Androgen responsiveness to simulated territorial intrusions in Allobates femoralis males: Evidence supporting the challenge hypothesis in a territorial frog.

Territoriality has been widely described across many animal taxa, where the acquisition and defence of a territory are critical for the fitness of an individual. Extensive evidence suggests that androgens are involved in the modulation of territorial behaviours in male vertebrates. Short-term increase of androgen following a territorial encounter appears to favour the outcome of a challenge. The "Challenge Hypothesis" proposed by Wingfield and colleagues outlines the existence of a positive feedback relationship between androgen and social challenges (e.g., territorial intrusions) in male vertebrates. Here we tested the challenge hypothesis in the highly territorial poison frog, Allobates femoralis, in its natural habitat by exposing males to simulated territorial intrusions in the form of acoustic playbacks. We quantified repeatedly androgen concentrations of individual males via a non-invasive water-borne sampling approach. Our results show that A. femoralis males exhibited a positive behavioural and androgenic response after being confronted to simulated territorial intrusions, providing support for the Challenge Hypothesis in a territorial frog.

Tadpoles of Central Amazonia (Amphibia: Anura).

Amazonian lowland rainforests epitomize, as few other biomes, the terrestrial and freshwater biological diversity of our planet. We provide here a comprehensive description of the larval anurans of Central Amazonia, and their natural history. We base our analyses on fieldwork conducted in six terra-firme rainforest and two várzea floodplain sites during ~60 months between 1990 and 2013, complemented with an examination of museum specimens and a review of published literature. Ninety-nine species of anurans are known to occur in Central Amazonia. Of these, 84 species (85%) have a free-swimming exotrophic larva, six species (6%) have a terrestrial endotrophic larva, one species has a terrestrial exotrophic larva (1%) and another seven species (7%) have direct development of eggs into froglets. The life cycle of one species (1%) remains unknown. We formally describe and illustrate the larval stage of 68 species (i.e., 75% of the species with a known larval stage), five of them previously unkown and nineteen never described for Central Amazonia. We compile, review and update information on species natural history including reproductive modes, habitat use, phenology, and species interactions including diet, predators, competitors and parasites. Finally, we assessed the ecomorphological diversity in the region by the application of a system inspired in Altig & Johnston (1989), later updated by Altig & McDiarmid (1999), which recognizes tadpole guilds based on a combination of morphological and natural history data. Basic scientific information presented in this monograph provide the essential background for further studies on the ecological and evolutionary forces shaping anuran Amazonian assemblages, and their conservation.

Sound radiation pattern of the advertisement call of the highly territorial poison frog Allobates femoralis.

Sound radiation patterns have ecological implications in the effective communication between conspecifics, like optimization of the sound propagation, increase the likelihood to reach mates and to mitigate effects of sound scattering by environmental factors like vegetation. The territorial frog Allobates femoralis advertises its territory against conspecific males and attract females with advertisement calls. Here we report the nearly omnidirectional sound-radiation pattern of the advertisement call of A. femoralis. This sound spreading pattern allows the males to attract mates and repel rivals in all directions. Furthermore, A. femoralis males direct the advertisement call to conspecific neighbours after phonotactic orientation.

Multimodal Signal Testing Reveals Gestural Tapping Behavior in Spotted Reed Frogs.

In the majority of anuran species, acoustic signals are the dominant mode of inter- and intrasexual communication. Male calls are always accompanied by the movement of a more or less conspicuous vocal sac-a potential visual cue. Reed frogs possess a striking vocal sac with a colorful patch of gland tissue clearly visible once the vocal sac is inflated during acoustic signaling. To investigate the visual signal function of vocal sac and gular gland, we presented male Spotted Reed Frogs (Hyperolius puncticulatus) with unimodal and multimodal signal playbacks of conspecific rivals in their natural habitat and recorded their behavioral responses. We found no difference in receiver response to unimodal advertisement call stimuli and to multimodal stimulus presentations of calls combined with visual signals of an artificial vocal sac with or without a gular patch, moving synchronously or asynchronously with the call playback. The inflations of a vocal sac with a colorful gular patch did not alter receiver response and neither increase nor decrease signal salience during male-male communication. Interestingly, males frequently displayed a novel hind and front foot-tapping behavior in response to all playbacks. Comparison of male responses to advertisement and aggressive call playbacks showed that Spotted Reed Frogs approached the sound source less during aggressive call presentations. Tapping behavior was not influenced by either call playback. We suggest that the gestural tapping behavior might act as vibrational signal and discuss its potential signal function in male contests and courtship for females.

Frogolide - An Unprecedented Sesquiterpene Macrolactone from Scent Glands of African Frogs.

Some amphibians use chemical signals in addition to optical and acoustical signals to transmit information. Males of mantellid frogs from Madagascar and hyperoliid frogs from Africa emit complex, species- and sex-specific bouquets of volatiles from their femoral or gular glands. We report here on the identification, synthesis, and determination of the absolute configuration of a macrocyclic lactone occurring in several species of both families, (S)-3,7,11-dodec-6,10-dien-12-olide (S-14, frogolide). Macrolides are a preferred compound class of frog volatiles. Nevertheless, frogolide is the first macrocyclic lactone obviously derived from the terpene pathway, in contrast to known frog macrolides that are usually formed via the fatty acid biosynthetic pathway.

Map-like navigation from distances exceeding routine movements in the three-striped poison frog (Ameerega trivittata).

Most animals move in dense habitats where distant landmarks are limited, but how they find their way around remains poorly understood. Poison frogs inhabit the rainforest understory, where they shuttle tadpoles from small territories to widespread pools. Recent studies revealed their excellent spatial memory and the ability to home back from several hundred meters. It remains unclear whether this homing ability is restricted to the areas that had been previously explored or whether it allows the frogs to navigate from areas outside their direct experience. Here, we used radio-tracking to study the navigational performance of three-striped poison frog translocated outside the area of their routine movements (200-800 m). Translocated frogs returned to their home territory via a direct path from all distances and with little difference in orientation accuracy, suggesting a flexible map-like navigation mechanism. These findings challenge our current understanding of both the mechanisms and the sensory basis of amphibian orientation.

Relying on known or exploring for new? Movement patterns and reproductive resource use in a tadpole-transporting frog.

Animals relying on uncertain, ephemeral and patchy resources have to regularly update their information about profitable sites. For many tropical amphibians, widespread, scattered breeding pools constitute such fluctuating resources. Among tropical amphibians, poison frogs (Dendrobatidae) exhibit some of the most complex spatial and parental behaviors-including territoriality and tadpole transport from terrestrial clutches to ephemeral aquatic deposition sites. Recent studies have revealed that poison frogs rely on spatial memory to successfully navigate through their environment. This raises the question of when and how these frogs gain information about the area and suitable reproductive resources. To investigate the spatial patterns of pool use and to reveal potential explorative behavior, we used telemetry to follow males of the territorial dendrobatid frog Allobates femoralis during tadpole transport and subsequent homing. To elicit exploration, we reduced resource availability experimentally by simulating desiccated deposition sites. We found that tadpole transport is strongly directed towards known deposition sites and that frogs take similar direct paths when returning to their home territory. Frogs move faster during tadpole transport than when homing after the deposition, which probably reflects different risks and costs during these two movement phases. We found no evidence for exploration, neither during transport nor homing, and independent of the availability of deposition sites. We suggest that prospecting during tadpole transport is too risky for the transported offspring as well as for the transporting male. Relying on spatial memory of multiple previously discovered pools appears to be the predominant and successful strategy for the exploitation of reproductive resources in A. femoralis. Our study provides for the first time a detailed description of poison frog movement patterns during tadpole transport and corroborates recent findings on the significance of spatial memory in poison frogs. When these frogs explore and discover new reproductive resources remains unknown.

Acoustic ranging in poison frogs-it is not about signal amplitude alone.

ABSTRACT: Acoustic ranging allows identifying the distance of a sound source and mediates inter-individual spacing and aggression in territorial species. Birds and mammals are known to use more complex cues than only sound pressure level (SPL), which can be influenced by the signaller and signal transmission in non-predictable ways and thus is not reliable by itself. For frogs, only SPL is currently known to mediate inter-individual distances, but we hypothesise that the strong territoriality of Dendrobatids could make the use of complex cues for ranging highly beneficial for this family. Therefore, we tested the ranging abilities of territorial males of Allobates femoralis (Dendrobatidae, Aromobatinae) in playback trials, using amplitude-normalized signals that were naturally degraded over distance, and synthetic signals that were masked with different levels of noise. Frogs responded significantly less to signals recorded from larger distances, regardless of SPL and signal-to-noise ratio (SNR), but showed no differential response to natural minimum and maximum SNRs across the typical communication range in wild populations. This indicates that frogs used signal amplitude and SNR only as ancillary cues when assessing the distance of sound sources and relied instead mainly on more complex cues, such as spectral degradation or reverberation. We suggest that this ability mediates territorial spacing and mate choice in A. femoralis. Good ranging abilities might also play a role in the remarkable orientation performance of this species, probably by enabling the establishment of a mental acoustic map of the habitat. SIGNIFICANCE STATEMENT: Acoustic ranging allows the distance of vocalizing competitors and mates to be identified. While birds and mammals are known to use complex cues such as temporal degradation, frequency-dependent attenuation and reverberation for ranging, previous research indicated that frogs rely only on signal amplitude (sound pressure level) to assess the distance of other callers. The present study shows for the first time that also poison frogs can make use of more complex cues, an ability which is likely to be highly beneficial in their territorial social organization and probably can also be used for orientation.

Increased androgenic sensitivity in the hind limb muscular system marks the evolution of a derived gestural display.

Physical gestures are prominent features of many species' multimodal displays, yet how evolution incorporates body and leg movements into animal signaling repertoires is unclear. Androgenic hormones modulate the production of reproductive signals and sexual motor skills in many vertebrates; therefore, one possibility is that selection for physical signals drives the evolution of androgenic sensitivity in select neuromotor pathways. We examined this issue in the Bornean rock frog (Staurois parvus, family: Ranidae). Males court females and compete with rivals by performing both vocalizations and hind limb gestural signals, called "foot flags." Foot flagging is a derived display that emerged in the ranids after vocal signaling. Here, we show that administration of testosterone (T) increases foot flagging behavior under seminatural conditions. Moreover, using quantitative PCR, we also find that adult male S. parvus maintain a unique androgenic phenotype, in which androgen receptor (AR) in the hind limb musculature is expressed at levels ∼10× greater than in two other anuran species, which do not produce foot flags (Rana pipiens and Xenopus laevis). Finally, because males of all three of these species solicit mates with calls, we accordingly detect no differences in AR expression in the vocal apparatus (larynx) among taxa. The results show that foot flagging is an androgen-dependent gestural signal, and its emergence is associated with increased androgenic sensitivity within the hind limb musculature. Selection for this novel gestural signal may therefore drive the evolution of increased AR expression in key muscles that control signal production to support adaptive motor performance.

Take the long way home: Behaviour of a neotropical frog, Allobates femoralis, in a detour task.

Detour behaviour, an individual's ability to reach its goal by taking an indirect route, has been used to test spatial cognitive abilities across a variety of taxa. Although many amphibians show a strong homing ability, there is currently little evidence of amphibian spatial cognitive flexibility. We tested whether a territorial frog, Allobates femoralis, can flexibly adjust its homing path when faced with an obstacle. We displaced male frogs from their calling sites into the centre of circular arenas and recorded their escape routes. In the first experiment we provided an arena with equally high walls. In the second experiment we doubled the height of the homeward facing wall. Finally, we provided a tube as a shortcut through the high wall. In the equal-height arena, most frogs chose to escape via the quadrant facing their former calling site. However, when challenged with different heights, nearly all frogs chose the low wall, directing their movements away from the calling site. In the "escape tunnel" experiment most frogs still chose the low wall. Our results show that displaced A. femoralis males can flexibly adjust their homing path and avoid (presumably energetically costly) obstacles, providing experimental evidence of spatial cognitive flexibility in an amphibian.

Identification, synthesis and mass spectrometry of a macrolide from the African reed frog Hyperolius cinnamomeoventris.

The contents of the gular glands of the male African reed frog Hyperolius cinnamomeoventris consist of a mixture of aliphatic macrolides and sesquiterpenes. While the known macrolide gephyromantolide A was readily identified, the structure of another major component was suggested to be a tetradecen-13-olide. The synthesis of the two candidate compounds (Z)-5- and (Z)-9-tetradecen-13-olide revealed the former to be the naturally occurring compound. The synthesis used ring-closing metathesis as key step. While the Hoveyda-Grubbs catalyst furnished a broad range of isomeric products, the (Z)-selective Grubbs catalyst lead to pure (Z)-products. Analysis by chiral GC revealed the natural frog compound to be (5Z,13S)-5-tetradecen-13-olide (1). This compound is also present in the secretion of other hyperoliid frogs as well as in femoral glands of male mantellid frogs such as Spinomantis aglavei. The mass spectra of the synthesized macrolides as well as their rearranged isomers obtained during ring-closing metathesis showed that it is possible to assign the location of the double bond in an unsaturated macrolide on the basis of its EI mass spectrum. The occurrence of characteristic ions can be explained by the fragmentation pathway proposed in the article. In contrast, the localization of a double bond in many aliphatic open-chain compounds like alkenes, alcohols or acetates, important structural classes of pheromones, is usually not possible from an EI mass spectrum. In the article, we present the synthesis and for the first time elucidate the structure of macrolides from the frog family Hyperoliidae.

The significance of spatial memory for water finding in a tadpole-transporting frog.

The ability to associate environmental cues with valuable resources strongly increases the chances of finding them again, and thus memory often guides animal movement. For example, many temperate region amphibians show strong breeding site fidelity and will return to the same areas even after the ponds have been destroyed. In contrast, many tropical amphibians depend on exploitation of small, scattered and fluctuating resources such as ephemeral pools for reproduction. It remains unknown whether tropical amphibians rely on spatial memory for effective exploitation of their reproductive resources. Poison frogs (Dendrobatidae) routinely shuttle their tadpoles from terrestrial clutches to dispersed aquatic deposition sites. We investigated the role of spatial memory for relocating previously discovered deposition sites in an experimental population of the brilliant-thighed poison frog, Allobates femoralis, a species with predominantly male tadpole transport. We temporarily removed an array of artificial pools that served as the principal tadpole deposition resource for the population. In parallel, we set up an array of sham sites and sites containing conspecific tadpole odour cues. We then quantified the movement patterns and site preferences of tadpole-transporting males by intensive sampling of the area and tracking individual frogs. We found that tadpole-carrier movements were concentrated around the exact locations of removed pools and most individuals visited several removed pool sites. In addition, we found that tadpole-transporting frogs were attracted to novel sites that contained high concentrations of conspecific olfactory tadpole cues. Our results suggest that A. femoralis males rely heavily on spatial memory for efficient exploitation of multiple, widely dispersed deposition sites once they are discovered. Additionally, olfactory cues may facilitate the initial discovery of the new sites.

Low reproductive skew despite high male-biased operational sex ratio in a glass frog with paternal care.

BACKGROUND: Reproductive skew, the uneven distribution of reproductive success among individuals, is a common feature of many animal populations. Several scenarios have been proposed to favour either high or low levels of reproductive skew. Particularly a male-biased operational sex ratio and the asynchronous arrival of females is expected to cause high variation in reproductive success among males. Recently it has been suggested that the type of benefits provided by males (fixed vs. dilutable) could also strongly impact individual mating patterns, and thereby affecting reproductive skew. We tested this hypothesis in Hyalinobatrachium valerioi, a Neotropical glass frog with prolonged breeding and paternal care. RESULTS: We monitored and genetically sampled a natural population in southwestern Costa Rica during the breeding season in 2012 and performed parentage analysis of adult frogs and tadpoles to investigate individual mating frequencies, possible mating preferences, and estimate reproductive skew in males and females. We identified a polygamous mating system, where high proportions of males (69 %) and females (94 %) reproduced successfully. The variance in male mating success could largely be attributed to differences in time spent calling at the reproductive site, but not to body size or relatedness. Female H. valerioi were not choosy and mated indiscriminately with available males. CONCLUSIONS: Our findings support the hypothesis that dilutable male benefits - such as parental care - can favour female polyandry and maintain low levels of reproductive skew among males within a population, even in the presence of direct male-male competition and a highly male-biased operational sex ratio. We hypothesize that low male reproductive skew might be a general characteristic in prolonged breeders with paternal care.

Flexible compensation of uniparental care: female poison frogs take over when males disappear.

Parental care systems are shaped by costs and benefits to each sex of investing into current versus future progeny. Flexible compensatory parental care is mainly known in biparental species, particularly where parental desertion or reduction of care by 1 parent is common. The other parent can then compensate this loss by either switching parental roles and/or by increasing its own parental effort. In uniparental species, desertion of the caregiver usually leads to total brood loss. In the poison frog, Allobates femoralis, obligatory tadpole transport (TT) is generally performed by males, whereas females abandon their clutches after oviposition. Nevertheless, in a natural population we previously observed 7.8% of TT performed by females, which we could link to the absence of the respective fathers. In the following experiment, under laboratory conditions, all tested A. femoralis females flexibly took over parental duties, but only when their mates were removed. Our findings provide clear evidence for compensatory flexibility in a species with unisexual parental care. Contrary to the view of amphibian parental care as being stereotypical and fixed, these results demonstrate behavioral flexibility as an adaptive response to environmental and social uncertainty. Behavioral flexibility might actually represent a crucial step in the evolutionary transition from uniparental to biparental care in poison frogs. We suspect that across animal species flexible parental roles are much more common than previously thought and suggest the idea of a 3-dimensional continuum regarding flexibility, parental involvement, and timing, when thinking about the evolution of parental care.

Ontogenetic Change of Signal Brightness in the Foot-Flagging Frog Species Staurois parvus and Staurois guttatus.

Adult individuals of several anuran species exhibit conspicuous visual displays during intraspecific communication. While signal properties in adults have been subject to an increasing number of studies, little is known about the variation of visual signals in juveniles and during ontogenetic changes. Foot-flagging signals of the Bornean frogs Staurois guttatus and S. parvus were observed in juveniles a few days after metamorphosis. We investigated color parameters of foot webbings and body coloration of individuals bred at the Vienna Zoo, and their relation to age and body size using spectrophotometry. Our results indicate that the brightness of foot webbings of S. guttatus and S. parvus increased with increasing age. Additionally, we compared the results with measurements of adult individuals from a population in Brunei and discuss possible differences related to diet and age as well as the habitat use of juveniles and adults. We suggest that the ontogenetic increase in foot-webbing brightness enhances visual conspicuousness and the signal-to-noise ratio of the visual signal with sexual maturity and potentially functions as cue to the age of the signaler.

Brood-partitioning behaviour in unpredictable environments: hedging the bets?

Spreading reproduction across time or space can optimize fitness by minimizing the risks for offspring survival in varying and unpredictable environments. Poison frogs (Dendrobatidae) are characterized by complex spatial and reproductive behaviour, such as territoriality, prolonged courtship and parental care. The partitioning of larvae from terrestrial clutches across several water bodies is mainly known from species with carnivorous tadpoles that allocate their tadpoles in very small pools, where limited food availability is accompanied by an increased risk of cannibalism. However, little is known about the deposition behaviour of non-carnivorous species that use medium-sized to large pools. In the present study, we investigated whether the Neotropical poison frog Allobates femoralis exhibits brood-partitioning behaviour when males transport tadpoles 3 weeks after oviposition. We sampled 30 artificial water bodies for tadpoles, which we genotyped at seven highly polymorphic microsatellite loci. Based on the reconstructed pedigree, we show that A. femoralis males distribute larvae of single and of successive clutches across several water bodies. The number of pools used was significantly associated with the number of clutches per male. Ninety-three percent of the males that were assigned to more than one clutch spread their tadpoles across several water bodies. Given the highly variable and unpredictable biotic and abiotic conditions in tropical rainforest, at the spatial scale of the study species' behaviour, we interpret this behaviour as bet-hedging to improve offspring survival.

Populations, pools, and peccaries: simulating the impact of ecosystem engineers on rainforest frogs.

"Ecosystem engineering" describes habitat alteration by an organism that affects another organism; such nontrophic interactions between organisms are a current focus in ecological research. Our study quantifies the actual impact an ecosystem engineer can have on another species by using a previously identified model system-peccaries and rainforest frogs. In a 4-year experiment, we simulated the impact of peccaries on a population of Allobates femoralis (Dendrobatidae) by installing an array of artificial pools to mimic a forest patch modified by peccaries. The data were analyzed using a gradual before-after control-impact (gBACI) model. Following the supplementation, population size almost doubled as a result of increased autochthonous recruitment driven by a higher per-capita reproduction of males and a higher proportion of reproducing females. The effect was evenly distributed across the population. The differential response of males and females reflects the reproductive behavior of A. femoralis, as only the males use the aquatic sites for tadpole deposition. Our study shows that management and conservation must consider nontrophic relationships and that human "ecosystem engineering" can play a vital role in efforts against the "global amphibian decline."

The anuran vocal sac: a tool for multimodal signalling.

Although in anurans the predominant mode of intra- and intersexual communication is vocalization, modalities used in addition to or instead of acoustic signals range from seismic and visual to chemical. In some cases, signals of more than one modality are produced through or by the anuran vocal sac. However, its role beyond acoustics has been neglected for some time and nonacoustic cues such as vocal sac movement have traditionally been seen as an epiphenomenon of sound production. The diversity in vocal sac coloration and shape found in different species is striking and recently its visual properties have been given a more important role in signalling. Chemosignals seem to be the dominant communication mode in newts, salamanders and caecilians and certainly play a role in the aquatic life phase of anurans, but airborne chemical signalling has received less attention. There is, however, increasing evidence that at least some terrestrial anuran species integrate acoustic, visual and chemical cues in species recognition and mate choice and a few secondarily mute anuran species seem to fully rely on volatile chemical cues produced in glands on the vocal sac. Within vertebrates, frogs in particular are suitable organisms for investigating multimodal communication by means of experiments, since they are tolerant of disturbance by observers and can be easily manipulated under natural conditions. Thus, the anuran vocal sac might be of great interest not only to herpetologists, but also to behavioural biologists studying communication systems.

Poison frogs rely on experience to find the way home in the rainforest.

Among vertebrates, comparable spatial learning abilities have been found in birds, mammals, turtles and fishes, but virtually nothing is known about such abilities in amphibians. Overall, amphibians are the most sedentary vertebrates, but poison frogs (Dendrobatidae) routinely shuttle tadpoles from terrestrial territories to dispersed aquatic deposition sites. We hypothesize that dendrobatid frogs rely on learning for flexible navigation. We tested the role of experience with the local cues for poison frog way-finding by (i) experimentally displacing territorial males of Allobates femoralis over several hundred metres, (ii) using a harmonic direction finder with miniature transponders to track these small frogs, and (iii) using a natural river barrier to separate the translocated frogs from any familiar landmarks. We found that homeward orientation was disrupted by the translocation to the unfamiliar area but frogs translocated over similar distances in their local area showed significant homeward orientation and returned to their territories via a direct path. We suggest that poison frogs rely on spatial learning for way-finding in their local area.

Characterisation of nine new polymorphic microsatellite loci in the reticulated glass frog Hyalinobatrachium valerioi (Centrolenidae).

Here we document the development of thirteen novel microsatellite markers for the reticulated glass frog Hyalinobatrachium valerioi (Centrolenidae). Nine of those markers were polymorphic and contained between 4 and 34 alleles per locus (mean = 20.3) in 138 individuals (91 males, 47 females) from the field site 'La Gamba', Costa Rica. Average observed heterozygosity was 0.76. Two loci (Hyval19 and Hyval21) significantly deviated from Hardy-Weinberg equilibrium. We did not find evidence for linkage disequilibrium among any of the loci. These markers will serve to identify the genetic mating system in H. valerioi, investigate gene flow between local populations, and reconstruct parent-offspring relationships for studies on individual mating and reproductive success. Therefore, these markers will serve to answer a wide range of scientific questions in conservation, behavioural ecology, and also evolutionary biology.