The hierarchical radiation of phyllostomid bats as revealed by adaptive molar morphology.

Adaptive radiations are bursts in biodiversity that generate new evolutionary lineages and phenotypes. However, because they typically occur over millions of years, it is unclear how their macroevolutionary dynamics vary through time and among groups of organisms. Phyllostomid bats radiated extensively for diverse diets-from insects to vertebrates, fruit, nectar, and blood-and we use their molars as a model system to examine the dynamics of adaptive radiations. Three-dimensional shape analyses of lower molars of Noctilionoidea (Phyllostomidae and close relatives) indicate that different diet groups exhibit distinct morphotypes. Comparative analyses further reveal that phyllostomids are a striking example of a hierarchical radiation; phyllostomids' initial, higher-level diversification involved an "early burst" in molar morphological disparity as lineages invaded new diet-affiliated adaptive zones, followed by subsequent lower-level diversifications within adaptive zones involving less dramatic morphological changes. We posit that strong selective pressures related to initial shifts to derived diets may have freed molars from morpho-functional constraints associated with the ancestral molar morphotype. Then, lineages with derived diets (frugivores and nectarivores) diversified within broad adaptive zones, likely reflecting finer-scale niche partitioning. Importantly, the observed early burst pattern is only evident when examining molar traits that are strongly linked to diet, highlighting the value of ecomorphological traits in comparative studies. Our results support the hypothesis that adaptive radiations are commonly hierarchical and involve different tempos and modes at different phylogenetic levels, with early bursts being more common at higher levels.

Cochlea development shapes bat sensory system evolution.

Sensory organs must develop alongside the skull within which they are largely encased, and this relationship can manifest as the skull constraining the organs, organs constraining the skull, or organs constraining one another in relative size. How this interplay between sensory organs and the developing skull plays out during the evolution of sensory diversity; however, remains unknown. Here, we examine the developmental sequence of the cochlea, the organ responsible for hearing and echolocation, in species with distinct diet and echolocation types within the ecologically diverse bat super-family Noctilionoidea. We found the size and shape of the cochlea largely correlates with skull size, with exceptions of Pteronotus parnellii, whose high duty cycle echolocation (nearly constant emission of sound pulses during their echolocation process allowing for detailed information gathering, also called constant frequency echolocation) corresponds to a larger cochlear and basal turn, and Monophyllus redmani, a small-bodied nectarivorous bat, for which interactions with other sensory organs restrict cochlea size. Our findings support the existence of developmental constraints, suggesting that both developmental and anatomical factors may act synergistically during the development of sensory systems in noctilionoid bats.

Disentangling Mechanical and Sensory Modules in the Radiation of Noctilionoid Bats.

AbstractWith diverse mechanical and sensory functions, the vertebrate cranium is a complex anatomical structure whose shifts between modularity and integration, especially in mechanical function, have been implicated in adaptive diversification. Yet how mechanical and sensory systems and their functions coevolve, as well as how their interrelationship contributes to phenotypic disparity, remain largely unexplored. To examine the modularity, integration, and evolutionary rates of sensory and mechanical structures within the head, we analyzed hard and soft tissue scans from ecologically diverse bats in the superfamily Noctilionoidea, a clade that ranges from insectivores and carnivores to frugivores and nectarivores. We identified eight regions that evolved in a coordinated fashion, thus recognizable as evolutionary modules: five associated with bite force and three linked to olfactory, visual, and auditory systems. Interrelationships among these modules differ between Neotropical leaf-nosed bats (family Phyllostomidae) and other noctilionoids. Consistent with the hypothesis that dietary transitions begin with changes in the capacity to detect novel food items followed by adaptations to process them, peak rates of sensory module evolution predate those of some mechanical modules. We propose that the coevolution of structures influencing bite force, olfaction, vision, and hearing constituted a structural opportunity that allowed the phyllostomid ancestor to take advantage of existing ecological opportunities and contributed to the clade's remarkable radiation.

Bat teeth illuminate the diversification of mammalian tooth classes.

Tooth classes are an innovation that has contributed to the evolutionary success of mammals. However, our understanding of the mechanisms by which tooth classes diversified remain limited. We use the evolutionary radiation of noctilionoid bats to show how the tooth developmental program evolved during the adaptation to new diet types. Combining morphological, developmental and mathematical modeling approaches, we demonstrate that tooth classes develop through independent developmental cascades that deviate from classical models. We show that the diversification of tooth number and size is driven by jaw growth rate modulation, explaining the rapid gain/loss of teeth in this clade. Finally, we mathematically model the successive appearance of tooth buds, supporting the hypothesis that growth acts as a key driver of the evolution of tooth number and size. Our work reveal how growth, by tinkering with reaction/diffusion processes, drives the diversification of tooth classes and other repeated structure during adaptive radiations.

Insights into the formation and diversification of a novel chiropteran wing membrane from embryonic development.

BACKGROUND: Through the evolution of novel wing structures, bats (Order Chiroptera) became the only mammalian group to achieve powered flight. This achievement preceded the massive adaptive radiation of bats into diverse ecological niches. We investigate some of the developmental processes that underlie the origin and subsequent diversification of one of the novel membranes of the bat wing: the plagiopatagium, which connects the fore- and hind limb in all bat species. RESULTS: Our results suggest that the plagiopatagium initially arises through novel outgrowths from the body flank that subsequently merge with the limbs to generate the wing airfoil. Our findings further suggest that this merging process, which is highly conserved across bats, occurs through modulation of the programs controlling the development of the periderm of the epidermal epithelium. Finally, our results suggest that the shape of the plagiopatagium begins to diversify in bats only after this merging has occurred. CONCLUSIONS: This study demonstrates how focusing on the evolution of cellular processes can inform an understanding of the developmental factors shaping the evolution of novel, highly adaptive structures.

Ecological constraints on highly evolvable olfactory receptor genes and morphology in neotropical bats.

Although evolvability of genes and traits may promote specialization during species diversification, how ecology subsequently restricts such variation remains unclear. Chemosensation requires animals to decipher a complex chemical background to locate fitness-related resources, and thus the underlying genomic architecture and morphology must cope with constant exposure to a changing odorant landscape; detecting adaptation amidst extensive chemosensory diversity is an open challenge. In phyllostomid bats, an ecologically diverse clade that evolved plant visiting from a presumed insectivorous ancestor, the evolution of novel food detection mechanisms is suggested to be a key innovation, as plant-visiting species rely strongly on olfaction, supplementarily using echolocation. If this is true, exceptional variation in underlying olfactory genes and phenotypes may have preceded dietary diversification. We compared olfactory receptor (OR) genes sequenced from olfactory epithelium transcriptomes and olfactory epithelium surface area of bats with differing diets. Surprisingly, although OR evolution rates were quite variable and generally high, they are largely independent of diet. Olfactory epithelial surface area, however, is relatively larger in plant-visiting bats and there is an inverse relationship between OR evolution rates and surface area. Relatively larger surface areas suggest greater reliance on olfactory detection and stronger constraint on maintaining an already diverse OR repertoire. Instead of the typical case in which specialization and elaboration are coupled with rapid diversification of associated genes, here the relevant genes are already evolving so quickly that increased reliance on smell has led to stabilizing selection, presumably to maintain the ability to consistently discriminate among specific odorants-a potential ecological constraint on sensory evolution.

Bat Dentitions: A Model System for Studies at The Interface of Development, Biomechanics, and Evolution.

The evolution of complex dentitions was a major innovation in mammals that facilitated the expansion into new dietary niches that imposed selection for tight form-function relationships. Teeth allow mammals to ingest and process food items by applying forces produced by a third-class lever system composed by the jaw adductors, the cranium, and the mandible. Physical laws determine changes in jaw adductor (biting) forces at different bite point locations along the mandible (outlever), thus individual teeth are expected to experience different mechanical regimes during feeding. If the mammal dentition exhibits functional adaptations to mandible feeding biomechanics, then teeth are expected to have evolved to develop mechanically-advantageous sizes, shapes, and positions. Here, we present bats as a model system to test this hypothesis and, more generally, for integrative studies of mammal dental diversity. We combine a field-collected dataset of bite forces along the tooth row with data on dental and mandible morphology across 30 bat species. We (1) describe, for the first time, bite force trends along the tooth row of bats, (2) use phylogenetic comparative methods to investigate relationships among bite force patterns, tooth and mandible morphology, and (3) hypothesize how these biting mechanics patterns may relate to the developmental processes controlling tooth formation. We find that bite force variation along the tooth row is consistent with predictions from lever mechanics models, with most species having the greatest bite force at the first lower molar. The cross-sectional shape of the mandible body is strongly associated with the position of maximum bite force along the tooth row, likely reflecting mandibular adaptations to varying stress patterns among species. Further, dental dietary adaptations seem to be related to bite force variation along molariform teeth, with insectivorous species exhibiting greater bite force more anteriorly, narrower teeth and mandibles, and frugivores/omnivores showing greater bite force more posteriorly, wider teeth and mandibles. As these craniodental traits are linked through development, dietary specialization appears to have shaped intrinsic mechanisms controlling traits relevant to feeding performance.

Unraveling the heritage of lost traits.

We synthesize ontogenetic work spanning the past century that show evolutionarily lost structures are rarely entirely absent from earlier developmental stages. We discuss morphological and genetic insights from developmental studies reveal about the evolution of trait loss and regain.

Find the food first: An omnivorous sensory morphotype predates biomechanical specialization for plant based diets in phyllostomid bats.

The role of mechanical morphologies in the exploitation of novel niche space is well characterized; however, the role of sensory structures in unlocking new niches is less clear. Here, we investigate the relationship between the evolution of sensory structures and diet during the radiation of noctilionoid bats. With a broad range of foraging ecologies and a well-supported phylogeny, noctilionoids constitute an ideal group for studying this relationship. We used diffusible iodine-based contrast enhanced computed tomography scans of 44 noctilionoid species to analyze relationships between the relative volumes of three sensory structures (olfactory bulbs, orbits, and cochleae) and diet. We found a positive relationship between frugivory and both olfactory and orbit size. However, we also found a negative relationship between nectarivory and cochlea size. Ancestral state estimates suggest that larger orbits and olfactory bulbs were present in the common ancestor of family Phyllostomidae, but not in other noctilionoid. This constellation of traits indicates a shift toward omnivory at the base of Phyllostomidae, predating their radiation into an exceptionally broad range of dietary niches. This is consistent with a scenario in which changes in sensory systems associated with foraging and feeding set the stage for subsequent morphological modification and diversification.

Non-model systems in mammalian forelimb evo-devo.

Mammal forelimbs are highly diverse, ranging from the elongated wing of a bat to the stout limb of the mole. The mammal forelimb has been a long-standing system for the study of early developmental patterning, proportional variation, shape change, and the reduction of elements. However, most of this work has been performed in mice, which neglects the wide variation present across mammal forelimbs. This review emphasizes the critical role of non-model systems in limb evo-devo and highlights new emerging models and their potential. We discuss the role of gene networks in limb evolution, and touch on functional analyses that lay the groundwork for further developmental studies. Mammal limb evo-devo is a rich field, and here we aim to synthesize the findings of key recent works and the questions to which they lead.

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.

Making a bat: The developmental basis of bat evolution.

Bats are incredibly diverse, both morphologically and taxonomically. Bats are the only mammalian group to have achieved powered flight, an adaptation that is hypothesized to have allowed them to colonize various and diverse ecological niches. However, the lack of fossils capturing the transition from terrestrial mammal to volant chiropteran has obscured much of our understanding of bat evolution. Over the last 20 years, the emergence of evo-devo in non-model species has started to fill this gap by uncovering some developmental mechanisms at the origin of bat diversification. In this review, we highlight key aspects of studies that have used bats as a model for morphological adaptations, diversification during adaptive radiations, and morphological novelty. To do so, we review current and ongoing studies on bat evolution. We first investigate morphological specialization by reviewing current knowledge about wing and face evolution. Then, we explore the mechanisms behind adaptive diversification in various ecological contexts using vision and dentition. Finally, we highlight the emerging work into morphological novelties using bat wing membranes.

The role of core and variable Gene Regulatory Network modules in tooth development and evolution.

Among the developmental processes that have been proposed to influence the direction of evolution, the modular organization of developmental gene regulatory networks (GRNs) has shown particular promise. In theory, GRNs have core modules comprised of essential, conserved circuits of genes, and sub-modules of downstream, secondary circuits of genes that are more susceptible to variation. While this idea has received considerable interest as of late, the field of evo-devo lacks the experimental systems needed to rigorously evaluate this hypothesis. Here, we introduce an experimental system, the vertebrate tooth, that has great potential as a model for testing this hypothesis. Tooth development and its associated GRN have been well studied and modeled in both model and non-model organisms. We propose that the existence of modules within the tooth GRN explains both the conservation of developmental mechanisms and the extraordinary diversity of teeth among vertebrates. Based on experimental data, we hypothesize that there is a conserved core module of genes that is absolutely necessary to ensure tooth or cusp initiation and development. In regard to tooth shape variation between species, we suggest that more relaxed sub-modules activated at later steps of tooth development, e.g., during the morphogenesis of the tooth and its cusps, control the different axes of tooth morphological variation.

Grand Challenges in Comparative Tooth Biology.

Teeth are a model system for integrating developmental genomics, functional morphology, and evolution. We are at the cusp of being able to address many open issues in comparative tooth biology and we outline several of these newly tractable and exciting research directions. Like never before, technological advances and methodological approaches are allowing us to investigate the developmental machinery of vertebrates and discover both conserved and excitingly novel mechanisms of diversification. Additionally, studies of the great diversity of soft tissues, replacement teeth, and non-trophic functions of teeth are providing new insights into dental diversity. Finally, we highlight several emerging model groups of organisms that are at the forefront of increasing our appreciation of the mechanisms underlying tooth diversification.

The Vertebrate Tooth Row: Is It Initiated by a Single Organizing Tooth?

Teeth are one of the most fascinating innovations of vertebrates. Their diversity of shape, size, location, and number in vertebrates is astonishing. If the molecular mechanisms underlying the morphogenesis of individual teeth are now relatively well understood, thanks to the detailed experimental work that has been performed in model organisms (mainly mouse and zebrafish), the mechanisms that control the organization of the dentition are still a mystery. Mammals display simplified dentitions when compared to other vertebrates with only a single tooth row positioned in the anterior part of the mouth, whereas other vertebrates exhibit tooth rows in many locations. As proposed 60 years ago, tooth rows can be formed sequentially from an initiator tooth. Recent results in zebrafish have now largely confirmed this hypothesis. Here this observation is generalized upon and it is suggested that in most vertebrates tooth rows could form sequentially from a single initiator tooth.

Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning.

When patterns are set during embryogenesis, it is expected that they are straightly established rather than subsequently modified. The patterning of the three mouse molars is, however, far from straight, likely as a result of mouse evolutionary history. The first-formed tooth signaling centers, called MS and R2, disappear before driving tooth formation and are thought to be vestiges of the premolars found in mouse ancestors. Moreover, the mature signaling center of the first molar (M1) is formed from the fusion of two signaling centers (R2 and early M1). Here, we report that broad activation of Edar expression precedes its spatial restriction to tooth signaling centers. This reveals a hidden two-step patterning process for tooth signaling centers, which was modeled with a single activator-inhibitor pair subject to reaction-diffusion (RD). The study of Edar expression also unveiled successive phases of signaling center formation, erasing, recovering, and fusion. Our model, in which R2 signaling center is not intrinsically defective but erased by the broad activation preceding M1 signaling center formation, predicted the surprising rescue of R2 in Edar mutant mice, where activation is reduced. The importance of this R2-M1 interaction was confirmed by ex vivo cultures showing that R2 is capable of forming a tooth. Finally, by introducing chemotaxis as a secondary process to RD, we recapitulated in silico different conditions in which R2 and M1 centers fuse or not. In conclusion, pattern formation in the mouse molar field relies on basic mechanisms whose dynamics produce embryonic patterns that are plastic objects rather than fixed end points.

The Ectodysplasin receptor EDAR acts as a tumor suppressor in melanoma by conditionally inducing cell death.

Ectodysplasin receptor EDAR is seen as a typical Tumor Necrosis Factor receptor (TNFR) family member known to interact with its ligand Eda-A1, and signaling mainly through the nuclear factor-kappaB (NF-κB) and c-jun N-terminal kinases pathways. Mutations in genes that encode proteins involved in EDAR transduction cascade cause anhidrotic ectodermal dysplasia. Here, we report an unexpected pro-apoptotic activity of EDAR when unbound to its ligand Eda-A1, which is independent of NF-κB pathway. Contrarily to other death receptors, EDAR does recruit caspase-8 to trigger apoptosis but solely upon ligand withdrawal, thereby behaving as the so-called dependence receptors. We propose that pro-apoptotic activity of unbound EDAR confers it a tumor suppressive activity. Along this line, we identified loss-of-pro-apoptotic function mutations in EDAR gene in human melanoma. Moreover, we show that the invalidation of EDAR in mice promotes melanoma progression in a B-Raf mutant background. Together, these data support the view that EDAR constrains melanoma progression by acting as a dependence receptor.

Multifactorial processes underlie parallel opsin loss in neotropical bats.

The loss of previously adaptive traits is typically linked to relaxation in selection, yet the molecular steps leading to such repeated losses are rarely known. Molecular studies of loss have tended to focus on gene sequences alone, but overlooking other aspects of protein expression might underestimate phenotypic diversity. Insights based almost solely on opsin gene evolution, for instance, have made mammalian color vision a textbook example of phenotypic loss. We address this gap by investigating retention and loss of opsin genes, transcripts, and proteins across ecologically diverse noctilionoid bats. We find multiple, independent losses of short-wave-sensitive opsins. Mismatches between putatively functional DNA sequences, mRNA transcripts, and proteins implicate transcriptional and post-transcriptional processes in the ongoing loss of S-opsins in some noctilionoid bats. Our results provide a snapshot of evolution in progress during phenotypic trait loss, and suggest vertebrate visual phenotypes cannot always be predicted from genotypes alone.

Assessing Soft-Tissue Shrinkage Estimates in Museum Specimens Imaged With Diffusible Iodine-Based Contrast-Enhanced Computed Tomography (diceCT).

The increased accessibility of soft-tissue data through diffusible iodine-based contrast-enhanced computed tomography (diceCT) enables comparative biologists to increase the taxonomic breadth of their studies with museum specimens. However, it is still unclear how soft-tissue measurements from preserved specimens reflect values from freshly collected specimens and whether diceCT preparation may affect these measurements. Here, we document and evaluate the accuracy of diceCT in museum specimens based on the soft-tissue reconstructions of brains and eyes of five bats. Based on proxies, both brains and eyes were roughly 60% of the estimated original sizes when first imaged. However, these structures did not further shrink significantly over a 4-week staining interval, and 1 week in 2.5% iodine-based solution yielded sufficient contrast for differentiating among soft-tissues. Compared to six "fresh" bat specimens imaged shortly after field collection (not fixed in ethanol), the museum specimens had significantly lower relative volumes of the eyes and brains. Variation in field preparation techniques and conditions, and long-term storage in ethanol may be the primary causes of shrinkage in museum specimens rather than diceCT staining methodology. Identifying reliable tissue-specific correction factors to adjust for the shrinkage now documented in museum specimens requires future work with larger samples.

Timing the developmental origins of mammalian limb diversity.

Mammals have highly diverse limbs that have contributed to their occupation of almost every niche. Researchers have long been investigating the development of these diverse limbs, with the goals of identifying developmental processes and potential biases that shape mammalian limb diversity. To date, researchers have used techniques ranging from the genomic to the anatomic to investigate the developmental processes shaping the limb morphology of mammals from five orders (Marsupialia, Chiroptera, Rodentia, Cetartiodactyla, and Perissodactyla). Results of these studies suggest that the differential expression of genes controlling diverse cellular processes underlies mammalian limb diversity. Results also suggest that the earliest development of the limb tends to be conserved among mammalian species, while later limb development tends to be more variable. This research has established the mammalian limb as a model system for evolutionary developmental biology, and set the stage for more in-depth, cross-disciplinary research into the genetic controls, tissue-level cellular behaviors, and selective pressures that have driven the developmental evolution of mammalian limbs. Ideally, these studies will be performed in a diverse suite of mammalian species within a comparative, phylogenetic framework.