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The contents of the digestive tracts of 21 loggerhead turtles (20 juveniles and 1 adult) collected along the French Mediterranean coast were extracted during necropsies. Hard and soft items were collected and identified by visual observation. A 15-mL sample of the liquid from the rinsed contents of each digestive tract was collected and dietary DNA (dDNA) was extracted and amplified with a 18S universal eukaryotic primers. The obtained reads were compared with taxonomic DNA reference database. Ten taxonomic groups (annelids, cnidarians, crustaceans, echinoderms, fish, insects, molluscs, plants, poriferans and tunicates) were identified with visual observation and eight (ctenophores, cnidarians, crustaceans, fish, molluscs, poriferans, tunicates and plants) with dDNA metabarcoding. Annelids, echinoderms and insects were detected only with visual observation, whereas ctenophores were detected only with dDNA. The two methods were complementary to cover the range of prey, with more soft-bodied organisms detected by dDNA and more hard-shelled organisms detected by visual observation. The increased use of the dDNA metabarcoding method will help compare sea turtle diets of the different stages such as juveniles and adults, or specimens living in different areas, despite the limitations and complexity associated with its use.

Sea turtles are considered as bio-indicators for monitoring the efficiency of restoration measures to reduce marine litter impacts on health. However, the lack of extended and standardised empirical data has prevented the accurate analysis of the factors influencing litter ingestion and the relationships with individual health. Historic data collected from 1988 and standard data collected from 2016 were harmonised to enable such analyses on necropsied loggerhead turtles (Caretta caretta) in eight Mediterranean and North-East Atlantic countries. Litter was found in 69.24 % of the 1121 individuals, mostly single-use and fishing-related plastics. Spatial location, sex and life history stage explained a minor part of litter ingestion. While no relationships with health could be detected, indicating that all individuals can be integrated as bio-indicators, the mechanistic models published in literature suggest that the high proportion of plastics in the digestive contents (38.77 % per individual) could have long-term repercussions on population dynamics. © 2022

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

The presence of marine litter is a complex, yet persistent, threat to the health and biodiversity of the marine environment, and plastic is the most abundant, and ubiquitous type of marine litter. To monitor the level of plastic waste in an area, and the prospect of it entering the food chain, bioindicator species are used extensively throughout Northern European Seas, however due to their distribution ranges many are not applicable to the Mediterranean Sea. Guidance published for the Marine Strategy Framework Directive suggests that the contents of fish stomachs may be analyzed to determine trends of marine plastic ingestion. In order to equate transnational trends in marine plastic ingestion, the use of standardized fish species that widely occur throughout the basin is favoured, however for the Mediterranean Sea, specific species are not listed. Here we propose a methodology to assess how effective Mediterranean fish species, that are known to have ingested marine plastic, are as bioindicators. A new Bioindicator Index (BI) was established by incorporating several parameters considered important for bioindicators. These parameters included species distribution throughout the Mediterranean basin, several life history traits, the commercial value of each species, and the occurrence of marine litter in their gut contents. By collecting existing data for Mediterranean fish, ranked scores were assigned to each trait and an average value (BI value) was calculated for each species. Based on their habitat preferences, Engraulis encrasicolus (pelagic), Boops boops (benthopelagic), three species of Myctophidae (Hygophum benoiti, Myctophum punctatum and Electrona risso) (mesopelagic), Mullus barbatus barbartus (demersal) and Chelidonichthys lucerna (benthic), were identified as currently, the most suitable fish for monitoring the ingestion of marine plastics throughout the Mediterranean basin. The use of standardized indicator species will ensure coherence in the reporting of marine litter ingestion trends throughout the Mediterranean Sea.

[EN] The infectious chytrid fungus Batrachochytrium salamandrivorans (Bsal) has been responsible for severe population declines of salamander populations in Europe. Serious population declines and loss of urodelan diversity may occur if appropriate action is not taken to mitigate against the further spread and impact of Bsal. We provide an overview of several potential mitigation methods, and describe their possible advantages and limitations. We conclude that long-term, context-dependent, multi-faceted approaches are needed to successfully mitigate adverse effects of Bsal, and that these approaches should be initiated pre-arrival of the pathogen. The establishment of ex situ assurance colonies, or management units, for species threatened with extinction, should be considered as soon as possible. While ex situ conservation and preventive measures aimed at improving biosecurity by limiting amphibian trade may be implemented quickly, major challenges that lie ahead are in designing in situ disease containment and mitigation post-arrival and in increasing public awareness., This work was supported by the European Commission (Tender ENV.B.3/SER/2016/0028, Mitigating a new infectious disease in salamanders to counteract the loss of biodiversity). E.V. and S.C. are supported by the Research Foundation Flanders (FWO grants 12E6616N and FWO16/PDO/019 respectively). D.S.S. and A.L. were supported by the Belmont Forum (DFG-SCHM 3059/6-1) and the Axa Research Fund through the project GloMEC. M.C.F. was supported by NERC (NE/K014455/1), the Leverhulme Trust (RPG-2014-273), the Morris Animal Foundation (D16ZO-022) and the Canadian CIFAR “Fungal Kingdom” programme. T.W.J.G. was supported by NERC (NE/S000992/1, NE/N009967/1). S.L., S.S., M. Vences, M. Veith and N.W. were supported by the Federal Agency for Nature Conservation (BfN) (R+D project “Monitoring und Entwicklung von Vorsorgemaßnahmen zum Schutz vor der Ausbreitung des Chytridpilzes Batrachochytriumsalamandrivorans (“Bsal”) im Freiland”). S.L., N.W., Michael Veith and Miguel Vences were also supported by a seed grant from Deutsche Bundesstiftung Umwelt (DBU).

This study aims to characterise the genetic structure and composition of 245 individuals of loggerhead sea turtles collected from stranding and bycatch events along the French Mediterranean coasts (Gulf of Lion, Provence and Corsica). We obtained sequences of the mitochondrial control region for two fragments (683 bp and 241 bp for 170 and 51 individuals, respectively). The analysis of the long fragment revealed that 163 samples (95.9%) are attributed to the haplogroup II (mainly Mediterranean) whereas only seven individuals are included in haplogroup IB (Atlantic Ocean). The mixed stock analysis performed on the same dataset indicated that the biggest rookeries from the eastern Mediterranean mainly contributed to the French stock, with major contributions being from Greece (36% and 56% for adults and juveniles, respectively), Crete (12% and 18%) and Western Turkey (14% and 4%). The thirteen microsatellite nuclear markers that have been analysed for 81 specimens did not reveal much genetic structure within sampled individuals, thus suggesting that the studied individuals could belong to the same genetic group. The microsatellite analyses revealed however that the nine individuals issuing from two nests sampled in Gulf of Lion and Provence are clearly differentiated from the remaining samples, thus suggesting a long-distance colonisation of the western Mediterranean. Our results allowed addressing the question of loggerhead turtle conservation in the western Mediterranean basin that until now is not considered as a Management Unit despite high densities of juveniles and recent nesting observed on French, Italian and Spanish coasts.

Starting in 2010, rapid fire salamander (Salamandra salamandra) population declines in northwestern Europe heralded the emergence of Batrachochytrium salamandrivorans (Bsal), a salamander-pathogenic chytrid fungus. Bsal poses an imminent threat to global salamander diversity owing to its wide host range, high pathogenicity, and long-term persistence in ecosystems. While there is a pressing need to develop further research and conservation actions, data limitations inherent to recent pathogen emergence obscure necessary insights into Bsal disease ecology. Here, we use a hierarchical modeling framework to describe Bsal landscape epidemiology of outbreak sites in light of these methodological challenges. Using model selection and machine learning, we find that Bsal presence is associated with humid and relatively cool, stable climates. Outbreaks are generally located in areas characterized by low landscape heterogeneity and low steepness of slope. We further find an association between Bsal presence and high trail density, suggesting that human-mediated spread may increase risk for spillover between populations. We then use distribution modeling to show that favorable conditions occur in lowlands influenced by the North Sea, where increased survey effort is needed to determine how Bsal impacts local newt populations, but also in hill- and mountain ranges in northeastern France and the lower half of Germany. Finally, connectivity analyses suggest that these hill- and mountain ranges may act as stepping stones for further spread southward. Our results provide initial insight into regional environmental conditions underlying Bsal epizootics, present updated invasibility predictions for northwestern Europe, and lead us to discuss a wide variety of potential survey and research actions needed to advance future conservation and mitigation efforts.

International audience; Linear transport infrastructure can impact wildlife directly or indirectly, and amphibians are particularly affected. In some cases, mitigation measures e such as tunnels below the linear transport infrastructure e are implemented, but an evaluation of their effectiveness is often lacking, especially for small animals such as amphibians. Few studies have focused on crossing behaviour in underpasses, yet this information may be crucial to improve mitigation measures. In this study, we conducted experiments in three tunnels below a high-speed railway with individually marked amphibians (great crested newt Triturus cristatus, marbled newt Triturus marmoratus, alpine newt Ichthyosaura alpestris, fire sala- mander Salamandra salamandra, spined toad Bufo spinosus, agile frog Rana dalmatina and edible/pool frog Pelophylax kl. esculentus/lessonae) which were released at the entrance of tunnels varying in length. Behaviours (e.g. completed crossing, U-turn, not moving, and stay) and crossing speed were recorded. R. dalmatina, S. salamandra and T. cristatus (but not B. spinosus) exhibited crossing rate differences between tunnels 21 m and 40 m in length. More individuals performed “complete crossing” through the shorter tunnel. The crossing speed of the S. salamandra and T. cristatus did not differ between the tunnels of different lengths. In a second experiment, we evaluated the effect of acoustic enrichment on the crossing behaviour of one frog and three newt species by broadcasting a soundtrack of mating calls of syntopic anuran species in the tunnels. Pelophylax kl exhibited a large in- crease in complete crossings and in speed. T. cristatus showed an increase in tunnel complete crossings (but not speed) in one of the acoustically enhanced tunnels. These results indicate that acoustic enrichment can improve the crossing of underpasses. This low cost and easily implementable method could help to inform the design of wildlife passages as mitigation measures for impacted amphibian populations, though more ex- periments on the effect of tunnel characteristics on amphibian behaviour would be valuable.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide., S.J.O., T.W.J.G., L.Br., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were supported through NERC (standard grant NE/K014455/1). S.J.O. acknowledges a Microsoft Azure for Research Sponsorship (subscription ID: ab7cd695-49cf-4a83-910a-ef71603e708b). T.W.J.G., A.Lo., A.A.C., D.S.S., E.A.C., C.M., J.B., D.M.A., F.C., and M.C.F. were also supported by the EU BiodivERsA scheme (RACE, funded through NERC directed grant NE/G002193/1 and ANR08-Biodiversa-002-03) and NERC (standard grant NE/K012509/1). M.C.F., E.A.C., and C.M. acknowledge the Nouragues Travel Grant Program 2014. R.A.F. was supported by an MIT/Wellcome Trust Fellowship. T.W.J.G. was supported by the People’s Trust for Endangered Species and the Morris Animal Foundation (D12ZO002). J.M.G.S. and M.C.F. were supported by the Leverhulme Trust (RPG-2014-273) and the Morris Animal Foundation (D16ZO-022). F.B. was supported by the ERC (grant ERC 260801–Big_Idea). D.M.A. was funded by Wellcome Trust grant 099202. J.V. was supported by the Hungarian Scientific Research Fund (OTKA K77841) and Bolyai János Research Scholarship, Hungarian Academy of Sciences (BO/00579/14/8). D.J.G. was supported by the Conservation Leadership Programme (grant 0134010) with additional assistance from F. Gebresenbet, R. Kassahun, and S. P. Loader. C.S.-A. was supported by Fondecyt Nº11140902 and 1181758. T.M.D.-B. was supported by the Royal Geographical Society and the Royal Zoological Society of Scotland with assistance from M. Hirschfeld and the Budongo Conservation Field Station. B.W. was supported by the National Research Foundation of Korea (2015R1D1A1A01057282). L.F.T. was supported by FAPESP (#2016/25358-3) and CNPq (#300896/2016-6). L.Be., L.F.S., and R.J.W. were supported by the Australian Research Council (FT100100375, DP120100811). A.A.C. was supported by a Royal Society Wolfson Research Merit award. J.H., A.La., and S.M. were funded by the Swedish Research Council Formas (grant no. 2013- 1389-26445-20). C.W. was funded by the National Research Foundation, South Africa. T.Y.J. and T.S.J. acknowledge NSF grant DEB-1601259. W.E.H. was funded by the NSERC Strategic and Discovery grant programs

This study assesses microplastic ingestion in Boops boops at different geographical areas in the Mediterranean Sea. A total of 884 fish were caught at 20 coastal sites in Spain, France, Italy and Greece and analyzed using a common methodological protocol. Microplastics were found in 46.8% of the sampled fish, with an average number of items per individual of 1.17 ± 0.07. Filaments were the predominant shape type, while polyethylene and polypropylene were indicated by FTIR as the most common polymer types of ingested microplastics. The frequency of occurrence, as well as the abundance and proportion of types (size, shape, color and polymer) of ingested microplastics, varied among geographical areas. The spatial heterogeneity of the abundance of ingested microplastics was mainly related to the degree of coastal anthropogenic pressure at the sampling sites. Our findings further support the suitability of B. boops as bioindicator of microplastic pollution in the Mediterranean Sea.

Background Mitigating the effect of linear transport infrastructure (LTI) on fauna is a crucial issue in road ecology. Wildlife crossing structures (tunnels or overpasses) are one solution that has been implemented to restore habitat connectivity and reduce wildlife mortality. Evaluating how these crossings function for small wildlife has often been recommended but, mainly due to technical limitations, is not often conducted in practice or only as short-term monitoring (less than 1 year). In this study, we developed and tested an automated device that records the detailed behaviour of animals when using wildlife tunnels. The method is based on marking and detecting individuals with RFID (radio-frequency identification) tags and allows small animals to be tracked. Composed of four antennas (detectors) placed at roughly 2 m intervals, the system was tested in a tunnel in northern France in the summer of 2017. One species of amphibian (the toad Bufo spinosus, n = 13) and two carabids (the ground beetles Carabus coriaceus and Carabus nemoralis, respectively, n = 3 and n = 4), marked beforehand with PIT (passive integrated transponder) tags, were recorded when they crossed these detectors. This allowed individual trajectories, including crossing speed, to be estimated. Results We found that 12 of the 13 toads and 3 of the 7 ground beetles successfully crossed the entire wildlife passage of 7 m long. The detection rate of each detector varied from 8.33 to 100%, with a mean of 52.08%. All individuals were recorded by at least one detector. We observed high variability in the crossing characteristics of toads (mean transit duration = 41 min and 15 s ± 25 min) and ground beetles (6 h 11 min ± 3 h 30 min). The system provided information on precise trajectories (e.g., crossing speed, U-turns, distance travelled in the tunnel, proportion of individuals reaching the exit, etc.) for each individual, in a context of tunnel crossing. Conclusion The system allowed us to record small animal behaviour in the context of tunnel crossing in which other types of tracking (e.g. radio-tracking) or detection (e.g. camera traps) are not effective. It also opens the possibility for a range of experiments that would contribute to a better understanding of the behaviour of small animals in tunnels, allowing a comparison of tunnel characteristics (such as size, building material, substrate, etc.) with the aim of increasing wildlife use of the tunnel and proposing guidelines for the construction and maintenance of these mitigation measures.

The following protocol is intended to respond to the requirements set by the European Union’s Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: “There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region”, where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: “There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region”. © 2019 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.

This work aims to illustrating how it is possible to measure different modalities of adult dispersal in two subdivided populations of the alpine newt (Triturus alpestris). Recent developments in capture-mark-recapture methods make it possible to estimate transience rates from individuals captured only once. In the context of subdivided newt populations, transience is assumed to express nomadic behaviour that contribute to breeding dispersal. Skeletochronology and recaptures within each pond system also made it possible to estimate emigration rates and local dispersal. Two subdivided populations of alpine newts were monitored over 4 and 5 years, respectively. Whereas population A is suspected to have been established for more than 100 years, population B was monitored when colonizing a newly created archipelago of ponds. Transience was detected in each population at similar rates (37% in population A and 35% in the population B). Annual apparent survival rates were estimated as 82% in population A vs. 33% in population B. Similarity of age structures between populations leads us to consider such low survival rates in population B as resulting from emigration. Emigration was thus negligible in population A and was estimated to reach 57·3% in population B. Conversely, high local dispersal (movements within a pond system) was detected in population A, but not in population B. Even though the causation of dispersal in newts (genetic polymorphism vs. phenotypic plasticity) remains unexplored, our study succeeded in identifying several dispersal components that could result from different selective pressures (habitat heterogeneity at different temporal scales). Experimental approaches are needed to investigate the causative bases of these traits.

Actuarial senescence (hereafter “senescence”) has been viewed for a long time as an inevitable and uniform process. However, the work on senescence has mainly focused on endotherms (especially mammals) with deterministic growth and low regeneration capacity at adult stages, leading to a strong taxonomic bias in the study of aging. Recent studies have highlighted that senescence could indeed display highly variable trajectory shape that correlates with species life history traits. Slow life histories and indeterminate growth seem to be associated with weak and late senescence. Furthermore, a few studies have suggested that high regenerative abilities could make senescence negligible in several ectotherms (e.g., hydra and salamanders). However, demographic data for species that would allow testing of these hypotheses are scarce and fragmented. Here, we investigated senescence patterns in a group of salamanders (i.e. “true salamanders”) from the Western Palearctic using capture-recapture data and Bayesian modeling. Our results showed that salamanders have slow life histories and that they experience negligible senescence. This pattern was consistent at both intra- and interspecific levels, suggesting that the absence of senescence may be a phylogenetically conserved trait. The regenerative capacities of true salamanders, and urodeles in general, likely explains why these small ectotherms have lifespans similar to that of large endotherms (e.g., ungulates, large birds) and undergo negligible senescence contrary to most amniotes including humans. Our study seriously challenges the idea that senescence is a ubiquitous phenomenon in the living world.

Este artículo contiene 9 páginas, 3 figuras, 2 tablas., Range expansions are an intrinsic part of the biogeography of most species. Among them, some invasive species represent a paradigmatic case in which local introductions are followed by rapid expansions in several directions. Theoretically, this creates a recursive founder effect, leading to sustained losses of genetic diversity and – through stochastic changes in allele frequency – to a population structure that mirrors invasion history. Here, we use restriction site-associated DNA (RAD) markers to detect these patterns in the invasive range of the Mediterranean Painted Frog (Discoglossus pictus) in Europe. We identify and genotype a large panel of loci using genotyping-by-sequencing in several sites along its two main directions of invasion – northwards and southwards. Although the frog has a few translocated isolated populations, most of the invasive range is contiguous, allegedly corresponding to a self-sustained expansion from a single introduction point in Banyuls de la Marenda (Southern France) more than a century ago. As expected, the highest genetic diversity was found in this location, with progressive losses of genetic diversity north- and southwards. Genetic similarities among sampling sites also coincided with predictions according to the documented process of expansion, showing a latitudinal population structure matching predictions according to geographic distance among locations. This poses the two opposite expansion directions as independent processes of loss of genetic diversity and creation of population structure, that is, two different cases of range expansion which are ready for further examinations., Part of the fieldwork for this study was done during the support of the Spanish Ministerio de Educaci on y Deporte to EPB (FPU grant: AP2010-5563).

The American bullfrog, native to the eastern half of North America including southern Quebec, is considered one of the most invasive species in the world. It has been introduced in America, Asia and Europe, mainly for food purposes. A study on the age and body size of this species was carried out on individuals obtained from four introduced populations (one from Western Canada and three from France), using the skeletochronological method. Adults did not differ between sexes in mean body size or mean age, with the exception of one population in France where males were younger than females. Several differences in the mean age and body size were observed, with the individuals from the population in Canada exhibiting the highest values for both sexes. The scarcity of studies in the native range prevents the comparison of introduced versus native populations, and we focus on comparisons between introduced populations.

Invasive species can be a threat to native species in several ways, including transmitting lethal infections caused by the parasites they carry. However, invasive species may also be plagued by novel and lethal infections they acquire when invading, making inferences regarding the ability of an invasive host to vector disease difficult from field observations of infection and disease. This is the case for the pathogenic fungus Batrachochytrium dendrobatidis (Bd) in Europe and one invasive host species, the North American bullfrog Lithobates catesbeianus, hypothesized to be responsible for vectoring lethal infection to European native amphibians. We tested this hypothesis experimentally using the alpine newt Ichthyosaura alpestris as our model native host. Our results show that infected bullfrog tadpoles are effective vectors of Bd. Native adult newts co-housed with experimentally infected bullfrog tadpoles became Bd infected (molecular and histological tests). Moreover, the exposed adult newts suffered mortality while the majority of infected bullfrog tadpoles survived until metamorphosis. These results cannot resolve the historical role of alien species in establishing the distribution of Bd across Europe or other regions in the world where this species was introduced, but they show its potential role as a Bd reservoir capable of transmitting lethal infections to native amphibians. Finally, our results also suggest that the removal of infected bullfrogs from aquatic environments may serve to reduce the availability of Bd in European amphibian communities, offering another justification for bullfrog eradication programmes that are currently underway or may be considered.

Detection is crucial in the study and control of invasive species but it may be limited by methodological issues. In amphibians, classical survey techniques exhibit variable detection probability depending on species and are often constrained by climatic conditions often requiring several site visits. Furthermore, detection may be reduced at low density because probability capture (passive traps), or activity (acoustic surveys) drop. Such limits may impair the study of invasive species because low density is typical of the onset of colonisation on a site. In the last few years, environmental DNA (eDNA) methods have proved their ability to detect the presence of aquatic species. We developed here an eDNA method to detectXenopus laevisin ponds. This austral African species is now present worldwide because of its use in biology and as a pet. Populations have settled and expanded on several continents so that it is now considered as one of the major invasive amphibians in the World. We detected the presence ofX. laevisat density as low as 1 ind/100 m2and found a positive relationship between density in ponds and rate of DNA amplification. Results show that eDNA can be successfully applied to survey invasive populations ofX. laeviseven at low density in order to confirm suspected cases of introduction, delimit the expansion of a colonized range, or monitor the efficiency of a control program.

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.

International audience; 1. In the Northern Hemisphere, an increase in both the frequency and magnitude of violent flooding events has been reported due to climate change. According to life history theory, one might postulate that in ‘slow’ species: (i) environmental canalisation may act as a selective force that minimises to some extent adult survival variations caused by catastrophic flood and (ii) extreme flooding events would cause important variations in recruitment and young survival. Hence, it may be hypothesised that (iii) the population growth rate of ‘slow’ species might be relatively insensitive to changes in the frequency of extreme climatic events if adult survival remains largely unaffected.2. In this study, we investigated how extreme rainfall events resulting in severe flood impact popula- tion dynamics of a long-lived endangered amphibian, the yellow-bellied toad (Bombina variegata: Bombinatoridae). To address this issue, we used capture–recapture (CR) data collected on two populations (768 and 1154 individuals identified) in southern France and developed multi-event CR models.3. Our results indicated that extreme flooding did not cause any variation in sub-adult or adult survival, whereas recruitment and juvenile survival were negatively impacted. Furthermore, our simulations indicated that the population growth rate was only marginally sensitive to potential changes in the frequency of extreme flooding in the future.4. Hence, we suggest that extreme flooding does not appear to be a proximal factor of extinction risk for this endangered amphibian species.

Debris impact on marine wildlife has become a major issue of concern. Mainy species have been identified as being threatened by collision, entanglement or ingestion of debris, generally plastics, which constitute the predominant part of the recorded marine debris. Assessing sensitive areas, where exposure to debris are high, is thus crucial, in particular for sea turtles which have been proposed as sentinels of debris levels for the Marine Strategy Framework Directive and for the Unep-MedPol convention. Our objective here was to assess sea turtle exposure to marine debris in the 3 metropolitan French fronts. Using aerial surveys performed in the Channel, the Atlantic and the Mediterranean regions in winter and summer 2011–2012, we evaluated exposure areas and magnitude in terms of spatial overlap, encounter probability and density of surrounding debris at various spatial scales. Major overlapping areas appeared in the Atlantic and Mediterranean fronts, concerning mostly the leatherback and the loggerhead turtles respectively. The probability for individuals to be in contact with debris (around 90% of individuals within a radius of 2 km) and the density of debris surrounding individuals (up to 16 items with a radius of 2 km, 88 items within a radius of 10 km) were very high, whatever the considered spatial scale, especially in the Mediterranean region and during the summer season. The comparison of the observed mean debris density with random distribution suggested that turtles selected debris areas. This may occur if both debris and turtles drift to the same areas due to currents, if turtles meet debris accidentally by selecting high food concentration areas, and/or if turtles actively seek debris out, confounding them with their preys. Various factors such as species-specific foraging strategies or oceanic features which condition the passive diffusion of debris, and sea turtles in part, may explain spatio-temporal variations in sensitive areas. Further research on exposure to debris is urgently needed. Empirical data on sea turtles and debris distributions, such as those collected aerially, are essential to better identify the location and the factors determining risks.

Age and size at sexual maturity are major life-history traits that affect growth rate and reproductive output. The differences in these life-history traits can be better assessed in syntopic populations of related species, where environmental variability in time and space is removed. The Spadefoot Toads (Pelobates spp.) are highly specialized burrowing and nocturnal species with a narrow ecological niche. We tested if age-related parameters are responsible for sexual size dimorphism and size differences in two syntopic populations from the Danube Delta Biosphere Reserve (Romania). The two populations differed significantly in size and body mass, and showed distinct sexual dimorphism, with females significantly larger in P. fuscus, and males slightly larger in P. syriacus. We also found differences in age structure: P. fuscus was on average 5.0 yr old, whereas P. syriacus was on average 7.4 yr old. Both species reached sexual maturity at a similar age. Different growth rates before sexual maturity...

International audience; Variation in the environment can induce different patterns of genetic and phenotypic differentiation among populations. Both neutral processes and selection can influence phenotypic differentiation. Altitudinal phenotypic variation is of particular interest in disentangling the interplay between neutral processes and selection in the dynamics of local adaptation processes but remains little explored. We conducted a common garden experiment to study the phenotypic divergence in larval life-history traits among nine populations of the common toad (Bufo bufo) along an altitudinal gradient in France. We further used correlation among population pairwise estimates of quantitative trait (QST) and neutral genetic divergence (FST from neutral microsatellite markers), as well as altitudinal difference, to estimate the relative role of divergent selection and neutral genetic processes in phenotypic divergence. We provided evidence for a neutral genetic differentiation resulting from both isolation by distance and difference in altitude. We found evidence for phenotypic divergence along the altitudinal gradient (faster development, lower growth rate and smaller metamorphic size). The correlation between pairwise QSTs–FSTs and altitude differences suggested that this phenotypic differentiation was most likely driven by altitude-mediated selection rather than by neutral genetic processes. Moreover, we found different divergence patterns for larval traits, suggesting that different selective agents may act on these traits and/or selection on one trait may constrain the evolution on another through genetic correlation. Our study highlighted the need to design more integrative studies on the common toad to unravel the underlying processes of phenotypic divergence and its selective agents in the context of environmental clines.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species’ activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Pathogenic fungi and viruses cause mortality outbreaks in wild amphibians worldwide. In the summer of 2012, dead tadpoles and adults of the European common frog Rana temporaria were reported in alpine lakes in the southwestern Alps (Mercantour National Park, France). A preliminary investigation using molecular diagnostic techniques identified a Ranavirus as the potential pathogenic agent. Three mortality events were recorded in the park, and samples were collected. The amphibian chytrid fungus Batrachochytrium dendrobatidis was not detected in any of the dead adult and juvenile frogs sampled (n=16) whereas all specimens were positive for a Ranavirus. The genome sequence of this Ranavirus was identical to previously published sequences of the common midwife toad virus (CMTV), a Ranavirus that has been associated with amphibian mortalities throughout Europe. We cultured virus from the organs of the dead common frogs and infecting adult male common frogs collected in another alpine region where no frog mortality had been observed. The experimentally infected frogs suffered 100% mortality (n=10). The alpine die-off is the first CMTV outbreak associated with mass mortality in wild amphibians in France. We describe the lesions observed and summarize amphibian populations affected by Ranaviruses in Europe. In addition, we discuss the ecologic specificities of mountain amphibians that may contribute to increasing their risk of exposure to and transmission of Ranaviruses.

International audience; Dispersal is a central component of life history evolution. An increasing number of studies suggest that spatiotemporally variable environments may promote the evolution of “dispersal syndromes,” consisting of covariation patterns between dispersal and morphologi- cal, physiological, behavioral, and life history traits. At the interspecific scale, the “colonizer syndrome” appears to be one of the most frequently recorded associations between dispersal and life history traits, linking a high dispersal rate, high fecundity, and a short lifespan as sys- tematically combined adaptations in spatiotemporally varying environments. However, few studies have highlighted the existence of a “colonizer syndrome” at the intraspecific scale, and none have investigated how different degrees of habitat stochasticity might shape covariation patterns between dispersal and life history traits. In this study, we examined this issue in free- ranging metapopulations of the yellow-bellied toad (Bombina variegata) using capture–recap- ture data. Combining the results of this study with another recent study, we found that a high dispersal rate, high fecundity, and a short lifespan are associated in metapopulations experi- encing unpredictable environments. In contrast, a very low dispersal rate (close to zero), low fecundity and a long lifespan are associated in metapopulations occupying predictable environ- ments. We discuss these results as well as their demographic and evolutionary consequences.

International audience; Weather fluctuations have been demonstrated to affect demographic traits in many species. In long-lived organisms, their impact on adult survival might be buffered by the evolution of traits that reduce variation in interannual adult survival. For example, skipping breeding is an effective behavioral mechanism that may limit yearly variation in adult survival when harsh weather conditions occur; however, this in turn would likely lead to strong variation in recruitment. Yet, only a few studies to date have examined the impact of weather variation on survival, recruitment and breeding probability simultaneously in different populations of the same species. To fill this gap, we studied the impact of spring temperatures and spring rainfall on survival, on reproductive skipping behavior and on recruitment in five populations of a long-lived amphibian, the yellow-bellied toad (Bombina variegata). Based on capture–recapture data, our findings demonstrate that survival depends on interactions between age, population and weather variation. Varying weather conditions in the spring result in strong variation in the survival of immature toads, whereas they have little effect on adult toads. Breeding probability depends on both the individual’s previous reproductive status and on the weather conditions during the current breeding season, leading to high interannual variation in recruit- ment. Crucially, we found that the impact of weather variation on demographic traits is largely context dependent and may thus differ sharply between populations. Our results suggest that studies predicting the impact of climate change on population dynamics should be taken with caution when the relationship between climate and demo- graphic traits is established using only one population or few populations. We therefore highly recommend further research that includes surveys replicated in a substantial number of populations to account for context-dependent variation in demographic processes.

Summary 1. Alien invasive species (AIS) are one of the major causes of biodiversity loss and global homogenization. Once an AIS becomes established, costs of control can be extremely high and complete eradication is not always achieved. The ability to detect a species at a low density greatly improves the success of eradication and decreases both the costs of control and the impact on ecosystems. 2. In this study, we compare the sensitivity of traditional field methods, based on auditory and visual encounter surveys, with an environmental DNA (eDNA) survey for the detection of the American bullfrog Rana catesbeiana = Lithobates catesbeianus, which is invasive in south-western France. 3. We demonstrate that the eDNA method is valuable for species detection and surpasses traditional amphibian survey methods in terms of sensitivity and sampling effort. The bullfrog was detected in 38 sites using the molecular method, compared with seven sites using the diurnal and nocturnal surveys, suggesting that traditional field surveys have strongly underestimated the distribution of the American bullfrog. 4. Synthesis and applications. The environmental DNA approach permits the early detection of alien invasive species (AIS), at very low densities and at any life stage, which is particularly important for the detection of rare and ⁄ or secretive aquatic species. This method can also be used to confirm the sensitivity of control operations and to better identify the distributions of vulnerable species, making this a very relevant tool for species inventory and management.

The delimitation of the spatial equivalent of isolated populations or interacting sets of local populations is crucial for conservation management. In Amphibia, spatial organization in metapopulations requires knowledge on the individual variation of annual migratory capacity within local populations to delimit core habitats around the breeding site and to estimate the connectivity among neighbouring populations. The migratory behaviour of 143 adult Bufo calamita was monitored using radio-telemetry at eight localities covering a latitudinal range from 41° to 54°N. Aims of the study were to assess the geographic variation of migratory distance moved by males and females and to model their capacity for dispersal assuming exclusively unidirectional movements. Migratory range was not sex-biased, but was three times lower in population inhabiting sandy areas than in those on clay soils, probably because of the scarcity of moist shelters causing more frequent and more distant movements. For conservation management of local natterjack populations, we propose to use the migratory capacity of the 50% most sedentary individuals to delimit the core area around a given breeding site. To estimate the potential genetic connectivity between neighbouring local population, we propose to use the minimum migratory capacity of the 5% of individuals that moved most. Estimates obtained for populations in central Europe and the UK indicate a core area of 600 m around the breeding site and a maximum distance of 2250 m between the breeding ponds to maintain connectivity. Thus, the principal conservation problem in the UK is that most populations are isolated by distance and prone to local extinction. In contrast, core areas of populations in Spain extend to distances of about 5 km and connectivity is maintained up to a distance 12 km between neighbouring breeding ponds.

Large-scale patterns of body size variation are described by well-known generalizations such as Bergmanns rule; the generality and underlying causes of these patterns have been much debated. Intraspecific extension of this rule was tested in various ectotherms, and evidence was found for both Bergmann and converse Bergmann clines. In this study, we explored spatial patterns of variation in a widespread amphibian, the Common toad (Bufo bufo), along a 2240 km latitudinal gradient across Europe. We tested for covariation of adult body size, age and growth parameters with latitude, altitude, length of activity period and mean temperature during this period using both original and literature data. We selected 13 European populations, representing a latitudinal range from 43 to 63� N and altitudinal range from 15 to 1850 m a.s.l. The length of activity period (12–33 weeks) and Tmean (6.6–15.6� C) significantly decreased as latitude and altitude of these populations increased. Mean body size decreased as latitude increased (not with altitude), and increased with Tmean (not with length of activity period). Mean and minimal adult age increased with latitude and altitude, longevity increased with altitude only. Age increased as length of activity period decreased (not with Tmean). The growth coefficient (0.32–0.92 in males, 0.18–0.74 in females, available for six populations) decreased as altitude increased, and increased as both length of activity period and Tmean increased; latitudinal trend was non-significant. Our analysis shows that B. bufo clearly exhibited a converse Bergmann cline along latitudinal gradient, but not along altitudinal gradient; the main effect of elevation was on age. The effects of ecological conditions also differed: body size increased with Tmean, while age parameters were related to the length of activity period. This study highlights that, to identify causal factors underlying general ecogeographical rules, we have to take into account different phases of the life cycle, co-variation among life history traits and ecological factors acting on each of these traits. In amphibians with complex life cycles, lack of appropriate demographical or ecological data may affect our understanding of the variety of observed body size patterns.

International audience; Solar ultraviolet-B (UV-B) radiation can be harmful for developing amphibians. As the UV-B dose increases with altitude, it has been suggested that high-altitude populations may have an increased tolerance to high levels of UV-B radiation as compared to lowland populations. We tested this hypothesis with the common frog (Rana temporaria) by comparing populations from nine altitudes (from 333 to 2450 in above sea level). Eggs collected in the field were used for laboratory experiments, i.e., exposed to high levels of artificial UV-B radiation. Eggs were reared at 14 +/- 2 degrees C and exposed to UV treatments until hatching. Embryonic developmental rates increased strongly and linearly with increasing altitude, suggesting a genetic capacity for faster development in highland than lowland eggs. Body length at hatching varied significantly with UV-B treatments, being lower when eggs developed under direct UV-B exposure. Body length at hatching also increased as the altitude of populations increased, but UV-B exposure times were shorter as altitude of population increased. However, the body length difference between exposed and non-exposed individuals in each population decreased as altitude of populations increased, suggesting a costly effect of UV exposure on growth. Type of UV exposure did not influence the mean rates of embryonic mortality and deformity, but both mortality and deformity rates increased as the altitude of populations increased (while UV-B exposure duration decreased). The effect of UV-B on body length at hatching, mortality, and deformities suggests that the sensitivity to UV-B varied among populations along the altitudinal gradient. These results are discussed in evolutionary terms, specifically the potential of R. temporaria high-altitude populations to develop local genetic adaptation to high levels of UV-B. (c) 2008 Elsevier GmbH. All rights reserved.

Propagule pressure is considered the main determinant of success of biological invasions: when a large number of individuals are introduced into an area, the species is more likely to establish and become invasive. Nevertheless, precise data on propagule pressure exist only for a small sample of invasive species, usually voluntarily introduced. We studied the invasion of the American bullfrog, Rana catesbeiana, into Europe, a species that is considered a major cause of decline for native amphibians. For this major invader with scarce historical data, we used population genetics data (a partial sequence of the mitochondrial cytochrome b gene) to infer the invasion history and to estimate the number of founders of non-native populations. Based on differences between populations, at least six independent introductions from the native range occurred in Europe, followed by secondary translocations. Genetic diversity was strongly reduced in non-native populations, indicating a very strong bottleneck during colonization. We used simulations to estimate the precise number of founders and found that most non-native populations derive from less than six females. This capability of invasion from a very small number of propagules challenges usual management strategies; species with such ability should be identified at an early stage of introduction.

We used skeletochronology to determine the ages of 149 (74 females, 44 males, and 31 juveniles) Mountain Yellow-legged Frogs (Rana muscosa) from 13 locations (elevation 1509–3501 m) throughout their current range in the Sierra Nevada mountains of California. Lines of arrested growth (LAGs) from excised toe bones were distinct in these high elevation frogs, and each LAG was assumed to represent one year of age. Females ranged in age from 0–10 years (mean = 4.1 years) and males from 0–8 years (mean = 4.0 years). The skeletochronological age was that of the post-metamorphic frog and did not include the tadpole stage. Mountain Yellow-legged Frogs spend 3–4 years as tadpoles, but no age markers are found in their cartilaginous skeletons; thus, their total age, if both tadpole and post-metamorphic stages were included, would range up to 14 years. Females were significantly longer (snout–vent length: SVL) than males and had greater mean mass, but there was no difference in the mean ages. Juvenile fr...

International audience; Predicting the probability of successful establishment and invasion of alien species at global scale, by matching climatic and land use data, is a priority for the risk assessment. Both large- and local-scale factors contribute to the outcome of invasions, and should be integrated to improve the predictions. At global scale, we used climatic and land use layers to evaluate the habitat suitability for the American bullfrog Rana catesbeiana, a major invasive species that is among the causes of amphibian decline. Environmental models were built by using Maxent, a machine learning method. Then, we integrated global data with information on richness of native communities and hunting pressure collected at the local scale. Global-scale data allowed us to delineate the areas with the highest suitability for this species. Predicted suitability was significantly related to the invasiveness observed for bullfrog populations historically introduced in Europe, but did not explain a large portion of variability in invasion success. The integration of data at the global and local scales greatly improved the performance of models, and explained > 57% of the variance in introduction success: bullfrogs were more invasive in areas with high suitability and low hunting pressure over frogs. Our study identified the climatic factors entailing the risk of invasion by bullfrogs, and stresses the importance of the integration of biotic and abiotic data collected at different spatial scales, to evaluate the areas where monitoring and management efforts need to be focused.

Times Cited: 1; International audience; In conservation biology genetic diversity is recognized as an important criterion to consider when prioritizing populations for protection. Today, population genomics offers the opportunity to evaluate both neutral and adaptive components of genetic diversity directly at the genome level with molecular tools. By screening the genome with many genetic markers, it is possible to detect loci supposedly under natural selection and thus of adaptive significance. We devised a new diversity index, the population adaptive index (PAI), which accounts for the adaptive value of the population it refers to. To estimate this index, we performed a genome scan with amplified fragment length polymorphism markers to identify neutral and selected loci in several populations of a widespread amphibian (common frog, Rana temporaria) and a threatened plant (Austrian dragonhead, Dracocephalum austriacum L.). We then investigated four different conservation strategies aimed at protecting the maximum amount of genetic diversity (neutral or selected). In particular we explored the relevance of the principle of complementarity, usually applied to the protection of species, in the management of intraspecific diversity. This principle advocates the conservation of sets of units that together maximize the species' orgenetic diversity, which is in opposition to the traditional approach of targeting populations that are the most diverse individually. Four major conclusions emerged from these results. First, the PAI seemed to be a valuable index to evaluate the adaptive diversities within populations. Second, in the two species, the neutral and adaptive diversities within and among populations were not correlated, so conservation strategies based on the neutral and adaptive indexes would not select the same populations for protection. Third, because of its efficiency in conserving genetic diversity, the principle of complementarity deserves to be used more often for this purpose. Fourth, when neutral and adaptive results conflict, additional arguments (e.g., demography, ecology, and geographic proximity) should be considered together with levels of genetic diversity to determine a conservation strategy.