Diverse aging rates in ectothermic tetrapods provide insights for the evolution of aging and longevity.

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.

Sex-related differences in aging rate are associated with sex chromosome system in amphibians.

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an 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 and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the "unguarded X/Z effect") or repeat-rich Y/W chromosome (the "toxic Y/W effect") could accelerate aging in the heterogametic sex in some vertebrate clades.

Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe.

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno- and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed-ploidy populations without any parental species (i.e. all-hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all-hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.

Assessing risk and guidance on monitoring of Batrachochytrium dendrobatidis in Europe through identification of taxonomic selectivity of infection.

Amphibians are globally threatened, but not all species are affected equally by different threatening processes. This is true for the threat posed by the chytridiomycete fungus (Batrachochytrium dendrobatidis). We compiled a European data set for B. dendrobatidis to analyze the trends of infection in European amphibians. The risk of infection was not randomly distributed geographically or taxonomically across Europe. Within countries with different prevalence, infection was nonrandom in certain amphibian taxa. Brown frogs of the genus Rana were unlikely to be infected, whereas frogs in the families Alytidae and Bombinatoridae were significantly more likely to be infected than predicted by chance. Frogs in the 2 families susceptible to B. dendrobatidis should form the core of attempts to develop spatial surveillance studies of chytridiomycosis in Europe. Ideally, surveys for B. dendrobatidis should be augmented by sampling the widespread genus Pelophylax because this taxon exhibits geographically inconsistent overinfection with B. dendrobatidis and surveillance of it may facilitate recognition of factors causing spatial variability of infection intensity. Several European amphibian taxa were not represented in our data set; however, surveillance of unsampled species should also occur when warranted.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma.

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax).

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size-differences in the length of the serum albumin intron-1 and the RanaCR1, a non-LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon-specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.

Coexistence of diploid and triploid hybrid water frogs: population differences persist in the apparent absence of differential survival.

UNLABELLED: The role of differential selection in determining the geographic distribution of genotypes in hybrid systems has long been discussed, but not settled. The present study aims to asses the importance of selection in structuring all-hybrid Pelophylax esculentus populations. These populations, in which the parental species (P. lessonae with genotype LL and P. ridibundus with genotype RR) are absent, have pond-specific proportions of diploid (LR) and triploid (LLR and LRR) genotypes. RESULTS: With data from 12 Swedish ponds, we first show that in spite of significant changes in genotype proportions over time, the most extreme ponds retained their differences over a six year study period. The uneven distribution of genotypes among ponds could be a consequence of differential selection varying among ponds (selection hypothesis), or, alternatively, of different gamete production patterns among ponds (gamete pattern hypothesis). The selection hypothesis was tested in adults by a six year mark-recapture study in all 12 ponds. As the relative survival and proportion of LLR, LR and LRR did not correlate within ponds, this study provided no evidence for the selection hypothesis in adults. Then, both hypotheses were tested simultaneously in juvenile stages (eggs, tadpoles, metamorphs and one year old froglets) in three of the ponds. A gradual approach to adult genotype proportions through successive stages would support the selection hypotheses, whereas the presence of adult genotype proportions already at the egg stage would support the gamete pattern hypothesis. The result was a weak preference for the gamete pattern hypothesis. CONCLUSIONS: These results thus suggest that selection is of little importance for shaping genotype distributions of all-hybrid populations of P. esculentus, but further studies are needed for confirmation. Moreover, the study provided valuable data on genotype-specific body lengths, adult survival and sex ratios.

Gamete types, sex determination and stable equilibria of all-hybrid populations of diploid and triploid edible frogs (Pelophylax esculentus).

BACKGROUND: Triploid individuals often play a key role in speciation by hybridization. An understanding of the gamete types (ploidy and genomic content) and stability of hybrid populations with triploid individuals is therefore of importance for exploring the role of hybridization in evolution. The all-hybrid populations of the edible frog, Pelophylax esculentus, are unique in their composition and genetic dynamics: Diploid (genotype LR) and triploid (LLR and LRR) hybrids depend on each other's different gamete contributions for successful reproduction and maintenance of the populations, as the parental genotypes P. lessonae (LL) and P. ridibundus (RR) are absent among adults. This study provides data and interpretations on gamete types and sex determination that are essential for understanding the function, evolutionary potential and threats of this intriguing system. RESULTS: Dissection of metamorphs from a crossing experiment confirmed that sex determination is an XX-XY system with the Y confined to the L genome. From microsatellite analysis of parents and offspring from the crossings, gamete frequencies could be deduced: Triploids of both sexes mostly made haploid gametes with the genome they had in double dose, however LLR females also made approximately 10% LL gametes by automixis. LR frogs showed much variation in their gamete production. In LRR-rich populations, their LR sperm production was sufficiently high (22%) to explain the observed proportion of LRR males, the formation of which has not previously been understood. A model was constructed to calculate equilibrium genotype proportions for different population types on the basis of the gamete proportions found. These equilibria agreed well with empirical literature data. CONCLUSION: If population differentiation with respect to genotype proportions is really driven by gamete patterns, as strongly suggested by the present study, all-hybrid populations constitute not one, but several intrinsically different breeding systems. Tetraploidization could occur if the survival or fertility of both males and females increased. Whether introduction of hybrid or parental species individuals would threaten the all-hybrid populations cannot be predicted without further knowledge on the mechanisms behind non-hybrid inviability, but at least R genomes with Y factor are predicted to be invasive, if introduced, and could bring the populations to collapse.

From clonal to sexual hybrids: genetic recombination via triploids in all-hybrid populations of water frogs.

Speciation via interspecific hybrids is very rare in animals, as compared to plants. Whereas most plants overcome the problem of meiosis between different chromosome sets by tetraploidization, animal hybrids often escape hybrid sterility by clonal reproduction. This comes at the expense of genetic diversity and the ability to purge deleterious mutations. However, here we show that all-hybrid populations of diploid (LR) and triploid (LLR and LRR) water frogs (Pelophylax esculentus) have secondarily acquired sexual reproduction. First, in a crossing experiment analyzed with microsatellite markers, triploid hybrids of both sexes and genotypes (LLR and LRR) recombined their homospecific genomes. Second, the great majority of natural populations investigated had low multilocus linkage disequilibrium, indicating a high recombination rate. As predicted from mating system models, the L genome had constant, low levels of linkage disequilibrium, whereas linkage disequilibrium in the R genome showed a significant reduction with increasing proportion of recombining triploids. This direct evidence of sexual reproduction in P. esculentus calls for a change of the conventional view of hybridogens as clonally reproducing diploids. Rather, hybridogens can be independent sexually reproducing units with an evolutionary potential.

Reproduction and hybrid load in all-hybrid populations of Rana esculenta water frogs in Denmark.

All-hybrid populations of the water frog, Rana esculenta, are exceptional in consisting of independently and to some extent sexually reproducing interspecific hybrids. In most of its range R. esculenta reproduces hemiclonally with one of the parental species, R. lessonae or R. ridibunda, but viable populations of diploid and triploid hybrids, in which no individuals of the parental species have been found, exist in the northern part of the range. We test the hypothesis that nonhybrids arise every year in these all-hybrid populations, but die during larval development. Microsatellite markers were used to determine the genotypes of adults and abnormal and healthy offspring in three all-hybrid populations of R. esculenta in Denmark. Of all eggs and larvae, 63% developed abnormally or died, with some being nonhybrid (genomes matching one of the parental species), many being aneuploid (with noninteger chromosome sets), a few being tetraploid, and many eggs possibly being unfertilized. The 37% surviving and apparently healthy froglets were all diploid or triploid hybrids. In all three populations, gametogenesis matched the pattern previously described for all-hybrid R. esculenta populations in which most triploid adults have two R. lessonae genomes. This pattern was surprising for the one population in which triploid adults had two R. ridibunda genomes, because here it leads to a deficiency of gametes with an R. lessonae genome and should compromise the stability of this population. We conclude that faulty gametogenesis and mating between frogs with incompatible gametes induce a significant hybrid load in all-hybrid populations of R. esculenta, and we discuss compensating advantages and potential evolutionary trajectories to reduce this hybrid load.