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Abstract The Balkan Peninsula hosts a great proportion of Europe’s biodiversity, and this is well illustrated by amphibian richness and endemism. Among them, the yellow-bellied toad Bombina variegata has been a model in ecology and evolution, but several aspects of its phylogeography and taxonomy remain surprisingly poorly understood. In this study, we combine cytochrome b DNA barcoding data (1238 individuals from 355 localities), mitogenome phylogenetics (17.2 kb), gene-based nuclear phylogenetics (3.7 kb from four gene fragments) and multilocus phylogenomics (4759 loci / ~554 kb obtained by double digest Restriction Associated DNA sequencing; ddRAD-seq) to re-assess the diversification of B. variegata, and revisit its nomenclatural history to assign scientific names to phylogeographic lineages. The analyses support four major lineages, one assigned to B. v. variegata (Carpathians and northwestern ranges), one assigned to B. v. pachypus (Apennine Peninsula), and two assigned to B. v. scabra (Dinarides, Hellenides and Balkanides vs. the Rhodope mountains). Spatiotemporal patterns of diversification suggest a role for a Late Miocene marine incursion in the Pannonian Plain (Paratethys) as the initial trigger of divergence, followed by a vicariance event in the Apennines and a “sky island” process of Pleistocene differentiation in the Balkan Peninsula. As it reached the Dinarides during the Late Pleistocene, B. v. variegata potentially hybridized with B. v. scabra and captured its mitochondrial DNA, which resulted in a massive cyto-nuclear discordance across all northwestern European populations. Finally, we show that the two lineages of B. v. scabra significantly differ in morphology and ventral coloration patterns, and describe the Rhodope lineage as a new subspecies.

In response to the current worldwide amphibian extinction crisis, conservation instances have encouraged the establishment of ex-situ collections for endangered species. The resulting assurance populations are managed under strict biosecure protocols, often involving artificial cycles of temperature and humidity to induce active and overwintering phases, which likely affect the bacterial symbionts living on the amphibian skin. However, the skin microbiota is an important first line of defense against pathogens that can cause amphibian declines, such as the chytrid Batrachochytrium dendrobatidis (Bd). Determining whether current husbandry practices for assurance populations might deplete amphibians from their symbionts is therefore essential to conservation success. Here, we characterize the effect of the transitions from the wild to captivity, and between aquatic and overwintering phases, on the skin microbiota of two newt species. While our results confirm differential selectivity of skin microbiota between species, they underscore that captivity and phase-shifts similarly affect their community structure. More specifically, the translocation ex-situ is associated with rapid impoverishment, decrease in alpha diversity and strong species turnover of bacterial communities. Shifts between active and overwintering phases also cause changes in the diversity and composition of the microbiota, and on the prevalence of Bd-inhibitory phylotypes. Altogether, our results suggest that current husbandry practices strongly restructure the amphibian skin microbiota. Although it remains to be determined whether these changes are reversible or have deleterious effects on their hosts, we discuss methods to limit microbial diversity loss ex-situ and emphasize the importance of integrating bacterial communities to applied amphibian conservation.

Abstract Background In species with complex life cycles, size at metamorphosis is a key life-history trait which reflects the complex interactions between costs and benefits of life in the aquatic and terrestrial environments. Whereas the effects of a deteriorating larval habitat (e.g. pond desiccation) on triggering an early metamorphosis have been extensively investigated in amphibians, the consequences of the resulting reduced size at metamorphosis on fitness in the post-metamorphic terrestrial stage remain poorly understood. We tested the hypothesis that a smaller size at metamorphosis negatively affects performance and survival in the ensuing terrestrial stage. Using as model a tropical amphibian (Ceratophrys stolzmanni) showing a large phenotypic plasticity in metamorphosing traits, we evaluated the effects of size at metamorphosis on fitness-related trophic and locomotor performance traits, as well as on growth and survival rates. Results Our results support the hypothesis that a larger size at metamorphosis is correlated with better survival and performance. The survival rate of large metamorphosing individuals was 95%, compared to 60% for those completing metamorphosis at a small size. Locomotor performance and gape size were positively correlated with body size, larger animals being more mobile and capable to ingest larger prey. However, smaller individuals achieved higher growth rates, thus reducing the size gap. Conclusions Overall, size at metamorphosis affected profoundly the chances of survival in the short term, but smaller surviving individuals partly compensated their initial disadvantages by increasing growth rates.

The genomic era contributes to update the taxonomy of many debated terrestrial vertebrates. In an accompanying work, we provided a comprehensive molecular assessment of spadefoot toads (Pelobates) using genomic data. Our results call for taxonomic updates in this group. First, nuclear phylogenomics confirmed the species-level divergence between the Iberian P. cultripes and its Moroccan relative P. varaldii. Second, we inferred that P. fuscus and P. vespertinus, considered subspecies until recently, feature partial reproductive isolation and thus deserve a specific level. Third, we evidenced cryptic speciation and diversification among deeply diverged lineages collectively known as Pelobates syriacus. Populations from the Near East correspond to the Eastern spadefoot toad P. syriacus sensu stricto, which is represented by two subspecies, one in the Levant (P. s. syriacus) and the other in the rest of the range (P. s. boettgeri). Populations from southeastern Europe correspond to the Balkan spadefoot toad, P. balcanicus. Based on genetic evidence, this species is also polytypic: the nominal P. b. balcanicus inhabits the Balkan Peninsula; a new subspecies P. b. chloeae ssp. nov. appears endemic to the Peloponnese. In this paper, we provide an updated overview of the taxonomy and distribution of all extant Pelobates taxa and describe P. b. chloeae ssp. nov.

Anurans have been introduced in many parts of the world and have often become invasive over large geographic areas. Although predation is involved in the declines of invaded amphibian populations, there is a lack of quantitative assessments evaluating the potential risk posed to native species. This is particularly true for Pelophylax water frogs, which have invaded large parts of western Europe, but no studies to date have examined their predation on other amphibians in their invaded range. Predation of native amphibians by marsh frogs (Pelophylax ridibundus) was assessed by stomach flushing once a month over four months in 21 ponds in southern France. Nine percent of stomachs contained amphibians. Seasonality was a major determinant of amphibian consumption. This effect was mediated by body size, with the largest invaders ingesting bigger natives, such as tree frogs. These results show that invasive marsh frogs represent a threat through their ability to forage on natives, particularly at the adult stage. The results also indicate that large numbers of native amphibians are predated. More broadly, the fact that predation was site- and time-specific highlights the need for repeated samplings across habitats and key periods for a clear understanding of the impact of invaders.

Abstract The genetic era has revolutionized our perception of biological invasions. Yet, it is usually too late to understand their genesis for efficient management. Here, we take the rare opportunity to reconstruct the scenario of an uprising invasion of the famous water frogs (Pelophylax) in southern France, through a fine-scale genetic survey. We identified three different taxa over less than 200 km2: the autochthonous P. perezi, along with the alien P. ridibundus and P. kurtmuelleri, which have suddenly become invasive. As a consequence, the latter hybridizes and may now form a novel hybridogenetic complex with P. perezi, which could actively promote its replacement. This exceptional situation makes a textbook application of genetics to early-detect, monitor and understand the onset of biological invasions before they pose a continental-wide threat. It further emphasizes the alarming rate of amphibian translocations, both at global and local scales, as well as the outstanding invasive potential of Pelophylax aliens.

Divergence in ecological niche offers organisms the opportunity of exploiting different food and habitat resources, scaling down competition and predation both among species, and within different age or size-classes of the same species. In harsh environments, where abiotic factors determine a clustering of resources during short timespans, competition and predation between organisms is likely to be enhanced. This is the case in tropical dry forests, where amphibians have limited opportunities to feed, their activity being restricted to the short rainy season. One way to maximize resource exploitation while avoiding predation risk is by adopting different diel activity patterns. We tested this hypothesis by comparing activity patterns in adults and recently metamorphosed juveniles of Pacific horned frogs (Ceratophrys stolzmanni) during field surveys and in an experimental study. Field surveys showed that the adults are strictly nocturnal, whereas freshly metamorphosed juveniles can be found active above ground at all hours, with a peak activity during daytime. The average body condition index of juveniles found active during the night was higher than that of juveniles found active during the day, suggesting that the weaker individuals may be constrained to being active during the day. On the other hand, in a laboratory experiment, juveniles that were visually exposed to adults moved less than those in the absence of adults. Both field and experimental observations indicate a temporal niche divergence between life stages. The results of the experiment offer support to the hypothesis that the juveniles in this species display an inverse activity pattern compared to adults, which can reduce competitive interactions and predation pressure from the larger conspecifics.

Divergence in ecological niche offers organisms the opportunity of exploiting different food and habitat resources, scaling down competition and predation both among species, and within different age or size-classes of the same species. In harsh environments, where abiotic factors determine a clustering of resources during short timespans, competition and predation between organisms is likely to be enhanced. This is the case in tropical dry forests, where amphibians have limited opportunities to feed, their activity being restricted to the short rainy season. One way to maximize resource exploitation while avoiding predation risk is by adopting different diel activity patterns. We tested this hypothesis by comparing activity patterns in adults and recently metamorphosed juveniles of Pacific horned frogs (Ceratophrys stolzmanni) during field surveys and in an experimental study. Field surveys showed that the adults are strictly nocturnal, whereas freshly metamorphosed juveniles can be found active above ground at all hours, with a peak activity during daytime. The average body condition index of juveniles found active during the night was higher than that of juveniles found active during the day, suggesting that the weaker individuals may be constrained to being active during the day. On the other hand, in a laboratory experiment, juveniles that were visually exposed to adults moved less than those in the absence of adults. Both field and experimental observations indicate a temporal niche divergence between life stages. The results of the experiment offer support to the hypothesis that the juveniles in this species display an inverse activity pattern compared to adults, which can reduce competitive interactions and predation pressure from the larger conspecifics.

The Pyrenees represent a natural laboratory for biogeographic, evolutionary and ecological research of mountain fauna as a result of the high variety of habitats and the profound effect of the glacial and interglacial periods. There is a paucity of studies providing a detailed insight into genetic processes and better knowledge on the patterns of genetic diversity and how they are maintained under high altitude conditions. This is of particular interest when considering the course of past climate conditions and glaciations in a species which is considered site tenacious, with long generation times. Here we analyzed the genetic patterns of diversity and structure of the endemic Pyrenean brook newt (Calotriton asper) along its distribution range, with special emphasis on the distinct habitat types (caves, streams, and lakes), and the altitudinal and geographical ranges, using a total set of 900 individuals from 44 different localities across the Pyrenean mountain range genotyped for 19 microsatellite loci. We found evidence for a negative longitudinal and positive altitudinal gradient of genetic diversity in C. asper populations. The fact that genetic diversity was markedly higher westwards is in accordance with other Pyrenean species. However, the impact of altitudinal gradient on the genetic diversity seems to differ from other species, and mostly from other amphibians. We found that lower altitudes can act as a barrier probably because the lowlands do not provide a suitable habitat for C. asper. Regarding the distinct habitat types, caves had significantly lower values of genetic diversity compared to streams or lakes. The mean FST value was relatively high (0.304) with maximum values as high as 0.771, suggesting a highly structured total population. Indeed, populations were grouped into five subclusters, the eastern populations (cluster 1) remained grouped into two subclusters and the central-western Pyrenees (cluster 2) into three subclusters. The increase of isolation with geographical distance is consistent with the population structure detected. In conclusion, C. asper seems to be adapted to high altitude mountain habitats, and its genetic diversity is higher in the western Pyrenees. In terms of conservation priority, we consider more relevant the populations that represent a reservoir of genetic diversity.

Alternative reproductive strategies are widespread in caudate amphibians. Among them, fire salamanders (Salamandra salamandra) usually rely on streams to give birth to aquatic larvae but also use ponds, whereas palmate newt larvae (Lissotriton helveticus) typically metamorphose into terrestrial juveniles, but can also reproduce in retaining their gills, a process known as paedomorphosis. Here we report repeated observations of an unusual case of coexistence of these two alternative traits in the same pond (Larzac, France). The prevalence of fire salamanders in Southern Larzac was very low (pond occupancy: 0.36%). The observed abundance of fire salamander larvae and paedomorphic newts was also low in the studied pond. On one hand, the rarity of this coexistence pattern may suggest that habitat characteristics may not be optimal or that competition or predation processes might be operating. However, these hypotheses remain to be tested. On the other hand, as this is the only known case of breeding in Southern Larzac, it could be considered to be at a high risk of extirpation.

The introduction of alien species is one of the major causes of current and global biodiversity loss. The introduction of fish can be a particular threat to native amphibian populations, which are declining worldwide. One way for amphibians to persist in such altered environments is to adopt anti-predator strategies especially at the behavioural level. However, although it has been shown that avoidance behaviour may decrease the probability of being detected by a potential predator, little is known on the consequences on sexual behaviour. In this study, we tested the hypothesis that adult Alpine newts (Ichthyosaura alpestris) use shelters more often and exhibit less sexual activity in the presence of goldfish (Carassius auratus) and that they reduce sexual activity more in risky micro-habitats than in safe environments. To this end, we assessed behavioural patterns of adult newts in a replicated laboratory design. Goldfish were present in direct contact with newts in half of the tanks. Consistently throughout the study period, significantly more newts used shelter in the presence of fish than in their absence. Newts also significantly decreased their sexual activity level overall, but specially outside the shelter when they were in direct contact with fish. These results show that fish presence can affect newts in complex ways, such as through inhibition of their reproduction. Our work highlights that integrating behaviour in conservation studies is essential to understanding the patterns of coexistence and exclusion between introduced fish and amphibians.

Although rare species are often the focus of conservation measures, more common species may experience similar decline and suffer from the same threatening processes. We tested this hypothesis by examining, through an information-theoretic approach, the importance of ecological processes at multiple scales in the great crested newt Triturus cristatus, regionally endangered and protected in Europe, and the more common smooth newt, Lissotriton vulgaris. Both species were similarly affected by the same processes, i.e. suitability of aquatic and terrestrial components of their habitat at different scales, connectivity among breeding sites, and the presence of introduced fish. T. cristatus depended more on water depth and aquatic vegetation than L. vulgaris. The results show that environmental pressures threaten both common and rare species, and therefore the more widespread species should not be neglected in conservation programs. Because environmental trends are leading to a deterioration of aquatic and terrestrial habitat features required by newt populations, populations of the common species may follow the fate of the rarest species. This could have substantial conservation implications because of the numerical importance of common species in ecosystems and because commonness could be a transient state moving towards rarity. On the other hand, in agreement with the umbrella species concept, targeting conservation efforts on the most demanding species would also protect part of the populations of the most common species.

Sound production is widespread among fishes and accompanies many social interactions. The literature reports twenty-nine cichlid species known to produce sounds during aggressive and courtship displays, but the precise range in behavioural contexts is unclear. This study aims to describe the various Oreochromis niloticus behaviours that are associated with sound production in order to delimit the role of sound during different activities, including agonistic behaviours, pit activities, and reproduction and parental care by males and females of the species.Sounds mostly occur during the day. The sounds recorded during this study accompany previously known behaviours, and no particular behaviour is systematically associated with sound production. Males and females make sounds during territorial defence but not during courtship and mating. Sounds support visual behaviours but are not used alone. During agonistic interactions, a calling Oreochromis niloticus does not bite after producing sounds, and more sounds are produced in defence of territory than for dominating individuals. Females produce sounds to defend eggs but not larvae.Sounds are produced to reinforce visual behaviours. Moreover, comparisons with O. mossambicus indicate two sister species can differ in their use of sound, their acoustic characteristics, and the function of sound production. These findings support the role of sounds in differentiating species and promoting speciation. They also make clear that the association of sounds with specific life-cycle roles cannot be generalized to the entire taxa.