DNA content and genome composition of diploid and triploid water frogs belonging to the Rana esculenta complex (Amphibia, Anura)

The Central European water frog Rana esculenta L., 1758 is a natural hybrid between Rana lessonae Camerano, 1882 (LL) and Rana ridibunda Pallas, 1771 (RR). Hybrids are usually diploid (RL) or triploid (LLR or RRL). Distinguishing LL from RL, RR from RL, and LLR from RRL according to external morphology is ambiguous. In this study we checked whether the DNA content in erythrocyte nuclei measured by image cytometry is useful in determination of the taxonomic status of diploids (LL, RR, and RL) and the genome composition of triploids (LLR and RRL). For exact and direct identification of parental species, as well as for determination of genome composition in hybrids, we applied actinomycin D – 4',6-diamidino-2-phenylindole chromosome staining to metaphase plates. We analyzed 43 LL, 12 RR, and 32 RL diploids, and 37 LLR and 19 RRL triploids. All diploid hybrids had 2n = 26 chromosomes, and all triploid hybrids had 3n = 39 chromosomes. Neither aneuploid nor mosaic hybrids were detected. The expected numbers of 13 R. lessonae (L) and 13 R. ridibunda (R) chromosomes in RL hybrids were recorded in about 31% of individuals. In the rest of the sample the composition was variable, ranging from 9 to 14 R chromosomes and the corresponding number of L chromosomes. The expected composition of 26 L and 13 R chromosomes was detected in about 32% of LLR triploids, whereas in the rest of the sample the composition of chromosomes ranged from 8 to 15 R chromosomes and the corresponding number of L chromosomes. The expected numbers of 26 R and 13 L chromosomes were detected in about 26% of RRL triploids, whereas in the rest of the sample the composition of chromosomes ranged from 19 to 28 R chromosomes and the corresponding number of L chromosomes. The DNA content densitometry showed that RR and RL diploids had 9.5% and 3.8% more DNA, respectively, than LL diploids. These differences, although statistically significant, were not sufficient to unequivocally discriminate LL from RL and RR from RL. Triploids had about 50% more DNA than LL diploids (49% in LLR and 51% in RRL), but these differences were too small for unequivocal determination of their genome composition.