Development of the hyolaryngeal architecture in horseshoe bats: insights into the evolution of the pulse generation for laryngeal echolocation.

BACKGROUND: The hyolaryngeal apparatus generates biosonar pulses in the laryngeally echolocating bats. The cartilage and muscles comprising the hyolarynx of laryngeally echolocating bats are morphologically modified compared to those of non-bat mammals, as represented by the hypertrophied intrinsic laryngeal muscle. Despite its crucial contribution to laryngeal echolocation, how the development of the hyolarynx in bats differs from that of other mammals is poorly documented. The genus Rhinolophus is one of the most sophisticated laryngeal echolocators, with the highest pulse frequency in bats. The present study provides the first detailed description of the three-dimensional anatomy and development of the skeleton, cartilage, muscle, and innervation patterns of the hyolaryngeal apparatus in two species of rhinolophid bats using micro-computed tomography images and serial tissue sections and compares them with those of laboratory mice. Furthermore, we measured the peak frequency of the echolocation pulse in active juvenile and adult individuals to correspond to echolocation pulses with hyolaryngeal morphology at each postnatal stage. RESULTS: We found that the sagittal crests of the cricoid cartilage separated the dorsal cricoarytenoid muscle in horseshoe bats, indicating that this unique morphology may be required to reinforce the repeated closure movement of the glottis during biosonar pulse emission. We also found that the cricothyroid muscle is ventrally hypertrophied throughout ontogeny, and that the cranial laryngeal nerve has a novel branch supplying the hypertrophied region of this muscle. Our bioacoustic analyses revealed that the peak frequency shows negative allometry against skull growth, and that the volumetric growth of all laryngeal cartilages is correlated with the pulse peak frequency. CONCLUSIONS: The unique patterns of muscle and innervation revealed in this study appear to have been obtained concomitantly with the acquisition of tracheal chambers in rhinolophids and hipposiderids, improving sound intensity during laryngeal echolocation. In addition, significant protrusion of the sagittal crest of the cricoid cartilage and the separated dorsal cricoarytenoid muscle may contribute to the sophisticated biosonar in this laryngeally echolocating lineage. Furthermore, our bioacoustic data suggested that the mineralization of these cartilages underpins the ontogeny of echolocation pulse generation. The results of the present study provide crucial insights into how the anatomy and development of the hyolaryngeal apparatus shape the acoustic diversity in bats.

A deep learning approach for morphological feature extraction based on variational auto-encoder: an application to mandible shape.

Shape measurements are crucial for evolutionary and developmental biology; however, they present difficulties in the objective and automatic quantification of arbitrary shapes. Conventional approaches are based on anatomically prominent landmarks, which require manual annotations by experts. Here, we develop a machine-learning approach by presenting morphological regulated variational AutoEncoder (Morpho-VAE), an image-based deep learning framework, to conduct landmark-free shape analysis. The proposed architecture combines the unsupervised and supervised learning models to reduce dimensionality by focusing on morphological features that distinguish data with different labels. We applied the method to primate mandible image data. The extracted morphological features reflected the characteristics of the families to which the organisms belonged, despite the absence of correlation between the extracted morphological features and phylogenetic distance. Furthermore, we demonstrated the reconstruction of missing segments from incomplete images. The proposed method provides a flexible and promising tool for analyzing a wide variety of image data of biological shapes even those with missing segments.

Evolution, conservatism and overlooked homologies of the mammalian skull.

In the last decade, studies integrating palaeontology, embryology and experimental developmental biology have markedly altered our homological understanding of the mammalian skull. Indeed, new evidence suggests that we should revisit and restructure the conventional anatomical terminology applied to the components of the mammalian skull. Notably, these are classical problems that have remained unresolved since the ninteenth century. In this review, I offer perspectives on the overlooked problems associated with the homology, development, and conservatism of the mammalian skull, aiming to encourage future studies in these areas. I emphasise that ossification patterns, bone fusion, cranial sutures and taxon-specific neomorphic bones in the skull are virtually unexplored, and further studies would improve our homological understanding of the mammalian skull. Lastly, I highlight that overlooked bones may exist in the skull that are not yet known to science and suggest that further search is needed. This article is part of the theme issue 'The mammalian skull: development, structure and function'.

Anatomy and homology of the caudal auricular muscles in greater short-nosed fruit bat (Cynopterus sphinx).

Bats can be phylogenetically classified into three major groups: pteropodids, rhinolophoids, and yangochiropterans. While rhinolophoids and yangochiropterans are capable of laryngeal echolocation, pteropodids lack this ability. Delicate ear movements are essential for echolocation behavior in bats with laryngeal echolocation. Caudal auricular muscles, especially the cervicoauricularis group, play a critical role in such ear movements. Previously, caudal auricular muscles were studied in three species of bats with laryngeal echolocation, but to our knowledge, there have been no studies on non-laryngeal echolocators, the pteropodids. Here, we describe the gross anatomy of the cervicoauricularis muscles and their innervation in Cynopterus sphinx by using diffusible iodine-based contrast-enhanced computed tomography and 3D reconstructions of immunohistochemically stained serial sections. A previous study on bats with laryngeal echolocation reported that rhinolophoids have four cervicoauricularis muscles and yangochiropterans have three. We observed three cervicoauricularis muscles in the pteropodid C. sphinx. The number of cervicoauricularis muscles and their innervation pattern were comparable to those of non-bat boreoeutherian mammals and yangochiropterans, suggesting that pteropodids, and yangochiropterans maintain the general condition of boreoeutherian mammals and that rhinolophoids have a derived condition. The unique nomenclature had been previously applied to the cervicoauricularis muscles of bats with laryngeal echolocation, but given the commonality between non-bat laurasiatherians and bats, with the exception of rhinolophoids, maintaining the conventional nomenclature (i.e., M. cervicoauricularis superficialis, M. cervicoauricularis medius, and M. cervicoauricularis profundus) is proposed for bats.

Caudal auricular muscle variations and the evolution of echolocation behavior in pteropodid bats.

Among bats, rhinolophoids and yangochiropterans, but not pteropodids, exhibit laryngeal echolocation. Although Rousettus has been regarded as the only pteropodid capable of echolocation using tongue clicks, recent evidence suggests that other species of pteropodids are also capable of echolocation using wing clicks. Studies on laryngeal echolocators suggest that delicate ear movements are essential for the echolocation behavior of bats and that the cervicoauricularis muscles play a critical role in such ear movements. In this study, we observed the gross anatomy of cervicoauricularis muscles in three species of pteropodids (Cynopterus sphinx, Eonycteris spelaea, and Rousettus leschenaultii) to examine whether ear muscle anatomy varies among pteropodids with different echolocation types and between pteropodids and laryngeal echolocating bats. We found that M. cervicoauricularis profundus originates from the nuchal crest in tongue-click echolocators (R. leschenaultii) and from the midline in wing-click echolocators (C. sphinx and E. spelaea). In general, tongue-click echolocation using high click rates is considered to be more sophisticated in terms of sonar performance than wing-click echolocation. M. cervicoauricularis profundus originating from the nuchal crest (CPNC) is not common in non-bat laurasiatherian mammals, but can be found in laryngeal echolocating bats. As it pulls the ear pinna caudally in the horizontal plane and increases the access to sound, CPNC found in R. leschenaultii and laryngeal echolocating bats may be a key characteristic of the sophisticated active echolocation behavior of bats.

Timing of organogenesis underscores the evolution of neonatal life histories and powered flight in bats.

Bats have undergone one of the most drastic limb innovations in vertebrate history, associated with the evolution of powered flight. Knowledge of the genetic basis of limb organogenesis in bats has increased but little has been documented regarding the differences between limb organogenesis in bats and that of other vertebrates. We conducted embryological comparisons of the timelines of limb organogenesis in 24 bat species and 72 non-bat amniotes. In bats, the time invested for forelimb organogenesis has been considerably extended and the appearance timing of the forelimb ridge has been significantly accelerated, whereas the timing of the finger and first appearance of the claw development has been delayed, facilitating the enlargement of the manus. Furthermore, we discovered that bats initiate the development of their hindlimbs earlier than their forelimbs compared with other placentals. Bat neonates are known to be able to cling continuously with their well-developed foot to the maternal bodies or habitat substrates soon after birth. We suggest that this unique life history of neonates, which possibly coevolved with powered flight, has driven the accelerated development of the hindlimb and precocious foot.

Synchondrosis fusion contributes to the progression of postnatal craniofacial dysmorphology in syndromic craniosynostosis.

Syndromic craniosynostosis (CS) patients exhibit early, bony fusion of calvarial sutures and cranial synchondroses, resulting in craniofacial dysmorphology. In this study, we chronologically evaluated skull morphology change after abnormal fusion of the sutures and synchondroses in mouse models of syndromic CS for further understanding of the disease. We found fusion of the inter-sphenoid synchondrosis (ISS) in Apert syndrome model mice (Fgfr2S252W/+ ) around 3 weeks old as seen in Crouzon syndrome model mice (Fgfr2cC342Y/+ ). We then examined ontogenic trajectories of CS mouse models after 3 weeks of age using geometric morphometrics analyses. Antero-ventral growth of the face was affected in Fgfr2S252W/+ and Fgfr2cC342Y/+ mice, while Saethre-Chotzen syndrome model mice (Twist1+/- ) did not show the ISS fusion and exhibited a similar growth pattern to that of control littermates. Further analysis revealed that the coronal suture synostosis in the CS mouse models induces only the brachycephalic phenotype as a shared morphological feature. Although previous studies suggest that the fusion of the facial sutures during neonatal period is associated with midface hypoplasia, the present study suggests that the progressive postnatal fusion of the cranial synchondrosis also contributes to craniofacial dysmorphology in mouse models of syndromic CS. These morphological trajectories increase our understanding of the progression of syndromic CS skull growth.

Evolution of the therian face through complete loss of the premaxilla.

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreERT2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.

Temporal and regulatory dynamics of the inner ear transcriptome during development in mice.

The inner ear controls hearing and balance, while the temporal molecular signatures and transcriptional regulatory dynamics underlying its development are still unclear. In this study, we investigated time-series transcriptome in the mouse inner ear from embryonic day 11.5 (E11.5) to postnatal day 7 (P7) using bulk RNA-Seq. A total of 10,822 differentially expressed genes were identified between pairwise stages. We identified nine significant temporal expression profiles using time-series expression analysis. The constantly down-regulated profiles throughout the development are related to DNA activity and neurosensory development, while the constantly upregulated profiles are related to collagen and extracellular matrix. Further co-expression network analysis revealed that several hub genes, such as Pnoc, Cd9, and Krt27, are related to the neurosensory development, cell adhesion, and keratinization. We uncovered three important transcription regulatory paths during mice inner ear development. Transcription factors related to Hippo/TGFß signaling induced decreased expressions of genes related to the neurosensory and inner ear development, while a series of INF genes activated the expressions of genes in immunoregulation. In addition to deepening our understanding of the temporal and regulatory mechanisms of inner ear development, our transcriptomic data could fuel future multi-species comparative studies and elucidate the evolutionary trajectory of auditory development.

Unique bone microanatomy reveals ancestry of subterranean specializations in mammals.

Acquiring a subterranean lifestyle entails a substantial shift for many aspects of terrestrial vertebrates' biology. Although this lifestyle is associated with multiple instances of convergent evolution, the relative success of some subterranean lineages largely remains unexplained. Here, we focus on the mammalian transitions to life underground, quantifying bone microanatomy through high-resolution X-ray tomography. The true moles stand out in this dataset. Examination of this family's bone histology reveals that the highly fossorial moles acquired a unique phenotype involving large amounts of compacted coarse cancellous bone. This phenotype exceeds the adaptive optimum seemingly shared by several other subterranean mammals and can be traced back to some of the first known members of the family. This remarkable microanatomy was acquired early in the history of the group and evolved faster than the gross morphology innovations of true moles' forelimb. This echoes the pattern described for other lifestyle transitions, such as the acquisition of bone mass specializations in secondarily aquatic tetrapods. Highly plastic traits-such as those pertaining to bone structure-are hence involved in the early stages of different types of lifestyle transitions.

Morphological association between muscle attachments and ossification sites in the late cartilaginous skull of tuatara embryos.

During development, the embryonic cartilaginous skull in most vertebrates is partially replaced by bones with endochondral and perichondral ossifications. Muscle attachments are thought to influence the patterns of ossification and, hence, the differentiation of the skull. To investigate the association between muscle attachments and early ossifications of reptilian embryos, we conducted digital 3D reconstructions of the cranium, the head, and the neck musculature from a histological section series of a late term embryonic tuatara, Sphenodon punctatus, with a total body length of 52 mm. As the sole living rhynchocephalian species, it is an important outgroup in comparative studies of squamate evolution. We found that head and neck muscles are largely associated with early ossification of the basal plate and the palatoquadrate, and with three other ossifications in an older specimen with a total body length of 72 mm. These results suggest that tensile forces resulting from embryonic muscle contraction are largely, but not exclusively, correlated with the area of endochondral ossification in the chondrocranium and palatoquadrate in tuatara. Beyond little-known genetic factors, the complexity of chondrocranial architecture, the progress of its development, and the effect of multiple muscle transmitting forces in the chondrocranium must be considered to provide a more comprehensive discussion of the mechanical properties of the embryonic skull.

On the sequence heterochrony of cranial ossification of bats in light of Haeckel's recapitulation theory.

Haeckel's recapitulation theory has been a controversial topic in evolutionary biology. However, we have seen some recent cases applying Haeckel's view to interpret the interspecific variation of prenatal ontogeny. To revisit the validity of Haeckel's recapitulation theory, we take bats that have undergone drastic morphological changes and possess a characteristic ecology as a case study. All members of Rhinolophoidea and Yangochiroptera can generate an ultrasonic pulse from the larynx to interpret surrounding objects (laryngeal echolocation) whereas Pteropodidae lacks such ability. It is known that the petrosal bone is particularly derived in shape and expanded in laryngeal echolocators. If Haeckel's recapitulation theory holds, the formation of this derived trait should occur later than those of other bones. Therefore, we compared the prenatal ossification timing of the petrosal in 15 bat species and five outgroup species. We found that the ossification of the petrosal is accelerated in laryngeal echolocators while it is the last bone to ossify in non-laryngeal echolocating bats and non-volant mammals, which runs counter to the prediction generated by Haeckel's recapitulation theory. We point out the evolutionarily labile nature of trait developmental timing and emphasize that Haeckel's recapitulation theory does not hold in many cases. We caution that generating predictions on ancestral conditions and evolutionary history leading from Haeckel's recapitulation theory is not well supported.

Mammalian face as an evolutionary novelty.

The anterior end of the mammalian face is characteristically composed of a semimotile nose, not the upper jaw as in other tetrapods. Thus, the therian nose is covered ventrolaterally by the "premaxilla," and the osteocranium possesses only a single nasal aperture because of the absence of medial bony elements. This stands in contrast to those in other tetrapods in whom the premaxilla covers the rostral terminus of the snout, providing a key to understanding the evolution of the mammalian face. Here, we show that the premaxilla in therian mammals (placentals and marsupials) is not entirely homologous to those in other amniotes; the therian premaxilla is a composite of the septomaxilla and the palatine remnant of the premaxilla of nontherian amniotes (including monotremes). By comparing topographical relationships of craniofacial primordia and nerve supplies in various tetrapod embryos, we found that the therian premaxilla is predominantly of the maxillary prominence origin and associated with mandibular arch. The rostral-most part of the upper jaw in nonmammalian tetrapods corresponds to the motile nose in therian mammals. During development, experimental inhibition of primordial growth demonstrated that the entire mammalian upper jaw mostly originates from the maxillary prominence, unlike other amniotes. Consistently, cell lineage tracing in transgenic mice revealed a mammalian-specific rostral growth of the maxillary prominence. We conclude that the mammalian-specific face, the muzzle, is an evolutionary novelty obtained by overriding ancestral developmental constraints to establish a novel topographical framework in craniofacial mesenchyme.

Prenatal Developmental Trajectories of Fluctuating Asymmetry in Bat Humeri.

Fluctuating asymmetry (random fluctuations between the left and right sides of the body) has been interpreted as an index to quantify both the developmental instabilities and homeostatic capabilities of organisms, linking the phenotypic and genotypic aspects of morphogenesis. However, studying the ontogenesis of fluctuating asymmetry has been limited to mostly model organisms in postnatal stages, missing prenatal trajectories of asymmetry that could better elucidate decoupled developmental pathways controlling symmetric bone elongation and thickening. In this study, we quantified the presence and magnitude of asymmetry during the prenatal development of bats, focusing on the humerus, a highly specialized bone adapted in bats to perform under multiple functional demands. We deconstructed levels of asymmetry by measuring the longitudinal and cross-sectional asymmetry of the humerus using a combination of linear measurements and geometric morphometrics. We tested the presence of different types of asymmetry and calculated the magnitude of size-controlled fluctuating asymmetry to assess developmental instability. Statistical support for the presence of fluctuating asymmetry was found for both longitudinal and cross-sectional asymmetry, explaining on average 16% of asymmetric variation. Significant directional asymmetry accounted for less than 6.6% of asymmetric variation. Both measures of fluctuating asymmetry remained relatively stable throughout ontogeny, but cross-sectional asymmetry was significantly different across developmental stages. Finally, we did not find a correspondence between developmental patterns of longitudinal and cross-sectional asymmetry, indicating that processes promoting symmetrical bone elongation and thickening work independently. We suggest various functional pressures linked to newborn bats' ecology associated with longitudinal (altricial flight capabilities) and cross-sectional (precocial clinging ability) developmental asymmetry differentially. We hypothesize that stable magnitudes of fluctuating asymmetry across development could indicate the presence of developmental mechanisms buffering developmental instability.

On the Embryonic Development of the Nasal Turbinals and Their Homology in Bats.

Multiple corrugated cartilaginous structures are formed within the mammalian nasal capsule, eventually developing into turbinals. Due to its complex and derived morphology, the homologies of the bat nasal turbinals have been highly disputed and uncertain. Tracing prenatal development has been proven to provide a means to resolve homological problems. To elucidate bat turbinate homology, we conducted the most comprehensive study to date on prenatal development of the nasal capsule. Using diffusible iodine-based contrast-enhanced computed tomography (diceCT), we studied in detail the 3D prenatal development of various bat species and non-bat laurasiatherians. We found that the structure previously identified as "maxilloturbinal" is not the true maxilloturbinal and is only part of the ethmoturbinal I pars anterior. Our results also allowed us to trace the evolutionary history of the nasal turbinals in bats. The turbinate structures are overall comparable between laurasiatherians and pteropodids, suggesting that pteropodids retain the ancestral laurasiatherian condition. The absence of the ethmoturbinal I pars posterior in yangochiropterans and rhinolophoids has possibly occurred independently by convergent evolution.

Variation in cross-sectional shape and biomechanical properties of the bat humerus under Wolff's law.

Bats use their forelimbs in different ways, but flight is the most notable example of morphological adaptation. Foraging and roosting specializations beyond flight have also been described in several bat lineages. Understanding postcranial evolution during the locomotory and foraging diversification of bats is fundamental to understanding bat evolution. We investigated whether different foraging and roosting behaviors influenced humeral cross-sectional shape and biomechanical variation, following Wolff's law of bone remodeling. The effect of body size and phylogenetic relatedness was also tested, in order to evaluate multiple sources of variation. Our results suggest strong ecological signal and no phylogenetic structuring in shape and biomechanical variation in humeral phenotypes. Decoupled modes of scaling of shape and biomechanical variation were consistently indicated across foraging and roosting behaviors, suggesting divergent allometric trajectories. Terrestrial locomoting and upstand roosting species showed unique patterns of shape and biomechanical variation across all our analyses, suggesting that these rare behaviors among bats place unique functional demands on the humerus, canalizing phenotypes. Our results suggest that complex and multiple adaptive pathways interplay in the postcranium, leading to the decoupling of different features and regions of skeletal elements optimized for different functional demands. Moreover, our results shed further light on the phenotypical diversification of the wing in bats and how adaptations besides flight could have shaped the evolution of the bat postcranium.

Embryonic staging of bats with special reference to Vespertilio sinensis and its cochlear development.

BACKGROUND: How bats deviate heterochronically from other mammals remains largely unresolved, reflecting the lack of a quantitative staging framework allowing comparison among species. The standard event system (SES) is an embryonic staging system allowing quantitative detection of interspecific developmental variations. Here, the first SES-based staging system for bats, using Asian parti-colored bat (Vespertilio sinensis) is introduced. General aspects of normal embryonic development and the three-dimensional development of the bat cochlea were described for the first time. Recoding the embryonic staging tables of 18 previously reported bat species and Mus musculus into the SES system, quantitative developmental comparisons were performed. RESULTS: It was found that limb bud development of V. sinensis is relatively late among 19 bat species and late limb development is a shared trait of vespertilionid bats. The inner ear cochlear canal forms before the semicircular canal in V. sinensis while the cochlear canal forms after the semicircular canal in non-volant mammals. CONCLUSIONS: The present approach using the SES system provides a powerful framework to detect the peculiarities of bat development. Incorporating the timing of gene expression patterns into the SES framework will further contribute to the understanding of the evolution of specialized features in bats.

Embryonic evidence uncovers convergent origins of laryngeal echolocation in bats.

Bats are the second-most speciose group of mammals, comprising 20% of species diversity today. Their global explosion, representing one of the greatest adaptive radiations in mammalian history, is largely attributed to their ability of laryngeal echolocation and powered flight, which enabled them to conquer the night sky, a vast and hitherto unoccupied ecological niche. While there is consensus that powered flight evolved only once in the lineage, whether laryngeal echolocation has a single origin in bats or evolved multiple times independently remains disputed. Here, we present developmental evidence in support of laryngeal echolocation having multiple origins in bats. This is consistent with a non-echolocating bat ancestor and independent gain of echolocation in Yinpterochiroptera and Yangochiroptera, as well as the gain of primitive echolocation in the bat ancestor, followed by convergent evolution of laryngeal echolocation in Yinpterochiroptera and Yangochiroptera, with loss of primitive echolocation in pteropodids. Our comparative embryological investigations found that there is no developmental difference in the hearing apparatus between non-laryngeal echolocating bats (pteropodids) and terrestrial non-bat mammals. In contrast, the echolocation system is developed heterotopically and heterochronically in the two phylogenetically distant laryngeal echolocating bats (rhinolophoids and yangochiropterans), providing the first embryological evidence that the echolocation system evolved independently in these bats.

Phylogeny and foraging behaviour shape modular morphological variation in bat humeri.

Bats show a remarkable ecological diversity that is reflected both in dietary and foraging guilds (FGs). Cranial ecomorphological adaptations linked to diet have been widely studied in bats, using a variety of anatomical, computational and mathematical approaches. However, foraging-related ecomorphological adaptations and the concordance between cranial and postcranial morphological adaptations remain unexamined in bats and limited to the interpretation of traditional aerodynamic properties of the wing (e.g. wing loading [WL] and aspect ratio [AR]). For this reason, the postcranial ecomorphological diversity in bats and its drivers remain understudied. Using 3D virtual modelling and geometric morphometrics (GMM), we explored the phylogenetic, ecological and biological drivers of humeral morphology in bats, evaluating the presence and magnitude of modularity and integration. To explore decoupled patterns of variation across the bone, we analysed whole-bone shape, diaphyseal and epiphyseal shape. We also tested whether traditional aerodynamic wing traits correlate with humeral shape. By studying 37 species from 20 families (covering all FGs and 85% of dietary guilds), we found similar patterns of variation in whole-bone and diaphyseal shape and unique variation patterns in epiphyseal shape. Phylogeny, diet and FG significantly correlated with shape variation at all levels, whereas size only had a significant effect on epiphyseal morphology. We found a significant phylogenetic signal in all levels of humeral shape. Epiphyseal shape significantly correlated with wing AR. Statistical support for a diaphyseal-epiphyseal modular partition of the humerus suggests a functional partition of shape variability. Our study is the first to show within-structure modular morphological variation in the appendicular skeleton of any living tetrapod. Our results suggest that diaphyseal shape correlates more with phylogeny, whereas epiphyseal shape correlates with diet and FG.

A 1.4-million-year-old bone handaxe from Konso, Ethiopia, shows advanced tool technology in the early Acheulean.

In the past decade, the early Acheulean before 1 Mya has been a focus of active research. Acheulean lithic assemblages have been shown to extend back to ∼1.75 Mya, and considerable advances in core reduction technologies are seen by 1.5 to 1.4 Mya. Here we report a bifacially flaked bone fragment (maximum dimension ∼13 cm) of a hippopotamus femur from the ∼1.4 Mya sediments of the Konso Formation in southern Ethiopia. The large number of flake scars and their distribution pattern, together with the high frequency of cone fractures, indicate anthropogenic flaking into handaxe-like form. Use-wear analyses show quasi-continuous alternate microflake scars, wear polish, edge rounding, and striae patches along an ∼5-cm-long edge toward the handaxe tip. The striae run predominantly oblique to the edge, with some perpendicular, on both the cortical and inner faces. The combined evidence is consistent with the use of this bone artifact in longitudinal motions, such as in cutting and/or sawing. This bone handaxe is the oldest known extensively flaked example from the Early Pleistocene. Despite scarcity of well-shaped bone tools, its presence at Konso shows that sophisticated flaking was practiced by ∼1.4 Mya, not only on a range of lithic materials, but also occasionally on bone, thus expanding the documented technological repertoire of African Early Pleistocene Homo.