Cytogenetic Analysis of the Bimodal Karyotype of the Common European Adder, Vipera berus (Viperidae)

Abstract

Vipera berus is the species with the largest range of snakes on Earth and one of the largest among reptiles in general. It is also the only snake species found in the Arctic Circle. Vipera berus is the most involved species of the genus Vipera in the process of interspecific hybridization in nature. The taxonomy of the genus Vipera is based on molecular markers and morphology and requires clarification using SC-karyotyping. This work is a detailed comparative study of the somatic and meiotic karyotypes of V. berus, with special attention to DNA and protein markers associated with synaptonemal complexes. The karyotype of V. berus is a remarkable example of a bimodal karyotype containing both 16 large macrochromosomes and 20 microchromosomes. We traced the stages of the asynchronous assembly of both types of bivalents. The number of crossing-over sites per pachytene nucleus, the localization of the nucleolar organizer, and the unique heterochromatin block on the autosomal bivalent 6-an important marker-were determined. Our results show that the average number of crossing-over sites per pachytene nucleus is 49.5, and the number of MLH1 sites per bivalent 1 reached 11, which is comparable to several species of agamas.

Keywords: NOR; bimodal karyotype; chiasma; crossing-over; heterochromatin; microchromosomes; nucleolar organizer; prophase I of meiosis; recombination rate; synaptonemal complex.

Figure A1

Chromosomal “bouquet” stage, V. berus spermatocyte I. Detection of the heterochromatin regions on the both chromosomes 6 before synapsis (HR6(I) and HR6(II)). (a) Chromatin stained with DAPI (blue), (b) Axial elements of chromosomes are immunostained with antibodies against the SYCP3 protein (green), and (c) merge. Bar—10 μm.

Figure A2

Late zygotene stage, V. berus spermatocyte I. Heterochromatin region on the bivalent 6 (HR6) after the synapsis of two homologous chromosomes 6. (a) Chromatin stained with DAPI (blue), (b) Axial elements of chromosomes are immunostained with antibodies against the SYCP3 protein (green), and (c) merge. Bar—10 μm.

Figure A3

Pachytene stage, V. berus spermatocyte I. Immunodetection of crossing-over marker, MLH1 protein (red). (a) 57 MLH1 sites in one SC-spread. (b) 49 MLH1 sites in one SC-spread and 11 MLH1 sites on one macrobivalent 1 (asterisk). Heterochromatin region on the bivalent 6 (HR6) is indicated. Axial elements of chromosomes are immunostained with antibodies against the SYCP3 protein (green). Chromatin stained with DAPI (blue). Bar—10 μm.

Figure 1

Mitotic karyotypes of V. berus. (a,b) DAPI staining and (c–f) conventional Giemsa staining. (a,c,d) Male karyotypes; (b,e,f) Female karyotypes. (a–c,e) Metaphase chromosome plates; (d,f) karyograms. ZZ/ZW—sex chromosomes. Scale bar—10 μm.

Figure 2

Markers of V. berus SC-karyotype, ideogram, and spermatids morphology. (a) Immunodetection of the crossing-over marker, MLH1 protein. A total of 52 MLH1 sites per nucleus, and 10 MLH1 sites on the bivalent 1 (asterisk). (b) Immunodetection of Nucleolar Organizer (NOR) on the microchromosome bivalent and (b`) as the DAPI-negative region. Axial elements of chromosomes are immunostained with antibodies against the SYCP3 protein (green); crossing-over sites are immunostained with antibodies against MLH1 protein (red); NOR with antibodies against Fibrillarin protein (violet). Chromatin stained with DAPI (blue). (c) Ideorgam of V. berus SC-karyotype. Blue—p arms; orange—q arms. Metacentric Z-chromosome, heterochromatin region (HR6), and NOR are indicated. Heterochromatin region on the chromosome pair 6 is indicated as HR6. (c`) Number of MLH1 foci per spermatocyte nucleus (mean ± SD). (d) Spermatids, DAPI staining (blue). Bar—10 μm.

Figure 3

Presynaptic stages in V. berus primary spermatocytes. (a) Leptotene, long axial elements are completely asynapted, centromeres distributed over all spread nuclei. (b) Chromosomal ‘bouquet’ stage, U-shaped axial elements of chromosomes, clusterization of telomere ends in the local region of the spread nucleus. (c) Mid zygotene, asynchronous synapsis of the long chromosomes and microchromosomes. MLH1 protein loaded in bivalents of microchromosomes as well as in the regions of local synapsis of long assembling bivalents. (d) Late zygotene, finalization of assembly of the long bivalents. Bivalents of microchromosomes are fully assembled. Heterochromatin region on the chromosome pair 6 is indicated as HR6. AE—axial elements of asynapted chromosomes. SC—assembled regions of synaptonemal complexes. Axial elements of chromosomes are immunostained with antibodies against the SYCP3 protein (green), centromeres with antikinetochore antibodies ACA (yellow). Mismatch repair protein sites are immunostained with antibodies against MLH1 protein (red). Chromatin stained with DAPI (blue). Bar—10 μm.

Figure 4

Alignment stage and postsynaptic stages in V. berus primary spermatocytes. (a) Alignment stage, spatial alignment of pairs of axial elements of homologous chromosomes located opposite each other. (b) Pachytene, complete synapsis of all 18 bivalents. (c) Mid diplotene, desynapsis of homologues, chiasmata. (d) Late diplotene, diffuse axial elements, unloading of the SYCP3, chiasmata. Heterochromatin region on the chromosome pair 6 is indicated as HR6. Axial elements of chromosomes are immunostained with antibodies against the SYCP3 protein (green), centromeres with antikinetochore antibodies ACA (yellow). Mismatch repair protein sites are immunostained with antibodies against MLH1 protein (red). Chromatin stained with DAPI (blue). Bar—10 μm.