Body-axis organization in tetrapods: a model-system to disentangle the developmental origins of convergent evolution in deep time

Convergent evolution is a central concept in evolutionary theory but the underlying mechanism has been largely debated since On the Origin of Species. Previous hypotheses predict that developmental constraints make some morphologies more likely to arise than others and natural selection discards those of the lowest fitness. However, the quantification of the role and strength of natural selection and developmental constraint in shaping convergent phenotypes on macroevolutionary timescales is challenging because the information regarding performance and development is not directly available. Accordingly, current knowledge of how embryonic development and natural selection drive phenotypic evolution in vertebrates has been extended from studies performed at short temporal scales. We propose here the organization of the tetrapod body-axis as a model system to investigate the developmental origins of convergent evolution over hundreds of millions of years. The quantification of the primary developmental mechanisms driving body-axis organization (i.e. somitogenesis, homeotic effects and differential growth) can be inferred from vertebral counts, and recent techniques of three-dimensional computational biomechanics have the necessary potential to reveal organismal performance even in fossil forms. The combination of both approaches offers a novel and robust methodological framework to test competing hypotheses on the functional and developmental drivers of phenotypic evolution and evolutionary convergence.     

Evo-Devo of the Human Vertebral Column: On Homeotic Transformations, Pathologies and Prenatal Selection

Homeotic transformations of vertebrae are particularly common in humans and tend to come associated with malformations in a wide variety of organ systems. In a dataset of 1,389 deceased human foetuses and infants a majority had cervical ribs and approximately half of these individuals also had missing twelfth ribs or lumbar ribs. In?~10?% of all cases there was an additional shift of the lumbo-sacral boundary and, hence, homeotic transformations resulted in shifts of at least three vertebral boundaries. We found a strong coupling between the abnormality of the vertebral patterns and the amount and strength of associated malformations, i.e., the longer the disturbance of the vertebral patterning has lasted, the more associated malformations have developed and the more organ systems are affected. The germ layer of origin of the malformations was not significantly associated with the frequency of vertebral patterns. In contrast, we find significant associations with the different developmental mechanisms that are involved in the causation of the malformations, that is, segmentation, neural crest development, left-right patterning, etc. Our results, thus, suggest that locally perceived developmental signals are more important for the developmental outcome than the origin of the cells. The low robustness of vertebral A-P patterning apparent from the large number of homeotic transformations is probably caused by the strong interactivity of developmental processes and the low redundancy of involved morphogens during early organogenesis. Additionally, the early irreversibility of the specification of the A-P identity of vertebrae probably adds to the vulnerability of the process by limiting the possibility for recovery from developmental disturbances. The low developmental robustness of vertebral A-P patterning contrasts with a high robustness of the A-P patterning of the vertebral regions. Not only the order is invariable, also the variation in the number of vertebrae per region is small. This robustness is in agreement with the evolutionary stability of vertebral regions in tetrapods. Finally, we propose a new hypothesis regarding the constancy of the presacral number of vertebrae in mammals.     

The Development and Evolution of the Turtle Body Plan: Inferring Intrinsic Aspects of the Evolutionary Process from Experimental Embryology

The body plan of turtles is unique among tetrapods in the presenceof the shell. The structure of the carapace involves a uniquerelationship between the axial and the appendicular skeletons.A common developmental mechanism, an epithelial-mesenchymalinteraction, has been identified in the early stages of carapacedevelopment by means of basic histological and immunofluorescencetechniques. By analogy to other structures initiated by epithelial-mesenchymalinteractions, it is hypothesized that carapace development isdependent on this interaction in the body wall. Surgical perturbationswere designed to test the causal connection between the epithelial-mesenchymalinteraction in the body wall and the unusual placement of theribs in turtles. By comparison to data available on body wallformation in avian embryos, these experiments also shed lighton the segregation of somitic and lateral plate cell populationsand the embryonic origin of the scapula in turtles. This study specifically addresses the ontogeny of a unique tetrapodbody plan. The ontogenetic information can be used to make inferencesabout the phytogeny of this body plan and how it could haveevolved from the more typical primitive tetrapod. On a moregeneral level this studyexplores the potential role of commondevelopmental mechanisms in the generation of evolutionary novelties,and the developmental incongruities between homologous skeletalelements in different groups of tetrapods.     

New information on Bobosaurus forojuliensis (Reptilia: Sauropterygia): implications for plesiosaurian evolution

Preparation of the holotype specimen of Bobosaurus forojuliensis, a large sauropterygian from the lower Carnian of northeastern Italy, revealed new morphological data relevant in establishing its phylogenetic affinities among pistosauroid taxa and its relationships with plesiosaurians. Inclusion of B. forojuliensis in two phylogenetic analyses focusing, respectively, on sauropterygians and pistosauroids agreed in placing the Italian taxon as closer to plesiosaurians than to other pistosauroids. The phylogenetic interpretation of Bobosaurus was not biased by assumptions on character weighting, is consistent with its relatively younger age compared to most pistosauroids, extends the fossil record of the plesiosaurian basal lineage back to the Carnian and supports the earliest diversification of the clade during the Late Triassic in agreement with the record of several distinct lineages of rhomaleosaurids, plesiosauroids and pliosauroids in the lowermost Jurassic. Bobosaurus shows that the evolution of the plesiosaurian body plan from the ancestral pistosauroid grade was a step-wise process, and that some of the vertebral and appendicular specialisations of Jurassic and Cretaceous plesiosaurians had already developed in the earliest Late Triassic.     

Habitat transitions alter the adaptive landscape and shape phenotypic evolution in needlefishes (Belonidae)

Habitat occupancy can have a profound influence on macroevolutionary dynamics, and a switch in major habitat type may alter the evolutionary trajectory of a lineage. In this study, we investigate how evolutionary transitions between marine and freshwater habitats affect macroevolutionary adaptive landscapes, using needlefishes (Belonidae) as a model system. We examined the evolution of body shape and size in marine and freshwater needlefishes and tested for phenotypic change in response to transitions between habitats. Using micro‐computed tomographic (µCT) scanning and geometric morphometrics, we quantified body shape, size, and vertebral counts of 31 belonid species. We then examined the pattern and tempo of body shape and size evolution using phylogenetic comparative methods. Our results show that transitions from marine to freshwater habitats have altered the adaptive landscape for needlefishes and expanded morphospace relative to marine taxa. We provide further evidence that freshwater taxa attain reduced sizes either through dwarfism (as inferred from axial skeletal reduction) or through developmental truncation (as inferred from axial skeletal loss). We propose that transitions to freshwater habitats produce morphological novelty in response to novel prey resources and changes in locomotor demands. We find that repeated invasions of different habitats have prompted predictable changes in morphology.     

Mosaic evolution,preadaptation, and the evolution of evolvability in apes

A major goal in postsynthesis evolutionary biology has been to better understand how complex interactions between traits drive movement along and facilitate the formation of distinct evolutionary pathways. I present analyses of a character matrix sampled across the haplorrhine skeleton that revealed several modules of characters displaying distinct patterns in macroevolutionary disparity. Comparison of these patterns to those in neurological development showed that early ape evolution was characterized by an intense regime of evolutionary and developmental flexibility. Shifting and reduced constraint in apes was met with episodic bursts in phenotypic innovation that built a wide array of functional diversity over a foundation of shared developmental and anatomical structure. Shifts in modularity drove dramatic evolutionary changes across the ape body plan in two distinct ways: (1) an episode of relaxed integration early in hominoid evolution coincided with bursts in evolutionary rate across multiple character suites; (2) the formation of two new trait modules along the branch leading to chimps and humans preceded rapid and dramatic evolutionary shifts in the carpus and pelvis. Changes to the structure of evolutionary mosaicism may correspond to enhanced evolvability that has a “preadaptive” effect by catalyzing later episodes of dramatic morphological remodeling.     

Development of a chordate anterior-posterior axis without classical retinoic acid signaling

Developmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean urochordate Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception. This suggests the hypothesis that the RA-machinery was lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior-posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments and analyses of developmental molecular markers to investigate whether RA acts in anterior-posterior axial patterning in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. We discuss possible relationships of altered roles of RA in urochordate development to genomic events, such as rupture of the Hox-cluster, in the context of a new understanding of chordate phylogeny.     

The vertebrate segmentation clock and its role in skeletal birth defects

The segmental structure of the vertebrate body plan is most evident in the axial skeleton. The regulated generation of somites, a process called somitogenesis, underlies the vertebrate body plan and is crucial for proper skeletal development. A genetic clock regulates this process, controlling the timing of somite development. Molecular evidence for the existence of the segmentation clock was first described in the expression of Notch signaling pathway members, several of which are expressed in a cyclic fashion in the presomitic mesoderm (PSM). The Wnt and fibroblast growth factor (FGF) pathways have also recently been linked to the segmentation clock, suggesting that a complex, interconnected network of three signaling pathways regulates the timing of somitogenesis. Mutations in genes that have been linked to the clock frequently cause abnormal segmentation in model organisms. Additionally, at least two human disorders, spondylocostal dysostosis (SCDO) and Alagille syndrome (AGS), are caused by mutations in Notch pathway genes and exhibit vertebral column defects, suggesting that mutations that disrupt segmentation clock function in humans can cause congenital skeletal defects. Thus, it is clear that the correct, cyclic function of the Notch pathway within the vertebrate segmentation clock is essential for proper somitogenesis. In the future, with a large number of additional cyclic genes recently identified, the complex interactions between the various signaling pathways making up the segmentation clock will be elucidated and refined.     

Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid.

Exposure of murine embryos to teratogenic doses of retinoic acid (RA) induced homeotic transformations of vertebrae. Posterior transformations occurred along the complete body axis after RA administration on day 7 of gestation and were accompanied by anterior shifts of Hox gene expression domains in embryos. Anterior transformations of vertebrae in the caudal half of the vertebral column were induced on day 8.5. We suggest that the identity of a vertebral segment is specified by a combination of functionally active Hox genes, a "Hox code." In this concept the sequential activation of Hox genes defines sequentially more posterior axial levels, while mesodermal cells leave the primitive streak. Exogenous RA interferes with the normal establishment of Hox codes and thus with axial specification.     

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.     

Anatomical transformation in mammals: developmental origin of aberrant cervical anatomy in tree sloths

SUMMARY Mammalian cervical count has been fixed at seven for more than 200 million years. The rare exceptions to this evolutionary constraint have intrigued anatomists since the time of Cuvier, but the developmental processes that generate them are unknown. Here we evaluate competing hypotheses for the evolutionary origin of cervical variants in Bradypus and Choloepus , tree sloths that have broken the seven cervical vertebrae barrier independently and in opposite directions. Transitional and mediolaterally disjunct anatomy characterizes the cervicothoracic vertebral boundary in each genus, although polarities are reversed. The thoracolumbar, lumbosacral, and sacrocaudal boundaries are also disrupted, and are more extreme in individuals with more extreme cervical counts. Hypotheses of homologous, homeotic, meristic, or associational transformations of traditional vertebral column anatomy are not supported by these data. We identify global homeotic repatterning of abaxial relative to primaxial mesodermal derivatives as the origin of the anomalous cervical counts of tree sloths. This interpretation emphasizes the strong resistance of the "rule of seven" to evolutionary change, as morphological stasis has been maintained primaxially coincident with the generation of a functionally longer ( Bradypus ) or shorter ( Choloepus ) neck.     

Heterochrony in somitogenesis rate in a model marsupial,Monodelphis domestica

Marsupial newborns are highly altricial and also show a wide array of shifts in the rate or timing of developmental events so that certain neonatal structures are quite mature. One particularly notable feature is the steep gradient in development along the anterior–posterior axis such that anterior structures are generally well developed relative to posterior ones. Here, we study somitogenesis in the marsupial, Monodelphis domestica, and document two heterochronies that may be important in generating the unusual body plan of the newborn marsupial. First, we demonstrate a 4‐fold change in somitogenesis rate along the anterior–posterior axis, which appears to be due to somitogenesis slowing posteriorly. Second, we show that somitogenesis, particularly in the cervical region, initiates earlier in Monodelphis relative to other developmental events in the embryo. The early initiation of somitogenesis may contribute to the early development of the cervical region and forelimbs. Other elements of somitogenesis appear to be conserved. When compared to mouse, we see similar expression of genes involved in the clock and wavefront, and genes of the Wnt, Notch, and fibroblast growth factor (FGF) pathways also cycle in Monodelphis. Further, we could not discern differences in somite maturation rate along the anterior–posterior axis in Monodelphis, and thus rate of maturation of the somites does not appear to contribute to the steep anterior–posterior gradient.     

Terrestriality constrains salamander limb diversification: Implications for the evolution of pentadactyly

Patterns of phenotypic evolution can abruptly shift as species move between adaptive zones. Extant salamanders display three distinct life cycle strategies that range from aquatic to terrestrial (biphasic), to fully aquatic (paedomorphic) and to fully terrestrial (direct development). Life cycle variation is associated with changes in body form such as loss of digits, limb reduction or body elongation. However, the relationships among these traits and life cycle strategy remain unresolved. Here, we use a Bayesian modelling approach to test whether life cycle transitions by salamanders have influenced rates, optima and integration of primary locomotory structures (limbs and trunk). We show that paedomorphic salamanders have elevated rates of limb evolution with optima shifted towards smaller size and fewer digits compared to all other salamanders. Rate of hindlimb digit evolution is shown to decrease in a gradient as life cycles become more terrestrial. Paedomorphs have a higher correlation between hindlimb digit loss and increases in vertebral number, as well as reduced correlations between limb lengths. Our results support the idea that terrestrial plantigrade locomotion constrains limb evolution and, when lifted, leads to higher rates of trait diversification and shifts in optima and integration. The basic tetrapod body form of most salamanders and the independent losses of terrestrial life stages provide an important framework for understanding the evolutionary and developmental mechanisms behind major shifts in ecological zones as seen among early tetrapods during their transition from water to land.     

Evolution of axial patterning in elongate fishes

Within the ray-finned fishes, eel-like (extremely elongate) body forms have evolved multiple times from deeper-bodied forms. Previous studies have shown that elongation of the vertebral column may be associated with an increase in the number of vertebrae, an increase in the length of the vertebral centra, or a combination of both. Because the vertebral column of fishes has at least two anatomically distinct regions (i.e. abdominal and caudal), an increase in the number and relative length of the vertebrae could be region-specific or occur globally across the length of the vertebral column. In the present study, we recorded vertebral counts and measurements of vertebral aspect ratio (vertebral length/width) from museum specimens for 54 species representing seven groups of actinopterygian fishes. We also collected, from published literature, vertebral counts for 813 species from 14 orders of actinopterygian and elasmobranch fishes. We found that the number of vertebrae can increase independently in the abdominal and caudal regions of the vertebral column, but changes in aspect ratio occur similarly in both regions. These findings suggest that abdominal vertebral number, caudal vertebral number, and vertebral aspect ratio are controlled by separate developmental modules. Based on these findings, we suggest some candidate developmental mechanisms that may contribute to vertebral column patterning in fishes. Our study is an example of how comparative anatomical studies of adults can generate testable hypotheses of evolutionary changes in developmental mechanisms.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 90 , 97–116.     

Decoupling of morphological disparity and taxic diversity during the adaptive radiation of anomodont therapsids

Adaptive radiations are central to macroevolutionary theory. Whether triggered by acquisition of new traits or ecological opportunities arising from mass extinctions, it is debated whether adaptive radiations are marked by initial expansion of taxic diversity or of morphological disparity (the range of anatomical form). If a group rediversifies following a mass extinction, it is said to have passed through a macroevolutionary bottleneck, and the loss of taxic or phylogenetic diversity may limit the amount of morphological novelty that it can subsequently generate. Anomodont therapsids, a diverse clade of Permian and Triassic herbivorous tetrapods, passed through a bottleneck during the end-Permian mass extinction. Their taxic diversity increased during the Permian, declined significantly at the Permo–Triassic boundary and rebounded during the Middle Triassic before the clade''s final extinction at the end of the Triassic. By sharp contrast, disparity declined steadily during most of anomodont history. Our results highlight three main aspects of adaptive radiations: (i) diversity and disparity are generally decoupled; (ii) models of radiations following mass extinctions may differ from those triggered by other causes (e.g. trait acquisition); and (iii) the bottleneck caused by a mass extinction means that a clade can emerge lacking its original potential for generating morphological variety.     

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.     

A Carapace-Like Bony ‘Body Tube’ in an Early Triassic Marine Reptile and the Onset of Marine Tetrapod Predation

Parahupehsuchus longus is a new species of marine reptile from the Lower Triassic of Yuan’an County, Hubei Province, China. It is unique among vertebrates for having a body wall that is completely surrounded by a bony tube, about 50 cm long and 6.5 cm deep, comprising overlapping ribs and gastralia. This tube and bony ossicles on the back are best interpreted as anti-predatory features, suggesting that there was predation pressure upon marine tetrapods in the Early Triassic. There is at least one sauropterygian that is sufficiently large to feed on Parahupehsuchus in the Nanzhang-Yuan’an fauna, together with six more species of potential prey marine reptiles with various degrees of body protection. Modern predators of marine tetrapods belong to the highest trophic levels in the marine ecosystem but such predators did not always exist through geologic time. The indication of marine-tetrapod feeding in the Nanzhang-Yuan’an fauna suggests that such a trophic level emerged for the first time in the Early Triassic. The recovery from the end-Permian extinction probably proceeded faster than traditionally thought for marine predators. Parahupehsuchus has superficially turtle-like features, namely expanded ribs without intercostal space, very short transverse processes, and a dorsal outgrowth from the neural spine. However, these features are structurally different from their turtle counterparts. Phylogeny suggests that they are convergent with the condition in turtles, which has a fundamentally different body plan that involves the folding of the body wall. Expanded ribs without intercostal space evolved at least twice and probably even more among reptiles.     

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.