Extraordinary centromeres: differences in the meiotic chromosomes of two rock lizards species Darevskia portschinskii and Darevskia raddei

Abstract

According to the synthesis of 30 years of multidisciplinary studies, parthenogenetic species of rock lizards of genus Darevskia were formed as a result of different combination patterns of interspecific hybridization of the four bisexual parental species: Darevskia raddei, D. mixta, D. valentini, and D. portschinskii. In particular, D. portschinskii and D. raddei are considered as the parental species for the parthenogenetic species D. rostombekowi. Here for the first time, we present the result of comparative immunocytochemical study of primary spermatocyte nuclei spreads from the leptotene to diplotene stages of meiotic prophase I in two species: D. portschinskii and D. raddei. We observed similar chromosome lengths for both synaptonemal complex (SC) karyotypes as well as a similar number of crossing over sites. However, unexpected differences in the number and distribution of anti-centromere antibody (ACA) foci were detected in the SC structure of bivalents of the two species. In all examined D. portschinskii spermatocyte nuclei, one immunostained centromere focus was detected per SC bivalent. In contrast, in almost every studied D. raddei nuclei we identified three to nine SCs with additional immunostained ACA foci per SC bivalent. Thus, the obtained results allow us to identify species-specific karyotype features, previously not been detected using conventional mitotic chromosome analysis. Presumably the additional centromere foci are result of epigenetic chromatin modifications. We assume that this characteristic of the D. raddei karyotype could represent useful marker for the future studies of parthenogenetic species hybrid karyotypes related to D. raddei.

Keywords: Darevskia lizards; Dicentric chromosomes; Meiosis; Neocentromere; Reticulate evolution; Synaptonemal complex.

Figure 1. The spread nuclei of the leptotene–zygotene spermatocytes.

D. portschinskii (A and C) and D. raddei (B and D). Synaptonemal complexes (SCs) immunostained with antibodies against the SYCP3 protein (green), centromeres - with anti-kinetochore ACA antibodies (red), chromatin stained with DAPI (blue). (A and C) D. portschinskii, on each of the 19 SCs only one centromere signal is visible. (B and D) D. raddei, additional anti-kinetochore proteins antibodies signals are indicated with arrows. Bar = five μm.

Figure 2. D. raddei synaptonemal complexes, zygotene stage.

SCs immunostained with antibodies against the SYCP3 protein (green), centromeres with anti-kinetochore antibodies ACA (red). Chromatin stained with DAPI (blue). (A) Early zygotene. SC-bivalent with incomplete synapsis of pericentromeric region. (B, C) Enlarged fragments of A. (D) Late zygotene. Additional ACA-signals indicated with red arrows. (E) Enlarged fragment of D, demonstrate additional ACA foci on each axial element prior to synapsis. Bar = five μm.

Figure 3. The spread nuclei of the pachytene–diplotene spermatocytes.

SCs immunostained with antibodies against the SYCP3 protein (green), centromeres - with anti-kinetochore antibodies ACA (red). Chromatin stained with DAPI (blue). (A and C) D. portschinskii. On each of the 19 SCs only one centromere signal is visible. (B and D) D. raddei. Additional anti-kinetochore proteins antibodies signals are indicated with arrows. Bar = five μm.

Figure 4. A comparison idiogram of average SC lengths of two species, distribution of additional ACA foci and immuno-FISH with telomere probes.

(A) A comparison idiogram of average SC lengths of two species: D. portschinskii (blue, 78 pachytene nuclei) and D. raddei (green, 54 pachytene nuclei). (B and C) Fragments of SC preparations D. portschinskii and D. raddei, immuno-FISH. SCs immunostained with antibodies against the SYCP3 protein (green), centromeres with anti-kinetochore antibodies ACA (red), telomeres labelled by PNA telomere FISH probe (white), chromatin stained with DAPI (blue). (B) Single ACA focus on each of D. portschinskii SC bivalents; (C) two ACA foci in D. raddei SC is (red and white arrows). Telomere FISH signals (white) are located at the ends of SC bivalents in both species and were not detected in the area of the additional ACA focus in D. raddei. (D and E) Distribution of additional ACA-foci on the 19 SC-bivalents of D. raddei in 54 spermatocyte nuclei studied. Bar = two μm.

Figure 5. Immunodetection of crossing over sites in D. portschinskii and D. raddei.

SC spreads of D. portschinskii (A) and D. raddei (B) immunostained with antibodies against the SYCP3 protein (green)—lateral elements of meiotic chromosomes, anti-kinetochore ACA antibodies (red). Sites of crossing over immunostained with antibodies against mismatch repair protein MLH1 (yellow). (C) Number of crossing over sites (MLH1 foci) per spermatocyte nucleus (mean ± SD) in D. portschinskii and D. raddei. Bar = five μm.