Tadpoles rely on mechanosensory stimuli for communication when visual capabilities are poor.

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species.

Canopy coverage, light, and moisture affect thermoregulatory trade-offs in an amphibian breeding habitat.

When amphibians thermoregulate, they face a fundamental trade-off between the ability to maintain activity and an increased rate of dehydration at higher temperatures. Canopy coverage affects both the thermal and hydric conditions of the environment and can therefore influence amphibian thermoregulation. Frogs require proper conditions to thermoregulate to successfully grow, survive, and reproduce. But while we know how canopy and environmental variables typically affect operative temperature, less is known about effects on amphibian water loss rates. In this study, we measure the effect of canopy coverage on the conditions available for thermoregulation at a breeding pond of the California red-legged frog, Rana draytonii. We use agar frog models to estimate the thermal and hydric capacities frogs would experience in locations with different canopy coverage and microhabitats. At each site, we deployed models under four microhabitat treatments: wet/sun, wet/shade, dry/sun, and dry/shade. We modeled how environmental variables affected operative temperature and evaporative water loss from agar frogs. We found positive effects of air temperature, the sun treatment, and reduced canopy cover on operative temperature, and negative direct or indirect effects of these variables on evaporative water loss, consistent with the hypothesized trade-off between thermoregulatory behavior to increase temperature and the increased desiccation risk due to higher water loss. Additionally, our results indicate that the availability of wet microhabitats can allow frogs to reduce water loss, potentially mitigating the risk of desiccation when thermoregulating to achieve higher operative temperatures. Our findings suggest, that with access to proper microhabitats, amphibians can mitigate the fundamental trade-off and receive benefits of thermoregulating at high temperatures.

Invading new climates at what cost? Ontogenetic differences in the thermal dependence of metabolic rate in an invasive amphibian.

Global warming can either promote or constrain the invasive potential of alien species. In ectotherm invaders that exhibit a complex life cycle, success is inherently dependent on the capacity of each developmental stage to cope with environmental change. This is particularly relevant for invasive anurans, which disperse on land while requiring water for reproduction. However, it remains unknown how the different life stages respond in terms of energy expenditure under different climate change scenarios. We here quantified the oxygen uptake of frogs at rest (a proxy of the standard metabolic rate) in the aquatic phase (at the tadpole and climax, i.e. during metamorphosis, stages) and in the terrestrial phase (metamorphosed stage) at three environmental temperatures. To do so, we used marsh frogs (Pelophylax ridibundus), an amphibian with the largest invasive range within the palearctic realm and for which their adaptation to global warming might be key to their invasion success. Beyond an increase of metabolic rate with temperature, our data show variation in thermal adaptation across life stages and a higher metabolic cost during metamorphosis. These results suggest that the cost to shift habitat and face changes in temperature may be a constraint on the invasive potential of species with a complex life cycle which may be particularly vulnerable during metamorphosis.

Urea and glucose modulation during freezing exposure in three temperate frogs reveals specific targets in relation to climate.

Amphibian diversity is most prominent in the warm and humid tropical and subtropical regions across the globe. Nonetheless, amphibians also inhabit high-altitude tropical mountains and regions at medium and high latitudes, exposing them to subzero temperatures and requiring behavioural or physiological adaptations to endure freezing events. While freeze tolerance has been predominantly reported in high-latitude zones where species endure prolonged freezing (several weeks or months), less is known about mid-latitudes amphibians exposed to occasional subzero temperatures. In this study, we employed a controlled ecological protocol, subjecting three frog species from the Iberian Peninsula (Rana parvipalmata, Epidalea calamita, and Pelobates cultripes) to a 2-h exposure to temperatures of -2 °C to investigate the accumulation of urea and glucose as physiological mechanisms associated with survival at freezing temperatures. Our results revealed a moderate response in the production of cryoprotectant metabolites under experimental freezing conditions, particularly urea, with notable findings in R. parvipalmata and E. calamita and no response in P. cultripes. However, no significant alterations in glucose concentrations were observed in any of the studied frog species. This relatively weak freezing tolerance response differs from the strong response exhibited by amphibians inhabiting high latitudes and enduring prolonged freezing conditions, suggesting potential reliance on behavioural adaptations to cope with occasional freezing episodes.

Effects of warming at embryonic and larval stages on tadpole fitness in high-altitude Rana kukunoris.

Global warming may affect the early developmental stages of high-altitude amphibians, thereby influencing their later fitness. Yet, this has been largely unexplored. To investigate whether and how the temperatures experienced by embryonic and larval stages affect their fitness at later developmental stages, we designed two experiments in which the embryos and larvae were treated with three temperatures (24, 18 and 12 °C), respectively. Then, the life history traits of the tadpoles during the metamorphotic climax in all treatments were evaluated, including growth rate, survival rate, morphology, thermal physiology, swimming performance, standard metabolic rate (SMR), oxidative and antioxidative system, and metabolic enzyme activities. The results revealed that elevated temperature accelerated metamorphosis but decreased body size at metamorphosis. Additionally, warming during the embryonic and larval stages decreased the thermal tolerance range and induced increased oxidative stress. Furthermore, high embryonic temperature significantly decreased the hatching success, but had no significant effect on swimming performance and SMR. Warming during larval periods was harmful to the survival and swimming performance of tadpoles. The effect size analysis revealed that the negative impacts of embryonic temperature on certain physiological traits, such as growth and development, survival and swimming performance, were more pronounced than those of larval temperature. Our results highlight the necessity for particular attention to be paid to the early stages of amphibians, notably the embryonic stages when evaluating the impact of global warming on their survival.

DPOAEs and tympanal membrane vibrations reveal adaptations of the sexually dimorphic ear of the concave-eared torrent frog, Odorrana tormota.

While most anuran species are highly vocal, few of them seem to be endowed with a complex call repertoire. Odorrana tormota, combines a remarkable vocalization complexity with auditory sensitivity over an extended spectral range spanning from audible to ultrasonic frequencies. This species is also exceptional for its ability to modify its middle ear tuning by closing the Eustachian tubes (ET). Using scanning laser Doppler vibrometry, the tympanal vibrations were measured to investigate if the tuning shift caused by the ET closure contributes to intraspecific acoustic communication. To gain insight into the inner ear frequency selectivity and sensitivity of this species, distortion product otoacoustic emissions were recorded at multiple frequency-level combinations. Our measurements of inner ear responses indicated that in O. tormota each sex is more sensitive to the frequencies of the other sex's vocalizations, female ears are more sensitive to 2-7 kHz, while male ears are more sensitive to 3-15 kHz. We also found that in both sexes the ET closure impacts the sensitivity of the middle and inner ear at frequencies used for communication with conspecifics. This study broadens our understanding of peripheral auditory mechanisms contributing to intraspecific acoustic communication in anurans.

Male antiphonal calls and phonotaxis evoked by female courtship calls in the large odorous frog (Odorrana graminea).

Acoustic communication plays a vital role in frog reproduction. In most anuran species, long-distance sound communication is one-way from males to females; during the reproductive season, males produce species-specific advertisement calls to attract gravid females, and females are generally silent but perform phonotactic movements that lead to amplexus. One exception is the concave-eared torrent frog (Odorrana tormota). In this species, females produce courtship calls that elicit antiphonal vocalizations by males, followed by precise phonotactic movements. The large odorous frog O. graminea (previously Odorrana livida) in southern China is subject to the same environmental constraints as O. tormota, with which it is sympatric; it is unclear whether their sound communication is one-way or bidirectional. Here, we provide the first data on female O. graminea vocalizations and their functions. Using playbacks of female calls, we conducted acoustic behavioral experiments in the laboratory in response to which males emitted single- or multi-note antiphonal calls with a varying fundamental frequency. Moreover, they were attracted to female call playbacks, exhibiting precise phonotaxis. The female courtship call-male response interaction thus forms a duet between partners of a receptive pair. These results demonstrate that this unique communication system likely reflects an adaptation to an environment in which short-distance communication is at a premium given the high levels of ambient noise.

Social regulation of androgenic hormones and gestural display behavior in a tropical frog.

Many animals use forms of gesture and dance to communicate with conspecifics in the breeding season, though the mechanisms of this behavior are rarely studied. Here, we investigate the hormone basis of such visual signal behavior in Bornean rocks frogs (Staurois parvus). Our results show that males aggregating at breeding waterfalls have higher testosterone (T) levels, and we speculate that this hormone increase is caused by social cues associated with sexual competition. To this end, we find that T levels in frogs at the waterfall positively predict the number waving gestures-or "foot flags"-that males perform while competing with rivals. By contrast, T does not predict differences in male calling behavior. In these frogs, vocal displays are used largely as an alert signal to direct a rival's attention to the foot flag; thus, our results are consistent with the view that factors related to reproductive context drive up T levels to mediate displays most closely linked to male-male combat, which in this case is the frog's elaborate gestural routine.

Developmental environment has lasting effects on amphibian post-metamorphic behavior and thermal physiology.

Environmental challenges early in development can result in complex phenotypic trade-offs and long-term effects on individual physiology, performance and behavior, with implications for disease and predation risk. We examined the effects of simulated pond drying and elevated water temperatures on development, growth, thermal physiology and behavior in a North American amphibian, Rana sphenocephala. Tadpoles were raised in outdoor mesocosms under warming and drying regimes based on projected climatic conditions in 2070. We predicted that amphibians experiencing the rapid pond drying and elevated pond temperatures associated with climate change would accelerate development, be smaller at metamorphosis and demonstrate long-term differences in physiology and exploratory behavior post-metamorphosis. Although both drying and warming accelerated development and reduced survival to metamorphosis, only drying resulted in smaller animals at metamorphosis. Around 1 month post-metamorphosis, animals from the control treatment jumped relatively farther at high temperatures in jumping trials. In addition, across all treatments, frogs with shorter larval periods had lower critical thermal minima and maxima. We also found that developing under warming and drying resulted in a less exploratory behavioral phenotype, and that drying resulted in higher selected temperatures in a thermal gradient. Furthermore, behavior predicted thermal preference, with less exploratory animals selecting higher temperatures. Our results underscore the multi-faceted effects of early developmental environments on behavioral and physiological phenotypes later in life. Thermal preference can influence disease risk through behavioral thermoregulation, and exploratory behavior may increase risk of predation or pathogen encounter. Thus, climatic stressors during development may mediate amphibian exposure and susceptibility to predators and pathogens into later life stages.

Neuroendocrine correlates of juvenile amphibian behaviors across a latitudinal cline.

We assessed the macrogeographic and neuroendocrine correlates of behavioral variation exhibited by juveniles, an important life stage for dispersal, across the expansive range of the wood frog. By rearing animals from eggs in a common garden then using a novel environment test, we uniquely demonstrated differential expression of juvenile behaviors among 16 populations spanning 8° latitude. On the individual level, cluster analysis indicated three major behavior profiles and principal component analysis resolved four unique axes of behavior, including escape, foraging, food intake, feeding efficiency. We found that increased escape behavior was associated with lower adrenocorticotropic hormone (ACTH)-induced circulating corticosterone (CORT) levels, however, foraging and food intake behaviors were not associated with either resting or ACTH-induced CORT. At the population level, the expression of foraging behaviors increased with latitude while food intake behaviors declined with latitude, which raised several hypotheses of eco-evolutionary processes likely driving this variation. Given that these behaviors covary along the same ecological gradient as locally adapted developmental traits, genomic studies in this species could provide deep insights into how HPA/I activity is associated with the eco-evolutionary processes that structure intraspecific variation in morphology and behavior.

DNA damage and repair responses to freezing and anoxia stresses in wood frogs, Rana sylvatica.

Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.

Naive poison frog tadpoles use bi-modal cues to avoid insect predators but not heterospecific predatory tadpoles.

For animals to survive until reproduction, it is crucial that juveniles successfully detect potential predators and respond with appropriate behavior. The recognition of cues originating from predators can be innate or learned. Cues of various modalities might be used alone or in multi-modal combinations to detect and distinguish predators but studies investigating multi-modal integration in predator avoidance are scarce. Here, we used wild, naive tadpoles of the Neotropical poison frog Allobates femoralis ( Boulenger, 1884) to test their reaction to cues with two modalities from two different sympatrically occurring potential predators: heterospecific predatory Dendrobates tinctorius tadpoles and dragonfly larvae. We presented A. femoralis tadpoles with olfactory or visual cues, or a combination of the two, and compared their reaction to a water control in a between-individual design. In our trials, A. femoralis tadpoles reacted to multi-modal stimuli (a combination of visual and chemical information) originating from dragonfly larvae with avoidance but showed no reaction to uni-modal cues or cues from heterospecific tadpoles. In addition, visual cues from conspecifics increased swimming activity while cues from predators had no effect on tadpole activity. Our results show that A. femoralis tadpoles can innately recognize some predators and probably need both visual and chemical information to effectively avoid them. This is the first study looking at anti-predator behavior in poison frog tadpoles. We discuss how parental care might influence the expression of predator avoidance responses in tadpoles.

Evaluation of antimicrobial and anticancer activities of three peptides identified from the skin secretion of Hylarana latouchii.

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.

Species groups distributed across elevational gradients reveal convergent and continuous genetic adaptation to high elevations.

Although many cases of genetic adaptations to high elevations have been reported, the processes driving these modifications and the pace of their evolution remain unclear. Many high-elevation adaptations (HEAs) are thought to have arisen in situ as populations rose with growing mountains. In contrast, most high-elevation lineages of the Qinghai-Tibetan Plateau appear to have colonized from low-elevation areas. These lineages provide an opportunity for studying recent HEAs and comparing them with ancestral low-elevation alternatives. Herein, we compare four frogs (three species of Nanorana and a close lowland relative) and four lizards (Phrynocephalus) that inhabit a range of elevations on or along the slopes of the Qinghai-Tibetan Plateau. The sequential cladogenesis of these species across an elevational gradient allows us to examine the gradual accumulation of HEA at increasing elevations. Many adaptations to high elevations appear to arise gradually and evolve continuously with increasing elevational distributions. Numerous related functions, especially DNA repair and energy metabolism pathways, exhibit rapid change and continuous positive selection with increasing elevations. Although the two studied genera are distantly related, they exhibit numerous convergent evolutionary changes, especially at the functional level. This functional convergence appears to be more extensive than convergence at the individual gene level, although we found 32 homologous genes undergoing positive selection for change in both high-elevation groups. We argue that species groups distributed along a broad elevational gradient provide a more powerful system for testing adaptations to high-elevation environments compared with studies that compare only pairs of high-elevation versus low-elevation species.

Systemic hormonal and immune regulation induced by intraperitoneal LPS injection in bullfrogs (Lithobates catesbeianus).

Host's defense against external challenges activates an inflammatory response regulated by a set of chemical signals, including hormones. These immunomodulatory hormones, such as corticosterone, testosterone, and melatonin, trigger the systemic immune responses responsible for inflammatory assembly and resolution. This study aimed to investigate the effects of an immune challenge on endocrine and innate immune responses in the bullfrog (Lithobates catesbeianus). Adult males were intraperitoneally injected with lipopolysaccharide (LPS; 2 mg/kg) or saline, and blood samples were collected 6 and 24 h after injection for measurement of neutrophil/lymphocyte ratio, blood leukocyte phagocytosis, plasma bacterial killing ability, and plasma levels of corticosterone, melatonin, and testosterone. Our results showed LPS-induced increased neutrophil/lymphocyte ratio and leukocyte phagocytosis, and decreased melatonin and testosterone plasma levels, which were more pronounced 24 h after injection. Overall, we conclude that LPS intraperitoneal injection can activate the innate immune response and modulate the hormonal profile of the bullfrogs, with effects more pronounced 24 h than 6 h after treatment.

Effects of age on sperm quality metrics in endangered Mississippi gopher frogs (Lithobates sevosus) from captive populations used for controlled propagation and reintroduction efforts.

A decline in sperm quality with age is a common prediction of senescence-based hypotheses and empirical studies. While widely studied across taxa, there is little known on the effect of ageing on sperm quality in amphibians, especially in captive populations used for controlled propagation and reintroduction efforts. Here, we investigated variation in sperm quality metrics (i.e., motility, concentration, and morphology) in the endangered Mississippi gopher frog (Lithobates sevosus) among males of three age categories using individuals from captive breeding populations housed at three different zoological institutions. Different aged males across the species expectant lifespan (1-9, 1-2, 3-4, and 8-9-year-old subcategories) were chosen in an attempt to identify an optimal breeding age relevant for captive breeding programs. Moreover, we explored and statistically controlled for potential differences in sperm quality which may be attributed to the type of induction hormones and source populations that differed among institutions. Results indicated that males of different ages did not differ in sperm motility or concentration. However, we did find that older males (8-9 years old) had significantly longer sperm than other age categories and younger males (1-2 years old) had significantly more atypical sperm than other age categories. Furthermore, we found no significant differences in any sperm quality metrics between the different induction hormones or source populations used at the different institutions. Within a captive breeding program, this information is especially valuable as our results indicate that males that have only recently sexually matured may not be ready to breed, while older males maintain sperm quality metrics presumably related to fertilization success.

Population demographic history and adaptability of the vulnerable Lolokou Sucker Frog.

Amphibians are experiencing worldwide declines due to increasing anthropogenetic disturbances. However, the genetic variability and hence adaptability are still unknown for most frogs. We integrated the mitochondrial (ND2 gene), nuclear (TYR gene) and major histocompatibility complex (MHC) loci, to clarify the demographic patterns and immune-gene diversity of the Lolokou Sucker Frog (Amolops loloensis). Demographic analysis of the ND2 and TYR genes suggested that the Lolokou Sucker Frog experienced a population expansion within the last 10,000 years. High MHC diversity was detected, which has likely resulted from positive selection, indicating the current diversity bodes well for the species' adaptive potential to pathogenic challenges. These findings broaden our knowledge on the population history and evolution adaptation of the reclusive torrent frog, and conservation implications are provided.

Environmental and morphological constraints interact to drive the evolution of communication signals in frogs.

Animals show a rich diversity of signals and displays. Among the many selective forces driving the evolution of communication signals, one widely recognized factor is the structure of the environment where animals communicate. In particular, animals communicating by sounds often emit acoustic signals from specific locations, such as high up in the air, from the ground or in the water. The properties of these different display sites may impose different constraints on sound production, and therefore drive signal evolution. Here, we used comparative phylogenetic analyses to assess the relationship between calling site (aquatic versus nonaquatic), body size and call dominant frequency of 160 frog species from the families Ranidae, Leptodactylidae and Hylidae. We found that the frequency of frogs calling from the water was lower than that of species calling outside of the water, a trend that was consistent across the three families studied. Furthermore, phylogenetic path analysis revealed that call site had both direct and indirect effects on call frequency. Indirect effects were mediated by call site influencing male body size, which in turn was negatively associated with call frequency. Our results suggest that properties of display sites can drive signal evolution, most likely not only through morphological constraints imposed on the sound production mechanism, but also through changes in body size, highlighting the relevance of the interplay between morphological adaptation and signal evolution. Changes in display site may therefore have important evolutionary consequences, as it may influence sexual selection processes and ultimately may even promote speciation.

Solving Donor Organ Shortage with Insights from Freeze Tolerance in Nature: Activating endogenous antioxidant systems with non-coding RNA to precondition donor organs.

The North American wood frog, Rana sylvatica, endures seasonal whole-body freezing during the winter and thawing during the spring without sustaining any apparent damage from ice or oxidative stress. Strategies from these frogs may solve the shortage of human donor organs, which is a multidisciplinary problem that can be alleviated by eliminating geographical boundaries. Rana sylvatica deploys an array of molecular and physiological responses, such as glucose production and microRNA regulation, to help it survive the cold. These strategies have been adapted in the lab to impart cryotolerance in liver cells, and the non-freezing supercooled storage of transplantable rat livers - milestones that have advanced the field toward cryopreserving human donor organs in the clinic. In this review, a case is presented for the use of non-coding RNAs to decrease oxidative damage of donor organs by activating endogenous antioxidant systems prior to procurement.

Phylogenomics reveals rapid, simultaneous diversification of three major clades of Gondwanan frogs at the Cretaceous-Paleogene boundary.

Frogs (Anura) are one of the most diverse groups of vertebrates and comprise nearly 90% of living amphibian species. Their worldwide distribution and diverse biology make them well-suited for assessing fundamental questions in evolution, ecology, and conservation. However, despite their scientific importance, the evolutionary history and tempo of frog diversification remain poorly understood. By using a molecular dataset of unprecedented size, including 88-kb characters from 95 nuclear genes of 156 frog species, in conjunction with 20 fossil-based calibrations, our analyses result in the most strongly supported phylogeny of all major frog lineages and provide a timescale of frog evolution that suggests much younger divergence times than suggested by earlier studies. Unexpectedly, our divergence-time analyses show that three species-rich clades (Hyloidea, Microhylidae, and Natatanura), which together comprise ∼88% of extant anuran species, simultaneously underwent rapid diversification at the Cretaceous-Paleogene (K-Pg) boundary (KPB). Moreover, anuran families and subfamilies containing arboreal species originated near or after the KPB. These results suggest that the K-Pg mass extinction may have triggered explosive radiations of frogs by creating new ecological opportunities. This phylogeny also reveals relationships such as Microhylidae being sister to all other ranoid frogs and African continental lineages of Natatanura forming a clade that is sister to a clade of Eurasian, Indian, Melanesian, and Malagasy lineages. Biogeographical analyses suggest that the ancestral area of modern frogs was Africa, and their current distribution is largely associated with the breakup of Pangaea and subsequent Gondwanan fragmentation.