Flying Fish Chromosomes Reveal Hidden Evolution in the Open Ocean

Flying fish are among the most recognizable animals of the open ocean, gliding for tens of meters above the waves on elongated, rigid pectoral fins to escape predators. Yet for all the attention their aerial feats have attracted, one fundamental layer of their biology has remained almost entirely unexplored: their chromosomes. A new study published in Comparative Cytogenetics delivers the first detailed cytogenetic analyses of three Atlantic flying fish species, revealing a surprising degree of chromosomal reorganization that hints at cryptic lineages and incipient speciation in the vast epipelagic zone, the sunlit surface layer of the ocean that constitutes the largest ecosystem on Earth.

The research team examined Hirundichthys affinis, the fourwing flyingfish, from two Atlantic populations, one at the São Pedro and São Paulo Archipelago, a remote set of islets in the central Atlantic, and another at Caiçara do Norte on the northeastern coast of Brazil. They also analyzed two species of the genus Cheilopogon, C. exsiliens, the bandwing flyingfish, and C. furcatus, the spotfin flyingfish, both sampled at the archipelago. Together, these species span a family of roughly 80 species distributed across the Pacific, Indian, and Atlantic Oceans, animals that serve simultaneously as an economically valuable fishery resource, as the basis of prized roe, and as a critical trophic link converting energy from phytoplankton into biomass consumed by marine mammals, seabirds, and commercially important predatory fish.

The cytogenetic toolkit deployed in the study combined classic and molecular techniques. The researchers prepared mitotic chromosomes from kidney cells, stained them with Giemsa, and applied C-banding to reveal heterochromatin, the tightly packed, largely repetitive DNA concentrated around centromeres. Silver nitrate staining, known as Ag-NOR, highlighted active ribosomal gene clusters, while fluorescent in situ hybridization, or FISH, allowed the team to physically map three classes of repetitive sequences onto the chromosomes: the 18S and 5S ribosomal DNA genes and a (CA)15 dinucleotide microsatellite probe, each tagged with differently colored fluorophores so that their locations could be visualized under an epifluorescence microscope.

The headline result is a study in contrast. All three species share the same diploid number of 48 chromosomes, a count considered ancestral across the Percomorpha, the huge lineage of spiny-rayed fishes to which flying fish belong. However, the architecture of those chromosomes diverges in telling ways. Hirundichthys affinis carries an exclusively acrocentric karyotype, meaning every chromosome has its centromere at one end, yielding a fundamental number of 48 chromosome arms. Cheilopogon exsiliens and C. furcatus each possess one pair of bi-armed chromosomes, subtelocentric in the former and metacentric in the latter, raising their fundamental numbers to 50. That single structural difference between the two Cheilopogon species likely arose through pericentric inversions, a recognized engine of karyotypic diversification in marine percomorph fishes, or conceivably through centromere repositioning, a mechanism still poorly understood in fish evolution.

The most striking findings concern the ribosomal DNA markers. In all species, the 18S rDNA and Ag-NOR signals occupied a single locus, but that locus sat on entirely different chromosomes in each species and, remarkably, in each population of H. affinis. Fish from the Brazilian coast carried their 18S rDNA on the long arm of the large acrocentric pair 2, while individuals from the mid-Atlantic archipelago carried it on the short arm of the much smaller pair 21. The 5S rDNA showed parallel divergence: one site on pair 22 in the Brazilian population versus two sites, on pairs 18 and 22, in the archipelago population. Such a magnitude of rDNA variation between populations is unusual in marine fishes and can only be explained by a complex sequence of chromosomal events, including transposition and translocation.

Because H. affinis already shows documented population structure across the central western Atlantic, with earlier studies distinguishing at least three unit stocks among the eastern Caribbean, the southern Netherlands Antilles, and northeastern Brazil, the cytogenetic differences between the two sampled populations point to a genuinely complex scenario of transatlantic chromosomal diversification. The authors argue that the pronounced divergence in ribosomal gene organization raises the possibility of cryptic speciation, the emergence of reproductively isolated lineages that remain morphologically indistinguishable. Confirming that possibility will require molecular taxonomic approaches, but the chromosomal data now provide a clear set of candidate markers for detecting such hidden diversity in a species with vast interoceanic ranges and enormous population sizes.

The study also mapped the distribution of heterochromatin and microsatellite repeats, adding further resolution to the species comparisons. Heterochromatin in all three species was concentrated in centromeric and pericentromeric regions, but each species and population carried diagnostic additional blocks: extra telomeric segments on specific chromosome pairs in the Brazilian H. affinis, an interstitial block on pair 3 in the archipelago population, combined centromeric and telomeric staining on the first pair of C. exsiliens, and a terminal block on the first pair of C. furcatus that coincides with the Ag-NOR site. The (CA)15 microsatellite repeats, by contrast, showed a predominantly diffuse distribution along the chromosome arms, occasionally clustering in pericentromeric regions, a pattern consistent with reports from many other fish species and apparently a common genomic feature of the group.

The broader evolutionary picture that emerges challenges expectations for a pelagic fish family. Marine biologists have often noted an inverse relationship between geographic range and karyotypic variation in oceanic species: animals capable of widespread dispersal, and therefore extensive gene flow, tend to resist the fixation of structural chromosomal rearrangements, whereas reef fishes with fragmented populations accumulate them readily. Flying fish, despite their transoceanic distributions, large population sizes, and conserved diploid number, display conspicuous divergences in rDNA organization and chromosome structure at the population, species, and genus levels. The genus Cheilopogon is polyphyletic, with C. exsiliens and C. furcatus belonging to separate evolutionary clades, which may account for the independent chromosomal rearrangements each lineage has undergone, including possible homeologous divergence of the Ag-NOR-bearing chromosome pair through loss or gain of a major ribosomal site.

The authors conclude that karyotypic evolution in flying fish is more dynamic than expected for a transoceanic pelagic group, and that cytogenetics offers a promising model for investigating speciation, reproductive isolation, and environmental adaptation in the epipelagic realm. Given the ecological centrality of flying fish to pelagic food webs and the commercial importance of their fisheries, the ability to detect cryptic lineages and stock boundaries through chromosomal markers carries practical implications for management and conservation. The study fills a long-standing gap in the cytogenetic record of Exocoetidae and positions these charismatic gliders as a valuable evolutionary model for one of the planet’s least understood habitats, where the interplay of gene flow, population structure, and chromosomal change continues to unfold across thousands of kilometers of open water.

Subject of Research: Cytogenomic and cytotaxonomic analysis of Atlantic flying fish species to investigate chromosomal evolution, population structure, and speciation in the epipelagic zone.

Article Title: Flying fish as useful model for evolutionary investigations in epipelagic zones. A cytogenomic and cytotaxonomic approach

Article References: Costa, G. W. W. F. D., Shibatta, O. A., Cioffi, M. D. B., Vidal, J. A. D., Bertollo, L. A. C., & Molina, W. F. (2026). Flying fish as useful model for evolutionary investigations in epipelagic zones. A cytogenomic and cytotaxonomic approach. Comparative Cytogenetics, 20, 141-155. https://doi.org/10.3897/compcytogen.20.190559

Image Credits: AI Generated

DOI: 10.3897/compcytogen.20.190559

Keywords: flying fish, Exocoetidae, cytogenetics, karyotype, rDNA, FISH, heterochromatin, microsatellites, speciation, epipelagic zone, population structure, Atlantic Ocean