Evolution of bird sex chromosomes: a cytogenomic approach in Palaeognathae species

Abstract

Background: Different patterns of sex chromosome differentiation are seen in Palaeognathae birds, a lineage that includes the ratites (Struthioniformes, Rheiformes, Apterygiformes, Casuariiformes, and the sister group Tinamiformes). While some Tinamiform species have well-differentiated W chromosomes, both Z and W of all the flightless ratites are still morphologically undifferentiated. Here, we conducted a comprehensive analysis of the ZW differentiation in birds using a combination of cytogenetic, genomic, and bioinformatic approaches. The whole set of satDNAs from the emu (Dromaius novaehollandiae) was described and characterized. Furthermore, we examined the in situ locations of these satDNAs alongside several microsatellite repeats and carried out Comparative Genomic Hybridizations in two related species: the greater rhea (Rhea americana) and the tataupa tinamou (Crypturellus tataupa).

Results: From the 24 satDNA families identified (which represent the greatest diversity of satDNAs ever uncovered in any bird species), only three of them were found to accumulate on the emu's sex chromosomes, with no discernible accumulation observed on the W chromosome. The W chromosomes of both the greater rhea and the emu did not exhibit a significant buildup of either C-positive heterochromatin or repetitive DNAs, indicating their large undifferentiation both at morphological and molecular levels. In contrast, the tataupa tinamou has a highly differentiated W chromosome that accumulates several DNA repeats.

Conclusion: The findings provide new information on the architecture of the avian genome and an inside look at the starting points of sex chromosome differentiation in birds.

Keywords: Birds; Evolution; Molecular cytogenetics; Nascent sex chromosomes; satDNAs.

Fig. 1

Geographic distribution of Palaeognathae, showing color-coded species based on their geographical occurrence: Struthio (purple), Apteryx (green), Casuarius (blue), Dromaius (orange), Crypturellus (light green), and Rhea (pink). The dated species tree was obtained from [20]. Representative idiograms for the Z (light blue) and W (light red) sex chromosomes are provided, together with information on their C-banding (black) patterns (except for Apteryx). Data was retrieved from [–27] and present data). A geological scale with key periods is depicted on the left (N = Neogene; P = Paleogene)

Fig. 2

Female metaphase plates of the emu highlighting the chromosomal location of 23 DnoSatDNAs. Their family names are indicated in the lower right corner, in green (Atto488-dUTP labeled) or red (Atto550-dUTP labeled). While the W chromosome was appropriately identified by a sequential hybridization with the microsatellite (GA)n, the Z chromosome could not be properly identified based on its morphology. Bar = 10 μm

Fig. 3

Female metaphase plates of the emu highlighting the chromosomal location of microsatellite repeats, indicated in the lower right corner in red. While the W chromosome was appropriately identified by a sequential hybridization with the microsatellite (GA)n, the Z chromosome could not be properly identified based on its morphology. Bar = 10 μm

Fig. 4

Female metaphases of the greater rhea highlighting the chromosomal location of microsatellite repeats. The microsatellites are indicated in the lower right corner in red. While the W chromosome was appropriately identified by a sequential hybridization with the microsatellite (GA)n, the Z chromosome could not be properly identified based on its morphology. Bar = 10 μm

Fig. 5

Female metaphases of the tataupa tinamou highlighting the chromosomal location of microsatellite repeats. The microsatellites are indicated in the lower right corner in red. While the W chromosome was appropriately identified by a sequential hybridization with the microsatellite (GA)n, the Z chromosome could not be properly identified based on its morphology. Bar = 10 μm

Fig. 6

Intraspecific genomic hybridization with emu male and female gDNA probes hybridized in female metaphase chromosomes (a-d). (a) DAPI-stained metaphases of the emu female, (b) hybridization pattern of the male-derived probe (green), (c) hybridization pattern of the female-derived probe (red), and (d) merged images of both genomic probes and DAPI staining. Interspecific genomic hybridization between the emu and the greater rhea (e-h). (e) DAPI-stained metaphases of the emu female, (f) hybridization pattern of the emu female-derived probe (green), (g) hybridization pattern of the greater rhea female-derived probe (red), and (h) merged images of both genomic probes and DAPI staining. While the W chromosome was appropriately identified by a sequential hybridization with the microsatellite (GA)n, the Z chromosome could not be properly identified based on its morphology. Bar = 5 μm