Standard methods for marking caudate amphibians do not impair animal welfare over the short term: An experimental approach.

Major advancements in ecology and biodiversity conservation have been made thanks to methods for marking and individually tracking animals. Marking animals is both widely used and controversial due to the potential consequences for animal welfare, which are often incompletely evaluated prior to implementation. Two outstanding knowledge gaps concerning the welfare consequences of individual marking are their short-term behavioural impacts and the relative impacts from marking versus the handling of animals while carrying out procedures. We addressed these knowledge gaps through an experimental study of alpine newts (Ichthyosaura alpestris) in which we varied handling and marking procedures. Examining individual responses to handling, toe clipping and visible implant elastomer (VIE) injection over 21 days showed that handling and marking elicited increased newt activity and hesitancy to feed compared to animals that did not get handled or marked. These effects were apparent even when animals were handled only (not marked), and marking did not further increase the magnitude of responses. Increases in newt activity and feeding hesitancy were transient; they were not observed in the weeks following handling and marking. While previous studies emphasise the welfare impacts of marking procedures themselves, these findings highlight that handling alone can elicit behavioural changes with possible costs to welfare. Yet, the transient nature of behavioural responses suggests that immediate costs of handling may be subsequently compensated for in the short term.

Relationship between two pathogens in an amphibian community that experienced mass mortalities.

Because host species tend to harbor multiple parasitic species, coinfection in a host is common. The chytrid fungus Batrachochytrium dendrobatidis (Bd) and the viruses in the genus Ranavirus (Rv) are responsible for the decline of amphibians worldwide. Despite wide geographical co-occurrence and the serious conservation problem that coinfection with these pathogens could represent, little is known about their possible synergistic interactions and effects in a host community. We investigated the occurrence and associations between these two pathogens in an amphibian community after Rv-driven disease outbreaks were detected in four populations of the Iberian ribbed newt (Pleurodeles waltl) in northwestern Spain. We collected tissue samples from amphibians and fish and estimated Bd and Rv infection loads by qPCR. A few months after the most recent mass mortality event, Rv infection parameters at the affected sites decreased significantly or were lower than such registered at the sites where no outbreaks were recorded. Both pathogens were simultaneously present in almost all sites, but coinfection in a single host was rare. Our findings suggest that the co-occurrence of Bd and Rv does not predict adverse outcomes (e.g., enhanced susceptibility of hosts to one pathogen due to the presence or infection intensity of the other) following an outbreak. Other variables (such as species identity or site) were more important than infection with a pathogen in predicting the infection status and severity of infection with the other pathogen. Our results highlight the importance of host-specific and environmental characteristics in the dynamics of infections, coinfection patterns, and their impacts.

Relaciones entre dos patógenos en una comunidad anfibia que experimentó mortalidad masiva Resumen La coinfección es común en especies hospederas ya que estas especies tienden a albergar muchas especies parasíticas. El hongo quitridio Batrachochytrium dendrobatidis (Bd) y los virus del género Ranavirus (Rv) son responsables de la declinación mundial de anfibios. A pesar de la amplia co-ocurrencia geográfica y el problema serio de conservación que podría representar la coinfección con estos patógenos, se conoce muy poco sobre sus posibles interacciones sinérgicas y sus efectos en una comunidad hospedera. Investigamos la incidencia y las asociaciones entre estos dos patógenos en una comunidad anfibia después de que se detectaron brotes de enfermedades causados por Rv en cuatro poblaciones del tritón estriado ibérico (Pleurodeles waltl) en el noroeste de España. Recolectamos muestras de tejido de anfibios y peces y estimamos la carga infecciosa de Bd y Rv con una qPCR. Unos meses después del evento de mortalidad masiva más reciente, los parámetros de infección de Rv en los sitios afectados disminuyeron significativamente o fueron más bajos que los registrados en sitios sin brotes. Ambos patógenos estuvieron presentes de forma simultánea en casi todos los sitios, pero fue raro encontrar la coinfección en un solo hospedero. Nuestros descubrimientos sugieren que la coocurrencia de Bd y Rv no pronostica resultados adversos (aumento en la susceptibilidad de los hospederos a un patógeno debido a la presencia o intensidad de infección del otro patógeno) después de un brote. Otras variables, como la identidad de la especie o el sitio, fueron más importantes que la infección con un patógeno en la predicción del estado de infección y la severidad de la infección con otro patógeno. Nuestros resultados resaltan la importancia de las características ambientales y aquellas específicas del hospedero en las dinámicas de infección, los patrones de coinfección y sus impactos.

Post-epizootic microbiome associations across communities of neotropical amphibians.

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

Differential effects of dietary protein on early life-history and morphological traits in natterjack toad (Epidalea calamita) tadpoles reared in captivity.

The production of high quality amphibian larvae through optimal diets is a critical component of amphibian conservation breeding programs. Larval period, survival, body weight and total length are frequently used as metrics of adequate nutrition. However, the effects of nutrition on tadpole and metamorph morphology are rarely tested in detail. In the present study, we analyzed the most common metrics and six other larval and post-metamorphic morphological traits in natterjack toads (Epidalea calamita) fed with three different commercial fish diets, varying in protein content (32.0%, 38.3%, and 46.2%). Our results suggest that early life-history (tadpole growth, development, and survival) and morphological traits of E. calamita tadpoles are differentially affected by the percentage of dietary protein. As protein content increased, tadpoles exhibited larger bodies along with shorter tail fins; however, with no significant differences in total length. Larval period was similar across treatments but mortality was lower in high-protein diet. At high-protein diets the metamorphs revealed significantly longer bodies, and wider heads and hind legs, but there was no significant difference in the average weight across all dietary treatments. Based on our results, feed containing 46.2% protein promotes growth, development and survival of E. calamita tadpoles better than either of the other two feeds tested. The use of other body measures beyond weight, tadpole total length, and snout-vent length in studies of amphibian nutrition in captivity may assist the selection of appropriate diets to optimize tadpole survival and metamorph fitness.

Spatial Ecology of an Arboreal Iguana (Oplurus cyclurus) in a Treeless Landscape.

Understanding the spatial ecology of species has important implications for conservation, as it helps identify suitable habitats and minimum requirements for biodiversity monitoring and management. The spiny-tailed lizard Oplurus cyclurus is a widespread endemic iguanid occurring in dry areas of southern and western Madagascar. While the species is known to be mostly arboreal, populations of the Isalo sandstone massif suggest local adaptation to a less forested savannah and a more exposed habitat. We radio-tracked 19 spiny-tailed lizards to investigate the species' rock-dwelling behaviour and spatial ecology at Isalo National Park. Tracked individuals showed high site and burrow fidelity, and a basking behaviour mostly tied to the accessibility of their burrow, the time of day, and their life stage. Activity peaked during the sunniest hours, while juveniles were more active than adults with unfavourable weather conditions. Despite high burrow fidelity, lizards used shelters non-exclusively, regularly changing (approx. once a week) with neighbouring burrows (average distance between burrows = 13.6 m). However, there was no obvious relation between lizards' body and/or tail size and the width and depth of selected burrows. Dynamic Brownian Bridge Movement Models estimated frequented areas over 247.8 m2 (95% isopleth), where territorial overlap is common. Our results challenge the notion that burrow-site fidelity is the sole driving factor behind space utilization in the studied population. We argue that the apparently unusual saxicolous habits imposed by habitat features (the absence of trees) may lead to local behavioural adjustments influencing antipredatory and foraging strategies, as well as intraspecific interactions.

Non-Lethal Detection of Ranavirus in Fish.

Emergent infectious diseases have an increasing impact on both farmed animals and wildlife. The ability to screen for pathogens is critical for understanding host-pathogen dynamics and informing better management. Ranavirus is a pathogen of concern, associated with disease outbreaks worldwide, affecting a broad range of fish, amphibian, and reptile hosts, but research has been limited. The traditional screening of internal tissues, such as the liver, has been regarded as the most effective for detecting and quantifying Ranavirus. However, such methodology imposes several limitations from ethical and conservation standpoints. Non-lethal sampling methods of viral detection were explored by comparing the efficacy of both buccal swabbing and fin clipping. The study was conducted on two Iberian, threatened freshwater fish (Iberochondrostoma lusitanicum and Cobitis paludica), and all samples were screened using qPCR. While for C. paludica both methods were reliable in detecting Ranavirus, on I. lusitanicum, there was a significantly higher detection rate in buccal swabs than in fin tissue. This study, therefore, reports that fin clipping may yield false Ranavirus negatives when in small-bodied freshwater fish. Overall, buccal swabbing is found to be good as an alternative to more invasive procedures, which is of extreme relevance, particularly when dealing with a threatened species.

Another step through the crux: a new microendemic rock-dwelling Paroedura (Squamata, Gekkonidae) from south-central Madagascar.

Using an integrative taxonomic approach including genetic and morphological data, we formally describe a new microendemic gecko species belonging to the Paroedurabastardi clade, previously referred to as P.bastardi D. We name this taxon currently known from Anja Reserve and Tsaranoro Valley Forest (south-central Madagascar), as P.manongavatosp. nov. The new species differs from other species of the P.bastardi clade by ≥ 12.4% uncorrected p-distance at the mitochondrial 16S rRNA gene and it forms a monophyletic group in the COI mtDNA phylogenetic tree. It lacks haplotype sharing at the nuclear KIAA1239 and CMOS genes with the other species of the same complex, including the syntopic P.rennerae. Given its limited extent of occurrence and high levels of habitat fragmentation linked to forest clearances and fires, we propose the IUCN Red List Category of Critically Endangered, based on the B1ab(iii) criterion. The conservation value of Anja Reserve and Tsaranoro Valley Forest is remarkable. Preserving the remaining deciduous forest habitat is of paramount importance to protect these narrow-range reptile species.

Climate warming triggers the emergence of native viruses in Iberian amphibians.

The number of epizootics in amphibian populations caused by viruses of the genus Ranavirus is increasing worldwide. Yet, causes for pathogen emergence are poorly understood. Here, we confirmed that the Common midwife toad virus (CMTV) and Frog virus 3 (FV3) are responsible for mass mortalities in Iberia since the late 1980s. Our results illustrate the Iberian Peninsula as a diversity hotspot for the highly virulent CMTV. Although this pattern of diversity in Europe is consistent with spread by natural dispersal, the exact origin of the emergence of CMTV remains uncertain. Nevertheless, our data allow hypothesizing that the Iberian Peninsula might harbor the ancestral population of CMTVs that could have spread into the rest of Europe. In addition, we found that climate warming could be triggering the CMTV outbreaks, supporting its endemic status in the Iberian Peninsula.

Living within fallen palm leaves: the discovery of an unknown Blommersia (Mantellidae: Anura) reveals a new reproductive strategy in the amphibians of Madagascar.

We describe a new mantelline frog of the genus Blommersia found in rainforest in North East Madagascar, from the protected areas of Ambatovaky, Betampona, Masoala, and Zahamena. Blommersia angolafa n.sp. is a small frog, with a body size of 17-21 mm, expanded finger and toe tips, and colouration ranging from yellow to dark brown, with pale-bluish spots on the flanks and light tips of fingers and toes. A peculiar aspect characterising this new species is its novel life history and reproductive mode. Both sexes live and breed in a phytotelmic habitat of water accumulated within fallen prophylls and fallen leaf sheaths of at least three species of Dypsis palms. Within these phytotelmata, egg laying and complete larval development occur. Thus, B. angolafa n.sp. represents a new evolutionary lineage of Malagasy frogs in which phytotelmy is known. Up to now, reproduction in phytotelmata in Malagasy frogs has been reported for many cophyline microhylids, most species of Guibemantis, Mantella laevigata, and possibly in a still-undescribed species belonging to the genus Spinomantis. We consider the reproductive mode of B. angolafa as a derived character, having evolved from the more typical reproduction in lentic water bodies. The general scarcity of lentic habitats in Malagasy rainforests may have provided the conditions that favoured the evolution of this phytotelmic breeding strategy. The new species, being specialised to a habitat represented by a few selected Dypsis species, potentially suffers the selective exploitation of these palms.

Resolving a taxonomic and nomenclatural puzzle in mantellid frogs: synonymization of Gephyromantis azzurrae with G. corvus, and description of Gephyromantis kintana sp. nov. from the Isalo Massif, western Madagascar.

The genus Gephyromantis belongs to the species-rich family Mantellidae and is currently divided in six subgenera. Among these is the subgenus Phylacomantis, which currently includes four described species: Gephyromantis pseudoasper, G. corvus, G. azzurrae, and G. atsingy. The latter three species are distributed in western Madagascar, and two of them (G. azzurrae and G. corvus) occur in the Isalo Massif. Based on the analysis of molecular data (a fragment of the 16S rRNA gene), morphological inspection of museum specimens, and photographic comparisons, G. azzurrae is synonymised with G. corvus and the second Phylacomantis lineage of Isalo is described as G. kintana sp. nov. This medium-sized frog species (adult snout-vent length 35-44 mm) is assigned to this subgenus according to genetic and morphological similarities to the other known species of Phylacomantis. Gephyromantis kintana sp. nov. is known only from the Isalo Massif, while new records for G. corvus extend its range to ca. 200 km off its currently known distribution. These two taxa seem to occur in syntopy in at least one locality in Isalo, and the easiest way to distinguish them is the inspection of the ventral colouration, dark in G. corvus and dirty white in G. kintana.

Unveiling patterns of viral pathogen infection in free-ranging carnivores of northern Portugal using a complementary methodological approach.

Pathogen surveillance in free-ranging carnivores presents challenges due to their low densitie and secretive nature. We combined molecular and serological assays to investigate infections by viral pathogens (Canine parvovirus (CPV), Canine distemper virus (CDV) and Canine coronavirus (CCoV)) in Portuguese carnivores (Canis lupus, Vulpes vulpes, Lutra lutra, Martes foina, M. martes, Meles meles, and Genetta genetta) over a period of 16 years. Additionally we explored spatio-temporal patterns of virus occurrence in Canis lupus. Our study identified CPV DNA in all carnivore species with an overall prevalence of 91.9 %. CPV was detected in all sampled years and seasons in Canis lupus, supporting its enzootic nature. CDV RNA was mainly detected in the Canidae family, with viral nucleic acid recorded between 2005 and 2008 with a peak prevalence of 67 % among the wolf population, followed by a sharp decline, suggesting an epizootic behaviour of the virus. Antibodies show that mustelids and viverrids were often exposed to CDV. CCoV was first recorded by molecular methods in wolf samples in 2002, remaining in the wolf populations with marked fluctuations over time. The dual serological and molecular approach provided important epidemiological data on pathogens of wild carnivores in Portugal. These programmes should also include monitoring of other potential reservoir hosts such as domestic cats and dogs.

A new stump-toed frog from the transitional forests of NW Madagascar (Anura, Microhylidae, Cophylinae, Stumpffia).

A new species of the miniaturised microhylid frog genus Stumpffia, from north-western Madagascar, is described. Stumpffia froschaueri sp. nov. differs from all other described Stumpffia species in colouration and morphology and is genetically divergent (≥ 7% uncorrected p-distance to all other nominal species of the genus) in a fragment of the mitochondrial 16S rRNA gene and in a segment of the nuclear Rag-1 gene. The new species is reliably known only from a few specimens collected in the Sahamalaza (and surroundings) region. Its known distribution is limited to three forest patches severely threatened by fire, drought and high levels of forest clearance, thus suggesting a classification of "Critically Endangered" according to IUCN Red List criteria.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

From fish to frogs and beyond: Impact and host range of emergent ranaviruses.

Ranaviruses are pathogens of ectothermic vertebrates, including amphibians. We reviewed patterns of host range and virulence of ranaviruses in the context of virus genotype and postulate that patterns reflect significant variation in the historical and current host range of three groups of Ranavirus: FV3-like, CMTV-like and ATV-like ranaviruses. Our synthesis supports previous hypotheses about host range and jumps: FV3s are amphibian specialists, while ATVs are predominantly fish specialists that switched once to caudate amphibians. The most recent common ancestor of CMTV-like ranaviruses and FV3-like forms appears to have infected amphibians but CMTV-like ranaviruses may circulate in both amphibian and fish communities independently. While these hypotheses are speculative, we hope that ongoing efforts to describe ranavirus genetics, increased surveillance of host species and targeted experimental assays of susceptibility to infection and/or disease will facilitate better tests of the importance of hypothetical evolutionary drivers of ranavirus virulence and host range.

Impact of asynchronous emergence of two lethal pathogens on amphibian assemblages.

Emerging diseases have been increasingly associated with population declines, with co-infections exhibiting many types of interactions. The chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses have extraordinarily broad host ranges, however co-infection dynamics have been largely overlooked. We investigated the pattern of co-occurrence of these two pathogens in an amphibian assemblage in Serra da Estrela (Portugal). The detection of chytridiomycosis in Portugal was linked to population declines of midwife-toads (Alytes obstetricans). The asynchronous and subsequent emergence of a second pathogen - ranavirus - caused episodes of lethal ranavirosis. Chytrid effects were limited to high altitudes and a single host, while ranavirus was highly pathogenic across multiple hosts, life-stages and altitudinal range. This new strain (Portuguese newt and toad ranavirus - member of the CMTV clade) caused annual mass die-offs, similar in host range and rapidity of declines to other locations in Iberia affected by CMTV-like ranaviruses. However, ranavirus was not always associated with disease, mortality and declines, contrasting with previous reports on Iberian CMTV-like ranavirosis. We found little evidence that pre-existing chytrid emergence was associated with ranavirus and the emergence of ranavirosis. Despite the lack of cumulative or amplified effects, ranavirus drove declines of host assemblages and changed host community composition and structure, posing a grave threat to all amphibian populations.

Host species vary in infection probability, sub-lethal effects, and costs of immune response when exposed to an amphibian parasite.

The amphibian parasite Batrachochytrium dendrobatidis (Bd) is regarded as an extreme generalist, infecting over 500 species, but amongst these hosts there exists a great deal of variation in the susceptibility to and the costs of parasite exposure. We use two infection experiments to determine whether inter-specific variation in the sublethal and lethal effects of parasite exposure exist in two host species. We then tested the relative roles of host density and diversity on infection probability of a focal susceptible host. Our results show significant heterogeneity in host species response to parasite exposure, and that both lethal and sub-lethal costs exist in individuals that are able to resist infection, indicating that successful immune response to infection comes at a cost. Further, we show that increasing host density significantly increased the likelihood of susceptible individuals becoming infected with Bd irrespective of host diversity and variation in host susceptibility. These results suggest that populations of resistant species are likely to suffer ill-effects of exposure to Bd regardless of their infection status, and that at the stage of initial infection there was no support for the dilution of transmission events, in contrast to other studies that focus on subsequent transmission of infection.

Phylogeny and differentiation of reptilian and amphibian ranaviruses detected in Europe.

Ranaviruses in amphibians and fish are considered emerging pathogens and several isolates have been extensively characterized in different studies. Ranaviruses have also been detected in reptiles with increasing frequency, but the role of reptilian hosts is still unclear and only limited sequence data has been provided. In this study, we characterized a number of ranaviruses detected in wild and captive animals in Europe based on sequence data from six genomic regions (major capsid protein (MCP), DNA polymerase (DNApol), ribonucleoside diphosphate reductase alpha and beta subunit-like proteins (RNR-α and -ß), viral homolog of the alpha subunit of eukaryotic initiation factor 2, eIF-2α (vIF-2α) genes and microsatellite region). A total of ten different isolates from reptiles (tortoises, lizards, and a snake) and four ranaviruses from amphibians (anurans, urodeles) were included in the study. Furthermore, the complete genome sequences of three reptilian isolates were determined and a new PCR for rapid classification of the different variants of the genomic arrangement was developed. All ranaviruses showed slight variations on the partial nucleotide sequences from the different genomic regions (92.6-100%). Some very similar isolates could be distinguished by the size of the band from the microsatellite region. Three of the lizard isolates had a truncated vIF-2α gene; the other ranaviruses had full-length genes. In the phylogenetic analyses of concatenated sequences from different genes (3223 nt/10287 aa), the reptilian ranaviruses were often more closely related to amphibian ranaviruses than to each other, and most clustered together with previously detected ranaviruses from the same geographic region of origin. Comparative analyses show that among the closely related amphibian-like ranaviruses (ALRVs) described to date, three recently split and independently evolving distinct genetic groups can be distinguished. These findings underline the wide host range of ranaviruses and the emergence of pathogen pollution via animal trade of ectothermic vertebrates.

Widespread presence of the pathogenic fungus Batrachochytrium dendrobatidis in wild amphibian communities in Madagascar.

Amphibian chytridiomycosis, an emerging infectious disease caused by the fungus Batrachochytrium dendrobatidis (Bd), has been a significant driver of amphibian declines. While globally widespread, Bd had not yet been reported from within Madagascar. We document surveys conducted across the country between 2005 and 2014, showing Bd's first record in 2010. Subsequently, Bd was detected in multiple areas, with prevalence reaching up to 100%. Detection of Bd appears to be associated with mid to high elevation sites and to have a seasonal pattern, with greater detectability during the dry season. Lineage-based PCR was performed on a subset of samples. While some did not amplify with any lineage probe, when a positive signal was observed, samples were most similar to the Global Panzootic Lineage (BdGPL). These results may suggest that Bd arrived recently, but do not exclude the existence of a previously undetected endemic Bd genotype. Representatives of all native anuran families have tested Bd-positive, and exposure trials confirm infection by Bd is possible. Bd's presence could pose significant threats to Madagascar's unique "megadiverse" amphibians.

A new species of the Boophis rappiodes group (Anura, Mantellidae) from the Sahamalaza Peninsula, northwest Madagascar, with acoustic monitoring of its nocturnal calling activity.

A new species of treefrog of the Boophis rappiodes group (Anura, Mantellidae) is described from the Sahamalaza - Iles Radama National Park in northwest Madagascar. This new species is green in colour with bright red speckling across its head and dorsum; similar in morphology to other species of this group including: B. bottae, B. rappiodes, B. erythrodactylus and B. tasymena. The new species can be distinguished by its advertisement call and by a genetic divergence of more than 4.9% in the analysed mitochondrial 16S rRNA gene fragment. Its call consists of two note types: a trill and a click; although similar sounding to B. bottae, the trill note of the new species has a faster pulse rate while the click note is predominantly two-pulsed rather than three. All individuals were detected from the banks of two streams in Ankarafa Forest. The new species represents the only member of the B. rappiodes group endemic to Madagascar's western coast, with the majority of other members known from the eastern rainforest belt. Despite its conspicuous call, it has not been detected from other surveys of northwest Madagascar and it is likely to be a local endemic to the peninsula. The ranges of two other amphibian species also appear restricted to Sahamalaza, and so the area seems to support a high level of endemicity. Although occurring inside a National Park, this species is highly threatened by the continuing decline in the quality and extent of its habitat. Due to these threats it is proposed that this species should be classified as Critically Endangered according to the IUCN Red List criteria.