Frog Species Research Articles

Navigating access and benefit sharing in international trade of endemic species: The case of Colombia's poison frogs (Dendrobatidae)

AbstractWildlife is an important global commodity that generates significant revenue along the supply chain, including economic benefits in range states for sought‐after species. However, international trade in wild species is often associated with over‐exploitation and illegal extraction. To regulate, monitor, and ensure the sustainability of such trade, over 180 countries have ratified the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Using national data collected through CITES processes, we undertook a network analysis to evaluate the structure and dynamics of international trade in six CITES‐listed species of Colombian endemic poison frogs (Dendrobatidae). We assessed the magnitude of the international trade and the countries that were reporting legal imports/exports, both before and after Colombia allowed legal exports of these species, to examine the changes associated with Colombia's export legalization and to draw conclusions about the country's role and benefits from trade in these species. Our analyses show that, for over two decades, international trade of CITES‐listed species likely in contravention of the Convention resulted in significant exports—and likely greater financial benefits—accruing in non‐range states through reported captive breeding programs. Although the market was reconfigured when Colombia legalized the export of certain endemic frog species, non‐range states with founder stocks obtained from unknown and possibly illegal sources continue to account for a significant portion of the trade volume of some species (mostly, Phyllobates spp.). We discuss the importance of enforcing regulations and collaborations that promote better traceability of international commerce of endemic species to ensure the legitimacy of transactions, economic benefits for origin countries, and the conservation of species in the wild.

Defying decline: Very low chytrid prevalence in tadpoles, yet high infection in adults in a naturally recovering frog species

AbstractAmphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is associated with declines in ~500 amphibian species globally. Decades after initial disease outbreaks, the trajectory of impacted species varies substantially; while some species continue to decline, there are instances of natural recovery, such as the whistling tree frog, Litoria verreauxii, in south‐eastern Australia. The decline and subsequent recovery of this species have been quantified through repeated surveys of historically occupied sites over the past 30 years; however, the underlying mechanisms driving this recovery remain unknown. In this study, we investigate the potential factors facilitating the recovery of L. verreauxii by examining Bd prevalence and intensity in both adults and tadpoles. Specifically, we addressed the following hypotheses: (1) Bd prevalence in tadpoles would be lower compared to adults at the same breeding sites, (2) Bd prevalence in tadpoles would decrease over the spring breeding season due to the increasing availability of warm water microhabitats where tadpoles could potentially avoid or clear Bd infections and (3) there would be a negative correlation between Bd prevalence in tadpoles and the abundance and diversity of microfauna, which may consume Bd zoospores. Our findings indicate that tadpole infection prevalence remained consistently low at 1.36% (95% CI: 0.6–2.47%) throughout our spring sampling period, across different developmental stages. Adults had moderate to high prevalence within the same ponds at 50.53% (95% CI: 43.19–57.84%). No effect of temperature or microfauna diversity and abundance was apparent. While the mechanisms driving the recovery of this species remain unknown, low infection prevalence in tadpoles is likely a key component to the species' recovery. Our results emphasize the need for comprehensive investigations in Bd dynamics across all life history stages within recovering and declining amphibian species.

Skull Ossification in the Andean Toad Rhinella spinulosa (Bufonidae) and the Genetic Model Organism Xenopus tropicalis (Pipidae) Reveals Heterochrony Phenomena and Frontoparietal Suture Modifications.

Anurans are famous for having evolved a highly simplified skull through bone loss and fusion events. Nevertheless, their skeleton displays a rich morphological diversity associated with adaptations to diverse lifestyles and ecological niches. Here, we report larval skull ossification in the Andean toad Rhinella spinulosa (Bufonidae), and compare it to the phylogenetically distant genetic model organism Xenopus tropicalis (Pipidae). We find that the ossification timing of most skull bones is conserved between both species, except for the prootic and the angulosplenial that ossify at much later stages in R. spinulosa than X. tropicalis. We propose that a delayed lower jaw ossification in R. spinulosa is tightly related to the more extensive metamorphosis process observed in this species where the ventrally oriented mouth opening shifts anteriorly. We also report two conspicuous notches in the R. spinulosa frontoparietal bone mineralization front which are absent in X. tropicalis, and presumably represent evolutionary remnants of the coronal suture that separates the frontal and parietal bones in most vertebrates. As such notches have not been overtly reported in the literature, we examined the X. tropicalis sibling species Xenopus laevis, and were able to identify similar, albeit transient, indentations in the forming frontoparietal bone, suggesting that vestigial coronal sutures might exist in more frog species than anticipated. Taken together, we show that R. spinulosa represents an ideal organism to study heterochronic shifts and the mechanisms underlying cranial suture loss which drove anuran skull simplification.

Rediscovery of the mountain frog Telmatobius ceiorum (Anura: Telmatobiidae) in Argentina, last seen 40 years ago

We report the rediscovery and the geographic occurrence extension of Telmatobius ceiorum, an endemic frog species from northern Argentina. This marks the first record of T. ceiorum since November 1984. The frogs were observed near the village of Hualinchay, Tucuman Province, at an elevation of 2060 m. The introduction of salmonids across much of its historical range is one of the main threats that may have contributed to the decline of T. ceiorum. Our findings emphasize the need for continued monitoring and conservation efforts for the species, particularly in light of the potential expansion of invasive fish species.

Alkaloids are associated with increased microbial diversity and metabolic function in poison frogs.

Shifts in host-associated microbiomes can impact both host and microbes.1,2,3,4,5,6 It is of interest to understand how perturbations, like the introduction of exogenous chemicals,7,8,9,10,11,12,13 impact microbiomes. In poison frogs (family Dendrobatidae), the skin microbiome is exposed to alkaloids that the frogs sequester for defense.14,15,16,17,18,19 These alkaloids are antimicrobial20,21,22; however, their effect on the frogs' skin microbiome is unknown. To test this, we characterized microbial communities from field-collected dendrobatid frogs. Then, we conducted a laboratory experiment to monitor the effect of the alkaloid decahydroquinoline (DHQ) on the microbiome of two frog species with contrasting alkaloid loads in nature. In both datasets, we found that alkaloid-exposed microbiomes were more phylogenetically diverse, with an increase in diversity among rare taxa. To better understand the isolate-specific response to alkaloids, we cultured microbial isolates from poison frog skin and found that many isolates exhibited enhanced growth or were not impacted by the addition of DHQ. To further explore the microbial response to alkaloids, we sequenced the metagenomes from high- and low-alkaloid frogs and observed a greater diversity of genes associated with nitrogen and carbon metabolism in high-alkaloid frogs. From these data, we hypothesized that some strains may metabolize the alkaloids. We used stable isotope tracing coupled to nanoSIMS (nanoscale secondary ion mass spectrometry), which supported the idea that some of these isolates are able to metabolize DHQ. Together, these data suggest that poison frog alkaloids open new niches for skin-associated microbes with specific adaptations, such as alkaloid metabolism, that enable survival in this environment.

Surviving in the urban jungle: the role of foam nests as thermal insulator in Pleurodema borellii (Anura: Leptodactylidae)

Urbanisation stands as a primary driver of biodiversity loss globally, reshaping natural landscapes and imposing novel environmental conditions upon organisms. This ecological novelty poses challenges, and species capable of thriving in urban environments are considered tolerant. Among vertebrates, anurans are the most susceptible to land-use changes, mainly due to their life history and morphological traits. They exhibit a great diversity of reproductive strategies and modes, among which the foam nest plays several important roles, such as moderating or buffering adverse conditions. In this study, we assessed the foam nest of Pleurodema borellii, an urban tolerant species, as a potential thermal insulator in the city of San Salvador de Jujuy, Argentina. We carried out our surveys within San Salvador and surrounding areas, focusing on urban and peri-urban environments. We measured the height, length, and internal temperature of 48 foam nests, as well as the temperature of their immediate environment (water and air at one cm above the nest), over consecutive days from the first day until the nest disappeared. Finally, we collected four to five embryos from each foam nest once a day to identify their developmental stage. Our results suggest that foam nests not only mitigate low temperatures but also buffer against the heat of urban environments. These findings suggest that the foam nest could be one of the traits that facilitates the occurrence of this anuran species in the city and therefore may explain its tolerance to urbanisation.

Increased Temperature and Exposure to Ammonium Alter the Life Cycle of an Anuran Species.

Amphibian populations are undergoing a major recession worldwide, likely triggered by global change components such as the global warming and pollutants, among which agrochemicals, in general, and fertilizers, in particular, play a central role given their relevance in agriculture. Potential synergies among these stressors could maximize their individual effects. In this work, we investigated the consequences of a controlled chronic exposure to increased temperature and a sublethal dose of ammonium during the larval stage of Pelophylax perezi frogs on the growth, development, and locomotor performance of tadpoles and the metamorphs they gave rise to. To that end, tadpoles were reared either in heated or nonheated tanks, with or without ammonium added. The parents of these tadpoles came from either a pine grove or an agrosystem. Survival was reduced in agrosystem tadpoles reared with ammonium. Increased temperature potentiated tadpole growth while giving way to smaller metamorphs. Faster growth could be a consequence of increased metabolism, whereas the smaller size could follow an accelerated development and metamorphosis, which reduced the growth period. Also, swimming speed was greater in tadpoles reared in heated tanks, while jumping distance was greater in metamorphs reared in nonheated tanks. The effects of temperature were more marked in agrosystem than in pine grove individuals, which could mirror reduced adaptability. Thus, the ability to withstand the effects of these stressors was lower in agrosystem tadpoles.

Drivers of Anuran Assemblage Structure in a Subtropical Montane Region.

Elevation gradients provide excellent semi-experimental conditions to investigate how the spatial distribution of biodiversity is shaped by the environment. Here, we investigate how temperature, productivity, and elevation are related to the diversity of anuran assemblages in a montane region of the southern Brazilian Atlantic Forest. We sampled 20 half-hectare plots between 2020 and 2021, distributed along a sharp elevation gradient. Anuran species richness and abundance decreased with increasing elevation. We show a positive relationship between ambient temperature and frog species richness and abundance, highlighting the importance of temperature in shaping assemblages along the elevation gradient. In contrast, productivity did not affect species richness and abundance, suggesting that available energy via resources does not limit local frog diversity. We further observed marked differences in the composition of anuran assemblages between low and high elevation areas, which were related to temperature. Beta diversity is mainly driven by species replacements among assemblages, which were likely related to environmental conditions. These findings highlight the importance of incorporating protected areas that encompass the entire range of elevations to capture the full complement of landscape-scale diversity. This is crucial as species showed limited distributions, and the marked effects of temperature should be explicitly considered under future scenarios of elevated upslope warming.

Genomic and bioacoustic variation in a midwife toad hybrid zone: A role for reinforcement?

Hybrid zones, i.e., geographic areas where diverging lineages meet, hybridize and eventually mix their genomes, offer opportunities to understand the mechanisms behind reproductive isolation and speciation. Hybrid zones are particularly well suited to study reinforcement, i.e., the process by which selection against hybridization increases reproductive barriers, which, in anuran amphibians, is typically expressed by increased divergence in advertisement calls-the main cue to assortative mating-in parapatric ranges. Using mitochondrial barcoding (16S sequences), population genomics (thousands of SNPs) and bioacoustic analyses (four call parameters), we examine the hybrid zone between two incipient species of midwife toads (Alytes obstetricans and A. almogavarii) in southern France, with the purposes of locating their transition, measuring genetic introgression, and documenting potential signatures of reinforcement. We map range boundaries in the Eastern Pyrenees and the southwestern foothills of the Massif Central, namely along the Ariège valley and the Montagne Noire area. Similarly to another transition between these species in Spain, we found the hybrid zone to be narrow, involving geographically restricted gene flow (~20 km wide allele frequency clines) and barrier loci (i.e., loci resisting introgression), both suggestive of partial post-zygotic isolation (hybrid incompatibilities). The calls of the species overlap less inside than outside the hybrid zone, due to a reduction of their standing variation rather than a shift towards distinctive variants. While neutral causes cannot be excluded, this pattern follows the general expectations of reinforcement, yet without reproductive character displacement. Our study highlights the potential of amphibian hybrid zones to assess the genetic and behavioral drivers of reproductive isolation in statu nascendi and under various evolutionary contexts.

Amphibian Diversity and Morphometric Analysis in the Kadayam Region of the Tirunelveli District, India

This study examines the morphometric and genetic diversity of frog species in the agro ecosystems of Kadayam region part of the Western Ghats, a biodiversity hotspot in India. By conducting an amphibian survey in Kadayam, Southern Western Ghats, eight species from three families were documented, with Dicroglossidae showing the highest species representation. Amphibians were sampled from various habitats-forests, water bodies, and cultivated lands between January and March 2017, with notable species Duttaphrynus melanostictus showing a high abundance in human occupied areas. Morphometric analyses were performed on features such as snout-vent length, head length, (SVL–SNOW-VENT LENGTH; HL - HEAD LENGTH; HW-HEAD WIDTH; LHU–LENGTH OF THE HUMERUS; FOL-FOREARM LENGTH; THL-THIGH LENGTH; TL-TIBIA LENGTH; IOD-INTERORBITAL DIAMETER; IND-INTER NOSTRIL DISTANCE; ED-EYE DIAMETER; END–EYE–NOSTRIL DISTANCE; and FL -FOOT LENGTH) and limb proportions across species, correlating these to understand habitat adaptations. Results indicated high endemism and diversity within frog populations of the Western Ghats, highlighting conservation needs due to threats like habitat modification and pollution. The high positive correlations among primary body dimensions (such as SVL, head length, and total length) suggest that these species exhibit proportional growth in these features, a common trait in morphometric analyses. This study underscores the significance of morphometric approaches in amphibian biodiversity assessments in ecologically sensitive regions.

Sensitivity of larval Dendropsophus haddadi (Bastos and Pombal 1996), an anuran species from the Brazilian Atlantic Forest, to acute exposure to nitrate.

Nitrogenous pollution has been reported to be a major threat to biodiversity and, therefore, it may be related to the decline of amphibians, the most threatened group of vertebrates in the world. In spite of this, and the widespread release of nitrogenous compounds into the environment, research on the impact of this pollution on Neotropical species remains limited. The aim of this study was to assess the sensitivity of one anuran species inhabiting in the Brazilian Atlantic Forest (Dendropsophus haddadi) to NaNO3 by an acute exposure laboratory experiment, addressing also whether carryover effects exist. Larval mortality increased as time went on in the case of the highly polluted treatments, which also affected swimming performance. Mean lethal concentration (LC50) values revealed that the sensitivity of the study species to NaNO3 was similar to that described for other amphibians, both tropical and temperate species. Additionally, larvae exposed to moderate levels of pollution significantly increased final mass. In spite of this, once larvae were transfered into clean water, no further mortality was recorded, and swimming performance improved. These results indicate that tropical species may not be more vulnerable to chemical pollution than temperate ones, and suggest that they are able to recover from acute short exposure to nitrogenous compounds, all of which represent a new addition to the knowledge of the vulnerability of Dendropsophus haddadi to pollution and, thus, to Neotropical ecotoxicology.

Transcriptomic analysis of liver immune response in Chinese spiny frog (Quasipaa spinosa) infected with Proteus mirabilis.

The expansion of Chinese spiny frog (Quasipaa spinosa) aquaculture has increased the prevalence and severity of diseases such as "skin rot" disease, which is triggered by harmful bacteria. Previous studies have mainly focused on pathogen identification and vaccine development. However, frog immune responses following pathogenic bacterial infection have hardly been investigated. We thus examined the immune response of Chinese spiny frog to skin rot disease caused by Proteus mirabilis. The liver transcriptomes of Chinese spiny frog infected with P. mirabilis were sequenced using the MGISEQ-2000 platform. We identified a total of 138,936 unigenes, of which 32.35% were known genes. After infection with P. mirabilis, 801 genes showed differential expression, with 507 upregulated and 294 downregulated genes. These differentially expressed genes were enriched in pathways related to cytokine-cytokine receptor interaction, TNF signaling, and toll-like receptor signaling, according to Kyoto Encyclopedia of Genes and Genomes analysis. Following P. mirabilis infection, immune genes, including H2-Aa, hamp1, LYZ, CXCL10, and IRAK3, were significantly upregulated, while NLRP3, ADAM19, TYK2, FETUB, and MSR1 were significantly downregulated. The results provide important information on how the immune system of Chinese spiny frog responds to P. mirabilis infection and help understand the development of skin rot in cultured frog species.

Skittering locomotion in cricket frogs: a form of porpoising.

Multiple species of frogs in the Ranidae family have been observed to 'skitter' across the water surface, but little is understood about the biomechanical or physical mechanisms that underlie this behavior. All documented descriptions are anecdotal, asserting simply that the frogs can cross the water surface without sinking. To study this form of interfacial locomotion, we recorded high-speed video of the northern cricket frog Acris crepitans and quantified its kinematics. We also compared its semi-aquatic behavior with the frogs' terrestrial locomotion. Contrary to expectations based on anecdotal knowledge, we found that cricket frogs do not maintain an above-surface position throughout the locomotor cycle. Instead, the frogs are completely submerged during both the launching and landing phase of a jump cycle, similar to porpoising in other animals. It is possible that leg-retraction time constrains these frogs from performing true surface-only locomotion.

Novel sounds, native responses: exploring the acoustic consequences of Eleutherodactylus johnstonei’s invasion in urban areas

BackgroundBiological invasions pose a critical threat to biodiversity, affecting ecological balance and native species’ communication. Eleutherodactylus johnstonei, an exotic anuran in São Paulo, vocalizes at intensities that could interfere with native anuran species, potentially causing acoustic masking.MethodsWe evaluated the effects of E. johnstonei's calls on the vocalizations of two native species, Scinax imbegue and Physalaemus cuvieri, both with and without spectral overlap with the invasive species. Field playbacks were conducted using six versions of stimuli, including E. johnstonei's calls, the native Boana bischoffi (as a control), and white noise. We recorded response calls and behavioral changes of S. imbegue and P. cuvieri males.ResultsThe calls of E. johnstonei did not affect the spectral or temporal parameters of the native species’ announcement calls. However, S. imbegue males displayed behavioral responses such as cessation of vocalization or movement away from the noise source. Additionally, B. bischoffi's calls and white noise influenced native species’ call parameters.DiscussionOur findings reveal that exotic species’ vocalizations may disrupt native anurans’ acoustic behavior. This impact varies with species and context, underlining the need for further research on anuran acoustic interactions across different frequencies and acoustic environments to fully understand the effects of exotic acoustic interference.

Establishment of a cell culture from the frog Leptodactylus fuscus as a model for (eco)toxicological assays

Leptodactylus fuscus is a frog species widely distributed in the Neotropical region, occurring in several biomes, which makes it a potential biomonitor of environmental conditions. To advance the establishment of this species in this field, we developed a fibroblast-like cell line derived from the digits of the forelimbs (LFUfd) and evaluated its response to possible environmental stressors. An adult male L. fuscus was collected, anesthetized, and euthanized with eugenol. Digits from the forelimbs were extracted, decontaminated, and dissociated. Cells were plated and cultivated in vented flasks with DMEM/F12+GlutaMAX medium and 20% FBS at 28 °C. On the 15th passage, we made the chromosome preparations, where we placed them on slides and stained them with Giemsa for karyotype analysis. The cellular response to multiple stressors (Temperature, Hydrogen Peroxide, and Aluminum) was evaluated through the MTT assay. The temperature test evaluated two groups (28 °C and 37 °C for 24 h). The effect of hydrogen peroxide (H2O2) was analyzed by treatment for 1 h at 28 °C in different concentrations (control, 2 μM, 20 μM, 50 μM, 100 μM and 200 μM). The effects of aluminum have been tested at three concentrations (0.5 mg/L, 1 mg/L, and 10 mg/L). The karyotype obtained showed 22 chromosomes, according to the description of the species, although a small proportion of aneuploid cells was observed (∼7%). At 37 °C there was a reduction in cell survival and no effects on cell viability at the H2O2 concentrations tested; however, there was a decrease in cell viability when exposed to aluminum in all the concentrations tested.. The growth behavior and karyotype observed indicate the establishment of a continuous cell culture with a stable genome that responds to environmental changes.

Temporal Dynamics of Species Richness and Composition in a Peri-Urban Tropical Frog Community in Central Brazil.

Analyzing the temporal dynamics of ecological communities can shed light on coexistence mechanisms and help understand how populations and communities will behave in the face of climate change. However, little is known about how frog communities respond to climate in urban ecosystems, especially in tropical countries. Here, we analyzed how frog species richness and abundance are influenced by weather variables both intra- and inter-annually. We surveyed a peri-urban area in central Brazil, monthly for 3 years. To test the effect of weather variables on species richness and abundance, we used Generalized Additive Mixed-effects Models. We assessed seasonality using circular statistics. We also tested for differences in temporal beta diversity within and among years by estimating species disappearance and temporal rank shift, in addition to a multivariate model-based method to test the effect of year on species composition. Finally, we tested how taxonomic and phylogenetic alpha diversity changed through time using a novel approach based on Hill numbers. We found that species richness varied little among years and was affected only by photoperiod, while species abundance was more variable both between and within years, being mostly affected by humidity, temperature, and photoperiod. Species composition varied little between years, mostly between the first and subsequent years. Conversely, beta diversity was highest within years. Only the effective number of species changed significantly through time. Our results help not only understand temporal mechanisms that allow species coexistence, but also allow to make inferences about the impact of urbanization on biodiversity in recently urbanized landscapes, showing that species composition in peri-urban sites remains unaltered in a mid-timescale, especially when climate conditions change little across years.

An insight into the bioacoustics of some anurans of Meghalaya, India (Amphibia: Anura)

This paper presents an analysis of the temporal and spectral acoustic variables of nine anuran species across six families living in Meghalaya. The species accounted for in the study include the Jerdon’s tree frog Hyla annectans, the Long-tongued Forest Frog Hylarana leptoglossa, Northern Pointed-Snout Frog Clinotarsus alticola, Mymensingh Chorus Frog Microhyla mymensinghensis, Beautiful Painted Frog Kaloula pulchra, Double-spotted Oranged Webbed Tree Frog Rhacophorus bipunctatus, Shillong Bush Frog Raorchestes shillongensis, Pierrei Cricket Frog Minervarya pierrei, and Smith’s Litter Toad Leptobrachium smithi. For four out of nine species (Hylarana leptoglossa, Clinotarsus alticola, Microhyla mymensinghensis, and Rhacophorus bipunctatus), this is the first account of their calls; the remnant five recordings add considerable knowledge to the species’ acoustic variability. This study opens up potential benefits for taxonomy, ecology, and conservation.

Predation on eggs and larvae of the Japanese brown frog Rana japonica Boulenger, 1879 by the invasive crayfish Procambarus clarkii (Girard, 1852) (Decapoda: Astacidea: Cambaridae) under laboratory conditions

Abstract We hypothesized that the North American invasive crayfish Procambarus clarkii (Girard, 1852) can negatively affect the populations of native frog species through predation. We conducted three laboratory experiments to examine whether P. clarkii can prey on eggs or larvae of the Japanese native frog Rana japonicaBoulenger, 1879: Experiment I crayfish + frog egg in an aquarium without a substrate or aquatic plants, Experiment II crayfish + frog larva in an aquarium without a substrate or aquatic plants, and Experiment III crayfish + frog larva in an aquarium with a substrate and aquatic plants as shelter for the larva. We used three experimental groups of crayfish individuals (small, intermediate, and large size), each of 10 individuals, and simultaneously placed one crayfish individual and one egg or frog larva in the same aquarium for 24 h and checked for predation by the crayfish. We found that 88.9% and 86.7% of eggs and larvae used in Experiments I and II were preyed upon, respectively. Predation rate of frog larvae was still high (77.8%) in Experiment III, suggesting that crayfish can potentially find and prey on frog larvae in nature. Predation on frog eggs or larvae was high regardless of the crayfish body size. Our results indicate that P. clarkii can potentially have a marked effect on the population of R. japonica by preying on eggs and larvae.

Can eDNA Present in Aquatic Environments of Rural Areas Help Identify Species Diversity in the Order Anura?

Paddy fields are classified as wetland environments, and they comprise freshwater ecosystems. They are ecologically important habitats and breeding grounds for many aquatic insects, amphibians, and reptiles. However, paddy field ecosystems are constantly threatened by climate change and the indiscriminate use of pesticides. The metabarcode analysis of eDNA (environmental DNA) method is highly effective at accumulating information on many organisms that inhabit paddy field ecosystems. It can indirectly identify the existence of taxa that are no longer found in the target ecosystem due to behavioral characteristics, such as those exhibited by amphibians. In the metabarcoding results of this study, genes of five species of frogs were found, but it was impossible to confirm all of the frogs’ taxa, morphological pictures, and croak sounds. On the other hand, some frog taxa were only found in the metabarcoding analysis. The eDNA of the frogs found only in the metabarcoding analysis is estimated to have been introduced from nearby areas inhabited by frogs rather than the target region. Due to the powerful analytical resolution of eDNA metabarcoding, this eDNA-based paddy field search is expected to help investigate the biodiversity in agricultural ecosystems.

Captive Breeding Reveals Insights Into the Ecology and Reproductive Biology of 11 Little-Known Malagasy Frog Species.

Amphibians are facing an extinction crisis, with ex situ programs increasingly being used as a tool for their conservation. However, conservation efforts are often limited because we do not understand the ecological, behavioral, and life history traits of many amphibian species. Here, we report on the seasonal breeding patterns, egg-laying behavior, clutch size, and development of 11 frog species maintained at a conservation breeding facility in Andasibe, Madagascar. The frogs exhibited diverse breeding strategies aligned with life history theory. Counting the eggs in 1239 egg masses across these 11 species, we found endotrophic microhylids and terrestrial-breeding species had the smallest clutch size yet completed metamorphosis quickly, whereas species that laid eggs above or in water with exotrophic larvae had larger clutch sizes and took longer to develop. Most reproduction in captivity occurred during the warm, rainy season and followed seasonal patterns in temperature variation. Yet, Mantidactylus betsileanus bred throughout the year, and Heterixalus betsileo required additional environmental stimuli to trigger reproduction. Notably, we confirmed that Gephyromantis mitsinjo lays eggs on land with tadpoles developing terrestrially within jelly, a behavior previously theorized but which until now remained unobserved. Such observations show how captive breeding programs can be used to gain valuable data on the life history traits of species that are otherwise challenging to observe in nature. Our findings can be used to assess threats to closely related species, helping inform conservation efforts in a country harboring exceptional amphibian species richness and endemism.