Evolutionary trade-offs between testes size and parenting in Neotropical glassfrogs.

In males, large testes size signifies high sperm production and is commonly linked to heightened sperm competition levels. It may also evolve as a response to an elevated risk of sperm depletion due to multiple mating or large clutch sizes. Conversely, weapons, mate or clutch guarding may allow individuals to monopolize mating events and preclude sperm competition, thereby reducing the selection of large testes. Herein, we examined how paternal care, sexual size dimorphism (SSD), weaponry and female fecundity are linked to testes size in glassfrogs. We found that paternal care was associated with a reduction in relative testes size, suggesting an evolutionary trade-off between testes size and parenting. Although females were slightly larger than males and species with paternal care tended to have larger clutches, there was no significant relationship between SSD, clutch size and relative testes size. These findings suggest that the evolution of testes size in glassfrogs is influenced by sperm competition risk, rather than sperm depletion risk. We infer that clutch guarding precludes the risk of fertilization by other males and consequently diminishes selective pressure for larger testes. Our study highlights the prominent role of paternal care in the evolution of testes size in species with external fertilization.

A pilot study to estimate the population size of endangered Galápagos marine iguanas using drones.

BACKGROUND: Large-scale species monitoring remains a significant conservation challenge. Given the ongoing biodiversity crisis, the need for reliable and efficient methods has never been greater. Drone-based techniques have much to offer in this regard: they allow access to otherwise unreachable areas and enable the rapid collection of non-invasive field data. Herein, we describe the development of a drone-based method for the estimation of population size in Galápagos marine iguanas, Amblyrhynchus cristatus. As a large-bodied lizard that occurs in open coastal terrain, this endemic species is an ideal candidate for drone surveys. Almost all Amblyrhynchus subspecies are Endangered or Critically Endangered according to the IUCN yet since several colonies are inaccessible by foot, ground- based methods are unable to address the critical need for better census data. In order to establish a drone-based approach to estimate population size of marine iguanas, we surveyed in January 2021 four colonies on three focal islands (San Cristobal, Santa Fe and Espanola) using three techniques: simple counts (the standard method currently used by conservation managers), capture mark-resight (CMR), and drone-based counts. The surveys were performed within a 4-day window under similar ambient conditions. We then compared the approaches in terms of feasibility, outcome and effort. RESULTS: The highest population-size estimates were obtained using CMR, and drone-based counts were on average 14% closer to CMR estimates-and 17-35% higher-than those obtained by simple counts. In terms of field-time, drone-surveys can be faster than simple counts, but image analyses were highly time consuming. CONCLUSION: Though CMR likely produces superior estimates, it cannot be performed in most cases due to lack of access and knowledge regarding colonies. Drone-based surveys outperformed ground-based simple counts in terms of outcome and this approach is therefore suitable for use across the range of the species. Moreover, the aerial approach is currently the only credible solution for accessing and surveying marine iguanas at highly remote colonies. The application of citizen science and other aids such as machine learning will alleviate the issue regarding time needed to analyze the images.

Cryptic lineages and standing genetic variation across independent cane toad introductions.

Widespread introduced species can be leveraged to investigate the genetic, ecological and adaptive processes underlying rapid evolution and range expansion, particularly the contributions of genetic diversity to adaptation. Rhinella marina, the cane toad, has been a focus of invasion biology for decades in Australia. However, their introduction history in North America is less clear. Here, we investigated the roles of introduction history and genetic diversity in establishment success of cane toads across their introduced range. We used reduced representation sequencing (ddRAD) to obtain 34,000 SNPs from 247 toads in native (French Guiana, Guyana, Ecuador, Panama, Texas) and introduced (Bermuda, southern Florida, northern Florida, Hawai'i, Puerto Rico) populations. Unlike all other cane toad introductions, we found that Florida populations were more closely related to native Central American lineages (R. horribilis), than to native Southern American lineages (R. marina). Furthermore, we found high levels of diversity and population structure in the native range, corroborating suggestions that R. marina is a species complex. We also found that introduced populations exhibit only slightly lower genetic diversity than native populations. Together with demographic analyses, this indicates founding populations of toads in Florida were larger than previously reported. Lastly, within R. marina, only one of 245 putatively adaptive SNPs showed fixed differences between native and introduced ranges, suggesting that putative selection in these introduced populations is based upon existing genetic variation. Our findings highlight the importance of genetic sequencing in understanding biological introductions and hint at the role of standing genetic variation in range expansion.

Temperature dependence of metabolic rate in tropical and temperate aquatic insects: Support for the Climate Variability Hypothesis in mayflies but not stoneflies.

A fundamental gap in climate change vulnerability research is an understanding of the relative thermal sensitivity of ectotherms. Aquatic insects are vital to stream ecosystem function and biodiversity but insufficiently studied with respect to their thermal physiology. With global temperatures rising at an unprecedented rate, it is imperative that we know how aquatic insects respond to increasing temperature and whether these responses vary among taxa, latitudes, and elevations. We evaluated the thermal sensitivity of standard metabolic rate in stream-dwelling baetid mayflies and perlid stoneflies across a ~2,000 m elevation gradient in the temperate Rocky Mountains in Colorado, USA, and the tropical Andes in Napo, Ecuador. We used temperature-controlled water baths and microrespirometry to estimate changes in oxygen consumption. Tropical mayflies generally exhibited greater thermal sensitivity in metabolism compared to temperate mayflies; tropical mayfly metabolic rates increased more rapidly with temperature and the insects more frequently exhibited behavioral signs of thermal stress. By contrast, temperate and tropical stoneflies did not clearly differ. Varied responses to temperature among baetid mayflies and perlid stoneflies may reflect differences in evolutionary history or ecological roles as herbivores and predators, respectively. Our results show that there is physiological variation across elevations and species and that low-elevation tropical mayflies may be especially imperiled by climate warming. Given such variation among species, broad generalizations about the vulnerability of tropical ectotherms should be made more cautiously.

Narrow thermal tolerance and low dispersal drive higher speciation in tropical mountains.

Species richness is greatest in the tropics, and much of this diversity is concentrated in mountains. Janzen proposed that reduced seasonal temperature variation selects for narrower thermal tolerances and limited dispersal along tropical elevation gradients [Janzen DH (1967) Am Nat 101:233-249]. These locally adapted traits should, in turn, promote reproductive isolation and higher speciation rates in tropical mountains compared with temperate ones. Here, we show that tropical and temperate montane stream insects have diverged in thermal tolerance and dispersal capacity, two key traits that are drivers of isolation in montane populations. Tropical species in each of three insect clades have markedly narrower thermal tolerances and lower dispersal than temperate species, resulting in significantly greater population divergence, higher cryptic diversity, higher tropical speciation rates, and greater accumulation of species over time. Our study also indicates that tropical montane species, with narrower thermal tolerance and reduced dispersal ability, will be especially vulnerable to rapid climate change.

Concentrations and stable isotopes of mercury in sharks of the Galapagos Marine Reserve: Human health concerns and feeding patterns.

The human ingestion of mercury (Hg) from sea food is of big concern worldwide due to adverse health effects, and more specifically if shark consumption constitutes a regular part of the human diet. In this study, the total mercury (THg) concentration in muscle tissue were determined in six sympatric shark species found in a fishing vessel seized in the Galapagos Marine Reserve in 2017. The THg concentrations in shark muscle samples (n = 73) varied from 0.73 mg kg-1 in bigeye thresher sharks (Alopias superciliosus) to 8.29 mg kg-1 in silky sharks (Carcharhinus falciformis). A typical pattern of Hg bioaccumulation was observed for all shark species, with significant correlation between THg concentration and shark size for bigeye thresher sharks, pelagic thresher sharks (Alopias pelagicus) and silky sharks. Regarding human health concerns, the THg mean concentration exceeded the maximum weekly intake fish serving in all the studied species. Mass-Dependent Fractionation (MDF, δ202Hg values) and Mass-Independent Fractionation (MIF, Δ199Hg values) of Hg in whitetip sharks (Carcharhinus longimanus) and silky sharks, ranged from 0.70‰ to 1.08‰, and from 1.97‰ to 2.89‰, respectively. These high values suggest that both species are feeding in the epipelagic zone (i.e. upper 200 m of the water column). While, blue sharks (Prionace glauca), scalloped hammerhead sharks (Shyrna lewini) and thresher sharks were characterized by lower Δ199Hg and δ202Hg values, indicating that these species may focus their foraging behavior on prey of mesopelagic zone (i.e. between 200 and 1000 m depth). In conclusion, the determination of THg concentration provides straight-forward evidence of the human health risks associated with shark consumption, while mercury isotopic compositions constitute a powerful tool to trace the foraging strategies of these marine predators. CAPSULE: A double approach combining Hg concentrations with stable isotopes ratios allowed to assess ontogeny in common shark species in the area of the Galapagos Marine Reserve and the human health risks concern associated to their consumption.

Vastly underestimated species richness of Amazonian salamanders (Plethodontidae: Bolitoglossa) and implications about plethodontid diversification.

We present data showing that the number of salamander species in Amazonia is vastly underestimated. We used DNA sequences of up to five genes (3 mitochondrial and 2 nuclear) of 366 specimens, 189 corresponding to 89 non-Amazonian nominal species and 177 Amazonian specimens, including types or topotypes, of eight of the nine recognized species in the region. By including representatives of all known species of Amazonian Bolitoglossa, except for one, and 73% of the currently 132 recognized species of the genus, our dataset represents the broadest sample of Bolitoglossa species, specimens, and geographic localities studied to date. We performed phylogenetic analyses using parsimony with tree-alignment and maximum likelihood (ML) with similarity alignment, with indels as binary characters. Our optimal topologies were used to delimit lineages that we assigned to nominal species and candidate new species following criteria that maximize the consilience of the current species taxonomy, monophyly, gaps in branch lengths, genetic distances, and geographic distribution. We contrasted the results of our species-delimitation protocol with those of Automated Barcode Gap Discovery (ABGD) and multi-rate Poisson Tree Processes (mPTP). Finally, we inferred the historical biogeography of South American salamanders by dating the trees and using dispersal-vicariance analysis (DIVA). Our results revealed a clade including almost all Amazonian salamanders, with a topology incompatible with just the currently recognized nine species. Following our species-delimitation criteria, we identified 44 putative species in Amazonia. Both ABGD and mPTP inferred more species than currently recognized, but their numbers (23-49) and limits vary. Our biogeographic analysis suggested a stepping-stone colonization of the Amazonian lowlands from Central America through the Chocó and the Andes, with several late dispersals from Amazonia back into the Andes. These biogeographic events are temporally concordant with an early land bridge between Central and South America (~10-15 MYA) and major landscape changes in Amazonia during the late Miocene and Pliocene, such as the drainage of the Pebas system, the establishment of the Amazon River, and the major orogeny of the northern Andes.

A new rainfrog of the Pristimantis myersi Group (Amphibia, Craugastoridae) from Volcán Pichincha, Ecuador.

A new frog of the Pristimantis myersi Group is described from a bamboo patch within the Reserva Ecológica Verdecocha (0°5'46.9"S, 78°36'15.3"W; 2851 m), located at northwestern flank of the Volcán Pichincha, in the vicinities of Quito, Ecuador. The new species is known from eight adult males, whereas the females remain unknown; it can be readily distinguished from all species of the P. myersi Group that inhabit the highlands of the Ecuadorian Andes by the unique combination of the following characters: body small (adult male SVL 14.9-19.7 mm; females unknown); dorsal skin shagreen, with a barely visible middorsal raphe, scapular and dorsolateral folds; tympanum small but well-defined; upper eyelid with one enlarged tubercle; males with prominent vocal slits, but without nuptial pads on thumbs; fold-like tarsal tubercles. With this new species, the number of Pristimantis assigned to the P. myersi Group raises to 16, of which, 12 are in Ecuador. We provide notes on morphology and color variation, advertisement call, and natural history of the new species.

New records of native and introduced fish species in a river basin of Western Ecuador, the Chocó-Darien Ecoregion, using DNA barcoding.

DNA barcoding, based on mitochondrial markers, is widely applied in species identification and biodiversity studies. The aim of this study was to establish a barcoding reference database of fishes inhabiting the Cube River from Western Ecuador in the Chocó-Darien Global Ecoregion (CGE), a threatened ecoregion with high diversity and endemism, and evaluate the applicability of using barcoding for the identification of fish species. Barcode sequences were obtained from seven orders, 17 families, 23 genera and 26 species, which were validated through phylogenetic analysis, morphological measurements, and literature review. Our results showed that 43% of fish species in this region are endemic, confirmed the presence of known species in the area, and included the addition of three new records of native (Hoplias microlepis, Rhamdia guatemalensis and Sicydium salvini) and an introduced species (Xiphophorus maculatus) to Ecuador. In addition, eight species were barcoded for the first time. Species identification based on barcoding and morphology showed discrepancy with species lists from previous studies in the CGE, suggesting that the current baseline of western fishes of Ecuador is still incomplete. Because this study analyzed fishes from a relatively small basin (165 km2), more molecular-based studies focusing on fish are needed to achieve a robust sequence reference library of species inhabiting Western Ecuador. The new sequences of this study will be useful for future comparisons and biodiversity monitoring, supporting the application of barcoding tools for studying fish diversity in genetically unexplored regions and to develop well-informed conservation programs.

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.

Explaining Andean megadiversity: the evolutionary and ecological causes of glassfrog elevational richness patterns.

The Tropical Andes are an important global biodiversity hotspot, harbouring extraordinarily high richness and endemism. Although elevational richness and speciation have been studied independently in some Andean groups, the evolutionary and ecological processes that explain elevational richness patterns in the Andes have not been analysed together. Herein, we elucidate the processes underlying Andean richness patterns using glassfrogs (Centrolenidae) as a model system. Glassfrogs show the widespread mid-elevation diversity peak for both local and regional richness. Remarkably, these patterns are explained by greater time (montane museum) rather than faster speciation at mid-elevations (montane species pump), despite the recency of the major Andean uplift. We also show for the first time that rates of climatic-niche evolution and elevational change are related, supporting the hypothesis that climatic-niche conservatism decelerates species' shifts in elevational distributions and underlies the mid-elevation richness peak. These results may be relevant to other Andean clades and montane systems globally.

A new yellow species of glassfrog (Centrolenidae: Nymphargus) from the Amazonian slopes of the Ecuadorian Andes.

I describe a new glassfrog from the cloud forest of the Andes of southwestern Ecuador (Plan de Milagro-Gualaceo road; 3.0077°S, 78.53318°W), at elevations between 2140-2160 m. The new species is distinguished mostly by having a pale yellow dorsal coloration instead of the green that characterizes most centrolenids. Morphological traits (i.e., reduced webbing between Fingers III and IV and lack of humeral spines) support the placement of the new species in the genus Nymphargus.

A new species of the Pristimantis orestes group (Amphibia: Strabomantidae) from the high Andes of Ecuador, Reserva Mazar.

We describe a new Pristimantis from La Libertad and Rumiloma, Reserva Mazar, Andes of Southeastern Ecuador, at elevations between 2895-3415 m. This species is assigned to the P. orestes group, from whose members it differs by its small body size (adult males ≤ 18.1 mm; adult females ≤ 23.7 mm), usually reticulated ventral pattern, and visible tympanum. The vocalization of the new species consists of a series of calls; each call is composed by a pulsed, non-modulated note in frequency, and with a dominant frequency of 3122-3171 Hz. A molecular phylogeny based on a fragment of the mitochondrial gene 12S shows that the new species is sister to Pristimantis simonbolivari.

To name or not to name: Criteria to promote economy of change in Linnaean classification schemes.

The Linnaean classification system provides the universal reference system for communicating about the diversity of life and its hierarchic history. Several limitations that challenge the stability of this system have been identified and, as a result, alternative systems have been proposed since its early inception. The revolution caused by molecular phylogenetics has, more than ever, exemplified that Linnaean classification schemes are subject to a degree of instability that may hamper their significance and communication power. Our analysis of recent changes in the classification of several groups of organisms, with a focus on amphibians and reptiles, reveals two main sources of instability: (i) revisionary, objective (empirical) changes based on the discovery of unambiguous instances of non-monophyly and on progress in the Globe's species inventory, and (ii) subjective changes based on author preferences or on a poor analysis of the advantages and limitations of new classification schemes. To avoid subjective taxonomic instability, we review and elaborate proposals for the assignment of Linnaean rank to clades, and thereby for the naming of these clades as Linnaean taxa (Taxon Naming Criteria: TNCs). These are drafted from the perspective of practicing taxonomists and can help choosing among alternative monophyly-based classifications under a premise of economy of change. We provide a rationale for each TNC along with real and theoretical examples to illustrate their practical advantages and disadvantages. We conclude that not all TNCs lead to equally informative and stable taxonomies. Therefore, we order the various TNCs by the generality of their implications and provide a workflow scheme to guide the procedure of taxonomic decisions concerning the creation or modification of supraspecific classifications. The following criteria are considered primary when naming taxa: (i) Mono phyly of the taxon in an inferred species tree; (ii) Clade Stability, i.e., the monophyly of a clade to be named as taxon should be as strongly supported as possible by various methods of tree inference, tests of clade robustness, and different data sets; and (iii) Phenotypic Diagnosability, i.e., ranked supraspecific taxa should be those that are phenotypically most conspicuous although in phenotypically cryptic groups of organisms it can be warranted to name taxa based on molecular differences alone. We consider various other criteria as secondary (i.e., the Time Banding, Biogeography, Adaptive Zone, and Hybrid Viability TNCs) and refute using them as sole arguments for the modification of established classifications or proposal of new ones. Taxonomists are encouraged to be explicit and consistent when applying TNCs for creating or modifying classifications. We emphasize that, except for monophyly, the priority TNCs are not proposed as mandatory requisites of a Linnaean taxon but as yardsticks to allow for an informed choice among various clades in a tree that could alternatively be named as Linnaean taxa. Despite a need for plurality, classifications should avoid deliberately violating any of the three primary TNCs because taxa of unstable monophyly or poor diagnosability reduce the information content and hence the utility of the Linnaean system.

Environment rather than character displacement explains call evolution in glassfrogs.

The acoustic adaptation hypothesis (AAH) and ecological character displacement (ECD) are two potential mechanisms shaping call evolution that can predict opposite trends for the differentiation of signals. Under AAH, signals evolve to minimize environmental degradation and maximize detection against background noise, predicting call homogenization in similar habitats due to environmental constraints on signals. In contrast, ECD predicts greater differences in call traits of closely related taxa in sympatry because of selection against acoustic interference. We used comparative phylogenetic analyses to test the strength of these two selective mechanisms on the evolution of advertisement calls in glassfrogs, a highly diverse family of neotropical anurans. We found that, overall, acoustic adaptation to the environment may outweigh effects of species interactions. As expected under the AAH, temporal call parameters are correlated with vegetation density, but spectral call parameters had an unexpected inverse correlation with vegetation density, as well as an unexpected correlation with temperature. We detected call convergence among co-occurring species and also across multiple populations from the same species in different glassfrogs communities. Our results indicate that call convergence is common in glassfrogs, likely due to habitat filtering, while character displacement is relatively rare, suggesting that costs of signal similarity among related species may not drive divergent selection in all systems.

Unveiling the evolutionary relationships and the high cryptic diversity in Andean rainfrogs (Craugastoridae: Pristimantis myersi group).

Background: Pristimantis is the most diverse genus of terrestrial frogs. Historically, it has been divided into several phenetic groups in order to facilitate species identification. However, in light of phylogenetic analysis, many of these groups have been shown to be non-monophyletic, denoting a high degree of morphological convergence and limited number of diagnostic traits. In this study, we focus on the Pristimantis myersi group, an assemblage of small rainfrogs distributed throughout the Andes of Ecuador and Colombia, whose external morphology is highly conserved, and its species diversity and evolutionary relationships largely unknown. Methods: We inferred a new phylogenetic hypothesis for the frog genus Pristimantis, including all available sequences of the mtDNA 16S rRNA, as well as new DNA sequences from 175 specimens. Our sampling included 19 of the 24 species currently recognized as part of the Pristimantis myersi group. Results: Our new evolutionary hypothesis recovered the P. myersi group as non-monophyletic and composed of 16 species. Therefore, we exclude P. albujai, P. bicantus, P. sambalan, and P. nelsongalloi in order to preserve the monophyly of the group. We discovered at least eight candidate species, most of them hidden under the names of P. leoni, P. hectus, P. festae, P. gladiator, and P. ocreatus. Discussion: Our results reveal the occurrence of a high level of cryptic diversity to the species level within the P. myersi group and highlight the need to redefine some of its species and reassess their conservation status. We suggest that the conservation status of six species within the group need to be re-evaluated because they exhibit smaller distributions than previously thought; these species are: P. festae, P. gladiator, P. hectus, P. leoni, P. ocreatus, and P. pyrrhomerus. Finally, given that the Pristimantis myersi group, as defined in this work, is monophyletic and morphologically diagnosable, and that Trachyphrynus is an available name for the clade containing P. myersi, we implement Trachyphrynus as a formal subgenus name for the Pristimantis myersi group.

Two new species of terrestrial frogs of the Pristimantisgladiator complex (Anura, Strabomantidae) from the Ecuadorian Andes, with insights on their biogeography and skull morphology.

The explosive diversity of rainfrogs (Pristimantis spp) reaches its highest levels in the mountains of the Tropical Andes, with remarkable cryptic species mainly in unexplored areas of Ecuador. Based on phylogenetics, morphometric traits, skull osteology and bioacoustics, we describe two new species of Pristimantis, previously confused with Pristimantisgladiator, that belong to the subgenus Trachyphrynus traditionally known as the Pristimantismyersi species group. The two new taxa are closely related, but have allopatric distributions. We discuss the importance of the Quijos and Pastaza River valleys in the diversification along Amazonian slopes of the Ecuadorian Andes.

Human impacts outpace natural processes in the Amazon.

Amazonian environments are being degraded by modern industrial and agricultural activities at a pace far above anything previously known, imperiling its vast biodiversity reserves and globally important ecosystem services. The most substantial threats come from regional deforestation, because of export market demands, and global climate change. The Amazon is currently perched to transition rapidly from a largely forested to a nonforested landscape. These changes are happening much too rapidly for Amazonian species, peoples, and ecosystems to respond adaptively. Policies to prevent the worst outcomes are known and must be enacted immediately. We now need political will and leadership to act on this information. To fail the Amazon is to fail the biosphere, and we fail to act at our peril.

Evolutionary genomics of oceanic island radiations.

A recurring feature of oceanic archipelagos is the presence of adaptive radiations that generate endemic, species-rich clades that can offer outstanding insight into the links between ecology and evolution. Recent developments in evolutionary genomics have contributed towards solving long-standing questions at this interface. Using a comprehensive literature search, we identify studies spanning 19 oceanic archipelagos and 110 putative adaptive radiations, but find that most of these radiations have not yet been investigated from an evolutionary genomics perspective. Our review reveals different gaps in knowledge related to the lack of implementation of genomic approaches, as well as undersampled taxonomic and geographic areas. Filling those gaps with the required data will help to deepen our understanding of adaptation, speciation, and other evolutionary processes.

Species limits in the Andean toad genus Osornophryne (Bufonidae).

As Darwin observed, the differentiation among varieties, subspecies, and species seems, often times, arbitrary. Nowadays, however, novel tools provide the possibility of testing hypotheses of species. Using the Andean toad genus Osornophryne, we address the following questions: (1) How many species are within the genus? (2) Are morphological and molecular traits congruent when delimiting species? (3) Which morphological traits are the most divergent among species? We use recently developed methods for testing species boundaries and relationships using a multilocus data set consisting of two mitochondrial genes (12S, 16S; 1647bp aligned matrix), one exon (RAG-1; 923 aligned matrix), and one intron (RPL3Int5; 1410bp aligned matrix). As another line of evidence for species delimitation, we integrated analyses of 12 morphometric variables and 10 discrete traits commonly used in amphibian systematics. The molecular and morphological approaches support the validity of most of the described species in Osornophryne. We find, however, contradictory lines of evidence regarding the status of O. angel. Within O. guacamayo, we found a genetically divergent population that, we argue, represents a new species. We consider that O. bufoniformis represents a species complex that deserves further study. We highlight the importance of incorporating morphological data when delimiting species, especially for lineages that have a recent origin and have not achieved reciprocal monophyly in molecular phylogenies. Finally, the most divergent morphological traits among Osornophryne species are associated with locomotion (finger, toes and limbs) and feeding (head), suggesting an association between morphology and the ecological habits of the species.