Cranial asymmetry arises later in the life history of the blind Mexican cavefish, Astyanax mexicanus

Abstract

As a consequence of adaptation to the cave environment, the blind Mexican cavefish, Astyanax mexicanus, has evolved several cranial aberrations including changes to bone sizes, shapes and presence of numerous lateral asymmetries. Prior studies of cranial asymmetry in cavefish focused strictly on adult specimens. Thus, the extent to which these asymmetries emerge in adulthood, or earlier in the life history of cavefish, was unknown. We performed a geometric morphometric analysis of shape variation in the chondrocranium and osteocranium across life history in two distinct cavefish populations and surface-dwelling fish. The cartilaginous skull in juveniles was bilaterally symmetric and chondrocranial shape was conserved in all three populations. In contrast, bony skull shapes segregated into significantly distinct groups in adults. Cavefish demonstrated significant asymmetry for the bones surrounding the collapsed eye orbit, and the opercle bone posterior to the eye orbit. Interestingly, we discovered that cavefish also exhibit directional "bends" in skull shape, almost always biased to the left. In sum, this work reveals that asymmetric craniofacial aberrations emerge later in the cavefish life history. These abnormalities may mirror asymmetries in the lateral line sensory system, reflect a 'handedness' in cavefish swimming behavior, or evolve through neutral processes.

Fig 1. Chondrocranial shape is bilaterally symmetric in juvenile Astyanax mexicanus.

Representative juvenile (8 dpf) specimens from surface fish (A), Tinaja (B) and Pachón (C) cavefish populations stained with Alcian blue and imaged at 80x magnification (scale bar = 1mm). Eight homologous landmarks (yellow; A-C) were placed on the ethmoid (e; green), trabecula (tr; orange), hyosymplectic (hs; red), and palatoquadrate (pq; blue; D-F). A Principal Components Analysis was used to compare the average shape of each individual on the right (blue) and left (red) sides for surface fish (G), Tinaja (H) and Pachón (I) cavefish. Confidence ellipses set to a significance threshold of p<0.05 overlap between the right (blue) and the left (red) shape for each population suggests shape quantification based on these landmarks is bilaterally symmetric. A student’s t-test revealed no significant differences in Procrustes distance between right and left sides for surface fish (p = 0.1598), Tinaja (p = 0.1645) and Pachón (p = 0.3532) cavefish.

Fig 2. Juvenile constraint of chondrocranial shape contrasts adult osteocranial shape segregation in Astyanax mexicanus.

Global chondrocranial shape was compared in n = 30 Pachón (red), Tinaja (green) and surface (blue) populations using a principal component analysis (A). Populations did not significantly differ from one another for juvenile chondrocranial shape (Procrustes ANOVA; p = 0.0892; PC1 28%). Analysis of global skull shape in n = 20 adults revealed divergence in shape between cave and surface fish populations (p = 0.0002; PC1 65%) (B). When the landmarks surrounding the eye were removed from the analysis, cave and surface populations were also segregated (C), suggesting global shape differences in the adult cranium are not strictly associated with eye regression and orbital collapse (p = 0.0082; PC1 56%). Confidence ellipses for each PCA were set to p<0.05.

Fig 3. Adult cavefish exhibit fluctuating asymmetry in osteocranial shape.

Cavefish demonstrate lateral asymmetry compared to surface fish (A; p = 0.0002). Lateral shape differences in PC1, accounting for 18% of shape asymmetry, occur in the supraorbital, SO3 and opercle bones (wireframe graphs in gray = average surface fish shape, and green = average cavefish shape). Cavefish demonstrate global 3D asymmetry as demonstrated in the frontal view of a Pachón cavefish (B; p = 0.0002). Shape differences in PC1, accounting for 16% of variation, indicate presence of fluctuating asymmetry in the shape of the osteocranium in cavefish.

Fig 4. The dorsal osteocranium demonstrates directional asymmetry in adult cavefish.

Surface-rendered microCT images of the dorso-cranium are presented for individual surface fish (A), Pachón (D) and Tinaja (E) cavefish. Landmark-based wireframe graphs for PC1 (capturing 38% of asymmetry shape variation) depict the average dorso-cranial shape for surface fish (B; black lines) and cavefish shape from both populations (F; green lines). Cavefish average shape (green) overlaid on surface fish average shape (black) reveals a dramatic dorso-cranial bend to the left (C). Procrustes ANOVA revealed significant and leftward-biased directional asymmetry in the cavefish dorsocranium (p = 0.0409). The bend in cavefish is most severe in the anterior dorso-cranium inclusive of the premaxilla, supraorbitals and the frontal bone, as well as the dorsal foramen, which extends posteriorly to the supraoccipital bone.

Fig 5. Timeline summary of cranial development in Astyanax mexicanus cavefish.

The formation of the chondrocranium is complete by 6–8dpf. Cranial bones begin to develop around 20mm standard length, and the osteocranium is completely ossified by 35mm standard length. Previous studies reported morphological asymmetry [–22] and genetic asymmetry [45] for individual cranial bones in adult cavefish. Gross et al. (2016) showed that cranial sensory neuromasts located on the suborbital #3 bone were more asymmetric in cavefish than surface fish [50]. Here, we demonstrate normal chondrocranial development, followed by divergence of cranial shape and fluctuating asymmetry at the level of individual bones, as well as directional asymmetry associated with dorso-cranial bending toward the left.