Sequential DAPI/PI and CMA staining followed by FISH using 28S rDNA (28S) and telomeric (TEL) probes on Brachidontes puniceus (BPU) and Brachidontes rodriguezi (BRO) chromosomes. Sequential staining of the same metaphase plates with DAPI/PI (a, e) and CMA (b, f) shows the presence of two GC-rich (DAPI dull/CMA bright) regions located at the ends of the long arms on one chromosome pair in B. puniceus (arrows in a, b, e, f). One of the B. puniceus specimens shows an additional GC-rich region on the short arm of one of the members of the chromosome pair bearing the standard band (arrowhead in e, f). In B. rodriguezi four GC-rich regions appear on the short arms of two chromosome pairs (arrows in i, j) but one of the mussels shows only three of these bands (arrows in m, n) lacking the fourth (arrowhead in m, n). FISH using a 28S rDNA probe (digoxigenin, rhodamine, red) gives signals at the CMA+ positions in both B. puniceus (arrows in c, g, arrowhead in g) and B. rodriguezi (arrows in k, o). Note the absence of the fourth major rDNA signal in o (arrowhead). FISH using a PNA-telomeric probe (fluorescein, green) gives signals at both ends of every sister chromatid. Telomeric signals situated on the GC-rich NORs are consistently bigger than the rest of the telomeric signals and their sizes are similar to the sizes of the 28S signals (arrows in d, h, l, p). Scale bars, 5 μm.