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Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.

RrS1-like sequences of water frogs (genus Pelophylax) display varied genomic organization, whereas the centromeric hybridization pattern reveals species-specific differences. Using fluorescent in situ hybridization, Pelophylax cf. bedriagae, Pelophylax kurtmuelleri, and Pelophylax ridibundus showed a hybridization signal at centromeres of chromosomes 1-5, but in P. kurtmuelleri the medium-small chromosome labeled was 10 rather than 8. Pelophylax cretensis had almost 16 of 26 centromeres labeled, as did Pelophylax lessonae from Poland when its chromosomes are hybridized with a homologous probe. When StuI-digested genomic DNA was hybridized with RrS1 probe, hybridization ladders for P. ridibundus from Poland have evenly spaced steps (about 100 bp) of uniform intensity from about 200 bp upward. Steps in hybridization ladders from circum-Aegean taxa vary in intensity: larger, odd-numbered steps are often fainter. A strong double band (800/900 bp) in Anatolian P. cf. bedriagae, emphasized by a weak 700 bp band, distinguishes them from P. kurtmuelleri from the Peloponnisos, in which the 900 bp band is almost absent. The ladder in P. cretensis lacks odd-numbered steps. A and B repeats, observed originally within the RrS1 satellite of P. ridibundus, occur also in the circum-Aegean frogs and in P. lessonae, Pelophylax epeiroticus, Pelophylax saharicus, and Pelophylax shqipericus. It is plausible that AB dimers or ABB trimers rather than A or B monomers correspond to functional/evolutionary units. The presence of regions similar to yeast CDEs and mammalian CENP-B boxes suggests a role for RrS1 sequences in centromere organization.

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.

Genotypic variation at six of 67 microsatellite loci we developed from Rana ridibunda, containing di- or trinucleotide simplesequence repeats, confirms the value of microsatellites as an evolutionary genetic tool for studying western Palearctic water frogs, a model system characterized by clonal reproduction in natural hybrid lineages. R. ridibunda, Rana lessonae. and their hemiclonal hybrid Rana esculenta, which transmits only its ridibunda genome to gametes, are highly variable in central Poland and northern Switzerland: with one exception (a locus that is part of a coding region), each locus was polymorphic in each taxon when scorable, and the number of alleles was high (in Swiss lessonae-esculenta populations, 1-6 per locus, total 13, for ridibunda genomes; 1-9 per locus, total 23, for lessonae genomes; 3 shared by both genomes). Estimated repeat numbers ranged from 3 to 14 for trinucleotide- and from 6 to 27 for dinucleotide-repeats. Nonamplifying (null) alleles, problematic because they underestimate genotypic variability if undetected, occur in ridibunda genomes of Swiss R. esculenta at one locus: apparent lessonae null alleles at another locus were subsequently resolved for several segregating alleles evidenced by bands of much lower intensity. Two loci had multiple ridibunda alleles in Swiss R. esculenta and thus discriminate between hemiclones. with a resolution superior to that of allozymes. Adding these two loci to our protein electrophoretic data (7 loci) more than doubled the number of hemiclones detected, showed that several allozyme-defined hemiclones are composites of multiple microsatellite hemiclones, and revealed clonal diversity in populations that are uniclonal by allozyme data. The extent to which microsatellite-defined hemiclones result from primary hybridizations or from subsequent mutations in ridibunda genomes of R. esculenta lineages remains to be determined. A sample of newly metamorphosed R. ridibunda from hybrid × hybrid matings collected at a Swiss population in the fall had a significant heterozygote excess at the two loci that varied in ridibunda genomes; this shows that most, probably all, of them originated from inter- rather than intrahemiclonal matings, in accord with the idea that the usual hybrid × hybrid inviability in lessonae-esculenta systems is caused by deleterious recessive alleles on the clonally inherited ridibunda genomes. Three of four microsatellite loci tested amplified in most species of western Palearctic water frogs.

Rana esculenta progeny from experimental crosses (using R. esculenta from natural lineages and F1 Ram ridihztncia x Rnrlo lessonarr), reared in artificial ponds and in cages immersed in natural breeding ponds, showed three different types of malformations reflecting developmental disturbances, each affecting few individuals overall. Giant tadpoles arrested development at the foot-paddle stage, independent from a size at metamorphosisitime to metamorphosis trade-off that may be under selection. The fused nostrils/eyeless syndrome probably reflects a disturbed gastrulation. The observed polymely, affecting the hind limbs, may result from infectious agents in pond water used to inoculate the tanks. Although known parentage and distribution among rearing tanks for each of the three malformations exclude a simple genetic basis and a direct influence of particular human-caused factors, it is not possible to further discriminate among genetic, biotically-induced, and non-biotically induced environmental causes of the developmental disturbances and their interactions. Direct experimental approaches will be needed to disentangle these possibilities and to assess their relative contributions to the observed decline of amphibian populations.

A family of interspersed repetitive elements, RlBamHI, with sequence similarity to the transposase of hAT DNA transposons, occurs in genomes of eight western Palaearctic water frog taxa and the brown frog Rana dalmatina, but was not detected in Xenopus laevis or Salamandra salamandra. RlBamHI elements are not tandemly arrayed, are dispersed across all chromosomes although not uniformly distributed, and based on dot-blot hybridizations may constitute as much as approximately 10% of the genomes of Rana lessonae, Rana ridibunda, and Rana perezi, but only approximately 1% of that of Rana saharica. Eleven nucleotide sequences of a 572 bp fragment from the nine taxa are very similar (pairwise differences 0.4–8.1% for nucleotides, 0.6–18.2% for amino acids), and all share a single open reading frame across the whole RlBamHI fragment. The reading frame is maintained despite several indels, most of which are multiples of 3 bp, but a pair of which in one species alters and, after 13 codons, restores the reading frame. It is possible that the reading frame is selectively maintained, suggesting recent or even present transposition capacity. The amino acid sequences encoded by RlBamHI elements, but not the nucleotide sequences themselves, reveal the similarity of RlBamHI to members of the widespread hAT superfamily of DNA transposons. Amino acid sequence comparisons permitted no convincing phylogenetic placement of RlBamHI among 32 representative hAT transposons across organisms, probably reflecting both the genetic divergence of RlBamHI elements and the paucity of aligned residues available for analysis. Among DNA transposons, a genomic fraction of 10% is extraordinarily high. Phylogenetic analyses of Rana RlBamHI sequences match poorly with independent genetic and molecular phylogenies; the elements compared probably are not orthologous, which renders their sequences inadequate for reconstructing organismal phylogenetic histories.

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.

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.

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.

Abstract Animal taxa of interspecies hybrid origin that have derived obligatory clonal reproductive modes leading to genealogical persistence demand clear, stable names as labels to refer to them. We recommend (1) that clonal hybrid taxa distinguished by the combination of parental genomes they contain be recognized; (2) that they be referred to, consistently for each group studied, either with formal names or informal ones; and (3) that the existing taxonomic species category be used for them rather than creating additional special nomenclatural categories for the several non-Mendelian reproductive modes.

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.

AIM: Our aims were to assess the phylogeographic patterns of genetic diversity in eastern Mediterranean water frogs and to estimate divergence times using different geological scenarios. We related divergence times to past geological events and discuss the relevance of our data for the systematics of eastern Mediterranean water frogs. LOCATION: The eastern Mediterranean region. METHODS: Genetic diversity and divergence were calculated using sequences of two protein-coding mitochondrial (mt) genes: ND2 (1038 bp, 119 sequences) and ND3 (340 bp, 612 sequences). Divergence times were estimated in a Bayesian framework under four geological scenarios representing alternative possible geological histories for the eastern Mediterranean. We then compared the different scenarios using Bayes factors and additional geological data. RESULTS: Extensive genetic diversity in mtDNA divides eastern Mediterranean water frogs into six main haplogroups (MHG). Three MHGs were identified on the Anatolian mainland; the most widespread MHG with the highest diversity is distributed from western Anatolia to the northern shore of the Caspian Sea, including the type locality of Pelophylax ridibundus. The other two Anatolian MHGs are restricted to south-eastern Turkey, occupying localities west and east of the Amanos mountain range. One of the remaining three MHGs is restricted to Cyprus; a second to the Levant; the third was found in the distribution area of European lake frogs (P. ridibundus group), including the Balkans. MAIN CONCLUSIONS: Based on geological evidence and estimates of genetic divergence we hypothesize that the water frogs of Cyprus have been isolated from the Anatolian mainland populations since the end of the Messinian salinity crisis (MSC), i.e. since c. 5.5-5.3 Ma, while our divergence time estimates indicate that the isolation of Crete from the mainland populations (Peloponnese, Anatolia) most likely pre-dates the MSC. The observed rates of divergence imply a time window of c. 1.6-1.1 million years for diversification of the largest Anatolian MHG; divergence between the two other Anatolian MHGs may have begun about 3.0 Ma, apparently as a result of uplift of the Amanos Mountains. Our mtDNA data suggest that the Anatolian water frogs and frogs from Cyprus represent several undescribed species.

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.

A 5′ truncated non-LTR CR1-like retrotransposon, named RanaCR1, was identified in the serum albumin intron-1 (SAI-1) of at least seven species of western Palearctic water frogs (WPWF). Based on sequence similarity of the carboxy-terminal region (CTR) of ORF2 and/or the highly conserved 3′ untranslated region (3′ UTR), RanaCR1-like elements occur also in the genome of Xenopus tropicalis and Rana temporaria. Unlike other CR1 elements, RanaCR1 contains a CA microsatellite in its 3′ UTR. The low nucleotide diversity of the 3′ UTR compared to the CTR and to SAI-1 suggests that this region still plays a role in WPWF, either as a structure-stabilizing element, or within a species-specific transcriptional network. Length variation of water frog SAI-1 sequences is caused by deletions that extend in some cases beyond the 5′ or 3′ ends of RanaCR1, probably a result of selection for structural and functional stability of the primary transcript. The impact of RanaCR1 on SAI-1 evolution is also indicated by the significant negative correlation between the length of both SAI-1 and RanaCR1 and the percentage GC content of RanaCR1. Both SAI-1 and RanaCR1 sequences support the sister group relationship of R. perezi and R. saharica, which are placed in the phylogenetic tree at a basal position, the sister clade to other water frog taxa. It also supports the monophyly of the R. lessonae group; of Anatolian water frogs (R. cf. bedriagae), which are not conspecific with R. bedriagae, and of the European ridibunda group. Within the ridibunda clade, Greek frogs are clearly separated, supporting the hypothesis that Balkan water frogs represent a distinct species. Frogs from Atyrau (Kazakhstan), the type locality of R. ridibunda, were heterozygous for a ridibunda and a cf. bedriagae specific allele.

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.

European water frog hybrids Rana esculenta (R. ridibunda×R. 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.

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.

European water frog hybrids Rana esculenta ( Rana ridibunda × Rana lessonae ) reproduce hemiclonally, transmitting only their ridibunda genome to gametes. We compared fitness-related larval life-history traits of natural R. esculenta from Poland with those of the two sympatric parental species and of newly generated F 1 hybrids. Compared with either parental species, F 1 hybrid offspring had higher survival, higher early growth rates, a more advanced developmental stage by day 49, and earlier metamorphosis, but similar mass at metamorphosis. R. esculenta from natural lineages had trait values intermediate between those of F 1 offspring and of the two parental species. The data support earlier observations on natural R. esculenta that had faster larval growth, earlier metamorphosis, and higher resistance to hypoxic conditions compared with either parental species. Observing larval heterosis in F 1 hybrids in survival, growth rate, and time to metamorphosis, however, at an even higher degree than in hybrids from natural lineages, demonstrates that heterosis is spontaneous and results from hybridity per se rather than from subsequent interclonal selection; in natural lineages the effects of hybridity and of clonal history are confounded. This is compelling evidence for spontaneous heterosis in hybrid clonals. Results on hemiclonal fish hybrids ( Poeciliopsis ) showed no spontaneous heterosis; thus, our frog data are not applicable to all hybrid clonals. Our data do show, however, that heterosis is an important potential source for the extensively observed ecological success of hybrid clonals. We suggest that heterosis and interclonal selection together shape fitness of natural R. esculenta lineages.

Hybrid water frogs Rana esculenta reproduce by hybridogenesis: one parental genome (of Rana lessonae) is excluded in the germ line, the other (of Rana 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 in Rana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed in Rana ridibunda genomes by the restriction endonuclease Stul, 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 all Rana ridibunda chromosomes. RrS1 represents about 2.5% of the genome of Rana ridibunda; it may represent as little as 0.2% of the genome of Rana lessonae, and cannot be detected in Xenopus laevis frogs or Salamandra salamandra and Triturus 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.

European water frog hybrids Rana esculenta reproduce hemiclonally, by hybridogenesis: In the germ line they exclude the genome of the parental species Rana lessonae and produce haploid, unrecombined gametes with a genome of the parental species Rana ridibunda. These hybrids coexist with and depend as sexual parasites on the host parental species R. lessonae (the L-E population system); matings with R. lessonae restore somatic hybridity in each generation of R. esculenta. We investigated 15 L-E system populations in northern Switzerland, which is outside R. ridibunda’s native range. Frequency of hybrids in samples varied from 8% in marsh ponds to 100% in gravel pits and forest ponds. Clonal diversity (variation among R. ridibunda genomes of hybrids), detected by six protein electrophoretic marker loci, revealed a total of eight hemiclones and locally ranged from uniclonal populations in southern parts of the survey region to six coexisting hemiclones in the north. All alleles distinguishing hemiclones occur commonly in the nearest native R. ridibunda populations of east-central Europe; the most probable source of clonal diversity in our samples is multiple clone formation by primary hybridizations in the sympatry area of R. ridibunda and R. lessonae and subsequent dispersal of hemiclonal lineages. A positive correlation between amount of clonal diversity and hybrid frequency, predicted by the Frozen Niche Variation (FNV) model (each hemiclone is characterized by a relatively narrow niche, coexistence is possible through niche partitioning), was not found; this contrasts with hemiclonally reproducing fish hybrids (Poeciliopsis). Historical factors, such as availability of different colonizing hemiclones may be strong enough to override the signal from operation of the FNV. Zusammenfassung Europaische Wasserfrosch-Hybriden Rana esculenta pflanzen sich hemiklonal fort, durch Hybridogenese: In der Keimbahn schliessen sie das Genom der Elternart Rana lessonae aus und produzieren haploide, unrekombinierte Gameten mit einem Genom der Elternart Rana ridibunda. Diese Hybriden koexistieren mit und hangen als Sexualparasiten ab von der Wirts-Elternart R. lessonae (das L-E-Populationssystem); Paarungen mit R. lessonae stellen somatische Hybriditat in jeder Generation von R. esculenta wieder her. Wir untersuchten 15 L-E-System-Populationen in der Nordschweiz, die ausserhalb des ursprunglichen R. ridibunda-Areals liegt. Hybrid-Frequenz in Stichproben variierte von 8% in Moortumpeln bis 100% in Kiesgruben und Waldweihern. Klonale Diversitat (Variation unter R. ridibunda-Genomen von Hybriden), entdeckt durch Protein-Elektrophorese von 6 Marker-Loci, zeigte ein Total von 8 Hemiklonen und variierte lokal von uniklonalen Populationen in sudlichen Teilen des Untersuchungsgebiets bis zu 6 koexistierenden Hemiklonen im Norden. Alle Allele, die Hemiklone unterscheiden, kommen haufig in den naheliegendsten ursprunglichen R. ridibunda-Populationen im ostlichen Zentraleuropa vor; die wahrscheinlichste Quelle klonaler Diversitat in unseren Stichproben ist multiple Klonbildung durch Primarhybridisierungen im Sympatrie-Areal von R. ridibunda und R. lessonae und nachfolgende Dispersion hemiklonaler Linien. Eine positive Korrelation zwischen dem Grad klonaler Diversitat und Hybrid-Frequenz, vorhergesagt vom Frozen Niche Variation-Modell (jeder Hemiklon ist durch eine relativ enge Nische charakterisiert, Koexistenz ist moglich durch Nischen-Aufteilung), wurde nicht gefunden; das kontrastiert zu Fisch-Hybriden mit hemiklonaler Fortpflanzung (Poeciliopsis). Historische Faktoren wie die Verfugbarkeit verschiedener kolonisierender Hemiklone konnten stark genug sein, um das Signal vom Operieren der Frozen Niche Variation zu uberdecken.

Spontaneous deleterious mutations are expected to accumulate through Muller's ratchet in clonally reproducing organisms and may lead to their extinction. We study deleterious mutations and their effects in a system of European frogs. Rana esculenta (RL), natural hybrids R. ridibunda (RR) X R. lessonae (LL), reproduce hemiclonally; both sexes exclude the L genome in the germ line and produce unrecombined R gametes; hybridity is restored each generation by matings of RL with coexisting LL. Different allozyme-defined hybrid hemiclones (R genome haplotypes) are thought to have originated independently from primary hybridizations RR x LL. Natural matings between two hybrids usually lead to inviable RR tadpoles. This inviability is thought to result from unmasked deleterious alleles on the clonally transmitted R genomes. Most simply it reflects homozygosity for recessive deleterious alleles at particular loci; alternatively (consistent with absence of RR adults in multiclonal populations) it may reflect hemiclone-specific sets of incompletely recessive deleterious mutations that cumulatively cause inviability when two such genomes are combined. If inviability results from the former, progeny of two hybrids of different hemiclones, whether allopatric or coexisting, should be viable, because it is improbable that their R genomes share recessive deleterious alleles at the same set of loci; if inviability results from the latter, progeny of hybrids of different hemiclones should be inviable, especially when hybrid lineages are old. We tested these hypotheses in artificial crosses, using frogs from three regions: hemiclonal hybrids outside R. ridibunda's range from northern Switzerland (two abundant coexisting allozyme-defined hemiclones; estimated lineage ageor = 5,000 generations) and from Sicily, Italy (one hemiclone; estimated ageor = 25,000 generations) and R. ridibunda from Poland. We generated RR progeny, which we reared under benign conditions in the laboratory, by crossing (1) two hybrids from the same region (H x H local); (2) two hybrids from different regions (H X H foreign); (3) hybrids and R. ridibunda (H X R); and (4) two R. ridibunda (R X R). Survival to metamorphosis was similar and high for R x R, H X H foreign, and H X R, whereas all tadpoles of H X H local died before metamorphosis. This supports the hypothesis that homozygosity for recessive deleterious mutations at particular loci causes inviability. Crosses within and between the two coexisting hemiclones from Switzerland were, however, equally inviable. This result may reflect episodic sexual recombination in RR progeny from exceptional successful interclonal hybrid X hybrid matings, followed by matings of such RR with LL. This process would both slow down or halt Muller's ratchet and disrupt genetic independence of coexisting hemiclones, so that the same remaining deleterious R alleles could exist in different allozyme-defined hemiclones. Whereas all data are consistent with the prediction of Muller's ratchet operating on clonally transmitted R genomes of natural hybrid lineages, they are insufficient to demonstrate such operation, because deleterious recessives that mutated after clone formation and those that preexisted in the R. ridibunda source populations that formed the hemiclonal lineages are not distinguished. The possibility of episodic sexual recombination must be carefully taken into account when studying Muller's ratchet in natural populations of this Rana system.

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 Giutighausen, 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 co- existence 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.