Shixia Xu

Circadian rhythms are fundamental biological processes that enhance survival and reproductive fitness by coordinating physiology and behavior with predictable environmental cycles. However, in some species, circadian rhythms are weakened, irregular, or even undetectable, likely reflecting evolutionary responses to extreme environments or specialized ecological niches. Nevertheless, the genetic basis of these atypical phenotypes remains incompletely understood. Here, we investigated the molecular evolution of 20 PER2‐associated genes across 71 representative mammalian species. We identified four genes under positive selection in mammalian lineages exhibiting atypical circadian phenotypes, three of which ( CDK5 , CSNK1E , and GSK3B ) are involved in the post‐translational regulation of PER2 phosphorylation, suggesting that PER2 phosphorylation regulators may contribute to lineage‐specific circadian variation. Notably, CSNK1E, a key kinase regulating PER2 phosphorylation, showed signatures of positive selection and accelerated evolution in cetaceans, which exhibit unihemispheric slow‐wave sleep (USWS). In vitro assays showed that dolphin CSNK1E increased phosphorylation at the stabilizing FASP site S662 of PER2 and extended the half‐life of the PER2 protein, a molecular feature closely linked to circadian oscillator amplitude and sleep regulation. Increased PER2 phosphorylation may contribute to altered sleep and locomotor activity associated with sustained wakefulness during USWS, as supported by our cellular and whole‐animal assays. Consistently, zebrafish overexpressing dolphin CSNK1E exhibited elevated locomotor activity and reduced sleep duration. Overall, our findings provide evidence that evolutionary changes in PER2 phosphorylation regulation are associated with atypical circadian phenotypes and offer novel insights into the genetic mechanisms underlying circadian plasticity in mammals.

The circadian rhythm is a fundamental adaptation that enhances survival and reproductive fitness by coordinating physiology and behavior with predictable environmental cycles. However, in some species, circadian rhythms are weakened, irregular or even absent, likely reflecting adaptations to extreme environments or specialized ecological niches, yet the genetic basis of these atypical phenotypes remains incompletely understood. Here, we investigated the molecular evolution of 20 PER2-asccociated genes across 71 representative mammalian species. We identified four genes under positive selection in atypical circadian lineages, three of which (CDK5, CSNK1E, and GSK3B) participate in PER2 post-translational phosphorylation, suggesting PER2 phosphorylation may be involved in the evolution of atypical circadian rhythms. Notably, CSNK1E, a key kinase for PER2 phosphorylation, showed signatures of positive selection and accelerated evolution in cetaceans, which exhibit unihemispheric slow-wave sleep (USWS). In vitro assays showed that dolphin CSNK1E increases PER2 phosphorylation at the stabilizing FASP site S662, extents the half-life of PER2 protein, which is closely linked to oscillator amplitude and sleep regulation. The increased PER2 phosphorylation, which was further demonstrated by our cellar and whole animal assays, is beneficial for allowing cetacean to maintain sustained wakefulness and movement during unihemispheric slow-wave sleep (USWS). Concordantly, zebrafish overexpressing dolphin CSNK1E exhibited elevated locomotor activity and reduced sleep duration. Overall, our findings demonstrate that adaptive changes in PER2 phosphorylation regulation contribute to the emergence of atypical circadian rhythms, providing novel insights into the genetic mechanisms underlying circadian plasticity in mammals.

Gene loss represents a powerful driver of adaptive evolution. Cetaceans, which underwent a profound transition from terrestrial to fully aquatic life, provide an excellent model for investigating this process. Comparative genomic analysis of the cold-sensitive ion channel transient receptor potential ankyrin 1 ( TRPA1) revealed lineage-specific patterns of degeneration across cetaceans. In toothed whales, the ancestral lineage exhibited extensive exon loss within the TRPA1 locus, whereas baleen whales showed signatures consistent with markedly reduced or absent TRPA1 expression. These molecular alterations were inferred to disrupt or abolish TRPA1 protein function across cetaceans. Integration of these findings with established experimental evidence from human and murine TRPA1 studies supported several adaptive hypotheses for TRPA1 gene loss, including tolerance to abrupt thermal fluctuations, attenuation of nociceptive responses in aquatic environments, specialization of integumentary sensory systems, and the emergence of echolocation-associated sensory trade-offs in toothed whales. Collectively, these findings expand the gene loss repertoire of cetaceans and provide novel insights into the molecular underpinnings of secondary aquatic adaptation in mammals.

The testicular descent leading to the exteriorization of male gonads is a complex sexual dimorphic process observed in numerous mammals, whereas some species, such as elephants and cetaceans, retain the lifelong ascrotal testes. Despite the implication of Wnt signaling in testicular development and descent, the evolutionary mechanisms underlying ascrotal testes have not been adequately addressed. Here, we examined selection signatures and unique amino acid substitutions in genes of the Wnt signaling pathway. We identified an ascrotal mammal-specific substitution (S406G) in zinc and ring finger 3 (ZNRF3) located within the disheveled (DVL)-interaction region. Functional assays showed that this substitution enhances the affinity of ascrotal ZNRF3 for DVLs and suppresses Wnt3a-induced Wnt signaling. Comparative transcriptomics of the gubernaculum between male fetal rats from wild-type and spontaneous cryptorchid (orl) strains revealed upregulation of Ctnnb1 and Gsk3b in orl rats, along with ascrotal-specific evolutionary changes in regulatory elements. Collectively, these findings suggest that Wnt signaling may be dampened in ascrotal mammals. This study provides insights into the pathogenesis of cryptorchidism in humans and domestic animals.

Limb morphology is particularly important for animals to inhabit different environments. Limb modifications (e.g., flipper-like forelimbs and hindlimb regression) are among the most critical secondary aquatic adaptation mechanisms enabling cetaceans to fully adapt to an aquatic environment. Exploring the molecular mechanisms underlying limb evolution in cetaceans has attracted considerable attention from evolutionary biologists. In the present study, conserved non-coding elements (CNEs) closely associated with limb development, which exhibited lineage-specific sequence divergence (nucleotide mutations and indels) in cetaceans, were identified using comparative genomics. These sequence divergences might have led to the loss of binding motifs for transcription factors involved in limb development and significant alterations in autoregulatory activity. A transgenic mouse was constructed to carry a cetacean-specific enhancer (i.e., hs1586), which exhibited a significant phenotypic difference in forelimb buds at embryonic day (E)10.5, supported by transcriptomic and epigenomic evidence. However, the phenotypic recovery after E11.5 suggested that enhancer redundancy in the mouse genome may have compensated for the effects caused by the incorporation of cetacean hs1586. This further suggests that the complex phenotypic changes of limbs in cetaceans are likely not driven by a single CNE but rather involve multiple CNEs and/or genes. In summary, our study supports the functional role of CNE sequence divergence and the complex mechanisms underlying limb morphology changes in cetaceans.

The evolution of limb morphology plays an important role in animal adaptation to different ecological niches. To fully adapt to aquatic life, cetaceans underwent hindlimb degeneration and forelimb transformed into flipper; however, the molecular mechanisms underlying the limb changes in cetaceans remain unclear. We previous study had shown that the Tbx4 hindlimb enhancer A (Tbx4-HLEA) in cetaceans exhibited specific deletions and nucleotide substitutions, with significantly reduced regulatory activity. To further investigate whether cetacean HLEA has a potential impact on hindlimb development in vivo, a knock-in mouse model was generated by knocking in the homologous cetacean HLEA in the present study. Phenotypic analysis showed a significant reduction in hindlimb bud development in homozygous knock-in mice at embryonic day (E)10.5; however, the phenotypic difference was rescued after E11.5. Transcriptomic and epigenetic analyses indicated that the cetacean HLEA acts as an enhancer in the mouse embryos and significantly reduces the transcriptional expression levels of Tbx4 at E10.5, supporting that downregulation of cetaceans HLEA regulatory activity reduces the expression of Tbx4. Additionally, both the number of activated non-coding elements and chromatin accessibility near Tbx4 were increased in homozygous knock-in mice at E11.5. The functional redundancy of enhancers compensated for the functional defect of cetacean HLEA, rescuing the expression level of Tbx4, and may account for the phenotype restoration after E11.5. In conclusion, our study suggested that the evolution of cetacean HLEA may be an important link with relevant molecular mechanism for the hindlimb degeneration.

Protamines (PRMs) and transition nuclear proteins (TNPs) are two key classes of sperm nuclear basic proteins that regulate chromatin reorganization and condensation in the spermatozoon head, playing crucial roles in mammalian spermatogenesis. In scrotal mammals, such as humans, cryptorchidism, the failure of the testes to descend into the scrotal sac is generally associated with higher rates of defective spermatozoon quality and function. However, ascrotal mammals, such as cetaceans, with naturally undescended testes, produce normal spermatozoa similar to their scrotal counterparts. This study investigates the evolutionary pattern and functional changes in PRMs and TNPs to explore the potential molecular mechanisms underlying spermatogenesis in naturally ascrotal mammals. Although we found a conserved genomic arrangement for PRM and TNP genes across mammals, the coevolutionary loss of intact PRM2 and TNP2 was observed in several species, correlating significantly with diverse testicular positions. Notably, in cetaceans, which lack intact PRM2 and TNP2, we detected enhanced thermostability and DNA binding in PRM1, along with superior DNA repair capability in TNP1. These findings suggest that gene loss of PRM2 and TNP2, combined with functional enhancements in PRM1 and TNP1 proteins, evolved in response to physiological challenges posed by natural cryptorchidism in most ascrotal lineages. This evolutionary strategy enhances chromatin condensation efficiency and promotes DNA repair during spermatogenesis in natural cryptorchid mammals, supporting the Black Queen Hypothesis.

Dichromatic color vision is mediated by two cone visual pigments in many eutherian mammals. After reentry into the sea, early cetaceans lost their violet-sensitive visual pigment (short wavelength-sensitive 1) independently in the baleen and toothed whale ancestors and thus obtained only monochromatic cone vision. Subsequently, losses of the middle/long wavelength-sensitive (M/LWS) pigment have also been reported in multiple whale lineages, leading to rhodopsin (RH1)-mediated rod monochromatic vision. To further elucidate the phenotypic evolution of whale visual pigments, we assessed the spectral tuning of both M/LWS and RH1 from representative cetacean taxa. Interestingly, although the coding sequences for M/LWS are intact in both the pygmy right whale and the Baird's beaked whale, no spectral sensitivity was detected in vitro. Pseudogenization of other cone vision-related genes is observed in the pygmy right whale, suggesting a loss of cone-mediated vision. After ancestral sequence reconstructions, ancient M/LWS pigments from cetacean ancestors were resurrected and functionally measured. Spectral tuning of M/LWS from the baleen whale ancestor shows that it is green sensitive, with a 40-nm shift in sensitivity to a shorter wavelength. For the ancestor of sperm whales, although no spectral sensitivity could be recorded for its M/LWS pigment, a substantial sensitivity shift (20 to 30 nm) to a shorter wavelength may have also occurred before its functional inactivation. The parallel phenotypic evolution of M/LWS to shorter wavelength sensitivity might be visual adaptations in whales allowing more frequent deep-sea activities, although additional ecological differentiations may have led to their subsequent losses.

Jerboas is a lineage of small rodents displaying atypical mouse-like morphology with elongated strong hindlimbs and short forelimbs. They have evolved obligate bipedal saltation and acute senses, and been well-adapted to vast desert-like habitats. Using a newly sequenced chromosome-scale genome of the Mongolian five-toed jerboa (Orientallactaga sibirica), our comparative genomic analyses and in vitro functional assays showed that the genetic innovations in both protein-coding and non-coding regions played an important role in jerboa morphological and physiological adaptation. Jerboa-specific amino acid substitutions, and segment insertions/deletions (indels) in conserved non-coding elements (CNEs) were found in components of proteoglycan biosynthesis pathway (XYLT1 and CHSY1), which plays an important role in limb development. Meanwhile, we found specific evolutionary changes functionally associated with energy or water metabolism (e.g., specific amino acid substitutions in ND5 and indels in CNEs physically near ROR2) and senses (e.g., expansion of vomeronasal receptors and the FAM136A gene family) in jerboas. Further dual-luciferase reporter assay verified that some of the CNEs with jerboa-specific segment indels exerted a significantly different influence on luciferase activity, suggesting changes in their regulatory function in jerboas. Our results revealed the potential molecular mechanisms underlying jerboa adaptation since the divergence from the Eocene-Oligocene transition, and provided more resources and new insights to enhance our understanding of the molecular basis underlying the phenotypic diversity and the environmental adaptation of mammals.

Photic niche shifts of mammals are associated with changing visual capabilities, primarily mediated by three visual pigments, two (SWS1 and M/LWS) of them for color vision and rhodopsin (RH1) for dim-light vision. To further elucidate molecular mechanisms of mammalian visual adaptations to different light environments, a systematic study incorporating evolutionary analyses across diverse groups and in vitro assays have been carried out. Here, we collected gene sequences for the three opsins from 220 species covering all major mammalian clades. After screening for cone opsin gene losses, we estimated selective pressures on each of the three genes and compared the levels of selection experienced by species living in bright- and dim-light environments. SWS1 pigment is shown to experience accelerated evolution in species living in bright-light environments as has RH1 in aquatic cetaceans, indicating potential shifts for ecological adaptations. To further elucidate the functional mechanisms for these two pigments, we then carried out site-directed mutagenesis in representative taxa. For SWS1, violet and ultraviolet sensitivities in the pika and mouse are mainly affected by substitutions at the critical sites 86 and 93, which have strong epistatic interaction. For RH1, the phenotypic difference between the sperm whale and bovine sequences is largely contributed by a substitution at site 195, which could be critical for dim-light sensation for deep-diving species. Different evolutionary patterns for the visual pigments have been identified in mammals, which correspond to photic niches, although additional phenotypic assays are still required to fully explain the functional mechanisms.