Premeiotic chromosome doubling after genome elimination during spermatogenesis of the species hybrid Rana esculenta

Summary Gamete production in the hybridogenetic species hybrid Rana esculenta (Rana ridibunda X Rana lessonae) is preceded by a premeiotic elimination of the R. lessonae genome and subsequent duplication of the remaining R. ridibunda genome, so that only ridibunda chromosomes enter a quasi normal meiosis, and only ridibunda gametes are formed. This is demonstrated by differences in genome specific centromere fluorescence and electrophoretic patterns between somatic and gonadal tissue.     

A genetic mechanism of species replacement in European waterfrogs?

Introduced Rana ridibunda currentlyreplace the native waterfrogs R. lessonaeand R. esculenta in several areas ofcentral Europe. The unusual reproductive systemin waterfrogs of the Rana esculentacomplex suggests that this replacement may bedriven by a genetic mechanism: Ranaesculenta, a hybrid between R. ridibundaand R. lessonae, eliminates the lessonae genome from the germline and clonallytransmits the ridibunda genome(hybridogenesis). Hybrids form mixedpopulations with R. lessonae (L-E-system)in which they persist by backcrossing with theparental species. Matings between hybrids areunsuccessful, because their ridibundagenomes contain fixed recessive deleteriousmutations. When introduced into a L-E-system,R. ridibunda can mate with both nativetaxa, producing R. ridibunda offspringwith R. esculenta, and R. esculentaoffspring with R. lessonae (primaryhybridizations). If primary hybrids arehybridogenetic, they produce viable R.ridibunda offspring in matings with otherhybrids, because their clonal genomes areunlikely to share the deleterious allelespresent in the ancient clones. Thus, R.ridibunda will increase in the population atthe expense of both native taxa, eventuallyleaving a pure R. ridibunda population.We provide three lines of evidence for thisprocess from a currently invaded population inSwitzerland: (1) Primary hybridizations takeplace, as roughly 10% of hybrids in thepopulation possess ridibunda genomesderived from the introduced frogs. (2)Hybridogenesis occurs in primary hybrids,although at a low frequency. (3) Many hybrid ×hybrid matings in the population indeed produceviable offspring. Hence, the proposed geneticmechanism appears to contribute to the speciesreplacement, although its importance may belimited.     

FIXATION OF DELETERIOUS MUTATIONS IN CLONAL LINEAGES: EVIDENCE FROM HYBRIDOGENETIC FROGS

Abstract The hemiclonal waterfrog Rana esculenta (RL genotype), a bisexual hybrid between R. ridibunda (RR) and R. lessonae (LL), eliminates the L genome from its germline and clonally transmits the R genome (hybridogenesis). Matings between hybrids produce R. ridibunda offspring, but they generally die at an early larval stage. Mortality may be due to fixed recessive deleterious mutations in the clonally inherited R genomes that were either acquired through the advance of Muller's ratchet or else frozen in these genomes at hemiclone formation. From this hypothesis results a straightforward prediction: Matings between different hemiclones, that is, between R. esculenta possessing different R genomes of independent origin, should produce viable R. ridibunda offspring because it is unlikely that different clonal lineages have become fixed for the same mutations. I tested this prediction by comparing survival and larval performance of tadpoles from within‐ and between‐population crossings using R. esculenta from Seseglio (Se) in southern, Alpnach (Al) in central, and Elliker Auen (El) in northern Switzerland, respectively. Se is isolated from the other populations by the Alps. Enzyme electrophoresis revealed that parents from Se belonged to a single hemiclone that was different from all hemiclones found north of the Alps. Parents from Al also belonged to one hemiclone, but parents from El belonged to three hemiclones, one of which was indistinguishable from the one in Al. Rana esculenta from Se produced inviable tadpoles when crossed with other hybrids of their own population, but when crossed with R. esculenta from Al and El, tadpoles successfully completed metamorphosis, supporting the hypothesis I tested. Within‐population crosses from Al were also inviable, but some within‐population crosses from El, where three hemiclones were present, produced viable offspring. Only part of the crosses between Al and El were viable, but there was no consistent relationship between hemiclone combination and tadpole survival. When backcrossed with the parental species R. ridibunda, hybrids from all source populations produced viable offspring. Performance of these tadpoles with a sexual and a clonal genome was comparable to that of normal, sexually produced R. ridibunda tadpoles. Thus, in the heterozygous state, the deleterious mutations on the clonal R genomes did not appear to reduce tadpole fitness.     

Heterozygous fitness effects of clonally transmitted genomes in waterfrogs

The European waterfrog Rana esculenta (RL‐genotype) is a natural hybrid between R. ridibunda (RR) and R. lessonae (LL) and reproduces by hybridogenesis, i.e. it eliminates the L‐genome from the germline and produces gametes only containing the clonally transmitted R‐genome. Because of the lack of recombination, R‐genomes are prone to accumulate spontaneous deleterious mutations. The homozygous effects of such mutations become evident in matings between hybrids: their offspring possess two clonal R‐genomes and are generally inviable. However, the evolutionary fate of R. esculenta mainly depends on the heterozygous effects of mutations on the R‐genome. These effects may be hidden in the hybrid R. esculenta because it has been shown to benefit from spontaneous heterosis. To uncouple clonal inheritance from hybridity, I crossed R. esculenta with R. ridibunda to produce nonhybrid offspring with one clonal and one sexual R‐genome, and compared their survival and larval performance with normal, sexually produced R. ridibunda tadpoles. Because environmental stress can enhance the negative effects of mutation accumulation, I measured the performance at high and low food levels. There was no indication that tadpoles with a clonal genome performed worse at either food level, suggesting that at least in the larval stage, R. esculenta benefits from heterosis without incurring any costs because of heterozygous effects of deleterious mutations on the clonally transmitted R‐genome.     

Ecology of the Hybridogenetic Rana esculenta Complex: Differential Oxygen Requirements of Tadpoles

Because of intrinsic demographic load induced by hybridogenesis (infertility of homotypic hybrid matings), the maintenance of hybrid lineages supposes that they present better performances (heterosis) than their host species which allows them to coexist on a long-term basis. However, this necessity of high fitness can be relaxed if a relative niche partitioning occurs between the taxa, each of them differing in their ecological optima. In the waterfrog hybridogenetic complex (Rana esculenta complex), recent studies have revealed that hybrids show intermediate distribution between parental species across a gradient of river influence (that is related to a gradient of oxygen levels), and intermediate performances of their tadpoles with regard to oxygen availability (hypoxia). In investigating oxygen consumption rates, survival time in anoxia, and metabolite contents in the three forms of the complex, the present study confirms intermediate characteristics of hybrid tadpoles (R. esculenta) when compared to both parental lineages (R. lessonae and R. ridibunda). Whereas R. ridibunda tadpoles were the most sensitive to anoxia, R. lessonae tadpoles were the most tolerant. Because oxygen requirements of the hybrid proved to be intermediate, no heterosis was detected. These results confirm the hypothesis of the intermediate niche hypothesis to explain the coexistence of R. lessonae and R. esculenta and the success of the hybridogens.     

Morphometric and DNA investigations into European water frogs (Rana kl. esculenta Synklepton (Anura, Ranidae)) from different population systems

The population specific variability of diploid and triploid R. kl. esculenta individuals was investigated by means of morphometric methods (canonical discriminant analysis, UPGMA cluster analysis) and DNA fingerprinting. As a result of the morphometric investigations, as well as of the DNA investigations, a clear separation of single populations was possible. However, no correlations between the morphometry and different population systems could be recognized. Clear morphometric differences could be seen between diploid ♀♀ and ♂♂ and triploid ♀♀ on the one hand, and triploid ♂♂ on the other. While the diploid ♀♀ and ♂♂ and the triploid ♀♀ were located in the intermediate area between the parental species R. lessonae and R. ridibunda according to their morphometric parameters, the triploid ♂♂ showed a great overlap with R. lessonae. Up to now, this phenomenon has not been explained. The first results of the DNA investigations provided further hints at the high inter-individual and population-specific variability of R. kl. esculenta. R. kl. esculenta individuals of the R. lessonae/esculenta population Toter See could be distinguished from conspecific individuals of the R. ridibunda/esculenta-♀♀ population Alte Oder according to their fingerprint patterns. Moreover, in the R. lessonae / esculenta population, the fingerprints or the diploid R. kl. esculenta-♀♀ and the investigated R. lessonae-♀ were very similar. Furthermore, in this population, many R. kl. esculenta genotypes resemble R. lessonae in their morphometric features. This finding suggests the occurrence of recombination in R. kl. esculenta. In general, every population seems to have its own genetic background. A classification of water-frog populations according to population systems is only possible under certain conditions.     

Allele frequencies of the human platelet antigen-1 in the Egyptian population

Background

We report the expression pattern of 5S rDNA in the eggs of water frogs Rana lessonae, Rana ridibunda and Rana esculenta using the quantitative real-time PCR. This kind of research had never been performed before.

Results

5S rDNA relative expression of the Rana ridibunda oocytes is approximately six times higher in comparison to the Rana lessonae oocytes. The oocytes of the investigated Rana esculenta frogs, in respect of 5S rDNA relative expression ratio, were very similar to the Rana ridibunda oocytes.

Conclusion

We suggest the possibility of using 5S rDNA as the internal control gene, in the studies of relative mRNA quantitative assays in water frog oocytes, because of its characteristic specific expression pattern in the Rana lessonae, Rana ridibunda and Rana esculenta oocytes.     

Karyological and serological studies in Rana lessonae,R. ridibunda and in their hybrid R. ‘esculenta’ (Amphibia,Anura)

The karyotypes of the three water frog forms: Rana lessonae, R. ridibunda and R. esculenta were analysed from bone marrow cell preparations of animals captured in several localities of the GDR. In the three forms chromosome morphology was similar (5 large and 8 small pairs), although differences in the relative length of most elements were found; R. esculenta chromosomes were always intermediate.One of the small pairs (Chr. 12) was found to be metacentric in R. lessonae and submetacentric in R. ridibunda. Most R. esculenta individuals examined had one meta-and one submetacentric 12th element, indicating the hybrid nature of this form. However 16.6% esculenta proved to be homozygous for either the metacentric or the submetacentric chromosome 12, while 13% lessonae individuals and 7.7% ridibunda were heterozygous for this element.By starch gel electrophoresis an analysis was undertaken of serum proteins from water frogs coming from regions in which the forms occur together (sympatric populations) and from zones in which only one of them lives (allopatric populations).In Rana lessonae, where only one allele had been previously described, two different alleles were found in animals coming from the GDR.If genetic polymorphism is excluded, between 6.1% and 9.1% individuals from sympatric lessonae and ridibunda populations showed introgression of an albumin allele. No gene introgression was found in allopatric lessonae populations from the Leningrad region or in ridibunda from Alma Ata and southern Bulgaria.     

A microsatellite‐based method for genotyping diploid and triploid water frogs of the Rana esculenta hybrid complex

Rana esculenta is a hybrid between Rana lessonae (LL) and Rana ridibunda (RR), and hybrids may be diploid (LR) or triploid (LLR or LRR). Genotypes can be roughly determined from erythrocyte size and morphometry in adult frogs, but accurate genotyping requires more labourious methods. Here I demonstrate that both the L and R genomes have specific microsatellite alleles, and that genotype and ploidy can be accurately inferred from the quantitative ratio of PCR‐amplified (polymerase chain reaction‐amplified) genome‐specific alleles. This method greatly facilitates genotyping in DNA studies of the R. esculenta complex and allows analysis of badly preserved samples and embryos.     

Variation in Sex Ratio and Evolutionary Rate of Hemiclonal Rana esculenta Populations

In many plant and animal taxa mutation rates are higher in males than in females. As a result, the evolutionary speed of genes depends on how much time they spend in either sex. Usually, this time differs between genes located on sex chromosomes but not between those on autosomes. Here we present an unusual system with a partially sex-linked inheritance of autosomes: the hemiclonal frog Rana esculenta (E) which is originally a hybrid between the sexual species R. lessonae (L) and R. ridibunda (R). Rana esculenta excludes the L genome prior to meiosis, produces eggs or sperm containing an unrecombined R genome and restores hybridity by mating with R. lessonae (‘hybridogenesis’). Matings between L males and E females result in offspring with an even sex ratio, whereas the reverse combination produces only daughters. The extent of the resulting female bias and the proportion that R alleles have spent in either sex depend on the relative survival (b) and the relative reproductive contribution (a) of E males vs. E females. In this paper, we analyze mathematically how different combinations of a and b influence the sex ratio in R. esculenta populations and, combined with the male/female mutation rate ratio (α), the evolutionary rate of the clonally transmitted R genome. We find that this rate is higher than in an asexual population and lower than in a sexual one. Hence, clonal diversity through new mutations is more easily achievable than in purely asexual species. In contrast, the occurrence and accumulation of deleterious mutations is lower than in a comparable sexual species. We conclude that these intermediate mutation rates improve the ecological and evolutionary potential of hemiclonal organisms, and we draw attention to the implications for the use of microsatellites. Co-ordinating editor: L. Hurst     

Vocal response of male European water frogs (Rana Esculenta complex) to mating and territorial calls

The responses of male European water frogs (the two species Rana lessonae and Rana ridibunda and their hybrid, Rana esculenta) to playback of their mating and territorial calls were studied during the mating season.In order to select biologically relevant intensities for the presentation of the recorded calls, the sound pressure of the calls produced by the frogs themselves was established prior to the experiment. At a distance of 1 m the most intense calls were those of R. ridibunda, with a sound pressure of 110 dB (peak SPL). The smaller males of R. esculenta gave calls about 5 dB lower in intensity. The calls of R. lessonae, the smallest phenotype, were still less intense, 10 dB lower than those of R. ridibunda.The territorial calls of all three phenotypes elicited territorial calls in all of the males tested, as a rule accompanied by approach to the sound source. The sound pressure required to elicit a vocal response was nearly the same for each of the three different territorial calls. Sometimes encounter calls and warning calls were given in addition to territorial calls.When the mating calls were presented at low intensity, in some cases the males responded with their own mating calls. Mating calls at higher intensity elicited the same behavior that appeared following presentation of territorial calls, but significantly higher sound pressures were required to elicit such a response to mating calls than to territorial calls. The males of R. lessonae and R. esculenta did not respond to the mating calls of R. ridibunda, and each of them had significantly lower thresholds to the mating call of its own phenotype than to that of the other. The males of R. ridibunda responded only to conspecific mating calls.The vocal-response thresholds are compared with those of the electrodermal response reacting to the same stimuli. The significance of the different calls of the European water frogs is discussed.     

Allozyme variation among Rana balcanica,R. levantina,and R. ridibunda (Amphibia: Anura)1

The allozyme variation among water frogs of the species R. balcanica, R. levantina and R. ridibunda, all formerly considered as one species (R. ridibunda Pallas, 1771), was studied using horizontal starch gel electrophoresis. Blood samples (N # 63) of frogs were collected from five populations in Greece and Israel. Samples (N = 9) of the hybrid frog R. esculenta collected from a locality in Germany were used as an outgroup for phylogenetic analyses. Fifteen enzymes controlled by twenty presumptive loci were identified. Thirteen loci were polymorphic within or among the studied populations. Genetic differentiation among the species was considerably greater than among populations of the same species. Even at Nestos River where R. ridibunda and R. balcanica occur in the same habitats, individuals could be assigned to either species due to characteristic differences of the genotypes (GPI1). This indicates reproductive isolation among these species. The reconstruction of phylogenetic relationships among the three species based on the allozyme data corroborated the model presented on the basis of bioacoustic data: R. ridibunda and R. balcanica (Nei's genetic distance D = 0.0820) are sibling species pertaining to an Eurasian lineage, whereas R. levantina (distance to the European species D = 0.1780 - 0.1955) together with R. perezi represent an independent afroasian lineage.     

Taxon composition and genetic variation of water frogs in the Mid-Rhone floodplain

Natural hemidonal hybrid lineages of water frogs reproduce by hybridogenesis, excluding one parental genome in the germ line and mating with the coexisting same parental species. Two such sexual hosthybridogen systems occur in the Rhône valley: the L-E system in the north, the P-G system in the south. Although these hybridogenetic complexes may overlap along the Rhône river, there is no evidence for a contact zone in our samples: only Rana ridibunda and R. esculenta were identified using protein electrophoresis. Whether the absence of R. perezi reflects a more southern distribution or its exclusive occurrence in other habitats, remains to be tested. Comparison of somatic and gonadal tissues reveals that gametogenesis of R. esculenta is of the L-E type: gametes carry ridibunda genomes. R. ridibunda apparently is not native, but was introduced by humans, and the R. esculenta in our samples is probably an immigrant from nearby L-E systems.     

Variation of plastic responses to oxygen availability within the hybridogenetic Rana esculenta complex

The hypothesis of superiority of hybrid phenotype to explain the maintenance of hybridogenesis (exclusion of one parental genome in the germline before meiosis) was investigated in the Rana esculenta complex. Survival and growth of progenies from each parental and hybrid lineages were compared across contrasted conditions of oxygen availability (this factor is suspected to influence habitat utilization of each form of the complex). Whereas growth and development were strongly affected by constant hypoxic conditions in the R. ridibunda lineage, they were not altered in the R. lessonae lineage. Because the performance of the hybrid (R. esculenta) lineage proved to be intermediate between the parental species, no heterosis effect was detected. These results supported the intermediate niche hypothesis as an alternative to the heterotic hybrid superiority hypothesis. The ecological and evolutionary implications are then discussed.     

Genetic compatibility between sexual and clonal genomes in local populations of the hybridogeneticRana esculenta complex

Summary Hybridogenetic species possess a hybrid genome: half is clonally inherited (hemiclonal reproduction) while the other half is obtained each generation by sexual reproduction with a parental species. We addressed the question of whether different hemiclones of the hybridogenetic water frogRana esculenta are locally adapted for genetic compatibility with their sexual parental hostRana lessonae. We artificially crossedR. esculenta females of three hemiclones (GUT1, GUT2 and GUT3) from a pond near Gütighausen, Switzerland and one hemiclone (HEL1) from near Hellberg, Switzerland each toR. lessonae males from both populations. We also created primary hybrids by crossing the sameR. lessonae males from both populations toR. ridibunda females from Pozna, Poland (POZ). Tadpoles were then reared in the laboratory at two food levels to assess their performance related to early larval growth rate, body size at metamorphosis and length of the larval period. Tadpoles from hemiclones GUT1, GUT3 and POZ had higher growth rates than those from hemiclones GUT2 and HEL1 at the low food level, but at the high food level all growth rates were higher and diverged significantly between hemiclones GUT2 and HEL1. Tadpoles from the intrapopulational crosses GUT2 × GUT and HEL1 × HEL were larger at metamorphosis than those from the interpopulational crosses GUT2 × HEL and HEL1 × GUT. A high food level increased the size at metamorphosis in all tadpoles. A high food level also decreased the days to metamorphosis and tadpoles from GUT1, GUT3 and POZ had the shortest larval period whereas those from GUT2 and HEL1 had the longest. These results indicate that the differential compatibility of clonal genomes may play an important role in hybridogenetic species successfully using locally adapted sexual genomes of parental species and that interclonal selection is likely important in determining the distribution of hemiclones among local populations.     

Distribution and habitat use of water frog hybrid complexes in France

Hybrid zones are either distributed along clines or in a mosaic of patches. This distribution may depend upon variation in taxon habitat use. Habitat use and distribution of diverse taxa of water frogs (Rana ridibunda, R. lessonae, R. perezi, R. kl. grafi and R. kl. esculenta) in France are analysed to determine whether water frog complexes conform to the mosaic or clinal model. Biogeographical scenarios may be invoked in order to explain the distribution of water frogs. However, the distribution of R. perezi and R. kl. grafi, being restricted to regions characterized by Mediterranean or Oceanic climatic conditions, suggests that these frogs do not endure cold winters. R. ridibunda is widespread in Southern France and its distribution suggests multiple introductions. It is concluded that water frogs conform to the mosaic zone model rather than to the tension zone model because: (i) taxa exhibited differences in habitat use, (ii) pure parental species were documented and (iii) hybrids are not unfit relative to parental species.     

Heat resistance of the skeletal muscle in Western Palearctic green frogs (<Emphasis Type="Italic">Rana esculenta</Emphasis> complex)

Heat resistance of the gastrocnemius muscle was studied in five species of the Rana esculenta complex. It was similar in R. bedriagae, R. lessonae, and in the European form of R. ridibunda; while North African R. saharica demonstrated a lower heat resistance. No heterosis was expressed in R. esculenta, a clonal hybrid of R. lessonae and R. ridibunda, for the heat resistance of the muscle. Moreover, this species demonstrated low heat resistance at the highest test temperature (42°C). Comparison of diploid and triploid R. esculenta syntopically occurring in the same water bodies demonstrated no differences between them, thus, suggesting that polyploidy has no effect on this parameter at least in this case.     

Neuroanatomical distribution of MCH in the brain and pituitary of submammalian vertebrates

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that has been initially characterized from a salmon pituitary extract and subsequently identified in various species from all classes of vertebrates. The present review summarizes the current knowledge regarding the neuroanatomical distribution of MCH-immunoreactive neurons in submammalian vertebrates. In all species examined, MCH-immunoreactive perikarya are confined to the hypothalamus, with the exception of the cyclostome Lampetra fluvialis and the lungfish Protopterus annectens, in which additional populations of MCH-immunoreactive cell bodies occur in the telencephalon, and the frogs Rana ridibunda and Rana esculenta which exhibit MCH-positive perikarya in thalamic nuclei. In teleosts, in the frog R. ridibunda and in the L. fluvialis, MCH is present in the classical hypothalamic-neurohypophysial system indicating that the peptide may play the role of a neurohormone. In other groups, MCH-immunoreactive nerve fibers are widely distributed in various brain regions suggesting that, in these species, MCH in the central nervous system may act as a neurotransmitter or/and a neuromodulator rather than a neurohormone.     

Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation

The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway in tissues of frog Rana ridibunda and land snail species Helix lucorum during seasonal hibernation. Moreover responses of glycolytic enzymes from cold acclimated R. ridibunda and H. lucorum were studied as well. The drop in Po₂ in the blood of hibernated frogs and land snails indicated lower oxygen consumption and a decrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had a differential effect on the glycolyis in the two species studied and also in the tissues of the same species. The activity of l-LDH decreased significantly in the skeletal muscle and heart of hibernated R. ridibunda indicating a low glycolytic potential. Similar biochemical responses were observed in the same tissues during cold acclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, might indicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda, the activities of the enzymes increased and remained at higher levels than those of the prehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating land snails.     

Degeneration of germ line cells in amphibian ovary

Ogielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91 : 319–327 We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.