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.

AbstractMuch of the world’s biodiversity is held within tropical rainforests, which are increasingly fragmented by agricultural practices. In these threatened landscapes, there are many organisms that acquire chemical defenses from their diet and are therefore intimately connected with their local food webs. Poison frogs (Family Dendrobatidae) are one such example, as they acquire alkaloid-based chemical defenses from their diet of leaf litter ants and mites. It is currently unknown how habitat fragmentation impacts chemical defense across trophic levels, from arthropods to frogs. We examined the chemical defenses and diets of the Diablito poison frog (Oophaga sylvatica), and the diversity of their leaf litter ant communities in secondary forest and reclaimed cattle pasture. Notably, this research was performed in collaboration with two high school science classrooms. We found that the leaf litter of forest and pasture frog habitats differed significantly in ant community structure. We also found that forest and pasture frogs differed significantly in diet and alkaloid profiles, where forest frogs contained more of specific alkaloids and ate more ants in both number and volume. Finally, ant species composition of frog diets resembled the surrounding leaf litter, but diets were less variable. This suggests that frogs tend to consume particular ant species within each habitat. To better understand how ants contribute to the alkaloid chemical profiles of frogs, we chemically profiled several ant species and found some alkaloids to be common across many ant species while others are restricted to a few species. At least one alkaloid (223H) found in ants from disturbed sites was also found in skins from pasture. Our experiments are the first to link anthropogenic land use changes to dendrobatid poison frog chemical defenses through variation in leaf litter communities, which has implications for conservation management of these threatened amphibians.

Highlights d South American and Malagasy poison frogs exhibit convergently evolved traits d Both clades are toxic and provide parental care via maternal egg provisioning d Egg-provisioning provides chemical defenses to developing tadpoles in both clades d Provisioning relies on shared brain regions but distinct molecular mechanisms

26understand the mechanisms underlying parental behavior and its evolution are comparative 51 studies across closely-related species that vary in parental care strategies. 52Parental care can be conceptualized as a complex set of inter-related behaviors controlled 53 by brain regions involved in the integration of sensory, social, motivational, and cognitive aspects 54 of care [6]. Across vertebrates, these functions are largely performed by the social decision-55 making network (SDMN; [7]), a highly interconnected group of evolutionarily ancient and 56 functionally conserved brain regions. Although studies on the neural mechanisms of parental 57 behavior are sparse outside mammals, and particularly lacking in amphibians and reptiles, the 58 SDMN provides an ideal starting point for this work as network nodes and connectivity are well 59 understood, highly conserved, and behaviorally important ligand/receptor complexes have been 60 extensively studied. 61Dendrobatid poison frogs show remarkable diversity in parental care across closely 62 related species, including male uniparental care, female uniparental care, and biparental care. 63Parental care in poison frogs involves egg attendance during embryo development, generally 64 followed by transportation of tadpoles "piggyback" to pools of water upon hatching [8][9][10]. In some 65 species, mothers regularly return to nourish growing tadpoles with unfertilized, trophic eggs until 66 the laboratory, allowing us to identify both parental individuals and their non-caregiving partners. 87To control for effects of experience, all pairs successfully reared at least one clutch from egg-88 laying through tadpole transport prior to the experiment. For the non-parental group, we collected 89 frog pairs between parental bouts when they were not caring for eggs or tadpoles, collecting 90 individuals of both the caregiving sex (non-transport; n=10 D. tinctorius, n=7 R. imitator) and their 91 opposite sex partners (non-transport partner; n=9 D. tinctorius, n=8 R. imitator). For the tadpole 92 5 transport group, when we found transporting frogs, we collected both the tadpole transporting 93 individual (tadpole transporter; n=13 D. tinctorius, n=7 R. imitator) and its opposite sex, non-94 transporting partner (transport partner; n=11 D. tinctorius, n=6 R. imitator). All brain tissue was 95 collected in an identical manner: frogs were captured, anesthetized with benzocaine gel, weighed 96 and measured, and euthanized by rapid decapitation. This entire process took less than 5 97 minutes. All procedures were approved by the Harvard University Animal Care and Use 98 Committee (protocol no. 12-10-1). 99 100 Field sample collection 101Oophaga sylvatica (Puerto Quito-Santo Domingo population) were collected in field 102 enclosures in Ecuador in April and May of 2016. We collected non-parental control females (N=8) 103 from enclosures containing only mature females to ensure that frogs were not currently caring for 104 eggs or tadpoles. We collected tadpole transporting females (N=5) fr...

Poison frogs acquire chemical defenses from the environment for protection against potential predators. These defensive chemicals are lipophilic alkaloids that are sequestered by poison frogs from dietary arthropods and stored in skin glands. Despite decades of research focusing on identifying poison frog alkaloids, we know relatively little about how environmental variation and subsequent arthropod availability impacts alkaloid loads in poison frogs. We investigated how seasonal environmental variation influences poison frog chemical profiles through changes in the diet of the Climbing Mantella (Mantella laevigata). We collected M. laevigata females on the Nosy Mangabe island reserve in Madagascar during the wet and dry seasons and tested the hypothesis that seasonal differences in rainfall is associated with changes in diet composition and skin alkaloid profiles of M. laevigata. The arthropod diet of each frog was characterized into five groups (i.e. ants, termites, mites, insect larvae, or ‘other’) using visual identification and cytochrome oxidase 1 DNA barcoding. We found that frog diet differed between the wet and dry seasons, where frogs had a more diverse diet in the wet season and consumed a higher percentage of ants in the dry season. To determine if seasonality was associated with variation in frog defensive chemical composition, we used gas chromatography / mass spectrometry to quantify alkaloids from individual skin samples. Although the assortment of identified alkaloids was similar across seasons, we detected significant differences in the abundance of certain alkaloids, which we hypothesize reflects seasonal variation in the diet of M. laevigata. We suggest that these variations could originate from seasonal changes in either arthropod leaf litter composition or changes in frog behavioral patterns. Although additional studies are needed to understand the consequences of long-term environmental shifts, this work suggests that alkaloid profiles are relatively robust against short-term environmental perturbations.

Much of the world's biodiversity is held within tropical rainforests, which are increasingly fragmented by agricultural practices. In these threatened landscapes, there are many organisms that acquire chemical defenses from their diet and are therefore intimately connected with their local food webs. Poison frogs (Family Dendrobatidae) are one such example, as they acquire alkaloid-based chemical defenses from their diet of leaf litter ants and mites. It is currently unknown how habitat fragmentation impacts chemical defense across trophic levels, from arthropods to frogs. We examined the chemical defenses and diet of the Diablito poison frog (Oophaga sylvatica), and the diversity of their leaf litter ant communities in secondary forest and reclaimed cattle pasture. Notably, this research was performed in collaboration with two high school science classrooms. We found that the leaf litter of forest and pasture frog habitats differed significantly in ant community structure. We also found that forest and pasture frogs differed significantly in diet and alkaloid profiles, where forest frogs contained more of specific alkaloids and ate more ants in both number and volume. Finally, ant species composition of frog diets resembled the surrounding leaf litter, but diets were less variable. This suggests that frogs tend to consume particular ant species within each habitat. To better understand how ants contribute to the alkaloid chemical profiles of frogs, we chemically profiled several ant species and found some alkaloids to be common across many ant species while others are restricted to a few species. At least one alkaloid (223H) found in ants from disturbed sites was also found in skins from pasture frogs. Our experiments are the first to link anthropogenic land use changes to dendrobatid poison frog chemical defenses through variation in leaf litter communities, which has implications for conservation management of these threatened amphibians.

The ability to use small molecule alkaloids as defensive chemicals has evolved in many organisms, often via trophic interactions due to dietary specialization. Animals with diet-derived defenses must balance food choices to maintain their defense reservoirs along with other physiological needs. However, environmental availability of prey and dietary preferences of vertebrate animals with acquired defenses remains largely unexplored. Here, we test the hypothesis that poison frogs that sequester alkaloids from their arthropod prey display prey preference. We collected alkaloid-defended dDiablito frogs (Oophaga sylvatica) and leaf litter samples in five localities in northwestern Ecuador. Additionally, we collected the undefended Chimbo rocket frogs (Hyloxalus infraguttatus) from one locality in which it is living in sympatry with O. sylvatica. We found that both diets and skin alkaloids of O. sylvatica frogs were distinct among localities, and that undefended and sympatric H. infraguttatus ate fewer ants and mites. Unexpectedly, across O. sylvatica populations, we found a negative correlation between their summed skin alkaloid content and number of ants and mites they consumed. Finally, we compared leaf litter ants to frog stomach contents, and found that frogs eat a small subset of the available ant genera found in surrounding leaf litter, as only 13% of all the ants recovered from leaf litter samples belong to the 16 ant genera consumed by the frogs. Our results suggest that the consumption of diet-acquired defenses depends on preference-informed food choices in addition to environmental availability. This impacts our understanding of chemical defenses as a whole, as behavioral reinforcement may be an understudied, yet important selection factor in the evolution of acquired defenses.

The ability to acquire chemical defenses through the diet has evolved across several major taxa. Chemically defended organisms may need to balance chemical defense acquisition and nutritional quality of prey items. However, these dietary preferences and potential trade-offs are rarely considered in the framework of diet-derived defenses. Poison frogs (Family Dendrobatidae) acquire defensive alkaloids from their arthropod diet of ants and mites, although their dietary preferences have never been investigated. We conducted prey preference assays with the Dyeing Poison frog (Dendrobates tinctorius) to test the hypothesis that alkaloid load and prey traits influence frog dietary preferences. We tested size preferences (big versus small) within each of four prey groups (ants, beetles, flies, and fly larvae) and found that frogs preferred interacting with smaller prey items of the fly and beetle groups. Frog taxonomic prey preferences were also tested as we experimentally increased their chemical defense load by feeding frogs decahydroquinoline, an alkaloid compound similar to those naturally found in their diet. Contrary to our expectations, overall preferences did not change during alkaloid consumption, as frogs across groups preferred fly larvae over other prey. Finally, we assessed the protein and lipid content of prey items and found that small ants have the highest lipid content while large fly larvae have the highest protein content. Our results suggest that consideration of toxicity and prey nutritional value are important factors in understanding the evolution of acquired chemical defenses and niche partitioning.