The chromosomes of the queen scallop Aequipecten opercularis were studied using conventional Giemsa staining, chromosome measurements, C-banding, silver staining, and fluorescent in situ hybridization (FISH) with 18S-28S rDNA and 5S rDNA probes. The karyotype (2n = 26) consists of large metacentric (pairs 1 and 2), telocentric (pairs 3, 4, 5, 6, 7, 8, and 9), and small metacentric chromosomes (pairs 10, 11, 12, and 13). The C-bands observed can be described as major and minor C-bands which are differentiated according to the intensity of the fluorescence and the frequency of the detection. Major C-bands were found on the long arm of the chromosome pairs 6, 7, 8, and 9 in an intercalary or subterminal position. Minor C-bands were located in the centromeric region in all chromosomes of the complement and also on one arm of pairs 12 and 13 in a terminal position. Silver spots were detected on the telomere of the long arms of one or two chromosomes of pair 7 in every case, although in two individuals up to four additional silver spots were detected. These were located on pairs 8 and 9 in the same position as the C-bands. 18S-28S ribosomal genes were found by FISH on the long arm of chromosome pair 7. 5S ribosomal genes were located subterminally on one arm of metacentric pair 1, but two sites were differentiated in the case of elongated chromosomes. The results obtained allow for the identification of at least six different chromosome pairs in A. opercularis and contribute to the construction of an idiogram that is suitable for gene mapping and establishing accurate interspecific comparisons in scallops.

In higher eukaryotes, the gene family encoding the 5S ribosomal RNA (5S rRNA) has been used (together with histones) to showcase the archetypal example of a gene family subject to concerted evolution. However, recent studies have revealed conspicuous features challenging the predictions of this model, including heterogeneity of repeat units, the presence of functional 5S gene variants as well as the existence of 5S rDNA divergent pseudogenes lacking traces of homogenization. In the present work, we have broadened the scope in the evolutionary study of ribosomal gene families by studying the 5S rRNA family in mussels, a model organism which stands out among other animals due to the heterogeneity it displays regarding sequence and organization. To this end, 48 previously unknown 5S rDNA units (coding and spacer regions) were sequenced in five mussel species, leading to the characterization of two new types of units (referred to here as small-beta 5S rDNA and gamma-5S rDNA) coexisting in the genome with alpha and beta rDNA units. The intense genetic dynamics of this family is further supported by the first description of an association between gamma-5S rDNA units and tRNA genes. Molecular evolutionary and phylogenetic analyses revealed an extensive lack of homology among spacer sequences belonging to different rDNA types, suggesting the presence of independent evolutionary pathways leading to their differentiation. Overall, our results suggest that the long-term evolution of the 5S rRNA gene family in mussels is most likely mediated by a mixed mechanism involving the generation of genetic diversity through birth-and-death, followed by a process of local homogenization resulting from concerted evolution in order to maintain the genetic identities of the different 5S units, probably after their transposition to independent chromosomal locations.

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The 5S rDNA repeat unit of the cockle Cerastoderma glaucum from the Mediterranean and Baltic coasts was PCR amplified and sequenced. The length of the units was 539-568 bp, of which 120 bp were assigned to the 5S rRNA gene and 419-448 bp to the spacer region, and the G/C content was 46%-49%, 54%, and 44%-47%, respectively. Two types of units (A and B), differing in the spacer, were distinguished based on the percentage of differences and clustering in phylogenetic trees. A PCR assay with specific primers for each unit type indicated that the occurrence of both units is not restricted to the sequenced individuals. The 5S rDNA units of C. glaucum were compared with new and previously reported sequences of Cerastoderma edule. The degree of variation observed in C. edule was lower than that in C. glaucum and evidence for the existence of units A and B in C. edule was not found. The two cockles have the same coding region but displayed numerous fixed differences in the spacer region and group separately in the phylogenetic trees. Digestion of the 5S rDNA PCR product with the restriction enzymes HaeIII and EcoRV revealed two RFLPs useful for cockle identification.