Alytes Research Articles

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.

Alien amphibian introductions via the plant trade: a breeding population of the Catalonian midwife toad (Alytes almogavarii) in Central Spain

Abstract The plant-trade is among the main sources of accidental introduction of alien biodiversity. This includes amphibians, although effective colonization pathways have rarely been reported except from tropical areas. We document the discovery of an established breeding population of the Catalonian midwife toad, Alytes almogavarii almogavarii Arntzen and García-París, 1995, in a plant nursery in Madrid (Central Spain), 400 km away from its natural distribution. During surveys in 2023 we found adults, juveniles, and tadpoles, confirming reproduction. We used mtDNA sequences to genotype six individuals for taxonomic assignment and tracing their putative origin. Two haplotypes were found, one of them showing a broad distribution, precluding precise identification of the source of the introduction. We discuss conservation concerns, including the potential spread of pathogens. Our results suggest unintentional amphibian introductions via the plant-trade may be more frequent than perceived, and call for rigorous monitoring and education efforts.

Ontogenetic and cohort estimates of tadpole survival for the threatened Betic midwife toad (Alytes dickhilleni) in two contrasting small waterbodies

Abstract Comprehensive knowledge on demographic parameters and early life history traits is essential for an effective amphibian management and conservation. Here, we assessed tadpole survival of Alytes dickhilleni in two contrasting small waterbodies (natural pool vs man-made drinking trough). For the first time in the genus Alytes, tadpole survival was quantified at ontogenetic and cohort level. Overall, low survival values were reported for both study sites, being values in the natural pool (0.16) twice than in the man-made drinking trough (0.07). Ontogenetic and cohort variation was congruent between both study sites, with survival rates decreasing in intermediate developmental stages, and highest values being observed in overwintering as compared to summer tadpoles. Owing to the ongoing decline in Alytes populations, these results can be particularly useful for informing future conservation schemes based on tadpole translocation or reintroduction from wild populations.

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.

Genomic phylogeography illuminates deep cyto-nuclear discordances in midwife toads (Alytes)

The advent of genomic methods allows us to revisit the evolutionary history of organismal groups for which robust phylogenies are still lacking, particularly in species complexes that frequently hybridize. In this study, we conduct RAD-sequencing (RAD-seq) analyses of midwife toads (genus Alytes), an iconic group of western Mediterranean amphibians famous for their parental care behavior, but equally infamous for the difficulties to reconstruct their evolutionary history. Through admixture and phylogenetic analyses of thousands of loci, we provide a comprehensive phylogeographic framework for the A. obstetricans complex, as well as a fully resolved phylogeny for the entire genus. As part of this effort, we carefully explore the influence of different sampling schemes and data filtering thresholds on tree reconstruction, showing that several, slightly different, yet robust topologies may be retrieved with small datasets obtained by stringent SNP calling parameters, especially when admixed individuals are included. In contrast, analyses of incomplete but larger datasets converged on the same phylogeny, irrespective of the reconstruction method used or the proportion of missing data. The Alytes tree features three Miocene-diverged clades corresponding to the proposed subgenera Ammoryctis (A. cisternasii), Baleaphryne (A. maurus, A. dickhilleni and A. muletensis), and Alytes (A. obstetricans complex). The latter consists of six evolutionary lineages, grouped into three clades of Pliocene origin, and currently delimited as two species: (1) A. almogavarii almogavarii and A. a. inigoi; (2) A. obstetricans obstetricans and A. o. pertinax; (3) A. o. boscai and an undescribed taxon (A. o. cf. boscai). These results contradict the mitochondrial tree, due to past mitochondrial captures in A. a. almogavarii (central Pyrenees) and A. o. boscai (central Iberia) by A. obstetricans ancestors during the Pleistocene. Patterns of admixture between subspecies appear far more extensive than previously assumed from microsatellites, causing nomenclatural uncertainties, and even underlying the reticulate evolution of one taxon (A. o. pertinax). All Ammoryctis and Baleaphryne species form shallow clades, so their taxonomy should remain stable. Amid the prevalence of cyto-nuclear discordance among terrestrial vertebrates and the usual lack of resolution of conventional nuclear markers, our study advocates for phylogeography based on next-generation sequencing, but also encourages properly exploring parameter space and sampling schemes when building and analyzing genomic datasets.

Richard Semon (1859-1918): expeditions, engrams and epigenetics

Richard Semon (1859-1918) was astudent of Ernst Haeckel and began his career as azoologist with work on sea urchins, starfish, chicken and lung fish, which he collected at the Mediterranean Sea and in Australia. After his return to Germany he was forced to leave Jena and the university due to private reasons, and settled in Munich, where Semon devoted most of his time to the more philosophical aspects of biology, developed the theory of "mneme" (1904), which he extended towards the inheritance of acquired characteristics (1912). Semon's concept of memory reached far beyond the brain and the individual person. In 1918 he took his life, despondent because of asurmised lack of scientific appreciation, the death of his beloved wife, the political turmoil at the end of WWI, and his-the memory researcher's-suspected loss of memory. Eight years later, the experimental biologist Paul Kammerer (1880-1926) from Vienna, Semon's must trusted source for the inheritance of acquired characteristics, also shot himself. Serious doubts increasingly overshadowed his work on salamanders and midwife toads. Epigenetics, the nature of memory, the fear of cognitive impairment, depression, the impact of private and political matters on scientific work, suspected scientific errors, fraud and ascientists' suicides are condensed in Semon's life and death.

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.

New insights on patterns of genetic admixture and phylogeographic history in Iberian high mountain populations of midwife toads.

Multiple Quaternary glacial refugia in the Iberian Peninsula, commonly known as "refugia within refugia", allowed diverging populations to come into contact and admix, potentially boosting substantial mito-nuclear discordances. In this study, we employ a comprehensive set of mitochondrial and nuclear markers to shed light onto the drivers of geographical differentiation in Iberian high mountain populations of the midwife toads Alytes obstetricans and A. almogavarii from the Pyrenees, Picos de Europa and Guadarrama Mountains. In the three analysed mountain regions, we detected evidence of extensive mito-nuclear discordances and/or admixture between taxa. Clustering analyses identified three major divergent lineages in the Pyrenees (corresponding to the eastern, central and central-western Pyrenees), which possibly recurrently expanded and admixed during the succession of glacial-interglacial periods that characterised the Late Pleistocene, and that currently follow a ring-shaped diversification pattern. On the other hand, populations from the Picos de Europa mountains (NW Iberian Peninsula) showed a mitochondrial affinity to central-western Pyrenean populations and a nuclear affinity to populations from the central Iberian Peninsula, suggesting a likely admixed origin for Picos de Europa populations. Finally, populations from the Guadarrama Mountain Range (central Iberian Peninsula) were depleted of genetic diversity, possibly as a consequence of a recent epidemic of chytridiomycosis. This work highlights the complex evolutionary history that shaped the current genetic composition of high mountain populations, and underscores the importance of using a multilocus approach to better infer the dynamics of population divergence.

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.

New insights on patterns of genetic admixture and phylogeographic history in Iberian high mountain populations of midwife toads

New insights on patterns of genetic admixture and phylogeographic history in Iberian high mountain populations of midwife toads

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

Benefits of paternal thermoregulation: male midwife toads select warmer temperature to shorten embryonic development

Benefits of paternal thermoregulation: male midwife toads select warmer temperature to shorten embryonic development

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