The annual number of breeding adults and the effective population size of syntopic newts (Triturus cristatus, T-marmoratus)

Pond-breeding amphibians are deme-structured organisms with a population genetic structure particularly susceptible to demographic threats. We estimated the effective number of breeding adults (N-b) and the effective population size (N-e) of the European urodele amphibians Triturus cristatus (the crested newt) and T. marmoratus (the marbled newt), using temporal shifts in microsatellite allele frequencies. Eight microsatellite loci isolated from a T. cristatus library were used, five of which proved polymorphic in T. marmoratus, albeit with high frequencies of null alleles at two loci. Three ponds in western France were sampled, situated 4-10 kilometres apart and inhabited by both species. Parent-offspring cohort comparisons were used to measure N-b; samples collected at time intervals of nine or 12 years, respectively, were used to measure N-e. The adult population census size (N) was determined by mark-recapture techniques. With one exception, genetic distances (F-ST) between temporal samples were lower than among populations. N-b ranged between 10.6 and 101.8 individuals, N-e ranged between 9.6 and 13.4 individuals. For the pond where both parameters were available, N-b/N (overall range: 0.10-0.19) was marginally larger than N-e/N (overall range: 0.09-0.16), which is reflected in the temporal stability of N. In line with the observed differences in reproductive life-histories between the species, N-b/N ratios for newts were about one order of magnitude higher than for the anuran amphibian Bufo bufo. Despite of the colonization of the study area by T. cristatus only some decades ago, no significant genetic bottleneck could be detected. Our findings give rise to concerns about the long-term demographic viability of amphibian populations in situations typical for European landscapes

A new vertebrate species native to the British isles: Bufo spinosus Daudin, 1803 in Jersey

Recent molecular and morphological studies have shown that Bufo bufo and B. spinosus are genetically distinct and morphologically diagnosable across a relatively narrow contact zone in northern France and should be regarded as different species. However, the species identity of the neighbouring populations of Bufo on the British Channel Island of Jersey has not been investigated. We here present new molecular (a mtDNA RFLP assay plus sequences of the nuclear RAG1 gene) and morphological evidence that these populations are to be assigned to B. spinosus, and can thus be considered an addition to the native British herpetofauna. Jersey toad populations are declining and have a distinct breeding ecology compared to other populations in mainland Britain. We discuss the results in the light of amphibian conservation efforts in Jersey.Fieldwork and sampling in Jersey was conducted under licence from the States of Jersey Environment Department and with support from Amphibian and Reptile Conservation. Other funds were 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).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

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

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

The crested newt Triturus cristatus recolonized temperate Eurasia from an extra-Mediterranean glacial refugium

We assess the role of the Carpathians as an extra-Mediterranean glacial refugium for the crested newt Triturus cristatus. We combine a multilocus phylogeography (one mitochondrial protein-coding gene, three nuclear introns, and one major histocompatibility complex gene) with species distribution modelling (projected on current and Last Glacial Maximum climate layers). All genetic markers consistently show extensive genetic variation within and genetic depletion outside the Carpathians. The species distribution model suggests that most of the current range was unsuitable at the Last Glacial Maximum, but a small suitable area remained in the Carpathians. Triturus cristatus dramatically expanded its postglacial range, colonizing much of temperate Eurasia from a glacial refugium in the Carpathians. Within the Carpathians, T.cristatus persisted in multiple geographically discrete regions, providing further support for a Carpathian refugia within refugia' scenario.(c) 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114, 574-587.Naturali

Data concatenation, Bayesian concordance and coalescent-based analyses of the species tree for the rapid radiation of Triturus newts

The phylogenetic relationships for rapid species radiations are difficult to disentangle. Here we study one such case, namely the genus Triturus, which is composed of the marbled and crested newts. We analyze data for 38 genetic markers, positioned in 3-prime untranslated regions of protein-coding genes, obtained with 454 sequencing. Our dataset includes twenty Triturus newts and represents all nine species. Bayesian analysis of population structure allocates all individuals to their respective species. The branching patterns obtained by data concatenation, Bayesian concordance analysis and coalescent-based estimations of the species tree differ from one another. The data concatenation based species tree shows high branch support but branching order is considerably affected by allele choice in the case of heterozygotes in the concatenation process. Bayesian concordance analysis expresses the conflict between individual gene trees for part of the Triturus species tree as low concordance factors. The coalescent-based species tree is relatively similar to a previously published species tree based upon morphology and full mtDNA and any conflicting internal branches are not highly supported. Our findings reflect high gene tree discordance due to incomplete lineage sorting (possibly aggravated by hybridization) in combination with low information content of the markers employed (as can be expected for relatively recent species radiations). This case study highlights the complexity of resolving rapid radiations and we acknowledge that to convincingly resolve the Triturus species tree even more genes will have to be consulted

Testing an hypothesis of hybrid zone movement for toads in France

Hybrid zone movement may result in substantial unidirectional introgression of selectively neutral material from the local to the advancing species, leaving a genetic footprint. This genetic footprint is represented by a trail of asymmetric tails and displaced cline centres in the wake of the moving hybrid zone. A peak of admixture linkage disequilibrium is predicted to exist ahead of the centre of the moving hybrid zone. We test these predictions of the movement hypothesis in a hybrid zone between common (Bufo bufo) and spined toads (B. spinosus), using 31 nuclear and one mtDNA SNPs along a transect in the northwest of France. Average effective selection in Bufo hybrids is low and clines vary in shape and centre. A weak pattern of asymmetric introgression is inferred from cline discordance of seven nuclear markers. The dominant direction of gene flow is from B. spinosus to B. bufo and is in support of southward movement of the hybrid zone. Conversely, a peak of admixture linkage disequilibrium north of the hybrid zone suggests northward movement. These contrasting results can be explained by reproductive isolation of the B. spinosus and B. bufo gene pools at the southern (B. spinosus) side of the hybrid zone. The joint occurrence of asymmetric introgression and admixture linkage disequilibrium can also be explained by the combination of low dispersal and random genetic drift due to low effective population sizes

Interspecific introgression of MHC genes in Triturus newts: evidence from multiple contact zones

The major histocompatibility complex (MHC) genes are central to the adaptive immune response in vertebrates. Selection generally maintains high MHC variation because the spectrum of recognized pathogens depends on MHC polymorphism. Novel alleles favoured by selection originate by interallelic recombination or de novo mutations but may also be acquired by introgression from related species. However, the extent and prevalence of MHC introgression remain an open question. In this study, we tested for MHC introgression in six hybrid zones formed by six Triturus newt species. We sequenced and genotyped the polymorphic second exons of the MHC class I and II genes and compared their interspecific similarity at various distances from the centre of the hybrid zone. We found evidence for introgression of both MHC classes in the majority of examined hybrid zones, with support for a more substantial class I introgression. Furthermore, the overall MHC allele sharing outside of hybrid zones was elevated between pairs of Triturus species with abutting ranges, regardless of the phylogenetic distance between them. No effect of past hybrid zone movement on MHC allele sharing was found. Finally, using previously published genome-wide data, we demonstrated that MHC introgression was more extensive than genome-wide introgression, supporting its adaptive potential. Our study thus provides evidence for the prevalence of MHC introgression across multiple Triturus hybrid zones, indicating that MHC introgression between divergent hybridizing species may be widespread and adaptive.Animal science

Corresponding Mitochondrial DNA and Niche Divergence for Crested Newt Candidate Species

Genetic divergence of mitochondrial DNA does not necessarily correspond to reproductive isolation. However, if mitochondrial DNA lineages occupy separate segments of environmental space, this supports the notion of their evolutionary independence. We explore niche differentiation among three candidate species of crested newt (characterized by distinct mitochondrial DNA lineages) and interpret the results in the light of differences observed for recognized crested newt species. We quantify niche differences among all crested newt (candidate) species and test hypotheses regarding niche evolution, employing two ordination techniques (PCA-env and ENFA). Niche equivalency is rejected: all (candidate) species are found to occupy significantly different segments of environmental space. Furthermore, niche overlap values for the three candidate species are not significantly higher than those for the recognized species. As the three candidate crested newt species are, not only in terms of mitochondrial DNA genetic divergence, but also ecologically speaking, as diverged as the recognized crested newt species, our findings are in line with the hypothesis that they represent cryptic species. We address potential pitfalls of our methodology