South American Frog Research Articles

Effect of water stress on locomotor performance in Pleurodema nebulosum (Anura: Leptodactylidae) a native frog from the Argentina Monte desert

Locomotion is a fundamental aspect in the life cycle of any organism, and plays a crucial role in survival, feeding, escaping from predators, territoriality, and reproduction. Locomotion in amphibian anurans includes swimming, walking, jumping, climbing, and gliding. Amphibians living in arid regions face a constant compromise between hydration balance and locomotor performance. We studied the effects of water stress on the locomotion of the frog Pleurodema nebulosum, a specialist from the Monte Desert. Locomotor performance was measured through different physical variables during the jump. The locomotor performance of P. nebulosum showed a significant decrease in the magnitudes associated with a decrease in body hydration. Our study demonstrated how a desert-adapted frog (P. nebulosum) showed changes in locomotion related to the loss of body water. Additionally, we observed an abrupt drop in locomotion with 15% desiccation, followed by a plateau where the evaluated parameters remained stable. Pleuroderma nebulosum showed a clear adaptation to extreme desiccation. The frogs are rarely found in optimal hydration conditions as the environment in which they develop is extremely stressful from a water perspective. The data evaluated here are the beginning of a deeper exploration of the desert adaptations of South American desert frogs.

Sexual dimorphism, female fertility, and diet of Physalaemus feioi (Anura: Leptodactylidae) from southeastern Brazil


 
 
 
 Physalaemus feioi is a South American frog endemic to the Brazilian Atlantic Forest that occurs in the states of Minas Gerais and São Paulo. We evaluated sexual dimorphism, female fertility, and diet of P. feioi from two locations in the state of Minas Gerais, including the type locality of the species. We collected 73 individuals (16 adult females and 57 adult males). The number of oocytes of P. feioi (x̅ = 1385 ±498.13) was higher than in other species of the genus, such as P. maculiventris, P. signifer, P. cuvieri, and P. ephippifer, but it resembles that of P. centralis, P. kroyeri, and P. marmoratus. The prey items with the highest index of importance were Coleoptera (IIR = 24.26) and Formicidae (IIR = 16.46). We found no sex differences regarding the number of ingested prey and the volume of the stomach contents. In summary, Physalaemus feioi was similar to other species of the genus regarding sexual dimorphism, fecundity, and feeding habits.
 
 
 

Bactericidal activities and action mechanism of the novel antimicrobial peptide Hylin a1 and its analog peptides against Acinetobacter baumannii infection

We developed an antimicrobial peptide (AMP) as a candidate substance for replacing antibiotics. Previously, a novel 18-amino acid antimicrobial peptide Hylin a1 was isolated from an electro-stimulated arboreal South American frog Hypsiboas albopunctatus, and was found to demonstrate antimicrobial activity and cytotoxicity. In a recent study, the analog peptides were designed based on the parent peptide Hylin a1 to decrease toxicity and to maintain antimicrobial efficacy. The analog peptides were substituted with alanine and lysine, resulting in the formation of amphipathic α-helical structures in membrane-mimicking environments and in the induction of hydrophobic moments and net charges. Moreover, the analog peptides showed lower hemolytic effects and mammalian cell selectivity than Hylin a1. In particularly Hylin a1-11K and Hylin a1-15K exhibited broad-spectrum antimicrobial activity and anti-biofilm activity against carbapenem-resistant Acinetobacter baumannii. Permeability assays indicated that analog peptides eliminated bacteria by binding to lipopolysaccharide and by disrupting the bacterial membrane. Hylin a1-11K and Hylin a1-15K reduced inflammation by suppressing pro-inflammatory cytokines expression by A. baumannii infection and effectively ameliorated carbapenem-resistant A. baumannii infection in mice. Therefore, our results suggest that the analog peptide substituted with several residues based on Hylin a1 have antibacterial and anti-inflammatory activity, and may be effective in the treatment of carbapenem-resistant A. baumannii infection.

A first look at the N- and O-glycosylation landscape in anuran skin secretions

Amphibians secrete a complex array of molecules that shape their interactions with coinhabiting microorganisms and macroscopic predators. Glycans are a rapidly evolving and complex class of biomolecules implicated in intrinsic and extrinsic recognition events. Despite the numerous studies aiming at the biochemical characterization of anuran skin secretions, little is known about protein-linked oligosaccharides, their synthesis pathways, and their homing secreted glycoproteins. In the present report, LC-MS/MS was used to investigate the diversity of N- and O-linked oligosaccharides in the skin secretion of two South American frogs, Pithecopus azureus and Boana raniceps. Additionally, the enzymes responsible for glycan synthesis pathways were evaluated based on their skin tissue transcriptome. Our analyses allowed the annotation of various N- and O-glycan structures commonly found in vertebrate proteins. Paucimannosidic glycans were abundant in the skin secretion of both amphibians; however, hybrid and complex N-glycan structures were detected only in B. raniceps. A good correlation between the structures discovered in glycomic analyses and transcripts encoding enzymes necessary for their synthesis was obtained. Some transcripts such as those of MAN1A2, FUT8, and ST6GALNAC were found solely in B. raniceps. Finally, secreted N- and O- linked glycoproteins were predicted from the transcriptomic data, indicating that proteases and protease inhibitors are putative sources of the glycans described herein. Overall, our results show the presence of oligosaccharides in amphibians skin secretions and suggest that their diversity is species-specific, paving the way for novel perspectives involving amphibian evolution and ecology.

Ecological divergence and synchronous Pleistocene diversification in the widespread South American butter frog complex

Phylogeographic studies primarily focus on the major role of landscape topography in driving lineage diversification. However, populational phylogeographic breaks may also occur as a result of either niche conservatism or divergence, in the absence of geographic barriers to gene flow. Furthermore, these two factors are not mutually exclusive and can act in concert, making it challenging to evaluate their relative importances on explaining genetic variation in nature. Herein, we use sequences of two mitochondrial and four nuclear genes to investigate the timing and diversification patterns of species pertaining to the Leptodactylus latrans complex, which harbors four morphologically cryptic species with broad distributions across environmental gradients in eastern South America. The origin of this species complex dates back to the late Miocene (ca. 5.5 Mya), but most diversification events occurred synchronically during the late Pleistocene likely as the result of ecological divergence driven by Quaternary climatic oscillations. Further, significant patterns of environmental niche divergences among species in the L. latrans complex imply that ecological isolation is the primary mode of genetic diversification, mostly because phylogenetic breaks are associated with environmental transitions rather than topographic barriers at both species and populational scales. We provided new insights about diversification patterns and processes within a species complex of broadly and continuously distributed group of frogs along South America.

Mountain radiations are not only rapid and recent: Ancient diversification of South American frog and lizard families related to Paleogene Andean orogeny and Cenozoic climate variations

Mountainous areas host a disproportionately large fraction of Earth's biodiversity, suggesting a causal relationship between mountain building and biological diversification. Mountain clade radiations are generally associated with changes in environment, climate, and the increase in heterogeneity therein during mountain building. However, examining the causal relationship between mountain building and diversification is a complex challenge, because isolating the effects of surface uplift from other abiotic (climate) or biotic variables is not straightforward. In this study, we investigate the relative contributions of abiotic climate-driven (temperature) and geology-driven (elevation) drivers on evolutionary rates of ancient groups of organisms in the South American Andes. We present regional curves of Andean elevation based on a recent compilation of paleo-elevational data back to the Late Cretaceous, and analyse the diversification history of six Andean frog and lizard families that originated equally far back in time. For two clades (Aromobatidae and Leptodactylidae), we find that they diversified most rapidly during the early phase of mountain building (Late Cretaceous - Paleogene), when the first high-elevation habitats emerged in South America. The diversification of one clade (Centrolenidae) is correlated with Cenozoic temperature variations, with higher speciation rates during warm periods. The last three clades (Dendrobatidae, Hemiphractidae and Liolaemidae) are best explained by environment-independent diversification, although for Liolaemidae, an almost equally strong positive correlation was found between speciation and Andean elevation since the late Eocene. Our findings imply that throughout the long-lived history of surface uplift in the Andes, mountain building drove the diversification of different clades at different times, while not directly affecting other clades. Our study illustrates the importance of paleogeographic reconstructions that capture the complexity and heterogeneity of mountain building in our understanding of the effects that a changing environment plays in shaping biodiversity patterns observed today.

Spatial proximity between polyploids across South American frog genera

AbstractAimPolyploids have been theorized to occur more frequently in environments that are subjected to severe conditions or sudden disruptions. Here we test the expectation that polyploid taxa occur more frequently in extreme or disrupted environments than their diploid counterparts, whether due to increased adaptive potential, environmental resilience or cross‐ploidy competition.LocationSouth America.TaxonAll frog genera in the area with both polyploid and diploid member species (Ceratophrys, Chiasmocleis, Odontophrynus, Phyllomedusa and Pleurodema).MethodsIn all, 13,556 occurrence records of 82 frog species were collected from the Global Biodiversity Information Facility. Species distribution models, range overlap estimates, statistical tests and principal component analyses were used to estimate and compare environments between diploid and polyploid species within and across genera using several categorical and quantitative variables taken from multiple publicly available sources.ResultsAlmost all polyploid occurrences are found within southeastern South America, largely to the exclusion of diploids. Polyploid species occur more closely with intergeneric polyploids than they do with congeneric diploids. Southeastern South America is more temperate, seasonal and less forested when compared to the tropical environments more commonly inhabited by diploids. The habitat ranges of polyploid species are subject to greater temperature fluctuations than diploid species. This region has also experienced major transformations in the modern era, owing to an agriculture boom over the last century. Polyploid occurrences are more likely to be found in areas with greater cropland usage, fertilizer application and pesticide application than diploids.Main ConclusionsAcross species, temperature seasonality was the only variable with strong statistical differences between diploids and polyploids. Greater annual fluctuations in temperature may lead to more established polyploid species due to reasons mentioned above; however, extreme temperature differences are also known to contribute to polyploid gamete formation, providing a possible non‐selective explanation. Polyploid occurrences are also more likely to be found in areas of high agricultural impact, providing support for the hypothesis that polyploids are more resilient to environmental disruptions than diploids.

A New Forest-Dwelling Frog Species of the Genus Adenomera (Leptodactylidae) from Northwestern Brazilian Amazonia

We describe a new species of the South American frog genus Adenomera, based on external morphology, color patterns, advertisement call, and mtDNA sequences. The new species was collected from the Japurá River basin in northwestern Brazilian Amazonia and is distinguished from all congeners by the combination of large snout–vent length (SVL), toe tips unexpanded, presence of antebrachial tubercle on underside of forearm, and by a multi-note advertisement call composed of non-pulsed notes. This new species is part of the A. lutzi clade together with a candidate new species known as Adenomera sp. P and A. lutzi. The three species have the largest SVL in the genus. The presence of toe tips fully expanded and a single-note advertisement call distinguish A. lutzi from the new species. Acoustic and morphological data are still required to assess the taxonomic identity of Adenomera sp. P. Our new species of Adenomera is the third anuran species described from the Solimões-Japurá interfluve. This flags this poorly known region of lowland forests as an important area of species richness in northwestern Amazonia.

Interaction of synthetic antimicrobial peptides of the Hylin a1 family with models of eukaryotic structures: Zwitterionic membranes and DNA

Antimicrobial peptides (AMPs) have been appointed as a possible alternative to traditional antibiotics in face of pathogens increasing resistance to conventional drugs. Hylin a1 (IFGAILPLALGALKNLIK), an AMP extracted from the skin secretion of a South American frog, Hypsiboas albopunctatus, was found to show a strong cytotoxicity against bacteria and fungus, but also a considerable hemolytic action. Considering the toxicity of the peptide in eukaryotic cells, this work focuses on investigating the effects of the interaction of the Hylin a1 analogues W6Hya1, D0W6Hya1 and K0W6Hya1 with models of eukaryotic structures, namely zwitterionic liposomes of dipalmitoyl phosphatidylcholine (DPPC) and calf-thymus DNA (CT DNA). Through intrinsic Trp fluorescence we determined that the peptide affinity for fluid DPPC bilayers follows the decreasing order: D0W6Hya1 (+2) > W6Hya1 (+3) » K0W6Hya1 (+4). Fluorescence data also indicate that the Trp residue in the more positively charged peptide, K0W6Hya1, is less deep in the bilayer than the residue in the other two peptides. This finding is supported by differential scanning calorimetry (DSC) data, which shows that both D0W6Hya1 and W6Hya1 disturb DPPC gel-fluid transition slightly more effectively than K0W6Hya1. DPPC DSC profiles are homogeneously disturbed by the three peptides, probably related to peptide-membrane diffusion. Surprisingly, the peptide that displays the lowest affinity for PC membranes and is located at the more superficial position in the bilayer, K0W6Hya1, is the most efficient in causing formation of pores on the membrane, as attested by carboxyfluorescein leakage assays. The three peptides were found to interact with CT DNA, with a deep penetration of the Trp residue into hydrophobic pockets of the double helix, as indicated by the significant blue shift on the Trp fluorescence, and the displacement of DNA-bound ethidium bromide by the peptides. The experiments of DNA electrophoresis confirm that Hylin peptides bind DNA in a concentration-dependent manner, inducing complete DNA retardation at the relative AMP/plasmid DNA weight ratio of ~17. These findings could help to better understand the AMPs toxic effects on eukaryotic cells, thus contributing to the design of healthier therapeutic agents.

A new species of Dendropsophus (Anura, Hylidae) from southwestern Amazonia with a green bilobate vocal sac.

Recent studies have shown that species diversity of the South American frog genus Dendropsophus is significantly underestimated, especially in Amazonia. Herein, through integrative taxonomy a new species of Dendropsophus from the east bank of the upper Madeira River, Brazil is described. Based on molecular phylogenetic and morphological analyses, the new species is referred to the D. microcephalus species group, where it is differentiated from its congeners mainly by having a green bilobate vocal sac and an advertisement call comprising 1–4 monophasic notes emitted with a dominant frequency of 8,979–9,606 Hz. Based on intensive sampling conducted in the study area over the last ten years, the new species is restricted to the east bank of the upper Madeira River, although its geographic range is expected to include Bolivian forests close to the type locality.

Biomarker and hematological fieldwork with amphibians: is it necessary to sample all night?

In the context of the global amphibian crisis, biomonitoring constitutes a valuable assessment tool to provide critical up to date information on the status and health of amphibians worldwide. The aim of the current study was to evaluate the possible confounding effects of sex, size, and time since capture on enzymatic biomarkers and hematologic parameters of the South American frog Leptodactylus latrans. Frogs were collected by hand between 9 pm and 12 am on two consecutive nights. On the first night, captured frogs were transported for 2h by car to laboratory installations, maintained overnight in plastic containers, and blood and tissue sampled on the next morning. In contrast, frogs collected on the second night were blood and tissue sampled in the field, immediately after the capture period. Hematological parameters were analyzed, and enzymatic activities of catalase, cholinesterase (ChE), and glutathione S-transferase (GST) were determined in the plasma, liver, kidney, and muscle. A sex difference was observed only for total white blood cell counts (WBC), females exhibiting significantly greater values than males. The packed cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), WBC, and muscle ChE activity were significantly correlated with snout-vent length (SVL). The correlation was inversed in the case of MCHC, WBC, and muscle ChE, while the correlation was positive between PCV and SVL. Most examined parameters presented similar values when frogs were sampled at night following capture or the next morning. Total red blood cells (RBCs) count, and plasma enzymatic activities of ChE and GST were the only parameters that presented significantly increased values in morning samplings compared with night samplings. Overall, the current study indicates that it is best to sample the frogs as soon as possible after capture if hematologic or plasmatic biomarkers are examined. Nevertheless, it is possible to sample on the next morning if tissular biomarkers are employed.

Effect of glyphosate (Roundup Active®) on liver of tadpoles of the colombian endemic frog Dendropsophus molitor (amphibia: Anura)

Glyphosate-based herbicide (GBH) using is increasing on a global scale. Few studies have investigated the sub-lethal effects of GBH in endemic amphibian species. The present work tested the GBH Roundup Active® on the tadpoles of Dendropsophus molitor. The exposure was in a range of plausible environmental concentrations (0–0.75 μg a.e./L) during a month. D. molitor is an endemic tropical frog of South America. The exposure from 325 μg a.e./L caused histological alterations in the liver. The high-resolution optical microscopy (HROM) detected sinusoidal dilatation and cytoplasmic vacuolization. The transmission electron microscopy (TEM) showed disorganization of the endoplasmic reticulum. Since the liver is essential for detoxification, these results suggest choric effects. Exposure to another GBH has caused histological alterations in liver tadpoles liver in a previous study, but, this study tested another endemic South-American frog for only 96h. The present work applied HROM to observe lipid alterations since it does not use organic solvents; and TEM for the ultrastructural observation of hepatocytes. Environmental risk of GBH can improve by including sub-lethal effects in endemic species.

Complex patterns of differentiation and gene flow underly the divergence of aposematic phenotypes in Oophaga poison frogs.

Hybridization and introgression can have complex consequences for both species evolution and conservation. Here, we investigated the origin and characteristics of a putative hybrid zone between two South American poison dart frog species, Oophaga anchicayensis and the critically endangered Oophaga lehmanni, which are heavily sought after on the illegal pet market. Using a combination of phenotypic (49 traits) and genomic (ddRADseq) data, we found that the putative hybrids are morphologically distinct from their parental species and confirmed genomic signatures of admixture in these populations. Several lines of evidence (hybrid indices, interspecific hybrid heterozygosity, genomic clines, comparisons with simulated hybrids and demographic modelling) support the conclusion that these populations are not comprised of early-generation hybrids and thus, they probably did not arise as a result of illegal translocations associated with wildlife trafficking. Instead, they probably represent an independent lineage which has persisted through isolation and has only relatively recently re-established gene flow with both parental species. Furthermore, we detected signals of differential introgression from parental species into these hybrid populations which suggest relaxed stabilizing selection on these aposematic colour morphs, potentially via context-dependent female choice. These populations thus provide a fascinating window into the role of hybridization, isolation and female choice in the diversification of South American poison dart frogs. In addition, our results underline the importance of landscape conservation measures to protect, not only known localities of nominal species, but also the phenotypic and genomic variation harbored by admixed lineages which represent crucial repositories for the impressive diversity in this system.

Comparative cytogenetics of the ground frogs Eupsophus emiliopugini Formas, 1989 and E. vertebralis Grandison, 1961 (Alsodidae) with comments on their inter- and intraspecific chromosome differentiation.

South American frogs of the genus Eupsophus Fitzinger, 1843 comprise 10 species. Two of them, Eupsophus vertebralis Grandison, 1961 and E. emiliopugini Formas, 1989 belong to the Eupsophus vertebralis group, exhibiting 2n = 28. Fundamental number differences between these species have been described using conventional chromosome staining of few specimens from only two localities. Here, classical techniques (Giemsa, C-banding, CMA3/DAPI banding, and Ag-NOR staining), and fluorescence in situ hybridization (FISH, with telomeric and 28S ribosomal probes), were applied on individuals of both species collected from 15 localities. We corroborate differences in fundamental numbers (FN) between E. vertebralis and E. emiliopugini through Giemsa staining and C-banding (FN = 54 and 56, respectively). No interstitial fluorescent signals, but clearly stained telomeric regions were detected by FISH using telomeric probe over spreads from both species. FISH with 28S rDNA probes and Ag-NOR staining confirmed the active nucleolus organizer regions signal on pair 5 for both species. Nevertheless, one E. emiliopugini individual from the Puyehue locality exhibited 28S ribosomal signals on pairs 4 and 5. Interestingly, only one chromosome of each pair showed Ag-NOR positive signals, showing a nucleolar dominance pattern. Chromosomal rearrangements, rRNA gene dosage control, mobile NORs elements, and/or species hybridization process could be involved in this interpopulation chromosomal variation.

Can a shade shed light on the monophyly of Cycloramphidae (Lissamphibia: Anura)?

We studied the morphology, development, and the terminology of a lobe over the iris present in three genera of South American cycloramphid frogs (Cycloramphus, Thoropa, and Zachaenus). This research is based on dissections and a macroscopic survey of the eyes of tadpoles, post-metamorphosed, and adults of all six species of Thoropa, nine species of Cycloramphus (3 with adults only, and 6 with tadpoles and adults), and Zachaenus parvulus and Zachaenus carvalhoi (only adults). Because at least three distinctive names are associated with a similar lobe on the iris of frogs from different families, we also discuss the properties that guarantee the observed variations of the involved morphologies as homologues. Lastly, we discuss the possibility that structure present in Cycloramphidae may be the first morphological synapomorphy for the family.

Skin transcriptional profiles in Oophaga poison frogs.

Aposematic organisms advertise their defensive toxins to predators using a variety of warning signals, including bright coloration. While most Neotropical poison frogs (Dendrobatidae) rely on crypsis to avoid predators, Oophaga poison frogs from South America advertise their chemical defenses, a complex mix of diet-derived alkaloids, by using conspicuous hues. The present study aimed to characterize the skin transcriptomic profiles of South American Oophaga poison frogs. Our analyses showed very similar transcriptomic profiles for these closely related species in terms of functional annotation and relative abundance of gene ontology terms expressed. Analyses of expression profiles of Oophaga and available skin transcriptomes of cryptic anurans allowed us to propose initial hypotheses for the active sequestration of alkaloid-based chemical defenses and to highlight some genes that may be potentially involved in resistance mechanisms to avoid self-intoxication and skin coloration. In doing so, we provide an important molecular resource for the study of warning signals that will facilitate the assembly and annotation of future poison frog genomes.

A new species of Rhabdias (Nematoda: Rhabdiasidae), a lung parasite of Pseudopaludicola pocoto (Anura: Leptodactylidae) from north-eastern Brazil: description and phylogenetic analyses.

Rhabdias pocoto n. sp. is herein described from the lungs of the swamp frog Pseudopaludicola pocoto Magalhães, Loebmann, Nogueira, Kokubum, Baptista, Haddad & Garda, 2014, from the Caatinga biome in the state of Ceará, in north-eastern Brazil. The new species is characterized by a body that dilates posteriorly, six small lips (protuberances) and two rounded lateral expansions of cuticular inflation on the anterior end, each containing an amorphous gland-like structure inside and a short and conical tail. Additionally, molecular analysis and comparison of the partial mitochondrial cytochrome c oxidase I sequence of R. pocoto n. sp. revealed genetic divergence between the new species and the sequences of Rhabdias spp. previously deposited in GenBank. Phylogenetic analysis grouped the new taxon into the R. pseudosphaerocephala species complex + R. glaurungi clade. The new discovery represents the 19th species of Rhabdias spp. described in the Neotropical region, the ninth in Brazil and the first species of Rhabdias found parasitizing South American frogs of the genus Pseudopaludicola, as well as the first Caatinga biome species of Rhabdias.

Mechanisms of Convergent Egg Provisioning in Poison Frogs.

Parental provisioning of offspring with physiological products (nursing) occurs in many animals, yet little is known about the neuroendocrine basis of nursing in non-mammalian species. Within amphibians, maternal provisioning has evolved multiple times, with mothers of some species feeding unfertilized eggs to their developing offspring until tadpoles complete metamorphosis [1-3]. We conducted field studies in Ecuador and Madagascar to ask whether convergence at the behavioral level provides similar benefits to offspring and relies on shared neural mechanisms in dendrobatid and mantellid poison frogs. At an ecological level, we found that nursing allows poison frogs to provide chemical defenses to their tadpoles in both species. At the neural level, nursing was associated with increased activity in the lateral septum and preoptic area, demonstrating recruitment of shared brain regions in the convergent evolution of nursing within frogs and across vertebrates [4]. In contrast, only mantellids showed increased oxytocin neuron activity akin to that in nursing mammals [5], suggesting evolutionary versatility in molecular mechanisms. Our findings demonstrate that maternal provisioning provides similar potential benefits to offspring and relies on similar brain regions in poison frog species with convergently evolved toxicity and maternal care. VIDEO ABSTRACT.

The neural basis of tadpole transport in poison frogs.

Parental care has evolved repeatedly and independently across animals. While the ecological and evolutionary significance of parental behaviour is well recognized, underlying mechanisms remain poorly understood. We took advantage of behavioural diversity across closely related species of South American poison frogs (Family Dendrobatidae) to identify neural correlates of parental behaviour shared across sexes and species. We characterized differences in neural induction, gene expression in active neurons and activity of specific neuronal types in three species with distinct care patterns: male uniparental, female uniparental and biparental. We identified the medial pallium and preoptic area as core brain regions associated with parental care, independent of sex and species. The identification of neurons active during parental care confirms a role for neuropeptides associated with care in other vertebrates as well as identifying novel candidates. Our work is the first to explore neural and molecular mechanisms of parental care in amphibians and highlights the potential for mechanistic studies in closely related but behaviourally variable species to help build a more complete understanding of how shared principles and species-specific diversity govern parental care and other social behaviour.

Poison frogs keep a mental map

Frogs aren't necessarily the first family of animals that springs to mind when you think of tree dwellers, but many South American poison frogs spend their early days in the forest canopy. After locating tiny pools of water up in the branches that could be home for individual tadpoles, the parents recall the location of each pool before delivering their freshly hatched tadpoles to their new abodes. But when Lainy Day from the University of Mississippi, USA, tested the place memory of frogs and lizards 20 years ago, none appeared to be capable of forming complex spatial memories; it seemed that the frogs’ brains were just too simple to carry a map. However, Sabrina Burmeister from the University of North Carolina recalls remarking at the time that Day should test the memories of poison frogs. So, when East Carolina University ecologist Kyle Summers recently sent some green-and-black poison dart frogs (Dendrobates auratus) to Burmeister, she and graduate student Yuxiang Liu decided to investigate the remarkable amphibian's spatial awareness.Yet neither Burmeister nor Liu figured on the frogs’ contrariness. Burmeister explains that most researchers test the spatial awareness of rodents using a platform submerged in a pool of water – known as a Morris water maze – surrounded by objects that the animal can use for orientation as they swim in search of the platform. ‘We knew that the frogs hate to swim – they are terrestrial after all’, says Burmeister, so she reasoned the frogs would be eager to locate the platform so they could hop out. However, when Liu introduced the amphibians to the test, they stubbornly refused to search for the platform, preferring instead to swim in never-ending circles around the rim of the pool. They even failed to locate the platform when Liu raised it above the water. ‘[Then] we knew the problem was with the maze and not necessarily the frogs’, says Burmeister. Taking advantage of the frogs’ aversion to swimming, Liu tried immersing a large platform 2 cm beneath the surface – deep enough for the frogs to hop around on while still submerged – and then located a second shallow platform (0.8 cm beneath the surface) on top in the southeast quadrant, which the frogs could clamber onto. He then encouraged the frogs that had successfully located the smaller platform to clamber out by slightly warming the water. Day also suggested that Liu included some moving objects to attract the frogs’ attention, so he decorated the walls of the pond with a red flashing light, an artificial yellow flower, a gently spinning blue fan and green artificial leaves, each located at one of the four points of the compass, to orient the amphibians.Over a two week period, Liu repeatedly released the frogs at various locations on the large platform and allowed them to explore until they reliably located the shallow platform; ‘they seem quick to learn’, says Burmeister. Then Liu played a trick on the unsuspecting amphibians; he switched the locations of the landmarks – effectively rotating their positions by 180 deg – removed the shallow platform and asked the frogs where they thought the platform should be. Filming the animals as they hopped about, Burmeister and Liu were delighted to see that instead of heading to the southeast quadrant, where the platform was located originally, the frogs were misled by the new positions of the landmarks, spending most of their time scouring the northwest quadrant.The frogs were definitely using a mental map of the platform's location relative to the landmarks on the side of the pool and Day adds, ‘Our results show that the ability to use a cognitive map can evolve even in frog brains’.