Phylogenomics and evolutionary history of Oreobates (Anura: Craugastoridae) Neotropical frogs along elevational gradients.

Mountain ranges offer opportunities for understanding how species evolved and diversified across different environmental conditions. Neotropical frogs of the genus Oreobates (Anura: Craugastoridae) are adapted to highland and lowland habitats along the Andes, but many aspects of their evolution remain unknown. We studied their evolutionary history using ~18,000 exons enriched by targeted sequence-capture. Since capture success was very variable across samples, we evaluated to what degree differing data filtering produced robust inferences. The inferred evolutionary framework evidenced phylogenetic discordances among lowland species that can be explained by taxonomic misidentification or admixture of ancestral lineages. Highland species showed smaller effective populations than lowland frogs, probably due to greater habitat fragmentation in montane environments. Stronger genetic drift likely decreased the power of purifying selection and led to an increased proportion of nonsynonymous mutations in highland populations that could play an important role in their adaptation. Overall, our work sheds light on the evolutionary history and diversification of this group of Neotropical frogs along elevational gradients in the Andes as well as on their patterns of intraspecific diversity.

Total evidence and sensitivity phylogenetic analyses of egg-brooding frogs (Anura: Hemiphractidae).

We study the phylogenetic relationships of egg-brooding frogs, a group of 118 neotropical species, unique among anurans by having embryos with large bell-shaped gills and females carrying their eggs on the dorsum, exposed or inside a pouch. We assembled a total evidence dataset of published and newly generated data containing 51 phenotypic characters and DNA sequences of 20 loci for 143 hemiphractids and 127 outgroup terminals. We performed six analytical strategies combining different optimality criteria (parsimony and maximum likelihood), alignment methods (tree- and similarity-alignment), and three different indel coding schemes (fifth character state, unknown nucleotide, and presence/absence characters matrix). Furthermore, we analyzed a subset of the total evidence dataset to evaluate the impact of phenotypic characters on hemiphractid phylogenetic relationships. Our main results include: (i) monophyly of Hemiphractidae and its six genera for all our analyses, novel relationships among hemiphractid genera, and non-monophyly of Hemiphractinae according to our preferred phylogenetic hypothesis; (ii) non-monophyly of current supraspecific taxonomies of Gastrotheca, an updated taxonomy is provided; (iii) previous differences among studies were mainly caused by differences in analytical factors, not by differences in character/taxon sampling; (iv) optimality criteria, alignment method, and indel coding caused differences among optimal topologies, in that order of degree; (v) in most cases, parsimony analyses are more sensitive to the addition of phenotypic data than maximum likelihood analyses; (vi) adding phenotypic data resulted in an increase of shared clades for most analyses.

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.

Phylogenetic relationships and systematics of the Amazonian poison frog genus Ameerega using ultraconserved genomic elements.

The Amazonian poison frog genus Ameerega is one of the largest yet most understudied of the brightly colored genera in the anuran family Dendrobatidae, with 30 described species ranging throughout tropical South America. Phylogenetic analyses of Ameerega are highly discordant, lacking consistency due to variation in data types and methods, and often with limited coverage of species diversity in the genus. Here, we present a comprehensive phylogenomic reconstruction of Ameerega, utilizing state-of-the-art sequence capture techniques and phylogenetic methods. We sequenced thousands of ultraconserved elements from over 100 tissue samples, representing almost every described Ameerega species, as well as undescribed cryptic diversity. We generated topologies using maximum likelihood and coalescent methods and compared the use of maximum likelihood and Bayesian methods for estimating divergence times. Our phylogenetic inference diverged strongly from those of previous studies, and we recommend steps to bring Ameerega taxonomy in line with the new phylogeny. We place several species in a phylogeny for the first time, as well as provide evidence for six potential candidate species. We estimate that Ameerega experienced a rapid radiation approximately 7-11 million years ago and that the ancestor of all Ameerega was likely an aposematic, montane species. This study underscores the utility of phylogenomic data in improving our understanding of the phylogeny of understudied clades and making novel inferences about their evolution.

Molecular systematics of teioid lizards (Teioidea/Gymnophthalmoidea: Squamata) based on the analysis of 48 loci under tree-alignment and similarity-alignment.

We infer phylogenetic relationships within Teioidea, a superfamily of Nearctic and Neotropical lizards, using nucleotide sequences. Phylogenetic analyses relied on parsimony under tree-alignment and similarity-alignment, with length variation (i.e. gaps) treated as evidence and as absence of evidence, and maximum-likelihood under similarity-alignment with gaps as absence of evidence. All analyses produced almost completely resolved trees despite 86% of missing data. Tree-alignment produced the shortest trees, the strict consensus of which is more similar to the maximum-likelihood tree than to any of the other parsimony trees, in terms of both number of clades shared, parsimony cost and likelihood scores. Comparisons of tree costs suggest that the pattern of indels inferred by similarity-alignment drove parsimony analyses on similarity-aligned sequences away from more optimal solutions. All analyses agree in a majority of clades, although they differ from each other in unique ways, suggesting that neither the criterion of optimality, alignment nor treatment of indels alone can explain all differences. Parsimony rejects the monophyly of Gymnophthalmidae due to the position of Alopoglossinae relative to Teiidae, whereas support of Gymnophthalmidae by maximum-likelihood was low. We address various nomenclatural issues, including Gymnophthalmidae Fitzinger, 1826 being an older name than Teiidae Gray, 1827. We recognize three families in the arrangement Alopoglossidae + (Teiidae + Gymnophthalmidae). Within Gymnophthalmidae we recognize Cercosaurinae, Gymnophthalminae, Rhachisaurinae and Riolaminae in the relationship Cercosaurinae + (Rhachisaurinae + (Riolaminae + Gymnophthalminae)). Cercosaurinae is composed of three tribes-Bachiini, Cercosaurini and Ecpleopodini-and Gymnophthalminae is composed of three-Gymnophthalmini, Heterodactylini and Iphisini. Within Teiidae we retain the currently recognized three subfamilies in the arrangement: Callopistinae + (Tupinambinae + Teiinae). We also propose several genus-level changes to restore the monophyly of taxa.

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.

Molecular phylogenetics, species diversity, and biogeography of the Andean lizards of the genus Proctoporus (Squamata: Gymnophthalmidae).

The family Gymnophthalmidae comprises ca. 220 described species of Neotropical lizards distributed from southern Mexico to Argentina. It includes 36 genera, among them Proctoporus, which contains six currently recognized species occurring across the yungas forests and wet montane grasslands of the Amazonian versant of the Andes from central Peru to central Bolivia. Here, we investigate the phylogenetic relationships and species limits of Proctoporus and closely related taxa by analyzing 2121 base pairs of mitochondrial (12S, 16S, and ND4) and nuclear (c-mos) genes. Our taxon sampling of 92 terminals includes all currently recognized species of Proctoporus and 15 additional species representing the most closely related groups to the genus. Maximum parsimony, maximum likelihood and Bayesian phylogenetic analyses recovered a congruent, fully resolved, and strongly supported hypothesis of relationships that challenges previous phylogenetic hypotheses and classifications, and biogeographic scenarios. Our main results are: (i) discovery of a strongly supported clade that includes all species of Proctoporus and within which are nested the monotypic Opipeuter xestus (a genus that we consider a junior synonym of Proctoporus), and two species of Euspondylus, that are therefore transferred to Proctoporus; (ii) the paraphyly of Proctoporus bolivianus with respect to P. subsolanus, which is proposed as a junior synonym of P. bolivianus; (iii) the detection of seven divergent and reciprocally monophyletic lineages (five of them previously assigned to P. bolivianus) that are considered confirmed candidate species, which implies that more candidate species are awaiting formal description and naming than currently recognized species in the genus; (iv) rejection of the hypothesis that Proctoporus diversified following a south to north pattern parallel to the elevation of the Andes; (v) species diversity in Proctoporus is the result of in situ diversification through vicariance in the grasslands of the high Andes, with at least five dispersals contributing to montane forest species.

A paradigm shift in our view of species drives current trends in biological classification.

Discontent about changes in species classifications has grown in recent years. Many of these changes are seen as arbitrary, stemming from unjustified conceptual and methodological grounds, or leading to species that are less distinct than those recognised in the past. We argue that current trends in species classification are the result of a paradigm shift toward which systematics and population genetics have converged and that regards species as the phylogenetic lineages that form the branches of the Tree of Life. Species delimitation now consists of determining which populations belong to which individual phylogenetic lineage. This requires inferences on the process of lineage splitting and divergence, a process to which we have only partial access through incidental evidence and assumptions that are themselves subject to refutation. This approach is not free of problems, as horizontal gene transfer, introgression, hybridisation, incorrect assumptions, sampling and methodological biases can mislead inferences of phylogenetic lineages. Increasing precision is demanded through the identification of both sister relationships and processes blurring or mimicking phylogeny, which has triggered, on the one hand, the development of methods that explicitly address such processes and, on the other hand, an increase in geographical and character data sampling necessary to infer/test such processes. Although our resolving power has increased, our knowledge of sister relationships - what we designate as species resolution - remains poor for many taxa and areas, which biases species limits and perceptions about how divergent species are or ought to be. We attribute to this conceptual shift the demise of trinominal nomenclature we are witnessing with the rise of subspecies to species or their rejection altogether; subspecies are raised to species if they are found to correspond to phylogenetic lineages, while they are rejected as fabricated taxa if they reflect arbitrary partitions of continuous or non-hereditary variation. Conservation strategies, if based on taxa, should emphasise species and reduce the use of subspecies to avoid preserving arbitrary partitions of continuous variation; local variation is best preserved by focusing on biological processes generating ecosystem resilience and diversity rather than by formally naming diagnosable units of any kind. Since many binomials still designate complexes of species rather than individual species, many species have been discovered but not named, geographical sampling is sparse, gene lineages have been mistaken for species, plenty of species limits remain untested, and many groups and areas lack adequate species resolution, we cannot avoid frequent changes to classifications as we address these problems. Changes will not only affect neglected taxa or areas, but also popular ones and regions where taxonomic research remained dormant for decades and old classifications were taken for granted.

An assessment of the taxonomic validity of three species of marsupial frogs (Anura: Hemiphractidae: Gastrotheca) from the Yungas of Bolivia based on external morphology and cranial osteology.

The genus Gastrotheca (Anura: Hemiphractidae) is a group of marsupial frogs particularly diverse in Andean regions. Several taxonomic studies of this genus have been conducted in the humid cloud forestsor Yungasof the Andean eastern slopes of central Bolivia (departments of Cochabamba and Santa Cruz). Yet, the distinction among three species that occur sympatrically in these forests, G. lauzuricae (proposed as a junior synonym of G. coeruleomaculatus in 2015), G. piperata, and G. splendens, remains unclear since the morphological characters that purportedly support their differentiation are variable and partly shared among them. We have carried out external morphological studies, including multivariate morphometric analyses, to assess how they support the taxonomic status of these three species. We also evaluated characters of the cranial osteology of a sample of six individuals using micro CT-scanning. Principal component and linear discriminant analyses resulted in a great overlap among the putative species. Cranial osteological comparisons did not reveal highly significant differences among them, but suggested that different degrees of hyperossification could be related to the developmental state of individuals. Our results indicate that most morphological and osteological reported differences between the three species likely represent intraspecific variation. Thus, we propose that the three nominal species belong to a single biological entity, for which the name Gastrotheca splendens (Schmidt, 1857) has priority. We also restrict the name Gastrotheca coeruleomaculatus (Werner, 1899) to externally similar congeneric populations from the Yungas forests of department of La Paz, but highlighting the need of a detailed evaluation of their taxonomic identity.

Discovery of an additional piece of the large gymnophthalmid puzzle: a new genus and species of stream spiny lizard (Squamata: Gymnophthalmidae: Cercosaurinae) from the western Guiana Shield in Venezuela.

Gymnophthalmids are a highly diverse group of Neotropical lizards and its species richness is still in process of discovery. The incorporation of molecular evidence and a noticeable increase in taxon and geographic sampling in systematic studies has led to the description of numerous new genera and species of gymnophthalmids (particularly in Cercosaurinae) in recent years. Herein we describe a new genus and species of cercosaurine lizard with crocodile-like morphology, from the Venezuelan Guiana Shield on the basis of molecular phylogenetic and morphological evidence. Kataphraktosaurus gen. nov. can be readily distinguished from all other genera of Cercosaurinae by a unique combination of morphological characters that includes heterogeneous dorsal scalation with enlarged and strongly keeled scales forming two paravertebral rows, ventral and subcaudal scales imbricated and strongly keeled, large and symmetrical cephalic scales, absence of postmental scale, palpebral disc translucent and divided, tail slightly compressed, all digits clawed, and only six femoral pores (three at each hindlimb) inserted in a clump of small scales. This genus is described as monotypic and only contains Kataphraktosaurus ungerhamiltoni sp. nov., which is known from one specimen and diagnosed by the same set of aforementioned characters. The secretive habits of this species and the remoteness of the locality may explain its singleton situation. Following the International Union for Conservation of Nature's criteria, we categorized the new species as Data Deficient.

The integrative future of taxonomy.

BACKGROUND: Taxonomy is the biological discipline that identifies, describes, classifies and names extant and extinct species and other taxa. Nowadays, species taxonomy is confronted with the challenge to fully incorporate new theory, methods and data from disciplines that study the origin, limits and evolution of species. RESULTS: Integrative taxonomy has been proposed as a framework to bring together these conceptual and methodological developments. Here we review perspectives for an integrative taxonomy that directly bear on what species are, how they can be discovered, and how much diversity is on Earth. CONCLUSIONS: We conclude that taxonomy needs to be pluralistic to improve species discovery and description, and to develop novel protocols to produce the much-needed inventory of life in a reasonable time. To cope with the large number of candidate species revealed by molecular studies of eukaryotes, we propose a classification scheme for those units that will facilitate the subsequent assembly of data sets for the formal description of new species under the Linnaean system, and will ultimately integrate the activities of taxonomists and molecular biologists.

Genetic analysis of post-epizootic amphibian chytrid strains in Bolivia: Adding a piece to the puzzle.

The evolutionary history and dispersal pattern of Batrachochytrium dendrobatidis (Bd), an emergent fungal pathogen responsible for the decline and extinctions of many species of amphibians worldwide, is still not well understood. In South America, the tropical Andes are known as an important site for amphibian diversification, but also for being a place where hosts are at greater risk of chytridiomycosis. In an attempt to understand the history and the geographic pattern of Bd-associated amphibian declines in Bolivia, we isolated Bd from hosts at two locations that differ in their chronology of Bd prevalence and host survival outcome, the cloud forests of the Amazonian slopes of the Andes and Lake Titicaca in the altiplano. We genotyped Bd from both locations and sequenced the genome from the cloud forest isolate and then compared them to reference sequences of other Bd strains across the world. We found that the Bolivian chytrid isolates were nearly genotypically identical and that they belong to the global panzootic lineage (Bd-GPL). The Bolivian Bd strain grouped with other tropical New World strains but was closest to those from the Brazilian Atlantic Forest. Our results extend the presence of Bd-GPL to the central Andes in South America and report this hypervirulent strain at Lago Titicaca, where Bd has been detected since 1863, without evidence of amphibian declines. These findings suggest a more complex evolutionary history for this pathogen in Bolivia and may point to the existence of an old lineage of Bd that has since been extirpated following the arrival of the panzootic Bd-GPL or that the timing of Bd-GPL emergence is earlier than generally acknowledged.

Linking behavioral thermoregulation, boldness, and individual state in male Carpetan rock lizards.

Mechanisms affecting consistent interindividual behavioral variation (i.e., animal personality) are of wide scientific interest. In poikilotherms, ambient temperature is one of the most important environmental factors with a direct link to a variety of fitness-related traits. Recent empirical evidence suggests that individual differences in boldness are linked to behavioral thermoregulation strategy in heliothermic species, as individuals are regularly exposed to predators during basking. Here, we tested for links between behavioral thermoregulation strategy, boldness, and individual state in adult males of the high-mountain Carpetan rock lizard (Iberolacerta cyreni). Principal component analysis revealed the following latent links in our data: (i) a positive relationship of activity with relative limb length and color brightness (PC1, 23% variation explained), (ii) a negative relationship of thermoregulatory precision with parasite load and risk-taking (PC2, 20.98% variation explained), and (iii) a negative relationship between preferred body temperature and relative limb length (PC3, 19.23% variation explained). We conclude that differences in boldness and behavioral thermoregulatory strategy could be explained by both stable and labile state variables. The moderate link between behavioral thermoregulatory strategy and risk-taking personality in our system is plausibly the result of differences in reproductive state of individuals or variation in ecological conditions during the breeding season.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Environmental temperatures shape thermal physiology as well as diversification and genome-wide substitution rates in lizards.

Climatic conditions changing over time and space shape the evolution of organisms at multiple levels, including temperate lizards in the family Lacertidae. Here we reconstruct a dated phylogenetic tree of 262 lacertid species based on a supermatrix relying on novel phylogenomic datasets and fossil calibrations. Diversification of lacertids was accompanied by an increasing disparity among occupied bioclimatic niches, especially in the last 10 Ma, during a period of progressive global cooling. Temperate species also underwent a genome-wide slowdown in molecular substitution rates compared to tropical and desert-adapted lacertids. Evaporative water loss and preferred temperature are correlated with bioclimatic parameters, indicating physiological adaptations to climate. Tropical, but also some populations of cool-adapted species experience maximum temperatures close to their preferred temperatures. We hypothesize these species-specific physiological preferences may constitute a handicap to prevail under rapid global warming, and contribute to explaining local lizard extinctions in cool and humid climates.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

The shadow of the past: Convergence of young and old South American desert lizards as measured by head shape traits.

Convergence is a pervasive phenomenon in the Tree of Life, and evolution of similar phenotypes sharing the same environmental conditions is expected in phylogenetically closely related species. In contrast, contingent factors are probably more influential in shaping phenotypic diversity for distantly related taxa. Here, we test putative convergent evolution of lizard head morphologies among relatively closely related desert dwelling Liolaemus species, and the very distantly related Ctenoblepharys adspersa. We estimated a multilocus time-calibrated phylogeny of 57 species of South American liolaemus lizards, based on seven molecular markers. We collected head shape data for 468 specimens, and used three phylogenetic comparative methods (SURFACE, CONVEVOL, and WHEATSHEAF index) to test for and estimate the strength of convergence. We found strong evidence for convergence among Pacific desert lizard C. adspersa, Liolaemus audivetulatus, Liolaemus insolitus, Liolaemus poconchilensis, Liolaemus stolzmanni, and a candidate species (Liolaemus "Moquegua"). Our results suggest that, despite the long divergence and phylogenetic distance of C. adspersa with respect to convergent Liolaemus species, natural selection was probably more important than historical contingency in shaping phenotypic evolution in these desert lizards.

Detection of the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis in Museum Specimens of Andean Aquatic Birds: Implications for Pathogen Dispersal.

The occurrence of the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) in the feet of live waterfowl has been documented, but the potential role of birds as dispersers has not been studied. We report the presence of Bd in the feet of preserved aquatic birds in the Bolivian high Andes during the time of drastic amphibian declines in the country. We sampled 48 aquatic birds from the Bolivian Andes that were preserved in museum collections. Birds were sampled for the presence of Bd DNA by swabbing, taking small pieces of tissue from toe webbing, or both. We detected Bd by DNA using quantitative PCR in 42% of the birds sampled via toe tissue pieces. This method was significantly better than swabbing at detecting Bd from bird feet. We confirmed Bd presence by sequencing Bd -positive samples and found 91-98% homology with Bd sequences from GenBank. Our study confirms that aquatic birds can carry Bd and thus may serve as potential vectors of this pathogen across large distances and complex landscapes. In addition, we recommend using DNA from preserved birds as a novel source of data to test hypotheses on the spread of chytridiomycosis in amphibians.

A new species of Microkayla (Anura: Craugastoridae: Holoadeninae) from Department La Paz, Bolivia.

We describe a new species of direct-developing frog of the genus Microkayla from the Cordillera Real of the Bolivian Andes, in the Department of La Paz. The new species, Microkayla huayna sp. nov., is closely related to M. teqta and can be distinguished from other species of the genus by its brown dorsal skin and the presence of a large dark brown vocal sac in males. This is the second species of Microkayla known from the Zongo Valley, and the ninth in the Cordillera Real, contributing to a total of 22 described species in Bolivia. Given its small distribution range, we recommend to considering it as Vulnerable according to IUCN criteria.