An experimental assessment of detection dog ability to locate great crested newts (Triturus cristatus) at distance and through soil.

Detection dogs are increasingly used to locate cryptic wildlife species, but their use for amphibians is still rather underexplored. In the present paper we focus on the great crested newt (Triturus cristatus), a European species which is experiencing high conservation concerns across its range, and assess the ability of a trained detection dog to locate individuals during their terrestrial phase. More specifically, we used a series of experiments to document whether a range of distances between target newts and the detection dog (odour channelled through pipes 68 mm in diameter) affects the localisation, and to assess the ability and efficiency of target newt detection in simulated subterranean refugia through 200 mm of two common soil types (clay and sandy soil, both with and without air vents to mimic mammal burrows, a common refuge used by T. cristatus). The detection dog accurately located all individual T. cristatus across the entire range of tested distances (0.25 m- 2.0 m). The substrate trials revealed that the detection dog could locate individuals also through soil. Contrary to existing studies with detection dogs in human forensic contexts, however, detection was generally slower for T. cristatus under sandy soil compared to clay soil, particularly when a vent was absent. Our study provides a general baseline for the use of detection dogs in locating T. cristatus and similar amphibian species during their terrestrial phase.

N-mixture models provide informative crocodile (Crocodylus moreletii) abundance estimates in dynamic environments.

Estimates of animal abundance provide essential information for population ecological studies. However, the recording of individuals in the field can be challenging, and accurate estimates require analytical techniques which account for imperfect detection. Here, we quantify local abundances and overall population size of Morelet's crocodiles (Crocodylus moreletii) in the region of Calakmul (Campeche, Mexico), comparing traditional approaches for crocodylians (Minimum Population Size-MPS; King's Visible Fraction Method-VFM) with binomial N-mixture models based on Poisson, zero-inflated Poisson (ZIP) and negative binomial (NB) distributions. A total of 191 nocturnal spotlight surveys were conducted across 40 representative locations (hydrologically highly dynamic aquatic sites locally known as aguadas) over a period of 3 years (2017-2019). Local abundance estimates revealed a median of 1 both through MPS (min-max: 0-89; first and third quartiles, Q1-Q3: 0-7) and VFM (0-112; Q1-Q3: 0-9) non-hatchling C. moreletii for each aguada, respectively. The ZIP based N-mixture approach shown overall superior confidence over Poisson and NB, and revealed a median of 6 ± 3 individuals (min = 0; max = 120 ± 18; Q1 = 0; Q3 = 18 ± 4) jointly with higher detectabilities in drying aguadas with low and intermediate vegetation cover. Extrapolating these inferences across all waterbodies in the study area yielded an estimated ~10,000 (7,000-11,000) C. moreletii present, highlighting Calakmul as an important region for this species. Because covariates enable insights into population responses to local environmental conditions, N-mixture models applied to spotlight count data result in particularly insightful estimates of crocodylian detection and abundance.

Challenging a host-pathogen paradigm: Susceptibility to chytridiomycosis is decoupled from genetic erosion.

The putatively positive association between host genetic diversity and the ability to defend against pathogens has long attracted the attention of evolutionary biologists. Chytridiomycosis, a disease caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has emerged in recent decades as a cause of dramatic declines and extinctions across the amphibian clade. Bd susceptibility can vary widely across populations of the same species, but the relationship between standing genetic diversity and susceptibility has remained notably underexplored so far. Here, we focus on a putatively Bd-naive system of two mainland and two island populations of the common toad (Bufo bufo) at the edge of the species' range and use controlled infection experiments and dd-RAD sequencing of >10 000 SNPs across 95 individuals to characterize the role of host population identity, genetic variation and individual body mass in mediating host response to the pathogen. We found strong genetic differentiation between populations and marked variation in their susceptibility to Bd. This variation was not, however, governed by isolation-mediated genetic erosion, and individual heterozygosity was even found to be negatively correlated with survival. Individual survival during infection experiments was strongly positively related to body mass, which itself was unrelated to population of origin or heterozygosity. Our findings underscore the general importance of context-dependency when assessing the role of host genetic variation for the ability of defence against pathogens.

Sex-related differences in aging rate are associated with sex chromosome system in amphibians.

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females) systems. Taking advantage of a unique collection of capture-recapture datasets in amphibians, a vertebrate group where XY and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the "unguarded X/Z effect") or repeat-rich Y/W chromosome (the "toxic Y/W effect") could accelerate aging in the heterogametic sex in some vertebrate clades.

Genetic and morphological data demonstrate hybridization and backcrossing in a pair of salamanders at the far end of the speciation continuum.

Deeply diverged yet hybridizing species provide a system to investigate the final stages of the speciation process. We study a hybridizing pair of salamander species-the morphologically and genetically drastically different newts Triturus cristatus and T. marmoratus-with a panel of 32 nuclear and mitochondrial genetic markers. Morphologically identified hybrids are mostly of the F1 generation and mothered by T. cristatus. The sex ratio of the F1 hybrid class is reciprocally skewed, with a preponderance of females in T. cristatus-mothered hybrids and males in T. marmoratus-mothered hybrids. This amounts to the Haldane effect operating in one direction of the cross. Deeper generation hybrids are occasionally produced, possibly including F1 hybrid × backcross hybrid offspring. Interspecific gene flow is low, yet skewed toward T. cristatus. This asymmetry may be caused by hybrid zone movement, with the superseding species being predisposed to introgression. The persisting gene flow between deeply differentiated species supports the notion that full genetic isolation may be selected against. Conversely, published morphological data suggest that introgressive hybridization is detrimental, with digital malformations occurring more frequently in the area of sympatry. Finally, to assist field identification, both within the area of natural range overlap and concerning anthropogenic introductions elsewhere, we document the phenotypical variation of two generations of hybrids compared with both parental species. We suggest that fluctuating range boundaries, ecological segregation, cytonuclear incompatibilities and hybrid breakdown through Bateson-Dobzhansky-Muller incompatibilities all contribute to species integrity, despite incomplete isolation during secondary contact.

Fungal microbiomes are determined by host phylogeny and exhibit widespread associations with the bacterial microbiome.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

The Influence of Environmental Variation on the Genetic Structure of a Poison Frog Distributed Across Continuous Amazonian Rainforest.

Biogeographic barriers such as rivers have been shown to shape spatial patterns of biodiversity in the Amazon basin, yet relatively little is known about the distribution of genetic variation across continuous rainforest. Here, we characterize the genetic structure of the brilliant-thighed poison frog (Allobates femoralis) across an 880-km-long transect along the Purus-Madeira interfluve south of the Amazon river, based on 64 individuals genotyped at 7609 single-nucleotide polymorphism (SNP) loci. A population tree and clustering analyses revealed 4 distinct genetic groups, one of which was strongly divergent. These genetic groups were concomitant with femoral spot coloration differences, which was intermediate within a zone of admixture between two of the groups. The location of these genetic groups did not consistently correspond to current ecological transitions between major forest types. A multimodel approach to quantify the relative influence of isolation-by-geographic distance (IBD) and isolation-by-environmental resistance (IBR) nevertheless revealed that, in addition to a strong signal of IBD, spatial genetic differentiation was explained by IBR primarily linked to dry season intensity (r2 = 8.4%) and canopy cover (r2 = 6.4%). We show significant phylogenetic divergence in the absence of obvious biogeographical barriers and that finer-scaled measures of genetic structure are associated with environmental variables also known to predict the density of A. femoralis.

Stable species boundaries despite ten million years of hybridization in tropical eels.

Genomic evidence is increasingly underpinning that hybridization between taxa is commonplace, challenging our views on the mechanisms that maintain their boundaries. Here, we focus on seven catadromous eel species (genus Anguilla) and use genome-wide sequence data from more than 450 individuals sampled across the tropical Indo-Pacific, morphological information, and three newly assembled draft genomes to compare contemporary patterns of hybridization with signatures of past introgression across a time-calibrated phylogeny. We show that the seven species have remained distinct for up to 10 million years and find that the current frequencies of hybridization across species pairs contrast with genomic signatures of past introgression. Based on near-complete asymmetry in the directionality of hybridization and decreasing frequencies of later-generation hybrids, we suggest cytonuclear incompatibilities, hybrid breakdown, and purifying selection as mechanisms that can support species cohesion even when hybridization has been pervasive throughout the evolutionary history of clades.

High genetic diversity and lack of pronounced population structure in five species of sympatric Pacific eels.

Understanding the population structure of tropical anguillids residing in the Pacific is vital for their conservation management. Here, the population genetic structure of five sympatric freshwater eels (Anguilla marmorata Quoy & Gaimard, A. megastoma Kaup, A. obscura Steindachner, A. reinhardtii Günther and A. australis Richardson) across 11 western South Pacific (WSP) islands was investigated based on partial nucleotide sequences of the mtDNA control region and the nuclear GTH2b genes of 288 newly collected samples jointly with existing sequences. WSP anguillids are characterised by overall high levels of genetic diversity. Both mtDNA and nuclear sequences provided no evidence for distinct geographic clines or barriers in any of the species across the WSP. The occurrence of admixed individuals between A. marmorata and A. megastoma was confirmed, and a new possible occurrence of a further species was revealed (A. interioris Whitley on Bougainville Island). All species showed evidence for demographic population growth in the Pleistocene, and a subsequent population reduction for A. megastoma. Common spawning grounds and mixing of larvae by ocean currents could promote the lack of pronounced isolation by distance, a finding that has significant implications for the future management of anguillids in the area.

Vanishing refuge? Testing the forest refuge hypothesis in coastal East Africa using genome-wide sequence data for seven amphibians.

High-throughput sequencing data have greatly improved our ability to understand the processes that contribute to current biodiversity patterns. The "vanishing refuge" diversification model is speculated for the coastal forests of eastern Africa, whereby some taxa have persisted and diversified between forest refugia, while others have switched to becoming generalists also present in non-forest habitats. Complex arrangements of geographical barriers (hydrology and topography) and ecological gradients between forest and non-forest habitats may have further influenced the region's biodiversity, but elucidation of general diversification processes has been limited by lack of suitable data. Here, we explicitly test alternative diversification modes in the coastal forests using genome-wide single nucleotide polymorphisms, mtDNA, spatial and environmental data for three forest (Arthroleptis xenodactyloides, Leptopelis flavomaculatus and Afrixalus sylvaticus) and four generalist (Afrixalus fornasini, A. delicatus, Leptopelis concolor and Leptopelis argenteus) amphibians. Multiple analyses provide insight about divergence times, spatial population structure, dispersal barriers, environmental stability and demographic history. We reveal highly congruent intra-specific diversity and population structure across taxa, with most divergences occurring during the late Pliocene and Pleistocene. Although stability models support the existence of some forest refugia, dispersal barriers and demographic models point towards idiosyncratic diversification modes across taxa. We identify a consistent role for riverine barriers in the diversification of generalist taxa, but mechanisms of diversification are more complex for forest taxa and potentially include topographical barriers, forest refugia and ecological gradients. Our work demonstrates the complexity of diversification processes in this region, which vary between forest and generalist taxa, but also for ecologically similar species with shared population boundaries.

Soil and forest structure predicts large-scale patterns of occurrence and local abundance of a widespread Amazonian frog.

The distribution of biodiversity within the Amazon basin is often structured by sharp environmental boundaries, such as large rivers. The Amazon region is also characterized by subtle environmental clines, but how they might affect the distributions and abundance of organisms has so far received less attention. Here, we test whether soil and forest characteristics are associated with the occurrence and relative abundance of the forest-floor dwelling Aromobatid frog, Allobates femoralis. We applied a structured sampling regime along an 880 km long transect through forest of different density. High detection probabilities were estimated for A. femoralis in each of the sampling modules. Using generalized linear mixed-effects models and simple linear regressions that take detectability into account, we show that A. femoralis is more abundant in open forests than in dense forests. The presence and relative abundance of A. femoralis is also positively associated with clay-rich soils, which are poorly drained and therefore likely support the standing water bodies required for reproduction. Taken together, we demonstrate that relatively easy-to-measure environmental features can explain the distribution and abundance of a widespread species at different spatial scales. Such proxies are of clear value to ecologists and conservation managers working in large inaccessible areas such as the Amazon basin.

Multiple paternity in a viviparous toad with internal fertilisation.

Anurans are renowned for a high diversity of reproductive modes, but less than 1 % of species exhibit internal fertilisation followed by viviparity. In the live-bearing West African Nimba toad (Nimbaphrynoides occidentalis), females produce yolk-poor eggs and internally nourish their young after fertilisation. Birth of fully developed juveniles takes place after 9 months. In the present study, we used genetic markers (eight microsatellite loci) to assign the paternity of litters of 12 females comprising on average 9.7 juveniles. In 9 out of 12 families (75 %), a single sire was sufficient; in three families (25 %), more than one sire was necessary to explain the observed genotypes in each family. These findings are backed up with field observations of male resource defence (underground cavities in which mating takes place) as well as coercive mating attempts, suggesting that the observed moderate level of multiple paternity in a species without distinct sperm storage organs is governed by a balance of female mate choice and male reproductive strategies.

High genetic diversity of common toad (Bufo bufo) populations under strong natural fragmentation on a Northern archipelago.

The last decades have shown a surge in studies focusing on the interplay between fragmented habitats, genetic variation, and conservation. In the present study, we consider the case of a temperate pond-breeding anuran (the common toad Bufo bufo) inhabiting a naturally strongly fragmented habitat at the Northern fringe of the species' range: islands offshore the Norwegian coast. A total of 475 individuals from 19 populations (three mainland populations and 16 populations on seven adjacent islands) were genetically characterized using nine microsatellite markers. As expected for a highly fragmented habitat, genetic distances between populations were high (pairwise F st values ranging between 0.06 and 0.33), with however little differences between populations separated by ocean and populations separated by terrestrial habitat (mainland and on islands). Despite a distinct cline in genetic variation from mainland populations to peripheral islands, the study populations were characterized by overall high genetic variation, in line with effective population sizes derived from single-sample estimators which were on average about 20 individuals. Taken together, our results reinforce the notion that spatial and temporal scales of fragmentation need to be considered when studying the interplay between landscape fragmentation and genetic erosion.

Geographic variation in life-history traits: growth season affects age structure, egg size and clutch size in Andrew's toad (Bufo andrewsi).

BACKGROUND: Environmental variation associated with season length is likely to promote differentiation in life-history traits, but has been little studied in natural populations of ectotherms. We investigated patterns of variation in egg size, clutch size, age at sexual maturity, maximum age, mean age, growth rate and adult body size in relation to growth season length among 17 populations of Andrew's toad (Bufo andrewsi) at different latitudes and altitudes in the Hengduan Mountains, western China. RESULTS: We found that egg size, age at sexual maturity, and mean age increased with decreasing length of the growth season, whereas clutch size showed a converse cline. Body size did not increase with decreasing length of the growth season, but was tightly linked to lifetime activity (i.e. the estimated number of active days during lifetime). Males and females differed in their patterns of geographic variation in growth rates, which may be the result of forces shaping the trade-off between growth and reproduction in different environments. CONCLUSIONS: Our findings suggest that growth season plays an important role in shaping variation in life-history traits in B. andrewsi across geographical gradients.

Sexual selection impacts brain anatomy in frogs and toads.

Natural selection is a major force in the evolution of vertebrate brain size, but the role of sexual selection in brain size evolution remains enigmatic. At least two opposing schools of thought predict a relationship between sexual selection and brain size. Sexual selection should facilitate the evolution of larger brains because better cognitive abilities may aid the competition for mates. However, it may also restrict brain size evolution due to energetic trade-offs between brain tissue and sexually selected traits. Here, we examined the patterns of selection on brain size and brain anatomy in male anurans (frogs and toads), a group where the strength of sexual selection differs markedly among species, using a phylogenetically controlled generalized least-squared (PGLS) regression analyses. The analysis revealed that in 43 Chinese anuran species, neither mating system, nor type of courtship, or testes mass was significantly associated with relative brain size. While none of those factors related to the relative size of olfactory nerves, optic tecta, telencephalon, and cerebellum, the olfactory bulbs were relatively larger in monogamous species and those using calls during courtship. Our findings support the mosaic model of brain evolution and suggest that while the investigated aspects of sexual selection do not seem to play a prominent role in the evolution of brain size of anurans, they do impact their brain anatomy.

Evolution of sperm morphology in anurans: insights into the roles of mating system and spawning location.

BACKGROUND: The degree of postcopulatory sexual selection, comprising variable degrees of sperm competition and cryptic female choice, is an important evolutionary force to influence sperm form and function. Here we investigated the effects of mating system and spawning location on the evolution of sperm morphology in 67 species of Chinese anurans. We also examined how relative testes size as an indicator of the level of sperm competition affected variation in sperm morphology across a subset of 29 species. RESULTS: We found a significant association of mating system and spawning location with sperm morphology. However, when removing the effects of body mass or absolute testes mass for species for which such data were available, this effect became non-significant. Consistent with predictions from sperm competition theory, we found a positive correlation between sperm morphology and relative testes size after taking phylogeny into account. CONCLUSIONS: Our findings suggest that sexual selection in Chinese anurans favors longer sperm when the level of sperm competition is high. Pre-copulatory male-male competition and spawning location, on the other hand, do not affect the evolution of sperm morphology after taking body mass and absolute testes mass into account.

Toxoplasma gondii: prevalence in species and genotypes of British bats (Pipistrellus pipistrellus and P. pygmaeus).

Few studies have investigated Toxoplasma gondii infections in bat populations and none have reported its presence in protected British bat species. Using a collection of dead/euthanased bats collected from Lancashire, UK, two species of bats (Pipistrellus pipistrellus and Pipistrellus pygmaeus) were tested using a highly sensitive SAG1-PCR method specific for detection of T. gondii DNA (n=77; 71 P. pipistrellus and 6 P. pygmaeus). Whilst some potential bias may exist in the sampling strategy, an overall prevalence of 10.39% (±6.06%; 95%CI) was detected. All P. pipistrellus, were also genotyped using eleven polymorphic microsatellite loci to determine their local population structure. The programme STRUCTURE revealed that the majority of individuals (83%) were derived from one interbreeding population, and the remaining individuals (17%) had mixed genetic origins. There was no significant difference in the frequency of T. gondii infection or geographical distribution between subclusters. As all British bats are insectivorous, the routes of infection with T. gondii remain elusive. However, the locally large and panmictic gene pool suggests that intraspecies transmission could be applicable.

Assessing risk and guidance on monitoring of Batrachochytrium dendrobatidis in Europe through identification of taxonomic selectivity of infection.

Amphibians are globally threatened, but not all species are affected equally by different threatening processes. This is true for the threat posed by the chytridiomycete fungus (Batrachochytrium dendrobatidis). We compiled a European data set for B. dendrobatidis to analyze the trends of infection in European amphibians. The risk of infection was not randomly distributed geographically or taxonomically across Europe. Within countries with different prevalence, infection was nonrandom in certain amphibian taxa. Brown frogs of the genus Rana were unlikely to be infected, whereas frogs in the families Alytidae and Bombinatoridae were significantly more likely to be infected than predicted by chance. Frogs in the 2 families susceptible to B. dendrobatidis should form the core of attempts to develop spatial surveillance studies of chytridiomycosis in Europe. Ideally, surveys for B. dendrobatidis should be augmented by sampling the widespread genus Pelophylax because this taxon exhibits geographically inconsistent overinfection with B. dendrobatidis and surveillance of it may facilitate recognition of factors causing spatial variability of infection intensity. Several European amphibian taxa were not represented in our data set; however, surveillance of unsampled species should also occur when warranted.

Sexual size dimorphism in anurans fails to obey Rensch's rule.

BACKGROUND: Sexual size dimorphism (SSD) is related to ecology, behaviour and life history of organisms. Rensch's rule states that SSD increases with overall body size in species where males are the larger sex, while decreasing with body size when females are larger. To test this rule, we analysed literature as well as own data on male and female body size in anurans (39 species and 17 genera). We also tested the hypothesis that SSD is largely a function of age difference between the sexes. RESULTS: Our data set encompassed 36 species with female-biased SSD, and three species with male-biased SSD. All considered species failed to support Rensch's rule, also when the analyses were phylogenetically corrected. However, SSD was significantly correlated with Sexual Age Difference (SAD) across species. We also found a significant correlation between SSD contrasts and SAD contrasts. CONCLUSIONS: Our study suggests that Rensch's rule does not accurately describe macroevolutionary patterns of SSD in anurans. That SAD can explain most of the variation in SSD among species when controlling for phylogenetic effects suggests that phylogeny is not responsible for the broad relationship between age and size across the sexes.

The female perspective of mating in A. femoralis, a territorial frog with paternal care--a spatial and genetic analysis.

The adaptive significance of sequential polyandry is a challenging question in evolutionary and behavioral biology. Costs and benefits of different mating patterns are shaped by the spatial distribution of individuals and by genetic parameters such as the pairwise relatedness between potential mating partners. Thus, females should become less choosy as costs of mating and searching for mates increase. We used parentage assignments to investigate spatial and genetic patterns of mating across a natural population of the Neotropical frog Allobates femoralis, a species characterized by male territoriality and care and female iteroparity. There was no correlation between genetic and spatial distances between adult individuals across the population. In 72% of cases, females mated with males available within a radius of 20 m. Mean pairwise relatedness coefficients of successful reproducers did not differ from random mating but had a lower variance than expected by chance, suggesting maximal reproductive output at intermediate genetic divergence. We also found evidence for selection in favor of more heterozygous individuals between the embryo and adult stage. The level of sequential polyandry significantly increased with the number of spatially available males. Females that had more candidate males also produced more adult progeny. We hypothesize that the benefits associated with female multiple mating outweigh the costs of in- and outbreeding depression, and consequently precluded the evolution of 'choosy' mate selection in this species.