The problem the team faced is a familiar one in coral biology. Coral chromosomes are typically small, roughly half the size of human chromosomes, and show little variation in size and shape. Under conventional staining, they appear morphologically undifferentiated, with poor banding patterns that make it nearly impossible to reliably distinguish individual chromosomes or identify homologous pairs. This uniformity has long frustrated karyotyping efforts and limited the use of chromosomal information in coral classification, even as climate change accelerates coral biodiversity loss and makes accurate species identification increasingly urgent for conservation.

To overcome this, the researchers turned to molecular cytogenetics, and specifically to FISH, a technique that uses fluorescently labeled DNA probes to bind complementary sequences on chromosomes, revealing gene locations and repetitive elements that clarify genome organization. Their target, M. amakusensis, is found in shallow sunlit waters around Japan, which hosts a wide variety of both tropical and temperate coral species. The team collected gametes at Nishidomari in Kochi Prefecture, where the simultaneously hermaphroditic coral releases eggs and sperm packed together into discrete bundles from the mouths of fertile polyps.

The collection itself was a feat of timing and improvisation. Between 21:00 and 21:45 on August 15, 2017, the researchers observed the release of gamete bundles and captured them using bundle collectors built from 15-centimeter funnels connected to 50-milliliter plastic test tubes, deployed at two separate sites to avoid fertilization between the same colony. The bundles were then broken apart into eggs and sperm by gentle shaking, eggs were rinsed with 0.2-micrometer-filtered seawater to remove contaminants, and sperm were collected by centrifugation at 2,000 × g. Fertilized eggs were produced by mixing sperm with eggs from different colonies, and embryos were treated with colchicine and a hypotonic solution to swell cells and spread chromosomes before fixation in methanol and acetic acid.

Conventional Giemsa staining of 50 metaphase spreads revealed a diploid karyotype of 2n = 28, arranged as seven submetacentric chromosome pairs (1–7) and seven metacentric pairs (8–14). More strikingly, the team identified a prominent homogeneously staining region, or HSR, on the long arm of chromosome 12, visible in roughly 50% of the cells examined. HSRs generally reflect genetic amplification at the chromosomal level, and while they have been observed in most of the seven stony coral species previously studied cytogenetically, this is the first report for a member of Lobophylliidae. The HSR made chromosome 12 easy to identify, but the other chromosomes remained difficult to distinguish precisely because of their similarities in length and centromere location.

The researchers then cloned, sequenced, and mapped five FISH markers designated MA-H3 for histone H3, MA-5S for 5S rRNA, MA-18/28S for 18S-28S rRNA, MA-13C for a centromeric region, and MA-TEL for a telomeric region. FISH analysis localized MA-H3 to the centromeric region of chromosome 1, MA-5S to the telomeric region of chromosome 4, MA-18/28S to the terminal region of chromosome 12 where it coincided exactly with the HSR, MA-13C to the centromere of chromosome 13, and MA-TEL to multiple telomeric regions across several chromosomes. Together, these markers enable the identification of at least 3 out of 14 chromosome pairs, a significant advance for precise karyotyping in a species where chromosomes were previously indistinguishable.

Sequence analysis confirmed the identities of the markers and revealed both conserved and novel repetitive elements. The MA-H3 marker, 282 base pairs long, closely matched the histone H3 gene and showed 99.25% identity with the Montastraea multipunctata histone H3 gene, reflecting the deep conservation of this gene across corals. The MA-5S marker, 920 base pairs, corresponded to part of the 5S rRNA gene and matched 5S U2RNA, with homology to the sea anemone Actinia equina and hits on multiple chromosomes of the Echinopora horrida genome. The MA-18/28S marker, 1,258 base pairs, matched the 18S-28S rRNA gene of M. amakusensis itself. The MA-13C marker, just 111 base pairs, produced hits exclusively with the E. horrida genome, while the 149-base-pair MA-TEL marker yielded no significant sequence similarities in any database, leaving its origin an open question for future study.

Beyond the five markers, the team applied genomic DNA hybridization (GDH), labeling whole-sperm DNA with Cy3-dUTP and hybridizing it to metaphase spreads. This revealed 6 to 10 signals collected predominantly at telomeric regions, suggesting the presence of repetitive sequences concentrated at chromosome ends. Similar telomeric signal distributions have been observed in other lobophylliid and merulinid corals such as Echinophyllia aspera and Trachyphyllia geoffroyi, whereas in Acropora species these repeats are primarily found in centromeric regions. Such species-specific chromosomal localizations of repeat DNA may prove helpful for taxonomic classification of stony corals, offering a cytogenetic signature that complements skeletal and DNA sequence data.

One intriguing observation was the variable behavior of the MA-TEL probe. While FISH signals from specific gene probes are generally observed in the same portions of homologous chromosomes, MA-TEL produced variable signal patterns between metaphase spreads, sometimes marking only one homolog of a chromosome pair. The researchers suggest this variability likely reflects differences in the number of MA-TEL-specific sequences present in each telomere region, and that the telomere regions of M. amakusensis may be unstable due to highly repeated sequences, similar to the amplified rDNA seen in HSRs. The functional activity of the over-amplified rDNA in the HSR also remains unclear, though its presence appears to be a characteristic feature of stony corals, with the notable exception of Acropora species.

The study demonstrates the utility of molecular cytogenetics in stony coral systematics at a moment when the field urgently needs new tools. Traditional taxonomy based on skeletal morphology often conflicts with molecular analyses using ribosomal and mitochondrial DNA, partly due to hybridization between closely related species and high morphological plasticity. Cytogenetics offers a complementary classification method based on chromosomal characteristics, and molecular cytogenetics is expected to bridge the gap between morphological and molecular approaches in coral taxonomy. By providing the first FISH markers for a lobophylliid coral and establishing a more accurate karyotype for M. amakusensis, the research opens the door for comparative genomic studies across the family and, ultimately, for resolving taxonomic ambiguities that have long clouded our understanding of coral genome evolution and reef biodiversity.

Subject of Research: Molecular cytogenetic characterization and FISH marker isolation in the stony coral Micromussa amakusensis

Article Title: Molecular cytogenetic study on the scleractinian coral Micromussa amakusensis (Veron, 1990) ( Hexacorallia , Anthozoa , Cnidaria ): isolation of five fluorescence in situ hybridization markers

Article References: Baldove, A. B., Ito, M., Taguchi, T., Mezaki, T., Saito, H., Manalili, S. E., Namura, Y., Nieves-Brutas, I. J., Tominaga, A., & Kubota, S. (2025). Molecular cytogenetic study on the scleractinian coral Micromussa amakusensis (Veron, 1990) (Hexacorallia, Anthozoa, Cnidaria): isolation of five fluorescence in situ hybridization markers. Comparative Cytogenetics, 19, 135-154. https://doi.org/10.3897/compcytogen.19.157310

Image Credits: AI Generated

DOI: 10.3897/compcytogen.19.157310

Keywords: stony corals, Micromussa amakusensis, fluorescence in situ hybridization, molecular cytogenetics, karyotype, Lobophylliidae, chromosome markers, rRNA genes, histone H3, telomeres, coral taxonomy, genome evolution