Hymenochirus sp.

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 : 1039

Cytogenetic characterization of the captive Hymenochirus sp.

The number of chromosomes in Hymenochirus sp. is 21 (20A + 1B) with seven pairs of metacentric, one pair of submetacentric, and two pairs of subtelocentric chromosomes, and one extra B chromosome ( Fig. 3B ). All chromosomes are biarmed, FN = 42. The size of chromosome 1 is estimated to be 12–13 μm. Both males and females have the same number and general morphology of chromosomes. Sequential DAPI and CMA 3 analyses ( Fig. 5A, B ) show a single NOR on the p arm of submetacentric chromosome 4. DAPI consistently stains all 21 chromosomes but without staining of the NOR locus, which is CMA 3 -positive. C-banding highlights telomeric and pericentromeric bands on most chromosomes, which helps to identify homologous chromosome pairs ( Fig. 5C ). Interestingly, the NOR locus is not stained using C-banding. The B chromosome is entirely painted and shows a high degree of heterochromatization ( Fig. 5C, D, F ; Supporting Information, Fig. S1A, B, D, E ). FISH with rDNA probes identifies the 5S locus on the telomere and interstitial region of the q arm of chromosome 6. The 28S (NOR) locus is mapped to chromosome 4 ( Fig. 5D ) and is identical with CMA 3 bands. FISH with snDNA probes identifies the U1 locus on chromosome 1 and the U2 locus on chromosome 8 ( Fig. 5E ). GISH with the H. boettgeri WGP probe paints 20 chromosomes. Accessory B chromosome is stained with DAPI (DAPI-positive) but is not painted with the H. boettgeri WGP probe ( Fig. 5F ; Supporting Information, Fig. S1A ). We performed two control GISH experiments with the Hymenochirus sp. WGP probe: the first with (Supporting Information, Fig. S1B ) and the second without (Supporting Information, Fig. S1C ) Hymenochirus sp. competitor DNA. Surprisingly, the WGP probe does not paint the B chromosome of Hymenochirus sp. in either control experiment (no red signal on the B chromosome). DAPI staining shows no signal on the B chromosome in the GISH experiment without the competitor (Supporting Information, Fig. S1C, F ) compared to GISH with the competitor DNA where DAPI stains the entire B chromosome (Supporting Information, Fig. S1A, B, D, E ).

Figures

Figure 1. Dwarf clawed frogs, Hymenochirus sp. (captive population) and H. boettgeri. A, Hymenochirus sp., female (IVB-H-Hsp06) and male (IVB-H-Hsp02) in amplexus.B, Hymenochirus sp., female in dorsolateral view (IVB-H-Hsp04). In (A) and (B), note the relatively smooth flanks and hindlegs with homogeneous, unenlarged tubercles. C, Hymenochirus boettgeri from the north-western part of the Republic of the Congo (male, IVB-H-CG17-356). D, holotype of H. boettgeri, female (ZMB 11521). The area marked by the red rectangle is detailed in (E). F,Hymenochirus boettgeri from the same locality as the karyotyped individual (IVB-H-CG17-112, male).In (C–F), note the enlarged and spiny tubercles on the flanks and hindlegs typical for H. boettgeri.

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 5. Hymenochirus sp. (IVB-H-Hsp06, female), sequential fluorescent chromosome mapping (DAPI, CMA3, C-banding, rDNA FISH), non-sequential snDNA FISH, and whole-genome painting on metaphase spread.A, DAPI (B&W) counter-stained metaphase spread shows all 21 chromosomes.B, CMA3 banding in green shows NOR locus on the p arm of chromosome 4.CMA3 signal co-localizes with 28S locus. C, C-banding (B&W,brighter staining) highlights heterochromatic blocks on telomeric and pericentromeric regions of almost all chromosomes. In addition,the whole B chromosome is intensely banded (arrow). D, 5S (green) and 28S (red) rDNA loci are located on the q arm of chromosome 6 and p arm of chromosome 4, respectively.The 5S rDNA is situated on two different chromosomal loci within the single q arm. E, the snDNA loci U1 (red) and U2 (green) are located on the p arm of chromosome 1 and the q arm of chromosome 8, respectively. F,the GISH experiment of the H.boettgeri whole-genome painting probe, which hybridizes on Hymenochirus sp.chromosomes.All chromosomes are painted (red) except one, B chromosome, which shows no GISH signal and is DAPI-positive (arrow).Scale barsrepresent 10 μm.