POSTMORTEM FINDINGS IN EIGHT SPECIES OF CAPTIVE CAECILIAN (AMPHIBIA: GYMNOPHIONA) OVER A TEN-YEAR PERIOD.

Between July 2007 and June 2017 there were 86 deaths in the populations of eight caecilian species at the Zoological Society of London (ZSL) London Zoo. The mortality rate (deaths per animal-year at risk) ranged from 0.03 in the Congo caecilian (Herpele squalostoma) to 0.85 in Kaup's caecilian (Potomotyphlus kaupii). Among the 73 individuals examined post mortem, no cause of death or primary diagnosis could be established in 35 cases, but of the others the most common cause of death was dermatitis (22 cases). When all significant pathological findings were considered, skin lesions of varying types were again the commonest (56 cases), particularly among the aquatic species: Typhlonectes compressicauda (18 out of 21 cases), T. natans (8/10) and P. kaupii (12/14). Other common findings were poor gut-fill (35 cases), kidney and gastrointestinal lesions (10 cases each), generalized congestion (8 cases) and poor body condition (6 cases). This review adds to the growing body of knowledge regarding the presentations and causes of disease in captive caecilians.

Comparison of the nutritional content of the captive and wild diets of the critically endangered mountain chicken frog (Leptodactylus fallax) to improve its captive husbandry.

It is vital to provide appropriate nutrition to maintain healthy populations in conservation breeding programs. Knowledge of the wild diet of a species can be used to inform captive diet formulation. The nutritional content of the wild diet of the critically endangered mountain chicken frog (Leptodactylus fallax) is unknown, like that of most amphibians. In this study, we analyzed the nutritional content of food items that comprise 91% of the wild diet of L. fallax, by dry weight of food items, and all food items offered to captive L. fallax at ZSL London Zoo and Jersey Zoo. We subsequently compared the nutritional content of the wild diet and captive diet at ZSL London Zoo consumed by L. fallax. To the authors' knowledge, this is the first study to directly compare the nutritional content of the wild and captive diets of an anuran amphibian. The captive diet at ZSL London Zoo, without dusting of nutritional supplements, was higher in gross energy and crude fat and lower in ash, calcium and calcium:phosphorus ratio than the wild diet. Most of the food items in the captive diets had a high omega-6:omega-3 fatty acid ratio and in the wild diet had a low omega-6:omega-3 fatty acid ratio. We recommend a combination of modifications to the captive diets to better reflect the nutritional content of the wild diet. Nutritional analysis of captive and wild diets is recommended for other species in conservation breeding programs to improve captive husbandry and ultimately fitness.

Development of a body condition score for the mountain chicken frog (Leptodactylus fallax).

The Critically Endangered mountain chicken frog (Leptodactylus fallax) has undergone drastic population decline due to habitat loss, hunting, invasive species, and chytridiomycosis. In response, several partner institutions initiated a conservation breeding program. It is important to maintain the captive population in good health. Therefore the program partners have recommended establishment of protocols for health examination of the species, including body condition assessment. Visual body condition scoring is a useful means to assess body condition in zoo animals for which regular bodyweight measurements are impractical or associated with capture-related stress. In this study, the authors developed a visual body condition score for the mountain chicken frog based on an ordinal categorical scale from 1 to 5 (1 = lowest body condition, 5 = highest body condition) using anatomical features that vary with total body energy reserves. Veterinary staff, animal managers, keepers, researchers, and students subsequently used the body condition score to assign scores to 98 mountain chicken frogs (41 male, 57 female) aged between 8 months and 12 years housed in five zoos in the UK and Jersey between February and March 2016. Body condition scores showed moderate (rho = 0.54; males) to strong (rho = 0.6; females) correlation with the scaled mass index, an objective measure of total energy reserves. The majority of pairwise comparisons between scores showed slight to substantial intra-observer agreement (93.8%) and slight to almost perfect inter-observer agreement (97.2%). Cases of poor agreement were likely due to limited observer experience working with the species.

FERLAVIRUS-RELATED DEATHS IN A COLLECTION OF VIPERID SNAKES.

Between June and October 2013, 26 snakes of six viperid species kept in two adjoining rooms died ( n = 16) or were euthanized on medical (1) or welfare grounds (9). Two were from the main zoo collection, but the other 24 had been imported and quarantined for a minimum of 6 mo. Four of those that died and the single snake euthanized on medical grounds showed minor signs of respiratory disease prior to death, and five were weak, lethargic, and/or poor feeders. Frequent postmortem findings among all snakes were poor body condition (18) and respiratory disease (13). Seventeen cases were examined histologically, and pneumonia, sometimes with air sacculitis and/or tracheitis, was present in 15 individuals. Lung samples from 24 snakes were ferlavirus polymerase chain reaction (PCR) positive, and one of the two snakes for which only liver was available was also positive. The negative liver sample was from a snake that died of sepsis following anesthesia for surgical removal of a spindle cell sarcoma. Correlation with antemortem PCR testing of glottal and cloacal swabs in five cases was poor (sensitivity = 40%). Immunohistochemistry (IHC) for ferlaviruses on the tissues of 13 PCR-positive cases showed positive labeling in 7 only. Tissues samples from 22 ferlavirus PCR-positive snakes were examined for Chlamydia species by PCR, and 9 were positive, although DNA sequencing only confirmed two of three tested as Chlamydia pneumoniae. Immunohistochemistry for Chlamydia pneumoniae of seven cases (two Chlamydiales PCR positive, one of which was sequenced as C. pneumoniae, plus five negative) confirmed the Chlamydia PCR results. These two Chlamydiales PCR and IHC positive snakes were ferlavirus PCR positive, but IHC negative suggesting that, even though a ferlavirus was the predominant cause of the outbreak, in a few cases death may have been due to chlamydiosis with ferlavirus present, but not acting as the primary pathogen.

Meeting ultraviolet B radiation requirements of amphibians in captivity: a case study with mountain chicken frogs (Leptodactylus fallax) and general recommendations for pre-release health screening.

Conservation breeding programmes are a tool used to prevent amphibian extinctions. The husbandry requirements of amphibians are complex. Ongoing research is needed to ensure optimal management of those captive-bred animals destined, in particular, for reintroduction. The UV-B and vitamin D3 requirements of amphibians are largely unknown. Metabolic bone disease has been reported in a number of species. These include the Critically Endangered mountain chicken frog (Leptodactylus fallax) reared in captivity on diets supplemented with a high-calcium multivitamin and mineral supplement containing vitamin D3 but without UV-B provision. Captive-bred L. fallax being reared for reintroduction to Montserrat were provided with UV-B radiation from metamorphosis and were fed on insects supplemented with vitamins and minerals. Overlapping heat, light and UV-B gradients were provided, mimicking what we believe best represents the natural situation and thereby facilitated self-regulation of UV-B exposure. A subset of 10 frogs was periodically radiographed to assess skeletal health. Radiographic bone density and anatomical integrity appeared unremarkable when compared with a wild caught L. fallax. In addition to other routine health-screening, we recommend that radiography be performed to a structured schedule on a subset of all captive-bred and reared amphibians to assess skeletal health and to gauge the appropriateness of captive husbandry. We demonstrate here that, through the appropriate provision of a combination of both UV-B radiation and dietary supplementation, L. fallax can be bred and reared in captivity with healthy skeletal development.

The tadpole of the Lake Oku clawed frog Xenopus longipes (Anura; Pipidae).

Xenopus longipes Loumont and Kobel, 1991 is an aquatic polyploid frog endemic to the high altitude crater lake, Lake Oku in North West region, Cameroon (Loumont & Kobel 1991). The tadpole of X. longipes is currently undescribed. So far, only dead tadpoles have been found at Lake Oku during regular monitoring since 2008 (Doherty-Bone et al. 2013), with specimens too decomposed to make adequate descriptions. Captive breeding provides one opportunity to obtain fresh specimens for description.

Global conservation status of the jawed vertebrate Tree of Life.

Human-driven extinction threatens entire lineages across the Tree of Life. Here we assess the conservation status of jawed vertebrate evolutionary history, using three policy-relevant approaches. First, we calculate an index of threat to overall evolutionary history, showing that we expect to lose 86-150 billion years (11-19%) of jawed vertebrate evolutionary history over the next 50-500 years. Second, we rank jawed vertebrate species by their EDGE scores to identify the highest priorities for species-focused conservation of evolutionary history, finding that chondrichthyans, ray-finned fish and testudines rank highest of all jawed vertebrates. Third, we assess the conservation status of jawed vertebrate families. We found that species within monotypic families are more likely to be threatened and more likely to be in decline than other species. We provide a baseline for the status of families at risk of extinction to catalyse conservation action. This work continues a trend of highlighting neglected groups-such as testudines, crocodylians, amphibians and chondrichthyans-as conservation priorities from a phylogenetic perspective.

Iron-coated Komodo dragon teeth and the complex dental enamel of carnivorous reptiles.

Komodo dragons (Varanus komodoensis) are the largest extant predatory lizards and their ziphodont (serrated, curved and blade-shaped) teeth make them valuable analogues for studying tooth structure, function and comparing with extinct ziphodont taxa, such as theropod dinosaurs. Like other ziphodont reptiles, V. komodoensis teeth possess only a thin coating of enamel that is nevertheless able to cope with the demands of their puncture-pull feeding. Using advanced chemical and structural imaging, we reveal that V. komodoensis teeth possess a unique adaptation for maintaining their cutting edges: orange, iron-enriched coatings on their tooth serrations and tips. Comparisons with other extant varanids and crocodylians revealed that iron sequestration is probably widespread in reptile enamels but it is most striking in V. komodoensis and closely related ziphodont species, suggesting a crucial role in supporting serrated teeth. Unfortunately, fossilization confounds our ability to consistently detect similar iron coatings in fossil teeth, including those of ziphodont dinosaurs. However, unlike V. komodoensis, some theropods possessed specialized enamel along their tooth serrations, resembling the wavy enamel found in herbivorous hadrosaurid dinosaurs. These discoveries illustrate unexpected and disparate specializations for maintaining ziphodont teeth in predatory reptiles.

A new species of the genus Rhabdophis Fitzinger, 1843 (Squamata: Colubridae) from the Hoang Lien range, northwest Vietnam.

We describe a new species of the Natricinae genus Rhabdophis Fitzinger, 1843 from the Hoang Lien range, northwest Vietnam. The new species is distinct from all congeneric species on the grounds of morphometric and molecular data. The new species is most similar to Rhabdophis leonardi in terms of morphology but can be distinguished from it based on differences in maxillary tooth count, scalation, and genetic data. A sequence on GenBank from a Rhabdophis specimen collected in Honghe, Yunnan, China was identical to the species we describe, and it is likely that the new species is not restricted to Vietnam. As a priority, future work should focus on determining the distribution of this species, as well as understanding population and life history traits such as reproductive rate.

Microbiome function predicts amphibian chytridiomycosis disease dynamics.

BACKGROUND: The fungal pathogen Batrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood. METHODS: Microbiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection. RESULTS: Microbiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes. CONCLUSIONS: Bd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection. Video Abstract.

A new species of Leptobrachella (Anura, Megophryidae) from Mount Pu Ta Leng, northwest Vietnam.

The Hoang Lien Range in northwest Vietnam is known to harbour five species of Leptobrachella, a specious genus of terrestrial frogs. We collected specimens of Leptobrachella from Mount Pu Ta Leng on the second highest peak in the Hoang Lien Range and use morphological and molecular data to show that this population represents a previously undescribed species which we name Leptobrachella graminicola sp. nov. after the abundance of calling males on sedge-like plants. This new species is closely related to L. bourreti but the new species is distinguished from L. bourreti and other congeneric species by a combination of the following characters: (1) a body size range of 23.124.6 mm in six adult males and 28.632.9 mm in five adult females; (2) skin on dorsum smooth, with many tubercles and lacking dermal ridges; (3) toes with rudimentary webbing and broad lateral fringes; (4) belly white with brown spots; (5) throat dark brown with light grey-brown flecks and spots; (6) a row of large white spots on the outer edge of the tarsus extending from the heel to the inner metatarsal tubercle, sometimes forming a long white stripe; and (7) a bicoloured iris. The new, high-elevation species is likely to be range-restricted and threatened by habitat loss and the activities of tourists.

Amolops caelumnoctis Rao amp; Wilkinson, 2007, a Junior Synonym of A. splendissimus Orlov amp; Ho, 2007 (Amphibia: Anura: Ranidae).

Amolops splendissimus Orlov and Ho, 2007 and A. caelumnoctis Rao and Wilkinson, 2007 were described almost simultaneously from either side of the China-Vietnam border. The two species share a strong morphological resemblance, and their taxonomic distinctiveness has been questioned, yet no one has confirmed the taxonomic relationship and status between the two taxa. To resolve this taxonomic issue, we collected additional topotypic and near-topotypic specimens of A. splendissimus and A. caelumnoctis from both China (A. caelumnoctis: Wenshan County, Yunnan Province; type locality Luchun County, Yunnan Province), and Vietnam (A. splendissimus: Tam Duong District, Lai Chau Province; type locality Mount Ky Quan San, Bat Xat, Lao Cai Province). Molecular analysis based on a 16S rRNA fragment revealed minimal genetic divergences between the two taxa (0.0%0.4% uncorrected p-distance), and both species are closely related to A. viridimaculatus (2.1%2.3%) and A. medogensis (3.5%3.7%). Morphological comparisons between the newly collected specimens and the original descriptions of both species further support the lack of distinctiveness of the two species, hence, we conclude that A. caelumnoctis is a junior synonym of A. splendissimus.

A description of the tadpole of Megophrys "Brachytarsophrys" intermedia (Smith, 1921).

Megophrys Kuhl and Van Hasselt are an Asian radiation of terrestrial frogs which are usually associated with montane forest (Mahony et al. 2017). The Annam horned frog (Megophrys intermedia) is reported from montane evergreen forest in Vietnam and Lao People's Democratic Republic (Stuart 2005; Hendrix et al. 2008; Nguyen et al. 2009; Tran 2013; Orlov et al. 2015; IUCN 2017; Mahony et al. 2017). The IUCN Red List assessment for the species reports an elevational range of 782-1500 m asl (IUCN 2017) although the type locality was reported to be above 1500 m asl (Smith 1921), and Tran (2013) collected a specimen at 1515 m asl. Megophrys intermedia is assessed as Least Concern (IUCN 2017) and little is known about the reproductive behaviour and life history of the species. In Lam Dong Province (Vietnam), male M. intermedia call in March (Tran 2013); on the Langbian Plateau (Vietnam), male specimens are reported to call by day and by night from March to May and specimens called from within deep crevices between boulders in streams (Smith 1921); in Xe Kong Province (Laos), males call or guard egg clutches that are laid in water in July (Stuart 2005).

A description of the tadpole of the Critically Endangered Botsford's leaf-litter frog (Leptobrachella botsfordi) with comments on the distribution and conservation status of the species.

We describe the tadpole of the Botsford's leaf-litter frog Leptobrachella botsfordi, a species endemic to Mount Fansipan in northwest Vietnam, for the first time. Tadpoles of this species were found in steep, fast-flowing streams at elevations between 2500-2600 m asl. We also report a previously unknown locality for this species on Mount Fansipan, which increases the species' known Extent of Occurrence from 8 km2 to 36 km2. Our findings will inform subsequent conservation initiatives for this poorly known and highly threatened species.

The tadpoles of five Megophrys Horned frogs (Amphibia: Megophryidae) from the Hoang Lien Range, Vietnam.

Frogs in the genus Megophrys are an Asian radiation of stream-breeding frogs. The tadpoles of many Megophrys species are undescribed; those that are described are often dubiously allocated to species by association with post metamorphic specimens at collection sites and without supportive molecular data. We provide detailed descriptions of the larvae of five species of Megophrys from the Hoang Lien Range in northwest Vietnam: Megophrys fansipanensis, M. gigantica, M. hoanglienensis, M. jingdongensis and M. maosonensis. Tadpoles from different subgenera differ from each other via a combination of patternation in life, oral disc shape and tail morphology but given the small sample size, and limited number of species it is unlikely that these differences can be applied more widely to delineate subgenera. Morphological differences between tadpoles from species within the subgenus Panophrys were insufficient to clearly delineate all species. The ability to identify tadpoles is likely to advance our understanding of the frog fauna in mainland southeast Asia.

Historical museum collections clarify the evolutionary history of cryptic species radiation in the world's largest amphibians.

Inaccurate taxonomic assessment of threatened populations can hinder conservation prioritization and management, with human-mediated population movements obscuring biogeographic patterns and confounding reconstructions of evolutionary history. Giant salamanders were formerly distributed widely across China, and are interpreted as a single species, Andrias davidianus. Previous phylogenetic studies have identified distinct Chinese giant salamander lineages but were unable to associate these consistently with different landscapes, probably because population structure has been modified by human-mediated translocations for recent commercial farming. We investigated the evolutionary history and relationships of allopatric Chinese giant salamander populations with Next-Generation Sequencing methods, using historical museum specimens and late 20th-century samples, and retrieved partial or near-complete mitogenomes for 17 individuals. Samples from populations unlikely to have been affected by translocations form three clades from separate regions of China, spatially congruent with isolation by either major river drainages or mountain ranges. Pliocene-Pleistocene divergences for these clades are consistent with topographic modification of southern China associated with uplift of the Qinghai-Tibet Plateau. General Mixed Yule Coalescent model analysis indicates that these clades represent separate species: Andrias davidianus (Blanchard, 1871) (northern Yangtze/Sichuan), Andrias sligoi (Boulenger, 1924) (Pearl/Nanling), and an undescribed species (Huangshan). Andrias sligoi is possibly the world's largest amphibian. Inclusion of additional reportedly wild samples from areas of known giant salamander exploitation and movement leads to increasing loss of biogeographic signal. Wild Chinese giant salamander populations are now critically depleted or extirpated, and conservation actions should be updated to recognize the existence of multiple species.

Reservoir frogs: seasonality of Batrachochytrium dendrobatidis infection in robber frogs in Dominica and Montserrat.

Emerging infectious diseases are an increasingly important threat to wildlife conservation, with amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis, the disease most commonly associated with species declines and extinctions. However, some amphibians can be infected with B. dendrobatidis in the absence of disease and can act as reservoirs of the pathogen. We surveyed robber frogs (Eleutherodactylus spp.), potential B. dendrobatidis reservoir species, at three sites on Montserrat, 2011-2013, and on Dominica in 2014, to identify seasonal patterns in B. dendrobatidis infection prevalence and load (B. dendrobatidis genomic equivalents). On Montserrat there was significant seasonality in B. dendrobatidis prevalence and B. dendrobatidis load, both of which were correlated with temperature but not rainfall. B. dendrobatidis prevalence reached 35% in the cooler, drier months but was repeatedly undetectable during the warmer, wetter months. Also, B. dendrobatidis prevalence significantly decreased from 53.2% when the pathogen emerged on Montserrat in 2009 to a maximum 34.8% by 2011, after which it remained stable. On Dominica, where B. dendrobatidis emerged seven years prior to Montserrat, the same seasonal pattern was recorded but at lower prevalence, possibly indicating long-term decline. Understanding the dynamics of disease threats such as chytridiomycosis is key to planning conservation measures. For example, reintroductions of chytridiomycosis-threatened species could be timed to coincide with periods of low B. dendrobatidis infection risk, increasing potential for reintroduction success.

Description and development of the tadpole of Rhacophorus feae (Anura; Rhacophoridae).

The genus Rhacophorus Kuhl Van Hasselt is currently known to contain 92 species of frogs (Frost 2018), distributed across south and south-east Asia. Rhacophorus feae Boulenger is a large member of this genus and has a seemingly expansive range been recorded from southern Yunnan in China, the Karen hills in Myanmar, northern Thailand, northern Laos and northern Vietnam (Chanard et al. 1999; Orlov et al. 2002; Nguyen et al. 2005; Stuart 2005; Yang 2008). It is currently included in the intrageneric R. dennysi group (Dubois 1986) along with R. duboisi Ohler, Marquis, Swan Grosjean , R. dennysi Blanford, R. dugritei (David), R. minimus Rao, Wilkinson Liu, R. hungfuensis Liu Hu, R. dorsoviridis Bourret, R. nigropunctatus Liu, Hu Yang, and R. smaragdinus (Blyth).

Two new and potentially highly threatened Megophrys Horned frogs (Amphibia: Megophryidae) from Indochina's highest mountains.

Megophrys are a group of morphologically conserved, primarily forest-dependent frogs known to harbour cryptic species diversity. In this study, we examined populations of small-sized Megophrys from mid- and high elevation locations in the Hoang Lien Range, northern Vietnam. On the basis of morphological, molecular and bioacoustic data, individuals of these populations differed from all species of Megophrys known from mainland Southeast Asia north of the Isthmus of Kra and from neighbouring provinces in China. Further, the newly collected specimens formed two distinct species-level groups. We herein describe two new species, Megophrys fansipanensis sp. nov. and Megophrys hoanglienensis sp. nov. Both new species are range restricted and likely to be highly threatened by habitat degradation. These discoveries highlight the importance of the Hoang Lien Range for Vietnam's amphibian diversity.

Imminent extinction in the wild of the world's largest amphibian.

Species with large geographic ranges are considered resilient to global decline [1]. However, human pressures on biodiversity affect increasingly large areas, in particular across Asia, where market forces drive overexploitation of species [2]. Range-wide threat assessments are often costly and thus extrapolated from non-representative local studies [3]. The Chinese giant salamander (Andrias davidianus), the world's largest amphibian, is thought to occur across much of China, but populations are harvested for farming as luxury food [4]. Between 2013 and 2016, we conducted field surveys and 2,872 interviews in possibly the largest wildlife survey conducted in China. This extensive effort revealed that populations of this once-widespread species are now critically depleted or extirpated across all surveyed areas of their range, and illegal poaching is widespread.