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Molecular tools have revolutionized assessments of blood parasites in freshwater turtles. In the Iberian Peninsula and North Africa, two native species of terrapins occur, Emys orbicularis (Linnaeus) and Mauremys leprosa (Schweigger). Both have been identified as hosts for the blood parasite Haemogregarina stepanowi Danilewsky, 1885, which has also been found in related species. However, recent assessments of M. leprosa have identified several distinct genetic lineages of these parasites in this host, while only three haemogregarine lineages were identified in E. orbicularis in Tunisia. Here, we screened 215 individuals of E. orbicularis from the Iberian Peninsula, Menorca Island and Morocco for haemogregarine parasites using partial 18S rRNA gene sequences to estimate relationships. Three unrelated lineages of parasites were detected, one presumed H. stepanowi and two lineages previously known from M. leprosa. A considerable undescribed diversity of parasites exists within these vertebrate host species, while mixed infection and host-sharing is also widespread. Considering that E. orbicularis is near threatened in this region, it is of great importance to identify the parasites infecting it, and to further assess the potential deleterious impact of these diverse parasites on their hosts. © 2022. The Author(s), under exclusive licence to Springer Nature B.V.

Adaptive genetic diversity is a key factor in conservation planning as it relates to the evolutionary potential of populations and their responses to environmental change. Developments in landscape genomics have fostered a proliferation of tests for selection that aim to identify candidate adaptive markers in natural populations. However, these tests rely on diferent assumptions and may produce contrasting results. Here we applied six tests for selection in a range-wide genomic scan of an Iberian amphibian, Pelobates cultripes, which shows spatially structured genetic variation across its range, encompassing diferent bioclimatic zones. As a cursory scan for selection, one test identifed candidate markers that describe a pattern of putatively adaptive genetic variation, highlighting coastal Atlantic localities as exhibiting putatively non-neutral patterns of genetic variation. While generalized genomic scans for selection in non-model species have limitations, exploratory searches can highlight areas to guide subsequent hypothesis-driven studies and conservation eforts., This work was developed under the project PTDC/BIA-BIC/3545/2014, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). AJDM, JOH, MCS, SBC, PT, GVA and AV were funded through national funds from Fundação para a Ciência e Tecnologia (CEECIND/01464/2017, ICETA/EEC2018/16, DL57/2016/CP1440/CT0008, CEECIND/00937/2018, and DL57/2016, respectively).

Investigation of the ecological and evolutionary mechanisms governing the origin and diversification of species requires integrative approaches that often have to accommodate strong discordance among datasets. A common source of conflict is the combination of morphological and molecular characters with different evolutionary rates. Resolution of these discordances is crucial to assess the relative roles of different processes in generating and maintaining biodiversity. Anuran amphibians provide many examples of morphologically similar, genetically divergent lineages, posing questions about the relative roles of phylogeny and ecological factors in phenotypic evolution. We focused on three circum-Mediterranean anuran genera (Hyla, Alytes and Discoglossus), characterizing morphological and environmental disparity and comparing diversity patterns across biological levels of organization. Using a comparative phylogenetic framework, we tested how shared ancestry and climatic factors come together to shape phenotypic diversity. We found higher morphological differentiation within Hyla and Alytes than in Discoglossus. Body size and limb morphology contributed most to inter- and intraspecific morphological variation in Hyla and Alytes, but there was no strong phylogenetic signal, indicating that shared ancestry does not predict patterns of phenotypic divergence. In contrast, we uncovered a significant association between morphology and climatic descriptors, supporting the hypothesis that morphological disparity between species results from adaptive evolution., This work was supported by FEDER Funds through the Operational Competitiveness Factors Program (COMPETE) and National Funds through the Foundation for Science and Technology (FCT) within the scope of the project ‘PTDC/BIA-EVL/28090/2017-POCI-01-0145-FEDER-028090’. Museum data collection was supported by four SYNTHESYS grants of the European Union to F.M.-F. (ES-TAF-5874, GB-TAF-5886, CZ-TAF-6627 and FR-TAF-6626). F.M.-F. and G.V.-A. are financed by the FCT (DL57/2016/CP1440/ CT0010 and IF/01425/2014—CEECIND/00937/2018, respectively). U.E.-U. is supported financially by the Institute of Vertebrate Biology of the Czech Academy of Sciences (RVO: 68081766). A.K. is supported by a Ramón y Cajal research grant co-funded by the Spanish State Research Agency and the European Social Fund (RYC2019-026688-I/AEI/10.13039/501100011033).

Multiple paternity is widespread in nature and despite costs, it has many associated benefits like increased genetic diversity and fertilization success. It has been described in many viviparous systems, suggesting the existence of some fitness advantages counteracting the inherent costs of viviparity, such as fecundity reduction and high parental investment. Reproductively polymorphic species, like the urodele Salamandra algira, which shows two types of viviparity: larviparity (i.e., delivering aquatic larvae), and pueriparity (i.e., delivering terrestrial metamorphosed juveniles), are suitable systems to study the relationship between reproductive modes and polygamous mating. Here, multiple paternity is confirmed in a pueriparous lineage of S. algira, as previously verified for the pueriparous lineages of the reproductively polymorphic species S. salamandra, suggesting polyandry is a successful mating strategy in pueriparous systems with reduced brood sizes. We discuss the potential benefits of polyandry in the context of viviparity evolution in urodeles.

Amphibians are subjected to an assortment of environmental stressors responsible for their population declines and malformations. Deciphering the underlying causes of amphibian deformities is challenging due to the complex nature and interplay among factors. We evaluated morphological deformities in 9 urban and 9 woodland populations of terrestrial-breeding fire salamanders. We report several types of malformations and higher incidences among urban populations. This model system allowed us to tease apart some of the common factors responsible for amphibian deformations, suggesting airborne/terrestrial pollutants, predation, and/or inbreeding as potential environmental stressors. Yet, the putative underlying factors of fire salamander deformities need to be properly addressed in thorough studies linking habitat quality and the prevalence of morphological abnormalities, as well as predator-prey interactions. Reporting deformation rates among amphibians is key to identify warning signals of population declines and preventing local extinctions.

The riverine barrier hypothesis (RBH) posits that rivers comprise geographical barriers to gene flow for terrestrial organisms, thus promoting genetic differentiation between populations. Here, we explored the RBH on larviparous and pueriparous populations of the live-bearing fire salamander (Salamandra salamandra). While larviparous fire salamanders exhibit a semi-aquatic life cycle (females deposit pre-metamorphic larvae on water), pueriparous salamanders present a fully terrestrial life cycle (females deliver terrestrial juveniles) and, therefore, a greater independence from water for survival and reproduction. We performed a fine-scale sampling of opposite transects in 11 rivers (six and five for larviparous and pueriparous populations, respectively) to test the hypothesis that rivers are more effective barriers for pueriparous salamanders due to their terrestrial life cycle. We carried out individual- and population-based genetic analyses using 14 microsatellites and a mitochondrial marker to examine the extent to which rivers hinder short- and long-term gene flow. We found that rivers are semi-permeable obstacles for both larviparous and pueriparous salamanders, although they appear to be more effective barriers for the latter when rivers with similar attributes are compared. We also found that river width and possibly the presence of crossing structures may influence the genetic barrier effects of rivers in fire salamanders. This is one of the very few studies in amphibians showing how different reproductive strategies influence the barrier effects imposed by rivers.

Explicitly accounting for phenotypic differentiation together with environmental heterogeneity is crucial to understand the evolutionary dynamics in hybrid zones. Species showing intra-specific variation in phenotypic traits that meet across environmentally heterogeneous regions constitute excellent natural settings to study the role of phenotypic differentiation and environmental factors in shaping the spatial extent and patterns of admixture in hybrid zones. We studied three environmentally distinct contact zones where morphologically and reproductively divergent subspecies of Salamandra salamandra co-occur: the pueriparous S. s. bernardezi that is mostly parapatric to its three larviparous subspecies neighbours. We used a landscape genetics framework to: (i) characterise the spatial location and extent of each contact zone; (ii) assess patterns of introgression and hybridization between subspecies pairs; and (iii) examine the role of environmental heterogeneity in the evolutionary dynamics of hybrid zones. We found high levels of introgression between parity modes, and between distinct phenotypes, thus demonstrating the evolution to pueriparity alone or morphological differentiation do not lead to reproductive isolation between these highly divergent S. salamandra morphotypes. However, we detected substantial variation in patterns of hybridization across contact zones, being lower in the contact zone located on a topographically complex area. We highlight the importance of accounting for spatial environmental heterogeneity when studying evolutionary dynamics of hybrid zones., This work was supported by National Funds through FCT – Foundation for Science and Technology (SALOMICS: PTDC/BIA-EVL/28475/2017); the authors also acknowledge funding from other FCT projects and FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE (EVOVIV: PTDC/BIA-EVF/3036/2012; FCOMP-01-0124-FEDER-028325 and UIDB/500027/2020); and by National/FEDER funds through Ministerio de Economía y Competitividad (CGL2012-40246-C02-02). G.V.-A., A.L. and P.T. were supported by FCT (IF/01425/2014 and CEECIND/00937/2018, PD/BD/106060/2015, DL57/2016/CP1440/CT0008, respectively).

Understanding the ecological and evolutionary processes driving biodiversity patterns and allowing their persistence is of utmost importance. Many hypotheses have been proposed to explain spatial diversity patterns, including water-energy availability, habitat heterogeneity, and historical climatic refugia. The main goal of this study is to identify if general spatial drivers of species diversity patterns of phylogenetic diversity (PD) and phylogenetic endemism (PE) at the global scale are also predictive of PD and PE at regional scales, using Iberian amphibians as a case study. Our main hypothesis assumes that topography along with contemporary and historical climate are drivers of phylogenetic diversity and endemism, but that the strength of these predictors may be weaker at the regional scale than it tends to be at the global scale. We mapped spatial patterns of Iberian amphibians' phylogenetic diversity and endemism, using previously published phylogenetic and distribution data. Furthermore, we compiled spatial data on topographic and climatic variables related to the water-energy availability, topography, and historical climatic instability hypotheses. To test our hypotheses, we used Spatial Autoregressive Models and selected the best model to explain diversity patterns based on Akaike Information Criterion. Our results show that, out of the variables tested in our study, water-energy availability and historical climate instability are the most important drivers of amphibian diversity in Iberia. However, as predicted, the strength of these predictors in our case study is weaker than it tends to be at global scales. Thus, additional drivers should also be investigated and we suggest caution when interpreting these predictors as surrogates for different components of diversity.

Reduced representation genome sequencing has popularized the application of single nucleotide polymorphisms (SNPs) to address evolutionary and conservation questions in nonmodel organisms. Patterns of genetic structure and diversity based on SNPs often diverge from those obtained with microsatellites to different degrees, but few studies have explicitly compared their performance under similar sampling regimes in a shared analytical framework. We compared range‐wide patterns of genetic structure and diversity in two amphibians endemic to the Iberian Peninsula: Hyla molleri and Pelobates cultripes, based on microsatellite (18 and 14 loci) and SNP (15,412 and 33,140 loci) datasets of comparable sample size and spatial extent. Model‐based clustering analyses with STRUCTURE revealed minor differences in genetic structure between marker types, but inconsistent values of the optimal number of populations (K) inferred. SNPs yielded more repeatable and less admixed ancestries with increasing K compared to microsatellites. Genetic diversity was weakly correlated between marker types, with SNPs providing a better representation of southern refugia and of gradients of genetic diversity congruent with the demographic history of both species. Our results suggest that the larger number of loci in a SNP dataset can provide more reliable inferences of patterns of genetic structure and diversity than a typical microsatellite dataset, at least at the spatial and temporal scales investigated., We compared patterns of genetic structure and diversity between microsatellites and SNPs in two amphibians from the Iberian Peninsula: Hyla molleri and Pelobates cultripes. Larger number of loci from SNP dataset can provide more reliable inferences of patterns of genetic structure and diversity than a typical microsatellite dataset (photo: Pelobates cultripes; credits: Guillermo Velo‐Antón).

Protected area systems should ideally maintain adaptive and neutral evolutionary processes. To achieve this, plans for expanding protected area systems (prioritizations) can improve coverage of related attributes (evolutionary attributes). However, long-standing challenges in mapping and operationalizing evolutionary attributes have prevented their widespread usage. We outline a novel framework for incorporating evolutionary processes into conservation planning. Using three amphibian species in the Iberian Peninsula (Hyla molleri, Pelobates cultripes and Rana iberica), we mapped a comprehensive range of adaptive and neutral evolutionary attributes to delineate places containing individuals with moderate to high heterozygosity, different neutral genetic clusters, different adaptive genetic clusters and climatic refugia. We overlaid these maps with boundaries of existing protected areas to quantify representation shortfalls and generated a prioritization to identify additional priority areas. To assess the performance of conventional approaches, we also generated a prioritization using only the species' distribution data—without the evolutionary attributes. We found that existing protected areas within the Iberian Peninsula are failing to adequately represent evolutionary attributes for the study species. Specifically, they are not adequately representing places predicted to contain individuals with moderate to high heterozygosity for any of the studied species, and neither are they adequately representing the species' potential climatic refugia. They also have poor coverage of the distinct adaptive and neutral genetic clusters that comprise each of the species' distributions. By incorporating the evolutionary attributes into the prioritization process, we identified priority areas that would address all of the shortfalls for only a minor increase in the size of the protected area system. In comparison, the prioritization generated following conventional approaches, despite encompassing a similar extent, did not substantially improve representation of the species' evolutionary attributes. Synthesis and applications. We introduce a framework for incorporating adaptive and neutral evolutionary processes into conservation planning. This framework can reveal weaknesses in the coverage of climatic refugia, genetic diversity and potential local adaptations by existing protected areas. Moreover, it can identify priority areas to improve conservation of evolutionary processes. Since neglecting evolutionary processes can impair conservation plans, we recommend using evolutionary data to inform decision-making where possible., This work was developed under the project PTDC/BIA-BIC/3545/2014, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Support by Portuguese National funds through Fundação para a Ciência e Tecnologia (FCT) was granted to AV (DL57/2016) and SBC (individual scientific employment program-contract CEECIND/01464/2017). GVA was supported by Investigador FCT contract (IF/01425/2014).

The global exponential growth in the number of exotic turtle species exploited in the pet trade market facilitates hybridization events between distantly related species. The 1997 EU trade ban on red-eared slider turtles (Trachemys scripta elegans), followed by the Spanish ban on all T. scripta subspecies in 2011, resulted in the importation of other chelonians (e.g. Graptemys spp., Pseudemys spp., Mauremys spp.), mainly from America and Asia to Spain. The importation of the Chinese stripe-necked pond turtle, Mauremys sinensis, via the pet trade market resulted in its release into natural environments across Europe, including the Iberian Peninsula where it co-occurs with the Western Mediterranean pond turtle, Mauremys leprosa. We maintained three M. leprosa females and two M. sinensis males in captivity, and analysed the obtained offspring (two hatchlings) with mitochondrial (cytochrome b) and nuclear (R35) genetic markers. Both morphological (i.e. with intermediate morphological characters) and genetic (heterozygous hatchlings) results confirmed the hybridization between these two species and raise concern about the negative effects of acclimatised Mauremys sinensis across the Mauremys leprosa range.

International audience; Aim: The biogeographic history of the Sahara-Sahel desert is tightly linked to its extreme and fluctuating palaeoclimate and diverse topography. For the mesic species inhabiting the region, coastal areas and the Nile Valley are perceived as the main pathways to disperse through desert habitats, but past connections may have also occurred throughout currently isolated mountain regions. Herein, we test the trans-Sahara mountain corridor hypothesis (i.e., mesic connectivity across Central Sahara highlands) and its role in the diversification of a small terrestrial vertebrate.Location: North Africa and Arabia.Taxon; Acanthodactylus boskianus (Squamata: Lacertidae).Methods: We integrated multi-locus mitochondrial and nuclear phylogenies with species’ climate-niche modelling, including palaeo-projections. Genetic analyses aimed to assess the species’ genetic structure, identify its main mitochondrial lineages and nuclear diversity, and reconstruct its ancestral biogeography. Species’ climate-niche stability was modelled independently for the Late Pleistocene-Holocene and the Plio-Pleistocene, to infer historical climatic refugia and dispersal corridors.Results: Four spatially structured mitochondrial lineages, integrating several parapatric sub-lineages, originated during the Plio-Pleistocene. Nuclear data revealed nine potential candidate species. Climatic refugia were located in mountains and desert fringes, remaining consistent for the Late Pleistocene-Holocene and the Plio-Pleistocene. Recurrent North-South climatic corridors were located along the desert periphery, while others less frequent were found across Central Sahara. Ancestral biogeography analyses recovered a recent Pleistocene colonization of the Sahel throughout eastern Sahara and either Sahara or Sahel origin for Central Sahara populations.Main conclusions: Species’ diversification was triggered by a combination of Plio-Pleistocene climatic cycles across a complex topographic region, where mountains acted as the main diversification hotspots. The historical role of Central Sahara highlands as main non-peripheral mesic refugia was corroborated. In addition, intermittent climatic connections linked Mediterranean and Sahel ecoregions with Central Sahara refugia, suggesting the existence of alternative trans-Sahara dispersal routes to the putative coastal and Nile corridors.

The recent introduction of the chytrid fungus Batrachochytrium salamandrivorans into north-eastern Spain threatens salamander diversity on the Iberian Peninsula. We assessed the current epidemiological situation with extensive field sampling of urodele populations. We then sought to delineate priority regions and identify conservation units for the Iberian Peninsula by estimating the susceptibility of Iberian urodeles using laboratory experiments, evidence from mortality events in nature and captivity and inference from phylogeny. None of the 1395 field samples, collected between 2015 and 2021 were positive for Bsal and no Bsal-associated mortality events were recorded, in contrast to the confirmed occurrence of Bsal outbreak previously described in 2018. We classified five of eleven Iberian urodele species as highly susceptible, predicting elevated mortality and population declines following potential Bsal emergence in the wild, five species as intermediately susceptible with variable disease outcomes and one species as resistant to disease and mortality. We identified the six conservation units (i.e., species or lineages within species) at highest risk and propose priority areas for active disease surveillance and field biosecurity measures. The magnitude of the disease threat identified here emphasizes the need for region-tailored disease abatement plans that couple active disease surveillance to rapid and drastic actions., This research was funded by the European Commission (Tender ENV.B.3/SER/2016/0028, Mitigating a new infectious disease in salamanders to counteract the loss of biodiversity), the Foundation for the Conservation of Salamanders (project “Monitoring the incidence of emerging pathogens on endemic urodeles from the Cantabrian range, Spain”). B.T. was supported by a ‘Doctorados Industriales’ grant of Comunidad de Madrid, Spain (ref. IND2020/AMB-17438). G.V.-A. was financed by the Portuguese Foundation for Science and Technology (FCT; CEECIND/00937/2018). B.C.S. was supported by an ARC DECRA (DE200100121).

Evolutionary changes in reproductive mode may affect co-evolving traits, such as dispersal, although this subject remains largely underexplored. The shift from aquatic oviparous or larviparous reproduction to terrestrial viviparous reproduction in some amphibians entails skipping the aquatic larval stage and, thus, greater independence from water. Accordingly, amphibians exhibiting terrestrial viviparous reproduction may potentially disperse across a wider variety of suboptimal habitats and increase population connectivity in fragmented landscapes compared to aquatic-breeding species. We investigated this hypothesis in the fire salamander (Salamandra salamandra), which exhibits both aquatic- (larviparity) and terrestrial-breeding (viviparity) strategies. We genotyped 426 larviparous and 360 viviparous adult salamanders for 13 microsatellite loci and sequenced a mitochondrial marker for 133 larviparous and 119 viviparous individuals to compare population connectivity and landscape resistance to gene flow within a landscape genetics framework. Contrary to our predictions, viviparous populations exhibited greater differentiation and reduced genetic connectivity compared to larviparous populations. Landscape genetic analyses indicate viviparity may be partially responsible for this pattern, as water courses comprised a significant barrier only in viviparous salamanders, probably due to their fully terrestrial life cycle. Agricultural areas and, to a lesser extent, topography also decreased genetic connectivity in both larviparous and viviparous populations. This study is one of very few to explicitly demonstrate the evolution of a derived reproductive mode affects patterns of genetic connectivity. Our findings open avenues for future research to better understand the eco-evolutionary implications underlying the emergence of terrestrial reproduction in amphibians.

Ongoing global warming is disrupting several ecological and evolutionary processes, spanning different levels of biological organization. Species are expected to shift their ranges as a response to climate change, with relevant implications to peripheral populations at the trailing and leading edges. Several studies have analyzed the exposure of species to climate change but few have explored exposure at the intraspecific level. We introduce a framework to forecast exposure to climate change at the intraspecific level. We build on existing methods by combining correlative species distribution models, a model of species range dynamics, and a model of phylogeographic interpolation. We demonstrate the framework by applying it to 20 Iberian amphibian and reptile species. Our aims were to: (a) identify which species and intraspecific lineages will be most exposed to future climate change; (b) test if nucleotide diversity at the edges of species ranges are significantly higher or lower than on the overall range; and (c) analyze if areas of higher species gain, loss, and turnover coincide with those predicted for lineages richness and nucleotide diversity. We found that about 80% of the studied species are predicted to contract their range. Within each species, some lineages were predicted to contract their range, while others were predicted to maintain or expand it. Therefore, estimating the impacts of climate change at the species level only can underestimate losses at the intraspecific level. Some species had significant high amount of nucleotide at the trailing or leading edge, or both, but we did not find a consistent pattern across species. Spatial patterns of species richness, gain, loss, and turnover were fairly concurrent with lineages richness and nucleotide diversity. Our results support the need for increased attention to intraspecific diversity regarding monitoring and conservation strategies under climate change.

Despite growing efforts to halt biodiversity loss, knowledge of species diversity and distribution is highly geographically biased, leaving some areas unexplored. Taxa distributed in remote, desert areas, such as hedgehogs (Mammalia; Eulipotyphla) in North Africa, are good examples of current knowledge gaps in systematics and biogeography. Here we studied the geographical distribution and intraspecific genetic diversity of hedgehogs in North Africa. Specimens belonging to North African and Eurasian species were analysed with mitochondrial (control region, CR) and nuclear (recombination activating gene 1, RAG1) gene fragments. This revealed a broader geographical distribution of Atelerix algirus in south-western Libya and of Paraechinus aethiopicus along the Atlantic Sahara. High intraspecific genetic differentiation was found in A. algirus and A. albiventris at the mitochondrial level, with nuclear haplotype sharing across their ranges. These findings suggest that biogeographical patterns of hedgehogs in North Africa are more complex than previously suggested, highlighting a need for further investigation in this remote and poorly known area.

Our study seeks to: (1) reduce biodiversity knowledge gaps in amphibians from desert and arid regions; (2) quantify species diversity and detect potential cryptic diversity; (3) identify environmental correlates of amphibian richness distribution, and (4) identify diversity hotspots. DNA barcoding and spatial modelling were integrated to map observed and predicted amphibian richness in Mauritania, Africa. We built two DNA barcoding libraries using one mitochondrial (COI) and one nuclear marker (RAG1). Taxon richness was calculated using species delimitation methods. A generalised linear model was employed to identify environmental correlates of amphibian richness and the distribution of diversity hotspots was predicted. We found a total of 16 anuran taxa with Afrotropic affinities, and two genera and four species were added for the amphibian list of Mauritania. Potential cryptic diversity was detected in Hoplobatrachus occipitalis. Amphibian richness was positively correlated with proximity to savannah, gravel floodplains, and with presence of seasonal wetlands, and negatively with proximity to dunes. Major diversity hotspots were predicted in southern Mauritania. Barcoding methods for both genetic markers successfully identified the taxonomic identity of specimens, also uncovering cryptic diversity in the amphibians of the Sahara-Sahel ecoregions. Amphibian richness in Mauritania increases southwards and it is mostly concentrated in areas under pressure due to habitat conversion, lacking legal protection. The combination of DNA barcoding with spatial modelling can be easily applied to any desert system to address pressing needs for research on biodiversity distribution., Funding provided by National Geographic Society (CRE-7629-04, CRE-8412-08, GEFNE-53-12), Mohammed bin Zayed Species Conservation Fund (11052709, 11052707, 13257467), Rufford Foundation (SG-15399-1, SG-17893-1), Fundação para a Ciência e Tecnologia (FCT: PTDC/BIA-BEC/099934/2008, PTDC/BIA-BIC/2903/2012), FEDER through COMPETE-Operational Programme for Competitiveness Factors (FCOMP-01-0124-FEDER-008917, -028276), and by AGRIGEN–NORTE-01-0145-FEDER-000007, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Research conducted in the scope of the LIA “Biodiversity and Evolution”. Individual support given by FCT (contracts DL57/2016/CP1440/CT0010, IF/01425/2014, and CEECINST/00014/2018/CP1512/CT0001).

The riverine barrier hypothesis (RBH) posits that rivers comprise geographical barriers to gene flow for terrestrial organisms, thus promoting genetic differentiation between populations. Here, we explored the RBH on larviparous and pueriparous populations of the live-bearing fire salamander (Salamandra salamandra). While larviparous fire salamanders exhibit a semi-aquatic life cycle (females deposit pre-metamorphic larvae on water), pueriparous salamanders present a fully terrestrial life cycle (females deliver terrestrial juveniles) and, therefore, a greater independence from water for survival and reproduction. We performed a fine-scale sampling of opposite transects in 11 rivers (six and five for larviparous and pueriparous populations, respectively) to test the hypothesis that rivers are more effective barriers for pueriparous salamanders due to their fully terrestrial life cycle. We carried out individual- and population-based genetic analyses using 14 microsatellites and a mitochondrial marker to examine the extent to which rivers hinder short- and long-term gene flow. We found that rivers are semi-permeable obstacles for both larviparous and pueriparous populations, although they appear to be more effective barriers for the latter when rivers with similar attributes are compared. We also found that river width and possibly the presence of crossing structures may influence the genetic barrier effects of rivers in fire salamanders. This is one of the very few studies in amphibians showing how different reproductive strategies influence the barrier effects imposed by rivers.

The salamander genus Salamandra is widespread across Europe, North Africa, and the Near East and is renowned for its conspicuous and polymorphic colouration and diversity of reproductive modes. The phylogenetic relationships within the genus, and especially in the highly polymorphic species S. salamandra, have been very challenging to elucidate, leaving its real evolutionary history and classification at species and subspecies levels a topic of debate and contention. However, the distribution of diversity and species delimitation within the genus are critically important for identifying evolutionarily significant units for conservation and management, espe-cially in light of threats posed by the pathogenic chytrid fungus Batrachochytrium salamandrivorans that is causing massive declines of S. salamandra populations in central Europe. Here, we conducted a phylogenomic analysis from across the taxonomic and geographic breadth of the genus Salamandra in its entire range. Bayesian, maximum likelihood and network-based phylogenetic analyses of up to 4905 ddRADseq-loci (294,300 nucleo-tides of sequence) supported the distinctiveness of all currently recognised species (Salamandra algira, S. atra, S. corsica, S. infraimmaculata, S. lanzai, and S. salamandra), and all five species for which we have multiple ex-emplars were confirmed as monophyletic. Within S. salamandra, two main clades can be distinguished: one clade with the Apenninic subspecies S. s. gigliolii nested within the Iberian S. s. bernardezi/fastuosa; and a second clade comprising all other Iberian, Central and East European subspecies. Our analyses revealed that some of the currently recognized subspecies of S. salamandra are paraphyletic and may require taxonomic revision, with the Central- and Eastern-European subspecies all being poorly differentiated at the analysed genomic markers. Salamandra s. longirostris – sometimes considered a separate species – was nested within S. salamandra, consistent with its subspecies status. The relationships identified within and between Salamandra species provide valuable context for future systematic and biogeographic studies, and help elucidate critical evolutionary units for conservation and taxonomy., This work was funded by a Natural Environment Research Council (NERC) research studentship NE/L501918/1 to JDB with KRE and BKM. Partial funding also came from a Royal Society Research Grant to KRE and a Systematics Research Fund award, from the Linnean Society of London and the Systematics Association, to JDB. GVA is supported by FCT (IF/01425/2014). SS received support by the German Research Foundation (DFG) (STE1130/8-1 and BL 1271/1-1) as part of a German- Israeli project cooperation (DIP) grant. DRV received support by a Spanish Ministerio de Economía y Competitividad and FEDER CGL2017- 89898-R (AEI/FEDER, UE) and LIFE18 NATES000121-LIFE DIVAQUA grants.

[EN] Protected-area systems should conserve intraspecific genetic diversity. Because genetic data require resources to obtain, several approaches have been proposed for generating plans for protected-area systems (prioritizations) when genetic data are not available. Yet such surrogate-based approaches remain poorly tested. We evaluated the effectiveness of potential surrogate-based approaches based on microsatellite genetic data collected across the Iberian Peninsula for 7 amphibian and 3 reptilian species. Long-term environmental suitability did not effectively represent sites containing high genetic diversity (allelic richness). Prioritizations based on long-term environmental suitability had similar performance to random prioritizations. Geographic distances and resistance distances based on contemporary environmental suitability were not always effective surrogates for identification of combinations of sites that contain individuals with different genetic compositions. Our results demonstrate that population genetic data based on commonly used neutral markers can inform prioritizations, and we could not find an adequate substitute. Conservation planners need to weigh the potential benefits of genetic data against their acquisition costs., [ES] Los sistemas de áreas protegidas deberían conservar la diversidad genética intraespecífica. Ya que para obtener datos genéticos se requieren recursos, se han propuesto distintas estrategias para generar los planes para los sistemas de áreas protegidas (priorizaciones) cuando los datos genéticos no están disponibles. A pesar de lo anterior, dichas estrategias basadas en sustitutos han sido poco evaluadas. Evaluamos la efectividad del potencial de las estrategias basadas en sustitutos cuya base son los datos genéticos de microsatélites obtenidos en toda la Península Ibérica y correspondientes a siete especies de anfibios y a tres de reptiles. La idoneidad ambiental a largo plazo no representó efectivamente los sitios que contienen una diversidad genética alta (riqueza de alelos). Las priorizaciones basadas en la idoneidad ambiental a largo plazo tuvieron un desempeño similar a las priorizaciones aleatorias. Las distancias geográficas y las distancias de resistencia basadas en la idoneidad ambiental contemporánea no siempre fueron sustitutos efectivos para la identificación de las combinaciones de sitios que contienen individuos con composiciones genéticas diferentes. Nuestros resultados demuestran que Hanson et al. 635 los datos genéticos de una población basados en marcadores neutrales de uso común pueden informar a las priorizaciones y que no pudimos encontrar un sustituto adecuado. Los planificadores de la conservación necesitan sopesar los beneficios potenciales de los datos genéticos contra sus costos de adquisición., This work was developed under the project PTDC/BIA-BIC/3545/2014, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and through the European Regional Development Fund (ERDF). Support by Portuguese National funds through Fundação para a Ciência e Tecnologia (FCT) was granted to A.V. (DL57/2016), F.S. (UIDB/50027/2020), and S.B.C. (individual scientific employment program-contract CEECIND/01464/2017). G.V.A. was supported by Investigador FCT contract (IF/01425/2014).

International audience

Landscape features shape patterns of geneflow among populations, ultimately determining where taxa lay along thecontinuum between panmixia to complete reproductive isolation. Geneflow can be restricted, leading to populationdifferentiation in two non-exclusive ways:“physical isolation”, in which geographic distance in combination with thelandscape features restricts movement of individuals promoting genetic drift, and“ecological isolation”, in which adaptivemechanisms constrain geneflow between different environments via divergent natural selection. In central Iberia, twofiresalamander subspecies occur in parapatry across elevation gradients along the Iberian Central System mountains, while inthe adjacent Montes de Toledo Region only one of them occurs. By integrating population and landscape genetic analyses,we show a ubiquitous role of physical isolation between and within mountain ranges, with unsuitable landscapes increasingdifferentiation between populations. However, across the Iberian Central System, we found strong support for a significantcontribution of ecological isolation, with low genetic differentiation in environmentally homogeneous areas, but highdifferentiation across sharp transitions in precipitation seasonality. These patterns are consistent with a significantcontribution of ecological isolation in restricting geneflow among subspecies. Overall, our results suggest that ecologicaldivergence contributes to reduce genetic admixture, creating an opportunity for lineages to follow distinct evolutionary trajectories., GV-A was supported by FCT–Foundation for Science and Technology (CEECIND/00937/2018). This research was supported by the European Science Foundation (Frontiers of Speciation Research, Exchange grant 3318), and by the European Commission (Synthesys grant ES-TAF-1486), granted to RJP. Partial funds were additionally provided by grants EVOVIV: PTDC/BIA-EVF/3036/2012 (FCT, Portugal) to GV-A, and CGL2017-83131-P (FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación, Spain) to IMS.

The ability to bear live offspring, viviparity, has evolved multiple times across the tree of life and is a remarkable adaptation with profound life-history and ecological implications. Within amphibians the ancestral reproductive mode is oviparity followed by a larval life stage, but viviparity has evolved independently in all three amphibian orders. Two types of viviparous reproduction can be distinguished in amphibians; larviparity and pueriparity. Larviparous amphibians deliver larvae into nearby ponds and streams, while pueriparous amphibians deliver fully developed juveniles and thus do not require waterbodies for reproduction. Among amphibians, the salamander genus Salamandra is remarkable as it exhibits both inter- and intraspecific variation in the occurrence of larviparity and pueriparity. While the evolutionary relationships among Salamandra lineages have been the focus of several recent studies, our understanding of how often and when transitions between modes occurred is still incomplete. Furthermore, in species with intraspecific variation, the reproductive mode of a given population can only be confirmed by direct observation of births and thus the prevalence of pueriparous populations is also incompletely documented. We used sequence capture to obtain 1,326 loci from 94 individuals from across the geographic range of the genus, focusing on potential reproductive mode transition zones. We also report additional direct observations of pueriparous births for 20 new locations and multiple lineages. We identify at least five independent transitions from the ancestral mode of larviparity to pueriparity among and within species, occurring at different evolutionary timescales ranging from the Pliocene to the Holocene. Four of these transitions occurred within species. Based on a distinct set of markers and analyses, we also confirm previous findings of introgression between species and the need for taxonomic revisions in the genus. We discuss the implications of our findings with respect to the evolution of this complex trait, and the potential of using five independent convergent transitions for further studies on the ecological context in which pueriparity evolves and the genetic architecture of this specialized reproductive mode., Financial support came from National Funds through FCT – Foundation for Science and Technology (SALOMICS: PTDC/BIA-EVL/28475/2017); the authors also acknowledge funding from other FCT projects and FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE (EVOVIV: PTDC/BIA- EVF/3036/2012; FCOMP-01-0124-FEDER-028325 and UIDB/500027/2020); and by the office of the Associate Director of Science at the Smithsonian’s National Museum of Natural History. KPM was funded by an FCT predoctoral grant (PD/BD/52604/2014) and GVA by FCT research contracts (IF/01425/2014 and CEECIND/00937/2018), all from the Portuguese “Fundacão para a Ciência e a Tecnologia,” funded by Programa Operacional Potencial Humano (POPH)-Quadro de Referência Estratégica Nacional (QREN) from the European Social Fund.

Reproduction is intimately linked with dispersal, but the effects of changes in reproductive strategies on dispersal have received little attention. Such changes have occurred in many taxonomic groups, resulting in profound alterations in life-history. In amphibians, many species shifted from oviparous/larviparous aquatic reproduction (deposition of eggs or pre-metamorphic larvae in water) to pueriparous terrestrial reproduction (parturition of terrestrial juveniles). The latter provides greater independence from water by skipping the aquatic larval stage; however, the eco-evolutionary implications of this evolutionary step have been underexplored, largely because reproductive modes rarely vary at the intraspecific level, preventing meaningful comparisons. We studied the effects of a transition to pueriparity on dispersal and fine-scale genetic structure in the fire salamander (Salamandra salamandra), a species exhibiting two co-occurring reproductive modes: larviparity and pueriparity. We performed genetic analyses (parentage and genetic spatial autocorrelation) using 11 microsatellite loci to compare dispersal and fine-scale genetic structure in three larviparous and three pueriparous populations (354 individuals in total). We did not find significant differences between reproductive modes, but in some larviparous populations movement patterns may be influenced by site-specific features (type of water bodies), possibly due to passive water-borne dispersal of larvae along streams. Additionally, females (especially larviparous ones) appeared to be more philopatric, while males showed greater variation in dispersal distances. This study also points to future avenues of research to better understand the eco-evolutionary implications of changes in reproductive modes in amphibians.

This study provides the first atlas of the distribution of amphibians and reptiles in the Diawling National Park (DNP), Mauritania. Unpublished fieldwork observations collected between 2010 and 2017 were combined with published records and museum data in a geographical information system to produce maps with the distribution of individual species and species richness. The taxonomic list includes 32 species, six amphibians and 26 reptiles, grouped in 17 families. Ten species are new records for the area. Species form distinct groups according to their distribution patterns and preliminary habitat selection trends: 1) species distributed in inland dunes, some extending their range to coastal dunes; 2) species distributed in inland dunes and floodplains; and 3) species associated with habitats that are permanently or seasonally flooded or that inhabit riparian forests. Three areas accumulating most of the species found are located in sand habitats of DNP and peripheral zones. Specific counts of Crocodylus suchus suggest strong seasonal fluctuations in population size inside DNP, which are likely a consequence of the human-mediated dynamics of water availability in the area

Phenotypic divergence can occur through both local adaptations and phenotypic plasticity in response to particular environmental pressures. Holocene land-bridge islands harbor recent isolated populations that offer an excellent natural framework to study the microevolutionary processes involved in rapid or ongoing phenotypic shifts. Two insular populations (Ons and San Martino) of Salamandra salamandra in NW Iberian Peninsula exemplify these phenotypic shifts with a rapid evolutionary transition in reproductive mode and genetic differentiation since their isolation ca. 8000 years ago. We evaluate parallel phenotypic changes in body size and behavior. In particular, we (1) collected observational fieldwork data during diurnal and nocturnal transects in both islands for over a decade (2004–2016); (2) investigated the climatic conditions associated to fire salamanders’ activity; (3) compared physical condition between insular and mainland populations; and (4) used plasticine models and camera trapping to identify potential predators that might cause the observed diurnal activity in San Martino Island. Our 13-year-long study shows that salamanders from San Martino show mainly diurnal activity and are significantly smaller than salamanders from Ons and nearby continental populations. Overall, we present an exceptional case of behavior and phenotypic differentiation of an insular population of fire salamanders and suggest that this unusual behavior in the small population of San Martino might be triggered by a response to predator pressure exerted by a dense population of Rattus rattus.

Urbanization is a severe form of habitat fragmentation that can cause many species to be locally extirpated and many others to become trapped and isolated within an urban matrix. The role of drift in reducing genetic diversity and increasing genetic differentiation is well recognized in urban populations. However, explicit incorporation and analysis of the demographic and temporal factors promoting drift in urban environments are poorly studied. Here, we genotyped 15 microsatellites in 320 fire salamanders from the historical city of Oviedo (Est. 8th century) to assess the effects of time since isolation, demographic history (historical effective population size; Ne ) and patch size on genetic diversity, population structure and contemporary Ne . Our results indicate that urban populations of fire salamanders are highly differentiated, most likely due to the recent Ne declines, as calculated in coalescence analyses, concomitant with the urban development of Oviedo. However, urbanization only caused a small loss of genetic diversity. Regression modelling showed that patch size was positively associated with contemporary Ne , while we found only moderate support for the effects of demographic history when excluding populations with unresolved history. This highlights the interplay between different factors in determining current genetic diversity and structure. Overall, the results of our study on urban populations of fire salamanders provide some of the very first insights into the mechanisms affecting changes in genetic diversity and population differentiation via drift in urban environments, a crucial subject in a world where increasing urbanization is forecasted.

The reduction in fecundity associated with the evolution of viviparity may have far-reaching implications for the ecology, demography, and evolution of populations. The evolution of a polygamous behaviour (e.g. polyandry) may counteract some of the effects underlying a lower fecundity, such as the reduction in genetic diversity. Comparing patterns of multiple paternity between reproductive modes allows us to understand how viviparity accounts for the trade-off between offspring quality and quantity. We analysed genetic patterns of paternity and offspring genetic diversity across 42 families from two modes of viviparity in a reproductive polymorphic species, Salamandra salamandra. This species shows an ancestral (larviparity: large clutches of free aquatic larvae), and a derived reproductive mode (pueriparity: smaller clutches of larger terrestrial juveniles). Our results confirm the existence of multiple paternity in pueriparous salamanders. Furthermore, we show the evolution of pueriparity maintains, and even increases, the occurrence of multiple paternity and the number of sires compared to larviparity, though we did not find a clear effect on genetic diversity. High incidence of multiple paternity in pueriparous populations might arise as a mechanism to avoid fertilization failures and to ensure reproductive success, and thus has important implications in highly isolated populations with small broods.

Identifying the evolutionary processes that underlie morphological variation at the intraspecific level is cornerstone for understanding the drivers of phenotypic diversity at higher macro-evolutionary scales. The fire salamander, Salamandra salamandra, exhibits exceptional intraspecific variation in multiple phenotypic traits (i.e. body size, head shape, coloration, reproductive modes). Pueriparity (females laying fully metamorphosed juveniles) in S. salamandra entails modifications during embryonic development in comparison to larviparity (females laying aquatic larvae), which is the ancestral reproductive mode. These heterochronic modifications involve a general acceleration of development and mainly focus on cephalic structures to facilitate intrauterine active feeding, which might impact head shape in the adult stage. In the present study, we (i) describe the main features of head shape variation in adults of the two distinct subspecies of Salamandra salamandra that independently evolved to pueriparity, and (ii) explore the morphological consequences of developmental and functional changes related to this major evolutionary shift. Our results show that evolutionary history, and not reproductive mode, is the main driver of head shape variation in S. salamandra. These results suggest different evolutionary processes acting differentially on each subspecies, at least at the adult stage. The present study highlights the importance of comparative studies integrating evolutionary histories and ontogenetic trajectories to explore the different sources of observed morphological diversification., This work is funded by the Ministerio de Economía y Competitividad (Grants Nos. CGL2012-40246-C02-02 and CGL2017-86924-P); by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE (FCOMP-01-0124-FEDER-028325 and POCI-01- 0145-FEDER-006821); and by National Funds through FCT – Foundation for Science and Technology (EVOVIV: PTDC/BIA-EVF/3036/2012; SALOMICS: PTDC/BIA-EVL/28475/2017). L.A.-R. was supported by a FPU grant (FPU14/03015) from the Ministerio de Educación, Cultura y Deporte (MECD, Spain) and and G.V.-A. and A.K. by IF contracts (IF/01425/2014 and IF/00641/2014, respectively). from Fundação para a Ciência e a Tecnologia (FCT, Portugal).

Chelonians (turtles and tortoises) are key, but highly endangered, ecological components. Pet trade is a pervasive threat that often results in the confiscation of specimens, or their release in non-native regions. Emys orbicularis is considered near threatened, with declining populations across its western Palaeartic range. Individuals of unknown origin (confiscated or wild-caught) are maintained in zoos and recovery centres, draining space and resources with no benefit to wild populations. Here, we applied genetic assignment methods to 61 captive and wild-caught individuals from Spain, Portugal and Morocco of unknown origin, using robust genetic databases constructed for the species (ca. 1600 individuals sequenced for the cytochrome b), and for the Ibero-Maghrebian E. o. occidentalis (453 individuals genotyped with microsatellites). We identified unknown individuals as assigned or ambiguous, based on their membership coefficient (q) for a total of 15 genetic clusters, and provide evidence of long-distance translocations, possible due to illegal trade. We advocate for wildlife forensic science as a fundamental tool for reintroductions, and highlight the significance of recovery centres in the management of endangered species.

The study of morphological variation among and within taxa can shed light on the evolution of phenotypic diversification. In the case of urodeles, the dorso-ventral view of the head captures most of the ontogenetic and evolutionary variation of the entire head, which is a structure with a high potential for being a target of selection due to its relevance in ecological and social functions. Here, we describe a non-invasive procedure of geometric morphometrics for exploring morphological variation in the external dorso-ventral view of urodeles' head. To explore the accuracy of the method and its potential for describing morphological patterns we applied it to two populations of Salamandra salamandra gallaica from NW Iberia. Using landmark-based geometric morphometrics, we detected differences in head shape between populations and sexes, and an allometric relationship between shape and size. We also determined that not all differences in head shape are due to size variation, suggesting intrinsic shape differences across sexes and populations. These morphological patterns had not been previously explored in S. salamandra, despite the high levels of intraspecific diversity within this species. The methodological procedure presented here allows to detect shape variation at a very fine scale, and solves the drawbacks of using cranial samples, thus increasing the possibilities of using collection specimens and alive animals for exploring dorsal head shape variation and its evolutionary and ecological implications in urodeles. J. Morphol. 278:475-485, 2017. © 2017 Wiley Periodicals, Inc.

Salamandra algirais one of the few species of vertebrates that displays intraspecific variation in reproductive strategies, making it a valuable study model in reproductive mode evolution. How pueriparity (viviparity) inS. algiraarose remains unclear mainly due to insufficient information on the distribution of both reproductive modes in the species. This note summarizes what is known about pueriparous reproduction inS. algiraand adds new data on the distribution and geographic limits of both reproductive strategies and mitochondrial lineages withinS. a. tingitanaacross the Western Rif in Morocco, with possible implications for our understanding of the ecological context of pueriparity. The occurrence of pueriparity in two mitochondrial sublineages suggests multiple events of pueriparity evolution inS. algira, or a model of mitochondrial capture through hybridization and introgression across the contact zone.

Estimating geographical ranges of intra-specific evolutionary lineages is crucial to the fields of biogeography, evolution, and biodiversity conservation. Models of isolation mechanisms often consider multiple distances in order to explain genetic divergence. Yet, the available methods to estimate the geographical ranges of lineages are based on direct geographical distances, neglecting other distance metrics that can better explain the spatial genetic structure. We extended the phylogeographical interpolation method (phylin) in order to accommodate user-defined distance metrics and to incorporate the uncertainty associated with genetic distance calculation. These new features were tested with simulated and empirical data sets. Multiple distance matrices were generated including geographical, resistance, and environmental distances to derive maps of lineage occurrence. The new additions to this method improved the ability to predict lineage occurrence, even with low sample size. We used a regression framework to quantify the relationship between the genetic divergence and competing distance matrices representing potential isolation processes that are subsequently used in the interpolation process. Including uncertainty in tree topology and the different distance matrices improved the robustness of the variograms, allowing a better fit of the theoretical model of spatial dependence. The improvements to the method increase its potential application in other fields. Accurately mapping genetic divergence can help to locate potential contact zones between lineages as well as barriers to gene flow, which has a broad interest in biogeographical and evolutionary studies. Additionally, conservation efforts could benefit from the integration of genetic variation and landscape features in a spatially explicit framework., Fundação para a Ciência e a Tecnologia. Grant Numbers: IF/01425/2014, PTDC/BIA-BIC/3545/2014, SFRH/BPD/74423/2010, SFRH/BPD/93473/2013. European Regional Development Fund (ERDF) 'Programa Operacional Factores de Competitividade – COMPETE