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Ngatiwai Trust Board. et al., The tuatara (Sphenodon punctatus)—the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2—is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which—at approximately 5 Gb—is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing., N.J.G. acknowledges the support of Ngatiwai iwi, Allan Wilson Centre, University of Otago, New Zealand Department of Conservation, New Zealand Genomics and Illumina. J.I.A. was supported by CONICYT National Doctoral Scholarship No. 21130515. M.W. was supported by NIH grant R35 GM124827. Ensembl annotation was supported by the Wellcome Trust (WT108749/Z/15/Z) and the European Molecular Biology Laboratory.

The tuatara (Sphenodon punctatus), the only living member of the archaic reptilian order Rhynchocephalia (Sphenodontia) once widespread across Gondwana, is an iconic and enigmatic terrestrial vertebrate endemic to New Zealand. A key link to the now extinct stem reptiles from which dinosaurs, modern reptiles, birds and mammals evolved, the tuatara provides exclusive insights into the ancestral amniotes. The tuatara genome, at ∼5 Gbp, is among the largest vertebrate genomes assembled. Analysis of this genome and comparisons to other vertebrates reinforces the uniqueness of the tuatara. Phylogenetic analyses indicate tuatara diverged from the snakes and lizards ∼250 MYA. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Genome sequence analysis identifies expansions of protein, non-protein-coding RNA families, and repeat elements, the latter of which show an extraordinary amalgam of reptilian and mammalian features. Sequencing of this genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. It also provides important insights into both the technical challenges and the cultural obligations associated with genome sequencing.

The fossil record provides direct empirical data for understanding macroevolutionary patterns and processes. Inherent biases in the fossil record are well known to confound analyses of this data. Sampling bias proxies have been used as covariates in regression models to test for such biases. Proxies, such as formation count, are associated with paleobiodiversity, but are insufficient for explaining species dispersal owing to a lack of geographic context. Here, we develop a sampling bias proxy that incorporates geographic information and test it with a case study on early tetrapodomorph biogeography. We use recently-developed Bayesian phylogeographic models and a new supertree of early tetrapodomorphs to estimate dispersal rates and ancestral habitat locations. We find strong evidence that geographic sampling bias explains supposed radiations in dispersal rate (potential adaptive radiations). Our study highlights the necessity of accounting for geographic sampling bias in macroevolutionary and phylogenetic analyses and provides an approach to test for its effect.

Phylogenetic comparative methods (PCMs) are commonly used to study evolution and adaptation. However, frequently used PCMs for discrete traits mishandle single evolutionary transitions. They erroneously detect correlated evolution in these situations. For example, hair and mammary glands cannot be said to have evolved in a correlated fashion because each evolved only once in mammals, but a commonly used model (Pagel’s Discrete) statistically supports correlated (dependent) evolution. Using simulations, we find that rate parameter estimation, which is central for model selection, is poor in these scenarios due to small effective (evolutionary) sample sizes of independent character state change. Pagel’s Discrete model also tends to favor dependent evolution in these scenarios, in part, because it forces evolution through state combinations unobserved in the tip data. This model prohibits simultaneous dual transitions along branches. Models with underlying continuous data distributions (e.g., Threshold and GLMM) are less prone to favor correlated evolution but are still susceptible when evolutionary sample sizes are small. We provide three general recommendations for researchers who encounter these common situations: i) create study designs that evaluate a priori hypotheses and maximize evolutionary sample sizes; ii) assess the suitability of evolutionary models—for discrete traits, we introduce the phylogenetic imbalance ratio; and iii) evaluate evolutionary hypotheses with a consilience of evidence from disparate fields, like biogeography and developmental biology. Consilience plays a central role in hypothesis testing within the historical sciences where experiments are difficult or impossible to conduct, such as many hypotheses about correlated evolution. These recommendations are useful for investigations that employ any type of PCM. [Class imbalance; consilience; correlated evolution; evolutionary sample size; phylogenetic comparative methods.]

Exaggerated cranial structures such as crests and horns, hereafter referred to collectively as ornaments, are pervasive across animal species. These structures perform vital roles in visual communication and physical interactions within and between species. Yet the origin and influence of ornamentation on speciation and ecology across macroevolutionary time scales remains poorly understood for virtually all animals. Here, we explore correlative evolution of osseous cranial ornaments with large body size in theropod dinosaurs using a phylogenetic comparative framework. We find that body size evolved directionally toward phyletic giantism an order of magnitude faster in theropod species possessing ornaments compared with unadorned lineages. In addition, we find a body mass threshold below which bony cranial ornaments do not originate. Maniraptoriform dinosaurs generally lack osseous cranial ornaments despite repeatedly crossing this body size threshold. Our study provides novel, quantitative support for a shift in selective pressures on socio-sexual display mechanisms in theropods coincident with the evolution of pennaceous feathers., Many of the theropod dinosaurs, the group including Tyrannosaurus rex, had bony ornamentation on their skulls. Here, Gates et al. show that such ornaments are associated with greater body size and accelerated body size evolution in theropods; however, these relationships are absent in the maniraptoriform dinosaurs, which had evolved pennaceous feathers.

Genome size spans over a 300-fold range among vertebrates (132 pg for Protopterus aethiopicus, the marbled lungfish, and 0.35 pg for Tetraodon nigroviridis, the green spotted pufferfish). While phylogenetic analysis of genome size has helped clarify how this variation evolved in multiple tetrapod groups, the ancestral tetrapod condition still remains poorly characterized, and this obscures our understanding of character state polarity and macroevolutionary trends in genome size. To address this problem, we used phylogenetic comparative methods to analyze paleohistological data from eight taxa of the Middle and Late Paleozoic to the Early Mesozoic: Eusthenopteron, Ichthyostega, Acheloma, Eryops, Trimerorhachis, Wetlugasaurus, an unidentified dissorophoid, and Chroniosaurus. Five other extinct taxa were included from previous studies to better frame our results, including Marmorerpeton, Cardiocephalus, Diplocaulus, an unidentified basal sauropsid, and Mycterosaurus. We augmented a previously reported histological and genome size data set (including data from 14 lissamphibians, three testudines, Sphenodon, five squamates, two crocodilians, 11 birds, and 22 mammals) with genome size and histological data from extant Latimeria and three extant actinopterygians. Our results suggest that all eight of the newly analyzed extinct taxa had genome sizes ranging between 3.2 and 3.9 pg. These results imply that basal tetrapods had genome sizes (and underlying genomic architectures) similar to extant mammals and lepidosaurs. We find no major shifts in genome size during the tetrapod water-to-land transition. Our analysis suggests that Eusthenopteron and Ichthyostega had genome sizes well within the range of extant actinopterygians and Latimeria, despite several whole-genome duplications in actinopterygians.

Urodeles have the largest genomes among extant tetrapods, varying greatly between metamorphic and neotenic species, which have the smallest and the largest genomes of the group, respectively. The evolutionary tempo and mode of genome size expansion in urodeles are poorly documented, especially because genome size does not directly fossilize. Consequently, the ancestral state for genome size, and therefore, the polarity of its evolution in urodeles are uncertain. However, recent studies have demonstrated that osteocyte (lacuna) size is correlated with genome size. Below, we present histological data, on osteocyte lacuna size from one of the oldest known stem-urodeles, Marmorerpeton, from the Middle Jurassic (Bathonian, 166–168 Ma), as well as on five extant urodele species. Our analysis of these taxa, coupled with previously published data, suggests that stem-urodeles had already evolved large genomes, typical of extant urodeles by the Bathonian.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana

Genome size has long been hypothesized to affect the metabolic rate in various groups of animals. The mechanism behind this proposed association is the nucleotypic effect, in which large nucleus and cell sizes influence cellular metabolism through surface area-to-volume ratios. Here, we provide a review of the recent literature on the relationship between genome size and metabolic rate. We also conduct an analysis using phylogenetic comparative methods and a large sample of extant vertebrates. We find no evidence that the effect of genome size improves upon models in explaining metabolic rate variation. Not surprisingly, our results show a strong positive relationship between metabolic rate and body mass, as well as a substantial difference in metabolic rate between endothermic and ectothermic vertebrates, controlling for body mass. The presence of endothermy can also explain elevated rate shifts in metabolic rate whereas genome size cannot. We further find no evidence for a punctuated model of evolution for metabolic rate. Our results do not rule out the possibility that genome size affects cellular physiology in some tissues, but they are consistent with previous research suggesting little support for a direct functional connection between genome size and basal metabolic rate in extant vertebrates. This article is part of the theme issue ‘Vertebrate palaeophysiology’.

The field of evolutionary developmental biology is broadly focused on identifying the genetic and developmental mechanisms underlying morphological diversity. Connecting the genotype with the phenotype means that evo-devo research often considers a wide range of evidence, from genetics and morphology to fossils. In this commentary, we provide an overview and framework for integrating fossil ontogenetic data with developmental data using phylogenetic comparative methods to test macroevolutionary hypotheses. We survey the vertebrate fossil record of preserved embryos and discuss how phylogenetic comparative methods can integrate data from developmental genetics and paleontology. Fossil embryos provide limited, yet critical, developmental data from deep time. They help constrain when developmental innovations first appeared during the history of life and also reveal the order in which related morphologies evolved. Phylogenetic comparative methods provide a powerful statistical approach that allows evo-devo researchers to infer the presence of nonpreserved developmental traits in fossil species and to detect discordant evolutionary patterns and processes across levels of biological organization.

Mammalian bipedalism has long been thought to have arisen in response to arid and open environments. Here, we tested whether bipedalism coevolved with environmental changes using molecular and paleontological data from the rodent superfamily Dipodoidea and statistical methods for reconstructing ancestral characteristics and past climates. Our results show that the post-Late Miocene aridification exerted selective pressures on tooth shape, but not on leg length of bipedal jerboas. Cheek tooth crown height has increased since the Late Miocene, but the hind limb/head-body length ratios remained stable and high despite the environmental change from humid and forested to arid and open conditions, rather than increasing from low to high as predicted by the arid-bipedalism hypothesis. The decoupling of locomotor and dental character evolution indicates that bipedalism evolved under selective pressure different from that of dental hypsodonty in jerboas. We reconstructed the habitats of early jerboas using floral and faunal data, and the results show that the environments in which bipedalism evolved were forested. Our results suggest that bipedalism evolved as an adaptation to humid woodlands or forests for vertical jumping. Running at high speeds is likely a by-product of selection for jumping, which became advantageous in open environments later on.

Allowing biogeographical data to evolve at varying rates on a globe, not a plane, reveals new insights into the origin and dispersal of dinosaurs. The method could also be applied to manifold organisms, from humans to influenza viruses.

Acquisition and storage of Latimeria chalumnae samples was supported by grants from the African Coelacanth Ecosystem Programme of the South African National Department of Science and Technology. Generation of the Latimeria chalumnae and Protopterus annectens sequences by the Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard University was supported by grants from the National Human Genome Research Institute (NHGRI). K.L.T. is the recipient of a EURYI award from the European Science Foundation. We would also like to thank the Genomics Sequencing Platform of the Broad Institute for sequencing the L. chalumnae genome and L. chalumnae and P. annectens transcriptomes, S. Ahamada, R. Stobbs and the Association pour le Protection de Gombesa (APG) for their help in obtaining coelacanth samples, Y. Zhao for the use of data from Rana chensinensis, and L. Gaffney, C. Hamilton and J. Westlund for assistance with figure preparation. 10; International audience; The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Live birth has evolved many times independently in vertebrates, such as mammals and diverse groups of lizards and snakes. However, live birth is unknown in the major clade Archosauromorpha, a group that first evolved some 260 million years ago and is represented today by birds and crocodilians. Here we report the discovery of a pregnant long-necked marine reptile (Dinocephalosaurus) from the Middle Triassic (∼245 million years ago) of southwest China showing live birth in archosauromorphs. Our discovery pushes back evidence of reproductive biology in the clade by roughly 50 million years, and shows that there is no fundamental reason that archosauromorphs could not achieve live birth. Our phylogenetic models indicate that Dinocephalosaurus determined the sex of their offspring by sex chromosomes rather than by environmental temperature like crocodilians. Our results provide crucial evidence for genotypic sex determination facilitating land-water transitions in amniotes., Although live birth evolved repeatedly in other clades, it has not been found in archosauromorphs, the group including modern birds and crocodilians. Here, the authors describe a fossilized pregnant Dinocephalosaurus from ∼245 million years ago, providing evidence of live birth in archosauromorphs.

The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments. Among amniotes, genome sequences are available for mammals and birds, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes. Also, A. carolinensis mobile elements are very young and diverse—more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations., National Science Foundation (U.S.) (NSF grant DEB-0920892), National Science Foundation (U.S.) (NSF grant DEB-0844624), National Human Genome Research Institute (U.S.)

Rugose projections on the anterior and posterior aspects of vertebral neural spines appear throughout Amniota and result from the mineralization of the supraspinous and interspinous ligaments via metaplasia, the process of permanent tissue-type transformation. In mammals, this metaplasia is generally pathological or stress induced, but is a normal part of development in some clades of birds. Such structures, though phylogenetically sporadic, appear throughout the fossil record of non-avian theropod dinosaurs, yet their physiological and adaptive significance has remained unexamined. Here we show novel histologic and phylogenetic evidence that neural spine projections were a physiological response to biomechanical stress in large-bodied theropod species. Metaplastic projections also appear to vary between immature and mature individuals of the same species, with immature animals either lacking them or exhibiting smaller projections, supporting the hypothesis that these structures develop through ontogeny as a result of increasing bending stress subjected to the spinal column. Metaplastic mineralization of spinal ligaments would likely affect the flexibility of the spinal column, increasing passive support for body weight. A stiff spinal column would also provide biomechanical support for the primary hip flexors and, therefore, may have played a role in locomotor efficiency and mobility in large-bodied species. This new association of interspinal ligament metaplasia in Theropoda with large body size contributes additional insight to our understanding of the diverse biomechanical coping mechanisms developed throughout Dinosauria, and stresses the significance of phylogenetic methods when testing for biological trends, evolutionary or not.

An unsolved question in evolutionary genomics is whether amniote genomes have been expanding or contracting since the common ancestor of this diverse group. Here, we report on the polarity of amniote genome size evolution using genome size estimates for 14 extinct tetrapod genera from the Paleozoic and early Mesozoic Eras using osteocyte lacunae size as a correlate. We find substantial support for a phylogenetically controlled regression model relating genome size to osteocyte lacunae size (P of slopes

Migration promotes utilization of seasonal resources, and the distance flown is associated with specific morphologies, yet these relationships can be confounded by environmental factors and phylogeny. Understanding adaptations associated with migration is important: although migration patterns change rapidly, it is unclear whether migratory traits track behavioural shifts. We studied morphometrics of four stonechat populations representing a migratory gradient and raised under common-garden conditions. With multivariate analyses, we identified wing traits that differed clearly from general size trends, and used phylogenetic comparative methods to test the prediction that these traits correlated with migratory distance in captive and wild populations. Pointedness differed among populations, changed independently from overall body size, and was correlated with migration distance. Migration in stonechats may lead to deviations from allometric size changes, suggesting that birds may adapt morphologically to selection pressures created by their own behaviour in response to changing environmental conditions.

The two living groups of flying vertebrates, birds and bats, both have constricted genome sizes compared with their close relatives. But nothing is known about the genomic characteristics of pterosaurs, which took to the air over 70 Myr before birds and were the first group of vertebrates to evolve powered flight. Here, we estimate genome size for four species of pterosaurs and seven species of basal archosauromorphs using a Bayesian comparative approach. Our results suggest that small genomes commonly associated with flight in bats and birds also evolved in pterosaurs, and that the rate of genome-size evolution is proportional to genome size within amniotes, with the fastest rates occurring in lineages with the largest genomes. We examine the role that drift may have played in the evolution of genome size within tetrapods by testing for correlated evolution between genome size and body size, but find no support for this hypothesis. By contrast, we find evidence suggesting that a combination of adaptation and phylogenetic inertia best explains the correlated evolution of flight and genome-size contraction. These results suggest that small genome/cell size evolved prior to or concurrently with flight in pterosaurs. We predict that, similar to the pattern seen in theropod dinosaurs, genome-size contraction preceded flight in pterosaurs and bats.

Reptiles (sauropsids) represent the sister group to mammals, and the basal members of Reptilia may provide a good model for the condition of the common ancestor of both groups. Sex-determining mechanisms (SDM) and organizations of sex chromosomes among genotypically sex-determining (GSD) species vary widely across reptiles. Birds and snakes, for example, are entirely GSD whereas other reptiles, like all crocodilians, exhibit temperature-dependent sex determination (TSD). Here we explore the evolution of sex chromosomes and SDM within reptiles, using family-level analyses of character evolution and applying parsimony, likelihood, Bayesian, and stochastic methods. We find support for the common ancestor of amphisbaenians and whiptail lizards (Laterata) possessing the XY (male heterogametic) GSD mechanism, while the ancestors of Testudines and Crocodylia, as well as the larger group Archosauromorpha (here containing turtles) are inferred to have exhibited TSD. We also find evidence consistent with the hypothesis that the XY system is more labile and evolves faster than does the ZW (female heterogametic) system. Phylogenetic-based speciation tests do not support an association between GSD and speciation, and reject the hypothesis that the presence of the XY system is associated with speciation in reptiles.

One of the less well known factors associated with flight in vertebrates is a reduction of the size of the genome. Birds have remarkably small genomes compared with other vertebrates, and bats tend to have smaller genomes than do non-flying mammals. But does flight cause genome loss, or does genome loss predispose animals to take flight? The latter seems to be the case, to judge from an extensive analysis of bone cell volume and genome size in dinosaurs. Genome reduction can be traced deep into the lineage of saurischian dinosaurs — the dinosaur group of which the birds are the only surviving members — but not in ornithischian dinosaurs. A correlate of flight in vertebrates is a reduction of the genome. But does flight cause genome loss, or does genome loss predispose animals to take flight? Histology and statistical techniques shows that genome reduction can be traced deep into the lineage of saurischian dinosaurs. Genome reduction thus joins the morphological features we now associate with birds that can be traced far back into the dinosaurian ancestry. Avian genomes are small and streamlined compared with those of other amniotes by virtue of having fewer repetitive elements and less non-coding DNA1,2. This condition has been suggested to represent a key adaptation for flight in birds, by reducing the metabolic costs associated with having large genome and cell sizes3,4. However, the evolution of genome architecture in birds, or any other lineage, is difficult to study because genomic information is often absent for long-extinct relatives. Here we use a novel bayesian comparative method to show that bone-cell size correlates well with genome size in extant vertebrates, and hence use this relationship to estimate the genome sizes of 31 species of extinct dinosaur, including several species of extinct birds. Our results indicate that the small genomes typically associated with avian flight evolved in the saurischian dinosaur lineage between 230 and 250 million years ago, long before this lineage gave rise to the first birds. By comparison, ornithischian dinosaurs are inferred to have had much larger genomes, which were probably typical for ancestral Dinosauria. Using comparative genomic data, we estimate that genome-wide interspersed mobile elements, a class of repetitive DNA, comprised 5–12% of the total genome size in the saurischian dinosaur lineage, but was 7–19% of total genome size in ornithischian dinosaurs, suggesting that repetitive elements became less active in the saurischian lineage. These genomic characteristics should be added to the list of attributes previously considered avian but now thought to have arisen in non-avian dinosaurs, such as feathers5, pulmonary innovations6, and parental care and nesting7.

The ornithischia and its sister group, the Saurischia, comprise the Dinosauria. Ornithischians (Weishampel, 2004) are a diverse group of primarily quadrupedal herbivores that include such familiar dinosaurs as the armored stegosaurs and ankylosaurs (Thyreophora), as well as the duckbilled hadrosaurs, pachycephalosaurs, and ceratopsians (Cerapoda). The Ceratopsia (You and Dodson, 2004) includes a number of relatively small basal forms such as psittacosaurs and Neoceratopsia, which comprises protoceratopsids and the larger, more derived horned Ceratopsidae. Ossified tendons occur throughout the body of many birds (Vanden Berge and Storer, 1995), but reports in other Saurischia are rare (but see Sereno et al., 2004). However, they occur in nearly all ornithischian dinosaurs except stegosaurs (Sereno and Dong, 1992). The presence of ossified tendons above the sacrum is thought to be a diagnostic character of the Ornithischia (Weishampel, 2004). Many taxa also develop them in association with the caudal and/or thoracic vertebrae. Ossified tendons usually occur in bundles along the epaxial region of the vertebral column and the hypaxial region of the tail in basal ornithopods such as Hypsilophodon Galton, 1974. Similar tendon bundles are known in pachycephalosaurs (Sues and Galton, 1987) and ankylosaurs possess tendons associated with the tail “club” (Coombs, 1995). The ornithopod group Iguanodontoidea (Hadrosauridae and Iguanodontidae plus Protohadros Head, 1998 and Ouranosaurus Taquet, 1976) developed a rhomboidal trellis of ossified tendons (Fig. 1), often considered to be synapomorphic (e.g., Sereno, 1999), along the dorsal side of their vertebral columns (Dollo, 1886; Brown, 1916; Parks, 1920; Lull and Wright, 1942; Norman, 1980, 1986; Prieto-Marquez, 2001; Horner et al., 2004). Note that Hadrosauriformes is synonymous with Iguanodontoidea, but the latter is preferred (Norman, 2004) and used here. Figure 1 — Brachylophosaurus canadensis Sternberg, 1953, MOR 794. Posterior thoracic vertebrae showing the ossified tendon trellis …

Spinal ossified tendons are a defining character for Ornithischia, one of the two major clades of dinosaurs. The function of these bony rods has remained a mystery since their first detailed description in 1886. Qualitative approaches to understand ossified tendon function have resulted in different ecological and behavioral interpretations for ornithopod dinosaurs. To evaluate ossified tendon function, this study constructed finite element models of the vertebral column for two ornithopod taxa:Tenontosaurus, which shows the plesiomorphic condition of longitudinally arrayed tendons along the spinous processes, andBrachylophosaurus, which exhibits a lattice of tendons along the spinous processes. Both models predict that ossified tendons stiffened the vertebral column, especially the tail, but the derived lattice of ossified tendons in iguanodontoidean dinosaurs, likeBrachylophosaurus, increased spinal stiffness more than the plesiomorphic condition. Caudofemoral muscles that retracted the hindlimb during locomotion attached the femur to the tail in ornithopods. Increased tail stiffness caused by intratendinous ossification may have influenced locomotion by rigidly anchoring M. caudofemoralis longus to the tail, thereby allowing a more forceful retraction of the hindlimb by reducing ventral flexion of the tail during muscle contraction. Ossified tendons may also have been important for storing elastic energy throughout the gait cycle. Moreover, the lattice of ossified tendons stiffened the trunk and tail nearly equally inBrachylophosaurus, indicating the evolution of a postural function by passively supporting the epaxial musculature in maintaining a horizontal vertebral column.

In reptiles, sex-determining mechanisms have evolved repeatedly and reversibly between genotypic and temperature-dependent sex determination. The gene Dmrt1 directs male determination in chicken (and presumably other birds), and regulates sex differentiation in animals as distantly related as fruit flies, nematodes and humans. Here, we show a consistent molecular difference in Dmrt1 between reptiles with genotypic and temperature-dependent sex determination. Among 34 non-avian reptiles, a convergently evolved pair of amino acids encoded by sequence within exon 2 near the DM-binding domain of Dmrt1 distinguishes species with either type of sex determination. We suggest that this amino acid shift accompanied the evolution of genotypic sex determination from an ancestral condition of temperature-dependent sex determination at least three times among reptiles, as evident in turtles, birds and squamates. This novel hypothesis describes the evolution of sex-determining mechanisms as turnover events accompanied by one or two small mutations.

The field of evolutionary developmental biology is broadly focused on identifying the genetic and developmental mechanisms underlying morphological diversity. Connecting the genotype with the phenotype means that evo-devo research often considers a wide range of evidence, from genetics and morphology to fossils. In this commentary, we provide an overview and framework for integrating fossil ontogenetic data with developmental data using phylogenetic comparative methods to test macroevolutionary hypotheses. We survey the vertebrate fossil record of preserved embryos and discuss how phylogenetic comparative methods can integrate data from developmental genetics and paleontology. Fossil embryos provide limited, yet critical, developmental data from deep time. They help constrain when developmental innovations first appeared during the history of life and also reveal the order in which related morphologies evolved. Phylogenetic comparative methods provide a powerful statistical approach that allows evo-devo researchers to infer the presence of nonpreserved developmental traits in fossil species and to detect discordant evolutionary patterns and processes across levels of biological organization.

Development and metaplasia of ossified tendons in two hadrosaurine dinosaurs (Maiasaura peeblesorum and Brachylophosaurus canadensis) are described by comparison with the known developmental processes of ossified tendons in turkeys (Meleagris gallopavo). Mineralized primary tendon tissue and replacement patterns suggest that ossified tendons in hadrosaurs grew initially in a manner similar to those in turkeys. That is, biomineralization begins with apatite deposition and is followed by resorption of mineralized primary tissue and subsequent bone formation. Earlier ontogenetic onset of tendon ossification and later offset among ossified tendons of hadrosaurs differentiates them from tendons of turkeys. In addition, an external fundamental system (EFS) in hadrosaurian tendons indicates that they grew radially beyond the original tendon boundary, a process unknown among avian tendons. Tendon histology indicates that hadrosaurian ossified tendons developed in step with the skeletal system and that ossification was not induced by biomechanical stresses.

Intratendinous ossification is widespread in dinosaurs (including birds). Although intratendinous ossification in living birds is well understood, the physiological process of tendon metaplasia and associated histological variability in Dinosauria are not. Therefore, ossified tendons were histologically sampled across extinct dinosaurian clades. Tendons of living birds and alligators were also sampled. Despite various anatomical locations and large differences in body size, ossified tendons were found to possess uniform microstructure even in specimens that do not normally experience intratendinous ossification (such as Spinostropheus and Camarasaurus). The ossified tendons of non-avian dinosaurs are largely indistinguishable from skeletal bone with respect to microanatomical features. However, ossified tendons in birds lack periosteal bone and associated fibrolamellar structures associated with ornithischian dinosaur tendons. Variation in periosteal bone occurs along the length of individual ten...

The meter-grid system, using spikes hammered into the quarry floor as grid nodes, is a common way to map paleontological and archaeological excavations. This method is invasive to the fossil-bearing horizon and can damage or even destroy buried specimens. This potential is greatest when the site is characterized by high bone concentrations, and/or specimen/matrix friability. A new portable mapping system based on a floating-grid was developed to circumvent these problems. This system also is well suited for excavations with uneven quarry floors. The floating-grid system extends over a quarry using retractable wires and mobile posts. It employs a mapping square that hangs from wires extended from a back wall or rebar posts.

Mammalian bipedalism has long been thought to have arisen in response to arid and open environments. Here, we tested whether bipedalism coevolved with environmental changes using molecular and paleontological data from the rodent superfamily Dipodoidea and statistical methods for reconstructing ancestral characteristics and past climates. Our results show that the post-Late Miocene aridification exerted selective pressures on tooth shape, but not on leg length of bipedal jerboas. Cheek tooth crown height has increased since the Late Miocene, but the hind limb/head-body length ratios remained stable and high despite the environmental change from humid and forested to arid and open conditions, rather than increasing from low to high as predicted by the arid-bipedalism hypothesis. The decoupling of locomotor and dental character evolution indicates that bipedalism evolved under selective pressure different from that of dental hypsodonty in jerboas. We reconstructed the habitats of early jerboas using floral and faunal data, and the results show that the environments in which bipedalism evolved were forested. Our results suggest that bipedalism evolved as an adaptation to humid woodlands or forests for vertical jumping. Running at high speeds is likely a by-product of selection for jumping, which became advantageous in open environments later on.

A premature stop codon in ACTN3 resulting in α-actinin-3 deficiency (the ACTN3 577XX genotype) is common in humans and reduces strength, muscle mass, and fast-twitch fiber diameter, but increases the metabolic efficiency of skeletal muscle. Linkage disequilibrium data suggest that the ACTN3 R577X allele has undergone positive selection during human evolution. The allele has been hypothesized to be adaptive in environments with scarce resources where efficient muscle metabolism would be selected. Here we test this hypothesis by using recently developed comparative methods that account for evolutionary relatedness and gene flow among populations. We find evidence that the ACTN3 577XX genotype evolved in association with the global latitudinal gradient. Our results suggest that environmental variables related to latitudinal variation, such as species richness and mean annual temperature, may have influenced the adaptive evolution of ACTN3 577XX during recent human history.

The timing of the origin and diversification of rodents remains controversial, due to conflicting results from molecular clocks and paleontological data. The fossil record tends to support an early Cenozoic origin of crown-group rodents. In contrast, most molecular studies place the origin and initial diversification of crown-Rodentia deep in the Cretaceous, although some molecular analyses have recovered estimated divergence times that are more compatible with the fossil record. Here we attempt to resolve this conflict by carrying out a molecular clock investigation based on a nine-gene sequence dataset and a novel set of seven fossil constraints, including two new rodent records (the earliest known representatives of Cardiocraniinae and Dipodinae). Our results indicate that rodents originated around 61.7–62.4 Ma, shortly after the Cretaceous/Paleogene (K/Pg) boundary, and diversified at the intraordinal level around 57.7–58.9 Ma. These estimates are broadly consistent with the paleontological record, but challenge previous molecular studies that place the origin and early diversification of rodents in the Cretaceous. This study demonstrates that, with reliable fossil constraints, the incompatibility between paleontological and molecular estimates of rodent divergence times can be eliminated using currently available tools and genetic markers. Similar conflicts between molecular and paleontological evidence bedevil attempts to establish the origination times of other placental groups. The example of the present study suggests that more reliable fossil calibration points may represent the key to resolving these controversies.

Characterization of reptilian genomes is essential for understanding the overall diversity and evolution of amniote genomes, because reptiles, which include birds, constitute a major fraction of the amniote evolutionary tree. To better understand the evolution and diversity of genomic characteristics in Reptilia, we conducted comparative analyses of online sequence data from Alligator mississippiensis (alligator) and Sphenodon punctatus (tuatara) as well as genome size and karyological data from a wide range of reptilian species. At the whole-genome and chromosomal tiers of organization, we find that reptilian genome size distribution is consistent with a model of continuous gradual evolution while genomic compartmentalization, as manifested in the number of microchromosomes and macrochromosomes, appears to have undergone early rapid change. At the sequence level, the third genomic tier, we find that exon size in Alligator is distributed in a pattern matching that of exons in Gallus (chicken), especially in the 101-200 bp size class. A small spike in the fraction of exons in the 301 bp-1 kb size class is also observed for Alligator, but more so for Sphenodon. For introns, we find that members of Reptilia have a larger fraction of introns within the 101 bp-2 kb size class and a lower fraction of introns within the 5-30 kb size class than do mammals. These findings suggest that the mode of reptilian genome evolution varies across three hierarchical levels of the genome, a pattern consistent with a mosaic model of genomic evolution.

New genes are thought to arise primarily through a process of gene duplication. Genes that are homologous as a result of divergence across lineages via speciation are said to be orthologous , whereas genes that are homologous as a result of gene duplication are paralogous (Li, 2006). Paralogous genes that are functionally redundant and selectively nearly neutral can result in one copy being mutated into a functionless sequence called a pseudogene, or they can be deleted altogether. On the other hand, some duplicated genes can be beneficial from their time of origin because of dosage effects (Kondrashov et al., 2002) and may ultimately be important for speciation. In Passeriformes (perching birds) this may be the case for growth hormone (GH) paralogs, which have undergone differential selection since their divergence (Yuri et al., 2008). In a process called subfunctionalization , each paralog adopts partial function of their ancestral gene (Nowak et al., 1997; Lynch and Force, 2000). Changes in gene expression immediately following duplication as a result of

Many noncoding regions of genomes appear to be essential to genome function. Conservation of large numbers of noncoding sequences has been reported repeatedly among mammals but not thus far among birds and reptiles. By searching genomes of chicken (Gallus gallus), zebra finch (Taeniopygia guttata), and green anole (Anolis carolinensis), we quantified the conservation among birds and reptiles and across amniotes of long, conserved noncoding sequences (LCNS), which we define as sequences ≥500 bp in length and exhibiting ≥95% similarity between species. We found 4,294 LCNS shared between chicken and zebra finch and 574 LCNS shared by the two birds and Anolis. The percent of genomes comprised by LCNS in the two birds (0.0024%) is notably higher than the percent in mammals (

The genomes of birds and nonavian reptiles (Reptilia) are critical for understanding genome evolution in mammals and amniotes generally. Despite decades of study at the chromosomal and single-gene levels, and the evidence for great diversity in genome size, karyotype, and sex chromosome diversity, reptile genomes are virtually unknown in the comparative genomics era. The recent sequencing of the chicken and zebra finch genomes, in conjunction with genome scans and the online publication of the Anolis lizard genome, has begun to clarify the events leading from an ancestral amniote genome—predicted to be large and to possess a diverse repeat landscape on par with mammals and a birdlike sex chromosome system—to the small and highly streamlined genomes of birds. Reptilia exhibit a wide range of evolutionary rates of different subgenomes and, from isochores to mitochondrial DNA, provide a critical contrast to the genomic paradigms established in mammals.

In this review, we describe the history of amniote sex determination as a classic example of Darwinian evolution. We suggest that evolutionary changes in sex determination provide a foundation for understanding important aspects of chromosome and genome organization that otherwise appear haphazard in their origins and contents. Species with genotypic sex determination often possess heteromorphic sex chromosomes, whereas species with environmental sex determination lack them. Through a series of mutations followed by selection at key genes, sex-determining mechanisms have turned over many times throughout the amniote lineage. As a consequence, amniote genomes have undergone gains or losses of sex chromosomes. We review the genomic and ecological contexts in which either temperature-dependent or genotypic sex determination has evolved. Once genotypic sex determination emerges in a lineage, viviparity and heteromorphic sex chromosomes become more likely to evolve. For example, in extinct marine reptiles, genotypic sex determination apparently led to viviparity, which in turn facilitated their pelagic radiation. Sex chromosomes comprise genome regions that differ from autosomes in recombination rate, mutation rate, levels of polymorphism, and the presence of sex-determining and sexually antagonistic genes. In short, many aspects of amniote genome complexity, life history, and adaptive radiation appear contingent on evolutionary changes in sex-determining mechanisms.

Sauropodomorph dinosaurs include the largest land animals to have ever lived, some reaching up to 10 times the mass of an African elephant. Despite their status defining the upper range for body size in land animals, it remains unknown whether sauropodomorphs evolved larger-sized genomes than non-avian theropods, their sister taxon, or whether a relationship exists between genome size and body size in dinosaurs, two questions critical for understanding broad patterns of genome evolution in dinosaurs. Here we report inferences of genome size for 10 sauropodomorph taxa. The estimates are derived from a Bayesian phylogenetic generalized least squares approach that generates posterior distributions of regression models relating genome size to osteocyte lacunae volume in extant tetrapods. We estimate that the average genome size of sauropodomorphs was 2.02 pg (range of species means: 1.77–2.21 pg), a value in the upper range of extant birds (mean = 1.42 pg, range: 0.97–2.16 pg) and near the average for extant non-avian reptiles (mean = 2.24 pg, range: 1.05–5.44 pg). The results suggest that the variation in size and architecture of genomes in extinct dinosaurs was lower than the variation found in mammals. A substantial difference in genome size separates the two major clades within dinosaurs, Ornithischia (large genomes) and Saurischia (moderate to small genomes). We find no relationship between body size and estimated genome size in extinct dinosaurs, which suggests that neutral forces did not dominate the evolution of genome size in this group.

The Birds and the Dinosaurs The extent to which primary tissues are preserved in ancient fossils remains controversial. Schweitzer et al. (p. 626 ; see the news story by Service ) describe well-preserved tissues and primary collagen sequences from the femur of an 80-million-year-old hadrosaur. The fossil preserved structures resembling primary bone tissues and vessels. Both extracts and tissue pieces were analyzed in multiple laboratories by mass spectrometry, which revealed ancient collagen sequences that support a close relation between birds and dinosaurs.

Land vertebrates have returned to the sea many times over the ages. Modern seals and whales, as mammals, are live-bearing, and their sex is determined by genotype. But what of the many reptiles that once thronged the seas, such as the mosasaurs, ichthyosaurs and plesiosaurs? Sex determination might be environmentally or genotypically determined, and birth might be live, or through eggs. Based on phylogenetic analysis, Organ et al. propose that the sea-going reptiles of the past were not only live-bearers (which we know from the fossil record) but had genotypic sex determination. This would have freed them from the need to return to land to give birth (amniote eggs perish under water) and enabled their morphological transformation into highly evolved fish-like forms. Adaptive radiations often follow the evolution of key traits. The mechanism by which a species determines the sex of its offspring has been linked to critical ecological and life-history traits but not to major adaptive radiations. A coevolutionary relationship is now established in 94 amniote species between the sex-determining mechanism and whether a species bears live young or lays eggs. This is used to predict the evolution of genotypic sex determination before the acquisition of live birth in three extinct marine reptiles. Adaptive radiations often follow the evolution of key traits, such as the origin of the amniotic egg and the subsequent radiation of terrestrial vertebrates. The mechanism by which a species determines the sex of its offspring has been linked to critical ecological and life-history traits1,2,3 but not to major adaptive radiations, in part because sex-determining mechanisms do not fossilize. Here we establish a previously unknown coevolutionary relationship in 94 amniote species between sex-determining mechanism and whether a species bears live young or lays eggs. We use that relationship to predict the sex-determining mechanism in three independent lineages of extinct Mesozoic marine reptiles (mosasaurs, sauropterygians and ichthyosaurs), each of which is known from fossils to have evolved live birth4,5,6,7. Our results indicate that each lineage evolved genotypic sex determination before acquiring live birth. This enabled their pelagic radiations, where the relatively stable temperatures of the open ocean constrain temperature-dependent sex determination in amniote species. Freed from the need to move and nest on land4,5,8, extreme physical adaptations to a pelagic lifestyle evolved in each group, such as the fluked tails, dorsal fins and wing-shaped limbs of ichthyosaurs. With the inclusion of ichthyosaurs, mosasaurs and sauropterygians, genotypic sex determination is present in all known fully pelagic amniote groups (sea snakes, sirenians and cetaceans), suggesting that this mode of sex determination and the subsequent evolution of live birth are key traits required for marine adaptive radiations in amniote lineages.

Chris L. Organ, Mary H. Schweitzer, Wenxia Zheng, Lisa M. Freimark, Lewis C. Cantley, John M. Asara (2008): Molecular phylogenetics of Mastodon and Tyrannosaurus rex. Science 320: 499, DOI: 10.1126/science.1154284

Protein sequences from bone-derived collagen as old as 68 million years have been detected by using mass spectrometry (1, 2). A BLAST search of the resulting peptide sequences found the highest similarities between the collagen peptides obtained from extracts of Tyrannosaurus rex fossil bone and those ofbirds (Gallus gallus) and between mastodon (Mammut americanum) and other mam­ mals, including elephant (Loxodonta africana). We performed phylogenetic analyses to infer the evolutionary relationships of the T. rex [Museum of the Rockies (MOR) 1125] and mastodon (MOR 605). The results extend our knowledge of trait evo­ lution within nonavian dinosaurs into the macro­ molecular level of biological organization. We used 21 extant organisms in the analyses. Collagen a 1(I) and a2(I) protein sequences from 19 extant organisms (3) were obtained from the National Center for Biotechnology Information (NCBI) and ENSEMBL databases. Collagen a1(I) and a2(I) peptide sequences from a metacarpal of Alligator mississippiensis were obtained by mi­ crocapillary liquid chromatography tandem mass spectrometry (LC/MS/MS) with an ion trap mass spectrometer using the Sequest algorithm (1, 2) and the Paragon algorithm (4). Ostrich collagen se­ quences were determined elsewhere (1, 2). Sequences from alligator (Crocodilia) and ostrich (Aves) rep­ resent 63% and 43% of the full-length collagen a1(I) protein available for other organisms, respec­ tively. The collagen a1(I) and a2(I) sequences for elephant (L. africana), tenrec (Echinops telfairi), and green anole (Anolis carolinensis) were ob­ tained by using gene translations from online databases (5). P ' Bayesian, likelihood, parsimony, and dis­ tance methods were used to generate evolu­ tionary trees (6). In the Bayesian analysis, the posterior distribution of trees reconstructed all extant groups in generally agreed-upon rela­ tionships (the posterior probability of clades ranged from 0.80 to 1.00), with the exception of green anole (A. carolinensis), which is in­ ferred here to lie at the base of amniotes in­ stead ofgrouping as the sister taxon to alligator and birds (archosaurs) (Fig. 1). LC/MS/MS from tryptic digests produces fragmentary pro­ tein sequence data; however, we found unequivocal support (posterior probability of 1.00) uniting mastodon with elephant as members of Elephantinae, which together group with tenrec (E. telfairi) as members of the mam­ malian group Afrotheria (7). Maximum likeli­ hood produces the same groupings, although with less support (approximate likelihood ratio test; aLRT = 0.855 for Elephantinae and 0.872 for Afrotheria). Maximum parsimony analysis also groups mastodon, elephant, and tenrec together (fig. S1, B to D). For the T. rex sample, we used five peptide sequences from collagen a1(I) and one from collagen a2(I) (1, 2, 8) for a total of 89 amino acids (Fig. 1). The T. rex clusters within the Archosauria (posterior probability of 0.92), more closely related to birds (chicken and ostrich, 0.9) than alligator, although a lack of informative sites in the ostrich and T. rex leaves Dinosauria unresolved. The likelihood tree is identical to the Bayesian tree, except for higher support at these locations in the tree (aLRT = 0.969 for Archosauria and 0.907 for Dinosauria). Branch lengths (expected rates of change per site) indicate a relatively stable and uniform rate of evolution, lacking evidence for a deviation from a molecular clock. Maximum par­ simony analysis also groups the T. rex with the chicken and ostrich, although bootstrap support is low (fig. S1, B to D). Neighbor joining groups the T. rex with the birds, but miscalculates the branching order and misplaces alligator, mastodon, and several extant organisms (fig. S1, B to D). The slight disagreement between the distance results compared with the Bayesian, likelihood, and parsimony results (all three of which are concordant) are predictable given that distance methods perform poorly for taxa with large amounts of missing data (9). Nevertheless, there is congruence between three out of the four methods for the mastodon and four out of four methods for the T. rex despite the problem of missing sequence data. The recovered sequences contain informative phylogenetic signal consistent with predictions based on genetic and morphological data for mastodon (10, 11) and on morphological data for T. rex (12, 13) These results support the endogenous origin of the preserved collagen molecules and confirm the prediction based on morphology that, ifbiomolecules could be retrieved from a nonavian dinosaur, they would share a higher degree of similarity with birds than with other extant vertebrates. Our findings suggest that molecular data from long-extinct organisms may have the potential for resolving relationships at critical areas of the vertebrate evolutionary tree that have, so far, been intractable. The findings presented here also bolster the use ofmorphology in phylogenetics because our results are consistent with studies on the evolutionary relationships of fossil forms that rely on morphology (12, 13).