Molecular cytogenetic evidence of rearrangements on the Y chromosome of the threespine stickleback fish

Abstract

To identify the processes shaping vertebrate sex chromosomes during the early stages of their evolution, it is necessary to study systems in which genetic sex determination was recently acquired. Previous cytogenetic studies suggested that threespine stickleback fish (Gasterosteus aculeatus) do not have a heteromorphic sex chromosome pair, although recent genetic studies found evidence of an XY genetic sex-determination system. Using fluorescence in situ hybridization (FISH), we report that the threespine stickleback Y chromosome is heteromorphic and has suffered both inversions and deletion. Using the FISH data, we reconstruct the rearrangements that have led to the current physical state of the threespine stickleback Y chromosome. These data demonstrate that the threespine Y is more degenerate than previously thought, suggesting that the process of sex chromosome evolution can occur rapidly following acquisition of a sex-determining region.

Figure 1.—

Correspondence between genetic and sequence maps of the threespine stickleback X chromosome. The three supercontigs (SC, shaded regions) are oriented by marker order on the genetic map (Peichel et al. 2004). The sequence coordinates of markers and features (solid horizontal lines) are given on the left. Coordinates in smaller text give the sequence coordinates of the SC boundaries (dotted lines). Positions of BAC clones used as FISH probes (open horizontal bars) encompassing markers or genes of interest are shown to scale. The genetic positions of markers are from Peichel et al. (2004). Stn235 has not been genetically mapped; its position was determined by BLAST (Altschul et al. 1990).

Figure 2.—

The Y chromosome is heteromorphic. (a) Idh probe (green) hybridized to a male (XY) threespine stickleback metaphase spread (2n = 42). The hybridized submetacentric chromosome is identical to the X chromosomes in Figure 2b. The other chromosome with terminal hybridization signal is metacentric, identifying it as the Y chromosome. (b) Idh probe (green) hybridized to a female (XX) threespine stickleback metaphase spread (2n = 42). Hybridization signals identify the two submetacentric X chromosomes.

Figure 3.—

Threespine stickleback male karyogram. The karyogram was produced from a male (XY) metaphase spread (2n = 42) shown in supplemental Figure 1. The chromosomes are aligned vertically by centromere position and ordered from largest (left) to smallest (right) within each class. There are two metacentric pairs, three telocentric pairs, nine submetacentric pairs, six acrocentric pairs, and the heteromorphic X (submetacentric) and Y (metacentric) pair.

Figure 4.—

Deletion on the Y chromosome. Idh probe (green) and Cyp19b probe (purple) hybridized to a male (XY) threespine stickleback metaphase spread. The hybridized submetacentric chromosome at left exhibits the internal Idh hybridization signal of the X chromosome; Cyp19b signal is near the q arm telomere. The hybridized metacentric chromosome at right exhibits the terminal Idh hybridization signal of the Y chromosome; no Cyp19b staining is seen. In most metaphase spreads hybridized with the Idh probe, a diffuse signal can also be seen at the centromere of one submetacentric chromosome (arrowhead).

Figure 5.—

FISH-based cytogenetic maps of the threespine stickleback X and Y chromosomes. The sequence map of the X (left) is shown to scale with horizontal black lines representing positions of markers and features and horizontal white bars representing BAC clones used as probes (for clone identities, see Figure 1). The black circles represent the positions of the centromeres. Each FISH section (center) contains the X (left) and Y (right) from a single metaphase spread to which the Idh probe (green) and one additional probe (purple) were hybridized. The X and Y are vertically aligned by the position of Idh, and the position of the centromere is identified with a white arrowhead. Dashed lines joining the X map to the FISH sections indicate the BAC probe used in each section. Dashed lines leading from the FISH data to the Y cytogenetic map (right, not to scale) indicate the physical interval (pTEL-CEN or CEN-Idh) to which each FISH probe hybridizes.

Figure 6.—

Pericentric inversion on the Y chromosome. Idh probe (green), Stn187 probe (green), and Wt1a probe (purple) hybridized to a male (XY) threespine stickleback metaphase spread (2n = 42). While the X marker order is Wt1a (purple), centromere (white arrowhead), Stn187 (green), Idh (green; green arrowhead), the order is changed on the Y: Stn187 (green), centromere (white arrowhead), Wt1a (purple), and Idh (green; green arrowhead).

Figure 7.—

Parsimony model for the evolution of the threespine stickleback Y. This model is the most parsimonious way to use inversions and deletions to arrive at the physical order of markers on the Y (right) having started with the order on the X (left). We hypothesize that three inversions (crossing dashed lines) containing the centromere (solid circle) and one deletion (dotted lines that meet to the right) gave rise to the extant Y. Theoretical intermediate Y chromosome states are labeled Y_a and Y_b, although the order of inversions in this model is arbitrary. The relative timing of the deletion is also arbitrary and not necessarily concomitant with an inversion.