Development of assisted reproductive technologies for the conservation of Atelopus sp. (spumarius complex).

Amphibians are in peril, given the ongoing sixth mass extinction of wildlife. Thus, Conservation Breeding Programs (CBPs) are attempting to breed some species under laboratory conditions. The incorporation of assisted reproduction technologies (ARTs), such as hormonal stimulation, sperm collection and cryopreservation, and in vitro fertilization is contributing to successful CBPs. The objective of this study was to apply ARTs in sexually mature individuals of an undescribed species of Atelopus (spumarius complex) (harlequin frog). Our procedure involves hormonal induction of gametogenesis in this species. We were able to induce gamete release through administration of human chorionic gonadotropin (hCG) in males, and in females this has been achieved through the sequential administration of hCG (priming doses), and combinations of hCG with gonadotropin releasing hormone analogue, GnRHa (ovulary dose). We standardized sperm cryopreservation by performing toxicity tests of cryoprotectants, fast/slow freezing and thawing, as well as supplementation of non-penetrating cryoprotectants (sugars). Next, we performed in vitro fertilization, evaluated the fertilization capacity of the cryopreserved sperm, and describe external features of fresh and cryopreserved sperm. We found that 10 IU/g hCG induced the release of the highest sperm concentrations between 3 and 5 h post-injection, while 2.5 IU/g hCG induced the release of eggs in most treated females. Under cryopreservation conditions, the highest recovery of forward progressive motility or FPM was 26.3 ± 3.5%, which was obtained in cryosuspensions prepared with the 5% DMF and 2.5% sucrose. Cryopreserved sperm showed narrower mitochondrial vesicles after thawing, while in frozen samples without cryodiluent showed 31% of spermatozoa lost their tails. In most cases, our attempts of in vitro fertilization were successful. However, only ∼10% of embryos were viable. Overall, our study demonstrates that the development of ARTs in individuals of Atelopus sp. (spumarius complex) bred in laboratory can be successful, which result in viable offspring through in vitro fertilization. Our study provides a baseline for assisted breeding protocols applicable to other harlequin frogs of the genus Atelopus.

Whispers from vestigial nubbins: Arrested development provokes trait loss in toads.

Despite the use of acoustic communication, many species of toads (family Bufonidae) have lost parts of the tympanic middle ear, representing at least 12 independent evolutionary occurrences of trait loss. The comparative development of the tympanic middle ear in toads is poorly understood. Here, we compared middle ear development among two pairs of closely related toad species in the genera Atelopus and Rhinella that have (eared) or lack (earless) middle ear structures. We bred toads in Peru and Ecuador, preserved developmental series from tadpoles to juveniles, and examined ontogenetic timing and volume of the otic capsule, oval window, operculum, opercularis muscle, columella (stapes), and extracolumella in three-dimensional histological reconstructions. All species had similar ontogenesis of the otic capsule, oval window, operculum, and opercularis muscle. Moreover, cell clusters of primordial columella in the oval window appeared just before metamorphosis in both eared and earless lineages. However, in earless lineages, the cell clusters either remained as small nubbins or cell buds in the location of the columella footplate within the oval window or disappeared by juvenile and adult stages. Thus, columella growth began around metamorphosis in all species but was truncated and/or degenerated after metamorphosis in earless species, leaving earless adults with morphology typical of metamorphic anurans. Shifts in the timing or expression of biochemical pathways that regulate the extension or differentiation of the columella after metamorphosis may be the developmental mechanism underlying convergent trait loss among toad lineages.

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.

Molecular physiology of chemical defenses in a poison frog.

Poison frogs sequester small molecule lipophilic alkaloids from their diet of leaf litter arthropods for use as chemical defenses against predation. Although the dietary acquisition of chemical defenses in poison frogs is well documented, the physiological mechanisms of alkaloid sequestration has not been investigated. Here, we used RNA sequencing and proteomics to determine how alkaloids impact mRNA or protein abundance in the little devil frog (Oophaga sylvatica), and compared wild-caught chemically defended frogs with laboratory frogs raised on an alkaloid-free diet. To understand how poison frogs move alkaloids from their diet to their skin granular glands, we focused on measuring gene expression in the intestines, skin and liver. Across these tissues, we found many differentially expressed transcripts involved in small molecule transport and metabolism, as well as sodium channels and other ion pumps. We then used proteomic approaches to quantify plasma proteins, where we found several protein abundance differences between wild and laboratory frogs, including the amphibian neurotoxin binding protein saxiphilin. Finally, because many blood proteins are synthesized in the liver, we used thermal proteome profiling as an untargeted screen for soluble proteins that bind the alkaloid decahydroquinoline. Using this approach, we identified several candidate proteins that interact with this alkaloid, including saxiphilin. These transcript and protein abundance patterns suggest that the presence of alkaloids influences frog physiology and that small molecule transport proteins may be involved in toxin bioaccumulation in dendrobatid poison frogs.

Diversity within diversity: Parasite species richness in poison frogs assessed by transcriptomics.

Symbionts (e.g., endoparasites and commensals) play an integral role in their host's ecology, yet in many cases their diversity is likely underestimated. Although endoparasites are traditionally characterized using morphology, sequences of conserved genes, and shotgun metagenomics, host transcriptomes constitute an underused resource to identify these organisms' diversity. By isolating non-host transcripts from host transcriptomes, individual host tissues can now simultaneously reveal their endoparasite species richness (i.e., number of different taxa) and provide insights into parasite gene expression. These approaches can be used in host taxa whose endoparasites are mostly unknown, such as those of tropical amphibians. Here, we focus on the poison frogs (Dendrobatidae) as hosts, which are a Neotropical clade known for their bright coloration and defensive alkaloids. These toxins are an effective protection against vertebrate predators (e.g., snakes and birds), bacteria, and skin-biting ectoparasites (e.g., mosquitoes); however, little is known about their deterrence against eukaryotic endoparasites. With de novo transcriptomes of dendrobatids, we developed a bioinformatics pipeline for endoparasite identification that uses host annotated RNA-seq data and set of a priori parasite taxonomic terms, which are used to mine for specific endoparasites. We found a large community of helminths and protozoans that were mostly restricted to the digestive tract and a few systemic parasites (e.g., Trypanosoma). Contrary to our expectations, all dendrobatid frogs regardless of the presence of alkaloid defenses have endoparasites, with their highest species richness located in the frog digestive tract. Some of these organisms (e.g., roundworms) might prove to be generalists, as they were not found to be co-diversifying with their frog hosts. We propose that endoparasites may escape poison frogs' chemical defenses by colonizing tissues with fewer alkaloids than the frog's skin, where most toxins are stored.

Sensitive high-frequency hearing in earless and partially eared harlequin frogs (Atelopus).

Harlequin frogs, genus Atelopus, communicate at high frequencies despite most species lacking a complete tympanic middle ear that facilitates high-frequency hearing in most anurans and other tetrapods. Here, we tested whether Atelopus are better at sensing high-frequency acoustic sound compared with other eared and earless species in the Bufonidae family, determined whether middle ear variation within Atelopus affects hearing sensitivity and tested potential hearing mechanisms in Atelopus We determined that at high frequencies (2000-4000 Hz), Atelopus are 10-34 dB more sensitive than other earless bufonids but are relatively insensitive to mid-range frequencies (900-1500 Hz) compared with eared bufonids. Hearing among Atelopus species is fairly consistent, evidence that the partial middle ears present in a subset of Atelopus species do not convey a substantial hearing advantage. We further demonstrate that Atelopus hearing is probably not facilitated by vibration of the skin overlying the normal tympanic membrane region or the body lung wall, leaving the extratympanic hearing pathways in Atelopus enigmatic. Together, these results show Atelopus have sensitive high-frequency hearing without the aid of a tympanic middle ear and prompt further study of extratympanic hearing mechanisms in anurans.

Earless toads sense low frequencies but miss the high notes.

Sensory losses or reductions are frequently attributed to relaxed selection. However, anuran species have lost tympanic middle ears many times, despite anurans' use of acoustic communication and the benefit of middle ears for hearing airborne sound. Here we determine whether pre-existing alternative sensory pathways enable anurans lacking tympanic middle ears (termed earless anurans) to hear airborne sound as well as eared species or to better sense vibrations in the environment. We used auditory brainstem recordings to compare hearing and vibrational sensitivity among 10 species (six eared, four earless) within the Neotropical true toad family (Bufonidae). We found that species lacking middle ears are less sensitive to high-frequency sounds, however, low-frequency hearing and vibrational sensitivity are equivalent between eared and earless species. Furthermore, extratympanic hearing sensitivity varies among earless species, highlighting potential species differences in extratympanic hearing mechanisms. We argue that ancestral bufonids may have sufficient extratympanic hearing and vibrational sensitivity such that earless lineages tolerated the loss of high frequency hearing sensitivity by adopting species-specific behavioural strategies to detect conspecifics, predators and prey.

Ant and Mite Diversity Drives Toxin Variation in the Little Devil Poison Frog.

Poison frogs sequester chemical defenses from arthropod prey, although the details of how arthropod diversity contributes to variation in poison frog toxins remains unclear. We characterized skin alkaloid profiles in the Little Devil poison frog, Oophaga sylvatica (Dendrobatidae), across three populations in northwestern Ecuador. Using gas chromatography/mass spectrometry, we identified histrionicotoxins, 3,5- and 5,8-disubstituted indolizidines, decahydroquinolines, and lehmizidines as the primary alkaloid toxins in these O. sylvatica populations. Frog skin alkaloid composition varied along a geographical gradient following population distribution in a principal component analysis. We also characterized diversity in arthropods isolated from frog stomach contents and confirmed that O. sylvatica specialize on ants and mites. To test the hypothesis that poison frog toxin variability reflects species and chemical diversity in arthropod prey, we (1) used sequencing of cytochrome oxidase 1 to identify individual prey specimens, and (2) used liquid chromatography/mass spectrometry to chemically profile consumed ants and mites. We identified 45 ants and 9 mites in frog stomachs, including several undescribed species. We also showed that chemical profiles of consumed ants and mites cluster by frog population, suggesting different frog populations have access to chemically distinct prey. Finally, by comparing chemical profiles of frog skin and isolated prey items, we traced the arthropod source of four poison frog alkaloids, including 3,5- and 5,8-disubstituted indolizidines and a lehmizidine alkaloid. Together, the data show that toxin variability in O. sylvatica reflects chemical diversity in arthropod prey.

Response to Heethoff, Norton, and Raspotnig: Ant and Mite Diversity Drives Toxin Variation in the Little Devil Poison Frog and Erratum.

Our recent publication titled "Ant and Mite Diversity Drives Toxin Variation in the Little Devil Poison Frog" aimed to describe how variation in diet contributes to population differences in toxin profiles of poison frogs. Some poison frogs (Family Dendrobatidae) sequester alkaloid toxins from their arthropod diet, which is composed mainly of ants and mites. Our publication demonstrated that arthropods from the stomach contents of three different frog populations were diverse in both chemistry and species composition. To make progress towards understanding this trophic relationship, our main goal was to identify alkaloids that are found in either ants or mites. With the remaining samples that were not used for chemical analysis, we attempted to identify the arthropods using DNA barcoding of cytochrome oxidase 1 (CO1). The critique of Heethoff, Norton, and Raspotnig refers to the genetic analysis of a small number of mites. Here, we respond to the general argument of the critique as well as other minor issues detailed by Heethoff, Norton, and Raspotnig.

Aposematism increases acoustic diversification and speciation in poison frogs.

Multimodal signals facilitate communication with conspecifics during courtship, but they can also alert eavesdropper predators. Hence, signallers face two pressures: enticing partners to mate and avoiding detection by enemies. Undefended organisms with limited escape abilities are expected to minimize predator recognition over mate attraction by limiting or modifying their signalling. Alternatively, organisms with anti-predator mechanisms such as aposematism (i.e. unprofitability signalled by warning cues) might elaborate mating signals as a consequence of reduced predation. We hypothesize that calls diversified in association with aposematism. To test this, we assembled a large acoustic signal database for a diurnal lineage of aposematic and cryptic/non-defended taxa, the poison frogs. First, we showed that aposematic and non-aposematic species share similar extinction rates, and aposematic lineages diversify more and rarely revert to the non-aposematic phenotype. We then characterized mating calls based on morphological (spectral), behavioural/physiological (temporal) and environmental traits. Of these, only spectral and temporal features were associated with aposematism. We propose that with the evolution of anti-predator defences, reduced predation facilitated the diversification of vocal signals, which then became elaborated or showy via sexual selection.

A toxic environment selects for specialist microbiome in poison frogs.

Shifts in microbiome community composition can have large effects on host health. It is therefore important to understand how perturbations, like those caused by the introduction of exogenous chemicals, modulate microbiome community composition. In poison frogs within the family Dendrobatidae, the skin microbiome is exposed to the alkaloids that the frogs sequester from their diet and use for defense. Given the demonstrated antimicrobial effects of these poison frog alkaloids, these compounds may be structuring the skin microbial community. To test this, we first characterized microbial communities from chemically defended and closely related non-defended frogs from Ecuador. Then we conducted a laboratory experiment to monitor the effect of the alkaloid decahydroquinoline (DHQ) on the microbiome of a single frog species. In both the field and lab experiments, we found that alkaloid-exposed microbiomes are more species rich and phylogenetically diverse, with an increase in rare taxa. To better understand the strain-specific behavior in response to alkaloids, we cultured microbial strains from poison frog skin and found the majority of strains exhibited either enhanced growth or were not impacted by the addition of DHQ. Additionally, stable isotope tracing coupled to nanoSIMS suggests that some of these strains are able to metabolize DHQ. Taken together, these data suggest that poison frog chemical defenses open new niches for skin-associated microbes with specific adaptations, including the likely metabolism of alkaloids, that enable their survival in this toxic environment. This work helps expand our understanding of how exposure to exogenous compounds like alkaloids can impact host microbiomes.

Path for recovery: an ecological overview of the Jambato Harlequin Toad (Bufonidae: Atelopus ignescens) in its last known locality, Angamarca Valley, Ecuador.

The Jambato Harlequin toad (Atelopus ignescens), a formerly abundant species in the Andes of Ecuador, faced a dramatic population decline in the 1980s, with its last recorded sighting in 1988. The species was considered Extinct by the IUCN until 2016, when a fortuitous discovery of one Jambato by a local boy reignited hope. In this study, we present findings from an investigation conducted in the Angamarca parish, focusing on distribution, abundance, habitat preferences, ecology, disease susceptibility, and dietary habits of the species. In one year we identified 71 individuals at different stages of development in various habitats, with a significant presence in agricultural mosaic areas and locations near water sources used for crop irrigation, demonstrating the persistence of the species in a complex landscape, with considerable human intervention. The dietary analysis based on fecal samples indicated a diverse prey selection, primarily comprising arthropods such as Acari, Coleoptera, and ants. Amphibian declines have been associated with diseases and climate change; notably, our study confirmed the presence of the pathogen Batrachochytrium dendrobatidis (Bd), but, surprisingly, none of the infected Jambatos displayed visible signs of illness. When analyzing climatic patterns, we found that there are climatic differences between historical localities and Angamarca; the temporal analysis also exposes a generalized warming trend. Finally, in collaboration with the local community, we developed a series of management recommendations for terrestrial and aquatic environments occupied by the Jambato.

Binding and sequestration of poison frog alkaloids by a plasma globulin.

Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of 'free' plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms.

Amazonian amphibian diversity is primarily derived from late Miocene Andean lineages.

The Neotropics contains half of remaining rainforests and Earth's largest reservoir of amphibian biodiversity. However, determinants of Neotropical biodiversity (i.e., vicariance, dispersals, extinctions, and radiations) earlier than the Quaternary are largely unstudied. Using a novel method of ancestral area reconstruction and relaxed Bayesian clock analyses, we reconstructed the biogeography of the poison frog clade (Dendrobatidae). We rejected an Amazonian center-of-origin in favor of a complex connectivity model expanding over the Neotropics. We inferred 14 dispersals into and 18 out of Amazonia to adjacent regions; the Andes were the major source of dispersals into Amazonia. We found three episodes of lineage dispersal with two interleaved periods of vicariant events between South and Central America. During the late Miocene, Amazonian, and Central American-Chocoan lineages significantly increased their diversity compared to the Andean and Guianan-Venezuelan-Brazilian Shield counterparts. Significant percentage of dendrobatid diversity in Amazonia and Chocó resulted from repeated immigrations, with radiations at <10.0 million years ago (MYA), rather than in situ diversification. In contrast, the Andes, Venezuelan Highlands, and Guiana Shield have undergone extended in situ diversification at near constant rate since the Oligocene. The effects of Miocene paleogeographic events on Neotropical diversification dynamics provided the framework under which Quaternary patterns of endemism evolved.

Widespread amphibian extinctions from epidemic disease driven by global warming.

As the Earth warms, many species are likely to disappear, often because of changing disease dynamics. Here we show that a recent mass extinction associated with pathogen outbreaks is tied to global warming. Seventeen years ago, in the mountains of Costa Rica, the Monteverde harlequin frog (Atelopus sp.) vanished along with the golden toad (Bufo periglenes). An estimated 67% of the 110 or so species of Atelopus, which are endemic to the American tropics, have met the same fate, and a pathogenic chytrid fungus (Batrachochytrium dendrobatidis) is implicated. Analysing the timing of losses in relation to changes in sea surface and air temperatures, we conclude with 'very high confidence' (> 99%, following the Intergovernmental Panel on Climate Change, IPCC) that large-scale warming is a key factor in the disappearances. We propose that temperatures at many highland localities are shifting towards the growth optimum of Batrachochytrium, thus encouraging outbreaks. With climate change promoting infectious disease and eroding biodiversity, the urgency of reducing greenhouse-gas concentrations is now undeniable.

Contrasting parental roles shape sex differences in poison frog space use but not navigational performance.

Sex differences in vertebrate spatial abilities are typically interpreted under the adaptive specialization hypothesis, which posits that male reproductive success is linked to larger home ranges and better navigational skills. The androgen spillover hypothesis counters that enhanced male spatial performance may be a byproduct of higher androgen levels. Animal groups that include species where females are expected to outperform males based on life-history traits are key for disentangling these hypotheses. We investigated the association between sex differences in reproductive strategies, spatial behavior, and androgen levels in three species of poison frogs. We tracked individuals in natural environments to show that contrasting parental sex roles shape sex differences in space use, where the sex performing parental duties shows wider-ranging movements. We then translocated frogs from their home areas to test their navigational performance and found that the caring sex outperformed the non-caring sex only in one out of three species. In addition, males across species displayed more explorative behavior than females and androgen levels correlated with explorative behavior and homing accuracy. Overall, we reveal that poison frog reproductive strategies shape movement patterns but not necessarily navigational performance. Together this work suggests that prevailing adaptive hypotheses provide an incomplete explanation of sex differences in spatial abilities.

Red List assessment of amphibian species of Ecuador: A multidimensional approach for their conservation.

Ecuador is one of the most biodiverse countries in the world, but faces severe pressures and threats to its natural ecosystems. Numerous species have declined and require to be objectively evaluated and quantified, as a step towards the development of conservation strategies. Herein, we present an updated National Red List Assessment for amphibian species of Ecuador, with one of the most detailed and complete coverages for any Ecuadorian taxonomic group to date. Based on standardized methodologies that integrate taxonomic work, spatial analyses, and ecological niche modeling, we assessed the extinction risk and identified the main threats for all Ecuadorian native amphibians (635 species), using the IUCN Red List Categories and Criteria. Our evaluation reveals that 57% (363 species) are categorized as Threatened, 12% (78 species) as Near Threatened, 4% (26 species) as Data Deficient, and 27% (168 species) as Least Concern. Our assessment almost doubles the number of threatened species in comparison with previous evaluations. In addition to habitat loss, the expansion of the agricultural/cattle raising frontier and other anthropogenic threats (roads, human settlements, and mining/oil activities) amplify the incidence of other pressures as relevant predictors of ecological integrity. Potential synergic effects with climate change and emergent diseases (apparently responsible for the sudden declines), had particular importance amongst the threats sustained by Ecuadorian amphibians. Most threatened species are distributed in montane forests and paramo habitats of the Andes, with nearly 10% of them occurring outside the National System of Protected Areas of the Ecuadorian government. Based on our results, we recommend the following actions: (i) An increase of the National System of Protected Areas to include threatened species. (ii) Supporting the ex/in-situ conservation programs to protect species considered like Critically Endangered and Endangered. (iii) Focalizing research efforts towards the description of new species, as well as species currently categorized as Data Deficient (DD) that may turn out to be threatened. The implementation of the described actions is challenging, but urgent, given the current conservation crisis faced by amphibians.

A new species of Leucostethus (Anura: Dendrobatidae) from the Cordillera Mache-Chindul in northwestern Ecuador, with comments on similar Colostethus and Hyloxalus.

We describe a new species of dendrobatid frog, Leucostethus bilsa sp. nov., using molecular, morphological, and acoustic evidence. We also comment on the taxonomic status of four similar Hyloxalus and Colostethus. We provide an updated phylogeny of Leucostethus that corroborates previous hypotheses of relationships of nine species. Phylogenetic analysis using mitochondrial (i.e., 7095 bp of combined data from NADH1, NAHD2, cytochrome c oxidase I, cytochrome b and 12S-16S rRNA) and seven nuclear genes (i.e., 4739 bp) indicate a close relationship of L. bilsa to an undescribed species from Gorgona Island, Colombia, both of which apparently diverged in the Pliocene about 3 million years ago with about 6.25% (i.e., 146/2335 bp) differences for the section of 12S-16S mitochondrial fragment. Leucostethus bilsa is diurnal and riparian, characterized by distinctive bright mustard-yellow flash marks in the axillar and groin regions, posterior belly, and in the hindlimbs, the presence of dark gray lower labial stripe or marks, sexual dimorphism in ventral pattern, and by having male uniparental care. We describe its osteology and the male advertisement call, which is a series of peep notes. Osteological microCT images of representatives of each of the Colostethinae genera reveal a number of intriguing characters that may prove to be useful in phylogenetic studies. In terms of its distribution, Leucostethus bilsa is currently known only from a very small area within the Reserva Biológica Bilsa, located within the Cordillera Mache-Chindul in the Chocoan region of northwestern Ecuador, which was a Pliocene-Pleistocene refugium. This region is highly threatened with habitat degradation and remains as the last surviving refuge for a forest community known for a high proportion of endemic species of both flora and fauna.

Phylogenetic systematics, ecology, and conservation of marsupial frogs (Anura: Hemiphractidae) from the Andes of southern Ecuador, with descriptions of four new biphasic species.

We review the systematics of the species of Gastrotheca (Anura: Hemiphractidae) in the highlands of the southern Ecuadorian Andes. We analyzed phylogenetic, morphological, ecological, and acoustic data from populations in the region. We provide an updated phylogenetic hypothesis inferred from a database that contains 42 species of Gastrotheca, and sequences from mitochondrial (12S, 16S, and ND1) and nuclear (POMC and RAG-1) genes. We describe four new endemic biphasic species for Ecuador (Gastrotheca cuencana, G. elicioi, G. turnerorum, and G. yacuri). In addition, we redefine G. lojana, recently resurrected from the synonymy of G. monticola, and redescribe G. litonedis. Furthermore, we describe the tadpoles and ontogenetic color changes in six species (G. cuencana, G. elicioi, G. litonedis, G. lojana, G. pseustes, and G. turnerorum). Also, we describe the calls of seven species (G. cuencana, G. elicioi, G. litonedis, G. lojana, G. pseustes, G. testudinea, and G. yacuri). The phylogenetic relationships estimated here are congruent with previous phylogenetic hypotheses for the group, except for the placement of G. galeata, G. plumbea, G. orophylax, G. nicefori, and G. griswoldi. Because most sister species in Gastrotheca are allopatric, the evolutionarly scenarios that likely have intervened in shaping the diversity are the uplift of the Andes and the formation of cross-Andean river valleys, which probably promoted conditions that acted as dispersal barriers that led to speciation. Many species of Gastrotheca (44%) are highly threatened. Most monophasic species have not been seen since the late 1980s, whereas the highland biphasic species have survived the catastrophic events that led to the disappearance of many other sympatric anurans. Research and conservation actions are urgently needed for all surviving species treated herein, five of which are either Critically Endangered or Endangered because of their restricted distributions (10-1600 km2), habitat destruction and fragmentation, climate change, and pathogens. Two of the new species, G. cuencana, and G. elicioi, mostly are known from the cities of Cuenca and Loja, respectively, where urbanization threatens their survival. In order to call attention to conservation issues, we name one new species to refer to the Yacuri National Park, and the other three in honor of the people from Cuenca, the Turner family, and Elicio E. Tapia, whose actions are helping to halt amphibian extinctions.