Fossil biomolecules reveal an avian metabolism in the ancestral dinosaur.

Birds and mammals independently evolved the highest metabolic rates among living animals1. Their metabolism generates heat that enables active thermoregulation1, shaping the ecological niches they can occupy and their adaptability to environmental change2. The metabolic performance of birds, which exceeds that of mammals, is thought to have evolved along their stem lineage3-10. However, there is no proxy that enables the direct reconstruction of metabolic rates from fossils. Here we use in situ Raman and Fourier-transform infrared spectroscopy to quantify the in vivo accumulation of metabolic lipoxidation signals in modern and fossil amniote bones. We observe no correlation between atmospheric oxygen concentrations11 and metabolic rates. Inferred ancestral states reveal that the metabolic rates consistent with endothermy evolved independently in mammals and plesiosaurs, and are ancestral to ornithodirans, with increasing rates along the avian lineage. High metabolic rates were acquired in pterosaurs, ornithischians, sauropods and theropods well before the advent of energetically costly adaptations, such as flight in birds. Although they had higher metabolic rates ancestrally, ornithischians reduced their metabolic abilities towards ectothermy. The physiological activities of such ectotherms were dependent on environmental and behavioural thermoregulation12, in contrast to the active lifestyles of endotherms1. Giant sauropods and theropods were not gigantothermic9,10, but true endotherms. Endothermy in many Late Cretaceous taxa, in addition to crown mammals and birds, suggests that attributes other than metabolism determined their fate during the terminal Cretaceous mass extinction.

A Morrison stem gekkotan reveals gecko evolution and Jurassic biogeography.

Geckos are a speciose and globally distributed clade of Squamata (lizards, including snakes and amphisbaenians) that are characterized by a host of modifications for nocturnal, scansorial and insectivorous ecologies. They are among the oldest divergences in the lizard crown, so understanding the origin of geckoes (Gekkota) is essential to understanding the origin of Squamata, the most species-rich extant tetrapod clade. However, the poor fossil record of gekkotans has obscured the sequence and timing of the assembly of their distinctive morphology. Here, we describe the first North American stem gekkotan based on a three-dimensionally preserved skull from the Morrison Formation of western North America. Despite its Late Jurassic age, the new species already possesses several key characteristics of the gekkotan skull along with retained ancestral features. We show that this new stem gekkotan, and several previously named species of uncertain phylogenetic relationships, comprise a widespread clade of early crown lizards, substantiating faunal homogeneity in Laurasia during the Late Jurassic that extended across disparate ecological, body-size and physiological classes.

A transitional snake from the Late Cretaceous period of North America.

Snakes are the most diverse group of lizards, but their origins and early evolution remain poorly understood owing to a lack of transitional forms. Several major issues remain outstanding, such as whether snakes originated in a marine or terrestrial environment and how their unique feeding mechanism evolved. The Cretaceous Coniophis precedens was among the first Mesozoic snakes discovered, but until now only an isolated vertebra has been described and it has therefore been overlooked in discussions of snake evolution. Here we report on previously undescribed material from this ancient snake, including the maxilla, dentary and additional vertebrae. Coniophis is not an anilioid as previously thought a revised phylogenetic analysis of Ophidia shows that it instead represents the most primitive known snake. Accordingly, its morphology and ecology are critical to understanding snake evolution. Coniophis occurs in a continental floodplain environment, consistent with a terrestrial rather than a marine origin; furthermore, its small size and reduced neural spines indicate fossorial habits, suggesting that snakes evolved from burrowing lizards. The skull is intermediate between that of lizards and snakes. Hooked teeth and an intramandibular joint indicate that Coniophis fed on relatively large, soft-bodied prey. However, the maxilla is firmly united with the skull, indicating an akinetic rostrum. Coniophis therefore represents a transitional snake, combining a snake-like body and a lizard-like head. Subsequent to the evolution of a serpentine body and carnivory, snakes evolved a highly specialized, kinetic skull, which was followed by a major adaptive radiation in the Early Cretaceous period. This pattern suggests that the kinetic skull was a key innovation that permitted the diversification of snakes.

The origin of snakes: revealing the ecology, behavior, and evolutionary history of early snakes using genomics, phenomics, and the fossil record.

BACKGROUND: The highly derived morphology and astounding diversity of snakes has long inspired debate regarding the ecological and evolutionary origin of both the snake total-group (Pan-Serpentes) and crown snakes (Serpentes). Although speculation abounds on the ecology, behavior, and provenance of the earliest snakes, a rigorous, clade-wide analysis of snake origins has yet to be attempted, in part due to a dearth of adequate paleontological data on early stem snakes. Here, we present the first comprehensive analytical reconstruction of the ancestor of crown snakes and the ancestor of the snake total-group, as inferred using multiple methods of ancestral state reconstruction. We use a combined-data approach that includes new information from the fossil record on extinct crown snakes, new data on the anatomy of the stem snakes Najash rionegrina, Dinilysia patagonica, and Coniophis precedens, and a deeper understanding of the distribution of phenotypic apomorphies among the major clades of fossil and Recent snakes. Additionally, we infer time-calibrated phylogenies using both new 'tip-dating' and traditional node-based approaches, providing new insights on temporal patterns in the early evolutionary history of snakes. RESULTS: Comprehensive ancestral state reconstructions reveal that both the ancestor of crown snakes and the ancestor of total-group snakes were nocturnal, widely foraging, non-constricting stealth hunters. They likely consumed soft-bodied vertebrate and invertebrate prey that was subequal to head size, and occupied terrestrial settings in warm, well-watered, and well-vegetated environments. The snake total-group - approximated by the Coniophis node - is inferred to have originated on land during the middle Early Cretaceous (~128.5 Ma), with the crown-group following about 20 million years later, during the Albian stage. Our inferred divergence dates provide strong evidence for a major radiation of henophidian snake diversity in the wake of the Cretaceous-Paleogene (K-Pg) mass extinction, clarifying the pattern and timing of the extant snake radiation. Although the snake crown-group most likely arose on the supercontinent of Gondwana, our results suggest the possibility that the snake total-group originated on Laurasia. CONCLUSIONS: Our study provides new insights into when, where, and how snakes originated, and presents the most complete picture of the early evolution of snakes to date. More broadly, we demonstrate the striking influence of including fossils and phenotypic data in combined analyses aimed at both phylogenetic topology inference and ancestral state reconstruction.

Biogeography of worm lizards (Amphisbaenia) driven by end-Cretaceous mass extinction.

Worm lizards (Amphisbaenia) are burrowing squamates that live as subterranean predators. Their underground existence should limit dispersal, yet they are widespread throughout the Americas, Europe and Africa. This pattern was traditionally explained by continental drift, but molecular clocks suggest a Cenozoic diversification, long after the break-up of Pangaea, implying dispersal. Here, we describe primitive amphisbaenians from the North American Palaeocene, including the oldest known amphisbaenian, and provide new and older molecular divergence estimates for the clade, showing that worm lizards originated in North America, then radiated and dispersed in the Palaeogene following the Cretaceous-Palaeogene (K-Pg) extinction. This scenario implies at least three trans-oceanic dispersals: from North America to Europe, from North America to Africa and from Africa to South America. Amphisbaenians provide a striking case study in biogeography, suggesting that the role of continental drift in biogeography may be overstated. Instead, these patterns support Darwin and Wallace's hypothesis that the geographical ranges of modern clades result from dispersal, including oceanic rafting. Mass extinctions may facilitate dispersal events by eliminating competitors and predators that would otherwise hinder establishment of dispersing populations, removing biotic barriers to dispersal.

Mass extinction of lizards and snakes at the Cretaceous-Paleogene boundary.

The Cretaceous-Paleogene (K-Pg) boundary is marked by a major mass extinction, yet this event is thought to have had little effect on the diversity of lizards and snakes (Squamata). A revision of fossil squamates from the Maastrichtian and Paleocene of North America shows that lizards and snakes suffered a devastating mass extinction coinciding with the Chicxulub asteroid impact. Species-level extinction was 83%, and the K-Pg event resulted in the elimination of many lizard groups and a dramatic decrease in morphological disparity. Survival was associated with small body size and perhaps large geographic range. The recovery was prolonged; diversity did not approach Cretaceous levels until 10 My after the extinction, and resulted in a dramatic change in faunal composition. The squamate fossil record shows that the end-Cretaceous mass extinction was far more severe than previously believed, and underscores the role played by mass extinctions in driving diversification.

Toward consilience in reptile phylogeny: miRNAs support an archosaur, not lepidosaur, affinity for turtles.

Understanding the phylogenetic position of crown turtles (Testudines) among amniotes has been a source of particular contention. Recent morphological analyses suggest that turtles are sister to all other reptiles, whereas the vast majority of gene sequence analyses support turtles as being inside Diapsida, and usually as sister to crown Archosauria (birds and crocodilians). Previously, a study using microRNAs (miRNAs) placed turtles inside diapsids, but as sister to lepidosaurs (lizards and Sphenodon) rather than archosaurs. Here, we test this hypothesis with an expanded miRNA presence/absence dataset, and employ more rigorous criteria for miRNA annotation. Significantly, we find no support for a turtle + lepidosaur sister-relationship; instead, we recover strong support for turtles sharing a more recent common ancestor with archosaurs. We further test this result by analyzing a super-alignment of precursor miRNA sequences for every miRNA inferred to have been present in the most recent common ancestor of tetrapods. This analysis yields a topology that is fully congruent with our presence/absence analysis; our results are therefore in accordance with most gene sequence studies, providing strong, consilient molecular evidence from diverse independent datasets regarding the phylogenetic position of turtles.

Homology of the enigmatic nuchal bone reveals novel reorganization of the shoulder girdle in the evolution of the turtle shell.

The turtle shell represents a unique modification of the ancestral tetrapod body plan. The homologies of its approximately 50 bones have been the subject of debate for more than 200 years. Although most of those homologies are now firmly established, the evolutionary origin of the dorsal median nuchal bone of the carapace remains unresolved. We propose a novel hypothesis in which the nuchal is derived from the paired, laterally positioned cleithra-dorsal elements of the ancestral tetrapod pectoral girdle that are otherwise retained among extant tetrapods only in frogs. This hypothesis is supported by origin of the nuchal as paired, mesenchymal condensations likely derived from the neural crest followed by a unique two-stage pattern of ossification. Further support is drawn from the establishment of the nuchal as part of a highly conserved "muscle scaffold" wherein the cleithrum (and its evolutionary derivatives) serves as the origin of the Musculus trapezius. Identification of the nuchal as fused cleithra is congruent with its general spatial relationships to other elements of the shoulder girdle in the adult morphology of extant turtles, and it is further supported by patterns of connectivity and transformations documented by critical fossils from the turtle stem group. The cleithral derivation of the nuchal implies an anatomical reorganization of the pectoral girdle in which the dermal portion of the girdle was transformed from a continuous lateral-ventral arc into separate dorsal and ventral components. This transformation involved the reduction and eventual loss of the scapular rami of the clavicles along with the dorsal and superficial migration of the cleithra, which then fused with one another and became incorporated into the carapace.

MicroRNAs support a turtle + lizard clade.

Despite much interest in amniote systematics, the origin of turtles remains elusive. Traditional morphological phylogenetic analyses place turtles outside Diapsida-amniotes whose ancestor had two fenestrae in the temporal region of the skull (among the living forms the tuatara, lizards, birds and crocodilians)-and allied with some unfenestrate-skulled (anapsid) taxa. Nonetheless, some morphological analyses place turtles within Diapsida, allied with Lepidosauria (tuatara and lizards). Most molecular studies agree that turtles are diapsids, but rather than allying them with lepidosaurs, instead place turtles near or within Archosauria (crocodilians and birds). Thus, three basic phylogenetic positions for turtles with respect to extant Diapsida are currently debated: (i) sister to Diapsida, (ii) sister to Lepidosauria, or (iii) sister to, or within, Archosauria. Interestingly, although these three alternatives are consistent with a single unrooted four-taxon tree for extant reptiles, they differ with respect to the position of the root. Here, we apply a novel molecular dataset, the presence versus absence of specific microRNAs, to the problem of the phylogenetic position of turtles and the root of the reptilian tree, and find that this dataset unambiguously supports a turtle + lepidosaur group. We find that turtles and lizards share four unique miRNA gene families that are not found in any other organisms' genome or small RNA library, and no miRNAs are found in all diapsids but not turtles, or in turtles and archosaurs but not in lizards. The concordance between our result and some morphological analyses suggests that there have been numerous morphological convergences and reversals in reptile phylogeny, including the loss of temporal fenestrae.

Evolutionary origins of the prolonged extant squamate radiation.

Squamata is the most diverse clade of terrestrial vertebrates. Although the origin of pan-squamates lies in the Triassic, the oldest undisputed members of extant clades known from nearly complete, uncrushed material come from the Cretaceous. Here, we describe three-dimensionally preserved partial skulls of two new crown lizards from the Late Jurassic of North America. Both species are placed at the base of the skink, girdled, and night lizard clade Pan-Scincoidea, which consistently occupies a position deep inside the squamate crown in both morphological and molecular phylogenies. The new lizards show that several features uniting pan-scincoids with another major lizard clade, the pan-lacertoids, in trees using morphology were convergently acquired as predicted by molecular analyses. Further, the palate of one new lizard bears a handful of ancestral saurian characteristics lost in nearly all extant squamates, revealing an underappreciated degree of complex morphological evolution in the early squamate crown. We find strong evidence for close relationships between the two new species and Cretaceous taxa from Eurasia. Together, these results suggest that early crown squamates had a wide geographic distribution and experienced complicated morphological evolution even while the Rhynchocephalia, now solely represented by the tuatara, was the dominant clade of lepidosaurs.

Finding the frame shift: digit loss, developmental variability, and the origin of the avian hand.

The origin of the tridactyl hand of crown birds from the pentadactyl hand of those early theropod dinosaurs lying along the avian stem has become a classic, but at times seemingly intractable, historical problem. The point in question is whether the fingers of crown birds represent digits 1-3 as predicted by generalized trends in the fossil record; or digits 2-4, as evidenced by the topology of the embryonic mesenchymal condensations from which the digits develop. The frame shift hypothesis attempted to resolve this paradox by making these signals congruent by means of a homeotic transformation in digital identity, but recently the paleontological support for this hypothesis was questioned. Here, we reassess the frame shift from a paleontological perspective by addressing what predictions a frame shift makes for skeletal morphology, whether the frame shift remains a viable explanation of the known fossil data, and where on the theropod tree the frame shift most likely occurred. Our results indicate that the frame shift remains viable, and based on the inferred pattern of digit loss, the frame shift likely occurred at a deeper position in theropod phylogeny than previously proposed. A new evolutionary model of the frame shift is described in which the early history of the frame-shifted hand is marked by an extended zone of developmental polymorphism. This model provides a new conceptual framework for the role of developmental variability in communicating broad evolutionary patterns on a taxonomically inclusive phylogenetic tree.

Transitional fossils and the origin of turtles.

The origin of turtles is one of the most contentious issues in systematics with three currently viable hypotheses: turtles as the extant sister to (i) the crocodile-bird clade, (ii) the lizard-tuatara clade, or (iii) Diapsida (a clade composed of (i) and (ii)). We reanalysed a recent dataset that allied turtles with the lizard-tuatara clade and found that the inclusion of the stem turtle Proganochelys quenstedti and the 'parareptile' Eunotosaurus africanus results in a single overriding morphological signal, with turtles outside Diapsida. This result reflects the importance of transitional fossils when long branches separate crown clades, and highlights unexplored issues such as the role of topological congruence when using fossils to calibrate molecular clocks.

Noise and biases in genomic data may underlie radically different hypotheses for the position of Iguania within Squamata.

Squamate reptiles are a major component of vertebrate biodiversity whose crown-clade traces its origin to a narrow window of time in the Mesozoic during which the main subclades diverged in rapid succession. Deciphering phylogenetic relationships among these lineages has proven challenging given the conflicting signals provided by genomic and phenomic data. Most notably, the placement of Iguania has routinely differed between data sources, with morphological evidence supporting a sister relationship to the remaining squamates (Scleroglossa hypothesis) and molecular data favoring a highly nested position alongside snakes and anguimorphs (Toxicofera hypothesis). We provide novel insights by generating an expanded morphological dataset and exploring the presence of phylogenetic signal, noise, and biases in molecular data. Our analyses confirm the presence of strong conflicting signals for the position of Iguania between morphological and molecular datasets. However, we also find that molecular data behave highly erratically when inferring the deepest branches of the squamate tree, a consequence of limited phylogenetic signal to resolve this ancient radiation with confidence. This, in turn, seems to result from a rate of evolution that is too high for historical signals to survive to the present. Finally, we detect significant systematic biases, with iguanians and snakes sharing faster rates of molecular evolution and a similarly biased nucleotide composition. A combination of scant phylogenetic signal, high levels of noise, and the presence of systematic biases could result in the misplacement of Iguania. We regard this explanation to be at least as plausible as the complex scenario of convergence and reversals required for morphological data to be misleading. We further evaluate and discuss the utility of morphological data to resolve ancient radiations, as well as its impact in combined-evidence phylogenomic analyses, with results relevant for the assessment of evidence and conflict across the Tree of Life.

Fingerprinting snakes: paleontological and paleoecological implications of zygantral growth rings in Serpentes.

We introduce a new non-destructive source of skeletochronological data with applications to species identification, associating disarticulated remains, assessing minimum number of individuals (MNI), and collection management of fossil snakes, but with potential implications for all bony vertebrates, extinct or extant. Study of a diverse sample of Recent henophidian snakes confirms that annual growth cycles (AGCs) visible on the surface of the vertebral zygantrum correspond to lines of arrested growth in osteohistological thin sections and accordingly reflect chronological age. None of the specimens considered here showed signs of remodelling of the zygantrum, suggesting that a complete, unaltered age record is preserved. We tested potential influences on AGCs with a single experimental organism, a male Bogertophis subocularis, that was raised at a controlled temperature and with constant access to mice and water. The conditions in which this individual was maintained, including that it had yet to live through a full reproductive cycle, enabled us to determine that its AGCs reflect only the annual solar cycle, and neither temperature, nor resource availability, nor energy diversion to gametogenesis could explain that it still exhibited lines of arrested growth. Moreover, growth lines in this specimen are deposited toward the end of the growth season in the fall, and not in the winter, during which this individual continued to feed and grow, even though this mid-latitude species would normally be hibernating and not growing. This suggests that growth lines are not caused by hibernation, but reflect the onset of a physiological cycle preparing Bogertophis subocularis for winter rest. That being said, hibernation and reproductive cycle could still influence the amount of time represented by an individual growth line. Growth-line number and AGC spacing-pattern, plus centrum length, are used to estimate MNI of the Early Eocene fossil snake Boavus occidentalis collected from the Willwood Formation over two field seasons during the late 19th century. We identified eight or nine individuals among specimens previously parcelled among two specimen lots collected during those expeditions.

A tiny Triassic saurian from Connecticut and the early evolution of the diapsid feeding apparatus.

Following the Permo-Triassic Extinction, large-bodied diapsid reptiles-with a body length >1 m-rapidly expanded their ecological roles. This diversification is reflected in enormous disparity in the development of the rostrum and adductor chamber. However, it is unclear how marked the diversity of the feeding apparatus was in contemporary small-bodied diapsids. Here we describe the remarkably small skull (2.5 cm long) of a saurian reptile, Colobops noviportensis, gen. et sp. nov., from the Triassic New Haven Arkose of Connecticut, USA. The taxon possesses an exceptionally reinforced snout and strikingly expanded supratemporal fossae for adductor musculature relative to any known Mesozoic or Recent diapsid of similar size. Our phylogenetic analyses support C. noviportensis as an early diverging pan-archosaur. Colobops noviportensis reveals extraordinary disparity of the feeding apparatus in small-bodied early Mesozoic diapsids, and a suite of morphologies, functionally related to a powerful bite, unknown in any small-bodied diapsid.

Early Evolution of Modern Birds Structured by Global Forest Collapse at the End-Cretaceous Mass Extinction.

The fossil record and recent molecular phylogenies support an extraordinary early-Cenozoic radiation of crown birds (Neornithes) after the Cretaceous-Paleogene (K-Pg) mass extinction [1-3]. However, questions remain regarding the mechanisms underlying the survival of the deepest lineages within crown birds across the K-Pg boundary, particularly since this global catastrophe eliminated even the closest stem-group relatives of Neornithes [4]. Here, ancestral state reconstructions of neornithine ecology reveal a strong bias toward taxa exhibiting predominantly non-arboreal lifestyles across the K-Pg, with multiple convergent transitions toward predominantly arboreal ecologies later in the Paleocene and Eocene. By contrast, ecomorphological inferences indicate predominantly arboreal lifestyles among enantiornithines, the most diverse and widespread Mesozoic avialans [5-7]. Global paleobotanical and palynological data show that the K-Pg Chicxulub impact triggered widespread destruction of forests [8, 9]. We suggest that ecological filtering due to the temporary loss of significant plant cover across the K-Pg boundary selected against any flying dinosaurs (Avialae [10]) committed to arboreal ecologies, resulting in a predominantly non-arboreal post-extinction neornithine avifauna composed of total-clade Palaeognathae, Galloanserae, and terrestrial total-clade Neoaves that rapidly diversified into the broad range of avian ecologies familiar today. The explanation proposed here provides a unifying hypothesis for the K-Pg-associated mass extinction of arboreal stem birds, as well as for the post-K-Pg radiation of arboreal crown birds. It also provides a baseline hypothesis to be further refined pending the discovery of additional neornithine fossils from the Latest Cretaceous and earliest Paleogene.

Palaeoecology of triassic stem turtles sheds new light on turtle origins.

Competing hypotheses of early turtle evolution contrast sharply in implying very different ecological settings-aquatic versus terrestrial-for the origin of turtles. We investigate the palaeoecology of extinct turtles by first demonstrating that the forelimbs of extant turtles faithfully reflect habitat preferences, with short-handed turtles being terrestrial and long-handed turtles being aquatic. We apply this metric to the two successive outgroups to all living turtles with forelimbs preserved, Proganochelys quenstedti and Palaeochersis talampayensis, to discover that these earliest turtle outgroups were decidedly terrestrial. We then plot the observed distribution of aquatic versus terrestrial habits among living turtles onto their hypothesized phylogenies. Both lines of evidence indicate that although the common ancestor of all living turtles was aquatic, the earliest turtles clearly lived in a terrestrial environment. Additional anatomical and sedimentological evidence favours these conclusions. The freshwater aquatic habitat preference so characteristic of living turtles cannot, consequently, be taken as positive evidence for an aquatic origin of turtles, but must rather be considered a convergence relative to other aquatic amniotes, including the marine sauropterygians to which turtles have sometimes been allied.

Evolutionary origin of the turtle shell.

The origin of the turtle shell has perplexed biologists for more than two centuries. It was not until Odontochelys semitestacea was discovered, however, that the fossil and developmental data could be synthesized into a model of shell assembly that makes predictions for the as-yet unestablished history of the turtle stem group. We build on this model by integrating novel data for Eunotosaurus africanus-a Late Guadalupian (∼260 mya) Permian reptile inferred to be an early stem turtle. Eunotosaurus expresses a number of relevant characters, including a reduced number of elongate trunk vertebrae (nine), nine pairs of T-shaped ribs, inferred loss of intercostal muscles, reorganization of respiratory muscles to the ventral side of the ribs, (sub)dermal outgrowth of bone from the developing perichondral collar of the ribs, and paired gastralia that lack both lateral and median elements. These features conform to the predicted sequence of character acquisition and provide further support that E. africanus, O. semitestacea, and Proganochelys quenstedti represent successive divergences from the turtle stem lineage. The initial transformations of the model thus occurred by the Middle Permian, which is congruent with molecular-based divergence estimates for the lineage, and remain viable whether turtles originated inside or outside crown Diapsida.