Allozyme relationships among ten species of Rhodniini, showing paraphyly of Rhodnius including Psammolestes

Genetic relationships among 10 species of bugs belonging to the tribe Rhodniini (Hemiptera: Reduviidae), including some important vectors of Chagas disease, were inferred from allozyme analysis of 12 enzyme loci (out of 21 enzyme systems examined), using agarose gel electrophoresis. These species formed two clusters: one comprising Rhodnius brethesi, R. ecuadoriensis, R. pallescens and R. pictipes; the other with Psammolestes tertius, Rhodnius domesticus and the Rhodnius prolixus group comprising R. nasutus, R. neglectus, R. prolixus and R. robustus. The resulting tree was [((R. ecuadoriensis, R. pallescens) R. brethesi) R. pictipes], [R. domesticus (P. tertius [(R. nasutus, R. neglectus) (R. prolixus, R. robustus)])]. Rhodnius nasutus and R. neglectus differed by only one locus, whereas no diagnostic loci were detected between R. prolixus and R. robustus (22 loci were analysed for these four species), despite considerable DNA sequence divergence between species in each of these pairs. Allozymes of the R. prolixus group showed greater similarity with Psammolestes tertius than with other Rhodnius spp., indicating that Rhodnius is paraphyletic and might include Psammolestes.     

Attraction of Chagas disease vectors (Triatominae) to artificial light sources in the canopy of primary Amazon rainforest

Adult triatomines occasionally fly into artificially lit premises in Amazonia. This can result in Trypanosoma cruzi transmission to humans either by direct contact or via foodstuff contamination, but the frequency of such behaviour has not been quantified. To address this issue, a light-trap was set 45 m above ground in primary rainforest near Manaus, state of Amazonas, Brazil and operated monthly for three consecutive nights over the course of one year (432 trap-hours). The most commonly caught reduviids were triatomines, including 38 Panstrongylus geniculatus, nine Panstrongylus lignarius, three Panstrongylus rufotuberculatus, five Rhodnius robustus, two Rhodnius pictipes, one Rhodnius amazonicus and 17 Eratyrus mucronatus. Males were collected more frequently than females. The only month without any catches was May. Attraction of most of the known local T. cruzi vectors to artificial light sources is common and year-round in the Amazon rainforest, implying that they may often invade premises built near forest edges and thus become involved in disease transmission. Consequently, effective Chagas disease prevention in Amazonia will require integrating entomological surveillance with the currently used epidemiological surveillance.     

Feeding and defecation of Rhodnius (hemiptera: Reduviidae) fed human blood

Feeding and defecation behavior of Rhodnius prolixus Stal, 1859, R. robustus Larrousse, 1927, R. neivai Lent, 1953 and R. pictipes Stal, 1872, artificially fed on human blood, were studied under laboratory conditions. In all species, first instar nymphs did not defecate in the first 30 minutes after feeding. R. pictipes did not accept artificial feeding but fed directly on humans. Nymph and adult R. prolixus had a higher defecation index (DI) than other species; third instar nymphs had the highest DI = 1.62. In all instars, most individuals accepted the food in 3 Pounds minutes and finished feeding in less than 15 minutes.     

Rhodnius robustus in Bolivia identified by its wings.

Wings of a Rhodnius specimen from Alto Beni (Bolivia) was examined for identification and compared with R. stali, R. robustus, (certified Bolivian species), R. pictipes and R. prolixus (suspected Bolivian species). A projection of the unidentified wings as supplementary data into a discriminant analysis of shape revealed clear cut differences with R. stali and R. pictipes, less differences with R. prolixus, and none with R. robustus. Combining global size and shape of the wings, the unknown specimen was identified as R. robustus. Thus, this study confirmed the presence of R. robustus in Bolivia. It also highlighted the possibility of morphometrics to taxonomically interpret one individual, or even one piece of an individual, when related species data are available for comparison.     

Ribosomal DNA ITS-1 Intergenic Spacer Polymorphism in Triatomines (Triatominae, Heteroptera)

The length polymorphism of ribosomal DNA ITS-1 intergenic spacer was analyzed in eight species of triatomines belonging to Triatoma, Rhodnius, and Panstrongylus genera. The analyzed species were Rhodnius domesticus, R. neivai, R. robustus, Triatoma brasiliensis, T. infestans, T. vitticeps, Panstrongylus megistus, and P. herreri. These insects are vectors of Chagas' disease, one of the most prominent public health problems among South American countries. This work allowed the differentiation between species of the Triatomini and Rhodniini tribes through the analysis of ITS-1 length polymorphism by PCR and RFLP techniques. The species of the Triatoma and Panstrongylus genera presented an amplified ITS-1 fragment between 600 and 1000 bp, whereas Rhodnius presented a less variable ITS-1 length fragment, around 300 bp, which could reflect the monophyletic origin of the Rhodniini tribe. Species belonging to this genus were further differentiated by RFLP with HaeIII and AluI endonucleases. Our results corroborate the hypothesis of polyphyletic origin in this group of insects and contribute to knowledge about evolutionary relationships in triatomines.     

The triatomines of northern Peru,with emphasis on the ecology and infection by trypanosomes of Rhodnius ecuadoriensis (Triatominae)

Information on the distribution and synanthropic behaviour of triatomines is essential for Chagas disease vector control. This work summarises such information from northern Peru, and presents new data on Rhodnius ecuadoriensis - an important local vector infesting 10-35% of dwellings in some zones. Three species are strongly synanthropic and may be suitable targets for chemical control of domestic/peridomestic bug populations. Panstrongylus herreri, the main domestic vector in the area, is probably present in sylvatic ecotopes in the Mara?ón river system. R. ecuadoriensis and Triatoma dimidiata seem exclusively domestic; biogeographical and ecological data suggest they might have spread in association with humans in northern Peru. Confirmation of this hypothesis would result in a local eradication strategy being recommended. Presence of trypanosome natural infection was assessed in 257 R. ecuadoriensis; Trypanosoma rangeli was detected in 4% of bugs. Six further triatomine species are potential disease vectors in the region (T. carrioni, P. chinai, P. rufotuberculatus, P. geniculatus, R. pictipes, and R. robustus), whilst Eratyrus mucronatus, E. cuspidatus, Cavernicola pilosa, Hermanlentia matsunoi, and Belminus peruvianus have little or no epidemiological significance. A strong community-based entomological surveillance system and collaboration with Ecuadorian public health authorities and researchers are recommended.     

Population analysis using the nuclear white gene detects Pliocene/Pleistocene lineage divergence within Anopheles nuneztovari in South America

Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae), a locally important malaria vector in some regions of South America, has been hypothesized to consist of at least two cryptic incipient species. We investigated its phylogeographic structure in several South American localities to determine the number of lineages and levels of divergence using the nuclear white gene, a marker that detected two recently diverged genotypes in the primary Neotropical malaria vector Anopheles darlingi Root. In An. nuneztovari, five distinct lineages (1-5) were elucidated: (1) populations from northeastern and central Amazonia; (2) populations from Venezuela east and west of the Andes; (3) populations from Colombia and Venezuela west of the Andes; (4) southeastern and western Amazonian Brazil populations, and (5) southeastern and western Amazonian Brazil and Bolivian populations. There was a large amount of genetic differentiation among these lineages. The deepest and earliest divergence was found between lineage 3 and lineages 1, 2 and 4, which probably accounts for the detection of lineage 3 in some earlier studies. The multiple lineages within Amazonia are partially congruent with previous mtDNA and ITS2 data, but were undetected in many earlier studies, probably because of their recent (Pleistocene) divergence and the differential mutation rates of the markers. The estimates for the five lineages, interpreted as recently evolved or incipient species, date to the Pleistocene and Pliocene. We hypothesize that the diversification in An. nuneztovari is the result of an interaction between the Miocene/Pliocene marine incursion and Pleistocene climatic changes leading to refugial isolation. The identification of cryptic lineages in An. nuneztovari could have a significant impact on local vector control measures.     

Tree species composition and diversity gradients in white-water forests across the Amazon Basin

Aim Attention has increasingly been focused on the floristic variation within forests of the Amazon Basin. Variations in species composition and diversity are poorly understood, especially in Amazonian floodplain forests. We investigated tree species composition, richness and α diversity in the Amazonian white‐water (várzea) forest, looking particularly at: (1) the flood‐level gradient, (2) the successional stage (stand age), and (3) the geographical location of the forests. Location Eastern Amazonia, central Amazonia, equatorial western Amazonia and the southern part of western Amazonia. Methods The data originate from 16 permanent várzea forest plots in the central and western Brazilian Amazon and in the northern Bolivian Amazon. In addition, revised species lists of 28 várzea forest inventories from across the Amazon Basin were used. Most important families and species were determined using importance values. Floristic similarity between plots was calculated to detect similarity variations between forest types and over geographical distances. To check for spatial diversity gradients, α diversity (Fisher) of the plots was correlated with stand age, longitudinal and latitudinal plot location, and flood‐level gradient. Results More than 900 flood‐tolerant tree species were recorded, which indicates that Amazonian várzea forests are the most species‐rich floodplain forests worldwide. The most important plant families recorded also dominate most Neotropical upland forests, and c. 31% of the tree species listed also occur in the uplands. Species distribution and diversity varied: (1) on the flood‐level gradient, with a distinct separation between low‐várzea forests and high‐várzea forests, (2) in relation to natural forest succession, with species‐poor forests in early stages of succession and species‐rich forests in later stages, and (3) as a function of geographical distance between sites, indicating an increasing α diversity from eastern to western Amazonia, and simultaneously from the southern part of western Amazonia to equatorial western Amazonia. Main conclusions The east‐to‐west gradient of increasing species diversity in várzea forests reflects the diversity patterns also described for Amazonian terra firme. Despite the fine‐scale geomorphological heterogeneity of the floodplains, and despite high disturbance of the different forest types by sedimentation and erosion, várzea forests are dominated by a high proportion of generalistic, widely distributed tree species. In contrast to high‐várzea forests, where floristic dissimilarity increases significantly with increasing distance between the sites, low‐várzea forests can exhibit high floristic similarity over large geographical distances. The high várzea may be an important transitional zone for lateral immigration of terra firme species to the floodplains, thus contributing to comparatively high species richness. However, long‐distance dispersal of many low‐várzea trees contributes to comparatively low species richness in highly flooded low várzea.     

Response of Amazonian forests to mid-Holocene drought: A model-data comparison

There is a major concern for the fate of Amazonia over the coming century in the face of anthropogenic climate change. A key area of uncertainty is the scale of rainforest dieback to be expected under a future, drier climate. In this study, we use the middle Holocene (ca. 6000 years before present) as an approximate analogue for a drier future, given that palaeoclimate data show much of Amazonia was significantly drier than present at this time. Here, we use an ensemble of climate and vegetation models to explore the sensitivity of Amazonian biomes to mid-Holocene climate change. For this, we employ three dynamic vegetation models (JULES, IBIS, and SDGVM) forced by the bias-corrected mid-Holocene climate simulations from seven models that participated in the Palaeoclimate Modelling Intercomparison Project 3 (PMIP3). These model outputs are compared with a multi-proxy palaeoecological dataset to gain a better understanding of where in Amazonia we have most confidence in the mid-Holocene vegetation simulations. A robust feature of all simulations and palaeodata is that the central Amazonian rainforest biome is unaffected by mid-Holocene drought. Greater divergence in mid-Holocene simulations exists in ecotonal eastern and southern Amazonia. Vegetation models driven with climate models that simulate a drier mid-Holocene (100–150 mm per year decrease) better capture the observed (palaeodata) tropical forest dieback in these areas. Based on the relationship between simulated rainfall decrease and vegetation change, we find indications that in southern Amazonia the rate of tropical forest dieback was ~125,000 km² per 100 mm rainfall decrease in the mid-Holocene. This provides a baseline sensitivity of tropical forests to drought for this region (without human-driven changes to greenhouse gases, fire, and deforestation). We highlight the need for more palaeoecological and palaeoclimate data across lowland Amazonia to constrain model responses.     

Variation in tropical bird survival across longitude and guilds: a case study from the Amazon

Survival estimates of tropical birds have been used to examine life‐history variation across latitudes and dietary guilds. Here, we provide apparent survival estimates of 31 rainforest species from central Amazonian Brazil and compare our results with estimates from Ecuador, Peru (western Amazonia) and French Guiana (eastern Amazonia) to examine geographic variation in demography. Our averaged annual survival estimate for central Amazonian species (?= 0.59, SE = 0.10) was concordant with previously published estimates from the western Amazon (?= 0.58, SE = 0.02), and eastern Amazon (?= 0.63, SE = 0.06). Two predominate demographic patterns were detected across the study areas: within species, survival was highest or lowest in eastern or western Amazonia, but rarely in central Amazonia. The most striking demographic variation was exhibited by Pithys albifrons, for which apparent survival estimates were nearly twice as high in eastern Amazonia (?= 0.80, SE = 0.06) than in western Amazonia (?= 0.42, SE = 0.06) but intermediate in central Amazonia (?= 0.54, SE = 0.04). Although variation in survival may be associated with differences in life history characteristics, our analysis of flocking guild, body size, and nest architecture revealed only moderate differences in survival associated with nest architecture. These results suggest that geographic variation in population processes may be significant for widespread Amazonian species.     

Primate population densities in three nutrient-poor amazonian terra firme forests of south-eastern Colombia

We censused primate populations at three non-hunted 'terra firme' forests of south-eastern Colombian Amazonia. The aggregate biomass densities of diurnal primates at all sites were amongst the lowest recorded for any non-hunted forest in western Amazonia and elsewhere in the Neotropics. Densities of red howler monkeys were low, as is typical in Amazonian terra firme forests far removed from white-water rivers, and densities of woolly monkeys were 1.5-3.5 times lower than those estimated for this species in central-western Brazilian Amazonia. Densities of small to mid-sized primates except for brown capuchins (Cebus apella) and white-faced capuchins (Cebus albifrons) were similar to those of other oligotrophic Amazonian forest sites. Our results are in agreement with other studies showing that terra firme forests of lowland Amazonia typically sustain a low biomass density of primates and other mid-sized to large vertebrates. Large reserves are therefore required to assure the viability of primate populations in oligotrophic systems. Given the escalating negative impacts of human habitat disturbance and hunting in Colombian Amazonia, we urge that a baseline sampling protocol to quantify the abundance and distribution of the harvest-sensitive vertebrate fauna be established within protected areas and the large indigenous reserves so that conservation efforts can be defined and implemented.     

Alternative models of vertebrate speciation in Amazonia: an overview

The main hypotheses proposed to explain barrier formation separating populations and causing the differentiation of vertebrate species in Amazonia are based on different (mostly historical) factors, as follows. (1) Changes in the distribution of land and sea or in the landscape due to tectonic movements or sea-level fluctuations (Paleogeography hypothesis). (2) The barrier effect of Amazonian rivers (River hypothesis). (3) A combination of the barrier effect of broad rivers and vegetational changes in Northern and Southern Amazonia (River-refuge hypothesis). (4) The isolation of forest blocks near areas of surface relief in the periphery of Amazonia during dry climatic periods of the Tertiary and Quaternary (Refuge theory). (5) Competitive species interactions and local species isolations in peripheral regions of Amazonia due to invasion and counterinvasion during cold/warm periods of the Pleistocene (Disturbance-vicariance hypothesis). (6) Parapatric speciation across steep environmental gradients without separation of the representative populations (Gradient hypothesis). Several of these hypotheses are probably relevant to a different degree for the speciation processes in different faunal groups or during different geological periods. The paleogeography hypothesis refers mainly to faunal differentiation during the Tertiary and in combination with the Refuge hypothesis; Milankovitch cycles leading to global climatic-vegetational changes affected the biomes of the world not only during the Pleistocene but also during the Tertiary and earlier geological periods. New geoscientific evidence for the effect of dry climatic periods in Amazonia supports the predictions of the Refuge theory.     

From Amazonia to the Atlantic forest: Molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges

Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13Ma minimum. This striking biogeographic pattern coincides with major events that have shaped the face of the South American continent, as we know it today.     

The domestication of Amazonia before European conquest

During the twentieth century, Amazonia was widely regarded as relatively pristine nature, little impacted by human history. This view remains popular despite mounting evidence of substantial human influence over millennial scales across the region. Here, we review the evidence of an anthropogenic Amazonia in response to claims of sparse populations across broad portions of the region. Amazonia was a major centre of crop domestication, with at least 83 native species containing populations domesticated to some degree. Plant domestication occurs in domesticated landscapes, including highly modified Amazonian dark earths (ADEs) associated with large settled populations and that may cover greater than 0.1% of the region. Populations and food production expanded rapidly within land management systems in the mid-Holocene, and complex societies expanded in resource-rich areas creating domesticated landscapes with profound impacts on local and regional ecology. ADE food production projections support estimates of at least eight million people in 1492. By this time, highly diverse regional systems had developed across Amazonia where subsistence resources were created with plant and landscape domestication, including earthworks. This review argues that the Amazonian anthrome was no less socio-culturally diverse or populous than other tropical forested areas of the world prior to European conquest.     

Molecular systematics and phylogeography of Amazonian poison frogs of the genus Dendrobates

The study of Amazonian biodiversity requires detailed knowledge of the phylogenetic relationships of closely related taxa distributed across Amazonia. The Amazonian poison frogs of the genus Dendrobates have undergone many taxonomic revisions, but the phylogenetic relationships within this group remain poorly understood. Most previous classifications were based on morphology and skin toxin analyses, with limited use of DNA sequence data. Using mtDNA sequence data from four gene regions (cytochrome b, cytochrome oxidase I, 16S rRNA, and 12S rRNA), we present a molecular phylogenetic analysis of the evolutionary relationships within a representative group of Amazonian Dendrobates. We use the resulting phylogenetic hypothesis to investigate different biogeographic hypotheses concerning genetic divergence and species diversity in Amazonia. The results of the analysis support the presence of ancient paleogeographic barriers to gene flow between eastern and western Amazonia, and indicate substantial genetic divergence between species found in the northern and southern regions of western Amazonia.     

Chagas disease in the Amazon region

The risk that Chagas disease becomes established as a major endemic threat in Amazonia (the world's largest tropical biome, today inhabited by over 30 million people) relates to a complex set of interacting biological and social determinants. These include intense immigration from endemic areas (possibly introducing parasites and vectors), extensive landscape transformation with uncontrolled deforestation, and the great diversity of wild Trypanosoma cruzi reservoir hosts and vectors (25 species in nine genera), which maintain intense sylvatic transmission cycles. Invasion of houses by adventitious vectors (with infection rates > 60%) is common, and focal adaptation of native triatomines to artificial structures has been reported. Both acute (approximately 500) and chronic cases of autochthonous human Chagas disease have been documented beyond doubt in the region. Continuous, low-intensity transmission seems to occur throughout the Amazon, and generates a hypoendemic pattern with seropositivity rates of approximately 1-3%. Discrete foci also exist in which transmission is more intense (e.g., in localized outbreaks probably linked to oral transmission) and prevalence rates higher. Early detection-treatment of acute cases is crucial for avoiding further dispersion of endemic transmission of Chagas disease in Amazonia, and will require the involvement of malaria control and primary health care systems. Comprehensive eco-epidemiological research, including prevalence surveys or the characterization of transmission dynamics in different ecological settings, is still needed. The International Initiative for Chagas Disease Surveillance and Prevention in the Amazon provides the framework for building up the political and scientific cooperation networks required to confront the challenge of preventing Chagas disease in Amazonia.     

Three fishes in one: cryptic species in an Amazonian floodplain forest specialist

Accurately describing biodiversity in tropical regions such as Amazonia is difficult because of insufficient morphological inventories and the lack of studies on the distribution of genetic diversity. Aquatic organisms from Amazonian flooded forests are generally expected to move laterally along the forests during the annual inundation cycle, a behaviour that should promote admixture of populations and reduce within‐drainage speciation. We used an unprecedented fine‐scale sampling effort and multiple DNA markers to quantify region‐wide population differentiation in an Amazonian floodplain forest specialist, the black‐wing hatchet fish Carnegiella marthae ( Myers, 1927 ). Our study revealed three previously unsuspected and ancient cryptic species of black‐wing hatchet fish in the Rio Negro floodplain (RNF), in central Amazonia. Two species produce occasional first‐generation hybrids. The third and rarer species, although found in extreme sympatry with another species, appears to be reproductively isolated, and also differs in external morphology and dentition. Our findings have important implications for guiding conservation management because C. marthae is harvested commercially in the RNF ornamental fishery. They also suggest that the diversity of Amazonian ichthyofauna is vastly underestimated, including that found in landscapes lacking contemporary barriers to account for population divergence and speciation. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 391–403.     

Amazonian Triatomine Biodiversity and the Transmission of Chagas Disease in French Guiana: In Medio Stat Sanitas

The effects of biodiversity on the transmission of infectious diseases now stand as a cornerstone of many public health policies. The upper Amazonia and Guyana shield are hot-spots of biodiversity that offer genuine opportunities to explore the relationship between the risk of transmission of Chagas disease and the diversity of its triatomine vectors. Over 730 triatomines were light-trapped in four geomorphological landscapes shaping French-Guiana, and we determined their taxonomic status and infection by Trypanosoma cruzi. We used a model selection approach to unravel the spatial and temporal variations in species abundance, diversity and infection. The vector community in French-Guiana is typically made of one key species (Panstrongylus geniculatus) that is more abundant than three secondary species combined (Rhodnius pictipes, Panstrongylus lignarius and Eratyrus mucronatus), and four other species that complete the assemblage. Although the overall abundance of adult triatomines does not vary across French-Guiana, their diversity increases along a coastal-inland gradient. These variations unravelled a non-monotonic relationship between vector biodiversity and the risk of transmission of Chagas disease, so that intermediate biodiversity levels are associated with the lowest risks. We also observed biannual variations in triatomine abundance, representing the first report of a biannual pattern in the risk of Chagas disease transmission. Those variations were highly and negatively correlated with the average monthly rainfall. We discuss the implications of these patterns for the transmission of T. cruzi by assemblages of triatomine species, and for the dual challenge of controlling Amazonian vector communities that are made of both highly diverse and mostly intrusive species.     

Molecular phylogeny and morphometric analyses reveal deep divergence between Amazonia and Atlantic Forest species of Dendrophryniscus

Dendrophryniscus is an early diverging clade of bufonids represented by few small-bodied species distributed in Amazonia and the Atlantic Forest. We used mitochondrial (414 bp of 12S, 575 bp of 16S genes) and nuclear DNA (785 bp of RAG-1) to investigate phylogenetic relationships and the timing of diversification within the genus. These molecular data were gathered from 23 specimens from 19 populations, including eight out of the 10 nominal species of the genus as well as Rhinella boulengeri. Analyses also included sequences of representatives of 18 other bufonid genera that were publically available. We also examined morphological characters to analyze differences within Dendrophryniscus. We found deep genetic divergence between an Amazonian and an Atlantic Forest clade, dating back to Eocene. Morphological data corroborate this distinction. We thus propose to assign the Amazonian species to a new genus, Amazonella. The species currently named R. boulengeri, which has been previously assigned to the genus Rhamphophryne, is shown to be closely related to Dendrophryniscus species. Our findings illustrate cryptic trends in bufonid morphological evolution, and point to a deep history of persistence and diversification within the Amazonian and Atlantic rainforests. We discuss our results in light of available paleoecological data and the biogeographic patterns observed in other similarly distributed groups.