Artificial turf infill associated with systematic toxicity in an amniote vertebrate.

Artificial athletic turf containing crumb rubber (CR) from shredded tires is a growing environmental and public health concern. However, the associated health risk is unknown due to the lack of toxicity data for higher vertebrates. We evaluated the toxic effects of CR in a developing amniote vertebrate embryo. CR water leachate was administered to fertilized chicken eggs via different exposure routes, i.e., coating by dropping CR leachate on the eggshell; dipping the eggs into CR leachate; microinjecting CR leachate into the air cell or yolk. After 3 or 7 d of incubation, embryonic morphology, organ development, physiology, and molecular pathways were measured. The results showed that CR leachate injected into the yolk caused mild to severe developmental malformations, reduced growth, and specifically impaired the development of the brain and cardiovascular system, which were associated with gene dysregulation in aryl hydrocarbon receptor, stress-response, and thyroid hormone pathways. The observed systematic effects were probably due to a complex mixture of toxic chemicals leaching from CR, such as metals (e.g., Zn, Cr, Pb) and amines (e.g., benzothiazole). This study points to a need to closely examine the potential regulation of the use of CR on playgrounds and artificial fields.

The tissues and regulatory pattern of limb chondrogenesis.

Initial limb chondrogenesis offers the first differentiated tissues that resemble the mature skeletal anatomy. It is a developmental progression of three tissues. The limb begins with undifferentiated mesenchyme-1, some of which differentiates into condensations-2, and this tissue then transforms into cartilage-3. Each tissue is identified by physical characteristics of cell density, shape, and extracellular matrix composition. Tissue specific regimes of gene regulation underlie the diagnostic physical and chemical properties of these three tissues. These three tissue based regimes co-exist amid a background of other gene regulatory regimes within the same tissues and time-frame of limb development. The bio-molecular indicators of gene regulation reveal six identifiable patterns. Three of these patterns describe the unique bio-molecular indicators of each of the three tissues. A fourth pattern shares bio-molecular indicators between condensation and cartilage. Finally, a fifth pattern is composed of bio-molecular indicators that are found in undifferentiated mesenchyme prior to any condensation differentiation, then these bio-molecular indicators are upregulated in condensations and downregulated in undifferentiated mesenchyme. The undifferentiated mesenchyme that remains in between the condensations and cartilage, the interdigit, contains a unique set of bio-molecular indicators that exhibit dynamic behaviour during chondrogenesis and therefore argue for its own inclusion as a tissue in its own right and for more study into this process of differentiation.

Developmental plasticity and the origin of tetrapods.

The origin of tetrapods from their fish antecedents, approximately 400 million years ago, was coupled with the origin of terrestrial locomotion and the evolution of supporting limbs. Polypterus is a member of the basal-most group of ray-finned fish (actinopterygians) and has many plesiomorphic morphologies that are comparable to elpistostegid fishes, which are stem tetrapods. Polypterus therefore serves as an extant analogue of stem tetrapods, allowing us to examine how developmental plasticity affects the 'terrestrialization' of fish. We measured the developmental plasticity of anatomical and biomechanical responses in Polypterus reared on land. Here we show the remarkable correspondence between the environmentally induced phenotypes of terrestrialized Polypterus and the ancient anatomical changes in stem tetrapods, and we provide insight into stem tetrapod behavioural evolution. Our results raise the possibility that environmentally induced developmental plasticity facilitated the origin of the terrestrial traits that led to tetrapods.

Plastic Teabags Release Billions of Microparticles and Nanoparticles into Tea.

The increasing presence of micro- and nano-sized plastics in the environment and food chain is of growing concern. Although mindful consumers are promoting the reduction of single-use plastics, some manufacturers are creating new plastic packaging to replace traditional paper uses, such as plastic teabags. The objective of this study was to determine whether plastic teabags could release microplastics and/or nanoplastics during a typical steeping process. We show that steeping a single plastic teabag at brewing temperature (95 °C) releases approximately 11.6 billion microplastics and 3.1 billion nanoplastics into a single cup of the beverage. The composition of the released particles is matched to the original teabags (nylon and polyethylene terephthalate) using Fourier-transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The levels of nylon and polyethylene terephthalate particles released from the teabag packaging are several orders of magnitude higher than plastic loads previously reported in other foods. An initial acute invertebrate toxicity assessment shows that exposure to only the particles released from the teabags caused dose-dependent behavioral and developmental effects.

The evolution of the meatal chamber in crocodyliforms.

The unique outer ear of crocodylians consists of a large meatal chamber (MC) concealed by a pair of muscular earlids that shape a large part of the animal's head. This chamber is limited medially by the enlarged tympanic membrane. Yet, the anatomy of this distinctive and complex region is underexplored and its evolutionary history untraced. The osteology and soft tissues of the MC in extant crocodylians was analysed to describe it and establish osteological correlates within this region. A broad survey of the osteological correlates was conducted in major clades of fossil crocodyliforms to estimate evolutionary trends of the MC. The reorganization of the MC at the origin of crocodyliforms includes characters also present in more basal taxa such as 'sphenosuchians' as well as unique traits of crocodyliforms. Three major patterns are recognized in the MC of basal mesoeucrocodylians. The distinct 'thalattosuchian pattern' indicates that extensive modifications occurred in this clade of aquatic fossil crocodyliforms, even when multiple alternative phylogenetic positions are taken into account. Some traits already established in putative closely related clades are absent or modified in this group. The 'basal notosuchian/sebecian pattern' is widespread among basal metasuchians, and establishes for the first time characters maintained later in neosuchians and extant forms. The 'advanced notosuchian pattern' includes modifications of the MC possibly related to a terrestrial lifestyle and potentially a structure analogous to the mammalian pinna. The main variation in the MC of neosuchians is associated with the homoplastic secondary opening of the cranioquadrate passage. The inferred phylogenetic trends in the crocodyliform MC suggest the great anatomical disparity in this region followed a complex evolutionary pattern, and tympanic hearing played an important role in the origin and diversification of Crocodyliformes.

Among-character rate variation distributions in phylogenetic analysis of discrete morphological characters.

Likelihood-based methods are commonplace in phylogenetic systematics. Although much effort has been directed toward likelihood-based models for molecular data, comparatively less work has addressed models for discrete morphological character (DMC) data. Among-character rate variation (ACRV) may confound phylogenetic analysis, but there have been few analyses of the magnitude and distribution of rate heterogeneity among DMCs. Using 76 data sets covering a range of plants, invertebrate, and vertebrate animals, we used a modified version of MrBayes to test equal, gamma-distributed and lognormally distributed models of ACRV, integrating across phylogenetic uncertainty using Bayesian model selection. We found that in approximately 80% of data sets, unequal-rates models outperformed equal-rates models, especially among larger data sets. Moreover, although most data sets were equivocal, more data sets favored the lognormal rate distribution relative to the gamma rate distribution, lending some support for more complex character correlations than in molecular data. Parsimony estimation of the underlying rate distributions in several data sets suggests that the lognormal distribution is preferred when there are many slowly evolving characters and fewer quickly evolving characters. The commonly adopted four rate category discrete approximation used for molecular data was found to be sufficient to approximate a gamma rate distribution with discrete characters. However, among the two data sets tested that favored a lognormal rate distribution, the continuous distribution was better approximated with at least eight discrete rate categories. Although the effect of rate model on the estimation of topology was difficult to assess across all data sets, it appeared relatively minor between the unequal-rates models for the one data set examined carefully. As in molecular analyses, we argue that researchers should test and adopt the most appropriate model of rate variation for the data set in question. As discrete characters are increasingly used in more sophisticated likelihood-based phylogenetic analyses, it is important that these studies be built on the most appropriate and carefully selected underlying models of evolution.

Polypterus and the evolution of fish pectoral musculature.

Polypterus, a member of the most primitive living group of ray-finned fishes, has demonstrated the ability to perform fin-assisted terrestrial locomotion, a behavior that indicates a complex pectoral musculoskeletal system. Review of the literature reveals that many aspects of the pectoral muscular anatomy of Polypterus are still unclear, with a number of conflicting descriptions. We provide a new interpretation of the pectoral musculature using soft tissue-enhanced microCT scanning and gross anatomical dissection. The results demonstrate a complex musculature, with six independent muscles crossing the glenoid-fin joint. Comparisons with other bony-fish (Osteichthyes), including both ray-finned (Actinopterygii) and lobed-fin (Sarcopterygii) fish, indicate the presence of novel muscles within Polypterus: coracometapterygialis I+II and the zonopropterygialis medialis. Examination of these muscular additions in the context of osteichthyan phylogeny indicates that this represents a previously unrecognized event in the evolution of pectoral musculature in Osteichthyes. Despite its phylogenetic position as a basal actinopterygian, the musculature of Polypterus has more similarities both anatomically and functionally with that of sarcopterygians. This anatomy, along with other features of Polypterus anatomy such as lobed fins, ventral paired lungs, and a large spiracle, may make it a good model for inferences of stem tetrapod locomotion.

Quantitative analysis of microscopic X-ray computed tomography imaging: Japanese quail embryonic soft tissues with iodine staining.

Rapid three-dimensional imaging of embryos to better understand the complex process of morphogenesis has been challenging. Recently introduced iodine staining protocols (I2 KI and alcoholic iodine stains) combined with microscopic X-ray computed tomography allows visualization of soft tissues in diverse small organisms and tissue specimens. I2 KI protocols have been developed specifically for small animals, with a limited number of quantitative studies of soft tissue contrasts. To take full advantage of the low X-ray attenuation of ethanol and retain bound iodine while dehydrating the specimen in ethanol, we developed an ethanol I2 KI protocol. We present comparative microscopic X-ray computed tomography analyses of ethanol I2 KI and I2 KI staining protocols to assess the performance of this new protocol to visualize soft tissue anatomy in late stage Japanese quail embryos using quantitative measurements of soft tissue contrasts and sample shrinkage. Both protocols had only 5% shrinkage compared with the original harvested specimen, supporting the use of whole mounts to minimize tissue shrinkage effects. Discrimination within and among the selected organs with each staining protocol and microscopic X-ray computed tomography imaging were comparable to those of a gray scale histological section. Tissue discrimination was assessed using calibrated computed tomography values and a new discrimination index to quantify the degree of computed tomography value overlaps between selected soft tissue regions. Tissue contrasts were dependent on the depth of the tissue within the embryos before the embryos were saturated with each stain solution, and optimal stain saturations for the entire embryo were achieved at 14 and 28 days staining for I2 KI and ethanol I2 KI, respectively. Ethanol I2 KI provided superior soft tissue contrasts by reducing overstaining of fluid-filled spaces and differentially modulating staining of some tissues, such as bronchial and esophageal walls and spinal cord. Delineating the selected soft tissues using optimal threshold ranges derived from the quantitative analyses of the contrast enhancement in optimally stained embryos is possible. The protocols presented here are expected to be applicable to other organisms with modifications to staining time and contribute toward rapid and more efficient segmentation of soft tissues for three-dimensional visualization.

A new neosuchian with Asian affinities from the Jurassic of northeastern Brazil.

Phylogenetic relationships near the origin of extant crocodylians are weakly supported, and this lack of resolution makes for poor estimates of taxonomic and morphological diversity. Previously known taxa are found throughout the Cretaceous in Laurasia and at a few sites from Brazil, Australia, and northern Africa. Here, we report Batrachomimus pastosbonensis gen. et sp. nov. from the Late Jurassic of northeastern Brazil, which is deeply nested within Neosuchia and associated to the Cretaceous Asian paralligatorids. The new taxon predates all other members of Paralligatoridae and its immediate sister group (including Eusuchia) by 30 million years. A nearly complete skull, osteoderms, and limb bones were recovered, and autapomorphies of B. pastosbonensis include a scalloped lateral margin of the rostrum in dorsal view, unsculpted alveolar margin at the caudalmost portion of the maxilla, blunt lateral prongs on the jugal at the base of the postorbital bar, hourglass shaped choanae, and pterygoid choanal septum extended between the palatal shelves of the palatines. The crocodyloid-like skull proportions and the slender, conical teeth suggest an amphibious and piscivorous life history to this 1 m long animal.

Low beta diversity of Maastrichtian dinosaurs of North America.

Beta diversity is an important component of large-scale patterns of biodiversity, but its explicit examination is more difficult than that of alpha diversity. Only recently have data sets large enough been presented to begin assessing global patterns of species turnover, especially in the fossil record. We present here an analysis of beta diversity of a Maastrichtian (71-65 million years old) assemblage of dinosaurs from the Western Interior of North America, a region that covers approximately 1.5 x 10(6) km(2), borders an epicontinental sea, and spans approximately 20 degrees of latitude. Previous qualitative analyses have suggested regional groupings of these dinosaurs and generally concluded that there were multiple distinct faunal regions. However, these studies did not directly account for sampling bias, which may artificially decrease similarity and increase turnover between regions. Our analysis used abundance-based data to account for sampling intensity and was unable to support any hypothesis of multiple distinct faunas; earlier hypothesized faunal delineations were likely a sampling artifact. Our results indicate a low beta diversity and support a single dinosaur community within the entire Western Interior region of latest Cretaceous North America. Homogeneous environments are a known driver of low modern beta diversities, and the warm equable climate of the late Cretaceous modulated by the epicontenental seaway is inferred to be an underlying influence on the low beta diversity of this ancient ecosystem.

Ice Age effects on genetic divergence of the American crocodile (Crocodylus acutus) in Panama: reconstructing limits of gene flow and environmental ranges: a reply to O'Dea et al.

O'Dea et al. (2022) (Pleistocene sea level changes and crocodile population histories on the isthmus of panama: a comment on Avila-Cervantes et al. (2020). Evolution, 76(11), 2778-2783. https://doi.org/10.1111/evo.14610) question our hypothesis that sea-level changes during the past glaciation played a role in restricting gene flow between Pacific and Caribbean Crocodylus acutus in Panama. They argue that an error in sea-level high-stand reconstruction during the last interglacial period (118-130 ka) does not support our hypothesis. Although they are correct in our high-stand reconstruction error, overlooked the point in that we were presenting a model of restricted gene flow across the Panamanian Isthmus during low sea levels. We review the assumptions of gene demographic methods, emphasizing that we were focusing on times of genetic divergence. We expand here why gene flow between these coastal populations could have been restricted during the last glacial maximum (19-26.5 ka) and the 50,000 years preceding it when sea levels were lower than today. O'Dea et al. suggest local climates may have played larger roles than sea levels. We demonstrate that paleoclimate estimates for the past 3.3 Ma in Panama are within the bounds of extant C. acutus. The importance of Ice Age Sea-level dynamics on Neotropical species was likely profound and should be incorporated into evolutionary studies of these taxa.

Testing inferences in developmental evolution: the forensic evidence principle.

Developmental evolution (DE) examines the influence of developmental mechanisms on biological evolution. Here we consider the question: "what is the evidence that allows us to decide whether a certain developmental scenario for an evolutionary change is in fact "correct" or at least falsifiable?" We argue that the comparative method linked with what we call the "forensic evidence principle" (FEP) is sufficient to conduct rigorous tests of DE scenarios. The FEP states that different genetically mediated developmental causes of an evolutionary transformation will leave different signatures in the development of the derived character. Although similar inference rules have been used in practically every empirical science, we expand this approach here in two ways: (1) we justify the validity of this principle with reference to a well-known result from mathematical physics, known as the symmetry principle, and (2) propose a specific form of the FEP for DE: given two or more developmental explanations for a certain evolutionary event, say an evolutionary novelty, then the evidence discriminating between these hypotheses will be found in the most proximal internal drivers of the derived character. Hence, a detailed description of the ancestral and derived states, and their most proximal developmental drivers are necessary to discriminate between various evolutionary developmental hypotheses. We discuss how this stepwise order of testing is necessary, establishes a formal test, and how skipping this order of examination may violate a more accurate examination of DE. We illustrate the approach with an example from avian digit evolution.

Paratympanic sinuses in juvenile Alligator.

Crocodylia has an extensive epithelial pneumatic space in the middle ear, paratympanic sinus system. Although fossil and extant crocodylian paratympanic sinus systems have been studied recently using the computed tomography (CT) and three-dimensional (3D) reconstruction data, due to the soft tissue nature of the pneumatic system and presence of its surrounding soft tissue structures, some boundaries, and definitions of each extension remain ambiguous. We describe the comprehensive paratympanic sinus system in posthatched alligator using soft tissue enhanced CT data with 3D reconstructions. The data are compared to the available data to discuss the ontogenetic pattern in alligator. We introduce further divided entities of the pneumatic system based on their associated bony and soft tissue structures and epithelial membrane and clarify the pneumatic terminologies. We then re-visit the potential homology of the paratympanic sinus in Archosauria. Epithelial boundaries of the ventral portion of the pneumatic system from the histological data suggest that the dual origin of the basioccipital diverticulum derived from the tympanic sinus and basicranial diverticulum medially. The presence of the epithelial boundary and pneumatic changes in ontogeny suggests that the middle ear may function differently in developmental stages. Lastly, a morphogenetic tree is constructed to help future work of comparative developmental studies of the paratympanic sinus system between crocodiles and birds.

The Development of the Chimaeroid Pelvic Skeleton and the Evolution of Chondrichthyan Pelvic Fins.

Pelvic girdles, fins and claspers are evolutionary novelties first recorded in jawed vertebrates. Over the course of the evolution of chondrichthyans (cartilaginous fish) two trends in the morphology of the pelvic skeleton have been suggested to have occurred. These evolutionary shifts involved both an enlargement of the metapterygium (basipterygium) and a transition of fin radial articulation from the pelvic girdle to the metapterygium. To determine how these changes in morphology have occurred it is essential to understand the development of extant taxa as this can indicate potential developmental mechanisms that may have been responsible for these changes. The study of the morphology of the appendicular skeleton across development in chondrichthyans is almost entirely restricted to the historical literature with little contemporary research. Here, we have examined the morphology and development of the pelvic skeleton of a holocephalan chondrichthyan, the elephant shark (Callorhinchus milii), through a combination of dissections, histology, and nanoCT imaging and redescribed the pelvic skeleton of Cladoselache kepleri (NHMUK PV P 9269), a stem holocephalan. To put our findings in their evolutionary context we compare them with the fossil record of chondrichthyans and the literature on pelvic development in elasmobranchs from the late 19th century. Our findings demonstrate that the pelvic skeleton of C. milii initially forms as a single mesenchymal condensation, consisting of the pelvic girdle and a series of fin rays, which fuse to form the basipterygium. The girdle and fin skeleton subsequently segment into distinct components whilst chondrifying. This confirms descriptions of the early pelvic development in Scyliorhinid sharks from the historical literature and suggests that chimaeras and elasmobranchs share common developmental patterns in their pelvic anatomy. Alterations in the location and degree of radial fusion during early development may be the mechanism responsible for changes in pelvic fin morphology over the course of the evolution of both elasmobranchs and holocephalans, which appears to be an example of parallel evolution.

Permian tetrapods from the Sahara show climate-controlled endemism in Pangaea.

New fossils from the Upper Permian Moradi Formation of northern Niger provide an insight into the faunas that inhabited low-latitude, xeric environments near the end of the Palaeozoic era (approximately 251 million years ago). We describe here two new temnospondyl amphibians, the cochleosaurid Nigerpeton ricqlesi gen. et sp. nov. and the stem edopoid Saharastega moradiensis gen. et sp. nov., as relicts of Carboniferous lineages that diverged 40-90 million years earlier. Coupled with a scarcity of therapsids, the new finds suggest that faunas from the poorly sampled xeric belt that straddled the Equator during the Permian period differed markedly from well-sampled faunas that dominated tropical-to-temperate zones to the north and south. Our results show that long-standing theories of Late Permian faunal homogeneity are probably oversimplified as the result of uneven latitudinal sampling.

Effect of the Central American Isthmus on gene flow and divergence of the American crocodile (Crocodylus acutus).

The final formation of the Central American Isthmus (CAI) about 3.5 million years ago altered global ocean circulation, connected North and South America terrestrial biotas, and established the Caribbean Sea. The nature of this event creates a natural scenario to test vicariance, divergence, and speciation by allopatry. Studies have shown the effect of the CAI on marine and terrestrial species, but none have examined a large-bodied amphibious taxon. We used RAD sequencing on populations of the American crocodile (Crocodylus acutus) to study the genomic variation of C. acutus on both sides of the CAI, infer its demographic history, and measure the effect of the opening of the Panama Canal. Our results showed three genomic clusters: (1) Caribbean and the Panama Canal, (2) Pacific coast, and (3) Coiba island. The estimated divergence times between the Caribbean and Pacific populations are about 20,000 years ago, which is younger than the formation of the CAI, coinciding with the Last Glacial Maximum. We hypothesize the glacial/interglacial cycles facilitated gene flow between the Caribbean and Pacific crocodile populations after the formation of the CAI, masking any genomic divergence the CAI may have caused. There is no evidence of gene flow associated with the opening of the Panama Canal.

First fossil representative of Cerylonidae (Coleoptera: Coccinelloidea) described using X-ray micro-computed tomography, from Eocene Baltic amber.

Based on material originating from five amber collections of Eocene Baltic amber, Protostomopsis pandema gen. et sp. nov. is described and illustrated using X-ray micro-computed tomography. It is the first formally described extinct species of Cerylonidae, and the first known Palaearctic representative of the subfamily Ostomopsinae. As such, the new species extends the temporal range of the family Cerylonidae by approximately 45 Ma.

Response to Serrano and Chiappe.

In the recent study in Current Biology by Pei and colleagues1, we used two proxies - wing loading and specific lift - to reconstruct powered flight potential across the vaned feathered fossil pennaraptorans. The results recovered multiple origins of powered flight. We respectfully disagree with the criticism raised by Serrano and Chiappe2 that wing loading and specific lift, used in sequence, fail to discriminate between powered flight and gliding. We will explain this in reference to our original conservative approach.

Pentadactyl ground state of the manus of Alligator mississippiensis and insights into the evolution of digital reduction in Archosauria.

The three-fingered state of the avian manus poses intriguing questions about the evolution of digit reduction. Although digit reduction in most tetrapods appears to be the product of straightforward digit loss, avian digit reduction may have occurred with a dissociation of digit position from digit identity. The three digits of birds have the ancestral identities of I, II, and III but develop from an early pentadactyl ground state from digital anlage 2, 3, and 4. A series of hypotheses have been proposed in an attempt to explain this disparity, including a recent suggestion that the anteriormost condensation visible in the avian limb bud is in fact a vestigial structure from a hexadactyl ancestral ground state. We investigated this proposal by presenting sets of compatible evolutionary developmental trajectories starting from a hexadactyl state to test hypotheses of digit reduction. The development of skeletogenic mesenchymal condensations in a crocodylian, the closest extant relative to birds, is used to identify any extra precartiloginous digital vestiges. A developmental series of Alligator mississippiensis forelimb buds reveal only five digital anlagen, supports a pentadactyl ground state for the archosaurian manus, and rejects portions of the evolutionary developmental trajectories proposed. This condition lends further support to the contribution of a homeotic transformation during digit reduction in avian ancestry to account for the dissociation between digital identity and developmental position.