Convergent genomic signatures of high-altitude adaptation among six independently evolved mammals

doi:10.21203/rs.3.rs-2100857/v1

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Convergent genomic signatures of high-altitude adaptation among six independently evolved mammals

https://doi.org/10.21203/rs.3.rs-2100857/v1

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The species living in the Qinghai-Tibet Plateau provide an excellent model system for studying the relationship between molecular convergent evolution and adaptation. Distant species experiencing the same selection pressure (i.e., hypoxia, low temperature and strong ultraviolet radiation) are likely to evolve similar genetic adaptations independently. Here, we performed comparative genomics studies on six independently evolved high-altitude species. The results also showed that the convergent evolution of the six species was mainly reflected at the level of rapidly evolving genes, and the functions of these rapidly evolving genes were mainly related to hypoxia response and DNA damage repair. In addition, we found that high-altitude species had more gene family changes than their low-altitude relatives, except for the order Lagomorpha. The results also show that the convergence of gene family contraction of high-altitude species is much greater than that of expansion, revealing a possible pattern of species in adapting to high-altitude. Furthermore, we detected positive selection signature in four genes related to hypoxia response and ultraviolet radiation damage in these six species (FYCO1, ERBIN, SCAMP1 and CXCL10). Our study reveals that hypoxia response might play an important role in the adaptation of independently evolved species to high-altitude environment, providing a basic perspective for further exploring the high-altitude adaptation mechanism of different related species in the future.

comparative genomic

high-altitude

mammals

adaption

convergent evolution

As the highest plateau in the world, the average altitude of the Qinghai-Tibet Plateau exceeds 4000m. The harsh living environment of the Qinghai-Tibet Plateau exerts strong selection pressure (i.e., hypoxia, low temperature and strong ultraviolet radiation) on native species, which have driven striking phenotypic and genetic adaptations (Cai et al. 2013). A large number of high-altitude species has been explored since the whole genome sequencing was used to explore the high-altitude adaptation mechanism of Tibetans, which greatly enriched our understanding of the impact of high-altitude environment on the phenotype and genetic changes of species (Xu et al. 2011). For example, the genes showed positive selection signature of the Tibetan antelope were mainly associated with energy metabolism and oxygen transmission (Ge et al. 2013). The study on yak, another species belonging to the family Bovidae, showed that in addition to the positive selection signal detected in the genes related to hypoxia stress, the genes related to nutrition metabolism also changed significantly (Qiu et al. 2012). The genome of Tibetan fox, the landmark species on the Qinghai-Tibet Plateau, shows that VEGFA gene and MGME1 gene plays an important role in the adaptation mechanism of Tibetan fox to high-altitude (Lyu et al. 2022). The investigation of ectothermic snakes indicated that two genes (FEN1 and EPAS1) plays an important role in its response to hypoxia stress and DNA damage caused by ultraviolet radiation (Li et al. 2018). Independently evolved species might have different genetic alterations and mechanistic preferences in response to the same selection pressures. In addition to the well-known genes of hypoxia related HIF signaling pathway that can contribute species to cope with hypoxia stress, SLC25A14 gene and PTPN1 gene also play important roles in the hypoxia adaptation of the Himalayan marmot and Tibetan migratory locusts, respectively (Ding et al. 2018; Bai et al. 2019).

In addition to the individual adaptation mechanisms that species in different phylogenetic contexts develop when faced with the same selection pressure, the same selection pressure also leads to convergent evolution, including phenotypic convergence as well as genetic and functional convergence at the molecular level (Stern 2013; Houliez et al. 2021). The research on giant panda and red panda found that the common specialized bamboo diet led to the adaptive convergence of two genes (DYNC2H1 and PCNT) related to limb development (Hu et al. 2017). Not only could the same diet habits lead to the convergent evolution of different species, but also the same living habits could lead to the eco-morphological convergence of different species. Previous studies found that there was eco-morphological convergence in slow arboreal xenarthrans through the analysis of multiple omics of humeral and femoral internal structure (Alfieri et al. 2022). Independently evolving species might lead to convergent evolution of phenotypes in order to adapt to the same environment, but these convergent phenotypes do not necessarily result from convergent evolution at the molecular level. A comparative genomics study of three aquatic mammals (the killer whale, walrus, and manatee) have shown that although convergent phenotypic evolution can result from convergent molecular evolution, this is rare, and evolution more frequently utilizes different molecular pathways to achieve the same phenotypic outcome (Foote et al. 2015). Convergent evolution is not only reflected in the phenotypic convergence of species, but might also be reflected in the functional convergence at the molecular level. Previous analysis of the CDH23 gene and its ligand PCDH15 gene, as well as OTOF gene of neural signaling, found that these three genes had significantly convergent evolution in echolocation species (small bats and some whales), and CDH23 and PCDH15 genes had positive selection in echolocation species (Shen et al. 2012).

Up to now, a large number of studies have confirmed that the selection pressure exerted by the Qinghai-Tibet Plateau on indigenous species will also lead to the genetic changes of convergence at the molecular level. The genomes of Tibetan antelope showed that 247 positive selection genes related to hypoxia in Tibetan antelope and American pika had convergent evolution (Ge et al. 2013). Genomic analysis also identified six genes related to lung function, DNA repair, and angiogenesis in the high-altitude snub-nosed monkeys shared eight substitutions (Yu et al. 2016). The population genetic analysis showed that EPAS1 and HBB genes related to hypoxia adaptation have undergone significant convergent evolution in Tibetans and Tibetan Mastiffs (Wang et al. 2014). As the most well-known gene related to hypoxia response, EPAS1 gene also found positive selection signals of convergent evolution in six domestic animals living on the plateau (Wu et al. 2020). This study also reveals a fact, that is, the more species selected and the larger the genetic relationship span, the less evidence of convergent evolution at the molecular and genetic levels. Comparative transcriptomics is also applied to explore the convergent adaptation mechanism of species to high altitude. A previous comparative transcriptomic study detected a total of 218 convergent positive selection genes in three high-altitude passerine birds, but the substitution of amino acid sites of these genes was rarely convergent (Hao et al. 2019). Another comparative transcriptomic study of five high-altitude domestic animals and low-altitude related species showed that the differentially expressed genes at different altitudes might be related to the phenotypic differences caused by high-altitude adaptation (Tang et al. 2017). Although the convergent adaptation mechanism of a large number of high-altitude species has been explored, the coverage is still relatively limited, and most of them focus on the species with close relationship (same genus or family) or domesticated species. It is not clear whether the mammalian species that have evolved independently and adapted to the plateau environment for different times could produce convergent molecular level or functional level changes when coping with the same selection pressure (i.e., hypoxia, low temperature and strong ultraviolet radiation) brought by high-altitude.

In this study, we selected six representative mammals at the level of four orders, including Lagomorpha, Primates, Carnivora, and Artiodactyla, in order to compare these six mammals with their low-altitude relatives through the method of comparative genomics, and explored out their convergent evolution at the molecular or functional level when facing the selection pressure of high-altitude. This study provides a new perspective for the adaptation mechanism of mammals at high-altitude and expands the understanding of the impact of high-altitude on genetic changes in mammals.

Identification of orthologous gene set

In order to study the characteristics of high-altitude mammals to the maximum extent, the genomes of six high-altitude species (Rhinopithecus bieti, Pantholops hodgsonii, Ovis aries (high-altitude), Vulpes ferrilata, Ochotona curzoniae, and Bos mutus) and nine closely related low-altitude species (Canis lupus familiaris, Bos Taurus, Ovis aries (low-altitude), Capra hircus, Oryctolagus cuniculus, Bubalus bubalis, Trachypithecus francoisi, Vulpes lagopus, and Piliocolobus tephrosceles) were download from NCBI database (https://www.ncbi.nlm.nih.gov/) for subsequent analysis (Table S1). OrthoFinder v2.4.0 (default) was used to identify orthogroups and orthologs among these 15 species (Emms and Kelly 2019). Briefly, protein sets were collected from downloaded genomes of 15 species and the longest transcripts of each gene were extracted, in which miscoded genes and genes exhibiting premature termination were discarded. The extracted protein sequences were then aligned pair-wise to identify conserved orthologs using Blastp set to an e-value threshold of ≤ 1e-5, and orthologous inter-genome gene pairs, paralogous intra-genome gene pairs and single-copy gene pairs- were further identified. Proteins with no homologs were extracted as species-specific genes including unique genes and unclustered genes.

Phylogenetic Analysis and Divergence Time Estimation

On the basis of the identified orthologous gene sets with OrthoFinder v2.4.0, molecular phylogenetic analysis was performed using the shared single-copy genes. Briefly, the coding sequences were extracted from the single-copy families and each ortholog group were multiple aligned using Muscle v3.8.31 (Edgar 2004). Then combine all the alignment results to form a super alignment matrix. Finally, the phylogenetic relationship of 15 species was constructed using the maximum likelihood method (ML Tree) based on the sequence alignment results using RAxML v8.2.12 (-m PROTGAMMAAUTO -p 12345 -T 8 -f b) (Stamatakis 2014).

The estimation of divergence time was performed using MCMCTree package in PAML v4.8 (Yang 2007). The generated tree file was displayed using FigTree v1.4.4 and MEGA v10.1.8 (Sudhir et al. 2016). The silhouette of the species with no copyright were download from the http://phylopic.org/, and the rest use Adobe Photoshop CS6 to make silhouettes of representative species pictures. In this study, we used three secondary calibration points published in previous studies as references (i.e., the most recent common ancestors of C. l. familiaris and V. lagopus, V. ferrilata and C. l. familiaris, and B. mutus and B. taurus were calibrated as diverged between 8.8 and 17.95 Ma, 10.13 and 16.86 Ma, and 2.85 and 5.6 Ma, respectively) (Hassanin et al. 2013; Peng et al. 2021; Lyu et al. 2022).

Gene Family Expansion and Contraction Analysis

The expansion and contraction of homologous gene families were detected by CAFE v5.0.0 based on the results of OrthoFinder v2.4.0, which uses birth and death processes to simulate gene gains and losses during phylogeny (De Bie et al. 2006). Gene families that significantly expansion and contraction in high-altitude species were further analyzed by Gene Ontology (http://www.geneontology.org/) and KEGG (Kyoto Encyclopedia of Genes and Genomes) database enrichment to find gene families and functional categories that convergent evolved in the six high-altitude species.

Rapidly Evolving and Positive Selection Genes Analysis

According to the neutral theory of molecular evolution, the ratio of nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) of protein coding genes can be used to identify genes that show signatures of natural selection. We thus calculated average Ka/Ks values and conducted the two ratio model using Codeml implemented in the PAML package to identify rapidly evolving genes and positively selected genes in the six high-altitude species (Yang 2007). Genes with ω value of foreground branch > 1 and p value < 0.05 under the two ratio model were considered as positively selected genes. As for rapidly evolving genes, the genes with ω value of the background branch < ω value of foreground branch < 1 and p value < 0.05 were identified as rapidly evolving genes. The results were further analyzed by GO and KEGG pathway enrichment.

Identification of orthologous gene set and phylogenetic analysis

After data processing, an average of 8,601 single-copy genes were identified for each of the 15 species, ranging from 8,282 (O. curzoniae) to 8,814 (O. cuniculus). These single-copy genes accounted for 30.25% of the total number of genes in each species on average, of which B. mutus (44.59%) and P. hodgsonii (25.33%) had the highest and the lowest single-copy gene proportion, respectively. More information about gene sets of 15 species were shown in Table S1. These single-copy genes were further used to obtain the one-to-one single-copy orthologous genes. After alignment, a total of 6,008 single-copy orthologous genes were identified. After gap removal, 6,000 single-copy orthologous genes were used for subsequent analysis (Fig. S1).

According to the phylogenetic relationship constructed by single-copy orthologous genes, the six high-altitude species and their closest relatives belong to four different orders, including Lagomorpha, Primates, Carnivora and Artiodactyla (Fig. 1). Among them, Artiodactyla has the most high-altitude species, including B. mutus, P. hodgsonii and O. aries (high). Although these three high-altitude species belong to the same family and order (Bovidae and Artiodactyla), the phylogenetic relationship showed that the MRCA (most recent common ancestors) of these three species and their closest relatives were not high-altitude species. Thus, they might have independently evolved high-altitude adaptation mechanisms. According to the results of divergence time estimation, among the six high-altitude species, O. aries (high) and its low-altitude close relatives O. aries (low) had the closest relationship, with a divergence time of 3.99 Ma (95% CI: 6.30–2.30), while O. curzoniae and its low-altitude close relatives O. cuniculus had the longest divergence time of 45.15 Ma (95% CI: 65.5–24.9). In general, the results of phylogenetic relationships showed that these six high-altitude species belong to the high-altitude adaptation mechanism of independent evolution, which meets the requirements of convergent evolution research.

Gene Family Expansion and Contraction Analysis

According to the analysis results of gene family expansion and contraction, a total of 17,153 gene families were identified by MRCA (most recent common ancestors) in 15 species for subsequent analysis. Interestingly, except for the O. curzoniae, the remaining five high-altitude species had more gene family changes than their closest relatives (Fig. 2). It was worth noting that the expanded gene families of the remaining five high-altitude species, except O. curzoniae, were not significantly more than their closest relatives, or even less (i.e., 577 of R. bieti versus 685 of T. francoisi). Compared with low-altitude relatives, the gene family changes of these five high-altitude species had a significantly higher proportion of contracted gene families. At the same time, the number of contracted gene families of high-altitude species was also much larger than that of low-altitude relatives (i.e., 1,133 of R. bieti versus 600 of T. francoisi, 1,610 of P. hodgsonii versus 708 of C. hircus, 2,208 of O. aries (high) versus 357 of O. aries (low), 1,991 of V. ferrilata versus 482 of V. lagopus, and 976 of B. mutus versus 409 of B. taurus).

In order to explore the convergence pattern of gene family changes in high-altitude species, gene families that significantly (p < 0.05) expanded or contracted together in more than half of the high-altitude species were identified. The results showed that in these six high-altitude species, the convergence pattern of the significant contracted gene families was much larger than the significant expanded gene families (Fig. 3). As shown in Fig. 3a, 38 significantly expanded gene families were identified to exist in more than three high-altitude species and no significantly expanded gene families were found in all six high-altitude species. Furthermore, only one of these significantly expanded gene families existed in five high-altitude species (OG0000128), and five existed in four high-altitude species (OG0000068, OG0000304, OG0000398, OG0001349 and OG0001430). In contrast, 112 significantly contracted gene families were identified to exist in more than four high-altitude species (Fig. 3b, c). Among the 112 significantly contracted gene families, two gene families were identified in six species and 25 gene families were identified in five high-altitude species. In addition, among the 450 significantly expanded gene families, the number of species-specific gene families was much higher than that existing in more than two species (71 of P. hodgsonii, 66 of O. curzoniae, 49 of O. aries, 36 of V. ferrilata, 32 of R. bieti, and 29 of B. mutus, respectively) (Fig. S2a). As for significantly contracted gene families, compared with the number of gene families existing in more than two species, the species-specific gene families did not show a significantly higher trend. Only the number of species-specific gene family of one species was significantly higher than other types of gene families (82 of P. hodgsonii) (Fig. S2b).

To further explore the functional convergence pattern of the significantly changed gene families of these six high-altitude species, we performed GO enrichment analysis on the significantly expanded and contracted gene families of each species. Enriched GO terms that existed in at least five species were retained for subsequent analysis. As shown in Fig. 4a, a total of 29 enriched GO terms existed in at least five high-altitude species, of which 16 were enriched in all high-altitude species. In addition, the number of significantly expanded gene families in the same enriched GO term of each species also varied greatly (Table S2). Different from the results of gene families, the gene families that significantly contracted in the six high-altitude species did not enrich significantly more GO terms than the enrichment results of the significantly expanded gene families. A total of 37 enriched GO terms existed in at least five high-altitude species, of which 11 were enriched in all high-altitude species (Fig. 4b) (Table S3).

Rapidly Evolving and Positive Selection Genes

To better understand the effect of high-altitude environment on the nucleic acid sequence changes of genes of different species, identification of rapidly evolving gens (REGs) and positive selection genes (PSGs) were conducted under the two ratio model in PAML and a total of 1,713 REGs were identified in all six high-altitude species. After KEGG and GO enrichment analysis of these REGs, 40 significantly over-represented pathways and 43 significantly enriched GO terms were obtained (Fig. 5a, b). Notably, ultraviolet (UV) radiation response-, angiogenesis related- and hypoxia-related REGs showed significant pathway expansion, including the Melanoma (ko05218, 19 genes, p = 1.69E-06), MAPK signaling pathway (ko04010, 48 genes, p = 2.18E-06), Calcium signaling pathway (ko04020, 38 genes, p = 5.41E-05), EGFR tyrosine kinase inhibitor resistance (ko01521, 15 genes, p = 0.000886), Regulation of actin cytoskeleton (ko04810, 32 genes, p = 0.000986), Ubiquitin mediated proteolysis (ko04120, 24 genes, p = 0.001141), Ras signaling pathway (ko04014, 34 genes, p = 0.001142), mTOR signaling pathway (ko04150, 24 genes, p = 0.002045), PI3K-Akt signaling pathway (ko04151, 44 genes, p = 0.002641), Renal cell carcinoma (ko05211, 13 genes, p = 0.004766), and AMPK signaling pathway (ko04152, 18 genes, p = 0.008991). The 43 significantly enriched GO terms, including G protein-coupled receptor signaling pathway (GO:0007186, 28 genes, p = 1.13E-20), G protein-coupled receptor activity (GO:0004930, 26 genes, p = 2.69E-19), potassium ion transport (GO:0006813, 22 genes, p = 4.15E-06), protein phosphorylation (GO:0006468, 71 genes, p = 1.18E-05), voltage-gated potassium channel activity (GO:0005249, 14 genes, p = 0.000153), zinc ion binding (GO:0008270, 59 genes, p = 0.000341), protein tyrosine kinase activity (GO:0004713, 17 genes, p = 0.000551), voltage-gated potassium channel complex (GO:0008076, nine genes, p = 0.000814), intracellular signal transduction (GO:0035556, 20 genes, p = 0.004235), and ion transport (GO:0006811, 27 genes, p = 0.018482), were also related to angiogenesis and hypoxia response and might reflect the convergence adaptation mechanism of these six high-altitude species to the low-oxygen, high UV-radiation and extreme temperature conditions.

The number of PSGs was significantly less compared with REGs. Only seven PSGs were identified in all six high-altitude species. Four of these seven genes might reflect convergent coping mechanisms of high-altitude species when exposed to the stress of high-altitude environments, including CXCL10 gene, ERBIN gene, and SCAMP1 gene which promote angiogenesis under hypoxic conditions, as well as FYCO1 gene which is involved in UV-radiation damage response (Table 1).

Table 1

Basic information on four genes associated with high-altitude adaptation mechanisms that exhibit convergent positive selection features
Abbreviation	Full name	Function
ERBIN	Erbb2 interacting protein	Enhanced the expression of Hif-1α protein and promoted angiogenesis
CXCL10	C-X-C motif chemokine	Hypoxia response related gene
SCAMP1	Secretory carrier membrane protein 1	Vascular endothelial growth factor related gene
FYCO1	FYVE and coiled-coil domain-containing protein 1	UV-radiation damage repair related gene

Independent evolutionary species often evolve unique adaptive mechanisms when facing the same selection pressure, the previous studies on the mechanism of convergence adaptation at high-altitude were mainly limited to two species or species with close genetic relationship. Studies covering a wider range of species could help us to understand the most central convergent genetic changes in mammals when facing the selection pressure of high-altitude environment (i.e., low oxygen content, high UV-radiation, and extreme temperature). In this study, we first expanded the scope of research on the mechanism of convergence adaptation at high-altitude to six independently evolved plateau mammals belonging to four orders.

The Convergent Adaption Pattern of Gene Family Expansion and Contraction

During genome evolution, events of gene duplication and gene loss can lead to contraction and expansion of gene families (Rogers et al. 2022). Genome changes could be linked to evolutionary processes that cause environmental niche adaptation (Demuth and Hahn 2009). By increasing the amounts of protein synthesized or promoting evolutionary novelty of one of the duplicated genes through sub-functionalization or neo-functionalization, gene duplication might be beneficial (Szűcs et al. 2017). Previous studies on the convergent evolution of different species also indicated that when species face selection pressure such as environment or eating habits, the expansion and contraction of gene families are mainly the result of direction changes (Freitas and Nery 2020; Rogers et al. 2022). In this study, the result of gene family expansion and contraction analysis showed that five high-altitude species have more gene family changes than their closest relatives except for O. curzoniae, indicating that the strong selection pressure brought by the environment of the Qinghai-Tibet Plateau makes species have more gene family changes than their relatives to adapt to the plateau environment (Fig. 2). Besides, according to the hypothesis that "less is more", non-functionalization appears more frequently in evolutionary adaptive response (Albalat and Cañestro 2016). This might have special relevance when populations are faced with changes in selection pressure patterns due to rapid changes of environmental conditions, which also well explains that most high-altitude species have more contracted gene families than their low-altitude relatives. As to the contrary results obtained by the order Lagomorpha, this might be due to the distant genetic relationship between O. curzoniae and O. cuniculus. Ochotonidae and Leporidae have experienced multiple species evolution and differentiation since the common ancestor differentiation, which could also lead to more complex genetic changes (Mage et al. 2016). Therefore, the expansion and contraction of the O. cuniculus gene family might contain more complex cause, which also need to be confirmed by further studies.

Although a large number of significantly expanded and contracted gene families have been identified in each high-altitude species, there were few gene families with convergence changes, especially those with significant expansion (Fig. 3a). Previous studies have shown that mutation is more likely to lead to loss of function rather than acquisition of function, and gene loss has an important contribution to adaptive evolution, especially rapid response to environmental changes (Hottes et al. 2013). Another genomics study on yeast also confirmed that gene loss is the main adaptive mechanism to adapt to the growth-restricting environments (e.g., limited amounts of sugar) (Kvitek and Sherlock 2013). In addition, the experiments of bacterial population selection under different conditions showed that the adaptive function loss mutation of enzymes and regulatory functions played an important role in the adaptation of bacterial populations to new environments (Cooper et al. 2001). In this study, the number of convergent contracted gene families of six high-altitude species was significantly higher than that of convergent expanded gene families, which also indicates that convergent contracted gene families are the main adaptive strategies for high-altitude species to cope with the harsh plateau environment (Fig. 3b, c).

Interestingly, there was a large overlap between the GO terms enriched by the significantly expanded and contracted gene families, indicating that the gene families involved in these GO terms have changed greatly (Fig. 4a, b). It is worth noting that the gene family related to high-altitude adaptation had both significantly expanded and contracted, such as olfactory receptor activity (GO:0004984) related to hypoxia response (Zhou et al. 2022), glutathione metabolic process (GO:0006749) (Vats et al. 2008), and ATP hydrolysis activity (GO:0016887) related to energy metabolism and thermogenesis (Barata and de Meis 2002), and G protein-coupled receptor activity (GO:0004930 and GO:0007186) related to angiogenesis (Richard et al. 2001). In addition, the ion binding gene families (GO:0005509: calcium ion binding and GO:0008270: zinc ion binding) related to hypoxia response has significantly expanded without significant contraction, indicating that the high-altitude species are targeted to cope with hypoxia stress through the expansion of gene families related to calcium and zinc binding (Voelkel et al. 1980; Rawal et al. 1999).

The Convergent Adaption Pattern of REGs and PSGs

Previous studies proved that rapid adaptive evolution played a great driving role for species to adapt to new environments (Szűcs et al. 2017). Understanding what kind of genes rapidly convergent evolving when independently evolved high-altitude species face the same high-altitude selection pressure can provide valuable insights into mammalian convergence high-altitude adaptation mechanisms. In this study, a considerable number of REGs had been confirmed to be related to the high-altitude adaptation mechanism of species (Fig. 5a, b). For example, genes associated with melanoma have been shown to be associated with excessive ultraviolet radiation at high-altitudes (Aceituno-Madera et al. 2011). Previous studies confirmed that genes involved in MAPK signaling pathway, Calcium signaling pathway, Ubiquitin mediated proteolysis and Regulation of actin cytoskeleton are closely related to hypoxia response (Sang et al. 2003; Hui et al. 2006; Yanagawa and Komatsu 2012; Zieseniss 2014). The genes involved in EGFR tyrosine kinase inhibitor resistance, Ras signaling pathway, mTOR signaling pathway, PI3K-Akt signaling pathway and Renal cell carcinoma are related to angiogenesis, which has also been confirmed as an important way to cope with hypoxia pressure (Kranenburg et al. 2004; van Cruijsen et al. 2005; Chen et al. 2019). In addition, the gene contained in AMPK signaling pathway has been proved to be related to energy metabolism, which might be a coping mechanism of species to extreme temperature at high-altitude environment (Hardie et al. 2012). GO enrichment analysis of REGs yielded similar results, enrichment of GO terms for REGs related to G protein-coupled receptors and protein phosphorylation were strongly associated with angiogenesis and energy metabolism (Richard et al. 2001; Humphrey et al. 2015). The enrichment of GO terms associated with trace elements might be related to the species response to low oxygen pressure, such as potassium and zinc. Previous study has shown that exposure to hypoxia might cause decreased activity of voltage-gated potassium channel, leading to hypoxia pulmonary vasoconstriction (Ke et al. 2013). Another study also confirmed that plasma zinc contents significantly reduced upon induction to high-altitude (Rawal et al. 1999). In addition to this, the enrichment of REGs related to protein tyrosine kinases and cell signaling transduction might also reflected a convergent coping mechanism of species in the face of hypoxic stress (Mishra and Delivoria-Papadopoulos 2004; Nakayama 2009). In general, REGs associated with hypoxia stress response, UV-radiation damage repair, and energy metabolism might reflect convergent mechanisms underlying the adaptations of six high-altitude species to high-altitude environments.

As for PSGs, a total of seven genes were identified to show convergent positive selection signature in all high-altitude species. Among these seven genes, three genes might be related to the response of species to hypoxic pressure (Table 1). Previous study showed that Erbb2 interacting protein (ERBIN) gene enhanced the expression of Hif-1α protein and the activation of Hif-1α pathway by promoting angiogenesis (Chen et al. 2020). Another study indicated that hypoxia/ischemia can significantly increase the production of C-X-C motif chemokine (CXCL10) gene in myocardial microvascular endothelial cells (Xia et al. 2016). In addition, down-regulation of secretory carrier membrane protein 1 (SCAMP1) gene inhibited vascular endothelial growth factor (VEGF) levels in the conditioned medium of SCAMP1 siRNA transfected cells (Yang et al. 2013). In addition to genes related to hypoxia stress response, we found for the first time that FYVE and coiled-coil domain-containing protein 1 (FYCO1) gene might related to UV-radiation damage repair showed convergent positive selection signature in high-altitude species. The formation of cataract is closely related to long-term exposure to UV-radiation. Previous studies have shown that the incidence of cataract in high-altitude residents is much higher than that in plain residents, and FYCO1 gene plays a key role in preventing the formation of cataract. Therefore, the convergent positive selection signal of FYCO1 gene in high-altitude species might be one of the important mechanisms for species to cope with long-term excessive UV-radiation (Jha 2012; Satoh et al. 2021).

In this study, we conducted a comparative genomics approach to explore the convergent evolution of high-altitude adaptation mechanisms in six independently evolved mammals belonging to four orders. The results showed that the expanded gene families of these six species did not show significant convergence, while the contracted gene families showed much greater convergence than the expanded gene families, indicating that gene loss rather than gain might be an important adaptation mechanism for species in the face of extreme environments. Nonetheless, enrichment analysis indicated a convergence in the functions of significantly changed gene families across the six species. Besides, the 1,713 rapidly evolving genes identified were mainly related to hypoxia stress response, including activation of pathways and angiogenesis to increase oxygen delivery under hypoxic conditions. These results suggest that oxygen content might be one of the important reasons for the rapid evolution of genes in high-altitude species. In addition, pathways related to UV-radiation damage and energy metabolism might also reflect a coping mechanism of species in the face of excess UV-radiation and extreme temperatures in the plateau environment. Similar results were obtained for the analysis of positively selected genes. Among the four genes that might be related to high-altitude adaptation mechanisms, three positively selected genes are related to hypoxia stress response, and one positively selected gene was related to preventing cataract formation, which is closely related to UV-radiation at high altitude. These results reflected a convergent evolutionary adaptation mechanism of six independently evolving species in the face of the selective pressure of the plateau environment. In general, our study reveals that hypoxia response and UV-radiation might play an important role in the adaptation of independently evolved species to high-altitude environment, providing a basic perspective for further exploring the high-altitude adaptation mechanism of different related species in the future.

Funding

This work was supported by the National Natural Science Foundation of China (32270444, 32070405 and 32200349).

Author Contribution

This study was conceived by all authors. Data collection and analysis was performed by Tianshu Lyu and Shengyang Zhou. The figures in the manuscript were drawn by Jiaohui Fang. Writing of the initial draft of the manuscript was performed by Tianshu Lyu. All authors contributed to revising the manuscript and the acquisition of funding.

Availability of Data

The high-quality genomes of the 15 species used in this study were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/), and the accession numbers of the genomes are listed in Table S1.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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No competing interests reported.

FigS1.tif
Fig. S1 Statistics of the number of single copy genes of 15 species. The middle of the petal represents the one-to-one single copy homolog genes shared by 15 species.
FigS2.tif
Fig. S2 Upset statistical analysis of categories of significantly altered gene families in six high-altitude species. The black dots represent gene families that are significantly altered in the species, and the gray dots represent gene families that are not significantly altered in the species. Each bar represents the number of gene families in that category. a. Upset statistical analysis of categories of significantly expanded gene families in six high-altitude species. b.Upset statistical analysis of categories of significantly contracted gene families in six high-altitude species.
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Convergent genomic signatures of high-altitude adaptation among six independently evolved mammals

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Abstract

Figures

Introduction

Materials And Methods

Identification of orthologous gene set

Phylogenetic Analysis and Divergence Time Estimation

Gene Family Expansion and Contraction Analysis

Rapidly Evolving and Positive Selection Genes Analysis

Results

Identification of orthologous gene set and phylogenetic analysis

Gene Family Expansion and Contraction Analysis

Rapidly Evolving and Positive Selection Genes

Discussion

The Convergent Adaption Pattern of Gene Family Expansion and Contraction

The Convergent Adaption Pattern of REGs and PSGs

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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