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Contrasting genetic diversity and the level of clonality of parental genomes in a water frog population system with the prevalence of Pelophylax esculentus hybrids
Abstract
Pelophylax esculentus (genotype LR), an interspecific hybrid of P. ridibundus (genotype RR) and P. lessonae (genotype LL), is one of the most studied examples of hemiclonal hybridogenic vertebrates. In most populations, hybrids produce clonal gametes of one parental species and backcross with the other parental species, forming a new generation of hybrids. In this study, we focused on the population system of water frogs in the Siverskyi Donets river basin (eastern Ukraine) with the aim to determine the genetic diversity of parental genomes and clarify how such a system is maintained in nature. Of the 55 frogs analyzed, only one individual was P. ridibundus, 48 belonged to diploid LR and five to triploid LRR hybrids. Using 16 microsatellite loci, we found significantly lower genetic diversity and significantly higher clonality in the L-genome of hybrids compared to the R-genome. This contrasting diversity might be attributed to the absence of P. lessonae in the studied region and the clonal inheritance of the L-genome. Our population-genetic results are partially inconsistent with previous studies focusing on hybrid gametogenesis in the Siverskyi Donets river basin, according to which hybrids form both clonal R and L gametes. We hypothesize that discrepancies between the type of clonally formed gametes and the genotypic composition of the studied population may be due to the selective mortality of some genotypes during larval development and metamorphosis. Selective mortality thus may play an important role in the dynamics of the hybridogenetic systems in eastern Ukraine.
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Morphological-morphometric and genetic studies on water frogs (Pelophylax spp.) in the Müritz National Park (Mecklenburg-Western Pomerania) with special consideration of the pool frog (Pelophylax lessonae) The Müritz National Park (MNP), with its abundance of forests and water bodies, is one of the most important nature reserves in the federal state of Mecklenburg-Western Pomerania (M-V) and in the wider Federal Republic of Germany. The very numerous small water bodies and near-natural, extensively managed terrestrial ecosystems provide ideal conditions for amphibians, including water frogs, about which, however, hardly any data have been published so far. In 2021, the genomic structure of a water frog metapopulation system, inhabiting a structurally rich area of about 650 ha in the southern part of the MNP was investigated as part of the FFH monitoring of the pool frog (Pelophylax lessonae) in M-V. Among 247 water frogs, only six (2.4%) were P. lessonae; the other individuals were diploid (36%) or polyploid (61%) hybrids (Pelophylax esculentus). Among the polyploids, genotypes with two lessonae ge-nomes and one ridibundus genome (LLR) dominated (94,7%), only seven individuals (4,7%) had two ridibundus genomes and one lessonae genome (RRL); one animal was probably tetraploid (LLLR). Using univariate and multivariate statistics, based on mor-phometric data, all P. lessonae and triploid RRL genotypes could be correctly determined. Problems arose in distinguishing between P. lessonae and individual LLR genotypes , as the latter sometimes exhibited lessonae-like phenotypes because of gene dosage effects. Furthermore, an error-free differentiation between diploid (LR) and triploid LLR genotypes based on only morphological and/or morphometric characteristics was not possible, but required genetic methods. The data collected confirm the presence of P. lessonae in the MNP, although its origin and population dynamics in the study area remain unclear. Irrespective of this, the MNP bears special importance for the protection of so-called lessonae-esculentus populations simply because of its habitat configuration , especially the numerous, semi-natural small water bodies. Zusammenfassung Der Müritz-Nationalpark (MNP) ist mit seinem Wald-und Gewässerreichtum eines der bedeutendsten Schutzgebiete im Bundesland Mecklenburg-Vorpommern (M-V) © Laurenti-Verlag, Bielefeld, www.laurenti.de Wasserfrösche im Müritz-Nationalpark 185 und darüber hinaus in der Bundesrepublik Deutschland. Die sehr zahlreichen Klein-gewässer und naturnahen, extensiv bewirtschafteten Landökosysteme bieten ideale Bedingungen für Amphibien, darunter auch Wasserfrösche, über die jedoch bisher kaum Daten veröffentlicht worden sind. Im Jahr 2021 wurde im Rahmen des FFH-Monitorings des Kleinen Wasserfroschs (Pelophylax lessonae) in M-V die genomische Struktur eines Wasserfrosch-Metapopulationssystems untersucht, das ein struktur-reiches, etwa 650 ha großes Gebiet im südlichen Teil des MNP besiedelt. Von 247 Wasserfröschen waren nur sechs P. lessonae (2,4 %); bei den übrigen Individuen han-delte es sich um diploide (36 %) und polyploide (61 %) Hybriden (Pelophylax esculen-tus). Unter den Polyploiden dominierten Genotypen mit zwei lessonae-Genomen und einem ridibundus-Genom (LLR) (94,7 %), nur sieben Individuen (4,7 %) besaßen zwei ridibundus-Genome und ein lessonae-Genom (RRL); ein Tier war wahrscheinlich tet-raploid (LLLR). Mit Hilfe von univariaten und multivariaten statistischen Methoden, die auf morphometrischen Daten basierten, konnten alle P. lessonae-und triploiden RRL-Genotypen korrekt determiniert werden. Probleme traten bei der Differenzie-rung zwischen P. lessonae und einzelnen LLR-Genotypen auf, da letztere aufgrund von Gen-Dosis-Effekten mitunter lessonae-ähnliche Phänotypen aufwiesen. Darüber hinaus war eine fehlerfreie Unterscheidung zwischen diploiden und triploiden LLR-Genotypen allein auf der Grundlage morphologischer und/oder morphometrischer Merkmale nicht möglich, sondern erforderte genetische Methoden. Die erhobenen Daten bestätigen das Vorkommen von P. lessonae im MNP, wenngleich seine Her-kunft und Populationsdynamik im Untersuchungsgebiet unklar bleibt. Unabhängig davon kommt dem MNP allein schon aufgrund seiner Habitat-Konfiguration, insbe-sondere der zahlreichen naturnahen Kleingewässer, eine besondere Bedeutung für den Schutz sogenannter lessonae-esculentus-Populationen zu.
Hybridogenesis is a hemiclonal reproductive strategy in diploid and triploid hybrids. Our study model is a frog P. esculentus (diploid RL and triploids RLL and RRL), a natural hybrid between P. lessonae (LL) and P. ridibundus (RR). Hybridogenesis relies on elimination of one genome (L or R) from gonocytes (G) in tadpole gonads during prespermatogenesis, but not from spermatogonial stem cells (SSCs) in adults. Here we provide the first comprehensive study of testis morphology combined with chromosome composition in the full spectrum of spermatogenic cells. Using genomic in situ hybridization (GISH) and FISH we determined genomes in metaphase plates and interphase nuclei in Gs and SSCs. We traced genomic composition of SSCs, spermatocytes and spermatozoa in individual adult males that were crossed with females of the parental species and gave progeny. Degenerating gonocytes (24%–39%) and SSCs (18%–20%) led to partial sterility of juvenile and adult gonads. We conclude that elimination and endoreplication not properly completed during prespermatogenesis may be halted when gonocytes become dormant in juveniles. After resumption of mitotic divisions by SSCs in adults, these 20% of cells with successful genome elimination and endoreplication continue spermatogenesis, while in about 80% spermatogenesis is deficient. Majority of abnormal cells are eliminated by cell death, however some of them give rise to aneuploid spermatocytes and spermatozoa which shows that hybridogenesis is a wasteful process.
Interspecific hybridization can disrupt canonical gametogenic pathways, leading to the emergence of clonal and hemiclonal organisms. Such gametogenic alterations usually include genome endoreplication and/or premeiotic elimination of one of the parental genomes. The hybrid frog Pelophylax esculentus exploits genome endoreplication and genome elimination to produce haploid gametes with chromosomes of only one parental species. To reproduce, hybrids coexist with one of the parental species and form specific population systems. Here, we investigated the mechanism of spermatogenesis in diploid P. esculentus from sympatric populations of P. ridibundus using fluorescent in situ hybridization. We found that the genome composition and ploidy of germ cells, meiotic cells, and spermatids vary among P. esculentus individuals. The spermatogenic patterns observed in various hybrid males suggest the occurrence of at least six diverse germ cell populations, each with a specific premeiotic genome elimination and endoreplication pathway. Besides co-occurring aberrant cells detected during meiosis and gamete aneuploidy, alterations in genome duplication and endoreplication have led to either haploid or diploid sperm production. Diploid P. esculentus males from mixed populations of P. ridibundus rarely follow classical hybridogenesis. Instead, hybrid males simultaneously produce gametes with different genome compositions and ploidy levels. The persistence of the studied mixed populations highly relies on gametes containing a genome of the other parental species, P. lessonae.
Genome stability is a crucial feature of eukaryotic organisms because its alteration drastically affects the normal development and survival of cells and the organism as a whole. Nevertheless, some organisms can selectively eliminate part of their genomes from certain cell types during specific stages of ontogenesis. This review aims to describe the phenomenon of programmed DNA elimination, which includes chromatin diminution (together with programmed genome rearrangement or DNA rearrangements), B and sex chromosome elimination, paternal genome elimination, parasitically induced genome elimination, and genome elimination in animal and plant hybrids. During programmed DNA elimination, individual chromosomal fragments, whole chromosomes, and even entire parental genomes can be selectively removed. Programmed DNA elimination occurs independently in different organisms, ranging from ciliate protozoa to mammals. Depending on the sequences destined for exclusion, programmed DNA elimination may serve as a radical mechanism of dosage compensation and inactivation of unnecessary or dangerous genetic entities. In hybrids, genome elimination results from competition between parental genomes. Despite the different consequences of DNA elimination, all genetic material destined for elimination must be first recognised, epigenetically marked, separated, and then removed and degraded.
We review knowledge about the roles of sex chromosomes in vertebrate hybridization and speciation, exploring a gradient of divergences with increasing reproductive isolation (speciation continuum). Under early divergence, well-differentiated sex chromosomes in meiotic hybrids may cause Haldane-effects and introgress less easily than autosomes. Undifferentiated sex chromosomes are more susceptible to introgression and form multiple (or new) sex chromosome systems with hardly predictable dominance hierarchies. Under increased divergence, most vertebrates reach complete intrinsic reproductive isolation. Slightly earlier, some hybrids (linked in ‘the extended speciation continuum’) exhibit aberrant gametogenesis, leading towards female clonality. This facilitates the evolution of various allodiploid and allo- polyploid clonal (‘asexual’) hybrid vertebrates, where ‘asexuality’ might be a form of intrinsic reproductive isolation. A comprehensive list of ‘asexual’ hybrid vertebrates shows that they all evolved from parents with divergences that were greater than at the intraspecific level (K2P-distances of greater than 5–22% based on mtDNA). These ‘asexual’ taxa inherited genetic sex determi- nation by mostly undifferentiated sex chromosomes. Among the few known sex-determining systems in hybrid ‘asexuals’, female heterogamety (ZW) occurred about twice as often as male heterogamety (XY). We hypothesize that pre-/meiotic aberrations in all-female ZW-hybrids present Haldane- effects promoting their evolution. Understanding the preconditions to produce various clonal or meiotic allopolyploids appears crucial for insights into the evolution of sex, ‘asexuality’ and polyploidy.
This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)’.
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.
Background – Hybridogenesis can represent the first stage towards hybrid speciation where the hybrid taxon eventually weans off its parental species. In hybridogenetic water frogs, the hybrid Pelophylax kl. esculentus (genomes RL) usually eliminates one genome from its germline and relies on its parental species P. lessonae (genomes LL) or P. ridibundus (genomes RR) to perpetuate in so-called L-E and R-E systems. But not exclusively: some all-hybrid populations (E-E system) bypass the need for their parental species and fulfill their sexual cycle via triploid hybrid frogs. Genetic surveys are essential to understand the great diversity of these hybridogenetic dynamics and their evolution. Here we conducted such study using RAD-sequencing on Pelophylax from southern Switzerland (Ticino), a geographically-isolated region featuring different assemblages of parental P. lessonae and hybrid P. kl. esculentus.
Results – We found two types of hybridogenetic systems in Ticino: an L-E system in northern populations and a presumably all-hybrid E-E system in the closely-related southern populations, where P. lessonae was not detected. In the latter, we did not find evidence for triploid individuals from the population genomic data, but identified a few P. ridibundus (RR) as offspring from interhybrid crosses (LR×LR).
Conclusions – Assuming P. lessonae is truly absent from southern Ticino, the putative maintenance of all-hybrid populations without triploid individuals would require an unusual lability of genome elimination, namely that P. kl. esculentus from both sexes are capable of producing gametes with either L or R genomes. This could be achieved by the co-existence of L- and R- eliminating lineages or by “hybrid amphigamy”, i. e. males and females producing sperm and eggs among which both genomes are represented. These hypotheses imply that polyploidy is not the exclusive evolutionary pathway for hybrids to become reproductively independent, and challenge the classical view that hybridogenetic taxa are necessarily sexual parasites.
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.
Background
Interspecies animal hybrids can employ clonal or hemiclonal reproduction modes where one or all parental genomes are transmitted to the progeny without recombination. Nevertheless, some interspecies hybrids retain strong connection with the parental species needed for successful reproduction. Appearance of polyploid hybrid animals may play an important role in the substitution of parental species and in the speciation process.
Results
To establish the mechanisms that enable parental species, diploid and polyploid hybrids coexist we have performed artificial crossing experiments of water frogs of Pelophylax esculentus complex. We identified tadpole karyotypes and oocyte genome composition in all females involved in the crossings. The majority of diploid and triploid hybrid frogs produced oocytes with 13 bivalents leading to haploid gametes with the same genome as parental species hybrids usually coexist with. After fertilization of such gametes only diploid animals appeared. Oocytes with 26 bivalents produced by some diploid hybrid frogs lead to diploid gametes, which give rise to triploid hybrids after fertilization. In gonads of all diploid and triploid hybrid tadpoles we found DAPI-positive micronuclei (nucleus-like bodies) involved in selective genome elimination. Hybrid male and female individuals produced tadpoles with variable karyotype and ploidy even in one crossing owing to gametes with various genome composition.
Conclusions
We propose a model of diploid and triploid hybrid frog reproduction in R-E population systems. Triploid Pelophylax esculentus hybrids can transmit genome of parental species they coexist with by producing haploid gametes with the same genome composition. Triploid hybrids cannot produce triploid individuals after crossings with each other and depend on diploid hybrid females producing diploid eggs. In contrast to other population systems, the majority of diploid and triploid hybrid females unexpectedly produced gametes with the same genome as parental species hybrids coexist with.
Electronic supplementary material
The online version of this article (10.1186/s12862-017-1063-3) contains supplementary material, which is available to authorized users.
Background:
The ability to eliminate a parental genome from a eukaryotic germ cell is a phenomenon observed mostly in hybrid organisms displaying an alternative propagation to sexual reproduction. For most taxa, the underlying cellular pathways and timing of the elimination process is only poorly understood. In the water frog hybrid Pelophylax esculentus (parental taxa are P. ridibundus and P. lessonae) the only described mechanism assumes that one parental genome is excluded from the germline during metamorphosis and prior to meiosis, while only second genome enters meiosis after endoreduplication. Our study of hybrids from a P. ridibundus-P. esculentus-male populations known for its production of more types of gametes shows that hybridogenetic mechanism of genome elimination is not uniform.
Results:
Using comparative genomic hybridization (CGH) on mitotic and meiotic cell stages, we identified at least two pathways of meiotic mechanisms. One type of Pelophylax esculentus males provides supporting evidence of a premeiotic elimination of one parental genome. In several other males we record the presence of both parental genomes in the late phases of meiotic prophase I (diplotene) and metaphase I.
Conclusion:
Some P. esculentus males have no genome elimination from the germ line prior to meiosis. Considering previous cytological and experimental evidence for a formation of both ridibundus and lessonae sperm within a single P. esculentus individual, we propose a hypothesis that genome elimination from the germline can either be postponed to the meiotic stages or absent altogether in these hybrids.
The structure of hemiclonal population systems (HPS) of Pelophylax esculentus complex from the Iskov pond (Gaydary village, Zmiyiv District), the Lower Dobritskiy pond (the region of Gomolsha river floodplain, Dobritskiy yar, the territory of the National Park “Gomolshanski lisy”) and from the pond near the village Zhovtneve (Vovchanskiy District, Kharkiv Region) was studied. All studied HPS were defined as REt-type. We observed the triploid hybrids number increasing in the HPS from the Iskov pond. The large part of triploid hybrids was found in the HPS of the Lower Dobritskiy pond. It was observed that all triploid hybrids from the pond near the village Zhovtneve were females. There was significant bond between the diploid erythrocytes size and sex of the frogs. It seems that triploids in this HPS are originating in other way compared to other HPS. There were two groups of females differ in body size which possibly corresponds to different ontogenetic strategies of green frog females. The males have a normal size distribution.
Key words: hemiclonal population systems, Seversko-Donetskiy center of green frogs diversity, Pelophylax esculentus complex, species composition, triploid hybrids, size distribution.
Hybridization between two species usually leads to inviable or infertile offspring, due to endogenous or exogenous selection pressures. Yet, hybrid taxa are found in several plant and animal genera, and some of these hybrid taxa are ecologically and evolutionarily very successful. One example of such a successful hybrid is the water frog, Pelophylax esculentus which originated from matings between the two species P. ridibundus (genotype RR) and P. lessonae (LL). At the northern border of the distribution all-hybrid populations consisting of diploid (LR) and one or two triploid (LLR, LRR) frog types have been established. Here, the hybrid has achieved reproductive independence from its sexual ancestors and forms a self-sustaining evolutionary unit. Based on the gamete production of these hybrids, certain mating combinations should lead to LL and RR offspring, but these parental forms are absent among the adults.
In order to investigate the mechanisms that maintain such an all-hybrid system, we performed a field study and a crossing experiment. In the field we sampled several ponds for water frog larvae at different developmental stages. Genotype compositions were then analysed and life-history differences between the genotypes examined. In the experiment we crossed diploid and triploid males and females from different ponds and determined fertilization success as well as development speed and survival rates of the offspring under high, medium and low food availability. In both parts of the study, we found numerous LL and RR offspring during the egg and early larval stages; but the frequency of these parental genotypes decreased drastically during later stages. In natural ponds almost all of them had disappeared already before metamorphosis; under the more benign experimental conditions the last ones died as juveniles during the following year.
From the combined results we conclude that the absence of parental genotypes in all-hybrid populations is due to post-zygotic selection against them, rather than to pre-zygotic mechanisms that might prevent their formation in the first place. For this post-zygotic selection, genetic mechanisms resulting from low genetic diversity and fixation of deleterious mutations seem to be a more likely explanation than ecological factors.
Incompatibilities between parental genomes decrease viability of interspecific hybrids; however , deviations from canonical gametogenesis such as genome endoreplication and elimination can rescue hybrid organisms. To evaluate frequency and regularity of genome elimination and endoreplication during gametogenesis in hybrid animals with different ploi-dy, we examined genome composition in oocytes of di-and triploid hybrid frogs of the Pelo-phylax esculentus complex. Obtained results allowed us to suggest that during oogenesis the endoreplication involves all genomes occurring before the selective genome elimination. We accepted the hypothesis that only elimination of one copied genome occurs pre-meiotically in most of triploid hybrid females. At the same time, we rejected the hypothesis stating that the genome of parental species hybrid frogs co-exist with is always eliminated during oogenesis in diploid hybrids. Diploid hybrid frogs demonstrate an enlarged frequency of deviations in oogenesis comparatively to triploid hybrids. Typical for hybrid frogs deviations in gametogenesis increase variability of produced gametes and provide a mechanism for appearance of different forms of hybrids.
The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all-hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures.
In eastern Ukraine, the Rana esculenta complex consists of three species: R. lessonae, R. ridibunda, and hybrid R. esculenta. The first one was rare, whereas two latter frog taxa were very common. Based on DNA flow cyto-metry, mass occurrence of the triploidy in Rana esculenta has been revealed in 14 localities of Kharkov, Do-netsk, and Lugansk Provinces. One hybrid specimen from Kharkov Province was tetraploid. All polyploids were recorded along the middle part of Seversky Donets River (above 450 km). Triploids comprised two groups with different genome composition (LLR and LRR), and were found in three types of population systems (E, R–E, and L–E–R). Geographic distribution of polyploidy in European green frogs is briefly outlined. Different meth-ods of ploidy level identification are discussed. The chromosome count and nuclear DNA cytometry provide the most reliable data.
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.
Microsatellites are commonly used for mapping and population genetics because of their high heterozygosities and allelic variability
(i.e., polymorphism). Microsatellite markers are generally more polymorphic than other types of molecular markers such as
allozymes or SNPs because the insertions/deletions that give rise to microsatellite variability are relatively common compared
to nucleotide substitutions. Nevertheless, direct evidence of microsatellite mutation rates (MMRs) is lacking in most vertebrate
groups despite the importance of such estimates to key population parameters (e.g., genetic differentiation or θ=4N
e
μ). Herein, we present empirical data on MMRs in eastern tiger salamanders (Ambystoma tigrinum tigrinum). We conducted captive breeding trials and genotyped over 1,000 offspring at a suite of microsatellite loci. These data on
7,906 allele transfers provide the first direct estimates of MMRs in amphibians, and they illustrate that MMRs can vary by
more than an order of magnitude across loci within a given species (one locus had ten mutations whereas the others had none).
support for multiple independent origins of clones
Background
The role of differential selection in determining the geographic distribution of genotypes in hybrid systems has long been discussed, but not settled. The present study aims to asses the importance of selection in structuring all-hybrid Pelophylax esculentus populations. These populations, in which the parental species (P. lessonae with genotype LL and P. ridibundus with genotype RR) are absent, have pond-specific proportions of diploid (LR) and triploid (LLR and LRR) genotypes.
Results
With data from 12 Swedish ponds, we first show that in spite of significant changes in genotype proportions over time, the most extreme ponds retained their differences over a six year study period. The uneven distribution of genotypes among ponds could be a consequence of differential selection varying among ponds (selection hypothesis), or, alternatively, of different gamete production patterns among ponds (gamete pattern hypothesis). The selection hypothesis was tested in adults by a six year mark-recapture study in all 12 ponds. As the relative survival and proportion of LLR, LR and LRR did not correlate within ponds, this study provided no evidence for the selection hypothesis in adults. Then, both hypotheses were tested simultaneously in juvenile stages (eggs, tadpoles, metamorphs and one year old froglets) in three of the ponds. A gradual approach to adult genotype proportions through successive stages would support the selection hypotheses, whereas the presence of adult genotype proportions already at the egg stage would support the gamete pattern hypothesis. The result was a weak preference for the gamete pattern hypothesis.
Conclusions
These results thus suggest that selection is of little importance for shaping genotype distributions of all-hybrid populations of P. esculentus, but further studies are needed for confirmation. Moreover, the study provided valuable data on genotype-specific body lengths, adult survival and sex ratios.
Triploid individuals often play a key role in speciation by hybridization. An understanding of the gamete types (ploidy and genomic content) and stability of hybrid populations with triploid individuals is therefore of importance for exploring the role of hybridization in evolution. The all-hybrid populations of the edible frog, Pelophylax esculentus, are unique in their composition and genetic dynamics: Diploid (genotype LR) and triploid (LLR and LRR) hybrids
depend on each other`s different gamete contributions for successful reproduction and
maintenance of the populations, as the parental genotypes P. lessonae (LL) and P. ridibundus (RR) are
absent among adults. This study provides data and interpretations on gamete types and sex
determination that are essential for understanding the function, evolutionary potential and threats
of this intriguing system.
The performance of three genotypes (LL, LR, RR) of tadpoles resulting from the hybrid mating system of Rana lessonae (phenotype L, genotype LL) and Rana esculenta (phenotype E, genotype LR) was determined in artificial ponds. The effects of interspecific competition and pond drying on growth, development, and survival of tadpoles were used to measure the performance of genotypes and the relative fitness of offspring. Among the three genotypes, tadpoles from the homogametic mating RR had the lowest survival, growth, and development under all environmental conditions. Body size of the LL and LR genotype tadpoles at metamorphosis was reduced by competition and pond drying. Days to metamorphosis were also higher for the LL and LR genotype tadpoles in competition ponds. The proportion of individuals metamorphosing of each genotype was differentially lowered by competition and pond drying. The LL genotype produced more metamorphs than the LR genotype in the constant water level ponds, but the LR genotype produced more in drying ponds. In competition ponds, the LR genotype produced more metamorphs than the LL genotype, but the LL genotype produced more metamorphs in ponds without competition. The RR genotype produced no metamorphs in any of the experimental environments. Increased performance of LR offspring from the heterogametic mating, in harsh conditions, and reduced performance of RR offspring from the homogametic mating, even under favorable conditions, relative to the parental genotype (LL) suggests that the population dynamics of this hybridogenetic system is strongly dependent on mate choice in mixed populations and the subsequent pond environment females select for oviposition and larval development.
Hybridization and polyploidy play an important role in animal speciation. European water frogs of the Pelophylax esculentus complex demonstrate unusual genetic phenomena associated with hybridization, clonality and polyploidy which presumably indicate an initial stage of reticulate speciation. The Seversky Donets River drainage in north-eastern Ukraine is inhabited by both sexes of the diploid and triploid hybrid P. esculentus and only one parental species Pelophylax ridibundus. Based on the presence of various types of hybrids, all populations studied can be divided into three geographical groups: I) P. ridibundus-P. esculentus without triploids; II) P. ridibundus-P. esculentus without diploid hybrids; and III) P. ridibundus-P. esculentus with a mixture of diploids and triploids. A study of gametogenesis revealed that diploid P. esculentus in populations of the first type usually produced haploid gametes of P. ridibundus and a mixture of haploid gametes that carried one or another parental genome (hybrid amphispermy). In populations of the second type, hybrids are derived from crosses of P. ridibundus males with triploid hybrid females producing haploid eggs with a genome of P. lessonae. Therefore, we suggest that clonal genome duplication in these eggs might be the result of suppression of second polar body formation or extra precleavage endoreduplication. In populations of the third type, some diploid females can produce diploid gametes. Fertilization of these eggs with haploid sperm can result in triploid hybrids. Other hybrids here produce haploid gametes with one or another parental genome or their mixture giving rise to new diploid hybrids.
Sex is the queen of problems in evolutionary biology. Generations of researchers have investigated one of the last remaining evolutionary paradoxes: why sex exists at all. Given that sexual reproduction is costly from an evolutionary point of view, one could wonder why not all animals and plants reproduce asexually. Dozens of contemporary hypotheses attempt to explain the prevalence of sex and its advantages and predict the early extinction of fully asexual lineages. The major theme of this book is: what is the fate of animal and plant groups in which sex is lost? Initial chapters discuss theory behind asexual life: what major disadvantages do asexual groups have to face, what are the genetic and ecological consequences and what does this theory predict for more applied aspects of asexual life, for example in agricultural pests, diseases as well as in cultural crops such as grapes. Cases studies in many animals (focusing on both invertebrates and vertebrates) and plants reveal parallel, but also singularly novel adaptations to the absence of meiosis and syngamy. And last but not least, are asexuals really doomed to early extinction or do genuine ancient asexuals exist? This book assembles contributions from the most important research groups dealing with asexual evolution in eukaryotes. It is a milestone in research on parthenogenesis and will be useful to undergraduate as well as graduate students and to senior researchers in all fields of evolutionary biology, as the paradox of sex remains its queen of problems.
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.
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.Key words: all-hybrid population; sperm-dependent parthenogenesis, asexual reproduction; gamete production; hemiclone; hybridogenesis; hybridization; polyploidy; Rana; sexual parasite.This article is protected by copyright. All rights reserved.
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.
There are three taxons of central European water frogs of the Pelophylax esculentus complex: two morphologically distinct species, Pelophylax lessonae (LL) and Pelophylax ridibundus (RR), and hybrids Pelophylax esculentus, which can be either diploid (RL) or triploid (LLR or RRL). The morphology of hybrids is supposed to follow genome dosage effect. We describe colouration of water frogs with genome composition verified by chromosome analysis. Typical colouration features in LL were: spots on the ventral side, brown limbs, "weak" femur spotting pattern, brown dorsal folds and yellow-green colour in "waist". Typical RR had dark-green or olive-green limbs, "full" femur spotting pattern, no hip spot and no yellow colour in "waist". For all hybrids the most typical features were strong spots on the ventral side and a mosaic of green and brown colour on limbs. Typical LLR had brown-greenish dorsal folds underlined by a partial black line, "weak" femur spotting pattern and yellow-greenish colour in "waist" and on femur. Typical RL had greenish hind limbs, green dorsal folds, no yellow colour in "waist", and no hip spot. Typical RRL was similar to RL, but had a continuous black line under dorsal folds. There were, however, numerous exceptions to these trends, with the most prominent being much higher than expected variability of colouration of Pelophylax lessonae. Therefore caution must be advised when trying to estimate genome composition of water frogs solely on the basis of colouration.
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.
Sexual parasites offer unique insights into asexual and sexual reproduction. They mate with a 'host' whose genetic contribution is discarded either immediately (in androgenesis or gynogenesis) or after a delay of one generation (in hybridogenesis). The discarded genome can be maternal or paternal, implying that not only females but also males can reproduce asexually. The resulting lineages are often older than ecological or evolutionary theory predicts. Sexual parasites have links to a diverse set of concepts: selfish genetic elements, degradation of clonal genomes, evolution of sex, mate-choice theory, and host-parasite dynamics. We discuss the different sexually parasitic systems in both hermaphrodites and gonochoristic organisms, emphasizing their similarities and differences in ecological and evolutionary settings.
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.
Experiments designed to analyze the lethality and hybridogenesis in the European green frog complex have yielded the following results: 1. As a rule the inter-se cross ofRana esculenta is lethal, but several crosses have produced fully viable progeny. The frequency of such break-through crosses appears to be related to parental population structure. 2. Parabiotic joining of lethal to viable embryos indicates that manifestation of the lethal effect is autonomous. There is, however, a 16–18% increase in the life span of the lethal partner. 3. Studies of LDH isozyme patterns revealed that thelessonae-specific alleles coding for the Ba and Bc subunits can be passed to the F1 progeny from a parental female or male of theesculenta phenotype. This demonstrates that there is no total elimination of thelessonae genome in theesculenta germ cells. 4. Immunologically, offspring from the inter-se cross ofR. esculenta show a closer relationship to theridibunda than to theesculenta phenotype. Variations of antigenic protein patterns suggest the possibility of chromosomal recombination betweenlessonae andridibunda in theesculenta hybrid. These results are confirmed by two-dimensional electrophoretic analysis of proteins in the oocytes of the three frog phenotypes.
Abstract 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.
Water frogs are involved in several hybridogenetic complexes. However, investigations in Southern and Western France produced evidence of new hybridization events between R. lessonae and R. perezi (some new hybrids tadpoles were discovered), and the existence of new assemblages [R-G, L-P, R-P, P-R-G: (R, ridibunda; G, graft; L, lessonae; P, perezi)]. In respect to hybridogenesis, these assemblages offer opportunities for both primary and secondary hybridizations. Such peculiarities may be explained either by introductions or by relic populations and may influence water frog evolution.
GENALEX is a user-friendly cross-platform package that runs within Microsoft Excel, enabling population genetic analyses of codominant, haploid and binary data. Allele frequency-based analyses include heterozygosity, F statistics, Nei's genetic distance, population assignment, probabilities of identity and pairwise relatedness. Distance-based calculations include AMOVA, principal coordinates analysis (PCA), Mantel tests, multivariate and 2D spatial autocorrelation and TWOGENER. More than 20 different graphs summarize data and aid exploration. Sequence and genotype data can be imported from automated sequencers, and exported to other software. Initially designed as tool for teaching, GENALEX 6 now offers features for researchers as well. Documentation and the program are available at http://www.anu.edu.au/BoZo/GenAlEx/
Linkage disequilibrium is an ubiquitous biological phenomenon. However a common metric for disequilibrium — the index of association or I A - is dependent on sample size. In this paper we present a modification of I A that removes this dependency. This method has been implemented in a software package.
Collection of blood from amphibians, as in other classes of vertebrate animals, is essential to evaluate parameters of health, diagnose hemoparasitism, identify viral and bacterial pathogens, and measure antibodies. Various methods of blood collection have been described for amphibians. Most can be cumbersome (venipucture of femoral vein, ventral abdominal vein or lingual venus plexus) or result in pain or deleterious health consequences (cardiac puncture and toe-clipping). We describe an easy and practical technique to collect blood from frogs and toads that can be used in multiple species and is minimally invasive. The technique consists of puncturing either the facial or, less commonly, the musculo-cutaneous vein and collecting the blood with a capillary tube. These veins run dorsal and parallel to the maxillary bone and can be accessed by quick insertion and withdrawal of a needle through the skin between the upper jawline and the rostral or caudal side of the tympanum. The needle should be of 27 or 30 gauge for anurans weighing more or less than 25 g, respectively. Although the technique has been used by some amphibian researchers for years, it is little known by others and has never been fully described in a peer-reviewed publication.
In most parts of central Europe the hybridogenetic water frog Rana esculenta lives (and mates) in mixed populations with its parental species Rana lessonae. Ratios of parental and hybrid animals vary from 1:9 to 9:1 among ponds, depending on various biotic and abiotic factors. These factors may directly cause the ratio differences through species-specific effects on larval performance, but previous experimental studies under laboratory and seminatural conditions produced conflicting results as to how parental and hybrid tadpoles respond to these factors. We investigated larval development and relative recruitment of juveniles in a natural population at three sites that differed ecologically. Species ratios at the different stages (eggs, tadpoles, metamorphs) and body size of tadpoles and metamorphs were compared. Differences in timing of reproduction and larval development were more pronounced within species between ponds than between species within ponds. During the egg and tadpole stages, hybrids outnumbered parental larvae by 60:40, but this ratio reversed during metamorphosis. Higher mortality rates of hybrid froglets during metamorphosis as well as lower dispersal rates after metamorphosis may explain our findings.
Keywords: hybridogenesis;microsatellites;primers;Rana esculenta;Rana lessonae;water frogs
Pelophylax esculentus is a hybridogenetic frog originating from matings between P. ridibundus (RR) and P. lessonae (LL). Typically, diploid hybrids (LR) live in sympatry with one of their parental species, upon which they depend for successful reproduction. In parts of their range, however, pure hybrid populations can be found. These hybrid populations have achieved reproductive independence from their parental species by using triploid hybrids (LLR, LRR) rather than LL and RR as their sexual hosts. These different breeding systems also entail differences in reproduction (clonal versus sexual) and hence offer the opportunity to study how genetic diversity is affected by reproductive mode, population structure and geographic location. We investigated 33 populations in the Scania region (South Sweden) and 18 additional populations from Northern and Central Europe. Within both genomes (L, R), genetic variability increases with the potential for recombination and declines from the main species distribution area southeast of the Baltic Sea to the fringe populations northwest of the Baltic Sea. Within the main study area in Scania, genetic diversity is low and decreases from a core area to the periphery. Genetic differentiation between Scania populations is small but significant and best explained by 'isolation by distance'. Despite the low genetic variability within the discrete genomes, all-hybrid P. esculentus populations in southern Sweden are apparently not suffering from direct negative fitness effects. This is probably because of its somatic hybrid status, which increases diversity through the combination of genomes from two species.
Speciation via interspecific hybrids is very rare in animals, as compared to plants. Whereas most plants overcome the problem of meiosis between different chromosome sets by tetraploidization, animal hybrids often escape hybrid sterility by clonal reproduction. This comes at the expense of genetic diversity and the ability to purge deleterious mutations. However, here we show that all-hybrid populations of diploid (LR) and triploid (LLR and LRR) water frogs (Pelophylax esculentus) have secondarily acquired sexual reproduction. First, in a crossing experiment analyzed with microsatellite markers, triploid hybrids of both sexes and genotypes (LLR and LRR) recombined their homospecific genomes. Second, the great majority of natural populations investigated had low multilocus linkage isequilibrium, indicating a high recombination rate. As predicted from mating system models, the L genome had constant, low levels of linkage disequilibrium, whereas linkage disequilibrium in th
e R genome showed a significant reduction with increasing proportion of recombining triploids. This direct evidence of sexual reproduction in P. esculentus calls
for a change of the conventional view of hybridogens as clonally reproducing diploids. Rather, hybridogens can be independent sexually reproducing units with an evolutionary potential.