The chromosome number (2n = 28) of A. pruinosa agrees with those of other 18 species of Acropora and five other species from other coral genera (Montipora and Fungia)7. It is unclear whether the chromosome number 2n = 27 observed in this study was a result of missing one chromosome during mitotic preparations or it is another karyological characteristics in this coral species. Having two chromosome numbers (karyotypic mosaicism) is not uncommon in Acropora7,9. Acropora pruinosa Kochi was reported with chromosome numbers, 2n = 28 and 2n = 29, which was confirmed by the presence of an additional and unpaired chromosome in the case of 2n = 299.
Large-scale hybridization signals on a single chromosome were observed using WGH in this study on A. pruinosa (2n = 28) as well as in a previous study on A. pruinosa Kochi (2n = 29)9. However, for A. pruinosa with even number of chromosomes, the presence of a unique chromosome with no apparent pair based on length and hybridization pattern might indicate the presence of heteromorphic pairs. In most animals, these heteromorphic pairs are often associated with sex chromosomes. Although the sex-linked loci and genes have been identified in the gonochoric coral Corallium rubrum16, the role of heteromorphic chromosomes in the sexual characteristics of scleractinians has not been explored. This investigation is particularly important in Acropora because colonies of some coral species may contain male or female polyps, aside from the well-known co-sexual polyps17. The heteromorphic pairs observed in this study were present in all mitotic cells, and we propose two mechanisms how these cells maintained to carry this unusually long chromosome: (1) After meiotic segregation in the hermaphroditic gonads, either the eggs or the sperms exclusively receive this chromosome, (2) a cycle that involves translocation of the portion of chromosome from the autosomes, causing the chromosome that receives it the longest one. The second mechanism has been demonstrated in other organisms, which involves translocation of the nucleolar organizer region (NOR) containing repetitive tandem arrays of 18S and 28S rRNA genes from autosomes to the telomeric end of sex chromosomes18–20. This NOR in the sex chromosomes functions in the pairing of X-Y chromosomes during meiosis21. This is also supported by the presence of 18/28S rDNA loci at the telomere of one of the longest chromosome pairs in A. pruinosa Kochi9. Further work must be conducted to characterize the sequence arrays that constitute this hybridization signal on the longest chromosome and to confirm whether this chromosome is associated with functioning as a sex chromosome.
The loci of 5S rRNA and core histone genes showed intense hybridization signals on separate chromosome pairs. However, because the minimum sequence length of hybridization that can be readily detected in FISH is 6 kbp15, which is greater than the length of our probes (Table 2), it is possible that other loci composed of fewer or shorter arrays of the target genes exist. This is supported by the results of the experiment on the presence of several rDNA arrays obtained from subcloning, with shorter size of the target gene (LC557012, LC557015) that showed no hybridization signal. A sequence of similar length, but composed of indels (LC557016), compared with the identified repetitive histone array also showed no hybridization in FISH. Because these sequences were confirmed in the genome of A. pruinosa, we speculate that these arrays were either not repetitive (single-copy locus) or were short enough to be detected by FISH. Nonetheless, this study confirms the existence and chromosomal locations of highly clustered arrays of these genes. Studies have reported that this clustering of highly conserved genes is related to pseudogenes, which are acquired through hybridization of ancestral genes and have lost their coding potential22,23. Pseudogenes are implicated in the diversity of the nuclear ribosomal genes in Acropora, but only one rDNA sequence has been implicated to present across several species that are associated with pseudogenes24. It has also been reported previously that large clusters of pseudogenes consist of tRNAs and snRNAs on mammalian chromosomes25,26. Other identified pseudogenes that have repetitive gene copies in humans are the ribosome biogenesis protein gene (RLP24) and E3 ubiquitin-protein ligase gene (MDM2)27. Clustering of pseudogenes was also implicated in a mechanism to disable its function as a result of acquired mutations28,29. The arrangement of these genes in these clusters is tandemly repeated and lacks introns, and thus presumably arose from reverse transcription of mRNA, followed by multiple integration to specific regions in the chromosome29–31.
The linkage of 5S rRNA and snRNA genes and their tandemly repetitive characteristics observed in this study was first reported for mollusks32. The same linkage involving U1, U2, and U5 snRNA genes was also found in fish33 and crustaceans involving only U1 snRNA34. Here, we report for the first time a tandemly repetitive linkage of 5S rRNA and snRNA genes in the phylum Cnidaria. Although many FISH studies of single or multiple loci of repetitive 5S rRNA genes35–37 and snRNA genes38,39 have been reported, it is uncertain whether the loci observed in these studies may involve linkage to one another or to any other gene. We showed that repetitive linkage of both these two genes produced a single locus on the chromosomes. Conversely, in fish, the loci of these two repetitive genes were not linked and were located on different chromosomes40.
Only the H2A and H2B genes arranged in a typical manner were confirmed to constitute the observed loci. However, in cnidarians, various arrangements of repetitive core histone genes, including H1, H3, and H4, have been documented41. In Mytulis edulis, aside from the core histone genes, the sequence of the solitary linker H1 gene is also tandemly repeated42,43. The loci of these solitary H1 gene clusters were found to be located on chromosome pairs different from core histone genes44. This suggests the possible presence of other repetitive histone loci that can be observed in scleractinian chromosomes. Surprisingly, a unique arrangement of repetitive arrays involving linkage between histone and 5S rRNA genes was observed among crustaceans45 and fish46,47.
The varying hybridization patterns of core histone probes in other Acropora population might suggest chromosomal rearrangements during the evolutionary processes within Acropora. In the genus Mus, locations of clusters of conserved genes are shifted across different chromosomes, providing evidence of genome reshuffling that occurred during its evolution48. Variations in the number of histone loci within closely related taxonomic groups have also been observed in other taxa. In bivalves, loci of histone genes are in two chromosome pairs in the mussel, Mytilus galloprovinciali49, and in the scallop, Patinopecten yessoensis50, but there is only one locus in the mussel species, Perumytilus purpuratus51 and in three other species of scallops (Argopecten irradians, Chlamys farreri, and C. nobilis)50.
We demonstrated that single-sequence probes containing conserved genes produced a readily detectable hybridization signal on the chromosomes of A. pruinosa. These probes also hybridized on chromosomes of other Acropora population and species and thus have a potential for use as chromosomal markers within the taxa. In addition, the single-sequence probes revealed the presence of other loci in other species, which revealed the differences in chromosome organization. This study may provide a foundation for discovering the loci of other tandemly repetitive genes, such as 18 and 28S rDNA that can be used as additional chromosomal markers for improved karyotyping of Acropora.