Iberian Ribbed Newt Research Articles

Comparison of hypoxia- and hyperoxia-induced alteration of epigene expression pattern in lungs of Pleurodeles waltl and Mus musculus.

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.

Relationship between two pathogens in an amphibian community that experienced mass mortalities.

Because host species tend to harbor multiple parasitic species, coinfection in a host is common. The chytrid fungus Batrachochytrium dendrobatidis (Bd) and the viruses in the genus Ranavirus (Rv) are responsible for the decline of amphibians worldwide. Despite wide geographical co-occurrence and the serious conservation problem that coinfection with these pathogens could represent, little is known about their possible synergistic interactions and effects in a host community. We investigated the occurrence and associations between these two pathogens in an amphibian community after Rv-driven disease outbreaks were detected in four populations of the Iberian ribbed newt (Pleurodeles waltl) in northwestern Spain. We collected tissue samples from amphibians and fish and estimated Bd and Rv infection loads by qPCR. A few months after the most recent mass mortality event, Rv infection parameters at the affected sites decreased significantly or were lower than such registered at the sites where no outbreaks were recorded. Both pathogens were simultaneously present in almost all sites, but coinfection in a single host was rare. Our findings suggest that the co-occurrence of Bd and Rv does not predict adverse outcomes (e.g., enhanced susceptibility of hosts to one pathogen due to the presence or infection intensity of the other) following an outbreak. Other variables (such as species identity or site) were more important than infection with a pathogen in predicting the infection status and severity of infection with the other pathogen. Our results highlight the importance of host-specific and environmental characteristics in the dynamics of infections, coinfection patterns, and their impacts.

Amphibian newts as experimental models for studying weight gain after castration.

Vertebrate animals often exhibit sexual dimorphism in body shape. In mammals, decreases in sex hormones caused by testicular castration can affect body shape and occasionally lead to pathologies such as obesity. Post-castration obesity can also be problematic for the health of companion animals, including non-mammals. In order to understand the mechanism of post-castration obesity in vertebrates other than mammals, experimental models are required. We examined whether the Iberian ribbed newt, which has recently become a popular experimental model for amphibian research, could serve as a model for analyzing changes in body shape after castration. In newts, new testes can be regenerated after removal of differentiated testes. We analyzed changes in body shape by removing the testes under conditions in which they could regenerate or conditions in which they could not regenerate. Removal of the testes reduced blood testosterone levels. The body weight and abdominal girth of the newts were increased compared with normal male newts. Transcriptome analysis of the liver showed that a set of genes related to lipid metabolism was continuously up-regulated in castrated newts. Our study suggests that changes in body shape after castration are common in vertebrates. Iberian ribbed newts are thus a suitable model for comparative studies of the long-term physiologic- and endocrine-level effects of castration.

Development of an AI-Assisted Embryo Selection System Using Iberian Ribbed Newts for Embryo-Fetal Development Toxicity Testing.

The 3Rs (Reduction, Refinement, Replacement) principle is driving the need for alternative methods in animal testing. Despite advancements in in vitro testing, complex systemic toxicity tests still necessitate in vivo approaches. The aim of this study was to develop a developmental toxicity test protocol using the Iberian ribbed newt (Pleurodeles waltl) as a model organism, integrating AI image analysis for embryo selection to improve test accuracy and reproducibility. We established a developmental toxicity test protocol based on the zebrafish test. Gonadotropin was administered to induce ovulation, and in vitro fertilization was performed. Embryos were imaged at 5-6 and 6-7 h post-fertilization. AI image analysis was utilized to assess embryo viability. The test chemical was administered 24-48 h post-fertilization, and morphological changes were observed daily until day 8. Additionally, a time-lapse photography system was constructed to monitor embryonic development. Out of 24 cultured embryos, 75% developed normally to the late tail bud stage or initial hatching stage, whereas 25% experienced developmental arrest or death. AI image analysis achieved high accuracy in classifying embryos, with overall accuracies of 92.0% and 92.9% for two learning models. The AI system demonstrated higher precision in the selection of viable embryos compared to visual inspection. The Iberian ribbed newt presents a viable alternative model for developmental toxicity testing, adhering to the 3Rs principles. The integration of AI image analysis substantially enhances the accuracy and reproducibility of embryo selection, providing a reliable method for evaluating developmental toxicity in pharmaceuticals.

Biomechanical analysis of tendon regeneration capacity of Iberian ribbed newts following transection injury: Comparison to a mouse model.

Adult mammals are known for their poor ability to regenerate tissues, including tendons. On the other hand, urodeles have become an important model in regenerative studies for their remarkable ability to regenerate various body parts and organs throughout life, such as limbs, retinas, or even the brain. However, little is known about their capacity to regenerate injured tendons. If newts can also repair tendons without scar formation, they may be a suitable animal model for tendon regeneration studies in other adult vertebrates. Therefore, the present study used Iberian ribbed newts to characterize mechanical and structural regeneration of tendons following transection, using tensile tests and multiphoton microscopy. A digital flexor tendon in a hindlimb was transected either partially or completely, and regenerated tendon was examined 6 and 12 weeks after the operation. Tensile strength of regenerated tendons was significantly less than normal at 6 weeks, but was remarkably recovered at 12 weeks, reaching levels comparable to those of uninjured tendons. On the other hand, mouse tendons demonstrated poor recovery of strength even after 12 weeks. Multiphoton microscopy revealed that tendon-like collagenous tissue bridges residual tendon stubs in newts, but disorganized scar-like tissue filled the injured location in mice. These findings highlight the remarkable capacity of newts to recover from tendon injury and confirm the utility of newts as a model to study tendon regeneration.

Structural Mechanism of Tendon Regeneration in Iberian Ribbed Newt

Structural Mechanism of Tendon Regeneration in Iberian Ribbed Newt

Contextualising the bizarre: The integrated functioning of rib puncture as an antipredator defence in the Iberian ribbed newt (Pleurodeles waltl)

Abstract Numerous prey traits are the result of the pervasive evolutionary pressure imposed by predators. Mirroring their fundamental role in the fitness of prey, some antipredator defences can evolve into elaborate strategies. Such is the case of the Iberian ribbed newt (Pleurodeles waltl), a semiaquatic urodele that inhabits multiple types of inland waterbodies in central and southern Iberian Peninsula and north‐western Africa. It possesses sharp, protractile ribs, which can thwart predators by emerging through skin ‘warts’ they pierce, becoming impregnated with the toxicants these warts produce, and making the newts difficult to swallow. Plus, the orange colour of these warts contrasts with the predominantly dull dorsum. In this work, I studied the interactions between the number of these warts, the saliency of wart colour against dorsum colour of each individual (i.e., the difference in colour between the warts and the dorsum), rib response time (i.e., the time needed for the ribs to emerge in response to a standardised stimulus), body size, and locomotor performance in males and females in either their aquatic or terrestrial stage. I also conducted a field experiment to determine whether predators more frequently attacked plasticine models with conspicuous or inconspicuous warts. I found that wart colour saliency functions as a decoy, attracting predator attacks towards this well‐protected body part. Plus, many of the antipredator defences studied, namely the number of warts, their colour saliency, rib response time, body size, and locomotor performance, were interrelated. Accordingly, more salient warts indicated a faster rib response, suggesting that these defences are coordinated with each other. Plus, locomotion was considerably slower in the terrestrial than in the aquatic stage. Moreover, warts were more salient, and thus more efficient attracting predator attacks, in newts in the slower terrestrial stage. However, rib response was faster in the aquatic stage, which coincides with the newt's activity peak, and therefore with the height of its conspicuousness to predators. Remarkably, faster newts display less salient warts and a slower rib response, which suggests a trade‐off between both defences. Nonetheless, some of these trends are reversed in females, among which faster individuals also display a faster rib response. This could be a likely consequence of reduced predation pressure. These results further the understanding of how prey integrate and tune multiple antipredator defences according to factors that influence predation pressure, such as sex and life stage.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Cryo-injury procedure-induced cardiac regeneration shows unique gene expression profiles in the newt Pleurodeles waltl.

Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo-injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo-injury procedures are common in mouse and zebrafish. In the present study, we induced cryo-damage on the ventricle in Iberian ribbed newts using a liquid nitrogen-chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo-injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures. Notably, we established a cryo-injury procedure for the newt and confirmed that regeneration of the cryo-damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo-injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models.

Photoperiod-independent testicular development in the model newt Pleurodeles waltl.

Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P.waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P.waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P.waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P.waltl proceeds irrespective of day length. Our results show that P.waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P.waltl bioresources.

Are Iberian ribbed newts Pleurodeles waltl Michahelles, 1830 negatively affecting the populations of other amphibians?

AbstractTemporary ponds play a key role as amphibian breeding habitats, but predation and desiccation risks enforce major evolutionary pressures in these ecosystems. The newt Pleurodeles waltl is commonly found in Mediterranean ponds in the Iberian Peninsula, where it preys on a wide variety of organisms. We hypothesize that P. waltl may negatively influence populations of coexisting amphibian larvae in the natural environment. To test it, we monitored the amphibian populations of 21 temporary ponds in Eastern Spain, 10 of which included P. waltl. We visited each pond fortnightly on nine occasions, estimating the abundance of adult P. waltl and tadpoles of each amphibian species present. We built zero‐inflated Generalized Linear Mixed Models (GLMMs) to test for the effects of pond area and adult and larval P. waltl abundance on tadpole abundance of all amphibian species present. Our results show significant negative effects of P. waltl abundances on the anurans Alytes obstetricans, Epidalea calamita, and Pelodytes punctatus. Species adapted to shorter hydroperiods such E. calamita and P. punctatus show lower densities in the presence of P. waltl larvae. A. obstetricans, with preference for longer hydroperiod ponds, seems to better thrive in sites where the newt is absent. This is the first field work suggesting a negative impact of P. waltl on coexisting anurans, even though more studies are needed to disentangle other negative effects related to unmeasured environmental variables or biotic interactions, which can be important according to the large variation encompassed by the random variables of site and sampling period in the mixed models.

Altered metabolic state impedes limb regeneration in salamanders.

Salamanders are unique among tetrapods in their ability to regenerate the limbs throughout life. Like other poikilothermic amphibians, salamanders also show a remarkable capacity to survive long periods of starvation. Whether the physiological reserves necessary for tissue regeneration are preserved or sacrificed in starved salamanders is unknown. In the current study, we maintained Iberian ribbed newts (Pleurodeles waltl) under extreme physiological stress to assess the extent of regeneration and identify the molecular and cellular changes that may occur under such conditions. After 19 months of complete food deprivation, the animals exhibited extensive morphological and physiological adaptations but remained behaviorally active and vigilant. Autophagy was elevated in different tissues and the transformed gut microbiota indicated remodeling of the intestinal tract related to autophagy. Upon limb amputation in animals starved for 21 months, regeneration proceeded with progenitor cell proliferation and migration, leading to limb blastema formation. However, limb outgrowth and patterning were substantially attenuated. Blockage of autophagy inhibited cell proliferation and blastema formation in starved animals, but not in fed animals. Hence, tissue autophagy and the regenerative response were tightly coupled only when animals were under stress. Our results demonstrate that under adverse conditions, salamanders can exploit alternative strategies to secure blastema formation for limb regeneration.

Immune challenge does not impair short-distance escape speed in a newt

Animals may face trade-offs when several simultaneous functions or processes compete for limiting resources. In that context, allocation of resources to the most fitness-enhancing competing function can be prioritized. Immune response and locomotor performance are relevant fitness components, mainly due to their important roles as defence against pathogens and predators, respectively. However, both promote oxidative stress and consume energy and metabolites. Consistently, trade-offs between immune response and locomotion have been reported in some taxa. In this work, we examine whether an immune challenge hinders short-distance swimming speed of adult Iberian ribbed newts, Pleurodeles waltl. To that end, we inoculated Escherichia coli cell wall lipopolysaccharide (LPS), an innocuous antigen that triggers an immune response, in the sole pads of experimental newts, and phosphate buffer saline, with no physiological effect, in those of control newts. We estimated immune response as sole pad swelling and calculated swimming speed from videos of newts swimming towards a refuge nearby, 24 h and 7 days after inoculation. Sole pad swelling was greater in LPS-inoculated newts, but short-distance swimming speed was not affected by the immune challenge. These results could indicate priority allocation of resources to locomotion, which might be a consequence of compensatory mechanisms capable of avoiding locomotion impairment due to immune challenge. In any case, these findings could highlight the importance of short-distance escape as an antipredator strategy.

Larval newts adjust foraging rate to perceived predator and competitor proximity

Foraging is key for most animals, because their energy and matter income depend on food ingestion. However, animals may respond adaptively to perceived competitor and/or predator proximity by modifying their foraging rates. Increased foraging activity is expected to improve the chances of succeeding in competition. However, the benefits of increased foraging activity could be offset by the risks of increased conspicuousness to predators. Plus, starvation may have counteracting or additive effects on foraging behaviour. Here, we test these hypotheses in larval Iberian ribbed newts (Pleurodeles waltl). We estimated foraging rates of individuals haphazardly assigned to either a restricted or an unrestricted-feeding regime, in water with (1) self-odour as control, (2) predator Dytiscus sp. chemical cues, and (3) competitor conspecific larva chemical cues. Treatments were presented in a random sequence. Newt larvae responded to either predator or conspecific chemical cues by increasing foraging rates relative to the control. Conspecific chemical cues increased foraging rates over the other two treatments. Feeding regime did not modify larva response to perceived predator and competitor proximity. Increased foraging rates in the presence of conspecific chemical cues could simultaneously improve larval energetic status to face competition and reduce prey available for the competitor. Intermediate foraging rates in the presence of predator chemical cues suggest a trade-off between the benefits (likely improved energetic status or accelerated metamorphosis) and the costs (likely increased conspicuousness) of increased food ingestion. These results prove that larval newts adjust foraging rates to perceived predator and competitor proximity.

Unique high sensitivity to heat of axolotl TRPV1 revealed by the heterologous expression system

The thermosensation mechanism plays critical roles in various animals living in different thermal environment. We focused on an axolotl, which is a tailed amphibian originally from Lake Xochimilco area in the Vally of Mexico, and examined its behavior response to heat stimulation. Mild heat at 33 °C induced noxious locomotive activity to axolotls, but the noxious response of another tailed amphibian, Iberian ribbed newt, was not observed at 33 °C. To explore the mechanism for the temperature sensitivity of axolotls, we isolated a cDNA of TRPV1. Using the degenerate primer PCR method, we identified the DNA fragment encoding axolotl TRPV1 (axTRPV1), and then cloned a full-length cDNA. We studied the chemical and thermal sensitivities of axTRPV1 by two-electrode voltage clamp method using Xenopus oocyte expression system. Capsaicin, acid, and 2-aminoethoxydiphenylborane apparently activated axTRPV1 channels in a dose-dependent manner. The analysis of thermal sensitivity showed that axTRPV1 was significantly activated by heat but not by cold. The average temperature threshold for heat-activation was 30.95 ± 0.12 °C. This thermal activation threshold of axTRPV1 is unique and significantly low, when compared with the known thresholds of TRPV1s from various animals. Further, this threshold of axTRPV1 is well consistent with the observation of heat-induced behavior of axolotls at 33 °C, demonstrating that axolotl shows noxious response to mild heat mediated through axTRPV1.

A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology.

Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).

Modulation of Iberian Ribbed Newt Complement Component C3 by Stressors Similar to those Encountered during a Stay Onboard the International Space Station.

The complement system plays an important role in inflammation, innate and acquired immunity, as well as homeostasis. Despite these functions, the effects of spaceflight conditions on the complement system have not yet been intensively studied. Consequently, we investigated the effects of five types of chronic stressors, similar to those encountered during a stay onboard the International Space Station, on C3 expression in larvae of the urodele amphibian Pleurodeles waltl. We focused on C3 because it is a critical component of this system. These studies were completed by the analysis of adult mice exposed to two models of inflight stressors. Our data show that simulating space radiation, or combining a modification of the circadian rhythm with simulated microgravity, affects the amount of C3 proteins. These results suggest that C3 expression could be modified under real spaceflight conditions, potentially increasing the risk of inflammation and associated tissue damage.

Does predation risk outweigh the costs of lost feeding opportunities or does it generate a behavioural trade-off? A case study with Iberian ribbed newt larvae

PA was supported by a ‘Ramon y Cajal’ contract (RYC-2011–07670, MINECO). This study was partly funded by the project CGL2014-56416-P (MINECO).

Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeleswaltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.

Integrative inference of population history in the Ibero-Maghrebian endemic Pleurodeles waltl (Salamandridae)

Inference of population histories from the molecular signatures of past demographic processes is challenging, but recent methodological advances in species distribution models and their integration in time-calibrated phylogeographic studies allow detailed reconstruction of complex biogeographic scenarios. We apply an integrative approach to infer the evolutionary history of the Iberian ribbed newt (Pleurodeles waltl), an Ibero-Maghrebian endemic with populations north and south of the Strait of Gibraltar. We analyzed an extensive multilocus dataset (mitochondrial and nuclear DNA sequences and ten polymorphic microsatellite loci) and found a deep east-west phylogeographic break in Iberian populations dating back to the Plio-Pleistocene. This break is inferred to result from vicariance associated with the formation of the Guadalquivir river basin. In contrast with previous studies, North African populations showed exclusive mtDNA haplotypes, and formed a monophyletic clade within the Eastern Iberian lineage in the mtDNA genealogy. On the other hand, microsatellites failed to recover Moroccan populations as a differentiated genetic cluster. This is interpreted to result from post-divergence gene flow based on the results of IMA2 and Migrate analyses. Thus, Moroccan populations would have originated after overseas dispersal from the Iberian Peninsula in the Pleistocene, with subsequent gene flow in more recent times, implying at least two trans-marine dispersal events. We modeled the distribution of the species and of each lineage, and projected these models back in time to infer climatically favourable areas during the mid-Holocene, the last glacial maximum (LGM) and the last interglacial (LIG), to reconstruct more recent population dynamics. We found minor differences in climatic favourability across lineages, suggesting intraspecific niche conservatism. Genetic diversity was significantly correlated with the intersection of environmental favourability in the LIG and LGM, indicating that populations of P. waltl are genetically more diverse in regions that have remained environmentally favourable through the last glacial cycle, particularly southern Iberia and northern Morocco. This study provides novel insights into the relative roles of geology and climate on the biogeography of a biodiversity hotspot.