Different transformations underlie blowhole and nasal passage development in a toothed whale (Odontoceti: Stenella attenuata) and a baleen whale (Mysticeti: Balaenoptera physalus).

Reorientation of the nasal passage away from the anteroposterior axis has evolved rarely in mammals. Unlike other mammals, cetaceans (e.g., whales, dolphins, and porpoises) have evolved a "blowhole": posteriorly repositioned nares that open dorsad. Accompanying the evolution of the blowhole, the nasal passage has rotated dorsally. Neonatal cetaceans possess a blowhole, but early in development, cetacean embryos exhibit head morphologies that resemble those of other mammals. Previous workers have proposed two developmental models for how the nasal passage reorients during prenatal ontogeny. In one model, which focused on external changes in the whole body, dorsad rotation of the head relative to the body results in dorsad rotation of the nasal passage relative to the body. A second model, based on details of the cartilaginous nasal skull, describes dorsad rotation of the nasal passage itself relative to the palate and longitudinal axis of the skull. To integrate and revise these models, we characterized both external and internal prenatal changes in a longitudinal plane that are relevant to nasal passage orientation in the body and head of the pantropical spotted dolphin (Odontoceti: Stenella attenuata). These changes were then compared to those in a prenatal series of a baleen whale, the fin whale (Mysticeti: Balaenoptera physalus), to determine if they were representative of both extant cetacean suborders. In both species, the angle between the nasal passage and the sagittal axis of the foramen magnum decreased with age. In S. attenuata, this was associated with basicranial retroflexion and midfacial lordosis: the skull appeared to fold dorsad with the presphenoid as the vertex of the angle. In contrast, in B. physalus, alignment of the nasal passage and the sagittal axis of the plane of the foramen magnum was associated with angular changes within the posterior skull (specifically, the orientations of the supraoccipital and foramen magnum relative to the posterior basicranium). With these results, we propose a new developmental model for prenatal reorientation of the odontocete nasal passage and discuss ways in which mysticetes likely deviate from this model.

Morphological simulation tests the limits on phenotype discovery in 3D image analysis.

In the past few decades, advances in 3D imaging have created new opportunities for reverse genetic screens. Rapidly growing datasets of 3D images of genetic knockouts require high-throughput, automated computational approaches for identifying and characterizing new phenotypes. However, exploratory, discovery-oriented image analysis pipelines used to discover these phenotypes can be difficult to validate because, by their nature, the expected outcome is not known a priori . Introducing known morphological variation through simulation can help distinguish between real phenotypic differences and random variation; elucidate the effects of sample size; and test the sensitivity and reproducibility of morphometric analyses. Here we present a novel approach for 3D morphological simulation that uses open-source, open-access tools available in 3D Slicer, SlicerMorph, and Advanced Normalization Tools in R (ANTsR). While we focus on diffusible-iodine contrast-enhanced micro-CT (diceCT) images, this approach can be used on any volumetric image. We then use our simulated datasets to test whether tensor-based morphometry (TBM) can recover our introduced differences; to test how effect size and sample size affect detectability; and to determine the reproducibility of our results. In our approach to morphological simulation, we first generate a simulated deformation based on a reference image and then propagate this deformation to subjects using inverse transforms obtained from the registration of subjects to the reference. This produces a new dataset with a shifted population mean while retaining individual variability because each sample deforms more or less based on how different or similar it is from the reference. TBM is a widely-used technique that statistically compares local volume differences associated with local deformations. Our results showed that TBM recovered our introduced morphological differences, but that detectability was dependent on the effect size, the sample size, and the region of interest (ROI) included in the analysis. Detectability of subtle phenotypes can be improved both by increasing the sample size and by limiting analyses to specific body regions. However, it is not always feasible to increase sample sizes in screens of essential genes. Therefore, methodical use of ROIs is a promising way to increase the power of TBM to detect subtle phenotypes. Generating known morphological variation through simulation has broad applicability in developmental, evolutionary, and biomedical morphometrics and is a useful way to distinguish between a failure to detect morphological difference and a true lack of morphological difference. Morphological simulation can also be applied to AI-based supervised learning to augment datasets and overcome dataset limitations.

Challenges and advances in measuring phenotypic convergence.

Tests of phenotypic convergence can provide evidence of adaptive evolution, and the popularity of such studies has grown in recent years due to the development of novel, quantitative methods for identifying and measuring convergence. These methods include the commonly applied C1-C4 measures of Stayton (2015a), which measure morphological distances between lineages, and Ornstein-Uhlenbeck (OU) model-fitting analyses, which test whether lineages converged on shared adaptive peaks. We test the performance of C-measures and other convergence measures under various evolutionary scenarios and reveal a critical issue with C-measures: they often misidentify divergent lineages as convergent. We address this issue by developing novel convergence measures-Ct1-Ct4-measures-that calculate distances between lineages at specific points in time, minimizing the possibility of misidentifying divergent taxa as convergent. Ct-measures are most appropriate when focal lineages are of the same or similar geologic ages (e.g., extant taxa), meaning that the lineages' evolutionary histories include considerable overlap in time. Beyond C-measures, we find that all convergence measures are influenced by the position of focal taxa in phenotypic space, with morphological outliers often statistically more likely to be measured as strongly convergent. Further, we mimic scenarios in which researchers assess convergence using OU models with a priori regime assignments (e.g., classifying taxa by ecological traits) and find that multiple-regime OU models with phenotypically divergent lineages assigned to a shared selective regime often outperform simpler models. This highlights that model support for these multiple-regime OU models should not be assumed to always reflect convergence among focal lineages of a shared regime. Our new Ct1-Ct4-measures provide researchers with an improved comparative tool, but we emphasize that all available convergence measures are imperfect, and researchers should recognize the limitations of these methods and use multiple lines of evidence to test convergence hypotheses.

Cetacean Skull Telescoping Brings Evolution of Cranial Sutures into Focus.

Many modifications to the mammalian bauplan associated with the obligate aquatic lives of cetaceans-fusiform bodies, flukes, flippers, and blowholes-are evident at a glance. But among the most strikingly unusual and divergent features of modern cetacean anatomy are the arrangements of their cranial bones: (1) bones that are situated at opposite ends of the skull in other mammals are positioned close together, their proximity resulting from (2) these bones extensively overlapping the bones that ordinarily would separate them. The term "telescoping" is commonly used to describe the odd anatomy of modern cetacean skulls, yet its usage and the particular skull features to which it refers vary widely. Placing the term in historical and biological context, this review offers an explicit definition of telescoping that includes the two criteria enumerated above. Defining telescoping in this way draws attention to many specific biological questions that are raised by the unusual anatomy of cetacean skulls; highlights the central role of sutures as the locus for changes in the sizes, shapes, mechanical properties, and connectivity of cranial bones; and emphasizes the importance of sutures in skull development and evolution. The unusual arrangements of cranial bones and sutures referred to as telescoping are not easily explained by what is known about cranial development in more conventional mammals. Discovering the evolutionary-developmental processes that produce the extensive overlap characteristic of cetacean telescoping will give insights into both cetacean evolution and the "rules" that more generally govern mammalian skull function, development, and evolution. Anat Rec, 302:1055-1073, 2019. © 2019 Wiley Periodicals, Inc.

Anatomy and age estimation of an early blue whale (Balaenoptera musculus) fetus.

The external anatomy of a 130-mm blue whale fetus (Balaenoptera musculus) is described, and its internal anatomy is reconstructed noninvasively from microCT scans. The specimen lies developmentally at the junction of the embryonic and fetal periods. Similarly to the embryos of many odontocetes, it lacks a caudal fluke and dorsal fin, but it also exhibits an elongated rostrum, resorbed umbilical hernia, partially exposed cornea, and spatial separation of the anus and genitalia seen in early odontocete fetuses. Dermal ossification of the cranial bones has begun, but the endochondral skeleton is completely cartilaginous. The shape and position of the maxilla suggest that the earliest stages of anterior skull telescoping have begun, but there is no indication of occipital overlap posteriorly. The nasopharynx, larynx, and heart already display the distinctive morphology characteristic of Balaenoptera. This study develops a model of body length changes during blue whale development by integrating the large International Whaling Statistics (IWS) database, historical observations of blue whale migration and reproduction, and descriptions of fetal growth trends in other mammals. The model predicts an age of 65 days postconception for the specimen. The early developmental milestones of Balaenoptera mirror those of the odontocete Stenella to a remarkable extent, but the first appearance of the caudal fluke and dorsal fin are delayed relative to other morphological transitions. The accelerated prenatal growth characteristic of Balaenoptera occurs during fetal, not embryonic, development.