiple independent populations and used ancestral character state reconstructions to determine how many times scleral ossification has been lost. We then confirmed these results by assessing complementation of scleral ossification among the F1 hybrid progeny of two cavefish populations. Finally, we quantified the number of scleral ossicles present among the F2 hybrid progeny of a cross between surface fish and cavefish, and used this information to identify quantitative trait loci (QTL) responsible for this trait. Our results indicate that the loss of scleral ossification is common-but not ubiquitous-among Astyanax cavefish, and that this trait has been convergently lost at least three times. The presence of wild-type, ossified sclera among the F1 hybrid progeny of a cross between different cavefish populations confirms the convergent evolution of this trait. However, a strongly skewed distribution of scleral ossicles found among surface fish x cavefish F2 hybrids suggests that scleral ossification is a threshold trait with a complex genetic basis. Quantitative genetic mapping identified a single QTL for scleral ossification on Astyanax linkage group 1. We estimate that the threshold for this trait is likely determined by at least three genetic factors which may control the severity and onset of lens degeneration in cavefishes. We conclude that complex evolutionary and genetic patterns underlie the loss of scleral ossification in Astyanax cavefish.

Fig 1. Variation in scleral ossification among eyeless cave populations of the Mexican tetra, Astyanax mexicanus.

(A) Five populations exhibit degenerate or unossified sclera. Of these, four are members of the evolutionarily “Old” El Abra cavefishes (Chica, Curva, Sabinos, and Pachón; shown in red), and one is a member of the evolutionarily “Young” Guatemalan cavefishes (Molino; shown in blue). (B) Two populations exhibit wholly or partially ossified sclera. One is a member of the “Old” El Abra cavefishes (Tinaja) and another is a member of the additional lineage of “Young” cavefishes, the Micos cavefishes (Rio Subterráneo; shown in green). (C) The biogeographical distribution of the populations mentioned in A and B, along with 22 other known populations. Red = El Abra (A–E), Blue = Guatemalan (F), Green = Micos (G). (D) Ancestral character state reconstruction of scleral ossification using a recent molecular phylogeny of 11 surface fish populations and 5 of 7 cavefish populations used in this study [35]. The hypothetical positions of the remaining two populations are shown with a dashed line. Pie charts on internal nodes illustrate the ML support for ossified (white) vs. unossified (black) sclera at each ancestral position. Assuming that all surface fish populations exhibit ossified sclera, the ancestral character state reconstructions suggest that scleral ossification has been lost at least three times among the various cavefish populations.

Fig 2. Restoration of scleral ossification following complementation between two different cavefish populations.

(A) Diagram shows the results of crossing Pachón and Sabinos cavefishes. Diagram modified from Wilkens [54]. (B) A small ossified sclera develops in the F₁ progeny of a Pachón × Sabinos cross with restored eyes.

Fig 3. Limited phenotypic variation in scleral ossification among the progeny of a SF x CF F₂ hybrid cross.

(A) Illustrative examples of reduced and normal scleral ossicle formation among 196 hybrid F₂ progeny. Individual scleral ossicles are stained red with alizarin and highlighted with black arrows. (B) A ventral view of the same eyes as in (A). The length of the scleral ossicles varied dramatically, with some that occupy the entire circumference of the eye. White bars on the last image indicate where the width of the scleral ring and diameter of the eye were measured. (C) Distribution of the scleral phenotypes measured in this study. Both the number of scleral ossicles and the circumference of the sclera occupied by ossicles were highly skewed towards the wild-type phenotype of two ossicles that occupy >90% of the eye. Dotted line in the second panel indicates the threshold (90%) used to denote wild-type versus reduced scleral ossicles.

Fig 4. Quantitative trait loci (QTL) for Astyanax scleral ossification.

(A) Quantitative trait locus (QTL) mapping identified one QTL for the proportion or percent of the circumference of the eye occupied by scleral ossicles on linkage group (LG) 1, and another QTL for the relative width of the sclera on LG 2 that was marginally non-significant at P < 0.07. No QTL was found for ossicle number, though the power of this analysis was likely limited by the small number of F₂ individuals with only 0 or 1 scleral ossicles (see Fig 3). Please refer to O’Quin et al. [29] for the complete genetic linkage map used in this analysis. (B) Detailed view of the scleral QTL on LGs 1 and 2. Red line indicates P = 0.05; any value above that threshold is considered statistically significant. Shaded fields indicate the 95% Bayesian confidence interval for the location of the QTL. Inset boxes show the mean phenotypes for each genotypic class at the peak QTL marker. Black and white boxes at the top of the chart indicate the position of eye or lens size QTL found in this (black) or other (white) studies (see also S1 Fig).