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.

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. DN/dS 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.

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) that 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.

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. Electronic supplementary material The online version of this article (10.1186/s12862-019-1461-9) contains supplementary material, which is available to authorized users.

1Loss of tooth or enamel is widespread in multiple mammal lineages. Although several studies 2 have been reported, the evolutionary mechanisms of tooth / enamel loss are still unclear. 3Most previous studies have found that some tooth-related genes have been inactivated in 4 toothless and / or enamel-less mammals, such as ENAM, ODAM, C4orf26, AMBN, AMTN, 5 DSPP, etc. Here, we conducted evolutionary analyses on ACPT plays a key role in 6 amelogenesis, to interrogate the mechanisms. We obtained the ACPT sequences from 116 7 species, including edentulous and enamel-less mammals, then evolutionary analyses were 8 implemented. The results showed that variant ORF-disrupting mutations have been detected 9 in ACPT coding region among nine edentulous baleen whales and three enamel-less taxa 2 2 among toothless and enamel-less lineages during evolution. Our study further considered that 2 3ACPT is a novel gene for studying tooth evolution. 2 4

The oxidative phosphorylation (OXPHOS) pathway is an efficient way to produce energy via adenosine triphosphate (ATP), which is critical for sustaining an energy supply for cetaceans in a hypoxic environment. Several studies have shown that natural selection may shape the evolution of the genes involved in OXPHOS. However, how network architecture drives OXPHOS protein sequence evolution remains poorly explored. Here, we investigated the evolutionary patterns of genes in the OXPHOS pathway across six cetacean genomes within the framework of a functional network. Our results show a negative correlation between the strength of purifying selection and pathway position. This result indicates that downstream genes were subjected to stronger evolutionary constraints than upstream genes, which may be due to the dual function of ATP synthase in the OXPHOS pathway. Additionally, there was a positive correlation between codon usage bias and omega (ω = dN/dS) and a negative correlation with synonymous substitution rate (dS), indicating that the stronger selective constraint on genes (with less biased codon usage) along the OXPHOS pathway is attributable to an increase in the rate of synonymous substitution. Surprisingly, there was no significant correlation between protein-protein interactions and the evolutionary estimates, implying that highly connected enzymes may not always show greater evolutionary constraints. Compared with that observed for terrestrial mammals, we found that the signature of positive selection detected in five genes (ATP5J, LHPP, PPA1, UQCRC1 and UQCRQ) was cetacean-specific, reflecting the importance of OXPHOS for survival in hypoxic, aquatic environments.