Alytes Obstetricans Research Articles

Anthropogenic determinants of species presence in amphibian communities across a regional elevation gradient

AbstractIdentifying and quantifying the main factors that are jeopardizing amphibian communities worldwide is essential for planning effective amphibian conservation. We collected data on the presence/absence of salamanders and newts (Caudata) and frogs and toads (Anura) in >3600 water points from a large region (30,000 km2) in NW Spain during a 10‐year period (2004–2013). We contrasted a large set of hypotheses explaining presence/absence as a function of anthropogenic factors across the regional elevation gradient (1–2036 m). Logistic regression modeling revealed that salamanders and newts were mainly influenced by pollution and by land use changes, and that the effect of these factors changed with elevation. However, frogs and toads were affected by a larger set of factors acting synergistically, although not including land use changes, and their effect operated at all elevation ranges, except in the case of biological factors (invasive species and wild boar abundance) with higher effects at low elevation. Changes in land use was the most common factor at any elevation and included the abandonment of rural areas, which favors shrub and tree encroachment on former open land, and loss of water points. The most resilient species at any elevation were two frogs, Pelophylax perezi and Rana parvipalmata. The least resilient species were two salamanders, Chioglossa lusitanica and Lissotriton helveticus, followed closely by two toads, Alytes obstetricans and Pelobates cultripes. Unoccupied sites had higher frequencies of biological effects and of changes in land use in the first 5 years of study and lower frequencies of direct human influence factors in the second period. Overall, our results showed that the studied amphibian metacommunity was negatively influenced both by direct and indirect anthropogenic factors, but also that many amphibian species were not only capable of occupying sites which had been altered by human action, but were even favored by land uses generating open habitat, a habitat type that is increasingly uncommon in the region, in the entire Iberian Peninsula and in Europe.

The commensal skin microbiome of amphibian mountain populations and its association with the pathogen Batrachochytrium dendrobatidis.

Microbial assemblages naturally living on the skin are an integral part of immunity. In amphibians, this skin microbiota may hold a mitigation solution against the fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the panzootic disease chytridiomycosis. We used 16S rRNA gene metabarcoding to test the adaptive microbiome hypothesis. We compared the community composition, richness, and putative Bd-inhibitory function of the skin microbiome of three amphibian host species in the Pyrenees, as well as three species in Taiwan, in both Bd-positive and negative mountain populations. In both geographical regions, the amphibian host species played a decisive role in shaping the microbial assemblage and putative anti-Bd properties. In the Pyrenees, the species most susceptible to chytridiomycosis, Alytes obstetricans, had the lowest relative abundances of putative protective bacteria. In Bd-positive and negative sites, individuals had different skin microbiomes, with all anuran species showing increased relative abundances of potential anti-Bd bacteria, while the Taiwanese caudata Hynobius sonani showed the opposite pattern. Our results suggest that, in response to exposure to the pathogen, the skin microbiota shifted to a defensive state with increased anti-Bd function, which may contribute to promoting disease resistance, as proposed by the adaptive microbiome hypothesis.

A decision-making framework to maximise the evolutionary potential of populations - Genetic and genomic insights from the common midwife toad (Alytes obstetricans) at its range limits

Anthropogenic habitat modification and climate change are fundamental drivers of biodiversity declines, reducing the evolutionary potential of species, particularly at their distributional limits. Supportive breeding or reintroductions of individuals are often made to replenish declining populations, sometimes informed by genetic analysis. However, most approaches utilised (i.e. single locus markers) do not have the resolution to account for local adaptation to environmental conditions, a crucial aspect to consider when selecting donor and recipient populations. Here, we incorporate genetic (microsatellite) and genome-wide SNP (ddRAD-seq) markers, accounting for both neutral and putative adaptive genetic diversity, to inform the conservation management of the threatened common midwife toad, Alytes obstetricans at the northern and eastern edges of its range in Europe. We find geographically structured populations (n = 4), weak genetic differentiation and fairly consistent levels of genetic diversity across localities (observed heterozygosity and allelic richness). Categorising individuals based on putatively adaptive regions of the genome showed that the majority of localities are not strongly locally adapted. However, several localities present high numbers of private alleles in tandem with local adaptation to warmer conditions and rough topography. Combining genetic diversity and local adaptations with estimates of migration rates, we develop a decision-making framework for selecting donor and recipient populations which maximises the geographic dispersal of neutral and putatively adaptive genetic diversity. Our framework is generally applicable to any species, but especially to amphibians, so armed with this information, conservationists may avoid the reintroduction of unsuitable/maladapted individuals to new sites and increase the evolutionary potential of populations within species.

Gamete Collection, Artificial Fertilization and Captive-Rearing of Eggs in a Terrestrial-Breeding Anuran with Parental Care: Alytes obstetricans.

The genus Alytes consists of six primitive terrestrial-breeding species (five of them endangered or present in restricted distributions) with unique male parental care. Alytes obstetricans was used as a model for the development of assisted reproductive technologies for gamete collection, artificial fertilization and captive-rearing of embryos. Sperm collection was achieved with human chorionic gonadotropin (hCG), but only 17 to 50% of males responded to the hormone. In addition, an effect of captivity on sperm quality was observed. A combination of gonadotropin-releasing hormone agonist (GnRHa) and hCG was tested for induction of ovulation, and 100% of females responded to the treatment. Recently collected sperm was used in artificial fertilization, and 10% of eggs showed complete embryo development. To design a method for captive-rearing of embryos, natural clutches (n = 13) were removed from males and divided into two sub-clutches. One was kept with all eggs together and in the other, the egg string was cut and eggs were split individually. All sub-clutches with eggs kept separated presented embryo survival (ranging from 27 to 61%) that hatched normally, while only one sub-clutch with embryos maintained together had some embryo survival (36%). These results may contribute to the management of this and related species.

No amphibian chytrid fungus Batrachochytrium dendrobatidis detected in four introduced populations of the midwife toad Alytes obstetricans in eastern England

The amphibian chytrid fungus Batrachochytrium dendrobatidis threatens amphibian species globally as the causative agent of chytridiomycosis, with the introduction of non-native species being one of the pathways that the pathogen can spread to naive populations. We have monitored and screened the common midwife toad Alytes obstetricans in four separate populations in eastern England, to investigate the potential threats to local amphibians. Forty-eight toads across all life stages were swabbed between May 2018 and August 2020, and screened for the presence of B. dendrobatidis DNA using qPCR. None of the samples tested were positive, indicating that it is unlikely that any of the A. obstetricans swabbed were infected with B. dendrobatidis. The populations surveyed represent only a small part of the species range in Britain, consequently a more widespread survey is recommended to increase confidence that British A. obstetricans are free of B. dendrobatidis.

Сравнительная оценка биологического действия нативных и синтезированных пептидов

Biologically active peptides are considered as preventive and therapeutic agents for vari­ous diseases. Due to the high cost and complexity of isolating native peptides for use in phar­maceuticals, synthetically produced peptides are increasingly being used in dietary supple­ments. The aim of the research is to confirm the similarity and biological activity of synthe­sized peptides compared to native peptides. Synthesized and native peptides from bovine co­lostrum with the code names T1.1 and mpT2 were used as the objects of the study. The pep­tides were synthesized using the solid-phase method. Peptide T1.1 is similar to the peptide «POSSUM_01-POSSUM-C-EMBRYO-2KB», the biological activity of which has not been studied. Peptide mpT2 is similar to the anti-diabetic peptide «LL-16 Alytes obstetricans». It has been proven that the synthesized peptides do not differ from natural ones in terms of physical and chemical characteristics. Both synthesized and natural peptides are non-toxic. The anti-diabetic effect of natural and synthesized peptide mpT2 on animals with induced type 2 diabetes and the antioxidant activity of synthesized and natural peptide T1.1 have been demonstrated.

Effect of natural abiotic soil vibrations, rainfall and wind on anuran calling behavior: a test with captive-bred midwife toads (Alytes obstetricans).

Anurans are known to detect vibrations, but few studies explore relationships between vibrations and resultant behaviors. We studied the reaction of calling captive-bred male midwife toads (Alytes obstetricans) to the randomized playback of a vibrational crescendo stimulus train. We considered two sources of natural abiotic vibrational stimuli: rainfall and wind. Rainfall was expected to induce calling and wind was expected to inhibit it. Playback experiments with two synthetic tones (200Hz and 300Hz) tested the sensitivity to pure tones and could possibly reveal a hearing sensitivity trend between these frequencies. The toads did not increase call rate in response to rainfall vibrations and only one of the five wind stimulus levels caused a significant decrease in call rate. This limited response could be explained, because the tested toads came from a captive population, where emergence may not be mediated by rainfall vibrations. We found that A. obstetricans is highly sensitive to very low frequencies, which could explain the sensitivity observed to vibrational stimuli. Playback of a random crescendo stimulus train proves to be a valid approach for addressing behavioral questions. However, the use of a captive population may have been a limitation in the clarity of the results.

The common midwife toad, Alytes obstetricans, is able to learn a simple task under an appetitive stimulus

Studies of response learning of amphibians are scarce compared to other vertebrates. We conducted a simple experiment to evaluate response learning in a primitive anuran species, the common midwife toad (Alytes obstetricans) under an appetitive stimulus. We hypothesized that, despite the primitive character of the studied species, the animals would be able to rapidly associate a particular arm of a basic T-shaped experimental arena with the reward, and that the existing male parental care of the species would provide males greater cognitive abilities than females. The response obtained by 12 males and 13 females was statistically different than such expected by chance, suggesting a learning process. The sex of the animal, as well as the average time spent to select the correct choice, were key during the learning process. Contrary to expectations, females showed higher learning capacities than males, perhaps as a consequence of the elaborate mate selection process and the competition between females to access males in this group of amphibians. The obtained results indicate that a rapid learning capacity is highly conservative in the phylogeny, having evolved from early tetrapods to those existing today in reptiles, birds, and mammals.

The limits of mtDNA analysis for determining the provenance of invasive species: a midwife toad example

Abstract For most if not all European herpetofauna, range-wide mtDNA phylogeographies have been published. This facilitates establishing the provenance of introduced populations. However, precision is contingent on the spatial genetic structure across the range of the taxon under study and, in particular, from where within that range the introduction was sourced. In the Netherlands, the common midwife toad, Alytes obstetricans, only naturally occurs in the extreme southeast and is on the decline there. Yet, introduced populations thrive elsewhere in the country. We use mtDNA analysis to try to determine the origin of two introduced populations along the Dutch coast, in the city of The Hague and the dune area Meijendel. We compiled a database of hundreds of individuals from throughout the distribution range and added over 130 individuals from both native and introduced populations from the Netherlands, Belgium and Germany. The mtDNA haplotypes found in the introduced populations are associated with postglacial expansion. The main haplotype predominates in the natural range in the Netherlands, but also occurs much more widely across western Europe, north of the Pyrenees. A closely related haplotype, newly identified from The Hague, was not found in the native Netherlands range, suggesting an origin from abroad. The combination of low phylogeographic resolution and low sampling density in the postglacially colonized part of the range hampers our ability to determine the provenance of the introduced A. obstetricans populations.

Climate warming triggers the emergence of native viruses in Iberian amphibians.

The number of epizootics in amphibian populations caused by viruses of the genus Ranavirus is increasing worldwide. Yet, causes for pathogen emergence are poorly understood. Here, we confirmed that the Common midwife toad virus (CMTV) and Frog virus 3 (FV3) are responsible for mass mortalities in Iberia since the late 1980s. Our results illustrate the Iberian Peninsula as a diversity hotspot for the highly virulent CMTV. Although this pattern of diversity in Europe is consistent with spread by natural dispersal, the exact origin of the emergence of CMTV remains uncertain. Nevertheless, our data allow hypothesizing that the Iberian Peninsula might harbor the ancestral population of CMTVs that could have spread into the rest of Europe. In addition, we found that climate warming could be triggering the CMTV outbreaks, supporting its endemic status in the Iberian Peninsula.

Non-Lethal Detection of Frog Virus 3-Like (RUK13) and Common Midwife Toad Virus-Like (PDE18) Ranaviruses in Two UK-Native Amphibian Species.

Ranaviruses have been involved in amphibian mass mortality events worldwide. Effective screening to control this pathogen is essential; however, current sampling methods are unsuitable for the detection of subclinical infections. Non-lethal screening is needed to prevent both further spread of ranavirus and losses of at-risk species. To assess non-lethal sampling methods, we conducted two experiments: bath exposing common frogs to RUK13 ranavirus at three concentrations, and exposing common toads to RUK13 or PDE18. Non-lethal sampling included buccal, digit, body and tank swabs, along with toe clips and stool taken across three time-points post-exposure. The presence/load of ranavirus was examined using quantitative PCR in 11 different tissues obtained from the same euthanised animals (incl. liver, gastro-intestinal tract and kidney). Buccal swab screening had the highest virus detection rate in both species (62% frogs; 71% toads) and produced consistently high virus levels compared to other non-lethal assays. The buccal swab was effective across multiple stages of infection and differing infection intensities, though low levels of infection were more difficult to detect. Buccal swab assays competed with, and even outperformed, lethal sampling in frogs and toads, respectively. Successful virus detection in the absence of clinical signs was observed (33% frogs; 50% toads); we found no difference in detectability for RUK13 and PDE18. Our results suggest that buccal swabbing could replace lethal sampling for screening and be introduced as standard practice for ranavirus surveillance.

The role of habitat features in patterns of population connectivity of two Mediterranean amphibians in arid landscapes of central Iberia

ContextMediterranean wetland ecosystems are in continuous decline due to human pressure. Amphibians are key elements of biotic communities of Mediterranean temporary ponds and streams, and their persistence depends on the availability and inter-connectivity of breeding sites.ObjectivesWe investigated the role of different factors potentially driving functional connectivity patterns in two amphibian species at the landscape and local scales. We focused on two Mediterranean endemic pond-breeding amphibians inhabiting semi-arid landscapes of central Spain, the common parsley frog (Pelodytes punctatus) and the common midwife toad (Alytes obstetricans).MethodsWe genotyped 336 individuals of P. punctatus and 318 of A. obstetricans from 17 and 16 breeding populations at 10 and 17 microsatellite loci, respectively. We used remotely sensed vegetation/moisture indices and land use/cover data to derive optimized resistance surfaces and test their association with estimates of gene flow and migration rates across populations.ResultsWe found evidence for higher population connectivity in common midwife toads than in common parsley frogs, with a strong effect of water availability in patterns of population connectivity of both species. However, the two species differ in the role of landscape features on population connectivity, with the distance and spatial distribution of artificial land-use types positively influencing connectivity in A. obstetricans and meadows/pastureland favouring P. punctatus. This is in line with reported breeding site preferences for the two species, with A. obstetricans successfully breeding in artificial water bodies that P. punctatus generally avoid.ConclusionsThis study highlights the importance of assessing species–habitat relationships shaping connectivity when developing and implementing conservation and management actions to benefit fragmented amphibian populations in the Mediterranean region. Our results show that amphibian species respond differently, even contrastingly to landscape features and thus require alternative, complementary strategies to improve population connectivity and ensure long-term viability.

Artificialwater bodies as amphibian breeding sites: the case of the common midwife toad (Alytes obstetricans) in central Spain

Abstract Natural breeding sites for amphibians are decreasing in quantity and quality in temperate regions, resulting in local extinctions and increasing population fragmentation. Artificial water bodies (e.g., water tanks or cattle troughs) can represent suitable reproductive habitats for some amphibians, but demographic data are required to assess this assumption. We evaluated the role of artificial water bodies in the persistence of a species of population concern, the common midwife toad, Alytes obstetricans (Laurenti, 1768), at local and regional scales. We surveyed 275 water bodies to characterize the distribution of the species and detected 63 breeding populations of A. obstetricans where we estimated larval abundance. In addition, we monitored two populations for three consecutive breeding seasons using capture-mark-recapture methods based on photo-identification, assessing abundance, breeding success and the use of space of adult individuals captured on multiple occasions. Our results show that artificial sites are preferentially used as breeding sites in the region compared to natural aquatic habitats, providing key habitat for the species and hosting much larger numbers and densities of larvae than natural sites. At local scale, populations of A. obstetricans in artificial sites were abundant and characterized by high male breeding success. However, adults are spatially aggregated around breeding sites, with small home ranges, implying high vulnerability to population fragmentation. Our results suggest artificial breeding sites can sustain viable populations of A. obstetricans, provided measures promoting connectivity among breeding nuclei are considered.

Amphibian loss alters periphyton structure and invertebrate growth in montane streams.

Amphibians are declining worldwide due to a combination of stressors such as climate change, invasive species, habitat loss, pollution and emergent diseases. Although their losses are likely to have important ecological consequences on the structure and functioning of freshwater ecosystems, this issue has been scarcely explored. We conducted an experiment in three montane streams-where primary production is the main source of energy and carbon-to assess the effects of amphibian disappearance (i.e. presence or absence of the common midwife toad Alytes obstetricans, a common species found in pools of these streams) on several aspects of ecosystem functioning and structure: periphyton biomass and chlorophyll a concentration, algal assemblage structure, and growth of macroinvertebrate grazers. We compared four types of experimental enclosures: (i) without macroinvertebrates or amphibians; (ii) with larvae of the caddisfly Allogamus laureatus; (iii) with A. obstetricans tadpoles; and (iv) with both A. laureatus larvae and A. obstetricans tadpoles. The absence of tadpoles increased periphyton biomass, but did not cause differences on inorganic sediment accrual. The algal assemblage had a higher diversity in the absence of tadpoles, and their characteristic taxa differed from the assemblages in presence of tadpoles. A. laureatus presented higher mass in presence of tadpoles; however, tadpole length was not affected by presence of macroinvertebrates. Our results suggest that presence of tadpoles is a driver of periphyton accrual and assemblage structure, acting as top-down control and with key potential consequences on the functioning of montane stream ecosystems.

08. Limb malformations in introduced populations of midwife toad Alytes obstetricans in Great Britain

08. Limb malformations in introduced populations of midwife toad Alytes obstetricans in Great Britain

Microbiome function predicts amphibian chytridiomycosis disease dynamics

BackgroundThe fungal pathogenBatrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood.MethodsMicrobiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection.ResultsMicrobiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes.ConclusionsBd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection.6u4HXENqNsd5f8SBedLBdGVideo

Temperature and duration of exposure drive infection intensity with the amphibian pathogen Batrachochytrium dendrobatidis.

The intensity of a pathogen infection plays a key role in determining how the host responds to infection. Hosts with high infections are more likely to transmit infection to others, and are may be more likely to experience progression from infection to disease symptoms, to being physiologically compromised by disease. Understanding how and why hosts exhibit variation in infection intensity therefore plays a major part in developing and implementing measures aimed at controlling infection spread, its effects, and its chance of persisting and circulating within a population of hosts. To track the relative importance of a number of variables in determining the level of infection intensity, we ran field-surveys at two breeding sites over a 12 month period using marked larvae of the common midwife toad (Alyes obstetricans) and their levels of infection with the amphibian pathogen Batrachochytrium dendrobatidis (Bd). At each sampling occasion we measured the density of larvae, the temperature of the water in the 48 h prior to sampling, the period of time the sampled individual had been in the water body, the developmental (Gosner) stage and the intensity of Bd infection of the individual. Overall our data suggest that the temperature and the duration of time spent in the water play a major role in determining the intensity of Bd infection within an individual host. However, although the duration of time spent in the water was clearly associated with infection intensity, the relationship was negative: larvae that had spent less than 3–6 months in the water had significantly higher infection intensities than those that had spent over 12 months, although this infection intensity peaked between 9 and 12 months. This could be due to animals with heavier infections developing more quickly, suffering increased mortality or, more likely, losing their mouthparts (the only part of anuran larvae that can be infected with Bd). Overall, our results identify drivers of infection intensity, and potentially transmissibility and spread, and we attribute these differences to both host and pathogen biology.

Natural toxins leached from Eucalyptus globulus plantations affect the development and life‐history of anuran tadpoles

Abstract Rapid human‐induced transformation of habitats is presenting organisms with challenging novel environmental conditions. Nowadays, extensive areas of the world are covered by exotic plantations, and among these, eucalypt plantations stand out for their worldwide occurrence. These novel environments probably threaten the persistence of native species, with several studies reporting biodiversity losses in these transformed areas. However, the effects of such habitat alteration on life‐history traits that potentially constrain the ecology of individuals have been poorly explored. In a mesocosm experiment, we exposed tadpoles of three anuran species (Rana temporaria, Alytes obstetricans, and Pelophylax perezi) to the leachates of natural oak trees and exotic eucalypt trees to explore how the transformation of natural forests into eucalypt plantation affects the expression of behavioural and morphological traits and immune response. While anti‐predatory responses appeared not to be impacted by leachate type, tadpoles exposed to eucalypt leachates differed in morphology and growth, and exhibited weaker immune responses and smaller size at metamorphosis than those exposed to oak leachates. Some of the responses were also species‐specific, suggesting that some species are likely to be more sensitive than others to novel chemicals. We conclude that the poor quality of eucalypt leaf litter (e.g., its low nutrient content), low amount of food (e.g., periphyton), and the presence of toxic detritus and leachates can have important stress‐related consequences for the critical early stages of development in anurans.

Microplastics increase susceptibility of amphibian larvae to the chytrid fungus Batrachochytrium dendrobatidis

Microplastics (MPs), a new class of pollutants that pose a threat to aquatic biodiversity, are of increasing global concern. In tandem, the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causing the disease chytridiomycosis is emerging worldwide as a major stressor to amphibians. We here assess whether synergies exist between this infectious disease and MP pollution by mimicking natural contact of a highly susceptible species (midwife toads, Alytes obstetricans) with a Bd-infected reservoir species (fire salamanders, Salamandra salamandra) in the presence and absence of MPs. We found that MP ingestion increases the burden of infection by Bd in a dose-dependent manner. However, MPs accumulated to a greater extent in amphibians that were not exposed to Bd, likely due to Bd-damaged tadpole mouthparts interfering with MP ingestion. Our experimental approach showed compelling interactions between two emergent processes, chytridiomycosis and MP pollution, necessitating further research into potential synergies between these biotic and abiotic threats to amphibians.

Phylogeographic advances in midwife toads ( Alytes ) support the existence of a novel taxon endemic to the Central Pyrenees

Although necessary to promote conservation, defining evolutionary units and naming biodiversity remain a difficult task, especially in problematic species groups that experienced a dynamic biogeographic history. In this article, we undertake such task for midwife toads of the Alytes obstetricans complex by integrating recent molecular studies altogether—multilocus phylogenies and population genetic barcoding. Despite a partly unresolved phylogeny underlain by deep cyto-nuclear discordances, nuclear and mitochondrial evidence support the validity of six genuine lineages assigned to two different species (A. obstetricans and A. almogavarii), which could be accurately mapped across most of their ranges. In particular, we demonstrate the existence for an overlooked yet genetically distinct lineage previously confounded with A. almogavarii, confined to the northern part of Huesca Province in the Spanish Central Pyrenees. We describe this micro-endemic as the subspecies Alytes almogavarii inigoi ssp. nov., with reports on the mating call and the larvae. Conservation genetics of eight populations of this new taxon revealed two independent conservation units, separated by topographic barriers. In the wait for upcoming genomic analyses to unravel many elusive aspects of the evolution, diversity and systematics of Alytes, the present paper offers an integrative phylogeographic overview to guide future investigations and generally illustrates how multiple lines of molecular evidence can be combined to clarify the confusing taxonomy of complex species groups.