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Geographic peculiarities of structure and hemicloning reproduction of Pelophylax esculentus water frog complex (Anura, Ranidae) populations in the East European Plain within Ukraine
Abstract
Geographic peculiarities of population structure and hemicloning reproduction of the water frog Pelophylax esculentus complex of 904 samples within southern East European Plain were analyzed. The proportion of populations of P. ridibundus is 0.667 against those 0.042 of P. lessonae . The RE-type populations are the most common among mixed ones (0.153). The colonies of the two parental species and P. kl. esculentus (REL-type), as well as those of P. lessonae with P. kl. esculentus (LE-type), have the frequencies 0.072 and 0.046, respectively. All-hybrid populations (E-type) are not numerous across the region (0.017). In the populations of Central, Northern and Western Ukraine, the P. lessonae genome is eliminated during gametogenesis within hybrids while in the Lower Dnieper and Lower Danube drainages, genome of P. ridibundus is eliminated. In the Eastern Ukraine populations, hybrids usually produce diploid gametes or haploid gametes with the P. ridibundus genome, less often with the chromosome set of P. lessonae only, and even more rarely a mixture of different types of gametes. The predominance of P. ridibundus in hybrid communities and the elimination of the P. lessonae genome in hybrids inevitably leads to the transformation of hybrid populations into “pure” R-type populations. This circumstance makes hybridization with P. ridibundus a factor in the shrinking of P. lessonae populations. That trend is especially expressed in the Forest steppe zone, where hybridization proceeds most intensively, the P. lessonae genome is eliminated in hybrids, and unstable populations with a numerical superiority of P. ridibundus predominate.
... If it represents a sustainable introduction, the latter, which was detected opportunistically (Dufresnes et al., 2024b) may be worrisome as it could threaten the native Pelophylax communities inhabiting the Eastern European plains. For instance, Ukraine, which is home to well-studied hybridogenetic populations (Mezhzherin et al., 2023;Suriadna et al., 2020), seems to have so far been spared of alien marsh frog invasions. Given the high levels of Turkish exports, it is likely that the Turkish lineages may continue to spread across European populations. ...
While some biological invasions are well documented globally, others, more cryptic, are often underestimated despite multiple local warnings. This is the case of the marsh frog (Pelophylax ridibundus sensu lato), a Palearctic anuran amphibian distribured from Western Europe to Central Asia. Marsh frogs have been introduced into many European countries, where they pose threats to biodiversity, yet an integrative understanding of their invasion is lacking. Therefore, we combined diverse bibliographic sources with recent DNA barcoding to determine the extent and diversity of invasions in Europe and substantiate the causes and pathways of introductions. We document alien frog populations across 167 sub-regional administrative areas in 19 European countries, corresponding to nine phylogeographic lineages originating from three continents. Introduction pathways from the Balkans and the Pannonian Plain, Anatolia and the Levant coincide with the history of live frog imports into Europe, which involved the trade of hundreds of millions of individuals. Introductions were mainly associated with the consumption of frogs' legs, but also with ornamental, educational and research purposes. While some introductions date to the 18th century, most took place in the second half of the 20th century and new cases continue to emerge. Altogether these results show that the marsh frog is a widespread and complex invasive amphibian species in Europe, and ranks among the most worrying amphibian invaders in the world. Our assessment calls for the immediate ban on the commercial import of live water frogs, especially to prevent new lineage combinations in invaded areas, a Pandora's box that, if opened, could further boost the species' invasive potential.
... Accurate identification of waterfrogs (Pelophylax esculentus complex) using morphological traits is problematic in areas where hybrid frogs occur, requiring the use of molecular methods to distinguish between taxa (e.g. Herczeg et al. 2017, Jośko & Pabijan 2020, Mezhzherin et al. 2023. We used a molecular technique based on amplification of the nuclear serum albumin intron 1 (SAI-1) described by Hauswaldt et al. (2012) and modified by Ermakov et al. (2019). ...
The chytrid Batrachochytrium dendrobatidis
(Bd) is a widespread fungus causing amphibian
declines across the globe. Although data on Bd occurrence
in Eastern Europe are scarce, a recent species
distribution model (SDM) for Bd reported that western
and north-western parts of Ukraine are highly suitable
to the pathogen. We verified the SDM-predicted
range of Bd in Ukraine by sampling amphibians across
the country and screening for Bd using qPCR. A total
of 446 amphibian samples (tissue and skin swabs) from
11 species were collected from 36 localities. We obtained
qPCR-positive results for 33 samples including
waterfrogs (Pelophylax esculentus complex) and fireand
yellow-bellied toads (Bombina spp.) from 8
localities. We found that Bd-positive localities had
significantly higher predicted Bd habitat suitability
than sites that were pathogen-free. Amplification and
sequencing of the internal transcribed spacer (ITS)
region of samples with the highest Bd load revealed
matches with ITS haplotypes of the globally distributed
BdGPL strain, and a single case of the BdASIA-2/
BdBRAZIL haplotype. We found that Bd was nonrandomly
distributed across Ukraine, with infections
present in the western and north-central forested
peripheries of the country with a relatively cool, moist
climate. On the other hand, our results suggest that Bd
is absent or present in low abundance in the more continental
central, southern and eastern regions of Ukraine,
corroborating the model-predicted distribution
of chytrid fungus. These areas could potentially serve
as climatic refugia for Bd-susceptible amphibian hosts.
... The established mass aberrations of premeiotic elimination within hybrids from the Dnieper region are obviously not directly related to the local features of gametogenesis of hybrids. Strict elimination of the chromosome set of P. lessonae within hybrids that is characteristic of populations from this region (Mezhzherin et al. 2023), is a common situation for most of the hybridization zone of P. ridibundus and P. lessonae. ...
Reproduction of water frog hybrids Pelophylax esculentus (Pelophylax ridibundus x Pelophylax lessonae) is associated with hemiclonal reproduction and backcrossing. The hemiclonal mode of reproduction occurs within P. esculentus allodiploids. In this case, the unrecombined genome of one parental species is transmitted to the offspring after premeiotic elimination of the chromosome set of the second parental species. Usually, the chromosome set of P. lessonae is eliminated, and the altered genome of P. ridibundus is passed on to the progeny. The hemiclonal inheritance within diploid Pelophylax esculentus hybrids may be accompanied by certain aberrations of premeiotic elimination. As a result, the formation of P. ridibundus specimens with introgressions of the P. lessonae genetic material, or the formation of recombinant hybrids occurs, depending on which of the parental species backcrossing takes place. The aim of our study is to describe the aberration of premeiotic elimination within the water frog P. esculentus complex detected by the nuclear gene Ldh-B inheritance, with an attempt to find out the causes of this phenomenon. It has been established that aberrations of premeiotic elimination are widespread, but only within populations of water frog from the river system of Upper Dnieper within Ukraine. The highest level of introgression takes place in the water frog populations within Kiev metropolis under conditions of expressed anthropogenization, while the maximum frequency of recombinants was detected within populations from the basin of Desna River, that has preserved native ecosystems. It was demonstrated that the frequency of premeiotic aberrations does not correlate with the intensity of interspecific water frog hybridization. Populations with introgressions are more common than populations with recombinants, however, within the latter, the frequency of recombination events is higher. The primary factor of gametogenesis aberrations, most likely, is the genetic characteristics of the local populations of parental species, since unambiguous explanations of this phenomenon based on the action of environmental stress (pollution of water systems) are not obvious.
Ecological and evolutionary consequences of population-genetic processes that occur because of natural cross-species hybridization can show mechanisms of overcoming the reproductive barrier and obtaining the species status by a hybrid taxon. This is clearly seen in the population systems of Eurasian water frogs – Pelophylax esculentus complex. The P. esculentus (E) hybrid usually discards one of the parental genomes of P. lessonae (L) or P. ridibundus (R) and reproduces semi-clonally. The genetic structure and direction of gene flows precisely depend on the type and distribution of mixed or pure population systems of water frogs. Three population systems in the south of Ukraine were identified and confirmed as RR, RE and REL. The populations of P. ridibundus are most common (76.2%). A mixed population systems of P. ridibundus and P. esculentus (20.0%) are concentrated in the floodplains of large rivers where triploids were found and the unisexual hybrids (1.0♂ : 0.1♀) were proved. Parent species populations having different ploidy of P. esculentus such as 3n and for the first time 4n were found. A mixed system of three taxa (REL) is rare (3.8%) and locally concentrated in the lower Danube and Dnieper with the smallest proportion of P. lessonae. We did not find populations of P. lessonae (LL), P. esculentus (EE, very rare system of hybrids only), and two mixed populations of parental species RL and semi-clonal LE in the south of Ukraine, but they are known for northern areas. The high number of P. ridibundus tends to decrease; the scarce P. esculentus and the extremely rare P. lessonae require special conservation measures. P. ridibundus (RR) occupies a wide range of diverse natural, permanent, temporary, coastal, continental, and artificial freshwater bodies, including synanthropic ecosystems. Mixed population systems inhabit willow and poplar forests in the floodplains of large rivers. In the south of Ukraine rare and isolated populations of the water frogs occurring outside the main range can be relict. Biotopic preferences, ratio and number of constituent taxa are crucial for an adequate assessment of biological (taxonomic) diversity and development of an appropriate strategy for the population systems’ conservation. Such characteristics as unisexuality of hybrids, their spreading patterns, specific sex structure and ploidy in different population systems of the P. esculentus complex contribute to the understanding of the hybridogenetic dynamics; produce new tendencies of becoming independent hybridogenous taxa and emergence of new evolutionary relationships.
GIS modeling of 138 georeferenced point data of anomalies in amphibians in Ukraine (data from both the literature and personal field surveys) revealed a rather poor, but statistically significant correlation of the numbers of anomalies with the anthropogenic impact measured by the Human Footprint (r=0,268, p=0,0025). We consider that in certain cases the abundance of anomalies in amphibians could be used for purposes of bioindication.
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.
Hybridization may drive rare taxa to extinction through genetic swamping, where the rare form is replaced by hybrids, or by demographic swamping, where population growth rates are reduced due to the wasteful production of maladaptive hybrids. Conversely, hybridization may rescue the viability of small, inbred populations. Understanding the factors that contribute to destructive versus constructive outcomes of hybridization is key to managing conservation concerns. Here we survey the literature for studies of hybridization and extinction to identify the ecological, evolutionary, and genetic factors that critically affect extinction risk through hybridization. We find that while extinction risk is highly situation dependent, genetic swamping is much more frequent than demographic swamping. In addition, human involvement is associated with increased risk and high reproductive isolation with reduced risk. Although climate change is predicted to increase the risk of hybridization-induced extinction, we find little empirical support for this prediction. Similarly, theoretical and experimental studies imply that genetic rescue through hybridization may be equally or more probable than demographic swamping, but our literature survey failed to support this claim. We conclude that halting the introduction of hybridization-prone exotics and restoring mature and diverse habitats that are resistant to hybrid establishment should be management priorities. This article is protected by copyright. All rights reserved.
The structure of
the green frogs hybrid populations Rana esculenta complex from the Middle Dnepr basin located in the
gradient from the total urban to wildlife landscapes far from Kyiv was studied by the biochemical genetic
marking. Five genetic forms of green frog were detected: the marsh frog R. ridibunda (R-form), the pool
frog R. esculenta (= lessonae) (L-form), the allodiploid hybrids R. kl. esculenta (E form), the marsh frog
with the introgressions (Ri form) and the recombinant hybrid (Er from), that are creating 5 population
types (R, L, RE, LE, REL — types). In the total urban part of Kiev only the marsh frog populations
are detected. The Ri and Er forms presence is demonstrated only for R, RE, REL population types, the
frequency of the marsh frogs with introgression being decreasing from the total urban zone (up to 40%)
to the wildlife landscapes. It was detected that the sexual constitution of the hybrids depends on the
population system type. Hybrids in the RE and REL populations practically always are males, although
in the LE ones the hybrids are the males and the females in equal proportion. The presented results
demonstrates the two alternative hybrid population types existence: the unstable populations which
structure is reinforced by the permanent parental species hybridization (RE and REL types) and stable
ones (LE type), which reinforcement by the parental species hybridization is not obligatory. These
populations are distinguished by the genetic peculiarities of the hybrids.
n the basis of genetically identified series of green frogs complex Rana esculenta main d³agnostic indices of the hind legs were identified. The best diagnostics was achieved in distinguishing R. esculenta L., 1758 (=lessonaeCamerano, 1882) (90% – average percentage of identified specimens), somewhat less – R. ridibunda (87,6%), a less of all – hybrids (49,2%). The most reliable diagnosticsoccurs by using all the selectes indices (99,5%), also highly reliable are discriminant combinations of particular variables (95—93%) and the multiplicative index of Tarashchuk (93%); the use of single traits results in 93% to 50% of identified specimens. Traditionally used for diagnosis T./C.in. and D.p./C.in. provide the most accurate identification (93 and 88%respectively), the diagnostic value of the rest are much lower – from 77 to 50%.
The water-frog L–E system, widespread in Western Europe, comprises the pool frog Pelophylax lessonae and the hybridogenetic edible frog P. esculentus, which originated from hybridization between pool frogs and marsh frogs (P. ridibundus). In P. esculentus, the lessonae (L) genome is eliminated during meiosis and has to be gained anew each generation from a P. lessonae partner, while the ridibundus (R′) genome is transmitted clonally. It therefore accumulates deleterious mutations, so that R′R′ offspring from P.
esculentus×P.
esculentus crosses are normally unviable. This system is now threatened by invasive P. ridibundus (RR) imported from Eastern Europe and the Balkans. We investigated the genetic interactions between invasive marsh frogs and native water frogs in a Swiss wetland area, and used genetic data collected in the field to validate several components of a recently postulated mechanism of species replacement. We identified neo-ridibundus individuals derived from crosses between invasive ridibundus and native esculentus, as well as newly arisen hybridogenetic esculentus lineages stemming from crosses between invasive ridibundus (RR) and native lessonae (LL). As their ridibundus genomes are likely to carry less deleterious mutations, such lineages are expected to produce viable ridibundus offspring, contributing to species replacement. However, such crosses with invasive ridibundus only occurred at a limited scale; moreover, RR×LL crosses did not induce any introgression from the ridibundus to the lessonae genome. We did not find any ridibundus stemming from crosses between ancient esculentus lineages. Despite several decades of presence on the site, introduced ridibundus individuals only represent 15 % of sampled frogs, and their spatial distribution seems shaped by specific ecological requirements rather than history of colonization. We therefore expect the three taxa to coexist stably in this area.
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.
Poland is inhabited by the two water frog species Rana ridibunda (genotype RR) and Rana lessonae (LL) and their hybrido-genetic hybrid Rana esculenta. Besides diploid (RL) hybrids two triploid (RRL, RLL) forms were found. In most populations the hybrid occurs syntopically with only one of the parental species. Concerning their genotypic structure water frog populations can be subdivided into three systems: the L-E (lessonae-esculenta) system, the R-E (ridibunda-esculenta) system and the E-E (esculenta-esculenta) system. The composition of water frog populations in Poland is highly differentiated. Among 27 population types described from central Europe 18 types were found in Poland. The highest diversity of population types was seen in the R-E system (8 types). In the L-E system three and in the E-E system four different types were found. Among 11170 investigated adult water frogs 690 (6%) presumptive triploid R. esculenta individuals occurred. In western Poland the proportion of triploid individuals and the diversity of population types was higher (7.7% triploid individuals, 13 population types) than in the eastern parts of the country (0.2% triploid individuals, six population types). The distribution picture of triploid individuals in western Poland is similar to that seen in eastern Germany (as a rule a high proportion of triploids occurs) while in eastern Poland the distribution pattern of triploid genotypes resembles the situation observed in the territory of the former Soviet Union (nearly no triploids appear).
Until recently, one widespread species of the genus Cobitis was thought to be present in Europe, the common spined loach Cobitis taenia. Recent studies have shown that the diversity of spined loaches is considerably higher due to (1) presence of several species as well as C. taenia and (2) presence of hybrid biotypes, living together with species. Here knowledge about distribution and diversity of spined loaches in Europe is summarized by combining original with published data. In Southern Europe, a number of distinct species occur allopatrically, while in Central and Eastern Europe a few species have wide distribution areas. Hybrid biotypes were found exclusively in Central and Eastern Europe. They were not restricted to the contact areas between the parental species, but occur in almost the entire area. In total, 15 hybrid biotypes with different genomic compositions were recorded. In the most diverse complex, four hybrid biotypes were associated with one species. In general, a syntopic occurrence of different species of Cobitis can be considered as exceptional, while the association of species with hybrid biotypes is the rule in Central and Eastern Europe. The composition of complexes seems to be most strongly influenced by the kind of associated species and by local history. Implications for conservation are (1) not to disturb the natural patterns of diversity, (2) to consider the known richness in legislation, and (3) to consider the evolutionary significance of hybrid biotypes.
In many species, males and females mate with multiple partners, which gives rise to sperm competition and multiple paternity. The experiments on water frogs presented here demonstrate that such sperm competition can affect the structure and dynamics of mixed-species communities. The hybrid frog Rana esculenta ( LR) mates with one of its parental species, usually R. lessonae ( LL), although in some areas R. ridibunda ( RR) , to regain the premeiotically eliminated parental genome ("hybridogenesis"). Mixed LL/LR-populations are stable although hybrid numbers should continuously increase at the expense of parental animals, because of differences in female fecundity and other factors. This would finally lead to the extinction of the sexual host, followed by that of the sexual parasite, unless the reproductive superiority of R. esculenta is reduced by other factors, such as lower hybrid male fertility. Eggs from LL- and LR-females were fertilised in vitro by single- and multi-male sperm suspensions of LL-, LR- and RR-males. In all experiments, the proportion of offspring sired by R. esculenta sperm was significantly lower than that sired by R. lessonae or R. ridibunda sperm. Gonad mass, sperm morphology, sperm swimming velocity, and sperm survival did not explain these differences in fertilisation success; nor did gamete recognition and compatibility. Sperm density was the only trait that paralleled fertilisation success, but it offers no explanation either, because densities were equalised for the in-vitro fertilisations. In natural LL/LR populations, the significantly smaller amount, poorer competitive ability and lower long-term survival of R. esculenta compared to R. lessonae sperm will reduce the initial reproductive superiority of hybrids and contribute to the stabilisation of mixed water-frog populations. Differences in fertilisation ability are also likely to be relevant for the structure and dynamics of several other systems with encounters between eggs and sperm from different genotypes, ecotypes, ploidy levels and/or species.
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.
The complex study, including allozyme variability and cytometry of hybrid populations of green frogs Pelophylax esculentus (L., 1758) complex has confirmed that the only region of Ukraine where allodiploid are encountered frequently is the Severski
Donets basin (9% of all hybrids). In other areas, only two polyploidy hybrids (0.9%) and one probably autopolyploid individual
of each parental species have been registered. According to allozyme specters, all three polyploidy hybrids from the Severski
Donets basin were males and belonged to biotype P. esculentus (=lessonae) — 2 ridibundus, and their population in this region has halved during the past decade.
In this study, quantitative analysis of paternal genome inheritance by a hybrid form Rana “esculenta” (= Rana esculenta L., 1758 × Rana ridibunda Pall., 1881) (Amphibia, Ranidae) was examined. The hybrid form examined was characterized by a polymodal mode of inheritance
(genome of any of the parental species can be inherited). The absence of correlation between the proportion of normal gametes
and either sex or ploidity of the producer was demonstrated. The gametes produced could be both haploid and diploid (hybrid
or homozygous). The mechanism of alloploid reproduction is discussed.
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.
In species with internal fertilization, females can 'cryptically' choose (e.g. through sperm selection) which individuals sire their offspring, even when their overt preferences for copulatory partners are overrun by male-male competition and sexual coercion. The experiment presented here reveals that control of paternity after copulation has begun is also possible in species with external fertilization. Females of the hybridogenetic Rana essonae-Rana esculenta (LL-LR) waterfrog complex adjust their clutch size in response to mate type: they release fewer eggs when amplexed by hybrid LR males who--jeopardize successful reproduction--than when amplexed by parental LL males. This reduction in the number of eggs laid can increase a female's residual reproductive value through a second mating in the same breeding season or a larger clutch size in the next year. We argue that cryptic female choice through clutch size adjustment (i) may have evolved more often than previously assumed, and (ii) can arise even where females mate only once during a reproductive period.
The biochemical genetic marking and analysis of the ploidy and sexual structure of the green frog populations from the Transcarpathian lowlands have demonstrated that this region is inhabited by the unisexual populations composed of the allodiploid females only. Their genome includes a small portion of the marsh frog genic diversity. This phenomenon is discussed in relation to the unisexual population reproduction problem. The assumption is proposed that in the Transcarpathian hybrid populations the hybrids are spawned by the parthenogenesis.
To persist, unisexual and asexual eukaryotes must have reproductive modes that circumvent normal bisexual reproduction. Parthenogenesis, gynogenesis, and hybridogenesis are the modes that have generally been ascribed to various unisexuals. Unisexual Ambystoma are abundant around the Great Lakes region of North America, and have variously been described as having all 3 reproductive modes. Diploid and polyploid unisexuals have nuclear genomes that combine the haploid genomes of 2 to 4 distinct sexual species, but the mtDNA is unlike any of those 4 species and is similar to another species, Ambystoma barbouri. To obtain better resolution of the reproductive mode used by unisexual Ambystoma and to explore the relationship of A. barbouri to the unisexuals, we sequenced the mitochondrial control and highly variable intergenic spacer region of 48 ambystomatids, which included 28 unisexuals, representatives of the 4 sexual species and A. barbouri. The unisexuals have similar sequences over most of their range, and form a close sister group to A. barbouri, with an estimated time of divergence of 2.4-3.9 million years ago. Individuals from the Lake Erie Islands (Kelleys, Pelee, North Bass) have a haplotype that demonstrates an isolation event. We examined highly variable microsatellite loci, and found that the genetic makeup of the unisexuals is highly variable and that unisexual individuals share microsatellite alleles with sexual individuals within populations. Although many progeny from the same female had the same genotype for 5 microsatellite DNA loci, there was no indication that any particular genome is consistently inherited in a clonal fashion in a population. The reproductive mode used by unisexual Ambystoma appears to be unique; we suggest kleptogenesis as a new unisexual reproductive mode that is used by these salamanders.
The evolutionary ecology of unisexual fishes is distinguished from that of closely related sexual species by two peculiar aspects of their genetics: First, they reproduce, in effect, asexually, and second, their genomic constitutions are invariably identical to those of F1 hybrids. Thus, a major difficulty in analyzing the evolutionary ecology of these species is to determine the extent to which the distinct phenomena of asexuality and hybridity contribute to their successes and failures. That is, where abundance indicates the success of a particular unisexual fish species, is it successful there because it reproduces asexually or because it possesses a hybrid genotype? The ecological consequences of asexuality, in turn, can be more finely divided. Asexually reproducing organisms do not pay the cost of meiosis (Williams, 1975), and, hence, an asexual species has twice the intrinsic rate of increase r of a comparable sexual species (Maynard Smith, 1978). On the other hand, asexual species lack genetic recombination and cannot generate the multiplicity of genotypes possible in a sexual species. This paucity of genotypic variation may limit the effectiveness of selection and, hence, evolutionary potential or it may reduce the aggregate effectiveness of resource utilization because there cannot be a spectrum of “specialized” genotypes in the population.
From the results of Berger’s studies on European green frogs in Poland, it is known that Rana esculenta Linnaeus is a hybrid between Rana lessonae Camerano and Rana ridibunda Pallas (Berger 1964, 1966, 1967, 1968a, 1968b, 1970). When the current study was begun, only Rana esculenta had been reported for the greater part of Switzerland. Actually, three types of green frogs, Rana esculenta, Rana lessonae and Rana ridibunda occur in this region (Blankenhorn et al. 1971). The natural ratio of the three phenotypes is peculiar. Out of 24 frog populations explored in the course of the study, only three were pure ridibunda populations, whereas 21 populations were mixed populations of R. lessonae and R. esculenta (Blankenhorn 1973). The R. ridibunda populations are separated geographically from the R. lessonae and R. esculenta region, and there is strong evidence that R. ridibunda was introduced between 1950 and 1970 by medical institutes using R. ridibunda from eastern Europe for pregnancy tests (Blankenhorn 1973).
European green frogs include the phenotypically distinct, largely sympatric forms Rana lessonae Camerano 1882, R. esculenta Linnaeus 1758, and R. ridibunda Pallas 1771, the relationships of which is still unsufficiently known. Recent investigations on Polish green frog populations indicate that R. lessonae and R. ridibunda are taxa at species rank, whereas R. esculenta is a hybrid resulting from interspecific crosses between R. lessonae and R. ridibunda. All three forms of green frogs are easily crossable indicating close relationship, and lack of genetic isolation. Ecological isolating mechanisms were probably established during the Pleistocene. In spite of very frequent natural hybridization, very high number of F1 hybrids (= esculanta) in nature, and successful backcrosses especially with lessonae, introgression between lessonae and ridibunda seems not to occur owing to an incompletely understood hereditary mechanism.
The diploid hybridogenetic frog Rana esculenta, an interspecific hybrid of R. ridibunda and R. lessonae, usually transmits its ridibunda genome to progeny: the lessonae genome is lost before gametogenesis is completed. Samples of ovary reveal only ridibunda alleles when examined electrophoretically. Cytological and genetic evidence suggests that the lessonae genome is excluded premeiotically. Since recombination between the parental genomes occurs at a low frequency, such recombination should be detectable by examination of the alleles expressed in individual enlarged oocytes.
Examination of electrophoretic markers for four loci from 860 individual oocytes (100 each from eight females, ten each from six females) revealed no evidence of either heterozygosity or recombination, although the eight females for which 100 oocytes were examined were somatically heterozygous 29 times for these loci, so that there were 2,900 possibilities for detecting heterozygosity or recombination in the oocytes.
These data abundantly confirm the inactivity of the lessonae genome during vitellogenesis in Rana esculenta. They are consistent with a premeiotic exclusion of the lessonae genome, but do not require it. The lessonae genome may be present although inactive, which may permit occasional crossing over between the lessonae and ridibunda genomes. The exclusion of the lessonae genome possibly is a consequence of its inactivity. Exclusion of the non-ridibunda genome seems to be under control of loci located in the ridibunda genome. A regulatory endoduplication of the ridibunda genome may be necessary before an orderly meiosis can occur.
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.
The evolution and maintenance of female choice based on purely genetic differences is still a controversial issue, not only for theoretical reasons, but also because of the practical difficulty of demonstrating the fitness consequences of preferences and heritability of and genetic variability in the chosen traits. We argue that hybrid systems (broadly defined) offer suitable models for studying mate choice according to genetic differences. We present such a study for European water-frogs of the hybridogenetic Rana lessonae/Rana esculenta complex (L/E complex). R. esculenta, originally a hybrid between R. lessonae and R. ridibunda, eliminates the L genome premeiotically and only produces eggs and sperm containing only the R. ridibunda (R) genome. Consequently, the hybrid will only persist when it lives and mates with R. lessonae in mixed populations where it can regain the lost L genome. In such mixed populations, there is strong selection against E x E matings. because these will produce no viable offspring. We tested whether females of the hybrid R. esculenta do indeed avoid their own R. exculenta males and choose males of the parental species R. lessonae instead. Eleven E females were offered a simultaenous choice between one L and one E male. Females exhibited a significant preference for L males that was determined by the type of male, rather than by its size or activity. This choice is in the direction predicted from genetics. The question of why L males agree to mate with E females, but L females only rarely mate with E males, is answered by a sexual asymmetry in the cost/benefit ratios of mating with the wrong type and the right size. Our results are consistent with the mating pattern found in natural populations, but further studies are needed to show that female choice really causes this pattern.
Since their discovery in 1932, all-female ''species'' of fish have provided rich material for ecological and evolutionary studies. The significance of these rare organisms lies in the perspective they provide on what is considered normal (i.e. biparental sexuality). Study of these fish and their sexual relatives has contributed to our understanding of: (i) the origins and evolution of asexuality; (ii) the ecology of hybrids; (iii) genotypic and environmental effects on ecologically relevant traits; and (iv) the maintenance of sex in higher organisms. A consistent message emerging from these studies is the significance of genetic diversity for survival in spatially and temporally heterogeneous environments. Consequently, unisexual fish also serve as useful models for studying the role of genetic variation in the survival of small endangered populations. Finally, unisexual fish serve as genetically uniform systems for environmental and biomedical studies. The purpose of this review is to bring wider attention to the range of biological problems that can be attacked with these remarkable organisms.
The hemiclonal waterfrog Rana esculenta, a hybrid between R. ridibunda and R. lessonae, eliminates the lessonae genome from the germline and clonally transmits the ridibunda genome (hybridogenesis). Such genomes are prone to accumulate deleterious mutations, which may explain why offspring from matings between hybrids are typically inviable. Here I present field data from a population for which experimental crossings showed that some R. esculenta pairs produce viable R. ridibunda offspring. I demonstrate: (1) that R. ridibunda metamorphs are also produced and survive under natural conditions; (2) that their genotypes are consistent with combinations of clonal ridibunda genomes found in hybrids; and (3) that all R. ridibunda are female. These females possibly recombine the clonal genomes they inherited and, upon mating with syntopic R. lessonae, produce new hemiclones with novel combinations of alleles. Hence, occasional recombination between otherwise clonal ridibunda genomes seems plausible and may provide an escape from the evolutionary dead end they were proposed to be trapped in.
Cytological aspects of hemiclonal (meroclonal) inheritance in diploid and triploid males of the hybridogenetic frog Rana esculenta (Rana ridibunda x Rana lessonae) have been studied by DNA flow cytometry. The fact that the R. ridibunda genome contains 16% more DNA than the R. lessonae genome provides the ability to discern cells containing genomes of any species from the water-frog complex under study. Data are presented showing that elimination of the R. ridibunda genome occurs in hybridogenetic males from certain populations. In triploid males, the cytogenetic mechanism of hemiclonal inheritance is simpler than in diploids: after the elimination of a genome (always the genome in the minority in the triploid set; "homogenizing elimination"), no compensatory duplication of the remaining genetic material is necessary, as it is in diploids. The process of elimination can be visualized in triploid males by using DNA flow cytometry to identify cells in the special phase of the spermatogonial cell cycle that we termed the E phase.
Population-genetic analysis conducted in hybrid populations of Rana esculenta complex from neighbourhood of Kiev has shown a high portion of recombinant frogs within Rana esculenta specimens and limitation of gene flows from one species to the other. The mechanisms of limitations are connected with assortative crossings within R. ridibunda and R. lessonae and low viability of frogs with a recombined genotype.
Analysis of the genetic structure of two green frog Rana esculenta complex populations from Volyn' was performed for 7 diagnostical loci. Populations of R- and REL-type were detected in which the introgression of Ldh-B allelic gene locus from R. lessonae into genome of R. ridibunda, characteristic for hybrid populations of Dnieper basin, was absent. This may be associated with alterations in gene pools of parental species.
Population-genetic analysis of hybrid populations of green frogs from the Dnepr basin demonstrated limited gene introgression in diploid hybrids in addition to semiclonal reproduction, which is typical for Rana esculenta hybrids. Introgression is largely confined to the Ldh-B locus: the gene of R. lessonae is introduced into the R. ridibunda genome. This phenomenon is unstable as it is geographically restricted and absent in populations of the E-L type.
The Iberian minnow Leuciscus alburnoides represents a complex of diploid and polyploid forms with altered modes of reproduction. In the present paper, we review the recent data on the origin, reproductive modes, and inter-relationships of the various forms of the complex, in order to predict its evolutionary potential. The complex follows the hybrid-origin model suggested for most other asexual vertebrates. Diploid and triploid females from the southern river basins exhibit reproductive modes that cannot be conveniently placed into the categories generally recognised for these vertebrate complexes, which imply continuous shifting between forms, where genomes derived from both parental ancestors are cyclically lost, gained or replaced. Replacement of nuclear genomes allow the introduction of novel genetic material, that may compensate for the disadvantages of asexual reproduction. Contrasting with most other vertebrate complexes, L. alburnoides males are fertile and play an important role in the dynamics of the complex. Moreover, diploid hybrid males may have initiated a tetraploidization process, when a diploid clonal sperm fertilised a diploid egg. This direct route to tetraploidy by originating fish with the right constitution for normal meiosis (symmetric), may eventually lead to a new sexually reproducing polyploid species. This case-study reinforces the significance of hybridisation and polyploidy in evolution and diversification of vertebrates.
Technical improvements in the method for vertical acrylamide gel electrophoresis (Raymond) have resulted in improved resolution
and reproducibility. In a survey of approximately 200 normal human serums, ten lines were found to be common in all specimens.
Interposed with these constant lines and in addition to known variations in the pattern reflecting haptoglobin and transferrin
types, at least one line followed that of albumin and three followed that of transferrin; the presence or absence of these
varied from individual to individual. Based on these variations, the electrophoretic patterns of normal humans may be divided
into at least 12 discrete groups.
We examined the genetic composition, habitat use, and morphological variation of a Phoxinus eos-neogaeus unisexual hybrid complex and its sexually reproducing progenitor species inhabiting beaver-modified drainages of Voyageurs National Park, Minnesota. In addition to the single diploid P. eos-neogaeus gynogenetic clone, triploid and diploid-triploid mosaic biotypes were present at our study sites. Both P. eos and P. neogaeus, and all three hybrid biotypes were ubiquitous throughout one intensively surveyed drainage, but abundances and relative frequencies of the parental species and hybrids varied considerably within and among successional environments. Data from a large number of additional sites indicated that the proportion of polyploid hybrids within an environment was negatively related to hybrid relative frequency, implying that the genomic constitution of hybrids is an important determinant of clonal fitness among successional environments. Statistical comparisons of variation along size-free multivariate body shape axes indicated that despite its genetic uniformity, the P. eos-neogaeus clone is no less variable than its sexual progenitors, suggesting that a single genotype may actually respond to environmental variation with as much phenotypic variation as a genetically variable sexual population. The incorporation and expression of a third genome in triploid and diploid-triploid mosaic biotypes derived from the gynogenetic clone significantly expanded phenotypic variation of the clone. This additional variation results in greater similarities in habitat use and morphological overlap with the parental species, primarily P. eos, the predominant sperm donor for gynogenetic hybrid females in this complex. Polyploid augmentation of a diploid gynogenetic clone appears to be typical in the P. eos-neogaeus complex, and the additional genetic and phenotypic variation that it generates has potentially significant ecological and evolutionary consequences for the success and persistence of a single genotype in highly variable environments.
All-hybrid populations of the water frog, Rana esculenta, are exceptional in consisting of independently and to some extent sexually reproducing interspecific hybrids. In most of its range R. esculenta reproduces hemiclonally with one of the parental species, R. lessonae or R. ridibunda, but viable populations of diploid and triploid hybrids, in which no individuals of the parental species have been found, exist in the northern part of the range. We test the hypothesis that nonhybrids arise every year in these all-hybrid populations, but die during larval development. Microsatellite markers were used to determine the genotypes of adults and abnormal and healthy offspring in three all-hybrid populations of R. esculenta in Denmark. Of all eggs and larvae, 63% developed abnormally or died, with some being nonhybrid (genomes matching one of the parental species), many being aneuploid (with noninteger chromosome sets), a few being tetraploid, and many eggs possibly being unfertilized. The 37% surviving and apparently healthy froglets were all diploid or triploid hybrids. In all three populations, gametogenesis matched the pattern previously described for all-hybrid R. esculenta populations in which most triploid adults have two R. lessonae genomes. This pattern was surprising for the one population in which triploid adults had two R. ridibunda genomes, because here it leads to a deficiency of gametes with an R. lessonae genome and should compromise the stability of this population. We conclude that faulty gametogenesis and mating between frogs with incompatible gametes induce a significant hybrid load in all-hybrid populations of R. esculenta, and we discuss compensating advantages and potential evolutionary trajectories to reduce this hybrid load.
Rare recombination in otherwise asexually reproducing organisms is known to beneficially influence the fitness in small populations. In most of the investigated organisms, asexual and rare sexual generations with recombination follow each other sequentially. Here we present a case where clonal reproduction and rare recombination occur simultaneously in the same population. The hybridogenetic water frog Rana esculenta (E), a hybrid between R. lessonae (L) and R. ridibunda (R) produces gametes that only contain the unaltered maternal R part of their genome. New generations of R. esculenta usually arise from E x L matings. Intraspecific E x E matings produce mostly inviable offspring, but in rare cases, female R. ridibunda arise from such matings which are capable of recombination. In the absence of conspecific males, these R females have to mate with E males, which results in further R females, or with L males, which produces new E lineages. This indirect mechanism reintroduces recombination into the otherwise clonally transmitted R genomes in R. esculenta populations. In this study, we show through Monte Carlo simulations that, in most cases, it is sufficient that only between 1 % and 10 % of mixed water frog populations consist of R females to prevent or significantly reduce the fixation and accumulation of deleterious mutations.
An all-hybrid water frog population persisting in agrocenoses of central Poland (Amphibia, Salienta, Ranidae)
- L Berger
Berger, L. (1988): An all-hybrid water frog population
persisting in agrocenoses of central Poland (Amphibia,
Salienta, Ranidae). Proc. Acad. Nat. Sci. Phila. 140:
202-219. www.jstor.org/stable/4064925.
Vitality and growth of progeny from different egg size classes of Rana esculenta L. (Amphibia, Salientia)
- L Berger
- T Uzzell
Berger, L., Uzzell, T. (1977): Vitality and growth of progeny
from different egg size classes of Rana esculenta L.
(Amphibia, Salientia). Zool. Pol. 26: 291-317.
Drei Phänotypen von Grünfröschen aus dem Rana esculenta-Komplex in der Schweiz
- H J Blankenhorn
- H Heusser
- P Vogel
Blankenhorn, H.J., Heusser, H., Vogel, P. (1971): Drei
Phänotypen von Grünfröschen aus dem Rana esculenta-Komplex in der Schweiz. Rev. Suisse Zool. 78: 1242-1247.