Reproduction and hybrid load in all-hybrid populations of Rana esculenta water frogs in Denmark

All-hybrid populations of the water frog, Rana esculenta, are exceptional in consisting of independently and to some extent sexually reproducing interspecific hybrids. In most of its range R. esculenta reproduces hemiclonally with one of the parental species, R. lessonae or R. ridibunda, but viable populations of diploid and triploid hybrids, in which no individuals of the parental species have been found, exist in the northern part of the range. We test the hypothesis that nonhybrids arise every year in these all-hybrid populations, but die during larval development. Microsatellite markers were used to determine the genotypes of adults and abnormal and healthy offspring in three all-hybrid populations of R. esculenta in Denmark. Of all eggs and larvae, 63% developed abnormally or died, with some being nonhybrid (genomes matching one of the parental species), many being aneuploid (with noninteger chromosome sets), a few being tetraploid, and many eggs possibly being unfertilized. The 37% surviving and apparently healthy froglets were all diploid or triploid hybrids. In all three populations, gametogenesis matched the pattern previously described for all-hybrid R. esculenta populations in which most triploid adults have two R. lessonae genomes. This pattern was surprising for the one population in which triploid adults had two R. ridibunda genomes, because here it leads to a deficiency of gametes with an R. lessonae genome and should compromise the stability of this population. We conclude that faulty gametogenesis and mating between frogs with incompatible gametes induce a significant hybrid load in all-hybrid populations of R. esculenta, and we discuss compensating advantages and potential evolutionary trajectories to reduce this hybrid load.     

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

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

Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex

Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC<5 microM)), were identified. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships.     

Taxonomic reassessment of Middle Eastern water frogs: Bioacoustic variation among populations considered as Rana ridibunda, R. bedriagae or R. levantina

Temporal characteristics of advertisement calls of water frogs of 10 localities in Turkey, Syria, Israel and Egypt were compared with those of Rana ridibunda in Kazakhstan, Armenia and Greece (Thrace) as reference populations. These study sites include the type localities of R. ridibunda , R. r. caralitana , R. esculenta var. bedriagae und R. levantina . The temperature-dependent as well as the temperature-independent call parameters clearly revealed two species. R. ridibunda is represented by the three reference populations and, in addition, a population in central Turkey. The other populations (in Turkey, Syria, Israel and Egypt) represent R. bedriagae . R. bedriagae is the oldest available name for water frogs of these regions and was given priority over R. r. caralitana and R. levantina .     

Taxonomic reassessment of Middle Eastern water frogs: Morphological variation among populations considered as Rana ridibunda, R. bedriagae or R. levantina

Eight morphometric features of water frogs of 14 localities in Turkey, Syria, Jordan and Israel were compared with those of Rana ridibunda in Kazakhstan, Armenia and Greece (Thrace). These study sites include the type localities of R. ridibunda , R. r. caralitana , R. esculenta var. bedriagae and R. levantina . Multivariate comparisons (principal-component analysis, discriminant analysis) based on the log₁₀-transformed variables demonstrate that the data set includes only two taxa that differ significantly in size and shape. By applying a morphospecies criterion, R. ridibunda is represented exclusively by the three reference populations, whereas all other populations (in Turkey, Syria, Jordan and Israel) represent the same species , R. bedriagae .     

Photoreceptor layer composition in the retina of the frog (Rana esculenta)

Retinas of Rana esculenta frogs were studied by light and electron microscopy in order to establish the photoreceptor layer composition. We found 56% red rods, 9% green rods, 19% single cones, and 16% double cones. This work provides the morphological basis for electrophysiological investigations concerning the mass receptor potential of isolated Rana esculenta retinas.     

Seasonal changes in angiotensin converting enzyme activity in male and female frogs (Rana esculenta)

Gonad, lung, kidney and serum angiotensin converting enzyme (ACE) activities were determined by specific substrate hydrolysis in male and female Rana esculenta over 1 year. Ovary ACE activity showed the highest values among the different tissues, with a significant peak (223+/-52 nmol min(-1) mg protein(-1)) in late winter-early spring. Testis ACE activity followed a significant seasonal cycle, increasing from September to peak in April (2.5+/-0.8 nmol min(-1) mg protein(-1)) and then decreased in the post-reproductive period. Lung and kidney ACE activities were not correlated with the annual reproductive cycle phases. In serum a peak of activity was present in the post-reproductive period both in male and female frogs. The present data show a correlation between ACE and the annual reproductive cycle of R. esculenta.     

Variation of BM224 microsatellite in green frogs of the genus Rana

The variable microsatellite repeat BM224 has been discovered in the genomes of eight species of green frogs (Rana ridibunda, R. cf bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Earlier, this repeat had been observed in members of the genus Bufo. In this paper, a possibility of usage of this genetic marker for species identification is discussed.     

Rhabdias esculentarum n. sp. (Nematoda: Rhabdiasidae) from green frogs of the Rana esculenta species complex in Italy: molecular evidence, morphological description and genetic differentiation from its congeners in frogs and toads

A new taxon, Rhabdias esculentarum n. sp., is described based on DNA sequence analysis at multiple loci (i.e. mtDNA cox-1, 12S rRNA, ITS-1 and partial ITS-2 regions of the nuclear rDNA) and morphometric analysis carried out on specimens collected from the green frogs of the Rana esculenta species complex in Italy (i.e. R. lessonae Camerano and R. esculenta Linnaeus, identified genetically by diagnostic allozyme loci). Rhabdias esculentarum n. sp. was differentiated genetically, at both mitochondrial and nuclear levels, from Rh. bufonis (Schrank, 1788) (sensu Hartwich, 1972) and Rh. sphaerocephala Goodey, 1924 recovered from the toad Bufo bufo Linnaeus collected sympatrically with the specimens of Rana lessonae and R. esculenta examined in the present study. Moreover, the new taxon proved to be different from the other species of Rhabdias from anurans, which had previously been sequenced using the same genes and deposited in GeneBank. Phylogenetic analyses (MP and ML) inferred from mitochondrial (mtDNA cox-1 and 12S ribosomal RNA) and nuclear (ITS-1 and ITS-2 of the rDNA regions) sequences datasets were congruent in depicting Rh. esculentarum n. sp. as forming a highly supported clade distinct from the sympatric species Rh. bufonis, as well as from Rh. sphaerocephala, characterised on the basis of the same loci. Morphometric analysis and the differential diagnosis of genetically characterised specimens of the new species have revealed differences in several features in comparison with the type-species, Rh. bufonis. Material of the latter species included voucher specimens from Germany deposited by Hartwich (1972) and other specimens collected from B. bufo in Italy. Among the diagnostic characters, the particular cup-shaped buccal capsule characterising Rh. esculentarum is clearly different from the tear-shaped buccal capsule observed in material of R. bufonis obtained from Berlin Museum and collected in the same geographical area as the green frogs under study. Rh. esculentarum was also found to differ in some measurements and allometric characters from Rh. bufonis (sensu Moravec et al., 1997). The data so far collected appear to indicate a host-preference of Rh. esculentarum for Rana lessonae and R. esculenta, which belong to the R. esculenta hybridogenetic species complex in Italy.     

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

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

Effects of pH on K depolarization and contracture in frog muscles.

Membrane potential changes and mechanical tension provoked by 20 or 80 mM K at pH 8.0 or 4.5 were measured in muscle fiber bundles from Rana temporaria and Rana esculenta. The bathing solution contained Cl as the main anion. Low pH increased the depolarizing efficiency of high K to about the same degree in muscles of both species of frogs. The tension-[K] relationship was shifted toward lower [K] in muscles from R. temporaria and toward higher [K] in muscles from R. esculenta. It was concluded that the depolarization-contraction coupling was insensitive to pH changes in the former but not in the latter frog species.     

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

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

Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia,Anura)

A structural and ultrastructural study was carried out on the cutaneous glands in some species of Rana (R. dalmatina, R. iberica, R. italica, R. "esculenta," and R. perezi), giving particular attention to the mucous secretory units. Two different types of mucous glands occur in R. dalmatina, R. iberica, and R. italica. Besides the ordinary mucous units, which are randomly distributed over the body surface in both males and females, a further population of mucous glands was observed on the male dorsal skin. The latter is recognizable by the peculiar morphology of the epithelial cells and some characteristics of the secretory product. Specialized mucous glands are absent in both sexes of R. "esculenta" and R. perezi. The possible adaptive role of the specialized mucous glands is discussed in light of the absence of vocal sacs in males of R. dalmatina, R. iberica, and R. italica. Chemosignals released by sexually dimorphic mucous units may replace vocal communication during the breeding season and so play an important role in female attraction and/or territorial announcement. The morphology and possible function of the specialized mucous glands in the three species of Rana are compared with the breeding glands of other frogs and with the hedonic glands of some urodeles (Salamandridae and Plethodontidae), which are known to produce pheromonal substances during courtship.     

Cardiac morpho-dynamics in Rana esculenta: influence of sex and season

Morpho-dynamic aspects of the frog (Rana esculenta) heart were correlated to seasonal and sexual traits. The statistical analysis of the data (696 frog specimens: 448 males and 248 females), collected during 8 years of routine research on frog cardiac physiology, indicated that cardiac biology is characterised by sexual dimorphism. The relative cardiac weight of males was higher than that of females of similar size. With respect to the males of similar size, female frogs revealed an increased relative ventricular weight and ventricular contractile capacity. The morphometric analysis showed that the ventricular growth was achieved through significant myocardial enlargement accompanied by a reduction of the lacunary spaces. This is more pronounced in females during the breeding period. The correlation between sexual maturity and ventricular morpho-functional changes revealed that in male frogs sexual maturity correlates with a remarkable increment of the ventricular weight associated with higher values of pulse pressure. This data indicate that sex and season-related factors influence cardiac morpho-dynamics in R. esculenta.     

Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs

Interspecies transfer of mitochondrial (mt) DNA is a common phenomenon in plants, invertebrates and vertebrates, normally linked with hybridization of closely related species in zones of sympatry or parapatry. In central Europe, in an area north of 48 degrees N latitude and between 8 degrees and 22 degrees E longitude, western Palaearctic water frogs show massive unidirectional introgression of mtDNA: 33.7% of 407 Rana ridibunda possessed mtDNA specific for Rana lessonae. By contrast, no R. lessonae with R. ridibunda mtDNA was observed. That R. ridibunda with introgressed mitochondrial genomes were found exclusively within the range of the hybrid Rana esculenta and that most hybrids had lessonae mtDNA (90.4% of 335 individuals investigated) is evidence that R. esculenta serves as a vehicle for transfer of lessonae mtDNA into R. ridibunda. Such introgression has occurred several times independently. The abundance and wide distribution of individuals with introgressed mitochondrial genomes show that R. lessonae mt genomes work successfully in a R. ridibunda chromosomal background despite their high sequence divergence from R. ridibunda mtDNAs (14.2-15.2% in the ND2/ND3 genes). Greater effectiveness of enzymes encoded by R. lessonae mtDNA may be advantageous to individuals of R. ridibunda and probably R. esculenta in the northern parts of their ranges.     

Differential Esterase Expression in Leaves of Manihot esculenta Crantz Infected with Xanthomonas axonopodis pv. manihotis

The polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases in plants and to show a differential expression of esterases as markers of pathogenesis in cassava plants (Manihot esculenta Crantz). The characterization of alpha- and beta-esterases from leaves of M. esculenta by the PAGE system was possible using an extraction solution containing two phenol-complexing agents (PVP-40 and sodium metabisulfite), three antioxidant agents (EDTA, beta-mercaptoethanol, and DTT), and one quinone reducer (ascorbic acid). Fourteen esterase isozymes were detected in young unexpanded leaves of M. esculenta cultivars. The inhibition pattern of alpha- and beta-esterases of M. esculenta showed that Est-9 is an arylesterase, and in the unexpanded leaves of the M. esculenta plants infected with Xanthomonas axonopodis pv. manihotis, the Est-7 beta-esterase showed the characteristic staining of an alpha/beta-esterase. This diffrential expression of Est-7 isozyme in young unexpanded leaves of cassava plants can be used as a marker of pathogenesis after infection with X. axonopodis pv. manihotis.     

Rana/Pol III: a family of SINE-like sequences in the genomes of western Palearctic water frogs.

The highly repetitive Rana/Pol III family consists of short, tandemly arrayed sequences, scattered throughout the genomes of Palearctic green water frogs. The repeat unit is about 250 bp in length and is a composite element: it contains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats, and the occurrence of two internal repeats gives evidence that an additional transposition event may have inserted a segment within the already transposed element. Rana/Pol III family is present in the genomes of Rana lessonae, R. ridibunda, and their hybrid form R. esculenta, as well as in R. shqiperica. R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian R. perezi, although less abundant, but appear to be lacking in the north African species R. saharica. The distribution of Rana/Pol III in the genomes of Palearctic green frogs is in agreement with the phyletic history based on genetic data. The evolutionary pattern proposed for the genus Rana enables us to suppose that the hybridogenetic mechanism is one of the factors accounting for the possible horizontal transfer of Rana/Pol III elements from the central-north Europe species to R. perezi.     

Variations in call and morphology in male water frogs: taxonomic and evolutionary implications

In Anurans, the specific mate recognition system (SMRS), which contributes to prezygotic isolation, is mainly based on morphological and call features. This is why such criteria are used by zoologists for taxonomic identification. In hybridogenetic water frogs (Rana ridibunda, R. lessonae, R. perezi, R. kl. grafi and R. kl. esculenta), hybridization opens up the question of the efficiency of these criteria for taxon identification and of the specific recognition system in this context. Variation in both morphological and call features revealed both significant mean differences among taxa but also large overlapping among individuals belonging to different taxa. Thus, using call or morphometrical features in order to identify water frogs may be hazardous. Moreover, species differ poorly in their specific mate recognition system, facilitating hybridization events, and therefore suggesting that postzygotic mechanisms may be prevalent over prezygotic mechanisms.     

Variations in <Emphasis Type="Italic">BM224</Emphasis> microsatellite in green frogs of genus <Emphasis Type="Italic">Rana</Emphasis>

The variable microsatellite repeat BM224 has been revealed in the genomes of eight species of green frogs (Rana ridibunda, R. cf. bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Previously, this repeat was observed in members of the genus Bufo. In this paper, the possibility of using this genetic marker for species identification is discussed.     

Male vocalization and female choice in the hybridogenetic Rana lessonae/Rana esculenta complex

In many species, females can improve their fitness by preferring particular males over others. In Palaearctic water frogs of the Rana lessonae/R. esculenta complex the consequences of such mate choice are particularly pronounced. To produce viable offspring, the hybrid R. esculenta (genotype RL) must mate with the parental species R. lessonae (LL); but R. lessonae should avoid mating with R. esculenta, because the resulting hybrid offspring will eliminate the L genome from the germline (hybridogenesis). Hence, there exists a conflict between the sexual parasite (RL) and its sexual host (LL) over the best mating partner. Previous studies have shown a preference for LL males in LL and RL females; but they have also shown that females cannot usually realize their choice when in close proximity to males, because the males forcefully and indiscriminately amplex them. We tested whether females use male vocalizations as a long-distance signal to increase their chances of mating with the preferred LL males. We exposed female R. lessonae and R. esculenta to playbacks of single LL and RL mating calls (experiment 1) and to choruses with a 3:1 excess of LL and RL calls, respectively (experiment 2). In experiment 1, both female types were attracted more by the LL than by the RL calls. In experiment 2, no discrimination between LL- and RL-dominated choruses was observed. The results suggest that females do not use distant male vocalization to approach preferentially ponds or arenas within a pond that hold an excess of LL males. But once they have arrived in a chorus, mating calls from nearby males can direct them to the preferred LL mates. We discuss possible reasons for the failure to discriminate between choruses and the chances for successful choice between individuals within choruses. Copyright 2000 The Association for the Study of Animal Behaviour.