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

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

Impact of gamete production on breeding systems and population structure of hybridogenetic frogs of the Pelophylax esculentus complex: The evolutionary potential of interspecific hybridization

The European water frog Pelophylax esculentus (genome LR) is a natural hybrid between P. lessonae (LL) and P. ridibundus (RR). It presents a peculiar quasi-sexual reproductive mode known as hybridogenesis: the hybrid excludes one of the parental species’ genomes at a pre-meiotic stage of gametogenesis, thus producing gametes containing clonal copies of the other parental genome. By mating with the parental species whose genome has been excluded it re-establishes hybridity at each generation. Moreover, because of its hybrid nature and resulting problems of chromosome pairing at gametogenesis, P. esculentus also produces diploid gametes from time to time. These gametes often lead to the generation of triploid frogs which will allow, under certain ecological conditions, the establishment of all hybrid populations which are maintained without the genetic contribution of either parental species. Over the past decade, such populations have been well studied in the north-western part of Europe, but the presence of triploid water frogs has also been reported for various areas in Central Europe. However, for those localities details on the breeding system, i.e. the genetic contribution of the various frog types, are usually lacking. The major goals of this thesis were to (a) investigate the Central European populations more closely, (b) to compare the breeding systems there with that in all-hybrid populations from Northern Europe and (c) find out whether triploid water frogs in different areas are of mono- or polyphyletic origin. In chapter one I used microsatellite DNA analyses and crossing experiments to compare five populations (one in Poland, two in Germany and two in Slovakia) presenting different population structures. Indices of 2 heterozygosity and of genetic differentiation allowed to depict the genetic interactions between the different type of frogs (LL, LLR, LR, LRR and RR). I was then able to define and differentiate the breeding systems occurring in each of them and to propose an evolutionary scenario for the appearance and maintenance of the all-hybrid populations. Chapter two presents a collaborative study with Alexandra Hoffmann. Here we enlarged our survey to populations distributed all over Europe and used microsatellite DNA and mitochondrial DNA (mtDNA) analyses to find patterns of genetic structure among different breeding system types. We found that genetic diversity among hybrid populations is influenced by geographic location (latitude, longitude) and by the proportions of parental genotypes in the hybrid population. Furthermore, we identified genetic clusters from both microsatellites and mtDNA, which indicate that there are at least two separate polyploid hybrid clades existing today: one in Northern and East- Central Europe and one in Eastern Europe (Eastern Ukraine)

Contrasting reproductive strategies of triploid hybrid males in vertebrate mating systems

The scarcity of parthenogenetic vertebrates is often attributed to their ‘inferior’ mode of clonal reproduction, which restricts them to self-reproduce their own genotype lineage and leaves little evolutionary potential with regard to speciation and evolution of sexual reproduction. Here, we show that for some taxa, such uniformity does not hold. Using hybridogenetic water frogs (Pelophylax esculentus) as a model system, we demonstrate that triploid hybrid males from two geographic regions exhibit very different reproductive modes. With an integrative data set combining field studies, crossing experiments, flow cytometry and microsatellite analyses, we found that triploid hybrids from Central Europe are rare, occur in male sex only and form diploid gametes of a single clonal lineage. In contrast, triploid hybrids from north-western Europe are widespread, occur in both sexes and produce recombined haploid gametes. These differences translate into contrasting reproductive roles between regions. In Central Europe, triploid hybrid males sexually parasitize diploid hybrids and just perpetuate their own genotype–which is the usual pattern in parthenogens. In north-western Europe, on the other hand, the triploid males are gamete donors for diploid hybrids, thereby stabilizing the mixed 2n-3nhybrid populations. By demonstrating these contrasting roles in male reproduction, we draw attention to a new significant evolutionary potential for animals with nonsexual reproduction, namely reproductive plasticity

Mode of hybridogenesis and habitat preferences influence population composition of water frogs (Pelophylax esculentus complex, Anura: Ranidae) in a region of sympatric occurrence (western Slovakia)

Coexistence of sperm-dependent asexual hybrids with their sexual progenitors depends on genetic and ecological interactions between sexual and asexual forms. In this study, we investigate genotypic composition, modes of hybridogenetic gametogenesis and habitat preferences of European water frogs (Pelophylax esculentus complex) in a region of sympatric occurrence. Pelophylax esculentus complex comprises parental species P. ridibundus and P. lessonae, whose primary hybridization leads to hybridogenetic lineages of P. esculentus. Hybrids clonally transmit one parental genome and mate with the other parental species, forming a new generation of hybrids. In the region of western Slovakia, we found syntopic occurrence of diploid and triploid hybrids with P. lessonae, syntopic occurrence of all three taxa as well as the existence of pure P. ridibundus populations. All triploid hybrids were exclusively male possessing one ridibundus and two different lessonae genomes (RLL). Sex ratio in diploid hybrids was substantially female-biased. Irrespective of the population composition, diploid hybrids excluded the lessonae genome from their germ line and produced ridibundus gametes. Contrarily, RLL males unequivocally eliminated the ridibundus genome and produced diploid lessonae sperms. Perpetuation of RLL males in studied populations is most likely achieved by their mating with diploid hybrid females. The composition of water frog populations is also shaped by taxon-specific habitat preferences. While P. ridibundus preferred larger water bodies (gravelpits, fishery ponds, dead river arms), P. lessonae was most frequently found in marshes and smaller sandpits. Pelophylax esculentus occupied predominately similar habitats as its sexual host P. lessonae

Genetic and cytogenetic characteristics of pentaploidy in water frogs

We describe a pentaploid froglet (LLLRR; three Pelophylax lessonaeand two Pelophylax ridibundusgenomes) that has never been reported before within the Water Frog Pelophylax esculentus)hybrid complexes. The pentaploid specimen was found among almost all triploid siblings obtained from a diploid femaleP. esculentus(LR) crossed with a diploid male P. lessonae(LL). We confirmed ploidy levels of the parents and the offspring by karyotyping, microsatellite analysis (18 loci), and measurements of DNA content and erythrocyte size. Microsatellite analysis indicated that the pentaploid originated from a tetraploid ovum (LLRR) fertilized by a haploid sperm (L). Surprisingly, the erythrocytes of the pentaploid were not proportionally larger than in triploids, despite a higher DNA content. Only 6.7% of the erythrocytes were distinctly large, whereas the others varied strongly in shape and size; besides typical ovoid mature erythrocytes there were small, tear-shaped, or enucleated ones. We discuss the possibility of loss of some cytoplasm by large erythrocytes as a result of mechanical damages during circulation through the narrow vessels; when the erythrocytes achieve a relatively higher surface-to-volume ratio, they may function more effectively in a proper gas exchange

All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genetics of maintenance

Background Sexual parasites offer unique insights into the reproduction of unisexual and sexual populations. Because unisexuality is almost exclusively linked to the female sex, most studies addressed host-parasite dynamics in populations where sperm-dependent females dominate. Pelophylax water frogs from Central Europe include hybrids of both sexes, collectively named P. esculentus. They live syntopically with their parental species P. lessonae and/or P. ridibundus. Some hybrid lineages consist of all males providing a chance to understand the origin and perpetuation of a host-parasite (egg-dependent) system compared to sperm-dependent parthenogenesis. Methods We focused on P. ridibundus-P. esculentus populations where P. ridibundus of both sexes lives together with only diploid P. esculentus males. Based on 17 microsatellite markers and six allozyme loci, we analyzed (i) the variability of individual genomes, (ii) the reproductive mode(s) of all-male hybrids, and (iii) the genealogical relationships between the hybrid and parental genomes. Results Our microsatellite data revealed that P. esculentus males bear Mendelian-inherited ridibundus genomes while the lessonae genome represents a single clone. Our data indicate that this clone did not recently originate from adjacent P. lessonae populations, suggesting an older in situ or ex situ origin. Conclusions Our results confirm that also males can perpetuate over many generations as the unisexual lineage and successfully compete with P. ridibundus males for eggs provided by P. ridibundus females. Natural persistence of such sex-specific hybrid populations allows to studying the similarities and differences between male and female reproductive parasitism in many biological settings

DNA microsatelitte allele data set

DNA microsatelitte data file for the 16 loci used in the study (RlCA5 and Res16 were not used for any of the analyses). (txt tabulated format

Data from: Contrasting reproductive strategies of triploid hybrid males in vertebrate mating systems

The scarcity of parthenogenetic vertebrates is often attributed to their ‘inferior’ mode of clonal reproduction, which restricts them to self-reproduce their own genotype lineage and leaves little evolutionary potential with regard to speciation and evolution of sexual reproduction. Here, we show that for some taxa such uniformity does not hold. By using hybridogenetic water frogs (Pelophylax esculentus) as a model system, we demonstrate that triploid hybrid males from two geographic regions exhibit very different reproductive modes. With an integrative data set combining field studies, crossing experiments, flow cytometry and microsatellite analyses, we found that triploid hybrids from Central Europe are rare, occur in male sex only and form diploid gametes of a single clonal lineage. In contrast, triploid hybrids from North Western Europe are widespread, occur in both sexes and produce recombined haploid gametes. These differences translate into contrasting reproductive roles between regions. In Central Europe, triploid hybrid males sexually parasitize diploid hybrids and just perpetuate their own genotype – which is the usual pattern in parthenogens. In North Western Europe, on the other hand, the triploid males are gamete donors for diploid hybrids, thereby stabilizing the mixed 2n-3n hybrid populations. By demonstrating these contrasting roles in male reproduction, we draw attention to a new significant evolutionary potential for animals with non-sexual reproduction, namely reproductive plasticity

Balancing a cline by influx of migrants: a genetic transition in water frogs of Eastern Greece

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

Long-term study of an infection with ranaviruses in a group of edible frogs (Pelophylaxkl.esculentus) and partial characterization of two viruses based on four genomic regions

Several edible frogs (Pelophylaxkl.esculentus) collected into a single group from various ponds in Europe died suddenly with reddening of the skin (legs, abdomen) and haemorrhages in the gastrointestinal tract. Ranavirus was detected in some of the dead frogs using PCR, and virus was also isolated in cell culture. Over the following 3 years, another two outbreaks occurred with low to high mortality in between asymptomatic periods. In the first 2 years, the same ranavirus was detected repeatedly, but a new ranavirus was isolated in association with the second mass-mortality event. The two different ranaviruses were characterized based on nucleotide sequences from four genomic regions, namely, major capsid protein, DNA polymerase, ribonucleoside diphosphate reductase alpha and beta subunit genes. The sequences showed slight variations to each other or GenBank entries and both clustered to the Rana esculenta virus (REV-like) clade in the phylogenetic analysis. Furthermore, a quiescent infection was demonstrated in two individuals. By comparing samples taken before and after transport and caging in groups it was possible to identify the pond of origin and a ranavirus was detected for the first time in wild amphibians in Germany