Linking Comparative Genomics, Morphology and In Vitro Assays to Understand Deep Diving in Cetaceans

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Some cetaceans are capable of diving more than three kilometres below the ocean's surface (Baird 2005). Such deep diving sparks scientific curiosity because it requires remarkable behavioural and physiological adaptations to withstand asphyxia and high hydrostatic pressure. While diving physiology has long been studied at the organ and whole organism levels, particularly in pinnipeds, less is understood at the molecular level. This knowledge gap is important because some physiological and biochemical contributors to diving responses are likely conserved among all vertebrates (e.g. bradycardia as a plesiomorphic character among mammals (Hochachka and Somero 2002)). Studying the molecular underpinnings of diving in deep‐diving cetaceans, therefore, can help us understand the mechanisms of and potential for evolutionary change in complex traits across mammals. Thankfully, increasingly available high‐quality annotated whole genomes enable comparative work that integrates evolutionary, physiological and biochemical perspectives in elusive species. In a From the Cover article in Molecular Ecology, Zhang et al. (2025) performed a deep dive on the genomics of cetacean super‐divers, defined as species that remain deeper than 1,000 metres for at least one hour at a time. They identified morphological correlates of dive depth and selective pressures, analysed amino acid substitutions and performed in vitro tests of two key genes related to lung function and oxidative stress (Figure 1). Supporting analyses included tests for fatty acid content in blubber, in vitro gene expression changes from site‐directed mutagenesis of one regulator and in vitro uric acid concentration changes after site‐directed mutagenesis in an enzyme that generates superoxide anions under hypoxia. This battery of analyses was performed in the context of responses to water pressure and oxygen availability. The authors’ findings demonstrated protective molecular changes in cetacean super‐divers, in a substantial step towards a comprehensive understanding of diving biology.

FIGURE 1.

Conceptual diagram of diving animals, visualising the approximate deepest recorded dive depths with respect to the Eiffel Tower (Paris, France) at 330 m tall. The Dwarf sperm whale ( Kogia sima ) was the deepest diving cetacean in the dataset of Zhang et al. (2025), while the other species were not in the study. Note that while depth markers are to scale, animal sizes are not to scale and are for visualisation only. The HMS Beagle (~16 m tall) is shown at the top, while the overall approaches and results used in Zhang et al. (2025) are summarised to the right. Illustration by Evelien de Greef.

The results of this work are broadly divided into three categories: morphological and physiological correlates of diving capability, responses to physical water conditions and responses to a shift in oxygen availability. Of the morphological variables, brain mass was positively correlated with dive time, while relative heart mass was negatively correlated with dive time. Surprisingly, body size was not significantly correlated with dive time (p = 0.056), but the authors attributed the result to a limited sample size of n = 23 for that particular analysis. Muscle myoglobin, haemoglobin, blubber thickness and relative total lung capacity were significantly correlated with dive time and depth. These findings connect themes of oxygen availability and responses to hydrostatic pressure, which were then explored at the molecular level.

Using a comparative genomics framework, the authors identified 5810 one‐to‐one gene orthologs, sometimes called single‐copy orthologs, and defined rapidly evolving genes, positively selected genes and amino acid substitutions in three approaches. Positive selection was defined as genes showing d _N/d _S values > 1 in super‐divers versus others from a branch‐site model in the popular comparative genomics software PAML (Yang 2007), while rapidly evolving genes were defined from branch models where d _N/d _S was compared between the super‐divers and other cetaceans. Therefore, the genes defined as rapidly evolving in this work exhibited higher d _N/d _S in the super‐divers than in other cetaceans, but d _N/d _S itself could have been < 1. As a third computational approach, genes with amino acid substitutions unique to super‐divers versus other cetaceans were identified, followed by protein structure prediction to visualise the models. Molecular results were categorised into those that support either hydrostatic pressure or oxygen availability responses, based on their functional annotations.

Genes related to cholesterol regulation, outer blubber fatty acid composition and lung integrity were linked to hydrostatic pressure. Notably, amino acid changes in ELOV1 elongate long‐chain fatty acids, and changes in ECHS1 degrade valine for fatty acid synthesis. The outer layer of deep diving cetacean blubber is rigid, with a possible protective function for immense undersea pressures (Koopman 2018); therefore, these amino acid changes might be linked to wax esters supporting this tough outer blubber. A tool for human phenotype and disease genomics, Gene Organiser, was used to link 73 genes to functions in lungs. The gene ATP8B1 had a unique amino acid substitution in super‐divers, which was an exciting result because this gene is involved in surfactant buildup, and a mouse model with an amino acid change in this gene develops lung fibrosis (Westermann‐Clark et al. 2022). The authors overexpressed ATP8B1 in a human alveolar cell system in vitro, finding no difference in gene expression under normoxia, while under hypoxia, the sperm whale version of the gene caused decreased expression of several others, including injury‐mediating interleukins 6 and 8. To experimentally verify these findings, the authors then used site‐directed mutagenesis on the bottlenose dolphin ATP8B1 to match the sperm whale residue and found the same modulation of expression patterns. Therefore, ATP8B1, specifically residue 380 within it, may play a role in lung tissue function in diving cetaceans.

Muscle myoglobin and blood haemoglobin levels were correlated with dive capability, and consistent with a molecular response to oxygen availability. Blood pressure regulation is likely under selection in cetacean super‐divers, such as in the gene ACE, which was found to be under both positive selection and rapid evolution. Other genes in the renin‐angiotensin‐aldosterone system had amino acid substitutions specific to super‐divers, providing additional evidence that blood pressure is under evolutionary pressure from deep diving. One enzyme of interest for the authors was xanthine oxidoreductase (XOR), which under hypoxia converts to an XO form that generates dangerous superoxide anions (O₂ ⁻) (Battelli et al. 2016). They identify positive selection and amino acid substitutions specific to super‐divers that were consistent with lower uric acid levels in sperm whales after extended hypoxia. Site‐directed mutagenesis from the sperm whale form of XO to the bottlenose dolphin form led to an increased in vitro uric acid levels under hypoxia. Therefore, oxygen availability during diving may have influenced the evolution of oxidative stress responses in cetaceans.

This work demonstrates the power of combining in vitro and in silico approaches to study molecular ecology. Tagging, telemetry and biomonitoring technologies have helped researchers understand the physiology and behaviour of cetaceans in self‐directed dives, but molecular responses are not feasibly studied with such approaches. The in vitro techniques demonstrated here, such as site‐directed mutagenesis to experimentally confirm hypotheses about molecular pathways, will be key for experimentally validating hypotheses generated from comparative genomics while providing an opportunity to study the biochemistry and molecular physiology of genomic interactions.

Deep‐diving marine mammals are especially interesting systems because they have adapted to deep waters after transitioning from terrestrial to marine lifestyles. The genomic changes supporting diving physiology are thus ‘newer’ in evolutionary terms than adaptations supporting the initial marine transition, and potentially shared in the sense that all mammals share the underlying physiological toolkit for diving responses (Hochachka and Somero 2002). Future work will undoubtedly have access to more genomes, in addition to being able to study parallel molecular changes in other diving vertebrates. Structural genomic changes may underlie deep‐diver physiology as well, and highly contiguous reference genomes may add important context for how rearrangements and duplications interact with nucleotide and amino acid‐level changes studied here. Given that diving responses involve multiple interacting traits, the basic components of the trait are shared among mammals, and diving has evolved in multiple mammal lineages, comprehensive knowledge of diving biology could be a powerful guide for understanding the evolution of complex traits in living organisms.

Conflicts of Interest

The author declares they have no conflicts of interest.

Linked Articles

This article is linked to Zhang et al. paper. To view this article, visit https://doi.org/10.1111/mec.17678.

Data Availability Statement

Data is sharing not applicable to this article as no datasets were generated or during the current study.