Variability of NOR patterns in European water frogs of different genome composition and ploidy level

Abstract

We studied water frogs from a complex composed of two species: Pelophylax lessonae (Camerano, 1882) (genome LL, 2n = 26) and P. ridibundus (Pallas, 1771) (RR, 2 = 26), and their natural hybrid P. esculentus (Fitzinger, 1843) of various ploidy and genome composition (RL, 2n = 26, and RRL or RLL, 3n = 39). Tetraploids RRLL were found (4n = 52) in juveniles. We applied cytogenetic techniques: AgNO₃, chromomycin A_3, PI and fluorescent in situ hybridization with a 28S rDNA probe. Results obtained by silver staining corresponded well with those stained with CMA₃, PI and FISH. As a rule, NORs are situated on chromosomes 10. The number of Ag-NORs visible on metaphase plates was the same as the number of Ag-nucleoli present in interphase nuclei of the same individual. In all analyzed metaphases, NORs exhibited variations in size after AgNO₃ and CMA₃ stainings. Sixty-six individuals (out of 407 analyzed) were polymorphic for the localization and number of NORs. Fifty-one diploids had NORs only on one chromosome of pair 10. Three triploids (LLR and RRL) displayed two NORs, and two other triploid RRL individuals displayed one, instead of expected three NORs. In ten individuals extra NORs were detected on chromosomes other than 10 (chromosomes 2 and 9).

Keywords: NOR inheritance; Pelophylax esculentus complex; hybridogenesis; triploidy.

Figure 1.

A The variability of size of AgNORs (black bands) on chromosomes 10 a Pelophylax ridibundus b P. lessonae c P. esculentus RL d P. esculentus RRL e P. esculentus LLR B–E Interphase nuclei of P. esculentus with Ag-nucleoli visualized as black dots by AgNO₃ B diploid RL with 1 AgNOR C diploid RL with 2 AgNORs D triploid RRL with 3 AgNORs E triploid RRL with 4 AgNORs F metaphase chromosomes and interphase nuclei of the same tetraploid P. esculentus RRLL with 4 sites of hybridization with 28S rDNA probe (FISH) (arrows).

Figure 2.

Localization and number of NORs (arrows) in diploid and triploid water frogs. Left column represent diploids (A–C) and right column represent triploids (D–G). Each picture is composed of a diagram of karyotype with black dots representing NORs and metaphase plates stained with silver (AgNOR), chromamycin A₃ (CMA₃), propidium iodide after denaturation (DPI) or after fluorescent in situ hybridization with 28S rDNA probe (FISH). A P. lessonae LL with 2 NORs, AgNORs (left) and FISH (right) B P. esculentus RL with 4 NORs on chromosome 9, CMA₃ (left) and FISH (right) C P. esculentus RL with 1 NOR, AgNORs (left) and FISH (right) D P. esculentus LLR with 3 NORs, AgNORs (left) and FISH (right) E P. esculentus RRL with 4 NORs, AgNOR (left) and DPI (right) F P. esculentus RRL with 2 NORs, AgNORs (left) and FISH (right) G P. esculentus RRL with 1 NOR, CMA₃.

Figure 3.

Inheritance of NORs by progeny of triploid females RRL (right) and RLL (left). The chromosome set represented by one copy is eliminated before oogenesis (marked by X) and the double one is segregated into eggs (represented by ovals). After fertilization (in this case by ridibundus sperm), two types of progeny arises: with two (A and C) and with one (B and D) NOR. The lack of NORs are transmitted either by lessonae (white) or by ridibundus (dark grey) chromosome sets. NORs are represented by black dots.