Anna Bastian

Nuclear introns outperform mitochondrial DNA in inter-specific phylogenetic reconstruction: Lessons from horseshoe bats (Rhinolophidae: Chiroptera).

Despite many studies illustrating the perils of utilising mitochondrial DNA in phylogenetic studies, it remains one of the most widely used genetic markers for this purpose. Over the last decade, nuclear introns have been proposed as alternative markers for phylogenetic reconstruction. However, the resolution capabilities of mtDNA and nuclear introns have rarely been quantified and compared. In the current study we generated a novel ∼5kb dataset comprising six nuclear introns and a mtDNA fragment. We assessed the relative resolution capabilities of the six intronic fragments with respect to each other, when used in various combinations together, and when compared to the traditionally used mtDNA. We focused on a major clade in the horseshoe bat family (Afro-Palaearctic clade; Rhinolophidae) as our case study. This old, widely distributed and speciose group contains a high level of conserved morphology. This morphological stasis renders the reconstruction of the phylogeny of this group with traditional morphological characters complex. We sampled multiple individuals per species to represent their geographic distributions as best as possible (122 individuals, 24 species, 68 localities). We reconstructed the species phylogeny using several complementary methods (partitioned Maximum Likelihood and Bayesian and Bayesian multispecies-coalescent) and made inferences based on consensus across these methods. We computed pairwise comparisons based on Robinson-Foulds tree distance metric between all Bayesian topologies generated (27,000) for every gene(s) and visualised the tree space using multidimensional scaling (MDS) plots. Using our supported species phylogeny we estimated the ancestral state of key traits of interest within this group, e.g. echolocation peak frequency which has been implicated in speciation. Our results revealed many potential cryptic species within this group, even in taxa where this was not suspected a priori and also found evidence for mtDNA introgression. We demonstrated that by using just two introns one can recover a better supported species tree than when using the mtDNA alone, despite the shorter overall length of the combined introns. Additionally, when combining any single intron with mtDNA, we showed that the result is highly similar to the mtDNA gene tree and far from the true species tree and therefore this approach should be avoided. We caution against the indiscriminate use of mtDNA in phylogenetic studies and advocate for pilot studies to select nuclear introns. The selection of marker type and number is a crucial step that is best based on critical examination of preliminary or previously published data. Based on our findings and previous publications, we recommend the following markers to recover phylogenetic relationships between recently diverged taxa (<20 My) in bats and other mammals: ACOX2, COPS7A, BGN, ROGDI and STAT5A.

Phenotypic convergence in genetically distinct lineages of a Rhinolophus species complex (Mammalia, Chiroptera).

Phenotypes of distantly related species may converge through adaptation to similar habitats and/or because they share biological constraints that limit the phenotypic variants produced. A common theme in bats is the sympatric occurrence of cryptic species that are convergent in morphology but divergent in echolocation frequency, suggesting that echolocation may facilitate niche partitioning, reducing competition. If so, allopatric populations freed from competition, could converge in both morphology and echolocation provided they occupy similar niches or share biological constraints. We investigated the evolutionary history of a widely distributed African horseshoe bat, Rhinolophus darlingi, in the context of phenotypic convergence. We used phylogenetic inference to identify and date lineage divergence together with phenotypic comparisons and ecological niche modelling to identify morphological and geographical correlates of those lineages. Our results indicate that R. darlingi is paraphyletic, the eastern and western parts of its distribution forming two distinct non-sister lineages that diverged ~9.7 Mya. We retain R. darlingi for the eastern lineage and argue that the western lineage, currently the sub-species R. d. damarensis, should be elevated to full species status. R. damarensis comprises two lineages that diverged ~5 Mya. Our findings concur with patterns of divergence of other co-distributed taxa which are associated with increased regional aridification between 7-5 Mya suggesting possible vicariant evolution. The morphology and echolocation calls of R. darlingi and R. damarensis are convergent despite occupying different biomes. This suggests that adaptation to similar habitats is not responsible for the convergence. Furthermore, R. darlingi forms part of a clade comprising species that are bigger and echolocate at lower frequencies than R. darlingi, suggesting that biological constraints are unlikely to have influenced the convergence. Instead, the striking similarity in morphology and sensory biology are probably the result of neutral evolutionary processes, resulting in the independent evolution of similar phenotypes.

The influence of feeding on the evolution of sensory signals: a comparative test of an evolutionary trade-off between masticatory and sensory functions of skulls in southern African horseshoe bats (Rhinolophidae).

The skulls of animals have to perform many functions. Optimization for one function may mean another function is less optimized, resulting in evolutionary trade‐offs. Here, we investigate whether a trade‐off exists between the masticatory and sensory functions of animal skulls using echolocating bats as model species. Several species of rhinolophid bats deviate from the allometric relationship between body size and echolocation frequency. Such deviation may be the result of selection for increased bite force, resulting in a decrease in snout length which could in turn lead to higher echolocation frequencies. If so, there should be a positive relationship between bite force and echolocation frequency. We investigated this relationship in several species of southern African rhinolophids using phylogenetically informed analyses of the allometry of their bite force and echolocation frequency and of the three‐dimensional shape of their skulls. As predicted, echolocation frequency was positively correlated with bite force, suggesting that its evolution is influenced by a trade‐off between the masticatory and sensory functions of the skull. In support of this, variation in skull shape was explained by both echolocation frequency (80%) and bite force (20%). Furthermore, it appears that selection has acted on the nasal capsules, which have a frequency‐specific impedance matching function during vocalization. There was a negative correlation between echolocation frequency and capsule volume across species. Optimization of the masticatory function of the skull may have been achieved through changes in the shape of the mandible and associated musculature, elements not considered in this study.

To seek or speak? Dual function of an acoustic signal limits its versatility in communication

The perception of different attributes of conspecifics is an integral part of intraspecific communication. It can facilitate the recognition of interaction partners or the assessment of potential mates. Acoustic signals can encode fine-scaled information through the interplay of acoustic variability and specificity. A reliable vocal signature is both unique within a class and variable between classes. Therefore, acoustic complexity might be associated with the number of classes to be discriminated. We investigated the assumption that limitations to signal design may affect the communicative functionality of a signal. To do so, we chose a signal with potentially dual functionality which may therefore display such limitations. In bats, echolocation is used primarily for foraging and orientation but there is increasing support for its additional role in communication. An acoustic analysis of echolocation pulses of the bat Rhinolophus clivosus confirmed sex and individual vocal signatures in echolocation pulses. A habituation–dishabituation playback experiment suggested that bats perceived these signatures because listening bats clearly discriminated between the sexes (two classes) and between individuals (representatives of a multiclass category), although to different degrees. The simple acoustic structure of these vocalizations provides sufficient specificity for sex discrimination but has limitations for individual discrimination because pulse parameters of individuals increasingly overlapped with increasing group size. We conclude that selection for the primary function of echolocation restricts the acoustic space available for communication. However, we frequently observed echolocation pulses with conspicuous structural modifications. Statistical analyses revealed that these vocalizations yielded increased individual distinctiveness. Such added systematic variation may indicate a communicative function and perhaps a signalling intent of the emitter, although the latter has yet to be tested. The findings suggest that the required specificity for effective communication could be obtained through modification of echolocation variants when adaptations for orientation and foraging constrain the evolution of complex communication signatures.

Predator–Prey Interactions: Co-evolution between Bats and Their Prey

The precision of bat echolocation has continuously fascinated scientists since the eighteenth century. It enables bats to find prey as small as 0.05–0.2 mm in complete darkness and even to determine the kind of prey from the echo of its own call reflected off the prey. Besides active acoustic detection, some bat species also use passive listening for sounds generated by the prey itself, for example frog mating calls or the rustling sounds of crawling insects. Bats are therefore well adapted for hunting and can eat up to 25% of their body weight in insects each night, exerting much selection pressure on their prey. In response to this pressure, bat prey has evolved a range of defences both auditory and non-auditory. Frogs, for example, can listen for bat echolocation and cease their calling to female frogs. The evolution of audition in insects enables them to detect bats before the bats detect them, allowing insects to take evasive action. This interaction between predator and prey may represent a system of co-evolution. Here, we define co-evolution as a process in which the evolution of traits in the predator is in direct response to the evolution of traits in the prey which in turn evolved in direct response to the traits of the predator and so on. Co-evolution is thus an iterative process of both reciprocal (each lineage responds to the other) and specific (evolution of a trait in one lineage responds specifically to a trait in the other) adaptations.

Synthesis and Future Research

Insects have obviously responded to bat predation by evolving a range of defences that are specific to bat predation. However, apart from rare examples of stealth echolocation, only one of which appears to meet the criteria of co-evolution; there is no indication that bats have responded reciprocally and specifically to the defences of their prey. However, with the advent of new technologies, more examples of co-evolved stealth echolocation may be uncovered. This requires an increase in both the geographic and taxonomic coverage of bat–insect interactions. This would include the auditory thresholds of insects and the diets of bats at a level that would allow the determination of whether bats are eating tympanate or tympanate prey. Systems that are likely to yield examples of co-evolution would include those that involve some kind of trade-off, for example, low intensity and/or low-frequency echolocation calls in bats. Bat species in the family Molossidae would be good candidates for this. It is imperative that investigations of co-evolution between bats and their prey are done within a phylogenetic framework, so that the ancestral character states of both groups can be identified and the timing of emergence of suspected co-evolving traits can be determined. For this, we need dated phylogenies with excellent taxonomic coverage for suspected co-evolving lineages.

Bat Echolocation: Adaptations for Prey Detection and Capture

Bats have evolved a plethora of adaptations in response to the challenges of their diverse habitats and the physics of sound propagation. Such adaptations can confound investigations of adaptations that arise in response to prey. Here, we review the adaptations in the echolocation and foraging behaviour of bats. Bats use a variety of foraging modes including aerial hawking and gleaning. The main challenge to bats echolocating in clutter is increased resolution to detect small objects, be they insects or twigs, and overcoming the masking effects that result from the overlap of echoes from prey and the background. Low-duty-cycle echolocating bats that aerial hawk in clutter have evolved short, frequency-modulated calls with high bandwidth that increase resolution and minimizes masking effects. Bats that glean prey from the vegetation have similar adaptations but in addition use passive listening and/or 3-D flight to ensonify substrate-bound prey from different directions. High-duty-cycle echolocating bats have evolved Doppler-shift compensation which allows the detection of acoustic glints from the flapping wings of insects. Bats that aerial hawk in the open have to search large volumes of space efficiently and use narrowband, low-frequency (for decrease atmospheric attenuation) echolocation calls that maximize detection distance. The wings and echolocation of bats form an adaptive complex. Bats that forage in clutter where distances are short have short broad wings for slow, manoeuvrable flight. Bats that hunt in the open where detection distances are long have long narrow wings for fast, agile flight.

It’s not all about the Soprano: Rhinolophid bats use multiple acoustic components in echolocation pulses to discriminate between conspecifics and heterospecifics

Acoustic communication plays a pivotal role in conspecific recognition in numerous animal taxa. Vocalizations must therefore have discrete acoustic signatures to facilitate intra-specific communication and to avoid misidentification. Here we investigate the potential role of echolocation in communication in horseshoe bats. Although it has been demonstrated that echolocation can be used to discriminate among con- and hetero-specifics, the specific acoustic cues used in discrimination are still relatively unknown. Furthermore, the Acoustic Communication Hypothesis proposes that in multispecies assemblages, in which echolocation frequencies are likely to overlap, bats partition acoustic space along several dimensions so that each species occupies a discrete communication domain. Thus, multiple echolocation variables may be used in discrimination. The objective of this study was to investigate the potential of various echolocation variables to function as discriminatory cues in echolocation-based species discrimination. Using habituation–dishabituation playback experiments, we firstly tested the ability of Rhinolophus clivosus to discriminate between echolocation pulses of heterospecifics with either discrete or overlapping frequencies. Secondly, to determine whether R. clivosus could use echolocation variables other than frequency, we investigated its ability to discriminate among echolocation pulses differing in only one manipulated parameter. These test variables were identified by their contribution to the dissimilarity among pulses. Our results suggest that R. clivosus could discriminate readily between species using echolocation pulses with discrete frequencies. When frequencies overlapped, the ability of bats to discriminate was dependant on additional acoustic variables that defined the acoustic space occupied by the test signal. These additional acoustic variables included, but may not be restricted to, sweep rate of the FM and duty cycle. Thus, when echolocation pulses share a similar acoustic domain, bats use several cues to reliably discriminate among heterospecifics.

Aerial Warfare: Have Bats and Moths Co-evolved?

The interaction between bats and moths has been cited as an example of co-evolution. However, this is dependent on how well the diverse behavioural responses to prey detection and predator avoidance by bats and moths, respectively, satisfy the two major characteristics of co-evolution, specificity, and reciprocity. In general, co-evolution is an interaction between two species. Therefore, an interaction between multiple species of two orders as in the case of insects and bats may be a case of diffuse co-evolution. There is much evidence that moth anti-bat defences have evolved in direct response to bats. Such evidence includes the evolutionary origin of moth audition after bat echolocation, the close association between between moth hearing sensitivity and bat echolocation frequencies, the degeneration of hearing in moths that are no longer exposed to bat predation and the production of ultrasonic clicks by moths in direct response to bat echolocation. In contrast, evidence for reciprocity and specificity in the evolution of bat traits is confounded by the fact that these traits could also have evolved as adaptation for particular habitats and tasks. However, these requirements might be met by stealth echolocation, especially where these involve evolutionary trade-offs. For example, some bats use calls of low-intensity or low-frequency, sacrificing detection distance and the ability to detect small insects, respectively, that allow them to detect the moths before the moths detect them. Presumably, the decrease in detection distanced and the detectability is offset by an increased ability to catch eared/large moths.

Passive and Active Acoustic Defences of Prey Against Bat Predation

Perhaps the most effective and sophisticated defence evolved by insects in the context of bat predation is audition. Here, we review the evolution, ecology, and physiology of insect audition in the context of bat predation. Ears have evolved independently multiple times in insects and occur in almost half of extant moth species. In moths, these ears were used secondarily for intraspecific communication but in other orders (e.g. some crickets) the evolution of ears preceded bats and probably arose primarily for communication, the anti-bat function was a secondary. Insect ears are used as a primary defence mechanism to initiate a variety of acoustic startle responses involving the cessation of advertisement behaviours in response to bat echolocation. They are also used as a secondary defence mechanism to execute evasive manoeuvres after they have been detected by a bat. This requires that the insect is able to localize the bat. Localization of the predator by insect ears is achieved through difference in intensities amongst signals arriving at different ears. Bat predation has also influenced the advertisement behaviours of insects. For example, it is thought to be responsible for the evolution of tremulation and the complex songs in some insects. Finally, we provide a detailed review of the jamming, aposematic, and startle functions of moth clicks. A unique secondary defence in moths is the use of ultrasonic clicks to directly affect the foraging success of the bat. We discuss the three hypotheses advanced for the function of moth clicks—jamming, aposematism, and startle.