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.
There are three taxons of central European water frogs of the Pelophylax esculentus complex: two morphologically distinct species, Pelophylax lessonae (LL) and Pelophylax ridibundus (RR), and hybrids Pelophylax esculentus, which can be either diploid (RL) or triploid (LLR or RRL). The morphology of hybrids is supposed to follow genome dosage effect. We describe colouration of water frogs with genome composition verified by chromosome analysis. Typical colouration features in LL were: spots on the ventral side, brown limbs, “weak” femur spotting pattern, brown dorsal folds and yellow-green colour in “waist”. Typical RR had dark-green or olive-green limbs, “full” femur spotting pattern, no hip spot and no yellow colour in “waist”. For all hybrids the most typical features were strong spots on the ventral side and a mosaic of green and brown colour on limbs. Typical LLR had brown-greenish dorsal folds underlined by a partial black line, “weak” femur spotting pattern and yellow-greenish colour in “waist” and on femur. Typical RL had greenish hind limbs, green dorsal folds, no yellow colour in “waist”, and no hip spot. Typical RRL was similar to RL, but had a continuous black line under dorsal folds. There were, however, numerous exceptions to these trends, with the most prominent being much higher than expected variability of colouration of Pelophylax lessonae. Therefore caution must be advised when trying to estimate genome composition of water frogs solely on the basis of colouration.
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