Lampbrush and mitotic chromosomes of the hemiclonally reproducing hybrid Rana esculenta and its parental species

Mitotic chromosomes of the European water frogs Rana ridibunda and Rana lessonae, the parental species of Rana esculenta, differ significantly in their centromeric regions: when C-banded or when made fluorescent, the centromeres of R. ridibunda (and of ridibunda chromosomes in R. esculenta) are visible as a conspicuous dark granule or as a conspicuous fluorescent spot; the centromeres of R. lessonae (and of the lessonae chromosomes in R. esculenta) are inconspicuous or not fluorescent. Lampbrush chromosomes of these three taxa are described in detail for the first time; those of R. ridibunda and R. lessonae differ significantly in morphostructural characters such as conspicuousness of centromeres and number, form, and location of giant loops as well as in chiasma frequency. Chromosomes of the two parental species can thus be distinguished when present in lampbrush complements of hybrids. Reproduction in both sexes of natural R. esculenta lineages is hemiclonal: only the unrecombined genome of one parental species, usually R. ridibunda, is transmitted to haploid gametes (hybridogenesis). In 18 hybrids from natural populations of Poland, somatic tissues had allodiploid complements with chromosomes from each parental species. In contrast, spermatocytes I of five males and oocytes I of seven of eight females (221 of 222 oocytes) were autodiploid and contained only R. ridibunda chromosomes that formed n bivalents. These 12 hybrids thus were hybridogenetic. A single female hybrid had oocytes I (33 of 34) with genomes of both parental species; they showed various disturbances including tetraploidy, reduced number of chiasmata, and incomplete synapsis resulting in univalents. This individual thus was not hybridogenetic. The irregular lampbrush patterns indicate that such hybrids will have severely reduced fertility and most of their successful gametes will result in allotriploid progeny.     

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.     

Evolutionary history of the hybridogenetic hybrid frog Rana esculenta as deduced from mtDNA analyses [published erratum appears in Mol Biol Evol 1986 Sep;3(5):463]

mtDNA of the hybridogenetic hybrid frog Rana esculenta from Switzerland, Austria, and Poland was compared to mtDNA of the parental species R. ridibunda and R. lessonae using electrophoretic analysis of restriction enzyme fragments. Two mtDNA phenotypes, with 3.4% sequence divergence, are present in R. lessonae: type C is found in Poland, and type D is found in Switzerland. Rana ridibunda from Poland has either of two mtDNA phenotypes: type A is the typical ridibunda mtDNA, and type B is a lessonae mitochondrial genome, introgressed into R. ridibunda, that differs from type C mtDNA of R. lessonae by only 0.3%. Each of the three lessonae genomes differs from A, the typical ridibunda mtDNA, by approximately 8%. All four types of mtDNA (A and B of R. ridibunda, C and D of R. lessonae) are found in R. esculenta. Of 62 R. esculenta from Poland, 58 had type C, three had type A, and one had type B mtDNA. All nine R. esculenta from Switzerland had type D mtDNA. All three R. esculenta from Austria, from a population in which males of R. esculenta are rare, had ridibunda mtDNA, two having type B and one having type A. Both field observations and studies of mating preference indicate that the primary hybridizations that produce R. esculenta are between R. ridibunda females and R. lessonae males; thereafter, R. esculenta lineages are usually maintained by matings of R. esculenta females with R. lessonae males. The presence of ridibunda mtDNA in the three R. esculenta sampled from Austria, its occasional presence in R. esculenta populations in Poland, and its absence from R. esculenta in Switzerland support both the direction of the original hybridization and the rarity of formation of new R. esculenta lineages. The preponderance of R. esculenta individuals with lessonae mtDNA in our samples from central Europe suggests that most lineages have gone through at least one mating between an R. lessonae female and an R. esculenta male. This reveals a greater reproductive role for R. esculenta males than their partial sterility and infrequent matings would suggest.      

Mitochondrial DNA reveals formation of nonhybrid frogs by natural matings between hemiclonal hybrids.

The European water frog Rana esculenta (RL), a natural hybrid between R. ridibunda (RR) and R. lessonae (LL), reproduces by hybridogenesis: haploid gametes usually contain an intact chromosome set of R. ridibunda (R); the lessonae nuclear genome (L) is lost from the germ line. Hybridity is restored in the next generation, via fertilization by syntopic R. lessonae. Matings between two hybrids (RL x RL) usually give inviable R. ridibunda (RR) progeny. The adult R. ridibunda subpopulation of Trubeschloo, a gravel pit in northern Switzerland, consists only of females. Fragment patterns for mitochondrial DNA (mtDNA) of these R. ridibunda were identical with those of syntopic R. esculenta and of local populations of R. lessonae; they differed from the patterns in eastern European populations of R. lessonae and of R. ridibunda mtDNAs (3.7% and 9.3% estimated sequence divergence, respectively). In contrast, mtDNAs of two R. ridibunda from an introduced Swiss population with both sexes, although different (2.7% divergence) from each other, were typical R. ridibunda rather than R. lessonae mtDNAs. These data, together with unisexuality, demonstrate conclusively that the all-female R. ridibunda population at Trubeschloo originated from matings between two R. esculenta. The formation of independently reproducing R. ridibunda populations via such hybrid x hybrid matings is precluded because progeny of these matings are unisexual. Recombination in the regenerated fertile R. ridibunda females, followed by matings with R. lessonae, nevertheless provides a mechanism for meiotic reshuffling of genetic material in ridibunda haplotypes that is not typically available in hemiclonal lineages.     

Freezing tolerance of the European water frogs: the good, the bad, and the ugly

Survival and some physiological responses to freezing were investigated in three European water frogs (Rana lessonae, Rana ridibunda, and their hybridogen Rana esculenta). The three species exhibited different survival times during freezing (from 10 h for R. lessonae to 20 h for R. ridibunda). The time courses of percent water frozen were similar; however, because of the huge differences in body mass among species (from 10 g for Rana lessonae to nearly 100 g for Rana ridibunda), the ice mass accumulation rate varied markedly (from 0.75 +/- 0.12 to 1.43 +/- 0.11 g ice/h, respectively) and was lowest in the terrestrial hibernator Rana lessonae. The hybrid Rana esculenta exhibited an intermediate response between the two parental species; furthermore, within-species correlation existed between body mass and ice mass accumulation rates, suggesting the occurrence of subpopulations in this species (0.84 +/- 0.08 g ice/h for small R. esculenta and 1.78 +/- 0.09 g ice/h for large ones). Biochemical analyses showed accumulation of blood glucose and lactate, liver glucose (originating from glycogen), and liver alanine in Rana lessonae and Rana esculenta but not in Rana ridibunda in response to freezing. The variation of freeze tolerance between these three closely related species could bring understanding to the physiological processes involved in the evolution of freeze tolerance in vertebrates.     

Feeding behaviour, food consumption, and growth efficiency of hemiclonal and parental tadpoles of the Rana esculenta complex

1. Clonally reproducing species are often assumed to lack sufficient genetic variability to evolve specific local adaptations to cope with environmental perturbation and competition from sexual species. Yet, many asexuals are extremely successful judged by abundance and wide range, suggesting high competitive abilities in resource exploitation.
2. In this study, food use and its effects on larval growth in a water frog system consisting of the two parental sexual species, Rana lessonae (Camerano 1882) and Rana ridibunda (Pallas 1771), and three different coexisting hemiclones of their hybrid, Rana esculenta (Linnaeus 1758) were investigated.
3. R. esculenta tadpoles spent 18·6% more time feeding than did tadpoles of either parental species, but feeding time was not affected by interspecific mixture.
4. R. esculenta tadpoles consumed 50·8% more food over the whole test period than did tadpoles of the two parental species.
5. R. esculenta tadpoles exhibited higher growth rates than did tadpoles of either parental species.
6. R. lessonae tadpoles had the highest and R . ridibunda tadpoles the lowest growth efficiencies with the R. esculenta tadpoles ranging between the two parentals.
7. The results obtained indicate that hemiclonal hybridogenetic R . esculenta tadpoles display significant phenotypic variation among coexisting hemiclones as well as out-perform tadpoles of the parental sexual species R. lessonae and R . ridibunda. The primary mechanism for success of the hybrid tadpoles is probably behavioural, through increased feeding time and food consumption, and not physiological via growth efficiency.     

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.     

Hemiclone diversity in the hybridogenetic frog Rana esculenta outside the area of clone formation: the view from protein electrophoresis

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.     

Elimination of the genome of one of the parents prior to premeiotic DNA synthesis in a hybridogenic species of Rana esculenta

A DNA-cytometric study was made of spermatogenesis of the hybridogenic European green frog R. esculenta, whose somatic cells have the ridibunda + lessonae genome. The DNA amount in the ridibunda genome is by 16% more than the lessonae one. The DNA content of esculenta somatic cells is exactly intermediate between those of both the parental species. On the contrary, the sperms (1c) and the primary spermatocytes (4c) of R. esculenta have the DNA content which corresponds to the size of the ridibunda genome. These findings are in a good agreement with the hypothesis of semiclonal inheritance. Furthermore, some hybridogenic males have also spermatogonia (2c) with only the ridibunda genome size, whereas the others have altogether diploid cells with the esculenta (i.e. ridibunda + lessonae) genome size. So, it can be suggested that the selective elimination of the lessonae genome and compensatory doubling of the ridibunda one may occur in spermatogonia of R. esculenta males before the premeiotic DNA synthesis. Meiosis, as it can be inferred from the DNA-cytometry data, proceeds in a usual way on the basis of the ridibunda genome.     

The Effect of Assortative Mating on the Coexistence of a Hybridogenetic Waterfrog and Its Sexual Host

In central Europe, the hybridogenetic waterfrog Rana esculenta, a hybrid between Rana ridibunda and Rana lessonae, lives in sympatry with one of its parental species, the poolfrog Rana lessonae. As R. esculenta has to backcross constantly with R. lessonae in order to produce viable offspring, this coexistence is obligatory for R. esculenta. Since R. esculenta has a higher primary fitness than R. lessonae, a mechanism is required that prevents the hybrid from driving the parental species, and hence itself, to extinction. Here, we present an analytical model and a computer simulation that investigate whether assortative mating can operate as a such a control mechanism. Our results show that assortative mating is very effective in regulating coexistence in such a hybrid-host system. This is particularly true when choice is affected by the proportion of the two male types in the population. Furthermore, we could show that even if the species composition in a mixed hybrid-host population may be largely influenced by differences in life-history parameters, assortative mating still plays a very important role by stabilizing coexistence. Thus, mating behavior turns out to be more important for the populations dynamics of hybridogenetic waterfrog systems than previously assumed.     

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 degrees N latitude and between 8 degrees and 22 degrees 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.     

Non-hybrid offspring from matings between hemiclonal hybrid waterfrogs suggest occasional recombination between clonal genomes

The hemiclonal waterfrog Rana esculenta , a hybrid between R. ridibunda and R. lessonae , eliminates the lessonae genome from the germline and clonally transmits the ridibunda genome (hybridogenesis). Such genomes are prone to accumulate deleterious mutations, which may explain why offspring from matings between hybrids are typically inviable. Here I present field data from a population for which experimental crossings showed that some R. esculenta pairs produce viable R. ridibunda offspring. I demonstrate: (1) that R. ridibunda metamorphs are also produced and survive under natural conditions; (2) that their genotypes are consistent with combinations of clonal ridibunda genomes found in hybrids; and (3) that all R. ridibunda are female. These females possibly recombine the clonal genomes they inherited and, upon mating with syntopic R. lessonae , produce new hemiclones with novel combinations of alleles. Hence, occasional recombination between otherwise clonal ridibunda genomes seems plausible and may provide an escape from the evolutionary dead end they were proposed to be trapped in.     

Genetic diversity in mitochondrial 12S rDNA of western Palearctic water frogs (Anura, Ranidae) and implications for their systematics

The nucleotide sequence of a part of the mitochondrial 12S rRNA gene of eight western Palearctic water frog species was analysed. The results are consistent with the species status of Rana bedriagae, Rana bergeri, Rana epeirotica, Rana lessonae, Rana perezi, Rana ridibunda, Rana saharica and Rana shqiperica . The obtained DNA data suggest that lake frogs from Greece and Yugoslavia on the one hand and lake frogs from Georgia, Uzbekistan and Turkmenistan on the other hand represent two distinct species. However, it is not yet clear whether lake frogs from Georgia, Uzbekistan and Turkmenistan belong to R. ridibunda or represent a new species. The very high similarity between the analysed 12S rDNA segments of German R. ridibunda and R. lessonae confirm the finding that mtDNA of R. lessonae was transmitted into the mitochondrial gene pool of R. ridibunda probably as a result of backcrosses with the hybridogenetic hybrid R. kl. esculenta . The results of parsimony analyses speak in favour of very close phylogenetic relations between R. perezi and R. saharica ; with a high probability these species represent an adelphotaxon. Furthermore, the clades ( R. lessonae + R. shqiperica + R. bergeri ) and ( R. ridibunda + R. bedriagae ) are considered to be sister groups. According to the mt 12S rDNA data R. epeirotica seems to be more closely related to the supraspecific taxon ( R. ridibunda + R. bedriagae ) than to ( R. lessonae + R. shqiperica + R. bergeri ). Thus, it can be excluded that R. shqiperica and R. epeirotica represent sister species.     

LC-MS/MS with 2D mass mapping of skin secretions' peptides as a reliable tool for interspecies identification inside Rana esculenta complex

Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC-MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.     

Biochemical investigation of the Rana esculenta complex in the Kis-Balaton Nature Reserve, Hungary

In this study it has been proved by PAGE of serum proteins that all the three members of the Rana esculenta complex occur in the Kis-Balaton Nature Reserve (Hungary). On the basis of the LDH isoenzyme pattern which is characteristic in green frogs we could distinguish all three variations of R. ridibunda and R. lessonae and one type of R. esculenta. The mobility of serum albumins on SDS-PAGE implies that the R. esculenta comes from hybridization of the two other species. The PAGE methods provide a reliable basis for the rapid taxonomic identification of both adults and immature speciments of the three forms of frogs.     

Character convergence in advertisement call and mate choice in two genetically distinct water frog hybridogenetic lineages (Rana kl esculenta, Rana kl grafi)

Patterns of advertisement call were investigated in two genetically distinct water frog lineages ( Rana kl esculenta, Rana. kl grafi ), which were identified by starch gel electrophoresis – the aim being to determinate the role of vocalization in the hybridogenetic process. Both hybrids displayed major modifications from the basic structure of the Rana ridibunda call. In Rana kl grafi , the call structure tended to correspond to that of Rana perezi in most of the studied parameters (frequency, duration, number of pulses) whereas the call of Rana kl esculenta tended to resemble that of Rana lessonae . The ascendant hierarchical classification clearly revealed such a convergence toward parental species and accounted for a divergence between hybrids. Changes in call patterns might result from both the expression of the non- ridibunda genome and the sexual selective pressure through female mate choice. Only few non- ridibunda females exhibited a preference for hybrid calls which, however, allowed some hybridogenetic males to obtain successful mates. Thus, hybridogenesis induced character convergence in courtship signal with the non- ridibunda species in hybrid zones. In any case, these changes in the courtship signal favoured the particular hybridogenetic process, which constitutes a quasi-parasitism of the non- ridibunda genome.     

The effect of B chromosomes on homoeologous pairing in species hybrids

The effect of B chromosomes on meiosis is described in the diploid and tetraploid interspecific hybrid Lolium multiflorum x Lolium perenne. Although the parental species are very closely related, the presence of B chromosomes in the diploid hybrid reduced both chiasma frequency and the number of bivalents at meiosis by a small but significant amount. However at the tetraploid level the presence of B chromosomes did not seem to alter the pairing pattern and chiasma frequency in any way. The use of B chromosomes to stabilize meiosis in amphiploids of this type between closely related outbreeding species is therefore ruled out.     

Hemiclonal reproduction slows down the speed of Muller's ratchet in the hybridogenetic frog Rana esculenta

Rare recombination in otherwise asexually reproducing organisms is known to beneficially influence the fitness in small populations. In most of the investigated organisms, asexual and rare sexual generations with recombination follow each other sequentially. Here we present a case where clonal reproduction and rare recombination occur simultaneously in the same population. The hybridogenetic water frog Rana esculenta (E), a hybrid between R. lessonae (L) and R. ridibunda (R) produces gametes that only contain the unaltered maternal R part of their genome. New generations of R. esculenta usually arise from E x L matings. Intraspecific E x E matings produce mostly inviable offspring, but in rare cases, female R. ridibunda arise from such matings which are capable of recombination. In the absence of conspecific males, these R females have to mate with E males, which results in further R females, or with L males, which produces new E lineages. This indirect mechanism reintroduces recombination into the otherwise clonally transmitted R genomes in R. esculenta populations. In this study, we show through Monte Carlo simulations that, in most cases, it is sufficient that only between 1 % and 10 % of mixed water frog populations consist of R females to prevent or significantly reduce the fixation and accumulation of deleterious mutations.     

Genome reduction in a hemiclonal frog Rana esculenta from radioactively contaminated areas

A decrease in genome size was found in the hemiclonal hybridogenetic frog Rana esculenta (R. ridibunda x R. lessonae) from areas of radioactive contamination that resulted from the Chernobyl fallout. This genome reduction was of up to 4% and correlated with the background level of gamma-radiation (linear regression corresponded on average to -0.4% per doubling of radiation level). No change in genome size was observed in the coexisting parental species R. lessonae. There was no correlation between genome size and body mass in R. esculenta froglets, which have metamorphosed in the year of the study. The hemiclonal forms may become a suitable object for study on biological significance of individual DNA sequences (and of genome size as a whole) because mutant animals with deletions in a specified genome can arise after a low radiation dose. The proneness to genetic damage makes such forms also a prospective bioindicator of radioactive (and possibly other mutagenic) pollution with the effects of genetic damage conveniently and rapidly monitored by DNA flow cytometry.