Variable Craniofacial Shape and Development among Multiple Cave-Adapted Populations of Astyanax mexicanus

Abstract

Astyanax mexicanus is a freshwater fish species with blind cave morphs and sighted surface morphs. Like other troglodytic species, independently evolved cave-dwelling A. mexicanus populations share several stereotypic phenotypes, including the expansion of certain sensory systems, as well as the loss of eyes and pigmentation. Here, we assess the extent to which there is also parallelism in craniofacial development across cave populations. Since multiple forces may be acting upon variation in the A. mexicanus system, including phylogenetic history, selection, and developmental constraint, several outcomes are possible. For example, eye regression may have triggered a conserved series of compensatory developmental events, in which case we would expect to observe highly similar craniofacial phenotypes across cave populations. Selection for cave-specific foraging may also lead to the evolution of a conserved craniofacial phenotype, especially in regions of the head directly associated with feeding. Alternatively, in the absence of a common axis of selection or strong developmental constraints, craniofacial shape may evolve under neutral processes such as gene flow, drift, and bottlenecking, in which case patterns of variation should reflect the evolutionary history of A. mexicanus. Our results found that cave-adapted populations do share certain anatomical features; however, they generally did not support the hypothesis of a conserved craniofacial phenotype across caves, as nearly every pairwise comparison was statistically significant, with greater effect sizes noted between more distantly related cave populations with little gene flow. A similar pattern was observed for developmental trajectories. We also found that morphological disparity was lower among all three cave populations versus surface fish, suggesting eye loss is not associated with increased variation, which would be consistent with a release of developmental constraint. Instead, this pattern reflects the relatively low genetic diversity within cave populations. Finally, magnitudes of craniofacial integration were found to be similar among all groups, meaning that coordinated development among anatomical units is robust to eye loss in A. mexicanus. We conclude that, in contrast to many conserved phenotypes across cave populations, global craniofacial shape is more variable, and patterns of shape variation are more in line with population structure than developmental architecture or selection.

Fig. 1

Stage 3 cleared and double-stained Astyanax specimens are shown. Landmark (LM) schemes are illustrated on the Pachon individual at top (a, b), and surface fish at bottom (c, d). Landmarks are denoted as solid red dots, while curves along which semi-landmarks were placed are denoted as dashed white lines. In the ventral view, LM 1 depicts the mandibular–quadrate joint, LMs 2 and 3 define the anterior end of the hyoid, LM 4 is the hyoid–interhyal joint, LM 5 is the interhyal–hyomandibula joint, and LM 6 is the proximal end of the ventral most pectoral fin ray/pad (stage 1). Contralateral LMs were used to capture bilateral shape variation. In addition, we used nine semi-landmarks between the left and right mandibular LMs. In the lateral view, LMs 1 and 2 depict the dorsal and ventral-most ends of the cleithrum, LMs 3 and 4 define the posterior and anterior-most ends of the hyoid, LM 5 is the ventral tip of the retroarticular process, LM 6 is the mandibular–quadrate joint, LM 7 is where the coronoid meets the articular bone, LM 8 is the ventral edge of the distal end of the mandible, LM 9 is the tip of the premaxilla/upper jaw (stage 1), and LM 10 is the posterior end of the parietal bone (in stage 1 and 2 animals this landmark was placed at the posterior end of the dorsal cranium). In this view, we also used three semi-landmarks between LMs 9 and 10 to assess variation in cranial profile. Scale bars equal 500 μm in all panels.

Fig. 2

Principal component analysis (PCA) and associated deformation grids for the ventral landmark dataset. Data for stage 1 (a, b), stage 2 (c, d), and stage 3 (e, f) are shown separately. For each stage, the percent variation explained by each axis is provided, and the shape space is overlain by colored convex hulls for each locality. Deformation grids represent the predicted shape at minimum and maximum values along each axis as compared to mean shape. Anterior is to the left, posterior is to the right.

Fig. 3

Principal component analysis (PCA) and associated deformation grids for the lateral landmark dataset. Data for stage 1 (a, b), stage 2 (c, d), and stage 3 (e, f) are presented separately. For each stage, the percent variation explained by each axis is provided, and shape space is overlain by colored convex hulls for each locality. Deformation grids represent the predicted shape at minimum and maximum values along each axis as compared to mean shape. In b and d, anterior is facing the top left corner, while posterior is bottom right. In f, anterior is bottom left, while posterior is top right.

Fig. 4

Principal component analysis (PCA) and associated deformation grids for the trajectory analysis in the ventral view. All population: stages are depicted in (a), and the percent variation explained by each component is provided. Corresponding deformation grids are illustrated in (b) as minimum and maximum values relative to mean shape. The anterior–posterior axis runs left to right. For ease of interpretation, each population is also shown separately (c–f). Circles represent individual data points, while triangles represent group means. Populations are denoted by different colors, and increasingly older fish are represented by darker hues.

Fig. 5

Principal component analysis (PCA) and associated deformation grids for the trajectory analysis in the lateral view. All population: stages are depicted in (a), and the percent variation explained by each axis is provided. Corresponding deformation grids are illustrated in (b) as minimum and maximum values relative to mean shape. Here, the anterior–posterior axis runs top left to bottom right. For ease of interpretation, each population is also shown separately (c–f). Circles represent individual data points, while triangles are group means. Populations are denoted by different colors, with increasingly older fish represented by darker hues.