Review and experimental evaluation of the embryonic development and evolutionary history of flipper development and hyperphalangy in dolphins (Cetacea: Mammalia).

Cetaceans are the only mammals to have evolved hyperphalangy, an increase in the number of phalanges beyond the mammalian plesiomorphic condition of three phalanges per digit. In this study, cetaceans were used as a novel model to review previous studies of mammalian hyperphalangy and contribute new experimental evidence as to the molecular origins of this phenotype in embryos of the pantropical spotted dolphin (Stenella attenuata). Results show embryos of dolphins, mice, and pigs share similar spatiotemporal patterns of signaling proteins known to shape limbs of mammals (e.g., FGF8, BMP2/4, WNT, GREM). However, fetal dolphins differ in that their interdigital tissues are retained, instead of undergoing apoptosis, and that multiple waves of interdigital signals likely contribute to the patterning of supernumerary joints and phalanges in adjacent digits. Integration of fossil and experimental evidence suggests that the presence of interdigital webbing within the fossils of semi-aquatic cetaceans, recovered from the Eocene Epoch (49Ma), was probably the result of BMP-antagonists counteracting interdigital apoptosis during embryonic limb development. Modifications to signals originating in these interdigital tissues likely contributed to the origin of an incipient form of hyperphalangy in obligatorily aquatic cetaceans about 35Ma. Finally, an extreme form of hyperphalangy, with six or more phalanges per digit, evolved independently in rorqual whales (Balaenopteridae) and delphinids, and was probably associated with a wave of signaling within the interdigital tissues.

Macroevolutionary developmental biology: Embryos, fossils, and phylogenies.

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.

Integrative single-cell characterization of a frugivorous and an insectivorous bat kidney and pancreas.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we use integrative single-cell sequencing (scRNA-seq and scATAC-seq) on insectivorous (Eptesicus fuscus; big brown bat) and frugivorous (Artibeus jamaicensis; Jamaican fruit bat) bat kidneys and pancreases and identify key cell population, gene expression and regulatory differences associated with the Jamaican fruit bat that also relate to human disease, particularly diabetes. We find a decrease in loop of Henle and an increase in collecting duct cells, and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the Jamaican fruit bat kidney. The Jamaican fruit bat pancreas shows an increase in endocrine and a decrease in exocrine cells, and differences in genes and regulatory elements involved in insulin regulation. We also find that these frugivorous bats share several molecular characteristics with human diabetes. Combined, our work provides insights from a frugivorous mammal that could be leveraged for therapeutic purposes.

Whales originated from aquatic artiodactyls in the Eocene epoch of India.

Although the first ten million years of whale evolution are documented by a remarkable series of fossil skeletons, the link to the ancestor of cetaceans has been missing. It was known that whales are related to even-toed ungulates (artiodactyls), but until now no artiodactyls were morphologically close to early whales. Here we show that the Eocene south Asian raoellid artiodactyls are the sister group to whales. The raoellid Indohyus is similar to whales, and unlike other artiodactyls, in the structure of its ears and premolars, in the density of its limb bones and in the stable-oxygen-isotope composition of its teeth. We also show that a major dietary change occurred during the transition from artiodactyls to whales and that raoellids were aquatic waders. This indicates that aquatic life in this lineage occurred before the origin of the order Cetacea.

Comparing age- and bone-related differences in collagen fiber orientation: A case study of bats and laboratory mice using quantitative polarized light microscopy.

As bones age in most mammals, they typically become more fragile. This state of bone fragility is often associated with more homogenous collagen fiber orientations (CFO). Unlike most mammals, bats maintain mechanically competent bone throughout their lifespans, but little is known of positional and age-related changes in CFO within wing bones. This study tests the hypothesis that age-related changes in CFO in big brown bats (Eptesicus fuscus) differ from those of the standard mammalian model for skeletal aging, the C57BL/6 laboratory mouse. We used data from quantitative polarized light microscopy (qPLM) to compare CFO across the lifespan of long-lived big brown bats and age matched C57BL/6 mice. Eptesicus and C57BL/6 mice displayed idiosyncratic patterns of CFO. Consistent age-related changes were only apparent in the outer cortical bone of Eptesicus, where bone tissue is more longitudinally arranged and more anisotropic in older individuals. Both taxa displayed a ring of more transversely oriented bone tissue surrounding the medullary cavity. In Eptesicus, this tissue represents a greater proportion of the overall cross-section, and is more clearly helically aligned (arranged at 45° to the bone long axis) than similar bone tissue in mice. Bat wing bones displayed a proximodistal gradient in CFO anisotropy and longitudinal orientation in both outer and inner cortical bone compartments. This study lays a methodological foundation for the quantitative evaluation of bone tissue architecture in volant and non-volant mammals that may be expanded in the future.

Integrative single-cell characterization of frugivory adaptations in the bat kidney and pancreas.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we used integrative single-cell sequencing on insectivorous and frugivorous bat kidneys and pancreases and identified key cell population, gene expression and regulatory element differences associated with frugivorous adaptation that also relate to human disease, particularly diabetes. We found an increase in collecting duct cells and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the frugivore kidney. In the frugivorous pancreas, we observed an increase in endocrine and a decrease in exocrine cells and differences in genes and regulatory elements involved in insulin regulation. Combined, our work provides novel insights into frugivorous adaptation that also could be leveraged for therapeutic purposes.

Long-term spring through fall capture data of Eptesicus fuscus in the eastern USA before and after white-nose syndrome.

Emerging infectious diseases threaten wildlife populations. Without well monitored wildlife systems, it is challenging to determine accurate population and ecosystem losses following disease emergence. North American temperate bats present a unique opportunity for studying the broad impacts of wildlife disease emergence, as their federal monitoring programs were prioritized in the USA throughout the 20th century and they are currently threatened by the invasive fungal pathogen, Pseudogymnoascus destructans (Pd), which causes white-nose syndrome. Here we provide a long-term dataset for capture records of Eptesicus fuscus (big brown bat) across the eastern USA, spanning 16 years before and 14 years after Pd invasion into North America. These data represent 30,496 E. fuscus captures across 3,567 unique sites. We encourage the use of this dataset for quantifying impacts of wildlife disease and other threats to wildlife (e.g., climate change) with the incorporation of other available data. We welcome additional data contributions for E. fuscus captures across North and Central America as well as the inclusion of other variables into the dataset that contribute to the quantification of wildlife health.

Intraskeletal consistency in patterns of vascularity within bat limb bones.

Bats are the only mammals to have achieved powered flight. A key innovation allowing for bats to conquer the skies was a forelimb modified into a flexible wing. The wing bones of bats are exceptionally long and dynamically bend with wingbeats. Bone microarchitectural features supporting these novel performance attributes are still largely unknown. The humeri and femora of bats are typically avascular, except for large-bodied taxa (e.g., pteropodid flying foxes). No thorough investigation of vascular canal regionalization and morphology has been undertaken as historically it has been difficult to reconstruct the 3D architecture of these canals. This study augments our understanding of the vascular networks supporting the bone matrix of a sample of bats (n = 24) of variable body mass, representing three families (Pteropodidae [large-bodied, species = 6], Phyllostomidae [medium-bodied, species = 2], and Molossidae [medium-bodied, species = 1]). We employed Synchrotron Radiation-based micro-Computed Tomography (SRµCT) to allow for a detailed comparison of canal morphology within humeri and femora. Results indicate that across selected bats, canal number per unit volume is similar independent of body size. Differences in canal morphometry based on body size and bone type appear primarily related to a broader distribution of the canal network as cortical volume increases. Heavier bats display a relatively rich vascular network of mostly longitudinally-oriented canals that are localized mainly to the mid-cortical and endosteal bone envelopes. Taken together, our results suggest that relative vascularity of the limb bones of heavier bats forms support for nutrient exchange in a regional pattern.

Metabolism: Evolution of dolphin sperm endurance.

Mammalian sperm have long been known to use energy derived from the metabolism of sugars and fatty acids. A new study shows that sperm of dolphins and their relatives lost functionality of the glycolysis pathway and are fueled only by energy-rich fatty acids that are metabolized by extra-large mitochondria, giving them exceptional endurance.

DNA methylation predicts age and provides insight into exceptional longevity of bats.

Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Relative growth rates of predator and prey dinosaurs reflect effects of predation.

Hadrosaurs grew rapidly, and quantifying their growth is key to understanding life-history interactions between predators and prey during the Late Cretaceous. In this study, we longitudinally sampled a sequence of lines of arrested growth (LAGs) from an essentially full-grown hadrosaur Hypacrosaurus stebingeri (MOR 549). Spatial locations of LAGs in the femoral and tibial transverse sections of MOR 549 were measured and circumferences were calculated. For each bone, a time series of circumference data was fitted to several stochastic, discrete growth models. Our results suggest that the femur and the tibia of this specimen of Hypacrosaurus probably followed a Gompertz curve and that LAGs reportedly missing from early ontogeny were obscured by perimedullary resorption. In this specimen, death occurred at 13 years and took approximately 10-12 years to reach 95 per cent asymptotic size. The age at growth inflection, which is a proxy for reproductive maturity, occurred at approximately 2-3 years. Comparisons with several small and large predatory theropods reveal that MOR 549 grew faster and matured sooner than they did. These results suggest that Hypacrosaurus was able to partly avoid predators by outgrowing them.

Comparative metabolomics of aging in a long-lived bat: Insights into the physiology of extreme longevity.

Vespertilionid bats (Mammalia: Order Chiroptera) live 3-10 times longer than other mammals of an equivalent body size. At present, nothing is known of how bat fecal metabolic profiles shift with age in any taxa. This study established the feasibility of using a non-invasive, fecal metabolomics approach to examine age-related differences in the fecal metabolome of young and elderly adult big brown bats (Eptesicus fuscus) as an initial investigation into using metabolomics for age determination. Samples were collected from captive, known-aged big brown bats (Eptesicus fuscus) from 1 to over 14 years of age: these two ages represent age groups separated by approximately 75% of the known natural lifespan of this taxon. Results showed 41 metabolites differentiated young (n = 22) and elderly (n = 6) Eptesicus. Significant differences in metabolites between young and elderly bats were associated with tryptophan metabolism and incomplete protein digestion. Results support further exploration of the physiological mechanisms bats employ to achieve exceptional longevity.

Evidence of Wnt/ß-catenin alterations in brain and bone of a tauopathy mouse model of Alzheimer's disease.

Low bone mineral density (BMD) is a significant comorbidity in Alzheimer's disease (AD) and may reflect systemic regulatory pathway dysfunction. Low BMD has been identified in several AD mouse models selective for amyloid-ß or tau pathology, but these deficits were attributed to diverse mechanisms. In this study, we identified common pathophysiological mechanisms accounting for bone loss and neurodegeneration in the htau mouse, a tauopathy model with an early low BMD phenotype. We investigated the Wnt/ß-catenin pathway-a cellular signaling cascade linked to both bone loss and neuropathology. We showed that low BMD persisted in male htau mice aged from 6 to 14 months, remaining significantly lower than tau-null and C57BL/6J controls. Osteogenic gene expression in female and male htau mice was markedly reduced from controls, indicating impaired bone remodeling. In both the bone and brain, htau mice showed alterations in Wnt/ß-catenin signaling genes suggestive of increased inhibition of this pathway. These findings implicate dysfunctional Wnt signaling as a potential target for addressing bone loss in AD.

Aquatic Habits of Cetacean Ancestors: Integrating Bone Microanatomy and Stable Isotopes.

The earliest cetaceans were interpreted as semi-aquatic based on the presence of thickened bones and stable oxygen isotopes in tooth enamel. However, the origin of aquatic behaviors in cetacean relatives (e.g., raoellids, anthracotheres) remains unclear. This study reconstructs the origins of aquatic behaviors based on long bone microanatomy and stable oxygen isotopes of tooth enamel in modern and extinct cetartiodactylans. Our findings are congruent with published accounts that microanatomy can be a reliable indicator of aquatic behaviors in taxa that are obligatorily aquatic, and also highlight that some "semi-aquatic" behaviors (fleeing into the water to escape predation) may have a stronger relationship to bone microanatomy than others (herbivory in near-shore aquatic settings). Bone microanatomy is best considered with other lines of information in the land-to-sea transition of cetaceans, such as stable isotopes. This study extends our understanding of the progression of skeletal phenotypes associated with habitat shifts in the relatives of cetaceans.

Anthracobunids from the middle eocene of India and pakistan are stem perissodactyls.

Anthracobunidae is an Eocene family of large mammals from south Asia that is commonly considered to be part of the radiation that gave rise to elephants (proboscideans) and sea cows (sirenians). We describe a new collection of anthracobunid fossils from Middle Eocene rocks of Indo-Pakistan that more than doubles the number of known anthracobunid fossils and challenges their putative relationships, instead implying that they are stem perissodactyls. Cranial, dental, and postcranial elements allow a revision of species and the recognition of a new anthracobunid genus. Analyses of stable isotopes and long bone geometry together suggest that most anthracobunids fed on land, but spent a considerable amount of time near water. This new evidence expands our understanding of stem perissodactyl diversity and sheds new light on perissodactyl origins.

The evolution and development of mammalian flight.

Mammals have evolved a stunning diversity of limb morphologies (e.g., wings, flippers, hands, and paws) that allowed access to a wide range of habitats. Over 50 million years ago, bats (Order Chiroptera) evolved a wing (composed of a thin membrane encasing long digits) and thereby achieved powered flight. Unfortunately, the fossil record currently lacks any transitional fossils between a rodent-like ancestor and a winged bat. To reconstruct how this important evolutionary transition occurred, researchers have begun to employ an evolutionary developmental approach. This approach has revealed some of the embryological and molecular changes that have contributed to the evolution of the bat wing. For example, bat and mouse forelimb morphologies are similar during earliest limb development. Despite this, some key signaling centers for limb development are already divergent in bat and mouse at these early stages. Bat and mouse limb development continues to diverge, such that at later stages many differences are apparent. For example, at these later stages bats redeploy expression of toolkit genes (i.e., Fgf, Shh, Bmp, Grem) in a novel expression domain to inhibit apoptosis of the interdigital tissues. When results are taken together, a broad picture of the developmental changes that drove the transition from a hand to a wing over 50 million years ago is beginning to take shape. Moreover, studies seem to suggest that small changes in gene regulation during organogenesis can generate large evolutionary changes in phenotype.

Hydrodynamic performance of the minke whale (Balaenoptera acutorostrata) flipper.

Minke whales (Balaenoptera acutorostrata) are the smallest member of balaenopterid whales and little is known of their kinematics during feeding maneuvers. These whales have narrow and elongated flippers that are small relative to body size compared to related species such as right and gray whales. No experimental studies have addressed the hydrodynamic properties of minke whale flippers and their functional role during feeding maneuvers. This study integrated wind tunnel, locomotion and anatomical range of motion data to identify functional parameters of the cambered minke whale flipper. A full-sized cast of a minke whale flipper was used in wind tunnel testing of lift, drag and stall behavior at six speeds, corresponding to swimming speeds of 0.7-8.9 m s(-1). Flow over the model surface stalled between 10 degrees and 14 degrees angle of attack (alpha) depending on testing speed. When the leading edge was rotated ventrally, loss in lift occurred around -18 degrees alpha regardless of speed. Range of mobility in the fresh limb was approximately 40% greater than the range of positive lift-generating angles of attack predicted by wind tunnel data (+14 degrees alpha). Video footage, photographs and observations of swimming, engulfment feeding and gulping minke whales showed limb positions corresponding to low drag in wind tunnel tests, and were therefore hydrodynamically efficient. Flippers play an important role in orienting the body during feeding maneuvers as they maintain trim of the body, an action that counters drag-induced torque of the body during water and prey intake.