Iñigo Martínez-Solano

Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain

During the summers of 1997, 1998 and 1999 mass mortality episodes of post-metamorphic common midwife toads (Alytes obstetricans) occurred in a protected area in central Spain. The population suffered a sharp decline, disappearing from 86% of the ponds where they were known to reproduce some years ago. Scanning electron microscopy and histological techniques revealed the presence of a chytridiomycosis infection in the skin of the toads. This evidence supports chytridiomycosis as the most plausible cause of the decline of the species in the area. This is the first report of an apparent chytridium-caused amphibian decline in Europe.

Mitochondrial DNA phylogeography of Lissotriton boscai (Caudata, Salamandridae): evidence for old, multiple refugia in an Iberian endemic

In Europe, southern peninsulas served as refugia during cold periods in the Pleistocene, acting both as centres of origin of endemisms and as sources from which formerly glaciated areas were recolonized during interglacial periods. Previous studies have revealed that within the main refugial areas, intraspecific lineages often survived in allopatric refugia. We analysed two mitochondrial markers (nad4, control region, ∼1.4 kb) in 103 individuals representing the entire distribution of Lissotriton boscai, a newt endemic to the western Iberian Peninsula. We inferred the evolutionary history of the species through phylogenetic, phylogeographic and historical demographic analyses. The results revealed unexpected, deep levels of geographically structured genetic variability. We identified two main evolutionary lineages, each containing three well‐supported clades. The first historical split involved populations from central‐southwestern coastal Portugal and the ancestor of all the remaining populations around 5.8 million years ago. Both lineages were subsequently fragmented into different population groups between 2.5 and 1.2 million years ago. According to nested clade analysis, at lower hierarchical levels the patterns suggest restricted gene flow with isolation by distance, whereas at higher levels the clades exhibit signatures of contiguous range expansion. Bayesian Skyline Plots show recent bottlenecks, followed by demographic expansions in all lineages. The significant genetic structure found is consistent with long‐term survival of populations in allopatric refugia, supporting the ‘refugia‐within‐refugia’ scenario for southern European peninsulas. The comparison of our results with other co‐distributed species highlights the generality of this hypothesis for the Iberian herpetofauna and suggests that Mediterranean refuges had more relevance for the composition and distribution of present biodiversity patterns than currently acknowledged. We briefly discuss the taxonomic and conservation implications of our results.

Chytrid fungus infection related to unusual mortalities of Salamandra salamandra and Bufo bufo in the Peñalara Natural Park, Spain

Chytridiomycosis is a fatal disease associated with amphibian population declines and extinctions worldwide. In a protected area in central Spain, the Penalara Natural Park, the disease almost extirpated the population of Alytes obstetricans over only a few years, but did not apparently affect other amphibians. We present new observations documenting the occur- rence of the disease in other species. In 2001-2003 we collected over 400 larvae or recently metamorphosed individuals of Salamandra salamandra and also several dead individuals of Bufo bufo. The analysis of the skin of post-metamorphic specimens revealed the presence of chytrid sporangia and discharge tubes in both species. According to measures of larval abundances in 1999 and 2003 the population of S. salamandra has suffered a marked decline but no significant trend was observed for B. bufo. We discuss the possible role of chytridiomycosis in the decline of S. salamandra and comment on the differential susceptibility exhibited by various species in the amphibian community at Penalara.

Ever-Young Sex Chromosomes in European Tree Frogs

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.

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

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.

Factors Influencing Occupancy of Breeding Ponds in a Montane Amphibian Assemblage

Abstract We analyzed relationships between presence and absence of seven amphibian species and characteristics of breeding ponds (physical, vegetation, substrate, spatial and surrounding habitat variables) at a protected montane area in Central Spain. We used classification trees for description and prediction of amphibian selection of breeding sites. Some factors identified in our models represent general preferences of the species, whereas others are consistent with demographic processes, including recent expansions and local declines of some species, that had been previously detected in the area. We discuss implications of a detailed knowledge of local habitat determinants for conservation.

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.

Spatial scales for the management of amphibian populations

Conservation programs must be based on a detailed knowledge of the distribution of communities and populations, but this changes with the spatial scale of observation. In this context, we examined the multiscale patterns of spatial variation of a montane amphibian assemblage in Central Spain. The largest spatial scale examined (in a range of 1200 m) accounted for the maximum variability in both species richness and larval abundance, while the smallest scale examined (pond) was responsible for the maximum variation of larval abundances of many individual species. Habitat characteristics seemed to be more related to spatial variation of the amphibian assemblage at a particular spatial scale (in a range of 75 m), and distances among ponds were in part responsible for variation of larval abundance, but not species richness.

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.

Evolutionary history of Ichthyosaura alpestris (Caudata, Salamandridae) inferred from the combined analysis of nuclear and mitochondrial markers

Widespread species with morphologically and ecologically differentiated populations are key to understand speciation because they allow investigating the different stages of the continuous process of population divergence. The alpine newt, Ichthyosaura alpestris, with a range that covers a large part of Central Europe as well as isolated regions in all three European Mediterranean peninsulas, and with strong ecological and life-history differences among populations, is an excellent system for such studies. We sampled individuals across most of the range of the species, and analyzed mitochondrial (1442 bp) and nuclear (two nuclear genes -1554 bp- and 35 allozyme loci) markers to produce a time-calibrated phylogeny and reconstruct the historical biogeography of the species. Phylogenetic analyses of mtDNA data produced a fully resolved topology, with an endemic, Balkan clade (Vlasina) which is sister to a clade comprising an eastern and a western group. Within the former, one clade (subspecies I. a. veluchiensis) is sister to a clade containing subspecies I. a. montenegrina and I. a. serdara as well as samples from southern Romania, Bosnia-Herzegovina, Serbia and Bulgaria (subspecies I. a. reiseri and part of I. a. alpestris). Within the western group, populations from the Italian peninsula (subspecies I. a. apuana and I. a. inexpectata) are sister to a clade containing samples from the Iberian Peninsula (subspecies I. a. cyreni) and the remainder of the samples from subspecies I. a. alpestris (populations from Hungary, Austria, Poland, France, Germany and the larger part of Romania). Results of (∗)BEAST analyses on a combined mtDNA and nDNA dataset consistently recovered with high statistical support four lineages with unresolved inter-relationships: (1) subspecies I. a. veluchiensis; (2) subspecies I. a. apuana+I. a. inexpectata; (3) subspecies I. a. cyreni+part of subspecies I. a. alpestris (the westernmost populations, plus most Romanian populations); and (4) the remaining populations, including subspecies I. a. serdara, I. a. reiseri and I. a. montenegrina and part of subspecies I. a. alpestris, plus samples from Vlasina. Our time estimates are consistent with ages based on the fossil record and suggest a widespread distribution for the I. alpestris ancestor, with the split of the major eastern and western lineages during the Miocene, in the Tortonian. Our study provides a solid, comprehensive background on the evolutionary history of the species based on the most complete combined (mtDNA+nDNA+allozymes) dataset to date. The combination of the historical perspective provided by coalescent-based analyses of mitochondrial and nuclear DNA variation with individual-based multilocus assignment methods based on multiple nuclear markers (allozymes) also allowed identification of instances of discordance across markers that highlight the complexity and dynamism of past and ongoing evolutionary processes in the species.