Judit Vörös

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.

Surveying Europe’s Only Cave-Dwelling Chordate Species (Proteus anguinus) Using Environmental DNA

In surveillance of subterranean fauna, especially in the case of rare or elusive aquatic species, traditional techniques used for epigean species are often not feasible. We developed a non-invasive survey method based on environmental DNA (eDNA) to detect the presence of the red-listed cave-dwelling amphibian, Proteus anguinus, in the caves of the Dinaric Karst. We tested the method in fifteen caves in Croatia, from which the species was previously recorded or expected to occur. We successfully confirmed the presence of P. anguinus from ten caves and detected the species for the first time in five others. Using a hierarchical occupancy model we compared the availability and detection probability of eDNA of two water sampling methods, filtration and precipitation. The statistical analysis showed that both availability and detection probability depended on the method and estimates for both probabilities were higher using filter samples than for precipitation samples. Combining reliable field and laboratory methods with robust statistical modeling will give the best estimates of species occurrence.

Weak population structuring in the Danube crested newt, Triturus dobrogicus , inferred from allozymes

Nuclear genetic variation and population structure were assessed in 140 individuals from 16 populations across the range of the Danube crested newt (Triturus dobrogicus) using 40 enzyme loci. Intraspecific hybridization with other crested newt species (Triturus carnifex, T. cristatus, T. macedonicus and T. arntzeni) affected 33 individuals in 11 populations at the range edge and reduced operational sample size to 107 T. dobrogicus in 14 populations. Allele diversity was high, and we inferred a high level of gene flow among T. dobrogicus populations, possibly associated with flooding conditions and the relatively continous habitat along rivers. Triturus dobrogicus showed weak but significant genetic structure between tributaries of the three main river systems of Danube, Sava and Tisza. The highest genetic diversity was observed in the Sava drainage, suggesting that this area might have been a Pannonian refugium during the most recent glacial maximum. The relatively high level of genetic variation observed suggests that a genetic bottleneck during this period has not been extreme.

'Monster... -omics': on segmentation, re-segmentation, and vertebrae formation in amphibians and other vertebrates

BackgroundThe axial skeleton is one of the defining evolutionary landmarks of vertebrates. How this structure develops and how it has evolved in the different vertebrate lineages is, however, a matter of debate. Vertebrae and vertebral structures are derived from the embryonic somites, although the mechanisms of development are different between lineages.DiscussionUsing the anecdotal description of a teratological newt (Triturus dobrogicus) with an unusual malformation in its axial skeleton, we review, compare, and discuss the development of vertebral structures and, in particular, the development of centra from somitic cellular domains in different vertebrate groups. Vertebrae development through re-segmentation of the somitic sclerotomal cells is considered the general mechanism among vertebrates, which has been generalized from studies in amniotic model organisms. The prevalence of this mechanism among anamniotes is, however, controversial. We propose alternative developmental mechanisms for vertebrae formation that should be experimentally tested.SummaryResearch in model organisms, especially amniotes, is laying the foundations for a thorough understanding of the mechanisms of development of the axial skeleton in vertebrates, foundations that should expand the extent of future comparative studies. Although immersed in the ‘-omics’ era, we emphasize the need for an integrative and organismal approach in evolutionary developmental biology for a better understanding of the causal role of development in the evolution of morphological diversity in nature.

Distribution and hybridization of Anguis fragilis and A. colchica in Hungary

Slow worms (Anguis spp.) are widely distributed in Europe. Based on pronounced divergences in molecular markers the subspecies of the slow worm, Anguis fragilis, have been recently elevated to species level. In Hungary both A. fragilis and A. colchica are present in the mountainous areas with their range being separated by the Danube River with potential contact zones in the Danube valley. Based on morphology, hybridization of the two taxa has been described earlier from the Budai and Pilis Mountains. In order to reveal the exact distribution and confirm hybridization of Anguis taxa in Hungary we analyzed fragments of mitochondrial (ND2) and nuclear (Rag1) genes in 36 specimens from eight regions of Hungary and adjacent countries. The results confirmed the previously known distribution pattern with an east-west split along the Danube River and supported the morphological findings about hybridization in the Budai and Pilis Mountains.

Is the Danube crested newt Triturus dobrogicus polytypic? A review and new nuclear DNA data

The Danube crested newt Triturus dobrogicus has been proposed to comprise two subspecies: T. d. dobrogicus and T. d. macrosoma. Uncertainty exists in the literature over their distribution and diagnosability. We conduct a multilocus phylogeographical survey and review published data to determine whether a two taxon treatment is warranted. Newly produced and published nuclear DNA data suggest intraspecific variation in the Pannonian Plain part of the range, but with extensive genetic admixture, whereas mitochondrial DNA data shows a lack of geographical structuring in T. dobrogicus altogether. None of the studied morphological characters suggest the presence of two geographical groups in T. dobrogicus unequivocally. Although Danube Delta newts do have relatively short bodies compared to the remainder of the range (the Pannonian and Lower Danube Plains and the Dnepr Delta), we argue that this finding can be explained by phenotypic plasticity – particularly in light of the incongruent evolutionary scenario suggested by genetic data. We conclude that the total body of evidence does not support the two subspecies hypothesis and recommend that T. dobrogicus is treated as a monotypic species.

Non-congruent geographic patterns of genetic divergence across European species of Branchinecta (Anostraca: Branchinectidae)

Three of the five European species of Branchinecta have a disjunct distribution. In this study, we analyze populations of B. ferox and B. orientalis for mitochondrial (cox1) and nuclear (ITS2) molecular markers. We compare intraspecific genetic divergences between geographically distant populations of B. orientalis, from its only known Spanish population (originally described as B. cervantesi) and from a Hungarian population (assigned to B. orientalis since its discovery), with data from two relatively close Iberian populations of B. ferox. Results indicate that isolation between B. ferox and B. orientalis clades is ancient, and that the clade including the two Iberian populations of B. ferox is geographically structured. Conversely, Iberian and Hungarian populations of B. orientalis do not show geographical structure for the mitochondrial fragment. Lack of geographic structure coupled with very low genetic distances indicates that current Iberian and Hungarian populations of B. orientalis originated from a common population stock, and that the time elapsed since their separation has not been long enough to render the clades reciprocally monophyletic. We hypothesize that colonization of the Iberian Peninsula by B. orientalis is probably the consequence of a single recent dispersal event, and consequently we confirm the synonymy between B. cervantesi and B. orientalis.

Development and worldwide use of a non-lethal and minimal population-level impact protocols for the isolation of chytrids from amphibians

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile 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 interested researchers worldwide 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 widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. 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 important group of emerging fungal pathogens.

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.

Strong genetic subdivision in Leptobrachium hendricksoni (Anura: Megophryidae) in Southeast Asia

Many biodiversity hotspots are located in areas with a complex geological history, like Southeast Asia, where species diversity may still be far underestimated, especially in morphologically conservative groups like amphibians. Recent phylogenetic studies on the frog genus Leptobrachium from Southeast Asia revealed the presence of deeply divergent mitochondrial clades in Leptobrachium hendricksoni from Malaysia and Sumatra but populations from Thailand have not been studied so far. In this study, we re-evaluate patterns of intraspecific genetic diversity in L. hendricksoni based on the analysis of combined sequences of mitochondrial 12S and 16S genes (1310 base pairs) including for the first time samples from southern Thailand. Thai populations of L. hendricksoni formed a distinct clade with respect to populations from central and southern Malaysia and Sumatra. High sequence divergence between lineages from Thailand, Malaysia and Sumatra suggests the possible presence of cryptic species in L. hendricksoni . Divergence within L. hendricksoni dates back to the late Miocene, around 6 Mya, when lineages from Thailand, north Malaysia and Sumatra split from a lineage in south Malaysia, at about the same time as rising sea levels isolated the Thai-Malay peninsula. Subsequent splits took place later in the Pliocene, around 4.5 and 2.6 Mya. Our results highlight the role of geological history in promoting population divergence and speciation.