Phylogenetic, ecological and biomechanical constraints on larval form: A comparative morphological analysis of barnacle nauplii

Abstract

Barnacle naupliar larvae are differentiated from other zooplankton by their unique pair of frontal lateral horns, frontal filaments, and a pear-shaped cephalic shield. Their morphology impose constraints on their ecological functions and reflect their evolutionary history. To explore the potential functional basis underlying the similarities and differences in barnacle larval form, we conducted a meta-analysis on the shape of the barnacle nauplii's cephalic shield and examined its relation to larval size, trophic mode, pelagic larval duration and habitat. Nauplii cephalic shield morphology of 102 species were quantified with normalized elliptic Fourier analysis. Most of the species were distributed around the center of the morphospace but a few extreme groups occupied the periphery: nauplii that were large and lecithotrophic. Subsequent principal component regression analyses showed that larval size was a good predictor of the first shape variations axis (aspect ratio). After allometry adjustment, nauplii from different trophic modes differentiated along the second axis of the major shape variations (relative frontal horn length). Habitat was a poor predictor of variations in naupliar body form, but it could be used to differentiate extreme morphology groups from other nauplii. Our result suggests that size-related biomechanical or developmental constraints and feeding requirements are important in shaping the evolution of the naupliar body form. Within the limitations of these functional constraints, habitat drives the divergence of extreme morphology groups from the majority of species. Our comparative morphometrics analysis demonstrated how variations in larval body form can be quantitatively linked to the functional needs that constrain or drive their diversity, and inform further empirical experiments on larval functional morphology.

Fig 1. Outline processing and analysis on cephalic shield of stage II barnacle nauplius larva.

(A) Shape mask labeled with the outline sampling scheme and linear measurements used. start denotes standardized starting point of outline sampling at tip of dorsal shield spine; Fh = frontal horn; Fh width = distance between tips of frontal horns. (B) Outline reconstructed from elliptic Fourier analysis. Blue shades are the masks of the original outline while black lines are reconstructed outlines using different number of harmonics.

Fig 2. Major directions of nauplii shape variations.

(A) Plot of first two principal component scores from principal component analysis (PCA) on outline shape of stage II barnacle nauplii. Data points were colored by relative frontal horn length (ratio of frontal horn length to larval length) and their sizes were scaled with larval aspect ratio (both data layers came from manual measurements). Note that scale of the color gradient and size of the aspect ratio are logarithmic. (B) Reconstructed outline shapes at ± 2SD PC score values, plotted as thin-plate-spline deformation from the mean outlines, depict the trend of shape changes along the PC axes.

Fig 3. Allometry of nauplii shape.

(A) Histogram of size distribution of stage II barnacle nauplii. Arrows correspond to size used for later reconstruction of predicted allometric shape changes. Dashed line indicates larval length at 90^th percentile for all species. (B) Shape score associated with size estimated from multivariate regression of shape of nauplii outlines on size. Species of special interest to discussion were annotated. (C) Predicted shapes from regression of shape on size at 50% and 95% intervals, shown as thin-plate-spline deformation from the predicted shape at mean size.

Fig 4. Nauplii shape differentiation between trophic modes.

(A) Allometry free outlines projected back onto the original shape space as shown in Fig 2. Enlarged symbols represent the group means. Lines were drawn to connect the data points before and after adjustment for allometry, and they are parallel to the vector of size gradient (Note: the angle of these lines to PC1 is smaller than the calculated angle in Table 1 because of the distortion of high-dimensional data in 2-dimensional PC plot). (B) Bootstrapped allometry-free nauplii outlines between trophic modes reconstructed from residuals of regression of shape on size. Bootstrapped means are represented by colored outlines while the grey lines illustrates the variability of mean in the bootstrap samples. (C) Boxplot of bootstrapped means for relative frontal horn length. In all bootstrapping, n = 1000.

Fig 5. Roles of phylogeny and habitat on nauplii shape.

(A) Phylomorphospace (subset of phylogeny over PCA plot from Fig 2) incorporated with information of adult habitats (Note: borer refers to Acrothoracican barnacles, which bore on calcareous substrate and live in its own created burrows; deep sea habitat refers to hydrothermal vents and open ocean >200m deep). Nauplii of most species have similar morphology and are concentrated around the center (blue shade, ~77%) while species at the periphery are more correlated with certain habitats or taxonomic groups. Peripheral species not belonging to these taxonomic groups are annotated (Bh = Balanodytes habei; Cf = Chthamalus fragilis; Ll = Leucolepas longa, Ob = Octomeris brunnea; Ss = Scalpellum scalpellum; Tr = Tetraclita rufotincta). (B) Relationship between larval habitat and nauplii shapes. Larval habitat differs from adult habitat in that there is less emphasis on substrate type. (C) Relationship between pelagic larval duration (PLD) and nauplii shape. PLD was categorized into extreme and general groups, see Fig H in S1 Text. Data for species reared at low temperature were highlighted, as low rearing temperature may greatly prolong rearing duration.

Fig 6. Trophic modes and naupliar morphology.

Examples of naupliar cephalic shield outlines from Cirripedia (barnacles) categorized by their trophic mode. Larvae from Ascothoracida, the Thecostracan sister group to Cirripedia, were also included (outlines traced from photos in [46]). Frontal horns are only present in barnacle nauplii and are absent in other crustacean nauplii. Relative frontal horn length is likely related to feeding.