Isolation and characterization of sixteen polymorphic microsatellite loci in the Western Spadefoot, Pelobates cultripes (Anura: Pelobatidae) via 454 pyrosequencing

The Western Spadefoot, Pelobates cultripes (Anura, Pelobatidae), is endemic to the Iberian Peninsula and southeastern France, with isolated populations in the Atlantic coast of France. Its populations are fragmented and it is considered Near Threatened by the IUCN. Here we describe the development of sixteen polymorphic microsatellite loci in this species. Polymorphism was assessed in 95 individuals from five Iberian populations. The number of alleles and expected heterozygosity ranged from 3 to 14 and 0.20 to 0.76, respectively. Eight loci cross-amplified in the closely related and Endangered Moroccan Spadefoot toad, Pelobates varaldii. These markers will be useful to address questions about the ecology, population genetics and evolutionary history of P. cultripes, including information on effective population size, habitat use and dispersal patterns, which are essential for the efficient management of the fragmented populations characteristic of most of its range.This research was funded by grants CGL2008-04271-C02-01/BOS and CGL2011-28300 (Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, Spain, and FEDER) and PPII10-0097- 4200 (Junta de Comunidades de Castilla la Mancha) to IMS. JGR is supported by the Consejo Superior de Investigaciones Científicas of Spain (CSIC) and the European Social Fund (ESF) (JAE-pre PhD fellowship), and IMS is a ‘Ramón y Cajal’ postdoctoral fellow supported by the Spanish Ministerio de Ciencia e Innovación and the Universidad de Castilla la Mancha.Peer Reviewe

Ever-young sex chromosomes in European tree frogs

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.Non-recombining sex chromosomes are expected to undergo evolutionary decay, ending up genetically degenerated, as has happened in birds and mammals. Why are then sex chromosomes so often homomorphic in cold-blooded vertebrates? One possible explanation is a high rate of turnover events, replacing master sex-determining genes by new ones on other chromosomes. An alternative is that X-Y similarity is maintained by occasional recombination events, occurring in sex-reversed XY females. Based on mitochondrial and nuclear gene sequences, we estimated the divergence times between European tree frogs (Hyla arborea, H. intermedia, and H. molleri) to the upper Miocene, about 5.4–7.1 million years ago. Sibship analyses of microsatellite polymorphisms revealed that all three species have the same pair of sex chromosomes, with complete absence of X-Y recombination in males. Despite this, sequences of sex-linked loci show no divergence between the X and Y chromosomes. In the phylogeny, the X and Y alleles cluster according to species, not in groups of gametologs. We conclude that sex-chromosome homomorphy in these tree frogs does not result from a recent turnover but is maintained over evolutionary timescales by occasional X-Y recombination. Seemingly young sex chromosomes may thus carry old-established sex-determining genes, a result at odds with the view that sex chromosomes necessarily decay until they are replaced. This raises intriguing perspectives regarding the evolutionary dynamics of sexually antagonistic genes and the mechanisms that control X-Y recombination.Financial support was provided by the Swiss National Science Foundation (http://www.snf.ch/E/Pages/default.aspx; grants 3100A0-108100 and 31003A-129894 to NP) and the Ministerio de Ciencia e Innovación (http://www.micinn.es/portal/site/MICINN/; grant CGL2008-04271-C02/01/BOS to IMS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

The collection of amphibians from Madrid at the Museo Nacional de Ciencias Naturales and its utility in conservation

[EN] We present the revised catalogue of the amphibians deposited in the “Colección de Anfibios y Reptiles” of the Museo Nacional de Ciencias Naturales that have been collected in the Comunidad Autónoma of Madrid. It consists of 2272 records of 18 species, including all species present in Madrid except Hyla meridionalis. We evaluate the collection on the basis of the representation of the specimens collected in relation with: 1) the present distribution of each amphibian species in Madrid, considering the number of UTM 10x10 km grid cells represented in the collection in relation with the total number of grids occupied by each species in Madrid; 2) the number of intraspecific subunits that are relevant from a conservation perspective (subspecies or groups of populations that are differentiated on ecological or genetic grounds) that are represented in the collection. Representation values range from 0% (Hyla meridionalis) to 50% (Triturus alpestris). We also include information on the conservation status of some populations from historical collection sites and conclude that at least 10 breeding sites have disappeared as a consequence of human activities, with the subsequent local extinction of their associated amphibian populations. We have also documented the disappearance of some species from unaltered areas. Finally, we discuss the utility of scientific collections in facing practical aspects of the management and conservation of endangered groups like amphibians.[ES] Se presenta el catálogo revisado de los anfibios de la Colección de Anfibios y Reptiles del Museo Nacional de Ciencias Naturales colectados en la Comunidad de Madrid. En total, consta de 2272 entradas correspondientes a 18 especies, que incluyen todas las presentes en la Comunidad excepto Hyla meridionalis. Se valora la colección en función del grado de representatividad de los ejemplares de la colección en relación con: 1) la distribución actual de las especies de anfibios en Madrid, considerando el % de cuadrículas UTM de 10x10 km del total del área de distribución de cada especie que se encuentran representadas en la colección; 2) el número de unidades infraespecíficas relevantes desde el punto de vista de la conservación (subespecies o grupos de poblaciones diferenciados ecológica o genéticamente) representados en la colección para cada especie. Los valores de representatividad obtenidos oscilan entre el 0% (Hyla meridionalis) y el 50% (Triturus alpestris). Además, se aporta información acerca de algunas de las poblaciones asentadas en los lugares de colecta históricos, lo que permite concluir que al menos 10 puntos de reproducción han desaparecido durante los últimos años como resultado de la actividad humana, con la consiguiente extinción local de esas poblaciones de anfibios. Por otro lado, en ciertos casos también se ha documentado la desaparición de especies en áreas aparentemente inalteradas. Finalmente, se discute la utilidad de las colecciones museológicas para afrontar aspectos prácticos relacionados con la gestión y conservación de grupos amenazados como los anfibios.El trabajo de IMS estáfinanciado por una beca predoctoral MNCN-CSIC-CAM.Peer reviewe

A review of the palaeoclimatic inference potential of Iberian Quaternary fossil batrachians

Ecological suitability analysis aims to model the potential or probable distribution of species using environmental variables and available species occurrence information as predictors. Most of the European fossil Quaternary amphibians belong to extant species, and being physiologically ectothermic animals, their current ecological niches could become a reliable inference tool to infer past environmental conditions. However, this expectation has never been properly verified. The validity and accuracy of palaeoclimatic inferences based on batrachian species ranges is tested on the Iberian Peninsula, using both palaeofaunistic and Recent distribution data, and an updated database of georeferenced species occurrences is provided. The difficulties of using current geographic information to represent the full spectrum of environmental conditions at which a species occurs are critically examined. A palaeofaunistic review of the role played by historical factors as dispersal limitations for present amphibian species ranges is presented using the available phylogeographical scenarios. Virtual hypothetical taxa can be devised by considering the distributions of several species together and relating their known joint presences with the environmental conditions in these locations. Species distribution models based on these virtual taxa provide the best advisable inferential procedure. For direct raw inferences of the mutual climatic range method, contrary to expectations, the combined taxa sets do not increase their accuracy with the number of species included. This preliminary review shows that Iberian amphibian palaeoclimatic inferences are both valid and reliable enough, a sufficient approximation to complement other techniques. The precision, however, is quite variable among taxa, reflecting the effect of non-climaticPeer reviewe

Hybridization during altitudinal range shifts: nuclear introgression leads to extensive cyto-nuclear discordance in the fire salamander

Ecological models predict that, in the face of climate change, taxa occupying steep altitudinal gradients will shift their distributions, leading to the contraction or extinction of the high-elevation (cold-adapted) taxa. However, hybridization between ecomorphologically divergent taxa commonly occurs in nature and may lead to alternative evolutionary outcomes, such as genetic merger or gene flow at specific genes. We evaluate this hypothesis by studying patterns of divergence and gene flow across three replicate contact zones between high- and low-elevation ecomorphs of the fire salamander (Salamandra salamandra) that have experienced altitudinal range shifts over the current postglacial period. Strong population structure with high genetic divergence in mitochondrial DNA suggests that vicariant evolution has occurred over several glacial–interglacial cycles and that it has led to cryptic differentiation within ecomorphs. In current parapatric boundaries, we do not find evidence for local extinction and replacement upon postglacial expansion. Instead, parapatric taxa recurrently show discordance between mitochondrial and nuclear markers, suggesting nuclear-mediated gene flow across contact zones. Isolation with migration models support this hypothesis by showing significant gene flow across all five parapatric boundaries. Together, our results suggest that, while some genomic regions, such as the mitochondria, may follow morphologic species traits and retreat to isolated mountain tops, other genomic regions, such as nuclear markers, may flow across parapatric boundaries, sometimes leading to a complete genetic merger. We show that despite high ecologic and morphologic divergence over prolonged periods of time, hybridization allows for evolutionary outcomes alternative to extinction and replacement of taxa in response to climate change.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.During the preparation of this manuscript, RJP was funded by the European Union’s Horizon 2020 research and innovation programme, under the Marie Sklodowska-Curie grant agreement No 658706.Peer reviewe

How complex is the Bufo bufo species group?

Species delineation remains one of the most challenging tasks in the study of biodiversity, mostly owing to the application of different species concepts, which results in contrasting taxonomic arrangements. This has important practical consequences, since species are basic units in fields like ecology and conservation biology. We here review molecular genetic evidence relevant to the systematics of toads in the Bufo bufo species group (Anura, Bufonidae). Two studies recently published in this journal (Recuero et al., MPE 62: 71-86 and García-Porta et al., MPE 63: 113-130) addressed this issue but reached opposing conclusions on the taxonomy of the group (four versus two species). In particular, allozyme data in the latter paper were interpreted as evidence for hybridization across species (between B. bufo-B. spinosus and B. bufo-B. verrucosissimus). We tested claims for hybridization through re-analysis of allozyme data for individuals instead of populations, to be able to distinguish between sympatry with and without admixture, and found no evidence of hybridization across taxa. We propose alternative explanations for the observed patterns that García-Porta et al. (2012) failed to consider. In the absence of unequivocal evidence for hybridization and introgression, we reject the proposal to downgrade Bufo spinosus and Bufo verrucosissimus to the subspecies level.IMS is a "Ramón y Cajal" postdoctoral fellow supported by the Spanish Ministerio de Ciencia e Innovación and the Universidad de Castilla la Mancha. Partial funds for this work were provided by grants CGL2008-04271-C02-01/BOS and CGL2011-28300 (Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, FEDER) and PPII10-0097-4200 (Junta de Comunidades de Castilla la Mancha) to IMS.Peer Reviewe

On the geographic distribution of Nemognatha plaumanni Borchmann, 1942 (Coleoptera: Meloidae): new records from Venezuela, with a 4500 km range extension

Nemognatha plaumanni Borchmann, 1942 (Coleoptera: Meloidae: Nemognathinae) was described from a single specimen found in Brazil, and since its description no additional information has been published on the species. During a field survey in Venezuela, we found one individual morphologically assignable to the species. In this note, we report and discuss this finding, which, together with the revision of the entomological collections of the Magyar Természettudományi Múzeum (Hungarian Natural History Museum, HNHM, Budapest, Hungary), the Natural History Museum (NHM, London, UK), and the Museo del Instituto de Zoología Agrícola (MIZA, Maracay, Venezuela), has led us to acknowledge the presence of the species in Venezuela, therefore extending the known distribution range of the species by more than 4500 km. We discuss the possibility that N. plaumanni might in fact correspond to a complex of cryptic species distributed over this vast range, an hypothesis that has to be tested with further field- and lab-work.This work has been funded by the project grant CGL2010–15786 (Ministerio de Ciencia e Innovación, Spain).Peer reviewe

Multilocus species tree analyses resolve the radiation of the widespread Bufo bufo species group (Anura, Bufonidae)

et al.New analytical methods are improving our ability to reconstruct robust species trees from multilocus datasets, despite difficulties in phylogenetic reconstruction associated with recent, rapid divergence, incomplete lineage sorting and/or introgression. In this study, we applied these methods to resolve the radiation of toads in the Bufo bufo (Anura, Bufonidae) species group, ranging from the Iberian Peninsula and North Africa to Siberia, based on sequences from two mitochondrial and four nuclear DNA regions (3490. base. pairs). We obtained a fully-resolved topology, with the recently described Bufo eichwaldi from the Talysh Mountains in south Azerbaijan and Iran as the sister taxon to a clade including: (1) north African, Iberian, and most French populations, referred herein to Bufo spinosus based on the implied inclusion of populations from its type locality and (2) a second clade, sister to B. spinosus, including two sister subclades: one with all samples of Bufo verrucosissimus from the Caucasus and another one with samples of B. bufo from northern France to Russia, including the Apennine and Balkan peninsulas and most of Anatolia. Coalescent-based estimations of time to most recent common ancestors for each species and selected subclades allowed historical reconstruction of the diversification of the species group in the context of Mediterranean paleogeography and indicated a long evolutionary history in this region. Finally, we used our data to delimit the ranges of the four species, particularly the more widespread and historically confused B. spinosus and B. bufo, and identify potential contact zones, some of which show striking parallels with other co-distributed species.IMS is a “Ramón y Cajal” postdoctoral fellow supported by the Spanish Ministerio de Ciencia e Innovación and the Universidad de Castilla la Mancha. The Synthesis Project (http://www.synthesys.info/) of the European Union partially supported this study (Ref.: HU-TAF-181). Partial funds were also provided by the Spanish Ministerio de Ciencia e Innovación (Ref.: CGL2008-04271-C02-01/ BOS) and Junta de Comunidades de Castilla la Mancha (Ref.: PPII10-0097-4200) to IMS, Hungarian Scientific Research Fund (OTKA K77841) to JV and Ministry of Education and Science of Republic of Serbia (Grant No. 173025) to JCI.Peer Reviewe

Multilocus assessment of phylogenetic relationships in Alytes (Anura, Alytidae)

With the advent of large multilocus datasets, molecular systematics is experiencing very rapid progress, but important challenges remain regarding data analysis and interpretation. Midwife toads (genus Alytes) exemplify two of the most widespread problems for accurate phylogenetic reconstruction: discerning the causes of discordance between gene trees, and resolving short internodes produced during rapid, successive splitting events. The three species in subgenus Baleaphryne ( A. maurus, A. dickhilleni and A. muletensis), the sister group to A. obstetricans, have disjunct and highly restricted geographical ranges, which are thought to result from old vicariant events affecting their common ancestor, but their phylogenetic relationships are still unresolved. In this study we re-address the phylogeny of Alytes with a special focus on the relationships in Baleaphryne with a multilocus dataset including > 9000 base pairs of mitochondrial DNA and four nuclear markers (3142. bp) in all recognized taxa, including all subspecies of A. obstetricans. Both concatenation and species tree analyses suggest that A. muletensis, endemic to the Balearic island of Mallorca, is the sister taxon to a clade comprising the southeastern Iberian endemic A. dickhilleni and the North African A. maurus. This scenario is consistent with palaeogeological evidence associated with the fragmentation of the Betic-Rifean Massif, followed by the opening of the Strait of Gibraltar. On the other hand, analyses of intraspecific variation in A. obstetricans are inconclusive regarding relationships between major clades and conflict with current subspecific taxonomy.This work was supported through Project “Genomics and Evolutionary Biology” cofinanced by North Portugal Regional Operational Programme 2007/2013 (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF); the Program Operacional Factores de Competitividade (COMPETE), and by national funds from Fundação para a Ciência e a Tecnologia (FCT), through the research Project PTDC/BIA-BEC/099915/2008 to HG; and partial funds provided by grants CGL2008-04271-C02-01/BOS and CGL2011-28300 (Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, Spain, and FEDER) and PPII10-0097- 4200 (Junta de Comunidades de Castilla la Mancha and FEDER) to IMS, who is currently funded by Project “Biodiversity, Ecology and Global Change”, co-financed by North Portugal Regional Operational Programme 2007/2013 (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). BMC is supported by a PhD grant from FCT (SFRH/BD/60305/2009), and HG is supported by a postdoctoral grant from FCT (SFRH/BPD/26555/2006).Peer Reviewe

Morphological and molecular data to describe a hybrid population of the Common toad (Bufo bufo) and the Spined toad (Bufo spinosus) in western France

The use of hyper-variable markers across species is often hindered by low cross-species amplification success, a reduced level of polymorphism or a high frequency of null alleles. However, optimizing sets of reliable and informative markers that can be consistently amplified and scored across taxa is key to address questions about patterns of genetic diversity and structure, hybridization and speciation. Here we present 14 newly developed microsatellite markers in the Spined toad (Bufo spinosus), assess their polymorphism in two Iberian populations and test for cross-species amplification in the closely related Common toad (Bufo bufo). We then use the 12 loci co-amplifying in both species to the study of a morphologically intermediate population (Moyaux) from the contact zone in northwest France as well as reference populations of the two species from both sides of the contact zone. Individuals from Moyaux had mtDNA haplotypes of the two species and were identified as hybrids in analyses with software NewHybrids. These results provide solid evidence for ongoing hybridization between B. bufo and B. spinosus, with no apparent restrictions to gene flow.This research was funded by grants CGL2008-04271-C02-01/BOS and CGL2011-28300 (Ministerio de Ciencia e Innovación), Ministerio de Economía y Competitividad and FEDER) and PPII10-0097-4200 (Junta de Comunidades de Castilla la Mancha) to IMS. J. Gutiérrez-Rodríguez was supported by the Consejo Superior de Investigaciones Científicas of Spain (CSIC) and the European Social Fund (ESF) with a JAE-pre PhD fellowship. IMS was funded by the project ‘Biodiversity, Ecology and Global Change’, co-financed by North Portugal Regional Operational Programme 2007/2013 (ON.2–O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF) and by funding from the Spanish Severo Ochoa Program (SEV-2012-0262). IMS’s stay in Leiden was supported by a Naturalis Temminck fellowship.Peer reviewe