Genetic evidence for a high diversity and wide distribution of endemic strains of the pathogenic chytrid fungus Batrachochytrium dendrobatidis in wild Asian amphibians

Population declines and extinctions of amphibians have been attributed to the chytrid fungus Batrachochytrium dendrobatidis (Bd), especially one globally emerging recombinant lineage (‘Bd‐GPL’). We used PCR assays that target the ribosomal internal transcribed spacer region (ITS) of Bd to determine the prevalence and genetic diversity of Bd in South Korea, where Bd is widely distributed but is not known to cause morbidity or mortality in wild populations. We isolated Korean Bd strains from native amphibians with low infection loads and compared them to known worldwide Bd strains using 19 polymorphic SNP and microsatellite loci. Bd prevalence ranged between 12.5 and 48.0%, in 11 of 17 native Korean species, and 24.7% in the introduced bullfrog Lithobates catesbeianus. Based on ITS sequence variation, 47 of the 50 identified Korean haplotypes formed a group closely associated with a native Brazilian Bd lineage, separated from the Bd‐GPL lineage. However, multilocus genotyping of three Korean Bd isolates revealed strong divergence from both Bd‐GPL and the native Brazilian Bd lineages. Thus, the ITS region resolves genotypes that diverge from Bd‐GPL but otherwise generates ambiguous phylogenies. Our results point to the presence of highly diversified endemic strains of Bd across Asian amphibian species. The rarity of Bd‐GPL‐associated haplotypes suggests that either this lineage was introduced into Korea only recently or Bd‐GPL has been outcompeted by native Bd strains. Our results highlight the need to consider possible complex interactions among native Bd lineages, Bd‐GPL and their associated amphibian hosts when assessing the spread and impact of Bd‐GPL on worldwide amphibian populations.     

Archive Contributions to a Molecular Phylogeography of the Toad Bufo calamita in Britain

A complete phylogeographic analysis of anyspecies requires sampling throughout its biogeographicalrange. In the case of the natterjack toad Bufocalamita in Britain, recent local extinctions haveleft substantial areas of its historical rangewithout extant populations. We therefore obtained tissuesamples of archived Bufo calamita from fourmuseums in the United Kingdom. A range of tissues(tongue, liver, skin, lung, and larval tail) was sampledfrom a total of 33 individual animals. DNA was extractedand eight polymorphic microsatellite loci were scored.One or more loci were amplified successfully from 27 individuals, and sufficient data wereobtained from regions with few or no survivingpopulations to supplement a phylogeographic analysisbased on extant populations.     

High levels of genetic variation at MHC class II DBB loci in the tammar wallaby (<Emphasis Type="Italic">Macropus eugenii</Emphasis>)

High levels of MHC diversity are crucial for immunological fitness of populations, with island populations particularly susceptible to loss of genetic diversity. In this study, the level of MHC class II DBB diversity was examined in tammar wallabies (Macropus eugenii) from Kangaroo Island by genotyping class II-linked microsatellite loci and sequencing of DBB genes. Here we show that the tammar wallaby has at least four expressed MHC class II DBB loci and extensive genetic variation in the peptide-binding region of the DBB genes. These results contradict early studies which suggested that wallabies lacked MHC class II diversity and demonstrate that, in spite of the long-term isolation on an offshore island, this population of wallabies has a high level of DBB diversity.     

Microsatellite analysis of the natterjack toad (Bufo calamita) in Denmark: populations are islands in a fragmented landscape

The European natterjack toad (Bufo calamita) has declined rapidly in recent years, primarily due to loss of habitat, and in Denmark it is estimated that 50% of the isolated populations are lost each decade. To efficiently manage and conserve this species and its genetic diversity, knowledge of the genetic structure is crucial. Based on nine polymorphic microsatellite loci, the genetic diversity, genetic structure and gene flow were investigated at 12 sites representing 5–10% of the natterjack toad localities presently known in Denmark. The expected heterozygosity (H _E) within each locality was generally low (range: 0.18–0.43). Further analyses failed to significantly correlate genetic diversity with population size, degree of isolation and increasing northern latitude, indicating a more complex combination of factors in determining the present genetic profile. Genetic differentiation was high (overall θ = 0.29) and analyses based on a Bayesian clustering method revealed that the dataset constituted 11 genetic clusters, defining nearly all sampling sites as distinct populations. Contemporary gene flow among populations was undetectable in nearly all cases, and the failure to detect a pattern of isolation by distance within major regions supported this apparent lack of a gene flow continuum. Indications of a genetic bottleneck were found in three populations. The analyses suggest that the remaining Bufo calamita populations in Denmark are genetically isolated, and represent independent units in a highly fragmented gene pool. Future conservation management of this species is discussed in light of these results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Estimation of survival rate and extinction probability for stage-structured populations with overlapping life stages

The development of methods providing reliable estimates of demographic parameters (e.g., survival rates, fecundity) for wild populations is essential to better understand the ecology and conservation requirements of individual species. A number of methods exist for estimating the demographics of stage-structured populations, but inherent mathematical complexity often limits their uptake by conservation practitioners. Estimating survival rates for pond-breeding amphibians is further complicated by their complex migratory and reproductive behaviours, often resulting in nonobservable states and successive cohorts of eggs and tadpoles. Here we used comprehensive data on 11 distinct breeding toad populations (Bufo calamita) to clarify and assess the suitability of a relatively simple method [the Kiritani–Nakasuji–Manly (KNM) method] to estimate the survival rates of stage-structured populations with overlapping life stages. The study shows that the KNM method is robust and provides realistic estimates of amphibian egg and larval survival rates for species in which breeding can occur as a single pulse or over a period of several weeks. The study also provides estimates of fecundity for seven distinct toad populations and indicates that it is essential to use reliable estimates of fecundity to limit the risk of under- or overestimating the survival rates when using the KNM method. Survival and fecundity rates for B. calamita populations were then used to define population matrices and make a limited exploration of their growth and viability. The findings of the study recently led to the implementation of practical conservation measures at the sites where populations were most vulnerable to extinction.     

Patterns of synchrony in natterjack toad breeding activity and reproductive success at local and regional scales

Empirical studies of the spatiotemporal dynamics of populations are required to better understand natural fluctuations in abundance and reproductive success, and to better target conservation and monitoring programmes. In particular, spatial synchrony in amphibian populations remains little studied. We used data from a comprehensive three year study of natterjack toad Bufo calamita populations breeding at 36 ponds to assess whether there was spatial synchrony in the toad breeding activity (start and length of breeding season, total number of egg strings) and reproductive success (premetamorphic survival and production of metamorphs). We defined a novel approach to assess the importance of short‐term synchrony at both local and regional scales. The approach employs similarity indices and quantifies the interaction between the temporal and spatial components of populations using mixed effects models. There was no synchrony in the toad breeding activity and reproductive success at the local scale, suggesting that populations function as individual clusters independent of each other. Regional synchrony was apparent in the commencement and duration of the breeding season and in the number of egg strings laid (indicative of female population size). Regional synchrony in both rainfall and temperature are likely to explain the patterns observed (e.g. Moran effect). There was no evidence supporting regional synchrony in reproductive success, most likely due to spatial variability in the environmental conditions at the breeding ponds, and to differences in local population fitness (e.g. fecundity). The small scale asynchronous dynamics and regional synchronous dynamics in the number of breeding females indicate that it is best to monitor several populations within a subset of regions. Importantly, variations in the toad breeding activity and reproductive success are not synchronous, and it is thus important to consider them both when assessing the conservation status of pond‐breeding amphibians.     

Reciprocal translocation of small numbers of inbred individuals rescues immunogenetic diversity

Genetic rescue can reduce inbreeding depression and increase fitness of small populations, even when the donor populations are highly inbred. In a recent experiment involving two inbred island populations of the New Zealand South Island robin, Petroica australis, reciprocal translocations improved microsatellite diversity and individual fitness. While microsatellite loci may reflect patterns of genome‐wide diversity, they generally do not indicate the specific genetic regions responsible for increased fitness. We tested the effectiveness of this reciprocal translocation for rescuing diversity of two immunogenetic regions: Toll‐like receptor (TLR) and major histocompatibility complex (MHC) genes. We found that the relatively small number of migrants (seven and ten per island) effectively brought the characteristic TLR gene diversity of each source population into the recipient population. However, when migrants transmitted TLR alleles that were already present at high frequency in the recipient population, it was possible for offspring of mixed heritage to have decreased gene diversity compared to recipient population diversity prior to translocation. In contrast to TLRs, we did not observe substantial changes in MHC allelic diversity following translocation, with limited evidence of a decrease in differentiation, perhaps because most MHC alleles were observed at both sites prior to the translocation. Overall, we conclude that small numbers of migrants may successfully restore the diversity of immunogenetic loci with few alleles, but that translocating larger numbers of animals would provide additional opportunity for the genetic rescue of highly polymorphic immunity regions, such as the MHC, even when the source population is inbred.     

Batrachochytrium dendrobatidis shows high genetic diversity and ecological niche specificity among haplotypes in the Maya Mountains of Belize

The amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines around the globe. Although it has been found in most countries in Central America, its presence has never been assessed in Belize. We set out to determine the range, prevalence, and diversity of Bd using quantitative PCR (qPCR) and sequencing of a portion of the 5.8 s and ITS1-2 regions. Swabs were collected from 524 amphibians of at least 26 species in the protected areas of the Maya Mountains of Belize. We sequenced a subset of 72 samples that had tested positive for Bd by qPCR at least once; 30 samples were verified as Bd. Eight unique Bd haplotypes were identified in the Maya Mountains, five of which were previously undescribed. We identified unique ecological niches for the two most broadly distributed haplotypes. Combined with data showing differing virulence shown in different strains in other studies, the 5.8 s - ITS1-2 region diversity found in this study suggests that there may be substantial differences among populations or haplotypes. Future work should focus on whether specific haplotypes for other genomic regions and possibly pathogenicity can be associated with haplotypes at this locus, as well as the integration of molecular tools with other ecological tools to elucidate the ecology and pathogenicity of Bd.     

Six polymorphic microsatellite loci in the Natterjack toad,Bufo calamita

The Natterjack toad, Bufo calamita, is a locally endangered anuran endemic to Europe. It has been used as a model organism in several ecological studies. When studying the genetic structure of marginalized populations of the species, there was a need to involve more loci in the study than those previously published. Here we present the development of six polymorphic microsatellite loci in the Natterjack. The loci will be used for studying population genetic structure and gene flow between populations.     

Genetic Variation of Microsatellite Loci in the Major Histocompatibility Complex (MHC) Region in the Southeast Asian House Mouse (<Emphasis Type="Italic">Mus musculus castaneus</Emphasis>)

Major histocompatibility complex (MHC) genes are the most polymorphic loci known for vertebrates. Here we employed five microsatellite loci closely linked to the MHC region in an attempt to study the amount of genetic variation in 19 populations of the southeast Asian house mouse (Mus musculus castaneus) in Taiwan. The overall polymorphism at the five loci was high (H_e = 0.713), and the level of polymorphism varied from locus to locus. Furthermore, in order to investigate if selection is operating on MHC genes in natural mouse populations, we compared the extent and pattern of genetic variation for the MHC-linked microsatellite loci (the MHC loci) with those for the microsatellite loci located outside the MHC region (the non-MHC loci). The number of alleles and the logarithm of variance in repeat number were significantly higher for the MHC loci than for the non-MHC loci, presumably reflecting linkage to a locus under balancing selection. Although three statistical tests used do not provide support for selection, their lack of support may be due to low statistical power of the tests, to weakness of selection, or to a profound effect of genetic drift reducing the signature of balancing selection. Our results also suggested that the populations in the central and the southwestern regions of Taiwan might be one part of a metapopulation structure.     

Unlocking the story in the swab: A new genotyping assay for the amphibian chytrid fungus Batrachochytrium dendrobatidis

One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures have advanced our understanding of Bd phylogenetics, genomic architecture and mechanisms of virulence. However, pure cultures are laborious to obtain and whole‐genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus, we still know little about the genetic diversity of Bd in natural systems. The most common noninvasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field‐collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole‐genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, noninvasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.     

Genetic attributes of midwife toad (Alytes obstetricans) populations do not correlate with degree of species decline

Genetic diversity is crucial for long‐term population persistence. Population loss and subsequent reduction in migration rate among the most important processes that are expected to lead to a reduction in genetic diversity and an increase in genetic differentiation. While the theory behind this is well‐developed, empirical evidence from wild populations is inconsistent. Using microsatellite markers, we compared the genetic structure of populations of an amphibian species, the midwife toad (Alytes obstetricans), in four Swiss regions where the species has suffered variable levels of subpopulation extirpation. We also quantified the effects of several geographic factors on genetic structure and used a model selection approach to ascertain which of the variables were important for explaining genetic variation. Although subpopulation pairwise F_ST‐values were highly significant even over small geographic scales, neither any of the geographic variables nor loss of subpopulations were important factors for predicting spatial genetic structure. The absence of a signature of subpopulation loss on genetic differentiation may suggest that midwife toad subpopulations function as relatively independent units.     

A comparison of single‐sample effective size estimators using empirical toad (Bufo calamita) population data: genetic compensation and population size‐genetic diversity correlations

The accuracy and precision of four single‐sample estimators of effective population size, N_e (heterozygote excess, linkage disequilibrium, Bayesian partial likelihood and sibship analysis) were compared using empirical data (microsatellite genotypes) from multiple natterjack toad (Bufo calamita) populations in Britain (n = 16) and elsewhere in Europe (n = 10). Census size data were available for the British populations. Because toads have overlapping generations, all of these methods estimated the number of effective breeders N_b rather than N_e. The heterozygote excess method only provided results, without confidence limits, for nine of the British populations. Linkage disequilibrium gave estimates for 10 British populations, but only six had finite confidence limits. The Bayesian and sibship methods both produced estimates with finite confidence limits for all the populations. Although the Bayesian method was the most precise, on most criteria (insensitivity to locus number, correlation with other effective and census size estimates and correlation with genetic diversity) the sibship method performed best. The results also provided evidence of genetic compensation in natterjack toads, and highlighted how the relationship between effective size and genetic diversity can vary as a function of geographical scale.     

High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Breeding phenology and larval distribution of amphibians in a Mediterranean pond network with unpredictable hydrology

Understanding spatial and temporal breeding patterns in Mediterranean amphibian communities is urgent considering the rate of habitat loss. Breeding phenology and breeding habitat selection by amphibians were analysed through the monthly occurrence of larvae in a mosaic of 198 Mediterranean temporary ponds during three years. A generalized linear model (GLM) coupled with principal component analysis showed that, for almost all species, occurrence was significantly positively correlated to pond depth. In addition, pond openness negatively affected the presence of some species. Temporal breeding patterns varied among species. Some species exhibited flexibility in their breeding date (Pelobates cultripes, Pelodytes punctatus, Hyla meridionalis, Rana perezi), while others did not (Triturus marmoratus, Triturus helveticus, Bufo calamita). When faced with inter-annual hydrological variability, the first group had a more constant breeding success than the second. Variable hydrological conditions caused differential larval occurrence of species between years. These fluctuations might favour long-term persistence of the whole amphibian community. We finally discuss the implications of our results for the management of amphibian habitats in the Mediterranean region.     

The effects of multiple stressors on wetland communities: pesticides,pathogens and competing amphibians

1. Anthropogenic effects have propelled us into what many have described as the sixth mass extinction, and amphibians are among the most affected groups. The causes of global amphibian population declines and extinctions are varied, complex and context‐dependent and may involve multiple stressors. However, experimental studies examining multiple factors contributing to amphibian population declines are rare. 2. Using outdoor mesocosms containing zooplankton, phytoplankton, periphyton and tadpoles, we conducted a 2 × 2 × 3 factorial experiment that examined the separate and combined effects of an insecticide and the fungal pathogen Batrachochytrium dendrobatidis (Bd) on three different assemblages of larval pacific treefrogs (Pseudacris regilla) and Cascades frogs (Rana cascadae). 3. Larval amphibian growth and development were affected by carbaryl and the amphibian assemblage treatment, but only minimally by Bd. Carbaryl delayed metamorphosis in both amphibian species and increased the growth rate of P. regilla. Carbaryl also reduced cladoceran abundance, which, in turn, had positive effects on phytoplankton abundance but no effect on periphyton biomass. Substituting 20 intraspecific competitors with 20 interspecific competitors decreased the larval period but not the growth rate of P. regilla. In contrast, substituting 20 intraspecific competitors with 20 interspecific competitors had no effect on R. cascadae. Results of real‐time quantitative polymerase chain reaction (qPCR) analysis confirmed infection of Bd‐exposed animals, but exposure to Bd had no effects on either species in univariate analyses, although it had significant or nearly significant effects in several multivariate analyses. In short, we found no interactive effects among the treatments on amphibian growth and development. 4. We encourage future research on the interactive effects of pesticides and pathogens on amphibian communities.     

Investigating Differences across Host Species and Scales to Explain the Distribution of the Amphibian Pathogen Batrachochytrium dendrobatidis

Many pathogens infect more than one host species, and clarifying how these different hosts contribute to pathogen dynamics can facilitate the management of pathogens and can lend insight into the functioning of pathogens in ecosystems. In this study, we investigated a suite of native and non-native amphibian hosts of the pathogen Batrachochytrium dendrobatidis (Bd) across multiple scales to identify potential mechanisms that may drive infection patterns in the Colorado study system. Specifically, we aimed to determine if: 1) amphibian populations vary in Bd infection across the landscape, 2) amphibian community composition predicts infection (e.g., does the presence or abundance of any particular species influence infection in others?), 3) amphibian species vary in their ability to produce infectious zoospores in a laboratory infection, 4) heterogeneity in host ability observed in the laboratory scales to predict patterns of Bd prevalence in the landscape. We found that non-native North American bullfrogs (Lithobates catesbeianus) are widespread and have the highest prevalence of Bd infection relative to the other native species in the landscape. Additionally, infection in some native species appears to be related to the density of sympatric L. catesbeianus populations. At the smaller host scale, we found that L. catesbeianus produces more of the infective zoospore stage relative to some native species, but that this zoospore output does not scale to predict infection in sympatric wild populations of native species. Rather, landscape level infection relates most strongly to density of hosts at a wetland as well as abiotic factors. While non-native L. catesbeianus have high levels of Bd infection in the Colorado Front Range system, we also identified Bd infection in a number of native amphibian populations allopatric with L. catesbeianus, suggesting that multiple host species are important contributors to the dynamics of the Bd pathogen in this landscape.     

Fitness costs associated with low genetic variation are reduced in a harsher environment in amphibian island populations

A basic premise of conservation geneticists is that low levels of genetic variation are associated with fitness costs in terms of reduced survival and fecundity. These fitness costs may frequently vary with environmental factors and should increase under more stressful conditions. However, there is no consensus on how fitness costs associated with low genetic variation change under natural conditions in relation to the stressfulness of the environment. On the Swedish west coast, natterjack toad Bufo calamita populations show a strong population genetic structure and large variation in the amount of within-population genetic variation. We experimentally examined the survival of natterjack larvae from six populations with different genetic variation in three thermal environments corresponding to (a) the mean temperature of natural ponds (stable, laboratory), (b) a high temperature environment occurring in desiccating ponds (stable, laboratory) and (c) an outdoor treatment mimicking the natural, variable thermal conditions (fluctuating, semi-natural). We found that larvae in the outdoor treatment had poorer survival than larvae in the stable environments suggesting that the outdoor treatment was more stressful. Overall, populations with higher genetic variation had higher larval survival. However, a significant interaction between treatments and genetic variation indicated that fitness costs associated with low genetic variation were less severe in the outdoor treatment. Thus, we found no support for the hypothesis that fitness costs associated with low genetic variation increase under more stressful conditions. Our results suggest that natural thermal stress may mask fitness losses associated with low genetic variation in these populations.     

Genetic diversity and its effect on fitness in an endangered plant species, <Emphasis Type="Italic">Dracocephalum austriacum</Emphasis> L.

The aim of this study was to estimate genetic diversity and assess its importance for plant fitness in a species belonging to the most endangered species in Europe, Dracocephalum austriacum L., and to select the most valuable populations for conservation of genetic diversity within the species in the studied regions. We analyzed allozyme variation of 12 populations in three distinct regions (Czech Karst, Moravia and Slovak Karst) in Central Europe. The results showed high genetic diversity within populations (80.14%) and relatively low differentiation among populations within regions (9.42%) and between regions (10.45%). Seed production was significantly higher in larger, genetically more diverse and less inbred populations. The results suggest that genetic diversity has important effect on seed production in this species and thus can be expected to have strong direct consequences for plant fitness and vitality of the whole populations. They also show large variation in genetic diversity between populations and indicate which populations should get a priority in attempts to conserve all the genetic diversity within the region.     

Genetic structure in insular and mainland populations of house sparrows (Passer domesticus) and their hemosporidian parasites

Small and isolated populations usually exhibit low levels of genetic variability, and thus, they are expected to have a lower capacity to adapt to changes in environmental conditions, such as exposure to pathogens and parasites. Comparing the genetic variability of selectively neutral versus functional loci allows one to assess the evolutionary history of populations and their future evolutionary potential. The genes of the major histocompatibility complex (MHC) control immune recognition of parasites, and their unusually high diversity is genes which is likely driven by parasite‐mediated balancing selection. Here, we examined diversity and differentiation of neutral microsatellite loci and functional MHC class I genes in house sparrows (Passer domesticus), living in six insular and six mainland populations, and we aimed to determine whether their diversity or differentiation correlates with the diversity and the prevalence of infection of hemosporidian parasites. We found that island bird populations tended to have lower neutral genetic variability, whereas MHC variability gene was similar between island and mainland populations. Similarly, island populations tended to show greater genetic differentiation than mainland populations, especially at microsatellite markers. The maintenance of MHC genetic diversity and its less marked structure in the island populations could be attributed to balancing‐selection. The greater MHC differentiation among populations was negatively correlated with similarity in blood parasites (prevalence and diversity of parasite strains) between populations. Even at low prevalence and small geographical scale, haemosporidian parasites might contribute to structure the variability of immune genes among populations of hosts.