Corresponding author: Maria Ogielska ( firstname.lastname@example.org )
Academic editor: Larissa Kupriyanova
© 2017 Anna Zaleśna, Maria Florek, Mariusz Rybacki, Maria Ogielska.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zaleśna A, Florek M, Rybacki M, Ogielska M (2017) Variability of NOR patterns in European water frogs of different genome composition and ploidy level. Comparative Cytogenetics 11(2): 249-266. https://doi.org/10.3897/CompCytogen.v11i2.10804
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 A3, 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).
Pelophylax esculentus complex, hybridogenesis, triploidy, NOR inheritance
Nucleolus organizer regions (NORs) are sites of nucleoli formation owing to the presence of genes (rDNA) coding for 18S rRNA, 5.8S rRNA and 28S rRNA. They are the only genes that can be recognized in genomes on the basis of chromosome structure and thereby are useful as cytogenetic markers. These chromosomes differ from others by the presence of secondary constrictions where NORs are situated. The number and position of NORs are species specific, although inter-individual variability of these regions has also been observed within species. In anuran amphibians, NORs detected by silver staining (Ag-NOR) revealed that most species, both from primitive and derived families, have only one pair of NOR-bearing chromosomes in their diploid karyotypes (
In water frogs, NORs are located in secondary constrictions on long arms of chromosome pair 10, as observed after AgNO3 staining (
Central European water frogs form 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) (
We applied cytogenetic techniques, i.e. silver, chromomycin A3 and Propidium Iodide (PI) staining, and fluorescent in situ hybridization with 28S rDNA probe commonly used in comparative studies of cold-blooded vertebrates: fish (
In total, 407 individuals were analyzed: 272 adults and 135 newly metamorphosed juveniles. Juveniles were analyzed separately, as we expected a higher frequency of polyploids than in adults because tetraplois and pentaploids do not survive until adulthood (
Frogs were collected in years 2002–2009. All specimens used in this study were collected according to legal regulations concerning wild species protection under the following permits: Agency for Nature Conservation and Landscape Protection of the Czech Republic, Poodrí Protected Landscape Area, c.j. 0926/PO/2008/AOPK, permit S/0673/PO/2008/AOPK, and Polish Ministry of Environment Protection and Forestry for performing studies on protected species OP 4072/218 /96, OP 4072/218/98/4501, OP 4201/144/99, and II Local Commission for Ethics in Experiments on Animals 13/02.
Number, collection site, sex, taxonomic status and genotype of adult individuals.
|Population||Number of individuals|
|nr||Name||type||Coordinates of sampling sites||lessonae||esculentus||ridibundus|
|2||Barycz River valley||RE||51°25'42"N, 16°56'49"E||1||11||43||4||23||54|
|4||Horni Budlovice||LE||49°44'54"N, 18°26'30"E||2||1|
|5||Mewia Rewa||LE||54°38'54"N, 18°27'57"E||1||8||1|
Twenty-four hours before dissection, adults were injected intraperitoneally with 1 ml, and juveniles with 0.5 ml of 0.3% colchicine (Sigma- Aldrich, St. Louis, Mo., USA). Shortly before tissue preparation, the frogs were anesthetized with 0.25% water solution of 3-aminobenzoic acid ethyl ester (MS 222, Sigma-Aldrich). The intestine was dissected, hypotonized in distilled water (20 min for adults and 10 min for juveniles), and fixed in fresh-made fixative ethanol:glacial acetic acid (3:1) according to
Chromosome preparations of three P. esculentus individuals were made in a different way. The animals were injected intraperitoneally with 0.1% colchicine (10µl/g body weight) 2.5 h before dissection, femur bones were removed from euthanized frogs, epiphyses were clipped off, and bone marrow cavities were immediately flushed with 0.075 M KCl solution applied with a syringe at 37°C. Bone marrow tissue was pressed through a small-mesh gauze, flushed out with 0.075 M KCl solution, then placed in a centrifuge tube filled up to 7 ml with 0.075 M KCl solution, and incubated at 37°C for 20 min. Hypotonic treatment was stopped by fixation in absolute methanol:glacial acetic acid (3:1), and cell suspension was centrifuged at 1,500 rpm for 10 min. The supernatant containing fat droplets was discarded with a Pasteur pipette and fresh fixative was added up to 5 ml. The pellet was re-suspended by agitation and kept in a freezer for 20 min, then centrifuged again at 1,500 rpm for 10 min. The procedure (centrifugation, fixative exchange, and cooling) was repeated 3 times. The suspension was then transferred to a 1-ml syringe, dropped onto slides (previously cleaned in HCl:ethanol, 3:1), and finally air-dried. Chromosome preparations obtained with either protocol were suitable for all staining methods used in this study. However, chromosomes prepared from bone marrow cells were more uniformly condensed and thus more suitable for FISH than the chromosomes obtained from gut epithelium prints that varied in the degree of condensation (
The nucleolus organizer regions (NORs) were stained in all individuals by the silver nitrate technique (Ag-NOR). Chromosome slides of 15 randomly selected individuals were sequentially stained with chromomycin A3 (CMA3) and AgNO3. All individuals that displayed another number of NORs than one per a haploid set after Ag-NOR staining were examined by PI or CMA3. For 22 individual (9 RR, 3 LL, 5RL, 1 RRL, 3 LLR, 1 RRLL) we applied the FISH method with 28S rDNA as a probe.
Silver staining. We followed the protocol of
Chromomycin A3 (CMA3). The method was used according to
Propidium Iodode staining after denaturation of chromosomes (PI). Tissue prints were dehydrated in 70%, 85% and 95% ethanol (30 sec each wash), air-dried at room temperature and denatured in 70% deionized formamide in 2xSSC at 70°C for 3.5 min. Slides were immediately dehydrated in chilled 70% ethanol from a freezer for 2 min, then in 85% and 95% at room temperature for 30 sec. Chromosomes were stained with PI (200ng/ml).
We applied the FISH method with 28S rDNA as a probe, according to the protocol of
Slides were incubated in 100 µg/ml DNase-free RNase A in 2x SSC for 1 h at 37° C in a humid chamber and then washed twice in 2xSSC at room temperature for 5 min, dehydrated in ethanol series (70%, 85%, and 95%, 30 s each) and air-dried. Chromosome preparations were denatured in 70% deionized formamide, 2x SSC at 70° C for 3.5 min, dehydrated immediately for 2 min in ice-cold 70% ethanol, then in 85% and 95% ethanol for 30s at room temperature, and finally air-dried. The 28S rDNA probe was dissolved in hybridization mixture consisted of 100% deionized formamide and 20% dextran sulfate in proportion 1:1. The mixture was denatured at 90° C for 5 min and then immediately placed on ice for 3 min. 20 µl of the probe was applied to each slide and covered with a coverslip. Hybridization lasted overnight in a dark humid chamber at 37° C. After hybridization, the coverslip was removed by rinsing with 50% formamide in 2xSSC at 42°C twice for 7 min and the slide was washed 3 times (7 min each) in 1xSSC at 42°C and then in 2xSSC at room temperature for 30 s. Chromosomes were counterstained with DAPI in Vectashield antifade buffer (Cambio, Cambridge, UK).
Chromosomes were analyzed in Olympus Provis AX 70 or Carl Zeiss Axioskop 20 microscopes equipped with fluorescence lamp HBO50 and appropriate filters. Images were recorded with Olympus DP30BW CCD and cooled Carl Zeiss AxioCam HRc CCD cameras and processed using AxioVision and Lucia ver. 2.0 (Laboratory Imaging) softwares.
The number of NORs was visualized for all (407) studied individuals (see Tables
Results obtained by silver staining corresponded well with those stained with CMA3, PI or FISH (Fig.
Number, sex, taxonomic status and genotype of juvenile progeny of artificial crosses.
|Number of individuals|
Number of NORs in individuals of various ploidy level. The number of NORs that is in agreement with ploidy level is in bold.
|Ploidy and genome
|Number of individuals
(adults and juveniles)
|Number of NORs|
|2n (LL, RR, RL)||354||51||302||1|
|3n (RRL, LLR)||48||2||3||34||9|
In 66 individuals (16.2%, 13 adult and 53 juveniles), the number of NORs differed from the expected values. Lack of NORs was relatively more frequent (13.8%, 56 out of 407 individuals) than extra NORs (2.46%, 10 out of 407 individuals), regardless of genomic compositions (RR, LL, RRL and LLR) and origin of individuals. Eight diploid adult individuals (3 diploid P. esculentus, 3 P. lessonae and 2 P. ridibundus) and 43 diploid juveniles (11 P. esculentus, 6 P. lessonae, and 26 P. ridibundus) had NORs only on one chromosome of pair 10 (Fig.
Additional NORs were detected in ten individuals. The extra NORs were located within additional secondary constrictions on one of the homologs of chromosomes other than 10. Extra NORs were found in one adult P. ridibundus in the distal position of the long arm of chromosome 9 (Fig.
The lack of one NOR was also detected in diploid progeny of two triploid RRL females and one LLR female. After AMD/DAPI staining we discovered that in the case of the RRL females the lack of NORs was inherited together with the haploid set of the ridibundus chromosomes, whereas in the case of the LLR female it was inherited with the haploid set of the lessonae chromosomes. Among diploid progeny of these females (altogether 37 juveniles) we observed 16 individuals with two NORs and 21 individuals with only one NOR. Each individual was stained with AgNOR and seven of them were confirmed by FISH. Thus, despite that the genomes of ova were the same (R in the case of RRL and L in the case of LLR females), the females produced in fact two types of gametes – with and without NORs (Fig.
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 AgNO3B 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).
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.
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.
The majority of individuals displayed one NOR per a haploid set of chromosomes (n = 13) located within the secondary constriction on the long arm of chromosome 10 (named 9 in
Ag-NORs observed in metaphase chromosomes in water frogs were also recognized in interphase nuclei, as was reported by
The number of visualized NORs corresponded well with the ploidy level, as expected for individuals with only one pair of NOR-bearing chromosomes. Thus, diploids had two NORs, triploids - three NORs, and tetraploid - four NORs. However, number of NORs in interphase nuclei may be misleading in specific cases. As we demonstrated in this study, interphase nuclei in a diploid individual with an extra NOR on chromosome 9 displayed the same pattern as in a triploid individual with three NORs on the homologous chromosomes 10.
The variability of AgNOR sizes in individuals from the same populations may reflect different amount of rDNA (
Extra NORs were also reported in Rana catesbeiana (Lithobates catesbeianus) that displayed from two to seven Ag-positive small NORs per haploid karyotype, apart from one “standard” NOR on the chromosome 10 (
More common was the lack of NORs (13.8% of all animal studied herein). We observed a lack of one NOR (2n and 3n) or two NORs (only in 3n), both in metaphase plates and interphase nuclei. Lack of NORs in amphibians was also reported by Schimd (1982) in Bombina variegata, Xenopus laevis, and Bufo fowleri. According to
Intraspecific polymorphism of the location of extra NORs was also reported in Hyla chrysoscelis and H. versicolor (
The lack of one NOR was detected in both RRL and LLR triploid females and was associated with the ridibundus and lessonae chromosome sets, respectively. Triploid water frogs produce haploid gametes and transmit the chromosome set that is represented by two copies (R in RRL and L in LLR) (
Finally, we conclude that polymorphism concerning the number and localization of NORs in water frogs was characteristic of both lessonae and ridibundus genomes, and – as expected – was observed in individuals regardless of their taxonomic position (P. lessonae, P. ridibundus and P. esculentus), ploidy level (2n, 3n, 4n), genomic constitution (RR, LL, RRL, RLL and LLRR), and collection site. The number of active silver-stained NORs reflected ploidy levels: two in diploids, three in triploids, and four in tetraploids, and therefore we believe that there is no diploidization of polyploids in water frogs, as has been observed in some of polyploid amphibians (for review see
The study was financially supported by grants 3490/B/P01/2007/33 and 3946/B/P01/2009/36 provided by the Polish Ministry of Science and Higher Education. We are grateful to Katarzyna Lipiec-Sidor for technical help with figures arrangement and setup.