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1. Wetland ecosystems worldwide are threatened by habitat alteration, climate change and the introduction of invasive species, even within protected areas. Unravelling the reliance of sensitive wetland-dwelling species, such as amphibians, on habitat characteristics is thus essential to identify conservation targets. 2. Here we assess the distribution of genetic diversity of two strongly aquatic amphibians (Pelophylax perezi and Pleurodeles waltl) in association with habitat features across the most extensive, protected wetland of the Iberian Peninsula: Doñana National Park. 3. Despite inhabiting a protected area free from anthropogenic barriers, the genetic diversity of P. perezi and P. waltl is not homogeneously distributed across the wetland, but instead concentrates in core areas, mainly in the northern zone. Both genetic diversity and connectivity (as opposed to genetic differentiation) showed significant positive associations with the area of the breeding sites and the flooded area surrounding the breeding sites within the dispersal potential of either species, that is nearby pond availability. 4. Large water bodies connected to abundant temporary ponds are key for the maintenance of amphibian genetic diversity. Nevertheless, the core populations of our target species, which show markedly aquatic habits, are concentrated in areas colonised by invasive species, which could compromise their long-term viability. 5. Our results highlight that maintaining widely connected arrays of ponds of different hydroperiods, including large breeding sites free from invasive predators and competitors, is paramount for amphibian conservation in Mediterranean wetlands.

Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. JGR was supported by “Doctores Junta de Andalucía” postdoctoral fellowship (DOC_00668, FEDER EU/Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía). JG was funded by the Individual Scientific Employment Stimulus Program (2017) by the Portuguese Foundation for Science and Technology (FCT CEECIND/02331/2017/CP1423/CT0012). EC was supported by the FCT through a Postdoctoral Grant (SFRH/BPD/109182/2015). This study was funded by projects CGL2008-04271-C02-01/BOS, CGL2011-28300 (Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, Spain, and FEDER), PPII10-0097-4200 (Junta de Comunidades de Castilla la Mancha and FEDER), and CGL2017-83131-P (FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación, Spain)., Context Mediterranean 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. Objectives We 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). Methods We 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. Results We 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. Conclusions This 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 respo, Depto. de Biodiversidad, Ecología y Evolución, Fac. de Ciencias Biológicas, TRUE, pub

[Abstract] The rich genetic and phenotypic diversity of species complexes is best recognized through formal taxonomic naming, but one must first assess the evolutionary history of phylogeographic lineages to identify and delimit candidate taxa. Using genomic markers, mitochondrial DNA barcoding, and morphometric analyses, we examined lineage diversity and distribution in the Iberian endemic frog Rana parvipalmata. We confirmed two deep phylogeographic lineages, one relatively homogenous genetically, found in Asturias and adjacent areas (T2), and one more fragmented and locally genetically impoverished, restricted to Galicia (T1). Analyses of their hybrid zone suggested a shallow transition characterized by far-ranging admixture, which was modelled by a wide geographic cline (~60 km for the genome average) and no obvious barrier loci (i.e. loci showing disproportionally restricted introgression). The relatively young T1 and T2 have thus remained reproductively compatible, which argues against their distinction as biological species, and we accordingly describe T2 as a new subspecies, Rana parvipalmata asturiensis ssp. nov. Remarkably, we highlight striking discordances between mitochondrial and nuclear distributions across their hybrid zones, as well as between their genetic and morphological differentiation. Our study illustrates how genomic-based phylogeographic frameworks can help decipher the high genetic and phenotypic variation of species complexes and substantiate the taxonomic assessment of candidate lineages.

Assembling DNA barcode reference libraries for various taxonomic groups allows researchers to use metabarcoding or environmental DNA approaches to gain a rapid understanding of diversity in given environments. However, our ability to use reference libraries depends on how accurately DNA barcodes are able to recover taxonomic boundaries and identify species, which is rarely considered. We constructed an extensive COI barcoding library for amphibians of the Western Palearctic and successfully recovered barcodes from 60 urodele and 73 anuran species (representing 94% and 98% of the nominal anuran and urodele species in the Western Palearctic, respectively), covering the intraspecific diversity of the majority of species in this region. We tested the effectiveness of our assembled DNA barcode dataset for species identification using barcoding gap, efficiency analyses, and two phylogenetic species delimitation methods. We obtained DNA barcodes for 1251 specimens (691 anurans and 560 urodeles) with a high success rate (92-96%) of species identification. The absence of a barcoding gap in a number of samples was linked to species misidentifications, which suggest incipient speciation or cryptic diversity, or previously described mitochondrial introgression events. The phylogenetic species delimitation methodsresulted in substantial oversplitting of currently accepted taxonomy. This COI barcoding library provides an almost complete and reliable reference library for Western Palearctic amphibians. We highlight the importance of generating comprehensive and well curated reference libraries that include intra- and interspecific genetic variability and the need of detailed taxonomic revision when ambiguous or incorrect DNA barcodes exist.

[Context]: Functional connectivity across fragmented habitat patches is essential for the conservation of animal populations in humanized landscapes. Given their low dispersal capacity, amphibians in the Mediterranean region are threatened by habitat fragmentation and loss due to changes in land use, including agricultural intensification., [Objectives]: We assessed patterns of functional connectivity of a Near Threatened Mediterranean amphibian, the sharp ribbed newt (Pleurodeles waltl), in an agricultural landscape matrix in NW Spain subject to different intensification regimes., [Methods]: We sampled newts in 17 ponds embedded in a terrestrial habitat matrix dominated by agricultural land uses. Genome-wide molecular markers (1390 SNPs) were used to assess patterns of genetic diversity and gene flow among ponds. We tested the role of landscape features on functional connectivity using isolation by resistance models incorporating information on Normalized Difference Vegetation Index (NDVI) data., [Results]: We found low levels of genetic diversity in all sampled populations. Global FST estimates and cluster analyses revealed shallow but significant genetic structure in the study area, with NDVI-based resistance models showing that open areas (rainfed crops and grasslands) offer lower resistance to gene flow and thus promote functional connectivity among demes., [Conclusions]: Our study highlights the important role of landscape features, such as open areas resulting from traditional rainfed agriculture, in promoting functional connectivity between amphibian populations in Mediterranean agrosystems. Conservation policies must adopt a functional network strategy and protect groups of inter-connected temporary ponds across the traditional agricultural matrix to efficiently preserve their associated biotic communities.

Many herpetofauna species have been introduced outside of their native range. MtDNA barcoding is regularly used to determine the provenance of such populations. The alpine newt has been introduced across the Netherlands, the United Kingdom and Ireland. However, geographical mtDNA structure across the natural range of the alpine newt is still incompletely understood and certain regions are severely undersampled. We collect mtDNA sequence data of over seven hundred individuals, from both the native and the introduced range. The main new insights from our extended mtDNA phylogeography are that 1) haplotypes from Spain do not form a reciprocally monophyletic clade, but are nested inside the mtDNA clade that covers western and eastern Europe; and 2) haplotypes from the northwest Balkans form a monophyletic clade together with those from the Southern Carpathians and Apuseni Mountains. We also home in on the regions where the distinct mtDNA clades meet in nature. We show that four out of the seven distinct mtDNA clades that comprise the alpine newt are implicated in the introductions in the Netherlands, United Kingdom and Ireland. In several introduced localities, two distinct mtDNA clades co-occur. As these mtDNA clades presumably represent cryptic species, we urge that the extent of genetic admixture between them is assessed from genome-wide nuclear DNA markers. We mobilized a large number of citizen scientists in this project to support the collection of DNA samples by skin swabbing and underscore the effectiveness of this sampling technique for mtDNA barcoding.

[EN] Amphibians are the most endangered vertebrate group on the planet, with many species under extinction threat. Habitat destruction is one of the main drivers of their worldwide decline, causing fragmentation in their ranges of distribution and thus reducing functional connectivity among their populations and increasing their extinction risk. Consequently, conservation of amphibian populations requires the study of connectivity, which is largely driven by the process of dispersal. While dispersal has been thoroughly studied in some animal groups, the spatial ecology of amphibians remains largely undocumented. Several methodological approaches are available to address questions about amphibian dispersal, including its direct study using capture-mark-recapture techniques and the analysis of the indirect consequences of the movement of individuals on the distribution of genetic diversity using genetic markers. The integration of both direct and indirect approaches has been rarely carried out but has great potential for disentangling the effects of geographic, demographic and ecological variables on amphibian dispersal and for the robust characterization of functional connectivity among populations. In addition, dispersal is highly context-dependent, complicating comparisons between studies performed at different areas and highlighting the need for multi-species approaches conducted at spatial scales suited to target organisms. This thesis combines photoidentification-aided capture-recapture data from a two-year monitoring program and genomic markers (>1000 individuals of five species genotyped with thousands of SNPs) to characterize fine-scale dispersal in pondbreeding amphibian populations in a pondscape (64 ponds) located in the southern slopes of sierra de Guadarrama (Alpedrete, central Spain). We integrated probabilistic connectivity graphs derived from dispersal kernels with inference of genetic structure, quantification of gene flow and neighborhood areas to p, [ES] Los anfibios son el grupo de vertebrados más amenazado del planeta, con un gran número de especies en riesgo de extinción. La destrucción del hábitat es una de las principales causas de su declive mundial, ya que ésta causa la fragmentación de sus rangos de distribución y, por tanto, reduciendo la conectividad funcional entre sus poblaciones e incrementando el riesgo de extinción. Por ello, la conservación de las poblaciones de anfibios requiere el estudio de la conectividad, la cual está principalmente condicionada por el proceso de dispersión. Mientras que la dispersión ha sido estudiada exhaustivamente en algunos grupos animales, la ecología espacial de los anfibios sigue siendo, en su mayor parte, desconocida. Existen diversas aproximaciones metodológicas al alcance de los investigadores para abordar cuestiones relativas a la dispersión de anfibios; desde su estudio directo usando técnicas de captura-marcaje-recaptura hasta el análisis de los efectos indirectos del movimiento de individuos en el acervo genético de las poblaciones usando marcadores genéticos. La combinación de ambas aproximaciones, directa e indirecta, ha sido escasamente explorada, pero presenta un gran potencial para revelar los efectos de variables geográficas, demográficas y ecológicas en la dispersión de anfibios, así como para obtener una caracterización robusta de la conectividad funcional entre poblaciones. Además, la dispersión es un proceso altamente dependiente del contexto en el que ocurre, lo que hace difícil la comparación de resultados entre estudios realizados en diferentes localizaciones geográficas y subraya la necesidad de aproximaciones que incluyan el análisis de múltiples especies cohabitantes a escalas espaciales relevantes según el organismo objeto de estudio. En esta tesis, se combinan datos de marcaje-recaptura obtenidos mediante fotoidentificación durante dos años de monitoreo y marcadores genómicos (>1000 individuos de cinco e

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 t

Assessing patterns of functional connectivity among amphibian demes is crucial to unravel their population dynamics and prevent their isolation and eventual extinction. Integrative studies based on direct (capture-mark-recapture) and indirect (genetic) estimates of dispersal provide robust, biologically realistic inferences on population structure and connectivity, with applications for conservation efforts. We focused on two pond-breeding amphibians with contrasting life-history traits: the short-lived, semi-arboreal Hyla molleri and the long-lived, fossorial Pelobates cultripes. We PIT-tagged 2150 individuals of both species in two ponds (Laguna and Gravera, separated by 700 m) and monitored them from 2009 to 2021 to document the frequency and spatial extent of dispersal events. In addition, we genotyped individuals from these and two additional breeding populations at a maximum distance of 5 km with 15–16 microsatellites to characterize fine-scale patterns of genetic structure. We detected dispersal events connecting Laguna and Gravera in both species, albeit at low frequencies (4.8% and 7.7% of recaptured individuals of H. molleri and P. cultripes, respectively). However, both species were capable of covering long distances, with individual accumulated displacements up to 3.5 km (Hyla) and 1.8 km (Pelobates). Breeding populations > 2 km apart were genetically differentiated, indicating lower connectivity at this spatial scale. Estimates of pairwise mmigration rates differed between species and were asymmetrical, with different ponds representing “source” populations contributing more migrants to other populations in each species. We discuss the role of differences in life history traits and ecological preferences in shaping population dynamics in the two species and highlight management implications of our results.

Uno de los factores que amenaza la conservación de los anfibios a nivel mundial son las enfermedades emergentes. Sin embargo, poco se sabe de los efectos de otros parásitos que llevan más tiempo conviviendo con ellos. En este trabajo se han investigado los posibles costes fisiológicos de parásitos sanguíneos en la rana común ibérica (Pelophylax perezi). Para ello se ha investigado la variación de la masa corporal y la concentración de hemoglobina de las ranas con relación a la variación en sus cargas parasitarias y perfil leucocitario mediante un diseño de captura-recaptura en una población de la Sierra de Guadarrama (Madrid). Se recapturaron y estudió la covariación de variables en 25 ranas. El 97.8% del total de individuos muestreados estaban infectados. El género Trypanosoma fue el más frecuente. También se identificaron tres géneros de coccidios sanguíneos. Se observó que el cambio en la masa corporal de P. perezi entre capturas se relacionó negativamente con el aumento de Trypanosoma y positivamente con el aumento de los monocitos, sugiriendo que la infección por este parásito es costosa y que los individuos en mejor condición pueden incrementar la respuesta antiparasitaria mediada por monocitos. Se observaron también efectos negativos independientes de Trypanosoma y algunos coccidios sanguíneos sobre la concentración de hemoglobina de las ranas. Todo ello sugiere que los costes fisiológicos de las infecciones por parásitos sanguíneos son cuantificables en las ranas pese a que, presuntamente, llevan más tiempo coevolucionando bajo procesos coadaptativos que otras infecciones de más reciente aparición.

Las poblaciones de muchas especies de anfibios están sufriendo declives debido a diversos factores, entre los que destacan la pérdida y fragmentación de hábitats como consecuencia de la actividad humana, las enfermedades infecciosas y la introducción de especies invasoras. Los ecosistemas acuáticos como charcas y arroyos, necesarios para que los anfibios lleven a cabo su reproducción, han sufrido una disminución notable en número y calidad en las últimas décadas, por lo que la creación y mantenimiento de puntos de agua artificiales puede representar un buen recurso para la conservación de algunas especies. En la península ibérica, el sapo común (Bufo spinosus) es una especie generalista cuya presencia ha decrecido progresivamente en amplias zonas de su distribución, pero apenas existen datos demográficos que permitan cuantificar sus tendencias poblacionales y planificar medidas de gestión. En el presente estudio, se ha realizado un seguimiento de una población de B. spinosus que se reproduce en una charca artificial en el sureste de la Comunidad de Madrid (Fuente del Valle, en el municipio de Arganda del Rey) durante los años 2020, 2021 y 2022. A partir de la identificación de individuos mediante fotografías de sus patrones dorsales, se ha estudiado la fenología, abundancia y éxito reproductor de la especie en esta localidad. Los resultados muestran que la población de B. spinosus en Fuente del Valle supera los 200 individuos, con una proporción de 2 machos por cada hembra aproximadamente. La actividad reproductora está restringida a un corto periodo de tiempo, principalmente entre febrero y marzo, aunque se observaron grandes variaciones interanuales, tanto en la fenología como en la abundancia, dependiendo de las condiciones climatológicas. El éxito reproductor de los machos durante el periodo de estudio fue de aproximadamente el 57%, y algunos machos consiguieron aparearse con varias hembras, aunque es posible que no siempre lleguen a fecundar sus puestas. Los re

El conocimiento sobre la conectividad funcional entre poblaciones es esencial para la gestión de especies que viven en hábitats fragmentados, como es el caso del sapo común, Bufo spinosus. Esta especie, a pesar de encontrarse ampliamente distribuida por la Península Ibérica, experimenta una continua regresión a causa de múltiples factores, como las enfermedades infecciosas, la destrucción de su hábitat y los atropellos. Sin embargo, apenas existen datos acerca de los patrones de conectividad en sus poblaciones, lo que dificulta aplicar medidas dirigidas a la conservación de la especie. La utilización de marcadores moleculares, como los microsatélites, ha resultado ser una herramienta eficaz para caracterizar patrones de conectividad funcional en grupos para los que se dispone de pocos datos sobre su capacidad de dispersión. En este trabajo, se han utilizado 12 microsatélites polimórficos para caracterizar los patrones de diversidad genética y conectividad funcional en 11 poblaciones de B. spinosus en la sierra de Guadarrama, concretamente en el entorno de la sierra de la Cabrera. Los resultados obtenidos muestran que las poblaciones del área de estudio presentan una diversidad genética moderadamente alta, sin signos de endogamia. Si bien las poblaciones muestran cierta estructuración genética, también hay señales de buena conectividad funcional, especialmente en el núcleo central. A través de la caracterización de las tasas de migración por generación entre los dos núcleos de reproducción centrales de la zona de estudio, se ha revelado que la conectividad es asimétrica entre las poblaciones, y presenta ciertas diferencias entre machos y hembras. La información recopilada en este trabajo permite plantear que las medidas para mejorar la situación poblacional de la especie deben priorizar, además de la conservación de zonas de reproducción, la reducción de las barreras a la migración, como las carreteras, que ponen en riesgo el mantenimiento de la conectividad funciona

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determinationsystems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due toan apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females)systems. Taking advantage of a unique collection of capture–recapture datasets in amphibians, a vertebrate group where XY andZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sexdifferences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and notlifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but agingrates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome systemon sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleteriousmutations on the X/Z chromosome (the “unguarded X/Z effect”) or repeat-rich Y/W chromosome (the “toxic Y/W effect”) couldaccelerate aging in the heterogametic sex in some vertebrate clades.

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.

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.

Comparative studies of mortality in the wild are necessary to understand the evolution of aging; yet, ectothermic tetrapods are underrepresented in this comparative landscape, despite their suitability for testing evolutionary hypotheses. We present a study of aging rates and longevity across wild tetrapod ectotherms, using data from 107 populations (77 species) of nonavian reptiles and amphibians. We test hypotheses of how thermoregulatory mode, environmental temperature, protective phenotypes, and pace of life history contribute to demographic aging. Controlling for phylogeny and body size, ectotherms display a higher diversity of aging rates compared with endotherms and include phylogenetically widespread evidence of negligible aging. Protective phenotypes and life-history strategies further explain macroevolutionary patterns of aging. Analyzing ectothermic tetrapods in a comparative context enhances our understanding of the evolution of aging.

[Context] Robust assessment of functional connectivity in amphibian population networks is essential to address their global decline. The potential of graph theory to characterize connectivity among amphibian populations has already been confirmed, but the movement data on which modelled graphs rely are often scarce and inaccurate. While probabilistic methods that account for intraspecific variability in dispersal better reflect the biological reality of functional connectivity, they must be informed by systematically recorded individual movement data, which are difficult to obtain for secretive taxa like amphibians., [Objectives] Our aim is to assess the applied potential of probabilistic graph theory to characterize overall connectivity across amphibian pondscapes using finescale capture-recapture data, and to inform conservation management based on the role of ponds on functional connectivity., [Methods] We monitored an amphibian community in a pondscape located in a Spanish “dehesa” for 2 years. Photoidentification was used to build capture histories for individuals of six species, from which dispersal kernels and population sizes were estimated to model probabilistic graphs., [Results] We obtained kernels of variable robustness for six species. Node importance for connectivity varied between species, but with common patterns such as shared road crossing areas and the presence of coincident interconnected pond clusters., [Conclusions] The combination of photoidentification, capture-recapture data and graph theory allowed us to characterize functional connectivity across the pondscape of study accounting for dispersal variability and identify areas where conservation actions could be most efficient. Our results highlight the need to account for interspecific differences in the study and management of amphibian pondscapes.

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.

[Aim]: Discerning the relative role of geographical and ecological factors in promoting diversification is central to our understanding of the origin and maintenance of biodi-versity. We explore the roles of geology and ecological tolerance in the diversification of a group of wingless beetles with low dispersal potential. [Location]: Western Mediterranean (Iberian Peninsula and North Africa)., [Taxon]: Darkling beetles (Tenebrionidae: Misolampus)., [Methods]: We sequenced nine gene fragments from the mitochondrial and nuclear genomes in all extant Misolampus species to reconstruct their phylogeny, evaluate species boundaries and potential contact zones and estimate divergence times. We modelled species distributions for different time periods to infer ecological preferences and assess the effects of climatic changes since the last interglacial. We used a time-stratified process-based biogeographical model to estimate ancestral areas of origin and the evolution of geographical ranges., [Results]: The palaeoclimatic model projections show contractions of favourable areas during the last interglacial period and mid-Holocene, and wide stretches of suitable areas during the last glacial maximum. Analyses of ancestral bioclimatic preferences reveal ecological adaptations in isolated lineages within three species. The phylog-eny of Misolampus is strongly supported and unveils deep divergences within the six species. Two well-supported clades were recovered, one distributed in North Africa- Balearic Islands and another in the Iberian Peninsula. The divergence between the North African and Iberian clades occurred during the early Miocene. The geographical ranges of species are dynamic, shaped by biotic (e.g. life history, demography, species interactions) and abiotic (e.g. tectonic events, climatic variables) factors that vary through space and time. This variation ultimately dictates differential patterns of population viability, connectivity and growth through time in dif-ferent sections of species ranges, with important consequences for species diversification processes (Bellard et al., 2012; Gouveia et al., 2014; Thuiller et al., 2005; Wiens, 2011). Following climatic changes, species can respond by contracting their ranges into cli-matic refugia and/or dispersing to areas where their environmen-tal preferences are maintained (niche tracking), by adapting to new environmental conditions in situ (niche evolution), and often by undergoing extinction in environmentally unsuitable areas (Kozak & Wiens, 2006; Yesson & Culham, 2006a). The fragmentation of ancestral distributions because of tectonic or climatic changes pro-motes allopatric speciation, sometimes associated with niche evolution, whereas demographic expansion of populations tracking climatic changes can lead to range fusion and admixture between formerly isolated population groups. Discerning the relative role of biotic and abiotic factors in promoting diversification is central to our understa, [Main conclusions]: Our results favour a dual role of vicariance and dispersal in driving the historical biogeography and diversification of Misolampus since the early Miocene. We also found evidence for incipient speciation events, underscoring the role of tectonic events and adaptation to local climatic conditions in the diversification of the group.

[Context] Mediterranean 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., [Objectives] We 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 semiarid landscapes of central Spain, the common parsley frog (Pelodytes punctatus) and the common midwife toad (Alytes obstetricans)., [Methods] We 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., [Results] We 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., [Conclusions] This 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.

[Aim]: The contributions of historical biogeography, morphology and climatic niche evolution in shaping species diversification have been typically examined separately. To fill this gap, we assessed the relative role of geologic history, environment and phenotypic trait evolution in lineage diversification of green lizards in the Mediterranean biodiversity hotspot., [Location]: Eurasia and North Africa., [Taxon]: Green lizards (genera Timon and Lacerta)., [Methods]: For all green lizard lineages, we characterized distributional ranges and external morphological traits across discrete biogeographical areas, occupied macro-habitats and climatic niches using environmental variables that represent average and extreme climatic conditions. To assess the contribution of geographical factors in shaping diversity patterns, we evaluated the fit of 24 biogeographical models. We used BAMM and estimated phylogenetic signal to assess the rates of lineage diversification and of phenotypic and climatic niche evolution, and to determine whether these processes occurred steadily or at specific time periods as a response to palaeogeological or palaeoclimatic events. Finally, we tested for associations between phenotypic traits and lineage diversification using trait-dependent diversification analyses (QuaSSE, ES-sim and STRAPP)., [Results]: Biogeographical analyses favoured a dispersal–vicariance model explaining speciation patterns in green lizards, including jump dispersal and constrained dispersal by geographical distance. Lineages accumulated gradually towards the present, with minor divergence in morphological traits and conservatism of climatic niches. In contrast, in the Lacerta agilis lineage, niche evolution may have allowed expansion towards colder environments. Morphological and climatic niche evolution were uncoupled from diversification rates., [Main conclusions]: Biogeographical processes largely explain the constant lineage diversification of green lizards in the Mediterranean Basin since the Miocene, followed by gradual phenotypic divergence unrelated to cladogenesis. Climatic niche conservatism promoted the accumulation of lineages within the Mediterranean, except for L. agilis, where climatic niche evolution might underpin its range spread towards higher latitudes.