Freshwater fish and the Cretaceous/Palaeogene boundary: a critical assessment of survivorship patterns.

Mass extinctions are major influences on both the phylogenetic structure of the modern biota and our ability to reconstruct broad-based patterns of evolutionary history. The most recent mass extinction is also the most famous-that which implicates a bolide impact in defining the Cretaceous/Palaeogene boundary (K/Pg). Although the biotic effects of this event receive intensive scrutiny, certain ecologically important and diverse groups remain woefully understudied. One such group is the freshwater ray-finned fishes (Actinopterygii). These fish represent 25% of modern vertebrate diversity, yet the isolated and fragmentary nature of their K/Pg fossil record limits our understanding of their diversity dynamics across this event. Here, we address this problem using diversification analysis of molecular-based phylogenies alongside a morphotype analysis of fossils recovered from a unique site in the Denver Basin of western North America that provides unprecedented K/Pg resolution. Our results reveal previously unrecognized signals of post-K/Pg diversification in freshwater clades and suggest that the change was driven by localized and sporadic patterns of extinction. Supported inferences regarding the effects of the K/Pg event on freshwater fish also inform our expectations of how freshwater faunas might recover from the current biodiversity crisis.

Giant gar from directly above the Cretaceous-Palaeogene boundary suggests healthy freshwater ecosystems existed within thousands of years of the asteroid impact.

The Cretaceous-Palaeogene (K-Pg) mass extinction was responsible for the destruction of global ecosystems and loss of approximately three-quarters of species diversity 66 million years ago. Large-bodied land vertebrates suffered high extinction rates, whereas small-bodied vertebrates living in freshwater ecosystems were buffered from the worst effects. Here, we report a new species of large-bodied (1.4-1.5 m) gar based on a complete skeleton from the Williston Basin of North America. The new species was recovered 18 cm above the K-Pg boundary, making it one of the oldest articulated vertebrate fossils from the Cenozoic. The presence of this freshwater macropredator approximately 1.5-2.5 thousand years after the asteroid impact suggests the rapid recovery and reassembly of North American freshwater food webs and ecosystems after the mass extinction.

Evolutionary origin of the turtle skull.

Transitional fossils informing the origin of turtles are among the most sought-after discoveries in palaeontology. Despite strong genomic evidence indicating that turtles evolved from within the diapsid radiation (which includes all other living reptiles), evidence of the inferred transformation between an ancestral turtle with an open, diapsid skull to the closed, anapsid condition of modern turtles remains elusive. Here we use high-resolution computed tomography and a novel character/taxon matrix to study the skull of Eunotosaurus africanus, a 260-million-year-old fossil reptile from the Karoo Basin of South Africa, whose distinctive postcranial skeleton shares many unique features with the shelled body plan of turtles. Scepticism regarding the status of Eunotosaurus as the earliest stem turtle arises from the possibility that these shell-related features are the products of evolutionary convergence. Our phylogenetic analyses indicate strong cranial support for Eunotosaurus as a critical transitional form in turtle evolution, thus fortifying a 40-million-year extension to the turtle stem and moving the ecological context of its origin back onto land. Furthermore, we find unexpected evidence that Eunotosaurus is a diapsid reptile in the process of becoming secondarily anapsid. This is important because categorizing the skull based on the number of openings in the complex of dermal bone covering the adductor chamber has long held sway in amniote systematics, and still represents a common organizational scheme for teaching the evolutionary history of the group. These discoveries allow us to articulate a detailed and testable hypothesis of fenestral closure along the turtle stem. Our results suggest that Eunotosaurus represents a crucially important link in a chain that will eventually lead to consilience in reptile systematics, paving the way for synthetic studies of amniote evolution and development.

Calibration uncertainty in molecular dating analyses: there is no substitute for the prior evaluation of time priors.

Calibration is the rate-determining step in every molecular clock analysis and, hence, considerable effort has been expended in the development of approaches to distinguish good from bad calibrations. These can be categorized into a priori evaluation of the intrinsic fossil evidence, and a posteriori evaluation of congruence through cross-validation. We contrasted these competing approaches and explored the impact of different interpretations of the fossil evidence upon Bayesian divergence time estimation. The results demonstrate that a posteriori approaches can lead to the selection of erroneous calibrations. Bayesian posterior estimates are also shown to be extremely sensitive to the probabilistic interpretation of temporal constraints. Furthermore, the effective time priors implemented within an analysis differ for individual calibrations when employed alone and in differing combination with others. This compromises the implicit assumption of all calibration consistency methods, that the impact of an individual calibration is the same when used alone or in unison with others. Thus, the most effective means of establishing the quality of fossil-based calibrations is through a priori evaluation of the intrinsic palaeontological, stratigraphic, geochronological and phylogenetic data. However, effort expended in establishing calibrations will not be rewarded unless they are implemented faithfully in divergence time analyses.

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.

Updated cranial and mandibular description of the Late Cretaceous (Maastrichtian) baenid turtle Saxochelys gilberti based on micro-computed tomography scans and new information on the holotype-shell association.

Saxochelys gilberti is a baenid turtle from the Late Cretaceous Hell Creek Formation of the United States of America known from cranial, shell, and other postcranial material. Baenid turtles are taxonomically diverse and common fossil elements within Late Cretaceous through Eocene faunas. Detailed anatomical knowledge is critical to understanding the systematics and morphological evolution of the group. This is particularly important as baenids represent an important group of continental vertebrates that survived the mass extinction event associated with the Cretaceous/Paleogene boundary. High-resolution micro-computed tomography scanning of the holotype skull reveals additional anatomical details for the already well-known Saxochelys gilberti. This includes the revision of some anatomical statements from the original description, but also detailed knowledge on internal anatomical features of the braincase and the description of a well-preserved axis (cervical vertebra 2). Our new detailed description and previous work on the shell and postcrania make Saxochelys one of the best-described, nearly complete baenid turtles, which are often only known from either isolated shell or cranial material. A revised phylogenetic analysis confirms the position of Saxochelys gilberti as a derived baenid (Eubaeninae) more closely related to Baena arenosa than to Eubaena cephalica. Supplementary Information: The online version contains supplementary material available at 10.1186/s13358-023-00301-6.

The cranial and postcranial morphology of Hutchemys rememdium and its impact on the phylogenetic relationships of Plastomenidae (Testudinata, Trionychidae).

Hutchemys rememdium is a poorly understood softshell turtle (Trionychidae) from the mid Paleocene of the Williston Basin of North America previously known only from postcranial remains. A particularly rich collection of previously undescribed material from the Tiffanian 4 North American Land Mammal Age (NALMA) of North Dakota is here presented consisting of numerous shells that document new variation, some non-shell postcrania, and cranial remains, which are described based on 3D models extracted from micro-CT data. Although the observed shell variation weakens previously noted differences with the younger species Hutchemys arctochelys from the Clarkforkian NALMA, the two taxa are still recognized as distinct. Parsimony and Bayesian phylogenetic analyses reaffirm the previously challenged placement of Hutchemys rememdium within the clade Plastomenidae, mostly based on novel observations of cranial characters made possible by the new material and the micro-CT data. The new topology supports the notion that the well-ossified plastron of plastomenids originated twice in parallel near the Cretaceous/Paleogene boundary, once in the Hutchemys lineage and once in the Gilmoremys/Plastomenus lineage. Hutchemys rememdium is notable for being the only documented species of trionychid in the mid Paleocene of the Williston Basin. The presence of multiple individuals in a carbonaceous claystone indicates this taxon lived in swamps and lakes and its expanded triturating surface suggests it had a durophagous diet. Supplementary Information: The online version contains supplementary material available at 10.1186/s13358-024-00315-8.

The girdles of the oldest fossil turtle, Proterochersis robusta, and the age of the turtle crown.

BACKGROUND: Proterochersis robusta from the Late Triassic (Middle Norian) of Germany is the oldest known fossil turtle (i.e. amniote with a fully formed turtle shell), but little is known about its anatomy. A newly prepared, historic specimen provides novel insights into the morphology of the girdles and vertebral column of this taxon and the opportunity to reassess its phylogenetic position. RESULTS: The anatomy of the pectoral girdle of P. robusta is similar to that of other primitive turtles, including the Late Triassic (Carnian) Proganochelys quenstedti, in having a vertically oriented scapula, a large coracoid foramen, a short acromion process, and bony ridges that connect the acromion process with the dorsal process, glenoid, and coracoid, and by being able to rotate along a vertical axis. The pelvic elements are expanded distally and suturally attached to the shell, but in contrast to modern pleurodiran turtles the pelvis is associated with the sacral ribs. CONCLUSIONS: The primary homology of the character "sutured pelvis" is unproblematic between P. robusta and extant pleurodires. However, integration of all new observations into the most complete phylogenetic analysis that support the pleurodiran nature of P. robusta reveals that this taxon is more parsimoniously placed along the phylogenetic stem of crown Testudines. All current phylogenetic hypotheses therefore support the basal placement of this taxon, imply that the sutured pelvis of this taxon developed independently from that of pleurodires, and conclude that the age of the turtle crown is Middle Jurassic.

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.

The hooked element in the pes of turtles (Testudines): a global approach to exploring primary and secondary homology.

The hooked element in the pes of turtles was historically identified by most palaeontologists and embryologists as a modified fifth metatarsal, and often used as evidence to unite turtles with other reptiles with a hooked element. Some recent embryological studies, however, revealed that this element might represent an enlarged fifth distal tarsal. We herein provide extensive new myological and developmental observations on the hooked element of turtles, and re-evaluate its primary and secondary homology using all available lines of evidence. Digital count and timing of development are uninformative. However, extensive myological, embryological and topological data are consistent with the hypothesis that the hooked element of turtles represents a fusion of the fifth distal tarsal with the fifth metatarsal, but that the fifth distal tarsal dominates the hooked element in pleurodiran turtles, whereas the fifth metatarsal dominates the hooked element of cryptodiran turtles. The term 'ansulate bone' is proposed to refer to hooked elements that result from the fusion of these two bones. The available phylogenetic and fossil data are currently insufficient to clarify the secondary homology of hooked elements within Reptilia.

Evolution of the turtle bauplan: the topological relationship of the scapula relative to the ribcage.

The turtle shell and the relationship of the shoulder girdle inside or 'deep' to the ribcage have puzzled neontologists and developmental biologists for more than a century. Recent developmental and fossil data indicate that the shoulder girdle indeed lies inside the shell, but anterior to the ribcage. Developmental biologists compare this orientation to that found in the model organisms mice and chickens, whose scapula lies laterally on top of the ribcage. We analyse the topological relationship of the shoulder girdle relative to the ribcage within a broader phylogenetic context and determine that the condition found in turtles is also found in amphibians, monotreme mammals and lepidosaurs. A vertical scapula anterior to the thoracic ribcage is therefore inferred to be the basal amniote condition and indicates that the condition found in therian mammals and archosaurs (which includes both developmental model organisms: chickens and mice) is derived and not appropriate for studying the developmental origin of the turtle shell. Instead, among amniotes, either monotreme mammals or lepidosaurs should be used.

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.

No Consistent Shift in Leaf Dry Mass per Area Across the Cretaceous-Paleogene Boundary.

The Chicxulub bolide impact has been linked to a mass extinction of plants at the Cretaceous-Paleogene boundary (KPB; ∼66 Ma), but how this extinction affected plant ecological strategies remains understudied. Previous work in the Williston Basin, North Dakota, indicates that plants pursuing strategies with a slow return-on-investment of nutrients abruptly vanished after the KPB, consistent with a hypothesis of selection against evergreen species during the globally cold and dark impact winter that followed the bolide impact. To test whether this was a widespread pattern we studied 1,303 fossil leaves from KPB-spanning sediments in the Denver Basin, Colorado. We used the relationship between petiole width and leaf mass to estimate leaf dry mass per area (LMA), a leaf functional trait negatively correlated with rate of return-on-investment. We found no evidence for a shift in this leaf-economic trait across the KPB: LMA remained consistent in both its median and overall distribution from approximately 67 to 65 Ma. However, we did find spatio-temporal patterns in LMA, where fossil localities with low LMA occurred more frequently near the western margin of the basin. These western margin localities are proximal to the Colorado Front Range of the Rocky Mountains, where an orographically driven high precipitation regime is thought to have developed during the early Paleocene. Among these western Denver Basin localities, LMA and estimated mean annual precipitation were inversely correlated, a pattern consistent with observations of both fossil and extant plants. In the Denver Basin, local environmental conditions over time appeared to play a larger role in determining viable leaf-economic strategies than any potential global signal associated with the Chicxulub bolide impact.

Brawn before brains in placental mammals after the end-Cretaceous extinction.

Mammals are the most encephalized vertebrates, with the largest brains relative to body size. Placental mammals have particularly enlarged brains, with expanded neocortices for sensory integration, the origins of which are unclear. We used computed tomography scans of newly discovered Paleocene fossils to show that contrary to the convention that mammal brains have steadily enlarged over time, early placentals initially decreased their relative brain sizes because body mass increased at a faster rate. Later in the Eocene, multiple crown lineages independently acquired highly encephalized brains through marked growth in sensory regions. We argue that the placental radiation initially emphasized increases in body size as extinction survivors filled vacant niches. Brains eventually became larger as ecosystems saturated and competition intensified.

Spatial niche partitioning in dinosaurs from the latest cretaceous (Maastrichtian) of North America.

We examine patterns of occurrence of associated dinosaur specimens (n = 343) from the North American Upper Cretaceous Hell Creek Formation and equivalent beds, by comparing their relative abundance in sandstone and mudstone. Ceratopsians preferentially occur in mudstone, whereas hadrosaurs and the small ornithopod Thescelosaurus show a strong association with sandstone. By contrast, the giant carnivore Tyrannosaurus rex shows no preferred association with either lithology. These lithologies are used as an indicator of environment of deposition, with sandstone generally representing river environments, and finer grained sediments typically representing floodplain environments. Given these patterns of occurrence, we argue that spatial niche partitioning helped reduce competition for resources between the herbivorous dinosaurs. Within coastal lowlands ceratopsians preferred habitats farther away from rivers, whereas hadrosaurs and Thescelosaurus preferred habitats in close proximity to rivers, and T. rex, the ecosystem's sole large carnivore, inhabited both palaeoenvironments. Spatial partitioning of the environment helps explain how several species of large herbivorous dinosaurs coexisted. This study emphasizes that different lithologies can preserve dramatically dissimilar vertebrate assemblages, even when deposited in close proximity and within a narrow window of time. The lithology in which fossils are preserved should be recorded as these data can provide unique insights into the palaeoecology of the animals they preserve.

Dinosaur extinction: closing the '3 m gap'.

Modern debate regarding the extinction of non-avian dinosaurs was ignited by the publication of the Cretaceous-Tertiary (K-T) asteroid impact theory and has seen 30 years of dispute over the position of the stratigraphically youngest in situ dinosaur. A zone devoid of dinosaur fossils reported from the last 3 m of the Upper Cretaceous, coined the '3 m gap', has helped drive controversy. Here, we report the discovery of the stratigraphically youngest in situ dinosaur specimen: a ceratopsian brow horn found in a poorly rooted, silty, mudstone floodplain deposit located no more than 13 cm below the palynologically defined boundary. The K-T boundary is identified using three criteria: (i) decrease in Cretaceous palynomorphs without subsequent recovery, (ii) the existence of a 'fern spike', and (iii) correlation to a nearby stratigraphic section where primary extraterrestrial impact markers are present (e.g. iridium anomaly, spherules, shocked quartz). The in situ specimen demonstrates that a gap devoid of non-avian dinosaur fossils does not exist and is inconsistent with the hypothesis that non-avian dinosaurs were extinct prior to the K-T boundary impact event.

New Skull Material of Taeniolabis taoensis (Multituberculata, Taeniolabididae) from the Early Paleocene (Danian) of the Denver Basin, Colorado.

Taeniolabis taoensis is an iconic multituberculate mammal of early Paleocene (Puercan 3) age from the Western Interior of North America. Here we report the discovery of significant new skull material (one nearly complete cranium, two partial crania, one nearly complete dentary) of T. taoensis in phosphatic concretions from the Corral Bluffs study area, Denver Formation (Danian portion), Denver Basin, Colorado. The new skull material provides the first record of the species from the Denver Basin, where the lowest in situ specimen occurs in river channel deposits ~730,000 years after the Cretaceous-Paleogene boundary, roughly coincident with the first appearance of legumes in the basin. The new material, in combination with several previously described and undescribed specimens from the Nacimiento Formation of the San Juan Basin, New Mexico, is the subject of detailed anatomical study, aided by micro-computed tomography. Our analyses reveal many previously unknown aspects of skull anatomy. Several regions (e.g., anterior portions of premaxilla, orbit, cranial roof, occiput) preserved in the Corral Bluffs specimens allow considerable revision of previous reconstructions of the external cranial morphology of T. taoensis. Similarly, anatomical details of the ascending process of the dentary are altered in light of the new material. Although details of internal cranial anatomy (e.g., nasal and endocranial cavities) are difficult to discern in the available specimens, we provide, based on UCMP 98083 and DMNH.EPV 95284, the best evidence to date for inner ear structure in a taeniolabidoid multituberculate. The cochlear canal of T. taoensis is elongate and gently curved and the vestibule is enlarged, although to a lesser degree than in Lambdopsalis.

A new pelomedusoid turtle, Sahonachelys mailakavava, from the Late Cretaceous of Madagascar provides evidence for convergent evolution of specialized suction feeding among pleurodires.

The Maevarano Formation in northwestern Madagascar has yielded a series of exceptional fossils over the course of the last three decades that provide important insights into the evolution of insular ecosystems during the latest Cretaceous (Maastrichtian). We here describe a new genus and species of pelomedusoid turtle from this formation, Sahonachelys mailakavava, based on a nearly complete skeleton. A phylogenetic analysis suggests close affinities of Sahonachelys mailakavava with the coeval Madagascan Sokatra antitra. These two taxa are the only known representatives of the newly recognized clade Sahonachelyidae, which is sister to the speciose clade formed by Bothremydidae and Podocnemidoidae. A close relationship with coeval Indian turtles of the clade Kurmademydini is notably absent. A functional assessment suggests that Sahonachelys mailakavava was a specialized suction feeder that preyed upon small-bodied invertebrates and vertebrates. This is a unique feeding strategy among crown pelomedusoids that is convergent upon that documented in numerous other clades of turtles and that highlights the distinct evolutionary pathways taken by Madagascan vertebrates.

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.

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.