Amphibian diversity across three adjacent ecosystems in Área de Conservación Guanacaste, Costa Rica.

Amphibians are the most threatened species-rich vertebrate group, with species extinctions and population declines occurring globally, even in protected and seemingly pristine habitats. These 'enigmatic declines' are generated by climate change and infectious diseases. However, the consequences of these declines are undocumented as no baseline ecological data exists for most affected areas. Like other neotropical countries, Costa Rica, including Área de Conservación Guanacaste (ACG) in north-western Costa Rica, experienced rapid amphibian population declines and apparent extinctions during the past three decades. To delineate amphibian diversity patterns within ACG, a large-scale comparison of multiple sites and habitats was conducted. Distance and time constrained visual encounter surveys characterised species richness at five sites-Murciélago (dry forest), Santa Rosa (dry forest), Maritza (mid-elevation dry-rain forest intersect), San Gerardo (rainforest) and Cacao (cloud forest). Furthermore, species-richness patterns for Cacao were compared with historic data from 1987-8, before amphibians declined in the area. Rainforests had the highest species richness, with triple the species of their dry forest counterparts. A decline of 45% (20 to 11 species) in amphibian species richness was encountered when comparing historic and contemporary data for Cacao. Conservation efforts sometimes focus on increasing the resilience of protected areas, by increasing their range of ecosystems. In this sense ACG is unique containing many tropical ecosystems compressed in a small geographic space, all protected and recognised as a UNESCO world heritage site. It thus provides an extraordinary platform to understand changes, past and present, and the resilience of tropical ecosystems and assemblages, or lack thereof, to climate change.

Drivers of Batrachochytrium dendrobatidis infection load, with evidence of infection tolerance in adult male toads (Bufo spinosus).

Chytridiomycosis is affecting hundreds of amphibian species worldwide, but while in tropical areas, adult individuals have been the focus of most investigations, the exact role played by infection intensity of breeding adults is not well understood in temperate areas. We conducted mark-recapture-capture surveys during spiny common toad breeding seasons from 2006 to 2018 at the site of the first recorded outbreak of chytridiomycosis in Europe, the Peñalara Massif (Sierra de Guadarrama National Park, central Spain), and collected infection samples and several variables related to the reproductive effort of male individuals. We used general linear mixed models to evaluate the contribution of study variables on the infection loads of adult male toads exhibited at their capturing date. We also analysed the differences on several male characteristics between the pond with the largest breeding population against the rest of the ponds. We found that the duration of time spent in the waterbody and the condition of the host predicted infection loads. Animals of good physical condition, that spent longer in water, have higher infection levels than individuals with the opposite set of traits. The pond supporting the largest breeding population housed smaller male toads and in poorer condition. Our results are consistent with a shift in reproductive strategy in response to infection and potentially a strategy of tolerance, rather than resistance to infection. These findings have applications for disease mitigation and theoretical implications related to the trade-offs made and the evolution of traits in response to the disease.

Cold water and harmful algal blooms linked to coral reef collapse in the Eastern Tropical Pacific.

Background: With conventional coral reef conservation methods proving ineffective against intensifying climate change, efforts have focussed on augmenting coral tolerance to warmer water-the primary driver of coral declines. We document coral cover and composition in relation to sea surface temperature (SST) over 25-years, of six marginal reefs in an upwelling area of Costa Rica's Eastern Tropical Pacific. Methods: Using reef survey data and sea surface temperature (SST) dating back over 25-years, we document coral cover and composition of six marginal reefs in an upwelling area of Costa Rica's Eastern Tropical Pacific in relation to thermal highs and lows. Results: A ubiquitous and catastrophic coral die-off event occurred in 2009, driven by SST minima and likely by the presence of extreme harmful algal blooms. Coral cover was dramatically reduced and coral composition shifted from dominant branching Pocillopora to massive Pavona, Porites, and Gardineroseris. The lack of coral recovery in the decade since indicates a breach in ecosystem tipping-point and highlights a need for resilience-based management (RBM) and restoration. We propose a locally tailored and globally scalable approach to coral reef declines that is founded in RBM and informed by coral health dynamics.

Bioclimatic and anthropogenic variables shape the occurrence of Batrachochytrium dendrobatidis over a large latitudinal gradient.

Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused the greatest known loss of biodiversity due to an infectious disease. We used Bd infection data from quantitative real-time PCR (qPCR) assays of amphibian skin swabs collected across Chile during 2008-2018 to model Bd occurrence with the aim to determine bioclimatic and anthropogenic variables associated with Bd infection. Also, we used Bd presence/absence records to identify geographical Bd high-risk areas and compare Bd prevalence and infection loads between amphibian families, ecoregions, and host ecology. Data comprised 4155 Bd-specific qPCR assays from 162 locations across a latitudinal gradient of 3700 km (18º to 51ºS). Results showed a significant clustering of Bd associated with urban centres and anthropogenically highly disturbed ecosystems in central-south Chile. Both Bd prevalence and Bd infection loads were higher in aquatic than terrestrial amphibian species. Our model indicated positive associations of Bd prevalence with altitude, temperature, precipitation and human-modified landscapes. Also, we found that macroscale drivers, such as land use change and climate, shape the occurrence of Bd at the landscape level. Our study provides with new evidence that can improve the effectiveness of strategies to mitigate biodiversity loss due to amphibian chytridiomycosis.

Geographic range estimates and environmental requirements for the harpy eagle derived from spatial models of current and past distribution.

Understanding species-environment relationships is key to defining the spatial structure of species distributions and develop effective conservation plans. However, for many species, this baseline information does not exist. With reliable presence data, spatial models that predict geographic ranges and identify environmental processes regulating distribution are a cost-effective and rapid method to achieve this. Yet these spatial models are lacking for many rare and threatened species, particularly in tropical regions. The harpy eagle (Harpia harpyja) is a Neotropical forest raptor of conservation concern with a continental distribution across lowland tropical forests in Central and South America. Currently, the harpy eagle faces threats from habitat loss and persecution and is categorized as Near-Threatened by the International Union for the Conservation of Nature (IUCN). Within a point process modeling (PPM) framework, we use presence-only occurrences with climatic and topographical predictors to estimate current and past distributions and define environmental requirements using Ecological Niche Factor Analysis. The current PPM prediction had high calibration accuracy (Continuous Boyce Index = 0.838) and was robust to null expectations (pROC ratio = 1.407). Three predictors contributed 96% to the PPM prediction, with Climatic Moisture Index the most important (72.1%), followed by minimum temperature of the warmest month (15.6%) and Terrain Roughness Index (8.3%). Assessing distribution in environmental space confirmed the same predictors explaining distribution, along with precipitation in the wettest month. Our reclassified binary model estimated a current range size 11% smaller than the current IUCN range polygon. Paleoclimatic projections combined with the current model predicted stable climatic refugia in the central Amazon, Guyana, eastern Colombia, and Panama. We propose a data-driven geographic range to complement the current IUCN range estimate and that despite its continental distribution, this tropical forest raptor is highly specialized to specific environmental requirements.

Choice of molecular assay determines ranavirus detection probability and inferences about prevalence and occurrence.

Ranaviruses are emerging pathogens that can cause morbidity, mortality and population declines in ectothermic hosts; however, there is no standardized approach to diagnostics. Here, we compared the inter-assay variation and intra-assay precision among 2 commonly used quantitative PCRs (qPCRs), a conventional and a nested PCR assay (used as a gold standard), using laboratory-propagated ranavirus (FV3 and CMTV) and field-collected samples. A qPCR assay ('Leung') detected viral DNA in dilutions 2 orders of magnitude lower than other assays regardless of the viral lineage of the cultured isolate (FV3/CMTV). The second qPCR ('Brunner') was slightly more sensitive than the conventional PCR ('Mao' assay). For field samples, the Leung qPCR detected all known positives, while the Mao assay PCR only detected 2.5% of the positive samples. Amplicon sequences from the 2 conventional PCRs were shown to be useful for inferring viral lineage. Inaccurate results will bias estimates of the distribution and prevalence of ranaviruses, and together these findings emphasize that molecular assays should be chosen carefully in the context of study aims.

Molecular Detection of Leishmania spp. in Skin and Blood of Stray Dogs from Endemic Areas of Cutaneous Leishmaniasis in Saudi Arabia.

BACKGROUND: Dogs can act as reservoirs of canine leishmaniasis, caused by Leishmania species. The aims of this study were to determine the prevalence of canine leishmaniasis using a PCR technique among stray dogs living in three provinces of Saudi Arabia, Riyadh, Al-Ahsa Oasis and Al-Qaseem, where the disease is endemic; and to identify and document different Leishmania to species levels. METHODS: This cross-sectional investigation was conducted, from Mar 2016 to Apr 2018, in three parts of Saudi Arabia: Central province (Riyadh), Eastern province (Al-Ahsa Oasis) and Al-Qaseem province. Blood samples were collected from 526 dogs; 40 presented cutaneous nodules so were suspected clinically of cutaneous leishmaniasis. Biopsy tissue collections and parasite cultures were performed. A generic kDNA was performed using different primers for Leishmania differentiation. RESULTS: All blood samples were negative for Leishmania infantum infection by molecular analysis, though forty dogs had thick cutaneous lesions in different parts of their body. Four dogs' skin lesions were associated with dermatitis, splenomegaly and lymphadenomegaly. Parasite culture was used to diagnose cutaneous leishmaniasis, identifying 31/40 (77.5%) positive samples. Overall, of 526 samples, the prevalence of L. major and L. tropica was found to be 4% and 1.9%, respectively. Gender and age had a significant effect on Leishmania prevalence: (P=0.0212 and 0.0357), respectively. CONCLUSION: This was the first molecular study of dog leishmaniasis from Saudi Arabia of dogs confirmed to have cutaneous leishmaniasis. Further epidemiological and molecular investigations of domestic and wild canine infections with L. major, L. tropica and L. infantum in endemic and nonendemic areas of Saudi Arabia are required, for leishmaniasis control.

Cryptic diversity in Lithobateswarszewitschii (Amphibia, Anura, Ranidae).

Lithobateswarszewitschii is a species of ranid frog distributed from southern Honduras to Panama. This species suffered severe population declines at higher elevations (above 500 m a.s.l.) from the 1980s to early 1990s, but there is more recent evidence of recovery in parts of its range. Here we advocate for the status of Lithobateswarszewitschii as a candidate cryptic species complex based on sequence data from mitochondrial genes CO1 and 16S. Using concatenated phylogenies, nucleotide diversity (K2P-π), net between group mean distance (NBGMD) (πnet) and species delimitation methods, we further elucidate cryptic diversity within this species. All phylogenies display polyphyletic lineages within Costa Rica and Panama. At both loci, observed genetic polymorphism (K2P-π) is also high within and between geographic populations, surpassing proposed species threshold values for amphibians. Additionally, patterns of phylogeographic structure are complicated for this species, and do not appear to be explained by geographic barriers or isolation by distance. These preliminary findings suggest L.warszewitschii is a wide-ranging species complex. Therefore, we propose further research within its wider range, and recommend integrative taxonomic assessment is merited to assess species status.

Effects of historic and projected climate change on the range and impacts of an emerging wildlife disease.

The global trend of increasing environmental temperatures is often predicted to result in more severe disease epidemics. However, unambiguous evidence that temperature is a driver of epidemics is largely lacking, because it is demanding to demonstrate its role among the complex interactions between hosts, pathogens, and their shared environment. Here, we apply a three-pronged approach to understand the effects of temperature on ranavirus epidemics in UK common frogs, combining in vitro, in vivo, and field studies. Each approach suggests that higher temperatures drive increasing severity of epidemics. In wild populations, ranavirosis incidents were more frequent and more severe at higher temperatures, and their frequency increased through a period of historic warming in the 1990s. Laboratory experiments using cell culture and whole animal models showed that higher temperature increased ranavirus propagation, disease incidence, and mortality rate. These results, combined with climate projections, predict severe ranavirosis outbreaks will occur over wider areas and an extended season, possibly affecting larval recruitment. Since ranaviruses affect a variety of ectothermic hosts (amphibians, reptiles, and fish), wider ecological damage could occur. Our three complementary lines of evidence present a clear case for direct environmental modulation of these epidemics and suggest management options to protect species from disease.

Molecular detection of equine trypanosomiasis in the Riyadh Province of Saudi Arabia.

We conducted a cross-sectional study to detect trypanosome infections of horses and donkeys in the Riyadh Province of Saudi Arabia. DNA was extracted from blood samples collected from 368 horses and 142 donkeys, and subjected to universal first ribosomal internal transcribed spacer region (ITS1)-PCR followed by Trypanosoma evansi species-specific RoTat1.2-PCR. The universal ITS1-PCR revealed T. evansi infection in horses ( n = 12; 3.3%) and donkeys ( n = 4; 2.8%). There was no significant effect of sex or age on the prevalence of trypanosomiasis in horses or donkeys. Application of the RoTat1.2-PCR revealed that the RoTat1.2 VSG gene was absent from the positive ITS1-PCR samples of 3 horses and 1 donkey. This discrepancy could be explained by the circulation of T. evansi type B in Saudi Arabia; however, this suspicion requires confirmation.

Endemicity of chytridiomycosis features pathogen overdispersion.

Pathogens can be critical drivers of the abundance and distribution of wild animal populations. The presence of an overdispersed pathogen load distribution between hosts (where few hosts harbour heavy parasite burdens and light infections are common) can have an important stabilizing effect on host-pathogen dynamics where infection intensity determines pathogenicity. This may potentially lead to endemicity of an introduced pathogen rather than extirpation of the host and/or pathogen. Overdispersed pathogen load distributions have rarely been considered in wild animal populations as an important component of the infection dynamics of microparasites such as bacteria, viruses, protozoa and fungi. Here we examined the abundance, distribution and transmission of the model fungal pathogen Batrachochytrium dendrobatidis (Bd, cause of amphibian chytridiomycosis) between wild-caught Litoria rheocola (common mist frogs) to investigate the effects of an overdispersed pathogen load distribution on the host population in the wild. We quantified host survival, infection incidence and recovery probabilities relative to infectious burden, and compared the results of models where pathogen overdispersion either was or was not considered an important feature of host-pathogen dynamics. We found the distribution of Bd load between hosts to be highly overdispersed. We found that host survival was related to infection burden and that accounting for pathogen overdispersion allowed us to better understand infection dynamics and their implications for disease control. In addition, we found that the pattern of host infections and recoveries varied markedly with season whereby (i) infections established more in winter, consistent with temperature-dependent effects on fungal growth, and (ii) recoveries (loss of infection) occurred frequently in the field throughout the year but were less likely in winter. Our results suggest that pathogen overdispersion is an important feature of endemic chytridiomycosis and that intensity of infection determines disease impact. These findings have important implications for our understanding of chytridiomycosis dynamics and the application of management strategies for disease mitigation. We recommend quantifying individual infectious burdens rather than infection state where possible in microparasitic diseases.

A new species of glassfrog, genus Hyalinobatrachium (Anura: Centrolenidae), from the Caribbean foothills of Costa Rica.

We describe a new glassfrog belonging to the genus Hyalinobatrachium from the tropical wet forests and premontane rainforests of the Caribbean foothills of Costa Rica, at elevations between 400-800 m. The type locality is approximately 4km west of Santa Clara, ca. 400 m a.s.l. (N 10.219, W 83.949). This new taxon is distinguished by its morphological characteristics, distinct advertisement call, and genetic distance (COI mRNA). The new species can be distinguished from other species of the genus Hyalinobatrachium by the combination of the following characters: (1) snout truncate in dorsal and lateral views; (2) granular dorsal skin; (3) parietal and cardial peritonea transparent; (4) hand webbing formula III 2(-)-2(+) IV; (5) in life having a uniform lime green dorsal surface that lacks any evident light or dark spots; (6) iris coloration silvery-white with fine dark spots or reticulation; (7) advertisement call consisting of a single tonal long metallic whistle-like note with a duration of 0.40-0.55 s (average 0.501 s) and a dominant frequency of 3.35-3.44 kHz (average 3.39 kHz); (8) highly divergent DNA sequences for the mitochondrial gene COI from other analysed members of the genus Hyalinobatrachium, with 12.4% divergence from its closest known species, Hyalinobatrachium chirripoi.

Extremely low prevalence of Batrachochytrium dendrobatidis in frog populations from neotropical dry forest of Costa Rica supports the existence of a climatic refuge from disease.

Population declines and extinctions of numerous species of amphibians, especially stream-breeding frogs, have been linked to the emerging infectious disease chytridiomycosis, caused by the chytrid fungus, Batrachochytrium dendrobatidis. In Central America, most of the 34 species of the Craugastor punctariolus species group have disappeared in recent years in high- and low-elevation rainforests. Distribution models for B. dendrobatidis and the continuous presence of the extirpated stream-dwelling species, Craugastor ranoides, in the driest site of Costa Rica (Santa Elena Peninsula), suggest that environmental conditions might restrict the growth and development of B. dendrobatidis, existing as a refuge from chytridiomycosis-driven extinction. We conducted field surveys to detect and quantify the pathogen using Real-time PCR in samples from 15 species of frogs in two locations of tropical dry forest. In Santa Elena Peninsula, we swabbed 310 frogs, and only one sample of the species, C. ranoides, tested positive for B. dendrobatidis (prevalence <0.1%). In Santa Rosa Station, we swabbed 100 frogs, and nine samples from three species tested positive (prevalence = 9.0%). We failed to detect signs of chytridiomycosis in any of the 410 sampled frogs, and low quantities of genetic equivalents (between 0 and 1073) were obtained from the ten positive samples. The difference in the prevalence between locations might be due not only to the hotter and drier conditions of Santa Elena Peninsula but also to the different compositions of species in both locations. Our results suggest that B. dendrobatidis is at the edge of its distribution in these dry and hot environments of tropical dry forest. This study supports the existence of climatic refuges from chytridiomycosis and highlights the importance of tropical dry forest conservation for amphibians in the face of epidemic disease.

Do pathogens become more virulent as they spread? Evidence from the amphibian declines in Central America.

The virulence of a pathogen can vary strongly through time. While cyclical variation in virulence is regularly observed, directional shifts in virulence are less commonly observed and are typically associated with decreasing virulence of biological control agents through coevolution. It is increasingly appreciated, however, that spatial effects can lead to evolutionary trajectories that differ from standard expectations. One such possibility is that, as a pathogen spreads through a naive host population, its virulence increases on the invasion front. In Central America, there is compelling evidence for the recent spread of pathogenic Batrachochytrium dendrobatidis (Bd) and for its strong impact on amphibian populations. Here, we re-examine data on Bd prevalence and amphibian population decline across 13 sites from southern Mexico through Central America, and show that, in the initial phases of the Bd invasion, amphibian population decline lagged approximately 9 years behind the arrival of the pathogen, but that this lag diminished markedly over time. In total, our analysis suggests an increase in Bd virulence as it spread southwards, a pattern consistent with rapid evolution of increased virulence on Bd's invading front. The impact of Bd on amphibians might therefore be driven by rapid evolution in addition to more proximate environmental drivers.

There is no evidence for a temporal link between pathogen arrival and frog extinctions in north-eastern Australia.

Pathogen spread can cause population declines and even species extinctions. Nonetheless, in the absence of tailored monitoring schemes, documenting pathogen spread can be difficult. In the case of worldwide amphibian declines the best present understanding is that the chytrid fungus Batrachochytrium dendrobatidis (Bd) has recently spread, causing amphibian declines and extinction in the process. However, good evidence demonstrating pathogen arrival followed by amphibian decline is rare, and analysis of putative evidence is often inadequate. Here we attempt to examine the relationship between Bd arrival and amphibian decline across north-eastern Australia, using sites where a wave-like pattern of amphibian decline was first noticed and at which intensive research has since been conducted. We develop an analytical framework that allows rigorous estimation of pathogen arrival date, which can then be used to test for a correlation between the time of pathogen arrival and amphibian decline across sites. Our results show that, with the current dataset, the earliest possible arrival date of Bd in north-eastern Australia is completely unresolved; Bd could have arrived immediately before sampling commenced or may have arrived thousands of years earlier, the present data simply cannot say. The currently available data are thus insufficient to assess the link between timing of pathogen arrival and population decline in this part of the world. This data insufficiency is surprising given that there have been decades of research on chytridiomycosis in Australia and that there is a general belief that the link between Bd arrival and population decline is well resolved in this region. The lack of data on Bd arrival currently acts as a major impediment to determining the role of environmental factors in driving the global amphibian declines, and should be a major focus of future research.

Short-term exposure to warm microhabitats could explain amphibian persistence with Batrachochytrium dendrobatidis.

Environmental conditions can alter the outcomes of symbiotic interactions. Many amphibian species have declined due to chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), but many others persist despite high Bd infection prevalence. This indicates that Bd's virulence is lower, or it may even be a commensal, in some hosts. In the Australian Wet Tropics, chytridiomycosis extirpated Litoria nannotis from high-elevation rain forests in the early 1990 s. Although the species is recolonizing many sites, no population has fully recovered. Litoria lorica disappeared from all known sites in the early 1990 s and was thought globally extinct, but a new population was discovered in 2008, in an upland dry forest habitat it shares with L. nannotis. All frogs of both species observed during three population censuses were apparently healthy, but most carried Bd. Frogs perch on sun-warmed rocks in dry forest streams, possibly keeping Bd infections below the lethal threshold attained in cooler rain forests. We tested whether short-term elevated temperatures can hamper Bd growth in vitro over one generation (four days). Simulating the temperatures available to frogs on strongly and moderately warmed rocks in dry forests, by incubating cultures at 33°C for one hour daily, reduced Bd growth below that of Bd held at 15°C constantly (representing rain forest habitats). Even small decreases in the exponential growth rate of Bd on hosts may contribute to the survival of frogs in dry forests.

Fresh waters and fish diversity: distribution, protection and disturbance in tropical Australia.

BACKGROUND: Given the globally poor protection of fresh waters for their intrinsic ecological values, assessments are needed to determine how well fresh waters and supported fish species are incidentally protected within existing terrestrial protected-area networks, and to identify their vulnerability to human-induced disturbances. To date, gaps in data have severely constrained any attempt to explore the representation of fresh waters in tropical regions. METHODOLOGY AND RESULTS: We determined the distribution of fresh waters and fish diversity in the Wet Tropics of Queensland, Australia. We then used distribution data of fresh waters, fish species, human-induced disturbances, and the terrestrial protected-area network to assess the effectiveness of terrestrial protected areas for fresh waters and fish species. We also identified human-induced disturbances likely to influence the effectiveness of freshwater protection and evaluated the vulnerability of fresh waters to these disturbances within and outside protected areas. The representation of fresh waters and fish species in the protected areas of the Wet Tropics is poor: 83% of stream types defined by order, 75% of wetland types, and 89% of fish species have less than 20% of their total Wet Tropics length, area or distribution completely within IUCN category II protected areas. Numerous disturbances affect fresh waters both within and outside of protected areas despite the high level of protection afforded to terrestrial areas in the Wet Tropics (>60% of the region). High-order streams and associated wetlands are influenced by the greatest number of human-induced disturbances and are also the least protected. Thirty-two percent of stream length upstream of protected areas has at least one human-induced disturbance present. CONCLUSIONS/SIGNIFICANCE: We demonstrate the need for greater consideration of explicit protection and off-reserve management for fresh waters and supported biodiversity by showing that, even in a region where terrestrial protection is high, it does not adequately capture fresh waters.