A set of principles and practical suggestions for equitable fieldwork in biology.

Field biology is an area of research that involves working directly with living organisms in situ through a practice known as "fieldwork." Conducting fieldwork often requires complex logistical planning within multiregional or multinational teams, interacting with local communities at field sites, and collaborative research led by one or a few of the core team members. However, existing power imbalances stemming from geopolitical history, discrimination, and professional position, among other factors, perpetuate inequities when conducting these research endeavors. After reflecting on our own research programs, we propose four general principles to guide equitable, inclusive, ethical, and safe practices in field biology: be collaborative, be respectful, be legal, and be safe. Although many biologists already structure their field programs around these principles or similar values, executing equitable research practices can prove challenging and requires careful consideration, especially by those in positions with relatively greater privilege. Based on experiences and input from a diverse group of global collaborators, we provide suggestions for action-oriented approaches to make field biology more equitable, with particular attention to how those with greater privilege can contribute. While we acknowledge that not all suggestions will be applicable to every institution or program, we hope that they will generate discussions and provide a baseline for training in proactive, equitable fieldwork practices.

Selection on Visual Opsin Genes in Diurnal Neotropical Frogs and Loss of the SWS2 Opsin in Poison Frogs.

Amphibians are ideal for studying visual system evolution because their biphasic (aquatic and terrestrial) life history and ecological diversity expose them to a broad range of visual conditions. Here, we evaluate signatures of selection on visual opsin genes across Neotropical anurans and focus on three diurnal clades that are well-known for the concurrence of conspicuous colors and chemical defense (i.e., aposematism): poison frogs (Dendrobatidae), Harlequin toads (Bufonidae: Atelopus), and pumpkin toadlets (Brachycephalidae: Brachycephalus). We found evidence of positive selection on 44 amino acid sites in LWS, SWS1, SWS2, and RH1 opsin genes, of which one in LWS and two in RH1 have been previously identified as spectral tuning sites in other vertebrates. Given that anurans have mostly nocturnal habits, the patterns of selection revealed new sites that might be important in spectral tuning for frogs, potentially for adaptation to diurnal habits and for color-based intraspecific communication. Furthermore, we provide evidence that SWS2, normally expressed in rod cells in frogs and some salamanders, has likely been lost in the ancestor of Dendrobatidae, suggesting that under low-light levels, dendrobatids have inferior wavelength discrimination compared to other frogs. This loss might follow the origin of diurnal activity in dendrobatids and could have implications for their behavior. Our analyses show that assessments of opsin diversification in across taxa could expand our understanding of the role of sensory system evolution in ecological adaptation.

Deep divergences among inconspicuously colored clades of Epipedobates poison frogs.

Poison frogs (Dendrobatidae) are famous for their aposematic species, having a combination of diverse color patterns and defensive skin toxins, yet most species in this family are inconspicuously colored and considered non-aposematic. Epipedobates is among the youngest genus-level clades of Dendrobatidae that includes both aposematic and inconspicuous species. Using Sanger-sequenced mitochondrial and nuclear markers, we demonstrate deep genetic divergences among inconspicuous species of Epipedobates but relatively shallow genetic divergences among conspicuous species. Our phylogenetic analysis includes broad geographic sampling of the inconspicuous lineages typically identified as E. boulengeri and E. espinosai, which reveals two putative new species, one in west-central Colombia (E. sp. 1) and the other in north-central Ecuador (E. aff. espinosai). We conclude that E. darwinwallacei is a junior subjective synonym of E. espinosai. We also clarify the geographic distributions of inconspicuous Epipedobates species including the widespread E. boulengeri. We provide a qualitative assessment of the phenotypic diversity in each nominal species, with a focus on the color and pattern of inconspicuous species. We conclude that Epipedobates contains eight known valid species, six of which are inconspicuous. A relaxed molecular clock analysis suggests that the most recent common ancestor of Epipedobates is âˆ¼11.1 million years old, which nearly doubles previous estimates. Last, genetic information points to a center of species diversity in the Chocó at the southwestern border of Colombia with Ecuador. A Spanish translation of this text is available in the supplementary materials.

Multiple origins of green coloration in frogs mediated by a novel biliverdin-binding serpin.

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.

Phylogenomics Reveals Ancient Gene Tree Discordance in the Amphibian Tree of Life.

Molecular phylogenies have yielded strong support for many parts of the amphibian Tree of Life, but poor support for the resolution of deeper nodes, including relationships among families and orders. To clarify these relationships, we provide a phylogenomic perspective on amphibian relationships by developing a taxon-specific Anchored Hybrid Enrichment protocol targeting hundreds of conserved exons which are effective across the class. After obtaining data from 220 loci for 286 species (representing 94% of the families and 44% of the genera), we estimate a phylogeny for extant amphibians and identify gene tree-species tree conflict across the deepest branches of the amphibian phylogeny. We perform locus-by-locus genealogical interrogation of alternative topological hypotheses for amphibian monophyly, focusing on interordinal relationships. We find that phylogenetic signal deep in the amphibian phylogeny varies greatly across loci in a manner that is consistent with incomplete lineage sorting in the ancestral lineage of extant amphibians. Our results overwhelmingly support amphibian monophyly and a sister relationship between frogs and salamanders, consistent with the Batrachia hypothesis. Species tree analyses converge on a small set of topological hypotheses for the relationships among extant amphibian families. These results clarify several contentious portions of the amphibian Tree of Life, which in conjunction with a set of vetted fossil calibrations, support a surprisingly younger timescale for crown and ordinal amphibian diversification than previously reported. More broadly, our study provides insight into the sources, magnitudes, and heterogeneity of support across loci in phylogenomic data sets.[AIC; Amphibia; Batrachia; Phylogeny; gene tree-species tree discordance; genomics; information theory.].

Post-epizootic microbiome associations across communities of neotropical amphibians.

Microbiome-pathogen interactions are increasingly recognized as an important element of host immunity. While these host-level interactions will have consequences for community disease dynamics, the factors which influence host microbiomes at larger scales are poorly understood. We here describe landscape-scale pathogen-microbiome associations within the context of post-epizootic amphibian chytridiomycosis, a disease caused by the panzootic chytrid fungus Batrachochytrium dendrobatidis. We undertook a survey of Neotropical amphibians across altitudinal gradients in Ecuador ~30 years following the observed amphibian declines and collected skin swab-samples which were metabarcoded using both fungal (ITS-2) and bacterial (r16S) amplicons. The data revealed marked variation in patterns of both B. dendrobatidis infection and microbiome structure that are associated with host life history. Stream breeding amphibians were most likely to be infected with B. dendrobatidis. This increased probability of infection was further associated with increased abundance and diversity of non-Batrachochytrium chytrid fungi in the skin and environmental microbiome. We also show that increased alpha diversity and the relative abundance of fungi are lower in the skin microbiome of adult stream amphibians compared to adult pond-breeding amphibians, an association not seen for bacteria. Finally, stream tadpoles exhibit lower proportions of predicted protective microbial taxa than pond tadpoles, suggesting reduced biotic resistance. Our analyses show that host breeding ecology strongly shapes pathogen-microbiome associations at a landscape scale, a trait that may influence resilience in the face of emerging infectious diseases.

Diversification history of clown tree frogs in Neotropical rainforests (Anura, Hylidae, Dendropsophus leucophyllatus group).

General consensus emphasizes that no single biological process can explain the patterns of species' distributions and diversification in the Neotropics. Instead, the interplay of several processes across space and time must be taken into account. Here we investigated the phylogenetic relationships and biogeographic history of tree frogs in the Dendropsophus leucophyllatus species group (Amphibia: Hylidae), which is distributed across Amazonia and the Atlantic rainforests. Using Next Generation Sequencing (NGS) and double digest restriction-site associated DNA (ddRADseq), we inferred phylogenetic relationships, species limits, and temporal and geographic patterns of diversification relative to the history of these biomes. Our results indicate that the D. leucophyllatus species group includes at least 14 independent lineages, which are currently arranged into ten described species. Therefore, a significant portion of species in the group are still unnamed. Different processes were associated to the group diversification history. For instance, the Andes uplift likely caused allopatric speciation for Cis-Andean species, whereas it may also be responsible for changes in the Amazonian landscape triggering parapatric speciation by local adaptation to ecological factors. Meanwhile, Atlantic Forest ancestors unable to cross the dry diagonal biomes after rainforest's retraction, evolved in isolation into different species. Diversification in the group began in the early Miocene, when connections between Atlantic Forest and the Andes (Pacific Dominion) by way of a south corridor were possible. The historical scenario in Amazonia, characterized by several speciation events and habitat heterogeneity, helped promoting diversification, resulting in the highest species diversity for the group. This marked species diversification did not happen in Atlantic Forest, where speciation is very recent (late Pliocene and Pleistocene), despite its remarkable climatic heterogeneity.

The birth of aposematism: High phenotypic divergence and low genetic diversity in a young clade of poison frogs.

Rapid radiation coupled with low genetic divergence often hinders species delimitation and phylogeny estimation even if putative species are phenotypically distinct. Some aposematic species, such as poison frogs (Dendrobatidae), have high levels of intraspecific color polymorphism, which can lead to overestimation of species when phenotypic divergence primarily guides species delimitation. We explored this possibility in the youngest origin of aposematism (3-7 MYA) in poison frogs, Epipedobates, by comparing genetic divergence with color and acoustic divergence. We found low genetic divergence (2.6% in the 16S gene) despite substantial differences in color and acoustic signals. While chemical defense is inferred to have evolved in the ancestor of Epipedobates, aposematic coloration evolved at least twice or was lost once in Epipedobates, suggesting that it is evolutionarily labile. We inferred at least one event of introgression between two cryptically colored species with adjacent ranges (E. boulengeri and E. machalilla). We also find evidence for peripheral isolation resulting in phenotypic divergence and potential speciation of the aposematic E. tricolor from the non-aposematic E. machalilla. However, we were unable to estimate a well-supported species tree or delimit species using multispecies coalescent models. We attribute this failure to factors associated with recent speciation including mitochondrial introgression, incomplete lineage sorting, and too few informative molecular characters. We suggest that species delimitation within young aposematic lineages such as Epipedobates will require genome-level molecular studies. We caution against relying solely on DNA barcoding for species delimitation or identification and highlight the value of phenotypic divergence and natural history in delimiting species.

Passive accumulation of alkaloids in non-toxic frogs challenges paradigms of the origins of acquired chemical defenses.

Understanding the origins of novel, complex phenotypes is a major goal in evolutionary biology. Poison frogs of the family Dendrobatidae have evolved the novel ability to acquire alkaloids from their diet for chemical defense at least three times. However, taxon sampling for alkaloids has been biased towards colorful species, without similar attention paid to inconspicuous ones that are often assumed to be undefended. As a result, our understanding of how chemical defense evolved in this group is incomplete. Here we provide new data showing that, in contrast to previous studies, species from each undefended poison frog clade have measurable yet low amounts of alkaloids. We confirm that undefended dendrobatids regularly consume mites and ants, which are known sources of alkaloids. Further, we confirm the presence of alkaloids in two putatively non-toxic frogs from other families. Our data suggest the existence of a phenotypic intermediate between toxin consumption and sequestration-passive accumulation-that differs from active sequestration in that it involves no derived forms of transport and storage mechanisms yet results in low levels of toxin accumulation. We discuss the concept of passive accumulation and its potential role in the origin of chemical defenses in poison frogs and other toxin-sequestering organisms.

A new species of small tree frog, genus Dendropsophus (Anura: Hylidae) from the eastern Amazon lowlands of Ecuador.

We describe a new species of the hylid frog genus Dendropsophus from Amazonian Ecuador. The new species, Dendropsophus shiwiarum sp. nov., appears to be part of the D. microcephalus species group. Dendropsophus shiwiarum differs from its congeners by a combination of: (1) tympanic membrane non-differentiated and annulus evident only ventrally, (2) disc of Finger III and Toe IV with pointed tip, (3) dorsal surfaces of digital discs with a conical tubercle as result of projection of distal phalanx, and (4) small size (SVL < 19 mm). The new species is morphologically most similar to Dendropsophus riveroi. Examination of the type material of the latter suggests that some Ecuadorian populations reported as D. riveroi were misidentified and in fact belong to D. shiwiarum sp. nov.

The Phyllomedusa perinesos group (Anura: Hylidae) is derived from a Miocene Amazonian Lineage.

The Phyllomedusa perinesos group is composed of four species that inhabit cloud forests in the eastern Andean slopes. We estimated the phylogenetic relationships among them and their closest relatives using mitochondrial DNA sequences. Our results confirm the monophyly of the group and a close relationship with the Amazonian species Phyllomedusa atelopoides and Phyllomedusa tomopterna. A chronogram indicates that the group originated during the Miocene and the contemporary species diverged from their closest relatives during the Miocene and early Pliocene. The timing of the group's origin suggests that its evolution was linked to the rise of the eastern Andes. Based on the phylogeny we expand the species content of the group to include P. atelopoides and P. tomopterna.

Two new syntopic species of glassfrogs (Amphibia, Centrolenidae, Centrolene) from the southwestern Andes of Ecuador.

We describe two new species of glassfrogs of the genus Centrolene living in syntopy at La Enramada, province of Azuay, southwestern Ecuador. They were found in a small creek in montane evergreen forests at 2,900 m elevation. The first new species is distinguished from all other members of the genus Centrolene by having the following combination of characters: dentigerous process of vomer absent; sloping snout in lateral view; thick, white labial stripe and a faint white line between the lip and anterior » of body; humeral spine in adult males; parietal peritoneum covered by iridophores, visceral peritonea translucent (except pericardium); ulnar and tarsal ornamentation; dorsal skin shagreen with dispersed warts; uniform green dorsum with light yellowish green warts; and green bones. The new species is remarkable by being sister to a species from the opposite Andean versant, C. condor. The second new species is distinguished from all other Centrolene by having the following combination of characters: dentigerous process of vomer absent; round snout in lateral view; thin, yellowish labial stripe with a row of white tubercles between the lip and arm insertion, and a yellowish line between arm insertion and groin; uniform green dorsum; humeral spine in adult males; parietal peritoneum covered by iridophores, visceral peritonea translucent (except pericardium); dorsal skin shagreen with dispersed spicules; ulnar and tarsal ornamentation; and green bones. The second new species is sister to C. sabini and an undescribed species of Centrolene from southeastern Ecuador. Based on nuclear and mitochondrial DNA sequences, we present a new phylogeny for Centrolene and comment on the phylogenetic relationships inside the genus.

2b or not 2b? 2bRAD is an effective alternative to ddRAD for phylogenomics.

Restriction-site-associated DNA sequencing (RADseq) has become an accessible way to obtain genome-wide data in the form of single-nucleotide polymorphisms (SNPs) for phylogenetic inference. Nonetheless, how differences in RADseq methods influence phylogenetic estimation is poorly understood because most comparisons have largely relied on conceptual predictions rather than empirical tests. We examine how differences in ddRAD and 2bRAD data influence phylogenetic estimation in two non-model frog groups. We compare the impact of method choice on phylogenetic information, missing data, and allelic dropout, considering different sequencing depths. Given that researchers must balance input (funding, time) with output (amount and quality of data), we also provide comparisons of laboratory effort, computational time, monetary costs, and the repeatability of library preparation and sequencing. Both 2bRAD and ddRAD methods estimated well-supported trees, even at low sequencing depths, and had comparable amounts of missing data, patterns of allelic dropout, and phylogenetic signal. Compared to ddRAD, 2bRAD produced more repeatable datasets, had simpler laboratory protocols, and had an overall faster bioinformatics assembly. However, many fewer parsimony-informative sites per SNP were obtained from 2bRAD data when using native pipelines, highlighting a need for further investigation into the effects of each pipeline on resulting datasets. Our study underscores the importance of comparing RADseq methods, such as expected results and theoretical performance using empirical datasets, before undertaking costly experiments.

Global Protected Areas as refuges for amphibians and reptiles under climate change.

Protected Areas (PAs) are the cornerstone of biodiversity conservation. Here, we collated distributional data for >14,000 (~70% of) species of amphibians and reptiles (herpetofauna) to perform a global assessment of the conservation effectiveness of PAs using species distribution models. Our analyses reveal that >91% of herpetofauna species are currently distributed in PAs, and that this proportion will remain unaltered under future climate change. Indeed, loss of species' distributional ranges will be lower inside PAs than outside them. Therefore, the proportion of effectively protected species is predicted to increase. However, over 7.8% of species currently occur outside PAs, and large spatial conservation gaps remain, mainly across tropical and subtropical moist broadleaf forests, and across non-high-income countries. We also predict that more than 300 amphibian and 500 reptile species may go extinct under climate change over the course of the ongoing century. Our study highlights the importance of PAs in providing herpetofauna with refuge from climate change, and suggests ways to optimize PAs to better conserve biodiversity worldwide.

High levels of cryptic species diversity uncovered in Amazonian frogs.

One of the greatest challenges for biodiversity conservation is the poor understanding of species diversity. Molecular methods have dramatically improved our ability to uncover cryptic species, but the magnitude of cryptic diversity remains unknown, particularly in diverse tropical regions such as the Amazon Basin. Uncovering cryptic diversity in amphibians is particularly pressing because amphibians are going extinct globally at an alarming rate. Here, we use an integrative analysis of two independent Amazonian frog clades, Engystomops toadlets and Hypsiboas treefrogs, to test whether species richness is underestimated and, if so, by how much. We sampled intensively in six countries with a focus in Ecuador (Engystomops: 252 individuals from 36 localities; Hypsiboas: 208 individuals from 65 localities) and combined mitochondrial DNA, nuclear DNA, morphological, and bioacoustic data to detect cryptic species. We found that in both clades, species richness was severely underestimated, with more undescribed species than described species. In Engystomops, the two currently recognized species are actually five to seven species (a 150-250% increase in species richness); in Hypsiboas, two recognized species represent six to nine species (a 200-350% increase). Our results suggest that Amazonian frog biodiversity is much more severely underestimated than previously thought.

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.

Six new species of Pristimantis (Anura: Strabomantidae) from Llanganates National Park and Sangay National Park in Amazonian cloud forests of Ecuador.

We describe six new species of rainfrogs of the genus Pristimantis (Strabomantidae) from Amazonian cloud forests in Ecuador. We also present a phylogeny showing the relationships of the new species. The phylogeny is based on mitochondrial genes 16S rRNA (16S), 12 rRNA (12S), NADH-ubiquinone oxidoreductase chain 1 (ND1) and the nuclear gene recombination-activating 1 (RAG1). We also describe the osteology of two of the new species using high-resolution x-ray computed tomography. The new species belong to two clades. The first clade is sister to the subgenus Huicundomantis and includes P. tamia sp. nov., P. miktos, and P. mallii. Pristimantis tamia sp. nov. is morphologically similar to P. miktos, P. mallii, P. martiae, and P. incomptus, but differs from them by lacking vocal slits and tympanic membrane and by having light greenish blue iris. Based in our results we expand the subgenus Huicundomantis to include the P. miktos species group. The second clade is remarkable by being highly divergent and consisting exclusively of new species: P. anaiae sp. nov., P. glendae sp. nov., P. kunam sp. nov., P. resistencia sp. nov., and P. venegasi sp. nov. The new species resemble P. roni, P. yanezi, P. llanganati, P. katoptroides, P. verecundus, and P. mutabilis but can be distinguished from them by lacking vocal slits and tympanic membrane and by having large dark round areas with thin clear borders in the sacral region. All six new species occur in the eastern slopes of the Ecuadorian Andes and are known from a single locality in Llanganates or Sangay National Park. We recommend assigning all of them to the Data Deficient (DD) Red List category. Based in our high-resolution x-ray tomographies, we report the presence of structures that appear to be intercalary elements. This would be the first report of such structures in Terrarana.

Dietary data of a highly biodiverse anuran assemblage in the Ecuadorian Amazon.

This dataset reports the diet composition of a highly diverse anuran assemblage in the Ecuadorian Amazon region. In 2001 we examined the diet of an assemblage of frogs from Yasuní National Park. We describe the diet of 396 adult individuals, belonging to 35 species, based on their gastrointestinal contents. Using a stereoscopic microscope, we were able to identify 4085 prey items, and classified them in 71 categories. Also, we used a digital caliper to measure the size and estimate the volume of prey items that were found intact. In addition to diet composition, we provide information of all specimens that were examined including, museum number, family name, species name, and place and date of collection. Finally, we present an anuran-prey interaction network figure to visualize species interactions. This is the first report of the diet composition of an anuran assemblage from Yasuní National Park. It contributes to the understanding of trophic ecology of frog assemblages and the functional role of frogs in Amazonian ecosystems. In addition, our dataset helps to fill the great knowledge gap that exists about ecological interactions in the tropics.

Description of a new minute frog of the genus Pristimantis (Anura: Strabomantidae) from Cordillera del Condor, Ecuador.

We describe a new species of Pristimantis from southern Ecuador, province of Zamora Chinchipe. The new species is closely related to an undescribed species of Pristimantis from Reserva Tapichalaca, Ecuador and with species of a clade historically assigned to the P. unistrigatus species group, such as P. parvillus, P. luteolateralis, P. walkeri, among others. The new species of Pristimantis is a miniaturized new frog (females 17.11.1 mm; males 13.20.9 mm), characterized by the presence of -shaped scapular folds, with two subconical tubercles on the medial and posterior regions of folds; tympanic membrane and tympanic annulus present but not externally visible; a prominent rostral papilla present; upper eyelid with one elongated conical tubercle; a conical tubercle on heels; groin with orange or yellow spots. The new species of Pristimantis is distributed in a restricted area in the Cordillera del Condor, a highly-diverse mountain range threatened by multiple anthropogenic activities. We recommend assigning the new species to the Endangered IUCN threatened category because it is only known from three nearby localities within mining concessions.

A polymorphism in oocyte pigmentation in natural populations of the glass frog Espadarana prosoblepon (Centrolenidae).

The adaptive role of amphibian oocyte melanic pigmentation and its molecular control are still elusive. Here we present evidence of a polymorphism in egg pigmentation in the emerald glass frog Espadarana prosoblepon. In Ecuadorian natural populations of this species, females can lay dark brown or pale eggs that develop into normal pigmented tadpoles and adults. This trait is a sex-limited phenotype which is inherited like a recessive allele that we called pale eggs like (pel). The pel phenotype is exclusive of oocyte cortical melanic pigmentation, which is reduced in comparison to wild type (wt) dark pigmented oocytes. Consequently, pel early embryos are paler in appearance, with reduced melanic pigmentation distributed to early blastomeres and embryonic ectoderm. However, these embryos form normal melanocyte derived pigmentation. Finally, we discuss the origin of this polymorphism and propose the use of E. prosoblepon as a model to study the adaptive role of egg pigmentation.