Morphological integration and serial homology: A case study of the cranium and anterior vertebrae in salamanders

Serial homology or the repetition of equivalent developmental units and their derivatives is a phenomenon encountered in a variety of organisms, with the vertebrate axial skeleton as one of the most notable examples. Serially homologous structures can be viewed as an appropriate model system for studying morphological integration and modularity, due to the strong impact of development on their covariation. Here, we explored the pattern of morphological integration of the cranium and the first three serially homologous structures (atlas, first, and second trunk vertebrae) in salamandrid salamanders, using micro-CT scanning and three-dimensional geometric morphometrics. We explored the integration between structures at static and evolutionary levels. Effects of allometry on patterns of modularity were also taken into account. At the static level (within species), we analyzed inter-individual variation in shape to detect functional modules and intra-individual variation to detect developmental modules. Significant integration (based on inter-individual variation) among all structures was detected and allometry is shown to be an important integrating factor. The pattern of intra-individual, asymmetric variation indicates statistically significant developmental integration between the cranium and the atlas and between the first two trunk vertebrae. At the evolutionary level (among species), the cranium, atlas, and trunk vertebrae separate as different modules. Our results show that morphological integration at the evolutionary level coincides with morphological and functional differentiation of the axial skeleton, allowing the more or less independent evolutionary changes of the cranial skeleton and the vertebral column, regardless of the relatively strong integration at the static level. The observed patterns of morphological integration differ across levels, indicating different impacts of developmental and phylogenetic constraints and functional demands.     

Differential occupation of axial morphospace

The postcranial system is composed of the axial and appendicular skeletons. The axial skeleton, which consists of serially repeating segments commonly known as vertebrae, protects and provides leverage for movement of the body. Across the vertebral column, much numerical and morphological diversity can be observed, which is associated with axial regionalization. The present article discusses this basic diversity and the early developmental mechanisms that guide vertebral formation and regionalization. An examination of vertebral numbers across the major vertebrate clades finds that actinopterygian and chondrichthyan fishes tend to increase vertebral number in the caudal region whereas Sarcopterygii increase the number of vertebrae in the precaudal region, although exceptions to each trend exist. Given the different regions of axial morphospace that are occupied by these groups, differential developmental processes control the axial patterning of actinopterygian and sarcopterygian species. It is possible that, among a variety of factors, the differential selective regimes for aquatic versus terrestrial locomotion have led to the differential use of axial morphospace in vertebrates.     

Histology and structural integration of the major morphologies of the Cypriniform Weberian apparatus

The Weberian apparatus, a diagnostic feature of otophysan fishes, is a novel hearing adaptation integrating several developmental and morphological systems (ear-vertebral column-swim bladder). Otophysan fishes are one of the largest and most successful freshwater clades, with over 10,000 species across most continents. The largest otophysan order, Cypriniformes, dominates the freshwaters of Asia, Europe, North America, and Africa. Spanning such a wide variety of environments, the Weberian apparatus undergoes morphological modifications to maintain functionality. Within Cypriniformes, we propose three distinct morphological classes of the Weberian apparatus based on the level of skeletal expansion around the swim bladder: simple (typical of most Cyprinidae), anterior plate (found in families such as Gyrinocheilidae, Catostomidae, and Botiidae), and encapsulated (either single-capsule as found, e.g., in Gobionidae and Cobitidae, or double-capsule as found, e.g., in Nemacheilidae and Balitoridae). Little ontological or comparative data exists regarding the construction or integration of these different morphologies, and less is known about the tissue level integration and variation within these morphologies. We used paraffin histology to document the hard and soft tissue anatomy of the Weberian apparatus in six species representing all morphological classes. We found sites of similarity across the morphologies including size and structure of the saccule, aspects of ossicle ossification, and swim bladder tunica composition, indicating potential sites of developmental and functional constraint. In contrast, we found differences across both auditory and nonauditory features in otic chamber size, ossification within ossicles and other vertebral elements, and composition of ligaments, indicating likely sites of adaptability. Some of these changes are likely evolutionary (taxonomic), but may be influenced by the environmental niche occupied by the clade. These results show a clear need for increased ontological and comparative study of the complete cypriniform Weberian apparatus, particularly histologically, as well as increased auditory studies across morphological types.     

Correlation between Hox code and vertebral morphology in archosaurs

The relationship between developmental genes and phenotypic variation is of central interest in evolutionary biology. An excellent example is the role of Hox genes in the anteroposterior regionalization of the vertebral column in vertebrates. Archosaurs (crocodiles, dinosaurs including birds) are highly variable both in vertebral morphology and number. Nevertheless, functionally equivalent Hox genes are active in the axial skeleton during embryonic development, indicating that the morphological variation across taxa is likely owing to modifications in the pattern of Hox gene expression. By using geometric morphometrics, we demonstrate a correlation between vertebral Hox code and quantifiable vertebral morphology in modern archosaurs, in which the boundaries between morphological subgroups of vertebrae can be linked to anterior Hox gene expression boundaries. Our findings reveal homologous units of cervical vertebrae in modern archosaurs, each with their specific Hox gene pattern, enabling us to trace these homologies in the extinct sauropodomorph dinosaurs, a group with highly variable vertebral counts. Based on the quantifiable vertebral morphology, this allows us to infer the underlying genetic mechanisms in vertebral evolution in fossils, which represents not only an important case study, but will lead to a better understanding of the origin of morphological disparity in recent archosaur vertebral columns.     

Development of the dorsal and anal fin in Kneria stappersii (Otomorpha: Gonorynchiformes)

The order Gonorynchiformes was repeatedly studied to gain new insights into the evolution of its sister-taxon, the Otophysi, the most successful freshwater fish taxon worldwide. Previous ontogenetic studies of gonorynchiforms mainly focused on the anterior vertebral column to investigate the evolutionary origin of the Weberian apparatus. Herein, we highlight the ontogeny of a different skeletal complex, the dorsal and anal fins. We studied the development of the skeletal elements of both fins in the gonorynchiform Kneria stappersii. We gained new insights into the developmental and formation patterns of K. stappersii. We discuss these patterns as well as the development of certain elements like the fin stay in comparison to other gonorynchiforms and available otomorph data. In general, the fin development in K. stappersii is very similar to that of other gonorynchiforms and even otomorphs. Specific differences, however, reveal that much remains unknown about the evolution of median fin elements such as the fin stay.     

Axial variation in the three-spine stickleback: genetic and environmental factors

Subtle differences in the pattern of arrangement of types of vertebrae and associated median skeletal structures between a benthic and limnetic species pair of threespine stickleback from Paxton Lake, British Columbia, are typical of those found throughout the range of the Gasterosteus aculeatus species complex. We established laboratory colonies from just three individuals of each species, and studied the effect of three generations of inbreeding on axial morphology. There was sufficient divergence in the location of individual elements between families to regenerate close to the entire range of axial diversity seen in threespine sticklebacks worldwide. Analysis of the patterns of variance and covariance between the axial locations of elements provides evidence for the action of both meristic and homeotic processes in the generation of morphological divergence within each species. Hybrid sticklebacks produced by the cross of limnetic and benthic parents tend to have intermediate morphologies, with dominance of either parental type evident for some elements. Effects of temperature and salinity were found to be small in direct comparison with between-family effects, and varied according to genetic background. These results demonstrate that considerable genetic variation for axial morphology is maintained in natural populations of threespine stickleback, and that differences between populations may be brought about rapidly by changes in frequency of alleles that have coordinated effects along the body axis.     

Evolutionary developmental perspective for the origin of turtles: the folding theory for the shell based on the developmental nature of the carapacial ridge

The body plan of the turtle represents an example of evolutionary novelty for acquisition of the shell. Unlike similar armors in other vertebrate groups, the turtle shell involves the developmental repatterning of the axial skeleton and exhibits an unusual topography of musculoskeletal elements. Thus, the turtle provides an ideal case study for understanding changes in the developmental program associated with the morphological evolution of vertebrates. In this article, the evolution of the turtle-specific body plan is reviewed and discussed. The key to understanding shell patterning lies in the modification of the ribs, for which the carapacial ridge (CR), a turtle-specific embryonic anlage, is assumed to be responsible. The growth of the ribs is arrested in the axial part of the body, allowing dorsal and lateral oriented growth to encapsulate the scapula. Although the CR does not appear to induce this axial arrest per se, it has been shown to support the fan-shaped patterning of the ribs, which occurs concomitant with marginal growth of the carapace along the line of the turtle-specific folding that takes place in the lateral body wall. During the process of the folding, some trunk muscles maintain their ancestral connectivities, whereas the limb muscles establish new attachments specific to the turtle. The turtle body plan can thus be explained with our knowledge of vertebrate anatomy and developmental biology, consistent with the evolutionary origin of the turtle suggested by the recently discovered fossil species, Odontochelys.     

Morphological variation in the Weberian apparatus of Cypriniformes

Cypriniformes (which includes the minnows, carps, loaches, algae-eaters, stone loaches, and suckers) is a morphologically diverse and incredibly speciose order of teleosts. It has been suggested that a number of evolutionary innovations, key to improved hearing and feeding, have played an important role in cypriniform fishes' success. One such innovation, the Weberian apparatus, is a novel assemblage of vertebral elements and modified ribs that relay and amplify sound pressure changes from the gas bladder to the inner ear. The Weberian apparatus unites Cypriniformes with other major orders into an extremely species-rich group of fishes, the Otophysi. Together, otophysan fishes comprise one of the largest groups of fishes in the world, as well as the majority of freshwater fishes. Here we present a detailed comparison of the Weberian apparatus in a number of cypriniform families using cleared and stained specimens. We present data regarding inter- and intrafamilial morphological variation within Cypriniformes. With few, but evolutionarily important, exceptions we find that diagnostic features of the Weberian apparatus characterize each family. Interspecific variation within each of the families Balitoridae, Gyrinocheilidae, and Catostomidae is only slight, whereas variation among subfamilies within Cyprinidae and Cobitidae is far more significant. This comparative study identifies a number of distinct morphologies, some of which appear highly correlated with ecological niche. For example, inhabiting swift-moving waters appears to be a key factor in the encapsulation of the anterior gas bladder in some cobitids, balitorids, and gobionin cyprinids.     

Vertebral evolution and the diversification of squamate reptiles

Taxonomic, morphological, and functional diversity are often discordant and independent components of diversity. A fundamental and largely unanswered question in evolutionary biology is why some clades diversify primarily in some of these components and not others. Dramatic variation in trunk vertebral numbers (14 to >300) among squamate reptiles coincides with different body shapes, and snake-like body shapes have evolved numerous times. However, whether increased evolutionary rates or numbers of vertebrae underlie body shape and taxonomic diversification is unknown. Using a supertree of squamates including 1375 species, and corresponding vertebral and body shape data, we show that increased rates of evolution in vertebral numbers have coincided with increased rates and disparity in body shape evolution, but not changes in rates of taxonomic diversification. We also show that the evolution of many vertebrae has not spurred or inhibited body shape or taxonomic diversification, suggesting that increased vertebral number is not a key innovation. Our findings demonstrate that lineage attributes such as the relaxation of constraints on vertebral number can facilitate the evolution of novel body shapes, but that different factors are responsible for body shape and taxonomic diversification.     

Early development of marine catfishes (Ariidae): from mouth brooding to the release of juveniles in nursery habitats

The development and allometric growth patterns of the ariid catfishes Cathorops spixii and Cathorops agassizii were studied from neurula embryos to juveniles. The ontogenetic sequence revealed that prior to hatching, embryos of both species are well developed, and their axial and appendicular skeletons are well ossified. Embryos of both species grow slowly longitudinally, but positively allometric growth (growth coefficient, β₁ > 1) was observed in head width and eye diameter. It is hypothesized that these growth patterns might be related to functional priorities for the development of sensory organs, such as the inner ears (otoliths), the Weberian apparatus, eyes and nostrils, during the embryonic period. The first appearance of vertebrae and otoliths, as well as the ossification of otoliths and the Weberian apparatus, occur earlier in embryos of C. agassizii than in embryos of C. spixii. After hatching, mouth‐brooded free embryos of both species grow isometrically. Negatively allometric growth was observed in head width and eye diameter during the yolk‐sac period, which is expected given that the sensory organs are already formed. Free embryos of C. agassizii are distinguishable from those of C. spixii by their larger eyes, longer snouts, longer heads and heavier yolk sacs. The end of the yolk‐sac period is characterized by a direct change from free embryo to juvenile, without a true larval period. The juveniles of the two species can also be distinguished from each other by the larger eyes of C. agassizii compared with C. spixii, as in adult fishes.     

Vertebral evolution and ontogenetic allometry: The developmental basis of extreme body shape divergence in microcephalic sea snakes

Snakes exhibit a diverse array of body shapes despite their characteristically simplified morphology. The most extreme shape changes along the precloacal axis are seen in fully aquatic sea snakes (Hydrophiinae): “microcephalic” sea snakes have tiny heads and dramatically reduced forebody girths that can be less than a third of the hindbody girth. This morphology has evolved repeatedly in sea snakes that specialize in hunting eels in burrows, but its developmental basis has not previously been examined. Here, we infer the developmental mechanisms underlying body shape changes in sea snakes by examining evolutionary patterns of changes in vertebral number and postnatal ontogenetic growth. Our results show that microcephalic species develop their characteristic shape via changes in both the embryonic and postnatal stages. Ontogenetic changes cause the hindbodies of microcephalic species to reach greater sizes relative to their forebodies in adulthood, suggesting heterochronic shifts that may be linked to homeotic effects (axial regionalization). However, microcephalic species also have greater numbers of vertebrae, especially in their forebodies, indicating that somitogenetic effects also contribute to evolutionary changes in body shape. Our findings highlight sea snakes as an excellent system for studying the development of segment number and regional identity in the snake precloacal axial skeleton.     

Serial homology and the evolution of mammalian limb covariation structure

The tetrapod forelimb and hindlimb are serially homologous structures that share a broad range of developmental pathways responsible for their patterning and outgrowth. Covariation between limbs, which can introduce constraints on the production of variation, is related to the duplication of these developmental factors. Despite this constraint, there is remarkable diversity in limb morphology, with a variety of functional relationships between and within forelimb and hindlimb elements. Here we assess a hierarchical model of limb covariation structure based on shared developmental factors. We also test whether selection for morphologically divergent forelimbs or hindlimbs is associated with reduced covariation between limbs. Our sample includes primates, murines, a carnivoran, and a chiropteran that exhibit varying degrees of forelimb and hindlimb specialization, limb size divergence, and/or phylogenetic relatedness. We analyze the pattern and significance of between-limb morphological covariation with linear distance data collected using standard morphometric techniques and analyzed by matrix correlations, eigenanalysis, and partial correlations. Results support a common limb covariation structure across these taxa and reduced covariation between limbs in nonquadruped species. This result indicates that diversity in limb morphology has evolved without signficant modifications to a common covariation structure but that the higher degree of functional limb divergence in bats and, to some extent, gibbons is associated with weaker integration between limbs. This result supports the hypothesis that limb divergence, particularly selection for increased functional specialization, involves the reduction of developmental factors common to both limbs, thereby reducing covariation.     

Segmental relationship between somites and vertebral column in zebrafish

The segmental heritage of all vertebrates is evident in the character of the vertebral column. And yet, the extent to which direct translation of pattern from the somitic mesoderm and de novo cell and tissue interactions pattern the vertebral column remains a fundamental, unresolved issue. The elements of vertebral column pattern under debate include both segmental pattern and anteroposterior regional specificity. Understanding how vertebral segmentation and anteroposterior positional identity are patterned requires understanding vertebral column cellular and developmental biology. In this study, we characterized alignment of somites and vertebrae, distribution of individual sclerotome progeny along the anteroposterior axis and development of the axial skeleton in zebrafish. Our clonal analysis of zebrafish sclerotome shows that anterior and posterior somite domains are not lineage-restricted compartments with respect to distribution along the anteroposterior axis but support a 'leaky' resegmentation in development from somite to vertebral column. Alignment of somites with vertebrae suggests that the first two somites do not contribute to the vertebral column. Characterization of vertebral column development allowed examination of the relationship between vertebral formula and expression patterns of zebrafish Hox genes. Our results support co-localization of the anterior expression boundaries of zebrafish hoxc6 homologs with a cervical/thoracic transition and also suggest Hox-independent patterning of regionally specific posterior vertebrae.     

Wing serial homologues and the diversification of insect outgrowths: insights from the pupae of scarab beetles

Modification of serially homologous structures is a common avenue towards functional innovation in developmental evolution, yet ancestral affinities among serial homologues may be obscured as structure-specific modifications accumulate over time. We sought to assess the degree of homology to wings of three types of body wall projections commonly observed in scarab beetles: (i) the dorsomedial support structures found on the second and third thoracic segments of pupae, (ii) the abdominal support structures found bilaterally in most abdominal segments of pupae, and (iii) the prothoracic horns which depending on species and sex may be restricted to pupae or also found in adults. We functionally investigated 14 genes within, as well as two genes outside, the canonical wing gene regulatory network to compare and contrast their role in the formation of each of the three presumed wing serial homologues. We found 11 of 14 wing genes to be functionally required for the proper formation of lateral and dorsal support structures, respectively, and nine for the formation of prothoracic horns. At the same time, we document multiple instances of divergence in gene function across our focal structures. Collectively, our results support the hypothesis that dorsal and lateral support structures as well as prothoracic horns share a developmental origin with insect wings. Our findings suggest that the morphological and underlying gene regulatory diversification of wing serial homologues across species, life stages and segments has contributed significantly to the extraordinary diversity of arthropod appendages and outgrowths.     

An evo-devo view on the origin of the backbone: evolutionary development of the vertebrae

Vertebral columns are a group of diverse axial structures that define the vertebrates and provide supportive, locomotive, protective, and other important functions. The embryonic origin of the first vertebral element in this subphylum, the lamprey arcualia, has remained a puzzle for more than a century although much developmental and genetic progress has been made. The comparative approach is a very powerful tool for studying vertebrate morphological variation and understanding how the novel structures were generated during evolution. Here, I first briefly describe the vertebral structures and their developmental processes in major taxa, and then analyze the most recently published data on the basal vertebrates. Finally, an ontogenetic and phylogenetic origin is proposed. The lamprey may have already evolved a sclerotome, which gave rise to arcualia ontogenetically; whole genome duplications likely promoted the establishment of sclerotomal core genetic program by gene co-options.     

Skeletal development in the fossorial gymnophthalmids Calyptommatus sinebrachiatus and Nothobachia ablephara

The development of the cartilaginous and bony elements that form the skull and axial and appendicular skeleton is described in detail for the post-ovipositional embryonic development of the fossorial gymnophthalmid species Calyptommatus sinebrachiatus and Nothobachia ablephara. Both species have a snake-like morphology, showing an elongated body and reduced or absent limbs, as well as modifications in skull bones for burrowing, such as complex articulation surfaces and development of bony extensions that enclose and protect the brain. Similar morphological changes have originated independently in several squamate groups, including the one that led to the snake radiation. This study characterizes the patterns of chondrogenesis and osteogenesis, with special emphasis on the features associated with the burrowing habit, and may be used for future comparative analyses of the developmental patterns involved in the origin of the convergent serpentiform morphologies.     

Primitive versus derived traits in the developmental program of the vertebrate head: views from cyclostome developmental studies

Evolution can be viewed as a series of changes in the developmental program along the phylogenetic tree. To better understand the early evolution of the vertebrate skull, we can use the embryos of the cyclostome species as models. By comparing the cyclostome developmental patterns with those of gnathostomes, it becomes possible to distinguish the primitive and derived parts of the developmental program as taxon-specific traits. These traits are often recognizable as developmental constraints that define taxa by biasing the developmental trajectories within a certain limited range, resulting in morphological homologies in adults. These developmental constraints are distributed on the phylogenetic tree like the morphological character states of adult animals and are associated with specific regions of the tree. From this perspective, we emphasize the importance of considering gene expression and embryonic anatomy as the mechanistic bases that can result in homologous or nonhomologous morphological patterns at later developmental stages. Taking the acquisition of the jaw and trabecula cranii as examples, we demonstrate that a set of embryonic features can be coupled or decoupled during evolution and development. When they are coupled, they exert an ancestral developmental constraint that results in homologous morphological patterns, and when they are decoupled, the ancestral constraints tend to be abandoned, generating a new body plan. The heterotopy behind the specification of the oral domain is an example of decoupling, based on shifted tissue interactions. We also stress the importance of "developmental burden" in determining the sequential order of changes through evolution.     

Altered expression patterns of TCP and MYB genes relating to the floral developmental transition from initial zygomorphy to actinomorphy in Bournea (Gesneriaceae)

The shift from zygomorphy to actinomorphy has been intensively studied in molecular genetic model organisms. However, it is still a key challenge to explain the great morphological diversity of derived actinomorphy in angiosperms, since different underlying mechanisms may be responsible for similar external morphologies. Bournea (Gesneriaceae) is of particular interest in addressing this question, as it is a representative of primarily derived actinomorphy characteristic of a unique developmental transition from zygomorphy to actinomorphic flowers at anthesis. Using RNA in situ hybridization, the expression patterns were investigated of three different Bournea orthologues of TCP and MYB genes that have been shown to control floral symmetry in model species. Here, it is shown that the initial zygomorphic pattern in Bournea is likely a residual zygomorphy resulting from conserved expression of the adaxial (dorsal) identity gene BlCYC1. As a key novel event, the late downregulation of BlCYC1 and BlRAD and the correlative changes in the late specific expression of the abaxial (ventral) identity gene BlDIV should be responsible for the origin of the derived actinomorphy in Bournea. These results further indicate that there might be diverse pathways in the origin and evolution of derived actinomorphy through modifications of pre-existing zygomorphic developmental programs under dynamics of regulatory networks.