Cryptic recombination in the ever-young sex chromosomes of Hylid frogs.

Sex chromosomes are expected to evolve suppressed recombination, which leads to degeneration of the Y and heteromorphism between the X and Y. Some sex chromosomes remain homomorphic, however, and the factors that prevent degeneration of the Y in these cases are not well understood. The homomorphic sex chromosomes of the European tree frogs (Hyla spp.) present an interesting paradox. Recombination in males has never been observed in crossing experiments, but molecular data are suggestive of occasional recombination between the X and Y. The hypothesis that these sex chromosomes recombine has not been tested statistically, however, nor has the X-Y recombination rate been estimated. Here, we use approximate Bayesian computation coupled with coalescent simulations of sex chromosomes to quantify X-Y recombination rate from existent data. We find that microsatellite data from H. arborea, H. intermedia and H. molleri support a recombination rate between X and Y that is significantly different from zero. We estimate that rate to be approximately 10(5) times smaller than that between X chromosomes. Our findings support the notion that very low recombination rate may be sufficient to maintain homomorphism in sex chromosomes.

Density, climate and varying return points: an analysis of long-term population fluctuations in the threatened European tree frog.

Experimental research has identified many putative agents of amphibian decline, yet the population-level consequences of these agents remain unknown, owing to lack of information on compensatory density dependence in natural populations. Here, we investigate the relative importance of intrinsic (density-dependent) and extrinsic (climatic) factors impacting the dynamics of a tree frog (Hyla arborea) population over 22 years. A combination of log-linear density dependence and rainfall (with a 2-year time lag corresponding to development time) explain 75% of the variance in the rate of increase. Such fluctuations around a variable return point might be responsible for the seemingly erratic demography and disequilibrium dynamics of many amphibian populations.

Genetic bottlenecks driven by population disconnection.

Connectivity among populations plays a crucial role in maintaining genetic variation at a local scale, especially in small populations affected strongly by genetic drift. The negative consequences of population disconnection on allelic richness and gene diversity (heterozygosity) are well recognized and empirically established. It is not well recognized, however, that a sudden drop in local effective population size induced by such disconnection produces a temporary disequilibrium in allelic frequency distributions that is akin to the genetic signature of a demographic bottleneck. To document this effect, we used individual-based simulations and empirical data on allelic richness and gene diversity in six pairs of isolated versus well-connected (core) populations of European tree frogs. In our simulations, population disconnection depressed allelic richness more than heterozygosity and thus resulted in a temporary excess in gene diversity relative to mutation drift equilibrium (i.e., signature of a genetic bottleneck). We observed a similar excess in gene diversity in isolated populations of tree frogs. Our results show that population disconnection can create a genetic bottleneck in the absence of demographic collapse.

Stronger transferability but lower variability in transcriptomic- than in anonymous microsatellites: evidence from Hylid frogs.

A simple way to quickly optimize microsatellites in nonmodel organisms is to reuse loci available in closely related taxa; however, this approach can be limited by the stochastic and low cross-amplification success experienced in some groups (e.g. amphibians). An efficient alternative is to develop loci from transcriptome sequences. Transcriptomic microsatellites have been found to vary in their levels of cross-species amplification and variability, but this has to date never been tested in amphibians. Here, we compare the patterns of cross-amplification and levels of polymorphism of 18 published anonymous microsatellites isolated from genomic DNA vs. 17 loci derived from a transcriptome, across nine species of tree frogs (Hyla arborea and Hyla cinerea group). We established a clear negative relationship between divergence time and amplification success, which was much steeper for anonymous than transcriptomic markers, with half-lives (time at which 50% of the markers still amplify) of 1.1 and 37 My, respectively. Transcriptomic markers are significantly less polymorphic than anonymous loci, but remain variable across diverged taxa. We conclude that the exploitation of amphibian transcriptomes for developing microsatellites seems an optimal approach for multispecies surveys (e.g. analyses of hybrid zones, comparative linkage mapping), whereas anonymous microsatellites may be more informative for fine-scale analyses of intraspecific variation. Moreover, our results confirm the pattern that microsatellite cross-amplification is greatly variable among amphibians and should be assessed independently within target lineages. Finally, we provide a bank of microsatellites for Palaearctic tree frogs (so far only available for H. arborea), which will be useful for conservation and evolutionary studies in this radiation.

Recombination is suppressed and variability reduced in a nascent Y chromosome.

Several hypotheses have been elaborated to account for the evolutionary decay commonly observed in full-fledged Y chromosomes. Enhanced drift, background selection and selective sweeps, which are expected to result from reduced recombination, may all share responsibilities in the initial decay of proto-Y chromosomes, but little empirical information has been gathered so far. Here we take advantage of three markers that amplify on both of the morphologically undifferentiated sex chromosomes of the European tree frog (Hyla arborea) to show that recombination is suppressed in males (the heterogametic sex) but not in females. Accordingly, genetic variability is reduced on the Y, but in a way that can be accounted for by merely the number of chromosome copies per breeding pair, without the need to invoke background selection or selective sweeps.

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.

Sex-chromosome evolution of Palearctic tree frogs in space and time

Sexual reproduction is nearly universal in eukaryotes and genetic determination of sex prevails among animals. The astonishing diversity of sex-determining systems and sex chromosomes is yet bewildering. Some taxonomic groups possess conserved and dimorphic sex chromosomes, involving a functional copy (e.g. mammals' X, birds' Z) and a degenerated copy (mammals' Y, birds' W), implying that sex- chromosomes are expected to decay. In contrast, others like amphibians, reptiles and fishes yet maintained undifferentiated sex chromosomes. Why such different evolutionary trajectories? In this thesis, we empirically test and characterize the main hypotheses proposed to prevent the genetic decay of sex chromosomes, namely occasional X-Y recombination and frequent sex-chromosome transitions, using the Palearctic radiation of Hyla tree frogs as a model system. We take a phylogeographic and phylogenetic approach to relate sex-chromosome recombination, differentiation, and transitions in a spatial and temporal framework. By reconstructing the recent evolutionary history of the widespread European tree frog H. arborea, we showed that sex chromosomes can recombine in males, preventing their differentiation, a situation that potentially evolves rapidly. At the scale of the entire radiation, X-Y recombination combines with frequent transitions to prevent sex-chromosome degeneration in Hyla: we traced several turnovers of sex-determining system within the last 10My. These rapid changes seem less random than usually assumed: we gathered evidences that one chromosome pair is a sex expert, carrying genes with key role in animal sex determination, and which probably specialized through frequent reuse as a sex chromosome in Hyla and other amphibians. Finally, we took advantage of secondary contact zones between closely-related Hyla lineages to evaluate the consequences of sex chromosome homomorphy on the genetics of speciation. In comparison with other systems, the evolution of sex chromosomes in Hyla emphasized the existence of consistent evolutionary patterns within the chaotic diversity of flexibility of cold-blooded vertebrates' sex-determining systems, and provides insights into the evolution of recombination. Beyond sex-chromosome evolution, this work also significantly contributed to speciation, phylogeography and applied conservation research. -- La reproduction sexuée est quasi-universelle chez les eucaryotes et le sexe est le plus souvent déterminé génétiquement au sein du règne animal. L'incroyable diversité des systèmes de reproduction et des chromosomes sexuels est particulièrement étonnante. Certains groupes taxonomiques possèdent des chromosomes sexuels dimorphiques et très conservés, avec une copie entièrement fonctionnelle (ex : le X des mammifères, le Z des oiseaux) et une copie dégénérée (ex : le Y des mammifères, le W des oiseaux), suggérant que les chromosomes sexuels sont voués à se détériorer. Cependant les chromosomes sexuels d'autres groupes tels que les amphibiens, les reptiles et les poissons sont pour la plupart indifférenciés. Comment expliquer des trajectoires évolutives si différentes? Au cours de cette thèse, nous avons étudié empiriquement les processus évolutifs pouvant maintenir les chromosomes sexuels intacts, à savoir la recombinaison X-Y occasionnel ainsi que les substitutions fréquentes de chromosomes sexuels, en utilisant les rainettes Paléarctiques du genre Hyla comme modèle d'étude. Nous avons adopté une approche phylogéographique et phylogénétique pour appréhender les événements de recombinaison, de différenciation et de transitions de chromosomes sexuels dans un contexte spatio-temporel. En retraçant l'histoire évolutive récente de la rainette verte H. arborea, nous avons mis en évidence que les chromosomes sexuels pouvaient recombiner chez les mâles, empêchant ainsi leur différenciation, et que ce processus avait le potentiel d'évoluer très rapidement. A l'échelle plus globale de la radiation, il apparait que les phénomènes de recombinaison X-Y soient également accompagnés de substitutions de chromosomes sexuels, et participent de concert au maintien de chromosomes sexuels intacts dans les populations: le système de détermination du sexe des rainettes a changé plusieurs fois au cours des 10 derniers millions d'années. Ces transitions fréquentes ne semblent pas aléatoires: nous avons identifié une paire de chromosomes qui présente des caractéristiques présageant d'une spécialisation dans le déterminisme du sexe (notamment car elle possède des gènes importants pour cette fonction), et qui a été réutilisée plusieurs fois comme tel chez les rainettes ainsi que d'autres amphibiens. Enfin, nous avons étudié l'hybridation entre différentes espèces dans leurs zones de contact, afin d'évaluer si l'absence de différenciation entre X et Y jouaient un rôle dans les processus génétiques de spéciation. Outre son intérêt pour la compréhension de l'évolution des chromosomes sexuels, ce travail contribue de manière significative à d'autres domaines de recherche tels que la spéciation, la phylogéographie, ainsi que la biologie de la conservation.

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X-Y Recombination.

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

Energy substrate utilization during nightly vocal activity in three species of Scinax (Anura/Hylidae)

Anuran amphibians exhibit different patterns of energy substrate utilization that correlate with the intensity of vocal and locomotor activities. Given the remarkable differences among species in breeding and feeding strategies, and the different ways energy is used in the whole animal, the suggested correlations between calling and locomotor behavior and the level of energy substrates in the muscles responsible for such activities are more complex than previously reported. We explored the relationships between calling and locomotor behavior and energy supply to trunk and hindlimb muscles, respectively, within the ecologically diverse tree-frog genus Scinax. Specifically, we measured the relative amount of carbohydrates and lipids in these two groups of muscles, and in the liver of three species of Scinax that differ in vocal and locomotor performance, and compared our results with those of two other species for which comparable data are available. We also compared the contents of lipids and carbohydrates of conspecific males collected at the beginning and after 4 h of calling activity. The stomach content to potential feeding opportunities across species was also assessed in both groups of males. Scinax hiemalis and S. rizibilis exhibit comparatively low and episodic calling during long periods of activity whereas S. crospedospilus calls at higher rates over shorter periods. Male S. hiemalis had highest levels of trunk muscle glycogen followed by those of S. rizilbilis and S. crospedospilus, respectively. There was no correlation between total lipid content in trunk muscle and calling rate among different species, suggesting that other metabolic aspects may be responsible for the energetic support for vocal activity. The levels of lipids and carbohydrates in trunk and hindlimb muscles and liver of males collected at the beginning and 4 h into the calling period were similar across species, so the extent of energetic reserves does not appear to constrain vocal or locomotor activity. Finally, we found exceptionally high levels of carbohydrates and lipids in the liver of S. rizibilis, a trait perhaps related to a long and demanding breeding period.

The specialized reproductive mode of the treefrog Aplastodiscus perviridis (Anura : Hylidae)

Males of the South American treefrog Aplastodiscus perviridis construct concealed subterranean nests. Using a complex courtship behavior that involves tactile stimuli and vocalizations, males guide the females to the subterranean nests where eggs are laid. Embryos and facultatively oophagous tadpoles (at least in stage 25) stay in subterranean nests until flooding transports them to ponds or streams. This is a rare reproductive mode previously known for few species in the Hyla albosignata and H. albofrenata complexes. Based on similarities of reproductive mode we suggest a monophyletic origin for Aplastodiscus and these complexes of Hyla.

Hylin a1, the first cytolytic peptide isolated from the arboreal South American frog Hypsiboas albopunctatus ('spotted treefrog')

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Resource use and coexistence of two syntopic hylid frogs (Anura, Hylidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

B-chromosomes in two Brazilian populations of Dendropsophus nanus (Anura, Hylidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reproductive modes of Atlantic forest hylid frogs: A general overview and the description of a new mode

This paper reports a new reproductive mode in anurans, observed for the green treefrog, Hyla leucopygia, a species from the Atlantic Forest of Brazil and a member of the H. albosignata group. The reproductive mode of H. leucopygia is defined as follows: aquatic eggs and embryos in subterranean constructed nests; subsequent to flooding, feeding tadpoles in ponds or streams. We suggest that the new reproductive mode is widespread among the species in the H. albosignata group. The specialized courtship behaviors of H. leucopygia, characterized by vocalizations and a stereotyped sequence of mutual touches between male and female, are described. A review of the reproductive modes of hylid frogs is provided, with 11 modes recognized for the Atlantic Forest hylids and 12 modes For hylids around the world.

Cytochemistry and polarization microscopy of amphibian sperm cell nuclei presumed to differ in basic protein composition

The pattern of availability of free DNA phosphates, and the kind of DNA-protein complex arrangement, both induced by nuclear basic proteins, and the richness in arginine residues in these proteins were investigated cytochemically and cytophysically in spermatozoa of the South-American Hylidae species, Hyla fuscovaria and Hyla biobeba. The aim was to demonstrate differences at the level of sperm histones in two species of Hyla until recently considered to be congeneric. The results indicated differences in the spermatozoal nuclear basic proteins and DNA-protein complexes when the two species were compared. The spermatozoa of Hyla biobeba were assumed to be likely to contain a Bloch's ''type 3'' protein type (intermediate sperm basic protein), similarly to Hyla species of North and Central America. on the other hand, the data obtained for the spermatozoa of Hyla fuscovaria indicated that they contain a protamine or protamine-like protein, differing from Hyla biobeba and Hyla species of North and Central America. It is suggested that the differences reported here may be genus-specific, since Hyla fuscovaria has recently been reclassified as Scinax fuscovaria based on parameters other than sperm histone types. These findings are in agreement with the general view of a wide variability in sperm nuclear proteins in the Anura group.