Are Pericentric Inversions Reorganizing Wedge Shell Genomes?

Abstract

Wedge shells belonging to the Donacidae family are the dominant bivalves in exposed beaches in almost all areas of the world. Typically, two or more sympatric species of wedge shells differentially occupy intertidal, sublittoral, and offshore coastal waters in any given locality. A molecular cytogenetic analysis of two sympatric and closely related wedge shell species, Donax trunculus and Donax vittatus, was performed. Results showed that the karyotypes of these two species were both strikingly different and closely alike; whilst metacentric and submetacentric chromosome pairs were the main components of the karyotype of D. trunculus, 10-11 of the 19 chromosome pairs were telocentric in D. vittatus, most likely as a result of different pericentric inversions. GC-rich heterochromatic bands were present in both species. Furthermore, they showed coincidental 45S ribosomal RNA (rRNA), 5S rRNA and H3 histone gene clusters at conserved chromosomal locations, although D. trunculus had an additional 45S rDNA cluster. Intraspecific pericentric inversions were also detected in both D. trunculus and D. vittatus. The close genetic similarity of these two species together with the high degree of conservation of the 45S rRNA, 5S rRNA and H3 histone gene clusters, and GC-rich heterochromatic bands indicate that pericentric inversions contribute to the karyotype divergence in wedge shells.

Keywords: Donax; GC-rich heterochromatin; chromosome; fluorescent in situ hybridization; histone genes; pericentric inversions; ribosomal RNA genes.

Figure 1

Collection localities and pictures of representative Donax trunculus and Donax vittatus analyzed.

Figure 2

Mapping of telomeric sequence, 5S rDNA, 28S rDNA, and H3 histone gene clusters on Donax trunculus chromosomes. Sequential fluorochrome staining of mitotic metaphase plates shows 4′,6-diamidino-2-phenylindole (DAPI)-negative regions (a,f) that fluoresce yellow after chromomycin A3 (CMA) (b,g) and bright red after DAPI/ propidium iodide (PI) (c,h) staining in both Atlantic (a–c) and Mediterranean (f–h) specimens of Donax trunculus. Hybridization of the same metaphase plates (c,h) with a telomeric peptide nucleic acid PNA probe discloses signals (TEL, green) at both ends of every chromosome. Sequential fluorescent in situ hybridization (FISH) experiments using major and minor rDNA and H3 histone gene probes on the same metaphase plates (d,i), and the corresponding karyotypes (e,j), show H3 histone gene signals (H3, green) intercalated in the long arms of chromosome pair 17. Minor rDNA probes (5S, red) map to two locations, intercalated in the short arms of chromosome pair 3 and subterminal to the long arms of chromosome pair 10. Major rDNA signals (28S, magenta) are intercalated in the short arms of subtelocentric chromosome pair 6 in Atlantic specimens (d,e) and subcentromeric to the long arms of metacentric pair 6 in Mediterranean specimens (i,j). Note that none of the chromosome pairs is telocentric. Scale bars, 5 μm.

Figure 3

Mapping of telomeric sequence, 5S rDNA, 28S rDNA, and H3 histone gene clusters on Donax vittatus chromosomes. Sequential fluorochrome staining of mitotic metaphase plates shows DAPI-negative regions (a,f) that fluoresce yellow after CMA (b,g) and bright red after DAPI/PI (c, h) staining in Donax vittatus specimens from both Samil (a–c) and all the other Galician (f–h) populations. Hybridization of the same metaphase plates (c,h) with a telomeric PNA probe discloses signals (TEL, green) at both ends of every chromosome. Sequential FISH experiments using major and minor rDNA and H3 histone gene probes on the same metaphase plates (d,i), and the corresponding karyotypes (e,j), show H3 histone gene signals (H3, green) intercalated in the long arms of telocentric chromosome pair 17. Minor rDNA signals (5S, red) are subterminal to the long arms of subtelocentric chromosome pair 10. Major rDNA signals (28S, magenta) overlap the short arms of telocentric chromosome pair 6. Note the presence of 10 telocentric chromosome pairs in specimens from Samil (4, 6, 7, 8, 9, 14, 15, 16, 17 and 18) and 11 in those from the remaining Galician populations (6, 7, 8, 9, 11, 13, 14, 15, 16, 17 and 18). Scale bars, 5 μm.

Figure 4

Schematic representation of the wedge shell haploid chromosome complements. For comparative purposes, the schemas of chromosomes 1 to 19 in Donax trunculus (DTR) Mediterranean (M) and Atlantic (A) populations and Donax vittatus (DVI) Galician (G) and Samil (S) populations are represented in quartets. DAPI−/CMA+ bands (yellow), 5S rDNA (red), 45S rDNA (magenta) and H3 histone gene (green) are also represented. The chromosomes showing the most remarkable intraspecific differences are shadowed in light green for Donax trunculus and light pink for Donax vittatus.

Figure 5

Ideogrammatic representation of the pericentric inversion differentiating chromosome 6 in Mediterranean and Atlantic populations of Donax trunculus. Arrows point to inversion breakpoints.