Mutual maintenance of di- and triploid Pelophylax esculentus hybrids in R-E systems: results from artificial crossings experiments

Abstract

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.

Keywords: Gamete; Genome elimination; Hemiclonal reproduction; Hybrid population systems; Karyotype; Polyploid hybrid.

Fig. 1

Results of crossing experiments of triploid hybrid females with RRL or LLR genotypes with P. ridibundus and di- or triploid hybrid males. Tadpoles resulting from crosses were represented by P. ridibundus (RR) and P. esculentus (RL) individuals. Triploid hybrid females with RRL genotype produced oocytes with 13 bivalents corresponding to P. ridibundus chromosomes (orange). All observed oocytes of triploid hybrid female with LLR genotype included 13 bivalents corresponding to P. lessonae chromosomes (blue). Obtained results allow to assume that triploid hybrid females with RRL genotypes produce gametes with P. ridibundus genome (R), triploid hybrid females with LLR genotype produce gametes with P. ridibundus (R) and P. lessonae (L) genomes. Question marks (?) indicate discrepancy between oocyte chromosomal sets and gametes genome composition inferred from crossing experiments. Diploid hybrid males presumably produce haploid sperm with P. ridibundus (R) and P. lessonae (L) genomes and finally triploid hybrid males produce haploid sperm with P. ridibundus genome (R). Crossing numbers correspond to Additional file 1: Table S1

Fig. 2

Results of crossings experiments of P. ridibundus individuals and diploid hybrid females with P. ridibundus and diploid P. esculentus males. Tadpoles obtained after crossings were represented by triploid hybrids with RRL and LLR genotypes, P. ridibundus (RR) and P. esculentus (RL) individuals. Diploid hybrid females produced oocytes with 26 bi- and univalents corresponding to P. ridibundus (orange) and P. lessonae (blue) chromosomes, 13 bivalents corresponding to P. ridibundus (orange) chromosomes. Obtained results allow us to assume that diploid hybrid females produce unreduced gametes (RL) and gametes with P. ridibundus (R) or P. lessonae (L) genomes. Oocytes with 26 univalents presumably cannot overcome meiosis and give aneuploidy gametes. Diploid hybrid males presumably produce haploid sperm with P. ridibundus (R) and P. lessonae (L) genomes. Two P. ridibundus individuals produce haploid gametes with P. ridibundus genome. Question marks (?) indicate discrepancy between oocyte chromosomal sets and gametes genome composition inferred from crossing experiments. Crossing numbers correspond to Additional file 1: Table S1

Fig. 3

FISH mapping of interstitial (TTAGGG)_n repeat sites allows to identify karyotypes of tadpoles resulting from artificial crosses of hybrid animals. Metaphase chromosomes of tadpoles after FISH with (TTAGGG)₅. One or two interstitial (TTAGGG)_n repeat sites (indicated by arrows) are distinguished in parental NOR-bearing chromosomes. Karyotypes of tadpoles resulting from crosses of triploid hybrid female with RRL genotype and diploid hybrid male (a, b), triploid hybrid female with LLR genotype and triploid hybrid male with RRL genotype (c, d) and two diploid hybrid frogs (e, f, g, h). Tadpoles were identified as P. ridibundus (b, c, g), diploid hybrids (a, d, h) and triploid hybrids with RRL (e) and LLR (f) genotypes. Scale bars = 10 μm

Fig. 4

Lampbrush chromosome sets from growing oocytes of diploid and triploid hybrid frog with RRL genotype. Lampbrush chromosome sets from oocytes of triploid hybrid frog with RRL genotype represented by 13 bivalents corresponding to chromosomes of P.ridibundus (a, b). Lampbrush chromosome sets from oocytes of diploid hybrid frog represented by 26 bivalents (c, d) and 26 univalents (e, f) with 13 bi- or univalents corresponding to P. ridibundus chromosomes and 13 bi- univalents corresponding to P. lessonae chromosomes. Lampbrush chromosomes are numbered alphabetically; italic type indicates correspondence of the identified chromosome to genotype of parental species: l – to P. lessonae, r – to P. ridibundus. Chromosomes were counterstained with DAPI (a, c, e). Corresponding phase-contrast micrographs are shown (b, d, f). Scale bars = 50 μm

Fig. 5

Contribution of diploid and triploid hybrid frogs in maintenance of studied R-E systems. a Crossings of triploid hybrid females with RRL genotype and diploid hybrid males led to appearance of P. ridibundus and diploid P. esculentus tadpoles. Triploid hybrid females produced haploid eggs with P. ridibundus genome while diploid hybrid males produced haploid sperm with either P. ridibundus or P. lessonae genomes. b Crossings of two diploid hybrid frogs led to appearance of triploid tadpoles with RRL and LLR genotypes, P. ridibundus tadpoles and diploid P. esculentus tadpoles. Diploid hybrid females produced diploid eggs with both P. ridibundus and P. lessonae genomes, and haploid eggs with P. ridibundus genome. Diploid hybrid males presumably produced haploid sperm with either P. ridibundus or P. lessonae genomes

Fig. 6

Detection of micronuclei in gonads of hybrid tadpoles. Morphological analysis of gonads dissected from P. ridibundus (a, b) tadpoles, diploid (e, g, h) and triploid (c, d, f, i, j) P. esculentus tadpoles. Undifferentiated gonads (stage 3) from P. ridibundus tadpoles (a). Gonads at stage of high mitotic activity (stage 4) from P. ridibundus tadpoles (b). Scale bars = 50 μm. Micronuclei (arrows) were abundant in cytoplasm of germ cells in gonads from diploid (e, g, h) and triploid (c, d, f, i, j) hybrid tadpoles. Micronuclei vary in size and chromatin density. Anaphase stage of mitosis (h, i, j); no lagging chromosomes (arrowheads) was observed. Scale bar for c = 50 μm; d = 25 μm; e-j = 10 μm