Hymenochirus boettgeri, Tornier, 1896

Gvoždík, Václav, Knytl, Martin, Zassi-Boulou, Ange-Ghislain, Fornaini, Nicola R. & Bergelová, Barbora, 2024, Tetraploidy in the Boettger’s dwarf clawed frog (Pipidae: Hymenochirus boettgeri) from the Congo indicates non-conspecificity with the captive population, Zoological Journal of the Linnean Society 200 (4), pp. 1034-1047 : 1038

Cytogenetic characterization of Hymenochirus boettgeri from the Congo

For both species of the genus Hymenochirus , we carried out the same methods. Giemsa staining shows better results than DAPI, thus we present Giemsa-stained karyotypes with consistently stained chromosomes. Hymenochirus boettgeri possesses 36 chromosomes with eight pairs of metacentric, two pairs of submetacentric, and seven pairs of subtelocentric chromosomes ( Fig. 3A ). All chromosomes are biarmed, the fundamental arm number (FN) is 72. The size of chromosome 1 is shown as the vertical scale bar and is estimated to be less than 10 μm.

DAPI (negative black band) and CMA 3 (positive green band) highlight the NOR locus on a single homologous pair, which is morphologically similar to Hymenochirus sp. chromosome 4 ( Fig. 4A, B ). C-banding identifies 14 heterochromatic blocks (on seven homologous pairs) ( Fig. 4C ). The rDNA FISH analysis shows the 28S NOR locus on the same chromosome on which the CMA 3 is displayed. The 5S probe does not highlight any locus ( Fig. 4D ), probably because of low efficacy of the used 5S probe. Unfortunately, we could not have repeated FISH with the 5S probe because chromosome structure on slide is broken after several attempts of staining and de-staining. The U1 locus is identified in the pericentromeric region of the q arm of the largest chromosome 1 ( Fig. 4E ), which is not homologous to the U1 locus of Hymenochirus sp. (the pericentromeric region of the p arm of chromosome 1). The U2 locus is found in the telomeric region of the q arm of chromosome 8 ( Fig. 4E ), which is homologous to the U2 locus of Hymenochirus sp. GISH analysis with the Hymenochirus sp. WGP probe and competitor Hymenochirus sp. DNA paint all 36 chromosomes of H. boettgeri , with some chromosomes having less intense signals (arrows in Fig. 4F ). The difference in signal intensity is one of the other clues to determine the level of ploidy and homoeologous chromosomes. We find no heterochromatic B chromosomes in the karyotype of H. boettgeri .

Figures

Figure 3. Karyotypes of (A) Hymenochirus boettgeri (IVB-H-CG17-356, male) with 18 homologous chromosome pairs, and (B) Hymenochirus sp. (IVB-H-Hsp06, female) with 10 pairs of A chromosomes and one B chromosome, arranged from Giemsa-stained chromosomes.Chromosomes were cut from metaphase spreads on the left. Long lines in karyotype arrangements indicate the position of chromosome centromere.Short vertical and horizontal lines correspond to the scale = 10 μm.

Figure 4. Hymenochirus boettgeri (IVB-H-CG17-356, male), sequential fluorescent chromosome mapping—DAPI, CMA3, C-banding, ribosomal DNA (rDNA) FISH, small nuclear DNA (snDNA) FISH; and non-sequential whole-genome painting on metaphase spread. A, DAPI (black and white, B&W) consistently stains all 36 chromosomes. B, CMA3 banding in green shows nucleolar secondary constriction (NOR locus) on the p arm of chromosome 4 that co-localizes with 28S.C, C-banding (B&W, brighter staining) highlights heterochromatic blocks on telomeric and pericentromeric regions of seven homologous chromosomes (14 arrows). D, FISH with 28S (red) ribosomal probes shows the p arm of chromosome 4. E, FISH with U1 (red) and U2 (green) snDNA probes shows very weak signals. The U1 probe maps to the q arm of chromosome 1, the U2 probe maps to the q arm of chromosome 8. F, genomic in situ hybridization (GISH) with Hymenochirus sp. whole-genome painting DNA probe that hybridizes to all 36 chromosomes with different intensity. Arrows show the less intensely painted chromosome pair.Scale bars represent 10 μm.