Evidence for different origin of sex chromosomes in snakes, birds, and mammals and step-wise differentiation of snake sex chromosomes

Abstract

All snake species exhibit genetic sex determination with the ZZ/ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python (Python molurus bivittatus) and the habu (Trimeresurus flavoviridis), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata, respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis. Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus, whereas they are fully differentiated in T. flavoviridis, and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids.

Fig. 1.

FISH mapping of RAB5A (a–d), DMRT1 (e), and SOX9 (f) to snake chromosomes. Arrows indicate the hybridization signals. RAB5A is mapped on both the Z and W chromosomes of E. quadrivirgata (a) and P. molurus (c) and only on the Z chromosome of T. flavoviridis (d). Hoechst-stained G-banded pattern of the same metaphase as in a is shown in b. DMRT1 and SOX9 are mapped on the short arm of chromosome 2 (e) and on the long arm of chromosome 2 (f), respectively, in T. flavoviridis. (Scale bar, 10 μm.)

Fig. 2.

A comparative cytogenetic map of chromosomes 1–7 and the Z and W chromosomes of E. quadrivirgata. For chromosome mapping of CHD1, DMRT1, ACO1, and SOX9, cDNA fragments isolated by RT-PCR were used, and all other genes were mapped by using EST clones. The chromosomal locations of the genes are shown to the right of E. quadrivirgata chromosomes. The ideogram of G-banded chromosomes constructed in our previous study (4) was used here. The genes mapped to microchromosomes of E. quadrivirgata and the chromosomal locations of their human and chicken homologues are given in Table 1. The human and chicken chromosome segments with homology to the snake chromosomes and their chromosome numbers are indicated to the left of the snake chromosomes. Gene symbols are according to the human nomenclature.

Fig. 3.

G-banded karyotypes and C-banded sex chromosomes of three snake species, P. molurus (a and b), E. quadrivirgata (c and d), and T. flavoviridis (e and f). Macrochromosomes other than sex chromosomes are numbered according to size in each species.

Fig. 4.

Cytogenetic and molecular characterization of a sex chromosome-specific repetitive sequence. (a–c) Chromosomal localization of the BamHI repeat sequence to chromosomes of E. quadrivirgata (a), P. molurus (b), and T. flavoviridis (c). Arrows indicate the hybridization signals. (d) Southern blot hybridization of E. quadrivirgata genomic DNA probed with the BamHI H4 fragment. Each lane contained 5 μg of genomic DNA. A mixture of lambda DNA digested with HindIII and phiX174 phage DNA digested with HaeIII was used as a molecular size marker.

Fig. 5.

Comparative cytogenetic maps of sex chromosomes of P. molurus, E. quadrivirgata, and T. flavoviridis. The ideograms of the Z and W chromosomes are made according to the G-banded patterns. The Z chromosome of E. quadrivirgata is depicted upside down to make the gene order on the Z chromosome correspond to those of the other two species. The locations of the 11 genes and the BamHI repeat sequence are shown to the side of each chromosome.