Gaston-Denis Guex

COMPETITION AMONG TADPOLES OF COEXISTING HEMICLONES OF HYBRIDOGENETIC RANA ESCULENTA: SUPPORT FOR THE FROZEN NICHE VARIATION MODEL

Vertebrate animals reproducing without genetic recombination typically are hybrids, which have large ranges, are locally abundant, and live in disturbed or harsh habitats. This holds for the hemiclonal hybridogenetic frog Rana esculenta: it is widespread in Europe and commonly is found in disturbed habitats such as gravel pits. We hypothesize that its widespread occurrence may either be the result of natural selection for a single hemiclone acting as a broadly adapted “general‐purpose” genotype, or of interclonal selection, which maintains multiple hemiclones that each are relatively narrowly adapted and perform differently across environments, that is, the Frozen Niche Variation model. We tested these competing hypotheses using 1000‐L outdoor artificial ponds to rear tadpoles of the parental species (Rana lessonae [LL] and Rana ridibunda [RR]) alone, and each of three hemiclones of Rana esculenta (GUT1, GUT2, GUT3) alone, and in mixed hemiclonal populations from hatching to metamorphosis. Tadpoles of three coexisting hemiclones from a single natural population (near Gütighausen, Switzerland) were reared in both two‐ and three‐way mixtures in equal total numbers at high and low density. For each species and hemiclone, the proportion of tadpoles metamorphosing decreased as the density of tadpoles increased, with the three hemiclones spanning the range of values exhibited by the two parental species. LL and GUT1 tadpoles produced the highest proportion of metamorphs, whereas tadpoles of RR produced the fewest metamorphs at both densities. GUT1 tadpoles also produced the largest metamorphs at low density, GUT2 and GUT3 tadpoles produced smaller metamorphs than did GUT1 tadpoles at the low density, but the three hemiclones did not differ from each other at high density. The parental species (LL and RR) were intermediate in metamorphic size to the hemiclones at low density, but all genotypes converged on a similar size at high density. Length of the larval period also was affected by density, but its effect was dependent on genotype. GUT1 tadpoles had the shortest larval period at the low density, but larval period was longer and not different between GUT1, GUT3, and LL at high density. RR tadpoles had the longest larval period at both densities. The most dramatic results were that three genotypes (GUT1, GUT2, and RR) maintained rank order and increased days to metamorphosis from low to high density, whereas two genotypes (GUT3 and LL) changed rank order and decreased days to metamorphosis from low to high density. Mixtures of hemiclones in two‐ and three‐way combinations facilitated the proportion of tadpoles metamorphosing for GUT1 and GUT2 at both densities, but only at the low density for GUT3 tadpoles. Results from this experiment are incompatible with the General‐Purpose Genotype model as a global explanation of hybrid abundance in these frogs. Alternatively, the Frozen Niche Variation prediction of general performance superiority of clonal mixtures relative to single clone populations is strongly supported. The data confirm that fitness advantages of hemiclones change, depending on the environment, such that in temporally and spatially heterogeneous habitats like ponds, frequency‐dependent selection among hemiclones may promote coexistence in hemiclonal assemblages. Yet, differential dispersal or colonization ability and historical factors affecting hemiclone distribution may also be important in shaping patterns of clonal coexistence.

Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs

Interspecies transfer of mitochondrial (mt) DNA is a common phenomenon in plants, invertebrates and vertebrates, normally linked with hybridization of closely related species in zones of sympatry or parapatry. In central Europe, in an area north of 48°N latitude and between 8° and 22°E longitude, western Palaearctic water frogs show massive unidirectional introgression of mtDNA: 33.7% of 407 Rana ridibunda possessed mtDNA specific for Rana lessonae. By contrast, no R. lessonae with R. ridibunda mtDNA was observed. That R. ridibunda with introgressed mitochondrial genomes were found exclusively within the range of the hybrid Rana esculenta and that most hybrids had lessonae mtDNA (90.4% of 335 individuals investigated) is evidence that R. esculenta serves as a vehicle for transfer of lessonae mtDNA into R. ridibunda. Such introgression has occurred several times independently. The abundance and wide distribution of individuals with introgressed mitochondrial genomes show that R. lessonae mt genomes work successfully in a R. ridibunda chromosomal background despite their high sequence divergence from R. ridibunda mtDNAs (14.2–15.2% in the ND2/ND3 genes). Greater effectiveness of enzymes encoded by R. lessonae mtDNA may be advantageous to individuals of R. ridibunda and probably R. esculenta in the northern parts of their ranges.

OVERWINTER SURVIVAL OF RANA LESSONAE AND ITS HEMICLONAL ASSOCIATE RANA ESCULENTA

In central Europe, the hybridogenetic water frog Rana esculenta is a sexual parasite that only persists in the presence of the parental species, R. lessonae, with which it must mate in order to reproduce. R. esculenta is a superior larval competitor, and adult females are about three times more fecund than R. lessonae. This suggests that, in the absence of some balancing advantage to R. lessonae, R. esculenta should drive the parental species locally extinct, closely followed by itself. We measured annual survival rates over six years at two widely separated ponds using modern capture–mark–recapture methods to test whether differences in adult survival might contribute to the persistence of the water frog system. We marked 856 adult frogs and recovered 182 at least once. The data did not deviate significantly from the assumptions of the Cormack-Jolly-Seber model at either pond. There were no detectable differences in recapture probabilities between taxa, but females were, overall, less likely to be recaptured. Survival was higher for R. esculenta in all but one year. Therefore, some mechanism other than adult survival must be invoked to explain the persistence of this unusual breeding system. There was considerable year-to-year variation in survival, ranging from 6% over the winter of 1996–1997, to 98% over the winter of 1995–1996. Although the two ponds are separated by 35 km, pond identity did not contribute to the minimum models. A composite measure of winter severity constructed from a principal components analysis of weather data during the course of the study had a very close relationship with survival (R. lessonae, r = 0.905; R. esculenta, r = 0.889). Survival was lowest in winters with low minimum temperatures coupled with high and variable maximum temperatures. Corresponding Editor: B. Sinervo.

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frogRana esculenta and its parental species

Hybrid water frogsRana esculenta reproduce by hybridogenesis: one parental genome (ofRana lessonae) is excluded in the germ line, the other (ofRana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher inRana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed inRana ridibunda genomes by the restriction endonucleaseStul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1–5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes allRana ridibunda chromosomes. RrS1 represents about 2.5% of the genome ofRana ridibunda; it may represent as little as 0.2% of the genome ofRana lessonae, and cannot be detected inXenopus laevis frogs orSalamandra salamandra andTriturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

Gametogenesis of intergroup hybrids of hemiclonal frogs

European water frog hybrids Rana esculenta (R. ridibundaxR. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.

Genetic divergence and evolution of reproductive isolation in eastern Mediterranean water frogs.

Water frogs [genus Pelophylax (Rana)] that occur around the eastern Mediterranean Sea provide an opportunity to study early stages of speciation. The geography of the eastern Mediterranean region has changed dramatically since the Middle Miocene as a result of motions of adjoining lithospheric plates and regional-scale vertical crustal motions (uplift and subsidence). For several hundred thousand years between 6 and 5 million years ago (Mya), the Mediterranean basin was isolated from the Atlantic Ocean, and became desiccated (the Messinian Salinity Crisis; MSC). Geological data suggest that the endemic water frog lineage on Cyprus was isolated by the flooding of the Mediterranean basin by salt water at the end of the MSC, circa 5.5–5.3 Mya. This suggests a rate of uncorrected genetic divergence of approximately 1.1% per million years (My). Divergence time estimates based on this rate are in good agreement with the chronology of events in the history of crustal deformation and landscape development in the eastern Mediterranean region.

Bioaccumulation of organochlorine pesticides in frogs of the Rana esculenta complex in central Italy

Concentrations of commonly used organochlorine pesticides (OCPs) were determined in tissues of 23 adult and 24 larval water frogs of two coexisting species (Rana lessonae and the hemiclonal hybrid R. esculenta) and in the water of their breeding pond in an agricultural zone in Umbria, central Italy, where increased occurrence of infectious diseases and distinctly oversized tadpoles were recently observed. The concentrations of OCP in tissues of both species were lower than those in the water of their breeding pond, except for DDT, which was more concentrated in adult frogs than in pond water (bioaccumulation factor 7 for R. lessonae, 15 for R. esculenta). Total OCP concentration and adult body weight were positively correlated for both species, which is consistent with bioaccumulation. In accord, adults contained higher OCP concentrations than tadpoles. Oversized tadpoles had higher OCP concentrations than normal tadpoles. Mean OCP concentrations in individual organs were about an order of magnitude higher than those in whole-frog homogenates. They were highest in brain, higher in ventral than in dorsal skin, and moderately high in ovaries; transmission of bioaccumulation loads to the next generation is therefore possible. The observed OCP concentrations appear too low to directly cause mortality in water frogs, but effects of cumulative exposure to low-level pollutants and their synergistic interactions with the effects of other natural and anthropogenic environmental stressors are unknown.

Masked Damage: Mutational Load in Hemiclonal Water Frogs

Hemiclonal hybrids of Western Palearctic water frogs of the Rana esculenta complex transmit only one parental genome to their offspring without recombination (hybridogenesis). Such genomes are thus prone to accumulate deleterious mutations. The frog complex is unique among hybridogens in that hemiclonal hybrids occur in both sexes. This provides the opportunity of using experimental crosses to produce offspring possessing two clonal genomes of various origins and thereby study their homozygous and heterozygous effects on fitness. Here we review work that made use of this possibility to assess the evolutionary consequences of clonal inheritance in water frogs. Overall, these studies indicate that clonally transmitted genomes bear a substantial load of fixed deleterious mutations, yet these mutations appear to have minor effects on fitness in the heterozygous state. We also point out potential mechanisms for episodic recombination by which otherwise clonal genomes may be purged of deleterious alleles, and we present evidence for such episodic recombination to occur in natural populations of hybridogenetic frogs. Finally, we provide an outlook on work in progress that exploits the peculiarities of this system to obtain relevant estimates of the frequency of segregating lethal mutations in sexual populations of water frogs.

Balancing a Cline by Influx of Migrants: A Genetic Transition in Water Frogs of Eastern Greece

Variation patterns of allozymes and of ND3 haplotypes of mitochondrial DNA reveal a zone of genetic transition among western Palearctic water frogs extending across northeastern Greece and European Turkey. At the western end of the zone, allozymes characteristic of Central European frogs known as Pelophylax ridibundus predominate, whereas at the eastern end, alleles characteristic of western Anatolian water frogs (P. cf. bedriagae) prevail. The ND3 haplotypes reveal 2 major clades, 1 characteristic of Anatolian frogs, the other of European; the European clade itself has distinct eastern and western subclades. Both the 2 major clades and the 2 subclades overlap within the transition zone. Using Bayesian model selection methods, allozyme data suggest considerable immigration into the Nestos River area from eastern and western populations. In contrast, the ND3 data suggest that migration rates are so high among all locations that they form a single panmictic unit; the best model for allozymes is second best for mitochondrial DNA (mtDNA). Nuclear markers (allozymes), which have roughly 4 times as deep a coalescent history as mtDNA data and thus may reflect patterns over a longer time, indicate that eastern and western refugial populations have expanded since deglaciation (in the last 10,000 years) and have met near the Nestos River, whereas the mtDNA with its smaller effective population size has already lost the signal of partitioning into refugia.

Factors contributing to the maintenance of the genetic polymorphism at the locus LDH-B in the pool frog, Rana lessonae

We tested for environmental factors that may lead to balancing selection and to the maintenance of a genetic polymorphism at the enzyme locus lactate dehydrogenase B (LDH-B) in the pool frog, Rana lessonae. We raised tadpoles individually in a factorial experiment in which we manipulated temperature, food level, and food quality. The only statistically significant difference among LDH-B genotypes was in growth rate, with the heterozygote performing best. Although the difference was not significant, heterozygotes also tended to perform best for size at metamorphosis. However, heterozygotes did not perform best in terms of other traits (age at metamorphosis and rates of survival and metamorphosis), where differences among LDH-B genotypes were also not significant. The size of the effect of LDH-B genotype depended on the environment, which suggests that the locus may be selectively neutral in some environments. There were no genotype–environment interactions in the sense that reaction norms along environmental gradients did not cross. When we raised tadpoles in groups, e/e homozygotes had a significantly higher body mass and developed at the significantly highest rate. In addition, there may be a trade-off between larval and adult performance: adult frogs show a different ranking in performance of LDH-B genotypes than tadpoles do. These results suggest that this genetic polymorphism is maintained through heterozygote advantage, possibly in conjunction with antagonistic pleiotropy. Résumé : Nous avons tenté de déterminer quels facteurs écologiques peuvent contribuer à une sélection d’équilibre et au maintien du polymorphisme génétique au locus lactate dyshydrogénase B (LDH-B) chez la grenouille Rana lessonae. Nous avons élevé des têtards individuellement au cours d’une expérience de type factoriel où la température, la quantité de nourriture et la qualité de la nourriture étaient prédéterminées. Le taux de croissance s’est avéré la seule variable à différe r significativement d’un génotype LDH-B à l’autre et ce sont les hétérozygotes qui avaient la meilleure performance. De même, ce sont les hétérozygotes qui avaient la taille la plus élevée à la métamorphose, mais la différence avec les autres génotypes n’était pas significative. Cependant, les hétérozygotes n’étaient pas favorisés quant aux autres variables (âge à la métamorphose, survie, taux de métamorphose) puisque les différences entre les divers génotypes LDH-B n’étaient pas significatives. L’importance de l’influence du génotype dépend de l’environnement, ce qui semble indiquer que le locus peut n’avoir aucun effet sélectif en certains milieux. Il n’y a pas d’interactions génotype–environnement dans la mesure où les normes de réaction le long de gradients environnementaux ne s’entrecroisent pas. Au cours d’expériences d’élevage en groupe, les têtards homozygotes se sont avérés avoir une masse significativement plus élevée et se sont développés à un rythme significativement plus rapide. De plus, il se peut qu’il y ait un compromis entre les performances larvaire et adulte : chez le s grenouilles adultes, la performance des divers génotypes LDH-B n’est pas la même que chez les têtards. Ces résultats indiquent que ce polymorphisme génétique se maintient grâce à la performance supérieure des hétérozygotes, peut-être combinée à la pleiotropie antagoniste. [Traduit par la Rédaction]