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Most vertebrates develop distinct females and males, where sex is determined by repeatedly evolved environmental or genetic triggers. Undifferentiated sex chromosomes and large genomes have caused major knowledge gaps in amphibians. Only a single master sex-determining gene, the dmrt1 -paralogue ( dm-w ) of female-heterogametic clawed frogs ( Xenopus ; ZW♀/ZZ♂), is known across >8740 species of amphibians. In this study, by combining chromosome-scale female and male genomes of a non-model amphibian, the European green toad, Bufo ( tes ) viridis , with ddRAD- and whole genome pool-sequencing, we reveal a candidate master locus, governing a male-heterogametic system (XX♀/XY♂). Targeted sequencing across multiple taxa uncovered structural X/Y-variation in the 5′-regulatory region of the gene bod1l , where a Y-specific non-coding RNA (ncRNA-Y), only expressed in males, suggests that this locus initiates sex-specific differentiation. Developmental transcriptomes and RNA in-situ hybridization show timely and spatially relevant sex-specific ncRNA-Y and bod1l -gene expression in primordial gonads. This coincided with differential H3K4me -methylation in pre-granulosa/pre-Sertoli cells, pointing to a specific mechanism of amphibian sex determination. Genetic mechanisms of sex determination are still unknown in most amphibians. This study elucidates a mechanism behind amphibian sex determination by uncovering a sex-determining locus ( bod1l gene and a Y-specific non-coding RNA, ncRNA-Y) in European green toads ( Bufo(tes) viridis ) which initiates male development.

Urban areas are increasing worldwide, which poses threats to animal wildlife. However, in certain cases cities can provide refuges for endangered animals. The European green toad ( Bufotes viridis ) is one of such examples, which is known from cities throughout their distribution. In contrast, considerable areas of their former (primary) habitats have been degraded. The primary habitats of this species include steppes and wild river floodplains, both characterized by dynamic changes and the presence of open areas. We used available green toad observation data (2007–2020) to model the effects of land-use types on occurrence probability in the city of Vienna. Forest and densely populated areas were highly significantly negatively associated with green toad presence, while transformation/construction site areas showed a strong positive effect. Such occurrence pattern might be characteristic for early succession species, which depend on stochastic environmental disturbances (e.g., droughts and floods) in their primary habitats. We argue that urban landscape planning should appreciate the potential ecological value of open land in cities which is either in a transition phase or a permanent ‘wasteland’. Ecological managing of such landscape could vastly increase urban biodiversity.

Pesticides are one of the main drivers of the worldwide amphibian decline. Their actual toxicity depends on a number of factors, like the species in focus or the developmental stage of exposed individuals. As ectothermic species, the metabolism of amphibians is influenced by ambient temperature. Therefore, temperature also affects metabolic rates and thus processes that might enhance or reduce toxic effects. Studies about the interactive effect of temperature and toxicity on amphibians are rare and deliver contrasting results. To investigate the temperature-dependent pesticide sensitivity of larvae of two European species we conducted acute toxicity tests for the viticultural fungicide Folpan® 500 SC with the active ingredient folpet at different temperatures (6°C, 11°C, 16°C, 21°C, 26°C). Sensitivity of Rana temporaria and Bufotes viridis was highly affected by temperature: early larvae (Gosner stage 20) were about twice more sensitive to Folpan® 500 SC at 6°C compared to 21°C. Next to temperature, species and developmental stage of larvae had an effect on sensitivity. The most sensitive individuals (early stages of R . temporaria at 6°C) were 14.5 times more sensitive than the least sensitive ones (early stages of B . viridis at 26°C). Our results raise concerns about typical ecotoxicological studies with amphibians that are often conducted at temperatures between 15°C and 20°C. We suggest that future test designs should be performed at temperatures that reflect the temperature range amphibians are exposed to in their natural habitats. Variations in the sensitivity due to temperature should also be considered as an uncertainty factor in upcoming environmental risk assessments for amphibians.

Body condition is increasingly used to assess the status of populations and as a proxy for individual fitness. A common, quick and non-invasive approach is to estimate condition from the relation between body length and mass. Among the methods developed for this purpose, the Scaled Mass Index (SMI) appears best suited for comparisons among populations. We assembled data from 17 populations of European green toads (Bufotes viridis) with the aim of devising a standard formula applicable for monitoring this species. The mean value of the exponents describing length–mass allometry in these samples was 3.0047. Hence, we propose using 3 as a scaling coefficient for calculating the SMI in green toads. From the contrast of SMI values for both sexes within populations, estimated with either the population-specific or the standard coefficient, we conclude that applying the standard formula not only facilitates comparisons among populations but may also help to avoid misinterpretation of variation within populations.

ABSTRACT Microsatellites (SSRs) are reliable markers for population genetic analyses but often suffer from null alleles caused by mutations in primer binding sites. Exon‐primed intron‐crossing (EPIC) markers address these limitations and serve as a complementary tool. In this study, we developed SSR and EPIC markers for the green toad (Bufotes viridis). We demonstrated their effectiveness using genotyping by high‐throughput amplicon sequencing (GBAS) across urban and rural populations. Marker development followed an established in‐house protocol. We then tested their functionality in singleplex‐PCRs before employing them in multiplex reactions and preparing them for sequencing. We analyzed the resulting data using several variability measures as well as individual‐based clustering methods. From the initial set of 48 markers of each type, 35 SSR and 46 EPIC markers consistently amplified across samples both in singleplex and multiplex assays. Data analysis did not corroborate the expectation of a continuous reduction of diversity in urban populations compared to rural ones. Clustering methods using EPIC markers revealed ecologically coherent results, showing weaker genetic structure in rural environments, whereas the SSRs' signal reflected drift‐induced patterns. Our findings suggest that the green toad exhibits a degree of resilience to urban environments. Furthermore, EPIC markers not only complement SSRs by having fewer null alleles but also provide greater robustness to random drift events. We recommend their combined use, especially in fragmented environments prone to genetic drift, such as urban areas. We developed and tested SSR and EPIC markers for the green toad (Bufotes viridis), demonstrating their effectiveness using genotyping by high‐throughput amplicon sequencing (GBAS) across urban and rural populations. EPIC markers exhibited fewer null alleles and provided more ecologically coherent clustering results, while SSRs reflected drift‐induced patterns. Our findings indicate that B. viridis may retain genetic diversity in urban environments and highlight the value of combining EPIC and SSR markers, particularly in fragmented habitats prone to genetic drift.

The toxicity of aluminum oxide (Al 2 O 3 ), copper oxide (CuO), iron oxide (Fe 3 O 4 ), nickel oxide (NiO), zinc oxide (ZnO), and titanium dioxide (TiO 2 ) nanoparticles (NPs) on amphibians and their interaction with high temperatures, remain unknown. In this study, we investigated the survival, developmental, behavioral, and histological reactions of Bufotes viridis embryos and larvae exposed to different NPs for a duration of 10 days, using lethal concentrations (LC25%, LC50%, and LC75% mg/L) under both ambient (AT: 18 °C) and high (HT: 21 °C) temperatures. Based on LC, NiONPs > ZnONPs > CuONPs > Al 2 O 3 NPs > TiO 2 NPs > Fe 3 O 4 NPs showed the highest mortality at AT. A similar pattern was observed at HT, although mortality occurred at lower concentrations and Fe 3 O 4 NPs were more toxic than TiO 2 NPs. The results indicated that increasing concentrations of NPs significantly reduced hatching rates, except for TiO 2 NPs. Survival rates decreased, abnormality rates increased, and developmental processes slowed down, particularly for NiONPs and ZnONPs, under HT conditions. However, exposure to low concentrations of Fe 3 O 4 NPs for up to 7 days, CuONPs for up to 72 h, and NiO, ZnONPs, and TiO 2 NPs for up to 96 h did not have a negative impact on survival compared with the control group under AT. In behavioral tests with larvae, NPs generally induced hypoactivity at AT and hyperactivity at HT. Histological findings revealed liver and internal gill tissue lesions, and an increase in the number of melanomacrophage centers at HT. These results suggest that global warming may exacerbate the toxicity of metal oxide NPs to amphibians, emphasizing the need for further research and conservation efforts in this context.

In 2016, we studied the occurrence of amphibians in 231 selected ponds in the city of Wrocław (Lower Silesia, Poland) and confirmed the occurrence of 10 species: Bombina bombina, Bufo bufo, Bufotes viridis, Hyla arborea, Pelobates fuscus, Pelophylax esculentus complex, Rana arvalis, Rana temporaria, Lissotriton vulgaris and Triturus cristatus. Among all studied sites, the most common were P. esculentus complex (146 occupied sites, 63.2%) and B. bufo (119, 51.5%), while the rarest was P. fuscus (8, 3.5%). The number of species at a single site varied from 0 (28 sites, 12.1%) to 9 (1 site, 0.4%). Frequency and mean amphibian species richness reached 87.9% and 2.7 ± 1.9, respectively. The composition of amphibian species did not change in comparison to previous studies conducted in the years 1997–2009, but declining trends in five species (B. bombina, B. viridis, H. arborea, P. esculentus complex, P. fuscus) were observed (28 compared sites). The results revealed that the permanency of ponds, their occurrence in the vicinity of river valleys, and a high ratio of watercourse length and green area around ponds are positively correlated and have a significant influence on amphibian species richness within the city. Thus, these identified factors should be considered in the course of sustainable urban planning in order to avoid potential conflicts between nature conservation and urban development.

Stormwater ponds (SWPs) are built to collect and retain polluted runoff water from roads. Consequently, they are not perceived as suitable habitat for wetland species, such as many amphibians. However, given the drastic decline of wetland areas, SWPs may serve as a habitat for protected amphibian species, such as the European green toad ( Bufotes viridis ). The latter species is frequently found inside these artificial ponds, but their reproductive success is unknown. We assessed the suitability of SWPs as breeding habitat for European green toads by monitoring 8 SWPs and 8 semi-natural ponds (SNPs), which served as control sites. At each site, two groups of 30 tadpoles, originating at that site, were held inside two floating enclosures that contained sediment from the respective pond. During bi-weekly monitoring, tadpoles were counted and measured, allowing to estimate growth and mortality rates. A variety of biotic and abiotic factors were studied to determine the causes of potential differences in growth and mortality rates between the two pond types. While growth rate did not differ between pond types, mortality rates were significantly greater in SWPs than in SNPs. The extremely low survival rate observed in SWPs might be explained by the considerably greater pollutant concentration in their sediment and/or by the presence of leeches, which were found exclusively inside SWPs. Implementation of management measures, such as regular draining/dredging during winter, might help to lower the pollutant concentration in the sediment and reduce the density of leeches inside SWPs, improving their suitability as habitat for amphibians.

Genetic variation is often lower at high latitudes, which may compromise the adaptability and hence survival of organisms. Here we show that genetic variability is negatively correlated with northern latitude in European green toads (Bufotes viridis). The result holds true for both putatively neutral microsatellite variation and supposedly adaptive MHC Class IIB variation. In particular, our findings have bearing on the conservation status of this species in Sweden, on the northern limit of its distribution where local populations are small and fragmented. These genetically impoverished populations are closely related to other populations found around the Baltic Sea basin. The low neutral and adaptive variation in these fringe populations compared to population at central ranges confirms a pattern shared across all other amphibians so far studied. In Sweden, the situation of green toads is of concern as the remaining populations may not have the evolutionary potential to cope with present and future environmental challenges.

Heavily urbanized areas can hinder dispersal and gene flow between amphibian populations. Given the growth potential of urbanization, it is important to examine how this specific environment shapes their genetic patterns at the local scale. The ability of the European green toad to successfully colonize large human settlements has been convincingly confirmed in the recent past, but little is known about its population genetics under these new conditions. In this study, we examined the effects of the environment on genetic variation, population structure, and the level of gene flow in populations of this amphibian in the city of Košice and the adjacent rural area (eastern Slovakia) using a set of neutral genetic markers. We found that urban populations had lower genetic variability than populations in adjacent rural areas; however, the degree of inbreeding was relatively low in all samples. Genetic differentiation was higher, and gene flow was more restricted in urban area, although geographic distances between sites were significantly less than in rural area (2–4 km versus 6–13 km). Our analyses suggested genetic isolation of urban populations at sites with less suitable habitat for green toads. In contrast, admixture of the population inhabiting the large city park, established on a former floodplain, with all rural populations was likely the result of an intense historical gene flow. The densely developed environment of the other urban sites likely presents a strong barrier to gene flow. The lack of suitable wetland habitat prior to development suggests that these sites were only recently colonized by a limited number of founders. Thus, we found differential effects of the city on the population structure of the green toad. Understanding current local genetic variation and structure is important for future conservation plans in urban environments.