Gene Duplication and Loss of AANAT in Mammals Driven by Rhythmic Adaptations.

Arylalkylamine N-acetyltransferase (AANAT) plays a crucial role in synchronizing internal biological functions to circadian and circannual changes. Generally speaking, only one copy of AANAT gene has been found in mammals, however, three independent duplications of this gene were detected in several cetartiodactyl lineages (i.e., Suidae, Hippopotamidae, and Pecora), which originated in the middle Eocene, a geological period characterized with the increased climate seasonality. Lineage-specific expansions of AANAT and the associated functional enhancement in these lineages strongly suggest an improvement in regulating photoperiodic response to adapt to seasonal climate changes. In contrast, independent inactivating mutations or deletions of the AANAT locus were identified in the four pineal-deficient clades (cetaceans, sirenians, xenarthrans, and pangolins). Loss of AANAT function in cetaceans and sirenians could disrupt the sleep-promoting effects of pineal melatonin, which might contribute to increasing wakefulness, adapting these clades to underwater sleep. The absence of AANAT and pineal glands in xenarthrans and pangolins may be associated with their body temperature maintenance. The present work demonstrates a far more complex and intriguing evolutionary pattern and functional diversity of mammalian AANAT genes than previously thought and provides further evidence for understanding AANAT evolution as driven by rhythmic adaptations in mammals.

Insights into body size variation in cetaceans from the evolution of body-size-related genes.

BACKGROUND: Cetaceans exhibit an exceptionally wide range of body size, yet in this regard, their genetic basis remains poorly explored. In this study, 20 body-size-related genes for which duplication, mutation, or deficiency can cause body size change in mammals were chosen to preliminarily investigate the evolutionary mechanisms underlying the dramatic body size variation in cetaceans. RESULTS: We successfully sequenced 20 body-size-related genes in six representative species of cetaceans. A total of 46 codons from 10 genes were detected and determined to be under strong positive selection, 32 (69.6%) of which were further found to be under radical physiochemical changes; moreover, some of these sites were localized in or near important functional regions. Interestingly, positively selected genes were well matched with body size evolution: for small cetaceans, strong evidence of positive selection was detected at ACAN, OBSL1, and GRB10, within which mutations or duplications could cause short stature; positive selection was found in large cetaceans at CBS and EIF2AK3, which could promote growth, and at the PLOD1 gene, within which mutations could cause tall stature. Importantly, relationship analyses revealed that the evolutionary rate of CBS was positively related to body length and body mass with statistical significance. Additionally, we identified 32 cetacean-specific amino acid changes in 10 genes. CONCLUSIONS: This is the first study to investigate the molecular basis of dramatic body size variation in cetaceans. Our results provide evidence of the positive selection of several body-size-related genes in cetaceans, as well as divergent selection between large or small cetaceans, which suggest cetacean body size variation possibly associated with these genes. In addition, cetacean-specific amino acid changes might have played key roles in body size evolution after the divergence of cetaceans from their terrestrial relatives. Overall, the evolutionary pattern of these body-size-related genes could provide new insights into genetic mechanisms for the body size variation in cetaceans.

Adaptive changes in BMAL2 with increased locomotion associated with the evolution of unihemispheric slow-wave sleep in mammals.

Marine mammals, especially cetaceans, have evolved a very special form of sleep characterized by unihemispheric slow-wave sleep (USWS) and a negligible amount or complete absence of rapid-eye-movement sleep; however, the underlying genetic mechanisms remain unclear. Here, we detected unique, significant selection signatures in basic helix-loop-helix ARNT like 2 (BMAL2; also called ARNTL2), a key circadian regulator, in marine mammal lineages, and identified two nonsynonymous amino acid substitutions (K204E and K346Q) in the important PER-ARNT-SIM domain of cetacean BMAL2 via sequence comparison with other mammals. In vitro assays revealed that these cetacean-specific mutations specifically enhanced the response to E-box-like enhancer and consequently promoted the transcriptional activation of PER2, which is closely linked to sleep regulation. The increased PER2 expression, which was further confirmed both in vitro and in vivo, is beneficial for allowing cetaceans to maintain continuous movement and alertness during sleep. Concordantly, the locomotor activities of zebrafish overexpressing the cetacean-specific mutant bmal2 were significantly higher than the zebrafish overexpressing the wild-type gene. Subsequently, transcriptome analyses revealed that cetacean-specific mutations caused the upregulation of arousal-related genes and the downregulation of several sleep-promoting genes, which is consistent with the need to maintain hemispheric arousal during USWS. Our findings suggest a potential close relationship between adaptive changes in BMAL2 and the remarkable adaptation of USWS and may provide novel insights into the genetic basis of the evolution of animal sleep.

Genomic insights into adaptation to bipedal saltation and desert-like habitats of jerboas.

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.

ACPT gene is inactivated in mammalian lineages that lack enamel or teeth.

Loss of tooth or enamel is widespread in multiple mammal lineages. Although several studies have been reported, the evolutionary mechanisms of tooth/enamel loss are still unclear. Most previous studies have found that some tooth-related genes have been inactivated in toothless and/or enamel-less mammals, such as ENAM, ODAM, C4orf26, AMBN, AMTN, DSPP, etc. Here, we conducted evolutionary analyses on ACPT playing a key role in amelogenesis, to interrogate the mechanisms. We obtained the ACPT sequences from 116 species, including edentulous and enamel-less mammals. The results shows that variant ORF-disrupting mutations were detected in ACPT coding region among nine edentulous baleen whales and three enamel-less taxa (pygmy sperm whale, aardvark, nine-banded armadillo). Furtherly, selective pressure uncovered that the selective constraints have been relaxed among all toothless and enamel-less lineages. Moreover, our results support the hypothesis that mineralized teeth were lost or degenerated in the common ancestor of crown Mysticeti through two shared single-base sites deletion in exon 4 and 5 of ACPT among all living baleen whales. D N/d S values on transitional branches were used to estimate ACPT inactivation records. In the case of aardvark, inactivation of ACPT was estimated at ~23.60-28.32 Ma, which is earlier than oldest aardvark fossil record (Orycteropus minutus, ~19 Ma), suggesting that ACPT inactivation may result in degeneration or loss of enamel. Conversely, the inactivation time of ACPT estimated in armadillo (~10.18-11.30 Ma) is later than oldest fossil record, suggesting that inactivation of ACPT may result from degeneration or loss of enamel in these mammals. Our findings suggested that different mechanisms of degeneration of tooth/enamel might exist among toothless and enamel-less lineages during evolution. Our study further considered that ACPT is a novel gene for studying tooth evolution.

Adaptive Evolution of Energy Metabolism-Related Genes in Hypoxia-Tolerant Mammals.

Animals that are able to sustain life under hypoxic conditions have long captured the imagination of biologists and medical practitioners alike. Although the associated morphological modifications have been extensively described, the mechanisms underlying the evolution of hypoxia tolerance are not well understood. To provide such insights, we investigated genes in four major energy metabolism pathways, and provide evidence of distinct evolutionary paths to mammalian hypoxia-tolerance. Positive selection of genes in the oxidative phosphorylation pathway mainly occurred in terrestrial hypoxia-tolerant species; possible adaptations to chronically hypoxic environments. The strongest candidate for positive selection along cetacean lineages was the citrate cycle signaling pathway, suggestive of enhanced aerobic metabolism during and after a dive. Six genes with cetacean-specific amino acid changes are rate-limiting enzymes involved in the gluconeogenesis pathway, which would be expected to enhance the lactate removal after diving. Intriguingly, 38 parallel amino acid substitutions in 29 genes were observed between hypoxia-tolerant mammals. Of these, 76.3% were radical amino acid changes, suggesting that convergent molecular evolution drives the adaptation to hypoxic stress and similar phenotypic changes. This study provides further insights into life under low oxygen conditions and the evolutionary trajectories of hypoxia-tolerant species.