Ecomorphology and sensory biology of bats.

This special issue of The Anatomical Record is inspired by and dedicated to Professor Kunwar P. Bhatnagar, whose lifelong interests in biology, and long career studying bats, inspired many and advanced our knowledge of the world's only flying mammals. The 15 articles included here represent a broad range of investigators, treading topics familiar to Prof. Bhatnagar, who was interested in seemingly every aspect of bat biology. Key topics include broad themes of bat development, sensory systems, and specializations related to flight and diet. These articles paint a complex picture of the fascinating adaptations of bats, such as rapid fore limb development, ear morphologies relating to echolocation, and other enhanced senses that allow bats to exploit niches in virtually every part of the world. In this introduction, we integrate and contextualize these articles within the broader story of bat ecomorphology, providing an overview of each of the key themes noted above. This special issue will serve as a springboard for future studies both in bat biology and in the broader world of mammalian comparative anatomy and ecomorphology.

Neuronal scaling in the olfactory system of bats.

Comparative studies are a common way to address large-scale questions in sensory biology. For studies that investigate olfactory abilities, the most commonly used metric is olfactory bulb size. However, recent work has called into question the broad-scale use of olfactory bulb size. In this paper, we use three neuroanatomical measures with a more mechanistic link to olfactory function (number of olfactory sensory neurons (OSNs), number of mitral cells (MCs), and number of glomeruli) to ask how species with different diets may differ with respect to olfactory ability. We use phyllostomid bats as our study system because behavioral and physiological work has shown that fruit- and nectar-feeding phyllostomids rely on odors for detecting, localizing, and assessing potential foods, while insect-eating species do not. Therefore, we predicted that fruit- and nectar-feeding bats would have larger numbers of these three neuroanatomical measures than insect-eating species. In general, our results supported the predictions. We found that fruit-eaters had greater numbers of OSNs and glomeruli than insect-eaters, but we found no difference between groups in number of MCs. We also examined the allometric relationship between the three neuroanatomical variables and olfactory bulb volume, and we found isometry in all cases. These findings lend support to the notion that neuroanatomical measures can offer valuable insights into comparative olfactory abilities, and suggest that the size of the olfactory bulb may be an informative parameter to use at the whole-organism level.

Mapping odorant sensitivities reveals a sparse but structured representation of olfactory chemical space by sensory input to the mouse olfactory bulb.

In olfactory systems, convergence of sensory neurons onto glomeruli generates a map of odorant receptor identity. How glomerular maps relate to sensory space remains unclear. We sought to better characterize this relationship in the mouse olfactory system by defining glomeruli in terms of the odorants to which they are most sensitive. Using high-throughput odorant delivery and ultrasensitive imaging of sensory inputs, we imaged responses to 185 odorants presented at concentrations determined to activate only one or a few glomeruli across the dorsal olfactory bulb. The resulting datasets defined the tuning properties of glomeruli - and, by inference, their cognate odorant receptors - in a low-concentration regime, and yielded consensus maps of glomerular sensitivity across a wide range of chemical space. Glomeruli were extremely narrowly tuned, with ~25% responding to only one odorant, and extremely sensitive, responding to their effective odorants at sub-picomolar to nanomolar concentrations. Such narrow tuning in this concentration regime allowed for reliable functional identification of many glomeruli based on a single diagnostic odorant. At the same time, the response spectra of glomeruli responding to multiple odorants was best predicted by straightforward odorant structural features, and glomeruli sensitive to distinct odorants with common structural features were spatially clustered. These results define an underlying structure to the primary representation of sensory space by the mouse olfactory system.

Testing Morphological Relationships Between Female and Male Copulatory Structures in Bats.

The lower reproductive tract of female mammals has several competing functions including mating, tract health maintenance, and parturition. Diverse vaginal anatomy suggests interactions between natural and sexual selection, yet despite its importance, female copulatory morphology remains under-studied. We undertook a comparative study across the species-rich mammalian order Chiroptera (bats) with a focus on the suborder Yangochiroptera (Vespertilioniformes) to examine how female vaginal features may have coevolved with male penis morphology to minimize mechanical damage to their tissues during copulation. The penis morphology is diverse, presenting great potential for post-copulatory sexual selection and coevolution with the female morphology, but vaginas have not been carefully examined. Here we test the hypotheses that vaginal thickness and collagen density have coevolved with features of the male penis including the presence of spines and a baculum. We present histological data from females of 24 species from 7 families of bats, and corresponding data on male penis anatomy. We also examine the role of phylogenetic history in the morphological patterns we observe. We found evidence that female vaginal thickness has coevolved with the presence of penile spines, but not with baculum presence or width. Collagen density did not appear to covary with male penile features. Our findings highlight the importance of considering interactions between the sexes in influencing functional reproductive structures and examine how these structures have been under selection in bats.

Venous networks in the upper airways of bats: A histological and diceCT study.

Our knowledge of nasal cavity anatomy has grown considerably with the advent of micro-computed tomography (CT). More recently, a technique called diffusible iodine-based contrast-enhanced CT (diceCT) has rendered it possible to study nasal soft tissues. Using diceCT and histology, we aim to (a) explore the utility of these techniques for inferring the presence of venous sinuses that typify respiratory mucosa and (b) inquire whether distribution of vascular mucosa may relate to specialization for derived functions of the nasal cavity (i.e., nasal-emission of echolocation sounds) in bats. Matching histology and diceCT data indicate that diceCT can detect venous sinuses as either darkened, "empty" spaces, or radio-opaque islands when blood cells are present. Thus, we show that diceCT provides reliable information on vascular distribution in the mucosa of the nasal airways. Among the bats studied, a nonecholocating pteropodid (Cynopterus sphinx) and an oral-emitter of echolocation sounds (Eptesicus fuscus) possess venous sinus networks that drain into the sphenopalatine vein rostral to the nasopharynx. In contrast, nasopharyngeal passageways of nasal-emitting hipposiderids are notably packed with venous sinuses. The mucosae of the nasopharyngeal passageways are far less vascular in nasal-emitting phyllostomids, in which vascular mucosae are more widely distributed in the nasal cavity, and in some nectar-feeding species, a particularly large venous sinus is adjacent to the vomeronasal organ. Therefore, we do not find a common pattern of venous sinus distribution associated with nasal emission of sounds in phyllostomids and hipposiderids. Instead, vascular mucosa is more likely critical for air-conditioning and sometimes vomeronasal function in all bats.

Circuit Contributions to Sensory-Driven Glutamatergic Drive of Olfactory Bulb Mitral and Tufted Cells During Odorant Inhalation.

In the mammalian olfactory bulb (OB), mitral/tufted (MT) cells respond to odorant inhalation with diverse temporal patterns that are thought to encode odor information. Much of this diversity is already apparent at the level of glutamatergic input to MT cells, which receive direct, monosynaptic excitatory input from olfactory sensory neurons (OSNs) as well as a multisynaptic excitatory drive via glutamatergic interneurons. Both pathways are also subject to modulation by inhibitory circuits in the glomerular layer of the OB. To understand the role of direct OSN input vs. postsynaptic OB circuit mechanisms in shaping diverse dynamics of glutamatergic drive to MT cells, we imaged glutamate signaling onto MT cell dendrites in anesthetized mice while blocking multisynaptic excitatory drive with ionotropic glutamate receptor antagonists and blocking presynaptic modulation of glutamate release from OSNs with GABAB receptor antagonists. GABAB receptor blockade increased the magnitude of inhalation-linked glutamate transients onto MT cell apical dendrites without altering their inhalation-linked dynamics, confirming that presynaptic inhibition impacts the gain of OSN inputs to the OB. Surprisingly, blockade of multisynaptic excitation only modestly impacted glutamatergic input to MT cells, causing a slight reduction in the amplitude of inhalation-linked glutamate transients in response to low odorant concentrations and no change in the dynamics of each transient. The postsynaptic blockade also modestly impacted glutamate dynamics over a slower timescale, mainly by reducing adaptation of the glutamate response across multiple inhalations of odorant. These results suggest that direct glutamatergic input from OSNs provides the bulk of excitatory drive to MT cells, and that diversity in the dynamics of this input may be a primary determinant of the temporal diversity in MT cell responses that underlies odor representations at this stage.

Dynamics of Glutamatergic Drive Underlie Diverse Responses of Olfactory Bulb Outputs In Vivo.

Mitral/tufted (MT) cells of the olfactory bulb (OB) show diverse temporal responses to odorant stimulation that are thought to encode odor information. Much of this diversity is thought to arise from inhibitory OB circuits, but the dynamics of excitatory input to MT cells, which is driven in a feedforward manner by sensory afferents, may also be important. To examine the contribution of excitatory input dynamics to generating temporal diversity in MT cells, we imaged glutamate signaling onto MT cell dendrites in anesthetized and awake mice. We found surprising diversity in the temporal dynamics of these signals. Inhalation-linked glutamate transients were variable in onset latency and duration, and in awake mice the degree of coupling to inhalation varied substantially with odorant identity and concentration. Successive inhalations of odorant produced nonlinear changes in glutamate signaling that included facilitating, adapting and suppressive responses and which varied with odorant identity and concentration. Dual-color imaging of glutamate and calcium signals from MT cells in the same glomerulus revealed highly correlated presynaptic and postsynaptic signals across these different response types. Suppressive calcium responses in MT cells were nearly always accompanied by suppression in the glutamate signal, providing little evidence for MT cell suppression by lateral or feedforward inhibition. These results indicate a high degree of diversity in the dynamics of excitatory input to MT cells, and suggest that these dynamics may account for much of the diversity in MT cell responses that underlies OB odor representations.

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.

Differential Impacts of Repeated Sampling on Odor Representations by Genetically-Defined Mitral and Tufted Cell Subpopulations in the Mouse Olfactory Bulb.

Sniffing, the active control of breathing beyond passive respiration, is used by mammals to modulate olfactory sampling. Sniffing allows animals to make odor-guided decisions within ∼200 ms, but animals routinely engage in bouts of high-frequency sniffing spanning several seconds; the impact of such repeated odorant sampling on odor representations remains unclear. We investigated this question in the mouse olfactory bulb (OB), where mitral and tufted cells (MTCs) form parallel output streams of odor information processing. To test the impact of repeated odorant sampling on MTC responses, we used two-photon imaging in anesthetized male and female mice to record activation of MTCs while precisely varying inhalation frequency. A combination of genetic targeting and viral expression of GCaMP6 reporters allowed us to access mitral cell (MC) and superficial tufted cell (sTC) subpopulations separately. We found that repeated odorant sampling differentially affected responses in MCs and sTCs, with MCs showing more diversity than sTCs over the same time period. Impacts of repeated sampling among MCs included both increases and decreases in excitation, as well as changes in response polarity. Response patterns across simultaneously-imaged MCs reformatted over time, with representations of different odorants becoming more distinct. Individual MCs responded differentially to changes in inhalation frequency, whereas sTC responses were more uniform over time and across frequency. Our results support the idea that MCs and TCs comprise functionally distinct pathways for odor information processing, and suggest that the reformatting of MC odor representations by high-frequency sniffing may serve to enhance the discrimination of similar odors.SIGNIFICANCE STATEMENT Repeated sampling of odorants during high-frequency respiration (sniffing) is a hallmark of active odorant sampling by mammals; however, the adaptive function of this behavior remains unclear. We found distinct effects of repeated sampling on odor representations carried by the two main output channels from the mouse olfactory bulb (OB), mitral and tufted cells (MTCs). Mitral cells (MCs) showed more diverse changes in response patterns over time as compared with tufted cells (TCs), leading to odorant representations that were more distinct after repeated sampling. These results support the idea that MTCs contribute different aspects to encoding odor information, and they indicate that MCs (but not TCs) may play a primary role in the modulation of olfactory processing by sampling behavior.

A Novel Olfactometer for Efficient and Flexible Odorant Delivery.

Understanding how sensory space maps to neural activity in the olfactory system requires efficiently and flexibly delivering numerous odorants within single experimental preparations. Such delivery is difficult with current olfactometer designs, which typically include limited numbers of stimulus channels and are subject to intertrial and interchannel contamination of odorants. Here, we present a novel olfactometer design that is easily constructed, modular, and capable of delivering an unlimited number of odorants in air with temporal precision and no detectable intertrial or interchannel contamination. The olfactometer further allows for the flexible generation of odorant mixtures and flexible timing of odorant sequences. Odorant delivery from the olfactometer is turbulent but reliable from trial to trial, supporting operant conditioning of mice in an odorant discrimination task and permitting odorants and concentrations to be mapped to neural activity with a level of precision equivalent to that obtained with a flow dilution olfactometer. This novel design thus provides several unique advantages for interrogating olfactory perception and for mapping sensory space to neural activity in the olfactory system.

Artificial Inhalation Protocol in Adult Mice.

Research in the area of in vivo olfactory physiology benefits from having direct access to the nasal airways through which odorants can be presented. Ordinarily, the passage of odorants through the airways is controlled by respiratory rhythm. This fact makes it difficult to control the timing and strength of an olfactory stimulus, since animals must breathe regularly, and the act of breathing itself also controls odorant presentation. However, using an artificial inhalation preparation allows us to decouple breathing from olfaction. With this technique we present oxygen and anesthetic (if desired) to the lungs directly and independently control odorant access to the nasal passages. This technique allows for direct control of odorant presentation in vivo, enabling more precise control of parameters of stimulation when investigating olfactory processing. This technique may have additional applications, for example in aerosolized drug delivery.

How much does nasal cavity morphology matter? Patterns and rates of olfactory airflow in phyllostomid bats.

The morphology of the nasal cavity in mammals with a good sense of smell includes features that are thought to improve olfactory airflow, such as a dorsal conduit that delivers odours quickly to the olfactory mucosa, an enlarged olfactory recess at the back of the airway, and a clear separation of the olfactory and respiratory regions of the nose. The link between these features and having a good sense of smell has been established by functional examinations of a handful of distantly related mammalian species. In this paper, we provide the first detailed examination of olfactory airflow in a group of closely related species that nevertheless vary in their sense of smell. We study six species of phyllostomid bats that have different airway morphologies and foraging ecologies, which have been linked to differences in olfactory ability or reliance. We hypothesize that differences in morphology correlate with differences in the patterns and rates of airflow, which in turn are consistent with dietary differences. To compare species, we make qualitative and quantitative comparisons of the patterns and rates of airflow through the olfactory region during both inhalation and exhalation across the six species. Contrary to our expectations, we find no clear differences among species in either the patterns of airflow through the airway or in rates of flow through the olfactory region. By and large, olfactory airflow seems to be conserved across species, suggesting that morphological differences appear to be driven by other mechanical demands on the snout, such as breathing and feeding. Olfactory ability may depend on other aspects of the system, such as the neurobiological processing of odours that work within the existing morphology imposed by other functional demands on the nasal cavity.

Nasal morphometry in marmosets: loss and redistribution of olfactory surface area.

The two major groups of primates differ in internal nasal anatomy. Strepsirrhines (e.g., lemurs) have more numerous turbinals and recesses compared with haplorhines (e.g., monkeys). Since detailed quantitative comparisons of nasal surface area (SA) have not been made, we measured mucosa in serially sectioned monkeys (Callithrix jacchus, Cebuella pygmaea). Data were compared with previously published findings on the mouse lemur, Microcebus murinus. The nasal airways were digitally reconstructed using computed tomography scanned heads of Cebuella and Microcebus. In addition, morphometric and functional analyses were carried out using segmented photographs of the histological sections of Cebuella and Microcebus. The SA of the ethmoturbinal complex is about half as large in marmosets compared with Microcebus, and is covered with less olfactory mucosa (18%-24% in marmosets, compared with ∼ 50% in Microcebus). Whereas the ethmoturbinal complex of Microcebus bears half of the total olfactory mucosa in the nasal airway, most (∼ 80%) olfactory mucosa is distributed on other surfaces in the marmosets (e.g., nasal septum). A comparison to previously published data suggests all primate species have less olfactory surface area (OSA) compared with other similar-sized mammals, but this is especially true of marmosets. Taken together, these findings support the hypothesis that there is a reduced OSA in at least some haplorhines, and this can be linked to diminished posterosuperior dimensions of the nasal fossae. However, haplorhines may have minimized their olfactory loss by redistributing olfactory mucosa on non-turbinal surfaces. Our findings also imply that airflow patterns in the olfactory region differ among primates.

Olfactory epithelium in the olfactory recess: a case study in new world leaf-nosed bats.

The olfactory recess (OR) is a restricted space at the back of the nasal fossa in many mammals that is thought to improve olfactory function. Mammals that have an olfactory recess are usually described as keen-scented, while those that do not are typically thought of as less reliant on olfaction. However, the presence of an olfactory recess is not a binary trait. Many mammal families have members that vary substantially in the size and complexity of the olfactory recess. There is also variation in the amount of olfactory epithelium (OE) that is housed in the olfactory recess. Among New World leaf-nosed bats (family Phyllostomidae), species vary by over an order of magnitude in how much of their total OE lies within the OR. Does this variation relate to previously documented neuroanatomical proxies for olfactory reliance? Using data from 12 species of phyllostomid bats, we addressed the hypothesis that the amount of OE within the OR relates to a species' dependence on olfaction, as measured by two commonly used neuroanatomical metrics, the size of the olfactory bulb, and the number of glomeruli in the olfactory bulb, which are the first processing units within the olfactory signal cascade. We found that the percentage of OE within the OR does not relate to either measure of olfactory "ability." This suggests that olfactory reliance is not reflected in the size of the olfactory recess. We explore other roles that the olfactory recess may play.

Joined at the hip: linked characters and the problem of missing data in studies of disparity.

Paleontological investigations into morphological diversity, or disparity, are often confronted with large amounts of missing data. We illustrate how missing discrete data affect disparity using a novel simulation for removing data based on parameters from published datasets that contain both extinct and extant taxa. We develop an algorithm that assesses the distribution of missing characters in extinct taxa, and simulates data loss by applying that distribution to extant taxa. We term this technique "linkage." We compare differences in disparity metrics and ordination spaces produced by linkage and random character removal. When we incorporated linkage among characters, disparity metrics declined and ordination spaces shrank at a slower rate with increasing missing data, indicating that correlations among characters govern the sensitivity of disparity analysis. We also present and test a new disparity method that uses the linkage algorithm to correct for the bias caused by missing data. We equalized proportions of missing data among time bins before calculating disparity, and found that estimates of disparity changed when missing data were taken into account. By removing the bias of missing data, we can gain new insights into the morphological evolution of organisms and highlight the detrimental effects of missing data on disparity analysis.

The role of the olfactory recess in olfactory airflow.

The olfactory recess - a blind pocket at the back of the nasal airway - is thought to play an important role in mammalian olfaction by sequestering air outside of the main airstream, thus giving odorants time to re-circulate. Several studies have shown that species with large olfactory recesses tend to have a well-developed sense of smell. However, no study has investigated how the size of the olfactory recess relates to air circulation near the olfactory epithelium. Here we used a computer model of the nasal cavity from a bat (Carollia perspicillata) to test the hypothesis that a larger olfactory recess improves olfactory airflow. We predicted that during inhalation, models with an enlarged olfactory recess would have slower rates of flow through the olfactory region (i.e. the olfactory recess plus airspace around the olfactory epithelium), while during exhalation these models would have little to no flow through the olfactory recess. To test these predictions, we experimentally modified the size of the olfactory recess while holding the rest of the morphology constant. During inhalation, we found that an enlarged olfactory recess resulted in lower rates of flow in the olfactory region. Upon exhalation, air flowed through the olfactory recess at a lower rate in the model with an enlarged olfactory recess. Taken together, these results indicate that an enlarged olfactory recess improves olfactory airflow during both inhalation and exhalation. These findings add to our growing understanding of how the morphology of the nasal cavity may relate to function in this understudied region of the skull.

Roosting ecology and the evolution of pelage markings in bats.

Multiple lineages of bats have evolved striking facial and body pelage makings, including spots, stripes and countershading. Although researchers have hypothesized that these markings mainly evolved for crypsis, this idea has never been tested in a quantitative and comparative context. We present the first comparative study integrating data on roosting ecology (roost type and colony size) and pelage coloration patterns across bats, and explore the hypothesis that the evolution of bat pelage markings is associated with roosting ecologies that benefit from crypsis. We find that lineages that roost in the vegetation have evolved pelage markings, especially stripes and neck collars, which may function in crypsis through disruptive coloration and a type of countershading that might be unique to bats. We also demonstrate that lineages that live in larger colonies and are larger in size tend not to have pelage markings, possibly because of reduced predation pressures due to the predator dilution effect and a lower number of potential predators. Although social functions for pelage color patterns are also possible, our work provides strong support for the idea that roosting ecology has driven the evolution of pelage markings in bats.

Distribution of olfactory and nonolfactory surface area in the nasal fossa of Microcebus murinus: implications for microcomputed tomography and airflow studies.

The nasal fossa of most mammals exemplifies extreme skeletal complexity. Thin scrolls of bone (turbinals) that both elaborate surface area (SA) and subdivide nasal space are used as morphological proxies for olfactory and respiratory physiology. The present study offers additional details on the nasal fossa of the adult mouse lemur (Microcebus murinus), previously described by Smith and Rossie (Smith and Rossie [2008]; Anatomical Record 291:895-915). Additional, intervening histological sections of the specimen were used to map and quantify the distribution of olfactory and nonolfactory mucosa on the smaller turbinal of the frontal recess (FR; frontoturbinal) and those that occur between ethmoturbinals (ETs; interturbinals). A second adult Microcebus specimen, available as a dried skull, was scanned using microcomputed tomography (microCT) and reconstructed to infer the position of these turbinals within the nasal airway. Overall, turbinal bones comprise more than half of internal nasal SA. All ETs combined comprise about 30% of total nasal fossa SA, and contribute nearly half of all olfactory SA. Of these, the nasoturbinal (NT) is most completely covered with olfactory mucosa, whereas ET I is least covered with olfactory mucosa. The FR contributes significantly to total olfactory SA (ca. 20%). This recess and the single frontoturbinal within it lie in a more lateral pathway of airflow compared with interturbinals, which lie in more central zone just anterior to the olfactory recess of Microcebus. Variations in the turbinals and recesses that complicate central and paranasal in primates should be investigated further in light of zone-specific distributions of olfactory receptors (ORs) that differ between these regions in rodents.

Brief communication: histology and micro CT as methods for assessment of facial suture patency.

The extent of fusion in facial sutures has implications for topics ranging from biomechanics to phylogeny reconstruction. An unfortunate limitation of studying sutural fusion in skeletal specimens is that it is difficult to assess whether apparently patent sutures are in fact fused internally. Both histology and microcomputed tomography (CT) are potential tools for solving this, but relatively few studies have attempted to discern the limits of micro CT for visualization of microanatomical structures. We examined microanatomical aspects of facial sutures in adult cadaveric samples from captive bushbabies. Premaxillary and nasopremaxillary sutures were examined in serially sectioned snouts of four greater bushbabies (Otolemur garnettii) and four lesser bushbabies (Galago moholi). Sections containing sutures with osseous bridging were rated as "fused," and the presence or absence of grooves on the external side was recorded. One bushbaby was studied using micro CT prior to physical sectioning. O. garnettii and two of the G. moholi show multiple foci of fusion. Histological examination confirmed that sutural fusion is limited to the internal surface in numerous sections, resulting in an external notch. Such points of internal fusion could be clearly visualized in raw CT slices. The presence of such notches suggests that external examination can underestimate the degree of suture fusion. Thus, microanatomical evidence may be needed to fully assess biomechanical correlates and phylogenetic interpretations based on fusion of facial sutures. Our results also indicate micro CT may be a useful tool to obtain this evidence.