Habitat and respiratory strategy effects on hypoxia performance in anuran tadpoles.

A critical component of animal conservation in a changing world is an understanding of the physiological resilience of animals to different conditions. In many aquatic animals, hypoxia (low environmental oxygen levels) is a regular occurrence, but the likelihood and severity of hypoxia varies across habitats. Fast-flowing, stream-like habitats are never hypoxic, so long as flow is maintained. Do animals from such habitats retain the capacity to survive hypoxic conditions? We use aquatic frog tadpoles to test the effects of natural habitat on performance in hypoxia in an experimental framework, finding that stream-living tadpoles have reduced performance in hypoxia. Tadpoles also vary in lung presence, with some species able to breathe air during hypoxia. We found that among lunged tadpoles, air-breathing rates increase in hypoxia in pond-living species but not stream-living species. Lung presence was also found to influence hypoxia performance, as lungless, stream-living tadpoles were found to be especially vulnerable to hypoxia, while pond-living, lungless tadpoles appeared largely resilient to hypoxia. We consider the ramifications of our findings on conservation outlooks and strategies for frogs and their tadpoles, suggesting that stream-living tadpoles, and especially lungless, stream-living tadpoles, may be particularly at risk to factors that reduce stream flow. Thus, a primary goal for conservation and management of species with stream-living tadpoles should be the maintenance of year-round streamflow, which oxygenates waters and prevents hypoxia.

Convergent Anuran Middle Ear Loss Lacks a Universal, Adaptive Explanation.

INTRODUCTION: Shared selection pressures often explain convergent trait loss, yet anurans (frogs and toads) have lost their middle ears at least 38 times with no obvious shared selection pressures unifying "earless" taxa. Anuran tympanic middle ear loss is especially perplexing because acoustic communication is dominant within Anura and tympanic middle ears enhance airborne hearing in most tetrapods. METHODS: Here, we use phylogenetic comparative methods to examine whether particular geographic ranges, microhabitats, activity patterns, or aspects of acoustic communication are associated with anuran tympanic middle ear loss. RESULTS: Although we find some differences between the geographic ranges of eared and earless species on average, there is plenty of overlap between the geographic distributions of eared and earless species. Additionally, we find a higher prevalence of diurnality in earless species, but not all earless species are diurnal. We find no universal adaptive explanation for the many instances of anuran tympanic middle ear loss. CONCLUSION: The puzzling lack of universally shared selection pressures among earless species motivates discussion of alternative hypotheses, including genetic or developmental constraints, and the possibility that tympanic middle ear loss is maladaptive.

Developmental Plasticity in Anurans: Meta-analysis Reveals Effects of Larval Environments on Size at Metamorphosis And Timing of Metamorphosis.

Many anuran amphibians (frogs and toads) rely on aquatic habitats during their larval stage. The quality of this environment can significantly impact lifetime fitness and population dynamics. Over 450 studies have been published on environmental impacts on anuran developmental plasticity, yet we lack a synthesis of these effects across different environments. We conducted a meta-analysis and used a comparative approach to understand whether developmental plasticity in response to different larval environments produces predictable changes in metamorphic phenotypes. We analyzed data from 124 studies spanning 80 anuran species and six larval environments and showed that intraspecific variation in mass at metamorphosis and the duration of the larval period is partly explained by the type of environment experienced during the larval period. Changes in larval environments tended to reduce mass at metamorphosis relative to control conditions, with the degree of change depending on the identity and severity of environmental change. Higher temperatures and lower water levels shortened the duration of the larval period, whereas less food and higher densities increased the duration of the larval period. Phylogenetic relationships among species were not associated with interspecific variation in mass at metamorphosis plasticity or duration of the larval period plasticity. Our results provide a foundation for future studies on developmental plasticity, especially in response to global changes. This study provides motivation for additional work that links developmental plasticity with fitness consequences within and across life stages, as well as how the outcomes described here are altered in compounding environments.

We conducted a meta-analysis to identify how six different environments affect mass at metamorphosis and time to metamorphosis in larval anurans. We find that some, but not all, environmental conditions triggered predictable changes in size and timing of metamorphosis, and phylogenetic relatedness rarely explains developmental plasticity variation among species.

The continued importance of comparative auditory research to modern scientific discovery.

A rich history of comparative research in the auditory field has afforded a synthetic view of sound information processing by ears and brains. Some organisms have proven to be powerful models for human hearing due to fundamental similarities (e.g., well-matched hearing ranges), while others feature intriguing differences (e.g., atympanic ears) that invite further study. Work across diverse "non-traditional" organisms, from small mammals to avians to amphibians and beyond, continues to propel auditory science forward, netting a variety of biomedical and technological advances along the way. In this brief review, limited primarily to tetrapod vertebrates, we discuss the continued importance of comparative studies in hearing research from the periphery to central nervous system with a focus on outstanding questions such as mechanisms for sound capture, peripheral and central processing of directional/spatial information, and non-canonical auditory processing, including efferent and hormonal effects.

Ocular lens morphology is influenced by ecology and metamorphosis in frogs and toads.

The shape and relative size of an ocular lens affect the focal length of the eye, with consequences for visual acuity and sensitivity. Lenses are typically spherical in aquatic animals with camera-type eyes and axially flattened in terrestrial species to facilitate vision in optical media with different refractive indices. Frogs and toads (Amphibia: Anura) are ecologically diverse, with many species shifting from aquatic to terrestrial ecologies during metamorphosis. We quantified lens shape and relative size using 179 micro X-ray computed tomography scans of 126 biphasic anuran species and tested for correlations with life stage, environmental transitions, adult habits and adult activity patterns. Across broad phylogenetic diversity, tadpole lenses are more spherical than those of adults. Biphasic species with aquatic larvae and terrestrial adults typically undergo ontogenetic changes in lens shape, whereas species that remain aquatic as adults tend to retain more spherical lenses after metamorphosis. Further, adult lens shape is influenced by adult habit; notably, fossorial adults tend to retain spherical lenses following metamorphosis. Finally, lens size relative to eye size is smaller in aquatic and semiaquatic species than other adult ecologies. Our study demonstrates how ecology shapes visual systems, and the power of non-invasive imaging of museum specimens for studying sensory evolution.

The mechanics of air breathing in African clawed frog tadpoles, Xenopus laevis (Anura: Pipidae).

Frog larvae (tadpoles) undergo many physiological, morphological and behavioral transformations throughout development before metamorphosing into their adult form. The surface tension of water prevents small tadpoles from breaching the surface to breathe air (including those of Xenopus laevis), forcing them to acquire air using a form of breathing called bubble sucking. With growth, tadpoles typically make a behavioral/biomechanical transition from bubble sucking to breaching. Xenopus laevis tadpoles have also been shown to transition physiologically from conforming passively to ambient oxygen levels to actively regulating their blood oxygen. However, it is unknown whether these mechanical and physiological breathing transitions are temporally or functionally linked, or how both transitions relate to lung maturation and gas exchange competency. If these transitions are linked, it could mean that one biomechanical breathing mode (breaching) is more physiologically proficient at acquiring gaseous oxygen than the other. Here, we describe the mechanics and development of air breathing and the ontogeny of lung morphology in X. laevis throughout the larval stage and examine our findings considering previous physiological work. We found that the transitions from bubble sucking to breaching and from oxygen conforming to oxygen regulation co-occur in X. laevis tadpoles at the same larval stage (Nieuwkoop-Faber stages 53-56 and 54-57, respectively), but that the lungs do not increase significantly in vascularization until metamorphosis, suggesting that lung maturation, alone, is not sufficient to account for increased pulmonary capacity earlier in development. Although breach breathing may confer a respiratory advantage, we remain unaware of a mechanistic explanation to account for this possibility. At present, the transition from bubble sucking to breaching appears simply to be a consequence of growth. Finally, we consider our results in the context of comparative air-breathing mechanics across vertebrates.

Peripheral Auditory System Divergence Does Not Explain Species Differences in Call Preference.

Receiver sensory systems have long been cited as an important source of variation in mate preferences that could lead to signal diversification and behavioral isolation between lineages, with a general assumption that animals prefer the most conspicuous signals. The matched filter hypothesis posits that tuning of the frog peripheral auditory system matches dominant frequencies in advertisement calls used to attract mates. However, little work has characterized species with frequency modulation in their calls. In this study, we extend prior work characterizing the lack of correlated evolution between auditory tuning and spectral properties of male calls in Engystomops (=Physalaemus) frogs. We analyze auditory sensitivity of three cryptic species that differ consistently in female mate preferences for calls of different frequencies. The audiograms of these species differ, but the frequency at which the frog is maximally sensitive is not the most relevant difference in tuning of the auditory periphery. Rather, we identify species differences in overall sensitivity within specific frequency ranges, and we model the effects of these sensitivity differences on neural responses to natural calls. We find a general mismatch between auditory brainstem responses and behavioral preferences of these taxa and rule out the matched filter hypothesis as explaining species differences in male calls and mate preferences in this group.

Whispers from vestigial nubbins: Arrested development provokes trait loss in toads.

Despite the use of acoustic communication, many species of toads (family Bufonidae) have lost parts of the tympanic middle ear, representing at least 12 independent evolutionary occurrences of trait loss. The comparative development of the tympanic middle ear in toads is poorly understood. Here, we compared middle ear development among two pairs of closely related toad species in the genera Atelopus and Rhinella that have (eared) or lack (earless) middle ear structures. We bred toads in Peru and Ecuador, preserved developmental series from tadpoles to juveniles, and examined ontogenetic timing and volume of the otic capsule, oval window, operculum, opercularis muscle, columella (stapes), and extracolumella in three-dimensional histological reconstructions. All species had similar ontogenesis of the otic capsule, oval window, operculum, and opercularis muscle. Moreover, cell clusters of primordial columella in the oval window appeared just before metamorphosis in both eared and earless lineages. However, in earless lineages, the cell clusters either remained as small nubbins or cell buds in the location of the columella footplate within the oval window or disappeared by juvenile and adult stages. Thus, columella growth began around metamorphosis in all species but was truncated and/or degenerated after metamorphosis in earless species, leaving earless adults with morphology typical of metamorphic anurans. Shifts in the timing or expression of biochemical pathways that regulate the extension or differentiation of the columella after metamorphosis may be the developmental mechanism underlying convergent trait loss among toad lineages.

Two-hundred million years of anuran body-size evolution in relation to geography, ecology and life history.

Surprisingly, little is known about body-size evolution within the most diverse amphibian order, anurans (frogs and toads), despite known effects of body size on the physiological, ecological and life-history traits of animals more generally. Here, we examined anuran body-size evolution among 2,434 species with over 200 million years of shared evolutionary history. We found clade-specific evolutionary shifts to new body-size optima along with numerous independent transitions to gigantic and miniature body sizes, despite the upper limits of anuran body size remaining quite consistent throughout the fossil record. We found a weak, positive correlation between a species' body size and maximum latitude and elevation, including a dearth of small species at higher elevations and broader latitudinal and elevational ranges in larger anurans. Although we found modest differences in mean anuran body size among microhabitats, there was extensive overlap in the range of body sizes across microhabitats. Finally, we found that larger anurans are more likely to consume vertebrate prey than smaller anurans are and that species with a free-swimming larval phase during development are larger on average than those in which development into a froglet occurs within the egg. Overall, anuran body size does not conform to geographic and ecological patterns observed in other tetrapods but is perhaps more notable for variation in body size within geographic regions, ecologies and life histories. Here, we document this variation and propose target clades for detailed studies aimed at disentangling how and why variation in body size was generated and is maintained in anurans.

Evolution of the Unique Anuran Pelvic and Hind limb Skeleton in Relation to Microhabitat, Locomotor Mode, and Jump Performance.

Anurans (frogs and toads) have a unique pelvic and hind limb skeleton among tetrapods. Although their distinct body plan is primarily associated with saltation, anuran species vary in their primary locomotor mode (e.g., walkers, hoppers, jumpers, and swimmers) and are found in a wide array of microhabitats (e.g., burrowing, terrestrial, arboreal, and aquatic) with varying functional demands. Given their largely conserved body plan, morphological adaptation to these diverse niches likely results from more fine-scale morphological change. Our study determines how shape differences in Anura's unique pelvic and hind limb skeletal structures vary with microhabitat, locomotor mode, and jumping ability. Using microCT scans of preserved specimens from museum collections, we added 3D landmarks to the pelvic and hind limb skeleton of 230 anuran species. In addition, we compiled microhabitat and locomotor data from the literature for these species that span 52 of the 55 families of frogs and ∼210 million years of anuran evolution. Using this robust dataset, we examine the relationship between pelvic and hind limb morphology and phylogenetic history, allometry, microhabitat, and locomotor mode. We find pelvic and hind limb changes associated with shifts in microhabitat ("ecomorphs") and locomotor mode ("locomorphs") and directly relate those morphological changes to the jumping ability of individual species. We also reveal how individual bones vary in evolutionary rate and their association with phylogeny, body size, microhabitat, and locomotor mode. Our findings uncover previously undocumented morphological variation related to anuran ecological and locomotor diversification and link that variation to differences in jumping ability among species.

Anuran limbs reflect microhabitat and distal, later-developing bones are more evolutionarily labile.

Tetrapod limbs have been used as a model system to investigate how selective pressures and constraints shape morphological evolution. Anurans have had many independent transitions to various microhabitats, allowing us to dissect how these factors influence limb morphology. Furthermore, anurans provide a unique system to test the generality of developmental constraints proposed in mammals, namely that later-developing limb bones are under less constraint and show more variation. We used microcomputed tomography scans of 236 species from 52 of 55 families, geometric morphometrics, and modern phylogenetic comparative methods to examine how limb bones are related to microhabitat, phylogeny, allometry, and developmental timing. Although there was significant phylogenetic signal, anuran limb shape showed a relationship with microhabitat and to a lesser extent, body size. We found that distal bones had higher evolutionary rates than proximal bones, providing evidence that developmental constraints are reduced in later-developing bones. Distal bones also showed increased selection related to allometry and microhabitat, providing an additional explanation for higher evolutionary rates. By looking at the evolution of limb shape across a diverse clade, we demonstrated that multiple factors have shaped anuran limbs and that greater evolutionary lability in later-developing limb bones is likely a general trend among tetrapods.

Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Scheele et al (Reports, 29 March 2019, p. 1459) bring needed attention to the effects of amphibian infectious disease. However, the data and methods implicating the disease chytridiomycosis in 501 amphibian species declines are deficient. Which species are affected, and how many, remains a critical unanswered question. Amphibians are imperiled; protective actions require public support and robust science.

Invasive vegetation affects amphibian skin microbiota and body condition.

Invasive plants are major drivers of habitat modification and the scale of their impact is increasing globally as anthropogenic activities facilitate their spread. In California, an invasive plant genus of great concern is Eucalyptus. Eucalyptus leaves can alter soil chemistry and negatively affect underground macro- and microbial communities. Amphibians serve as excellent models to evaluate the effect of Eucalyptus invasion on ground-dwelling species as they predate on soil arthropods and incorporate soil microbes into their microbiotas. The skin microbiota is particularly important to amphibian health, suggesting that invasive plant species could ultimately affect amphibian populations. To investigate the potential for invasive vegetation to induce changes in microbial communities, we sampled microbial communities in the soil and on the skin of local amphibians. Specifically, we compared Batrachoseps attenuatus skin microbiomes in both Eucalyptus globulus (Myrtaceae) and native Quercus agriflolia (Fagaceae) dominated forests in the San Francisco Bay Area. We determined whether changes in microbial diversity and composition in both soil and Batrachoseps attenuatus skin were associated with dominant vegetation type. To evaluate animal health across vegetation types, we compared Batrachoseps attenuatus body condition and the presence/absence of the amphibian skin pathogen Batrachochytrium dendrobatidis. We found that Eucalyptus invasion had no measurable effect on soil microbial community diversity and a relatively small effect (compared to the effect of site identity) on community structure in the microhabitats sampled. In contrast, our results show that Batrachoseps attenuatus skin microbiota diversity was greater in Quercus dominated habitats. One amplicon sequence variant identified in the family Chlamydiaceae was observed in higher relative abundance among salamanders sampled in Eucalyptus dominated habitats. We also observed that Batrachoseps attenuatus body condition was higher in Quercus dominated habitats. Incidence of Batrachochytrium dendrobatidis across all individuals was very low (only one Batrachochytrium dendrobatidis positive individual). The effect on body condition demonstrates that although Eucalyptus may not always decrease amphibian abundance or diversity, it can potentially have cryptic negative effects. Our findings prompt further work to determine the mechanisms that lead to changes in the health and microbiome of native species post-plant invasion.

Larger Genomes Linked to Slower Development and Loss of Late-Developing Traits.

Genome size varies widely among organisms and is known to affect vertebrate development, morphology, and physiology. In amphibians, genome size is hypothesized to contribute to loss of late-forming structures, although this hypothesis has mainly been discussed in salamanders. Here we estimated genome size for 22 anuran species and combined this novel data set with existing genome size data for an additional 234 anuran species to determine whether larger genome size is associated with loss of a late-forming anuran sensory structure, the tympanic middle ear. We established that genome size is negatively correlated with development rate across 90 anuran species and found that genome size evolution is correlated with evolutionary loss of the middle ear bone (columella) among 241 species (224 eared and 17 earless). We further tested whether the development of the tympanic middle ear could be constrained by large cell sizes and small body sizes during key stages of tympanic middle ear development (metamorphosis). Together, our evidence suggests that larger genomes, slower development rate, and smaller body sizes at metamorphosis may contribute to the loss of the anuran tympanic middle ear. We conclude that increases in anuran genome size, although less drastic than those in salamanders, may affect development of late-forming traits.

Prolonged middle ear development in Rhinella horribilis.

Despite the benefit of the tympanic middle ear to airborne hearing sensitivity, anurans range in how soon they develop functional middle ears after transitioning to life on land. Previous evidence suggested that bufonids had particularly slow middle ear developmental rates, but precise timelines have not yet been published for this family. Here, we provide the first age-verified middle ear development timeline for a true toad species (family Bufonidae). We find that although middle ear development begins during metamorphosis in Rhinella horribilis, the middle ear remains incomplete 15 weeks after the transition from aquatic tadpole to land-dwelling toadlet. Using this new middle ear timeline, we discuss commonalities and differences in middle ear development among bufonids, as well as among Anura.

Sensitive high-frequency hearing in earless and partially eared harlequin frogs (Atelopus).

Harlequin frogs, genus Atelopus, communicate at high frequencies despite most species lacking a complete tympanic middle ear that facilitates high-frequency hearing in most anurans and other tetrapods. Here, we tested whether Atelopus are better at sensing high-frequency acoustic sound compared with other eared and earless species in the Bufonidae family, determined whether middle ear variation within Atelopus affects hearing sensitivity and tested potential hearing mechanisms in Atelopus We determined that at high frequencies (2000-4000 Hz), Atelopus are 10-34 dB more sensitive than other earless bufonids but are relatively insensitive to mid-range frequencies (900-1500 Hz) compared with eared bufonids. Hearing among Atelopus species is fairly consistent, evidence that the partial middle ears present in a subset of Atelopus species do not convey a substantial hearing advantage. We further demonstrate that Atelopus hearing is probably not facilitated by vibration of the skin overlying the normal tympanic membrane region or the body lung wall, leaving the extratympanic hearing pathways in Atelopus enigmatic. Together, these results show Atelopus have sensitive high-frequency hearing without the aid of a tympanic middle ear and prompt further study of extratympanic hearing mechanisms in anurans.

Trait independence primes convergent trait loss.

The repeated, independent evolution of traits (convergent evolution) is often attributed to shared environmental selection pressures. However, developmental dependencies among traits can limit the phenotypic variation available to selection and bias evolutionary outcomes. Here, we determine how changes in developmentally correlated traits may impact convergent loss of the tympanic middle ear, a highly labile trait within toads that currently lack adaptive explanation. The middle ear's lability could reflect evolutionary trade-offs with other skull features under selection, or the middle ear may evolve independently of the rest of the skull, allowing it to be modified by active or passive processes without pleiotropic trade-offs with other skull features. We compare the skulls of 55 species (39 eared, 16 earless) within the family Bufonidae, spanning six hypothesized independent middle ear transitions. We test whether shared or lineage-specific changes in skull shape distinguish earless species from eared species and whether earless skulls lack other late-forming skull bones. We find no evidence for pleiotropic trade-offs between the middle ear and other skull structures. Instead, middle ear loss in anurans may provide a rare example of developmental independence contributing to evolutionary lability of a sensory system.

Earless toads sense low frequencies but miss the high notes.

Sensory losses or reductions are frequently attributed to relaxed selection. However, anuran species have lost tympanic middle ears many times, despite anurans' use of acoustic communication and the benefit of middle ears for hearing airborne sound. Here we determine whether pre-existing alternative sensory pathways enable anurans lacking tympanic middle ears (termed earless anurans) to hear airborne sound as well as eared species or to better sense vibrations in the environment. We used auditory brainstem recordings to compare hearing and vibrational sensitivity among 10 species (six eared, four earless) within the Neotropical true toad family (Bufonidae). We found that species lacking middle ears are less sensitive to high-frequency sounds, however, low-frequency hearing and vibrational sensitivity are equivalent between eared and earless species. Furthermore, extratympanic hearing sensitivity varies among earless species, highlighting potential species differences in extratympanic hearing mechanisms. We argue that ancestral bufonids may have sufficient extratympanic hearing and vibrational sensitivity such that earless lineages tolerated the loss of high frequency hearing sensitivity by adopting species-specific behavioural strategies to detect conspecifics, predators and prey.

The complex evolutionary history of the tympanic middle ear in frogs and toads (Anura).

Most anurans possess a tympanic middle ear (TME) that transmits sound waves to the inner ear; however, numerous species lack some or all TME components. To understand the evolution of these structures, we undertook a comprehensive assessment of their occurrence across anurans and performed ancestral character state reconstructions. Our analysis indicates that the TME was completely lost at least 38 independent times in Anura. The inferred evolutionary history of the TME is exceptionally complex in true toads (Bufonidae), where it was lost in the most recent common ancestor, preceding a radiation of >150 earless species. Following that initial loss, independent regains of some or all TME structures were inferred within two minor clades and in a radiation of >400 species. The reappearance of the TME in the latter clade was followed by at least 10 losses of the entire TME. The many losses and gains of the TME in anurans is unparalleled among tetrapods. Our results show that anurans, and especially bufonid toads, are an excellent model to study the behavioural correlates of earlessness, extratympanic sound pathways, and the genetic and developmental mechanisms that underlie the morphogenesis of TME structures.

Better late than never: effective air-borne hearing of toads delayed by late maturation of the tympanic middle ear structures.

Most vertebrates have evolved a tympanic middle ear that enables effective hearing of airborne sound on land. Although inner ears develop during the tadpole stages of toads, tympanic middle ear structures are not complete until months after metamorphosis, potentially limiting the sensitivity of post-metamorphic juveniles to sounds in their environment. We tested the hearing of five species of toads to determine how delayed ear development impairs airborne auditory sensitivity. We performed auditory brainstem recordings to test the hearing of the toads and used micro-computed tomography and histology to relate the development of ear structures to hearing ability. We found a large (14-27 dB) increase in hearing sensitivity from 900 to 2500 Hz over the course of ear development. Thickening of the tympanic annulus cartilage and full ossification of the middle ear bone are associated with increased hearing ability in the final stages of ear maturation. Thus, juvenile toads are at a hearing disadvantage, at least in the high-frequency range, throughout much of their development, because late-forming ear elements are critical to middle ear function at these frequencies. We discuss the potential fitness consequences of late hearing development, although research directly addressing selective pressures on hearing sensitivity across ontogeny is lacking. Given that most vertebrate sensory systems function very early in life, toad tympanic hearing may be a sensory development anomaly.