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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Evolution and Adaptation of Aquaporin Genes in Rodents along a Habitat Moisture Gradient: A Case Study of Eothenomys miletus

L
Lijuan Cao1
0009-0006-5629-9250
W
Wenrong Gao2
https://orcid.org/0000-0001-9521-7613
W
Wanlong Zhu1,*
0000-0001-8261-4089
T
Tongtong Gu1,*
1School of Life Sciences, Yunnan Normal University, Kunming, 650500, China.
2School of Biological Resources and Food Engineering, Qujing Normal University, Qujing, 655011, China.

ABSTRACT

Background: To elucidate the adaptive evolution of aquaporin (AQP) genes in arid habitats, this study performed phylogenetic and selection pressure analyses on AQP1 to AQP11 across 15 rodent species, with subsequent experimental validation of candidate genes under positive selection.

Methods: In the present study, phylogenetic trees were constructed using MEGA-X and the iTOL online platform, while selection pressure analysis was performed via PAML and the Datamonkey website. Finally, using Eothenomys miletus inhabiting the Hengduan Mountains as the research subject, water stress experiments were conducted by measuring AQP expression levels through ELISA.

Result: Phylogenetic reconstruction using MEGA-X and iTOL indicated that species from humid environments clustered into a distinct clade, implying that moisture availability may have influenced the divergence of AQP genes. Selection analyses implemented in PAML identified one positively selected site each in AQP1, AQP5 and AQP7. Furthermore, signals of positive selection were detected in AQP10 and AQP11 among species from arid habitats. Given the substantial existing literature on AQP1, AQP5 and AQP7 and the limited phenotypic consequences of AQP10 deficiency in mice, this study focused on validating AQP11. After subjecting the endemic species of the Hengduan Mountains, E. miletus, to water deprivation treatment, the protein expression levels of AQP11 were measured using enzyme-linked immunosorbent assay (ELISA). Findings demonstrated significantly elevated AQP11 levels in seven tissues-kidney, brain, liver, heart, small intestine, white adipose tissue (WAT) and brown adipose tissue (BAT)-in the water-deprived group compared to controls. Based on existing evidence, we propose that AQP11 is involved in cellular water stress responses across multiple organs by mediating H2O2 transport across the endoplasmic reticulum membrane. Thus, it is hypothesized that the AQP11 gene may play a critical role in the adaptation of rodents to arid environments. This research offers a theoretical basis for further elucidating the mechanisms by which organisms adapt to environmental water stress.

ABBREVIATIONS

AQP: Aquaporin; BAT: Brown adipose tissue; E. miletus: Eothenomys miletus; ER: Endoplasmic reticulum; O. torridus: Onychomys torridus; WAT: White adipose tissue.

INTRODUCTION

Aquaporins (AQPs) constitute a class of membrane proteins ubiquitously distributed in both prokaryotic and eukaryotic cells (Liu et al., 2024; Wang et al., 2024). They function as specialized channels that facilitate the rapid transmembrane transport of water molecules, thereby playing a pivotal role in water secretion and absorption processes (Ishibashi et al., 2017). Beyond their fundamental water permeability, AQPs participate in a wide range of physiological functions, including cell proliferation, apoptosis and neural signal transduction (Verkman, 2012). To date, 13 AQP subtypes (designated AQP0 to AQP12) have been identified in mammals. These sub-types are categorized into three major functional groups: orthodox AQPs (selective for water), aquaglyceroporins (permeable to water and small solutes such as glycerol) and superaquaporins (also known as unorthodox AQPs) (Rojek et al., 2008; Zhang et al., 2012). The AQP gene family has undergone complex evolutionary processes across different species. Its structural and functional diversity is closely associated with adaptations to environments characterized by varying water availability, such as those with distinct precipitation regimes (Gui et al., 2021).
       
Global warming alters atmospheric circulation and ocean current patterns, leading to a redistribution of global precipitation and an exacerbation of regional water crises (Benz et al., 2024). Against this backdrop, understanding the genetic mechanisms underlying organismal adaptation to hydrological stress has become a critical research focus. Accumulating evidence indicates that environmental adaptive selection is a major driver of functional diversification in AQPs. This diversity in AQP genes may originate from various evolutionary mechanisms, including whole-genome duplication, local gene replication and horizontal gene transfer (Ishibashi et al., 2017). For instance, cucumber genome-wide scans have revealed that the promoter regions of AQP genes contain an abundance of stress-responsive elements, with some of these genes having undergone purifying selection, reflecting adaptation to environmental challenges (Lai et al., 2022). In reptiles, positive selection signals identified in the AQP genes of terrestrial species suggest a role in adapting to arid environments (Zang et al., 2019; Zhang et al., 2024). Additionally, research indicates that AQP11 and its orthologs may have played a pivotal role in the aquatic-to-terrestrial transition of vertebrates, as observed in amphibians and fish. Furthermore, AQP11 has been demonstrated to perform essential functions in terrestrial reptiles (Lorente-Martínez et al., 2023). Consequently, investigating the evolutionary patterns of AQP genes under water stress will enhance our understanding of the molecular mechanisms that enable organisms to cope with and adapt to environmental pressures, thereby providing insights into ecological resilience.
       
Rodentia, the most species-rich and widely distributed order of mammals, possess complex mechanisms for regulating water metabolism (Liu et al., 2014). Their diversification across ecological gradients has resulted in the evolution of diverse strategies for water acquisition and conservation. Given the central role of AQPs in maintaining water balance and facilitating environmental adaptation (Beall et al., 2004; Bo et al., 2016; Li and Guo, 2011; Wang et al., 2009), this study investigates adaptive changes in AQP genes across rodent species distributed along a habitat humidity gradient. Due to the limited research on AQP0 and AQP12 in rodents, we focus on the evolutionary patterns of AQP1 to AQP11 in arid-adapted species, followed by functional validation of positively selected AQP genes.
       
Since the Tertiary Period, the orogenic uplift of the Tibetan Plateau and the formation of the Hengduan Mountains have transformed the regional paleoclimate from warm and humid to arid and cold (Geng and Zhu, 2024; Li et al., 2014). This region is now characterized by mild annual temperature variation, high diurnal temperature range and pronounced vertical climatic zonation (Ren et al., 2023). These heterogeneous topographic and climatic conditions have likely acted as key drivers of genetic and phenotypic differentiation in local species (Luo et al., 2024). In rodents, different functional categories of AQPs may face distinct selection pressures and their evolutionary patterns may correlate with habitat humidity gradients. To address this research gap, we therefore conducted water-deprivation experiments on Eothenomys miletus, a species endemic to the Hengduan Mountains, to elucidate how AQP expression underpins its adaptability to varying rainfall regimes. The complex paleoclimatic changes in this region provide the context for studying adaptive evolution, while research on this specific species can directly uncover mechanisms of local adaptation, addressing a gap left by previous studies that often focused on widely distributed or model species. The findings of this study furnish valuable understanding of the potential mechanisms by which rodents may cope with growing water scarcity under global warming conditions.

MATERIALS AND METHODS

Data acquisition for AQP genes
 
The coding sequence (CDS) regions of AQP1 to AQP11 genes from 15 rodent species were obtained from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov). Ecological metadata, including species habitat characteristics, were obtained from the Animal Diversity Web (https://animaldiversity.org) (Table 1).

Table 1: NCBI numbers for AQP1-11 genes in 15 rodent species.


 
Phylogenetic analysis of AQP genes
 
The amino acid sequence information of the AQP genes were aligned using MUSCLE software embedded in MEGA X (Kulikov et al., 2024). The most appropriate amino acid substitution model was selected using ModelGenerator (Keane et al., 2004). Maximum likelihood (ML) phylogenetic analysis was performed in MEGA X, with 1000 bootstrap replicates employed to estimate node support (Kumar et al., 2016). Ultimately, the resulting tree was graphically presented and annotated through the Interactive Tree of Life (iTOL) online resource (https://itol.embl.de).
 
Selection pressure analysis of AQP genes
 
Position-specific selection pressures acting on AQP genes were assessed using the site models (M7 and M8) implemented in PAML 4.7. To enhance the reliability of positively selected site identification across the 15 rodent species, two additional models-the Fixed Effects Likelihood (FEL) and Single-Likelihood Ancestor Counting (SLAC) approaches-accessible via the Datamonkey web server (http://www.datamonkey.org), were also employed. The strength of natural selection was quantified by calculating the ratio of nonsynonymous (dN) to synonymous (dS) substitution rates (ω = dN/dS). Selective pressures were interpreted as follows: ω<1 indicates purifying selection; ω>1 indicates positive selection and ω = 1 suggests neutral evolution. Branch-specific models in PAML 4.7 were applied to test for positive selection acting along specific lineages of rodent AQP genes (Zhang et al., 2025). Finally, branch-site models were used to examine whether AQP genes in rodent species inhabiting arid environments have undergone positive selection.
 
Water deprivation experiment design
 
Wild-caught E. miletus individuals were collected from farmland and shrubland habitats in Xiaguan Town, Dali City, Yunnan Province, China (GPS coordinates: 25°34′54″ N, 100°14′6″E; altitude: 2061 m). Following capture, all animals were disinfected and cleared of ectoparasites by local health authorities prior to transportation. Thereafter, the animals were transferred to the laboratory animal facility of Yunnan Normal University. They were individually housed in standard cages (260 mm × 160 mm × 150 mm) under a 12-hour light:12-hour dark photoperiod and a consistent surrounding temperature held at 25±1°C. All animals were provided with a standard laboratory diet (produced by Kunming Medical University) and were acclimatized for four weeks with food and water available ad libitum. Only healthy, non-reproductive adult individuals were selected for the experiment. Post-acclimation, the animals were randomly assigned to one of two experimental groups: a water-deprived treatment group (n = 6), which received standard feed but no water access and a control group (n = 6), which maintained ad libitum access to both food and water. Pre-experiment body weights did not differ significantly between the two groups (p>0.05). At the conclusion of the experimental period, all animals were humanely euthanized via CO‚  asphyxiation. Target tissues were promptly dissected, forthwith flash-frozen in liquid nitrogen and preserved at -80°C for subsequent molecular assays. As AQP1, AQP5 and AQP7 have been extensively characterized in previous literature and because AQP10 deficiency has been reported to produce minimal phenotypic consequences in murine models (Morinaga et al., 2002; Rebez and Jacob, 2024), the present study focused on the functional validation of AQP11.
 
ELISA for AQP11 protein in E. miletus
 
AQP11 expression levels were quantified in seven tissue types-kidney, brain, liver, heart, small intestine, white adipose tissue (WAT) and brown adipose tissue (BAT)-using ELISA. Frozen tissue samples of E. miletus, stored at -80°C, were analyzed. The measurements were performed using a commercially available rat AQP11 ELISA Kit (Baika Biological Technology Co., Kunming, China), strictly following the manufacturer’s protocol.
 
Data processing and analysis
 
SPSS Statistics version 27.0 was employed for all statistical analyses. First, the distribution of the data was assessed using normality tests. With the exception of the AQP11 expression data in BAT, which deviated from a normal distribution, all other datasets satisfied the assumption of normality. Based on the distribution characteristics of the data, appropriate statistical methods were selected for between-group comparisons: For data meeting the normality assumption, Variations in the expression levels of AQP11 between the control and treatment groups within the same tissue type were assessed via an independent-samples t-test. In the case of BAT data with a non-normal distribution, the non-parametric Mann Whitney U test was employed. To compare AQP11 expression levels among different tissues within the same experimental group, one-way analysis of variance (one-way ANOVA) was used. Prior to conducting the ANOVA, the assumption of homogeneity of variance was verified. To identify specific group differences after a significant ANOVA, we applied Tukey’s HSD post hoc test for pairwise comparisons.
       
All boxplot figures were generated using OriginPro 2024 software.

RESULTS AND DISCUSSION

Phylogenetic analysis of AQP genes
 
AQPs are critical membrane proteins that regulate water homeostasis in organisms and are classified into three major categories: classical water-selective AQPs (AQP0, 1, 2, 4, 5 and 6). Among these, AQP2, 5 and 6 are functionally important in the skin, bladder, salt glands and kidneys across amphibians, sauropsids and mammals. (Morinaga et al., 2002; Nishimura and Yang, 2013). Deficiencies in these AQPs can result in severe physiological defects (Ikeda et al., 2002; Noda and Sasaki, 2006; Oshio et al., 2006). Classical aquaglyceroporins (AQP3, 7, 9 and 10) are involved in osmoregulation and energy metabolism through glycerol transport (Hara-Chikuma, 2005; Sohara et al., 2006) and play crucial roles in metalloid homeostasis (Bienert et al., 2008). Unorthodox AQPs (AQP11 and AQP12) are considered “superaquaporins” within the AQP subfamily (Ishibashi, 2006). Additionally, AQP8 is capable of transporting water, urea, ammonia and free radicals (Bienert et al., 2007).
       
A total of 164 AQP genes were phylogenetically clustered into three major groups: AQP1, 2, 4, 5, 6 and 8 formed one cluster; AQP3, 7, 9 and 10 grouped together; while AQP11 formed a distinct separate branch. This phylogenetic structure corresponds exactly to the three recognized AQP subfamilies (Fig 1). Furthermore, AQP genes within each subfamily exhibited clearly resolved, species-specific phylogenetic relationships. Notably, rodent species inhabiting humid environments consistently clustered together within the phylogenetic tree.

Fig 1: Phylogenetic tree of AQP1-11 genes.


       
Within this research, phylogenetic trees were reconstructed based on AQP genes from 15 rodent species. The resulting clustering patterns revealed a correlation with species ecotypes. Specifically, lineages from humid habitats frequently formed independent clades, suggesting that adaptation to arid environments may have driven unique evolutionary changes in rodent AQP genes to fulfill physiological requirements under water stress. Interestingly, O. torridus-which inhabits arid regions including deserts, sand dunes and savannas in the western and southwestern United States and northern Mexico, where rainfall is relatively low-was found to group within a clade comprising species from more humid environments across the AQP1-11 gene tree (Harold and Egoscue, 1960). This anomalous positioning suggests that O. torridus may have evolved alternative physiological mechanisms for coping with water scarcity, potentially involving regulatory changes in AQP expression or function rather than rapid gene evolution. This contrasts with the rapid evolutionary pattern observed in AQP genes among other drought-adapted species, such as the genomic data from the comparatively arid and humid groups of Liangzhou donkeys, where the CYP4A11 gene was identified as a key factor in their adaptation to arid environments (Wang et al., 2022). This suggests that rodents may employ diverse molecular evolutionary pathways in responding to drought stress. However, the specific reasons still require subsequent experimental verification.
 
Positively selected sites in rodent AQP genes
 
To explore the molecular basis of adaptation in arid-adapted rodents, we identified sites under positive selection. Analysis of positively selected sites across the AQP gene family in the 15 rodent species identified a single positively selected site in each of three genes: site 131 in AQP1, site 111 in AQP5 and site 8 in AQP7. No significant signals of positive selection were detected in the remaining AQP genes analyzed (Table 2).

Table 2: Site model testing of positive selection sites in reptile AQP genes.


       
Signatures of positive selection were detected in AQP1, AQP5 and AQP7. The selection on AQP1, AQP5 and AQP7 aligns with their established roles in mediating adaptive responses to environmental stress in other mammals, such as enhanced renal water reabsorption in camels (Wang et al., 2014) and thermoregulation in goats (Kaushik et al., 2024). Notably, AQP7-the sole glycerol channel in adipose tissue-is closely linked to adipocyte morphology and physiology, obesity development and the maintenance of energy balance and glucose homeostasis (Miranda et al., 2010). Therefore, the positively selected sites identified in AQP1, AQP5 and AQP7 in this study may therefore represent adaptive modifications that enhance responses to environmental water variability by regulating water metabolism, thermoregulatory capacity and energy balance.
 
Positive selection in AQP genes of rodents from arid habitats
 
Various ω ratio models were employed to assess whether positive selection has acted on AQP genes in rodents. Significant heterogeneity in ω ratios among branches was identified for AQP2 (p = 0.0002*), AQP3 (p = 0.0074*), AQP5 (p = 0.0203*), AQP6 (p = 0.0034*), AQP9 (p< 0.0001), AQP10 (p<0.0001) and AQP11 (p = 0.0001*). In contrast, the remaining AQP genes exhibited no significant branch-specific variation in ω ratios (Table 3).

Table 3: Phylogenetic analysis of positive selection in the AQP gene branch.


       
Branch-site model A revealed positive selection acting on rodent AQP genes. Specifically, sites under significant positive selection were identified in AQP10 (site 294, p = 0.0020*) and AQP11 (site 184, p = 0.0037*) in species inhabiting arid environments. No significant signals of positive selection were detected in the other AQP genes (Table 4). Consequently, in environments with lower water content, AQP10 and AQP11 assume a more significant role in rodents. However, the specific reasons for this phenomenon require further investigation.

Table 4: Selection pressure analysis of AQP gene orthologous sites in rodents.


 
AQP11 expression in tissues of E. miletus
 
AQP11 is an unconventional AQP that localises to the endoplasmic reticulum (ER) and is implicated in regulating subcellular water distribution among organelles, particularly in maintaining osmotic balance in critical organs such as the kidneys (Morishita et al., 2004). It functions as a peroxiporin, facilitating the transport of H2O2 across the ER membrane. Expressed in multiple tissues-including kidney, liver, intestine and brain-AQP11 contributes to organellar redox homeostasis and participates broadly in cellular stress responses (Bestetti et al., 2020; Markou et al., 2022; Yakata et al., 2007).
       
Compared with the control group, water-deprived E. miletus exhibited significantly higher AQP11 expression (p<0.05) in the kidney, brain, liver, heart, small intestine, WAT and BAT (Fig 2). Post-hoc analysis further revealed that expression in the small intestine was significantly lower than in the other six tissues (p<0.05), indicating the most pronounced differential expression among all tissues examined.

Fig 2: Box plot showing AQP11 expression levels in seven tissues of E. miletus.


       
We quantified AQP11 expression in E. miletus under water-restricted conditions and observed significant upregulation across all seven tissues examined, with the most pronounced increase in the kidney. In mammals, AQP11 deficiency is associated with ER-derived vacuolation in proximal tubules, polycystic kidney disease and early renal failure, highlighting its essential role in renal tubule development and ER homeostasis (Michałek and Grabowska, 2019; Morishita et al., 2005; Schwartz and Johnson, 1971). The marked renal upregulation suggests a protective mechanism against osmotic stress, potentially through enhanced H2O2 efflux from the ER, thereby mitigating redox imbalance and preventing ER stress-induced pathology.
       
Notable upregulation was also observed in the brain and heart, indicating a previously underappreciated role for AQP11 in supporting neural and cardiovascular function during dehydration. In the brain, AQP11 is localised to the choroid plexus epithelium and brain capillary endothelium, suggesting a possible role in water regulation at the blood-brain barrier (Benga and Huber, 2012).  Its activity may involve interplay with other AQPs, such as AQP4, in maintaining cerebral water balance (Zhang et al., 2022). Although the cardiac mechanism remains unclear, our data imply AQP11 involvement in systemic adaptation to osmotic stress.
       
In metabolic tissues, strong upregulation in BAT points to a role in alleviating ER stress via H2O2 transport, which may be critical for sustaining thermogenesis and energy balance under metabolic challenge (Calamita and Delporte, 2021). Similarly, hepatic upregulation likely supports the clearance of H2O2 generated during oxidative protein folding, thereby promoting hepatocyte function under high ER load (Ishibashi et al., 2021). By contrast, AQP11 expression in the small intestine showed only a mild response to water stress, suggesting that other mechanisms may predominately regulate hydric balance in this organ under drought conditions.

CONCLUSION

AQPs are phylogenetically widespread across most species and investigations of AQP genes in rodents are likely to provide valuable insights for related human studies. Focusing on rodents with adaptations to arid habitats, this research identified a positively selected site within AQP11, suggesting that the gene may be critical for adaptive responses to xeric conditions. In E. miletus, AQP11 expression was significantly up-regulated across all seven tissues examined, supporting the hypothesis that AQP11 acts as a key physiological regulator of endoplasmic reticulum redox homeostasis-mediating H2O2 efflux and facilitating adaptive responses to water deprivation and other stressors in multiple organ systems. This study has certain limitations. Specifically, as the research focused on particular rodent species, the generalizability of the conclusions to a broader range of species requires further verification. Additionally, the relatively limited sample size may affect the statistical power of some analyses. Future studies could employ gain- and loss-of-function experiments while expanding both sample sizes and the range of species examined to more directly validate the specific role of AQP11 in adaptation to arid environments.

ACKNOWLEDGEMENT

This work was supported by the National Natural Scientific Foundation of China (No. 32560262; 32500436), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (NO. 202301BA070001-076).
 
Ethical approval
 
All animal procedures were within the rules of Animals Care and Use Committee of School of Life Sciences, Yunnan Normal University. This study was approved by the committee (13-0901-011).

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Evolution and Adaptation of Aquaporin Genes in Rodents along a Habitat Moisture Gradient: A Case Study of Eothenomys miletus

    L
    Lijuan Cao1
    0009-0006-5629-9250
    W
    Wenrong Gao2
    https://orcid.org/0000-0001-9521-7613
    W
    Wanlong Zhu1,*
    0000-0001-8261-4089
    T
    Tongtong Gu1,*
    1School of Life Sciences, Yunnan Normal University, Kunming, 650500, China.
    2School of Biological Resources and Food Engineering, Qujing Normal University, Qujing, 655011, China.

    ABSTRACT

    Background: To elucidate the adaptive evolution of aquaporin (AQP) genes in arid habitats, this study performed phylogenetic and selection pressure analyses on AQP1 to AQP11 across 15 rodent species, with subsequent experimental validation of candidate genes under positive selection.

    Methods: In the present study, phylogenetic trees were constructed using MEGA-X and the iTOL online platform, while selection pressure analysis was performed via PAML and the Datamonkey website. Finally, using Eothenomys miletus inhabiting the Hengduan Mountains as the research subject, water stress experiments were conducted by measuring AQP expression levels through ELISA.

    Result: Phylogenetic reconstruction using MEGA-X and iTOL indicated that species from humid environments clustered into a distinct clade, implying that moisture availability may have influenced the divergence of AQP genes. Selection analyses implemented in PAML identified one positively selected site each in AQP1, AQP5 and AQP7. Furthermore, signals of positive selection were detected in AQP10 and AQP11 among species from arid habitats. Given the substantial existing literature on AQP1, AQP5 and AQP7 and the limited phenotypic consequences of AQP10 deficiency in mice, this study focused on validating AQP11. After subjecting the endemic species of the Hengduan Mountains, E. miletus, to water deprivation treatment, the protein expression levels of AQP11 were measured using enzyme-linked immunosorbent assay (ELISA). Findings demonstrated significantly elevated AQP11 levels in seven tissues-kidney, brain, liver, heart, small intestine, white adipose tissue (WAT) and brown adipose tissue (BAT)-in the water-deprived group compared to controls. Based on existing evidence, we propose that AQP11 is involved in cellular water stress responses across multiple organs by mediating H2O2 transport across the endoplasmic reticulum membrane. Thus, it is hypothesized that the AQP11 gene may play a critical role in the adaptation of rodents to arid environments. This research offers a theoretical basis for further elucidating the mechanisms by which organisms adapt to environmental water stress.

    ABBREVIATIONS

    AQP: Aquaporin; BAT: Brown adipose tissue; E. miletus: Eothenomys miletus; ER: Endoplasmic reticulum; O. torridus: Onychomys torridus; WAT: White adipose tissue.

    INTRODUCTION

    Aquaporins (AQPs) constitute a class of membrane proteins ubiquitously distributed in both prokaryotic and eukaryotic cells (Liu et al., 2024; Wang et al., 2024). They function as specialized channels that facilitate the rapid transmembrane transport of water molecules, thereby playing a pivotal role in water secretion and absorption processes (Ishibashi et al., 2017). Beyond their fundamental water permeability, AQPs participate in a wide range of physiological functions, including cell proliferation, apoptosis and neural signal transduction (Verkman, 2012). To date, 13 AQP subtypes (designated AQP0 to AQP12) have been identified in mammals. These sub-types are categorized into three major functional groups: orthodox AQPs (selective for water), aquaglyceroporins (permeable to water and small solutes such as glycerol) and superaquaporins (also known as unorthodox AQPs) (Rojek et al., 2008; Zhang et al., 2012). The AQP gene family has undergone complex evolutionary processes across different species. Its structural and functional diversity is closely associated with adaptations to environments characterized by varying water availability, such as those with distinct precipitation regimes (Gui et al., 2021).
           
    Global warming alters atmospheric circulation and ocean current patterns, leading to a redistribution of global precipitation and an exacerbation of regional water crises (Benz et al., 2024). Against this backdrop, understanding the genetic mechanisms underlying organismal adaptation to hydrological stress has become a critical research focus. Accumulating evidence indicates that environmental adaptive selection is a major driver of functional diversification in AQPs. This diversity in AQP genes may originate from various evolutionary mechanisms, including whole-genome duplication, local gene replication and horizontal gene transfer (Ishibashi et al., 2017). For instance, cucumber genome-wide scans have revealed that the promoter regions of AQP genes contain an abundance of stress-responsive elements, with some of these genes having undergone purifying selection, reflecting adaptation to environmental challenges (Lai et al., 2022). In reptiles, positive selection signals identified in the AQP genes of terrestrial species suggest a role in adapting to arid environments (Zang et al., 2019; Zhang et al., 2024). Additionally, research indicates that AQP11 and its orthologs may have played a pivotal role in the aquatic-to-terrestrial transition of vertebrates, as observed in amphibians and fish. Furthermore, AQP11 has been demonstrated to perform essential functions in terrestrial reptiles (Lorente-Martínez et al., 2023). Consequently, investigating the evolutionary patterns of AQP genes under water stress will enhance our understanding of the molecular mechanisms that enable organisms to cope with and adapt to environmental pressures, thereby providing insights into ecological resilience.
           
    Rodentia, the most species-rich and widely distributed order of mammals, possess complex mechanisms for regulating water metabolism (Liu et al., 2014). Their diversification across ecological gradients has resulted in the evolution of diverse strategies for water acquisition and conservation. Given the central role of AQPs in maintaining water balance and facilitating environmental adaptation (Beall et al., 2004; Bo et al., 2016; Li and Guo, 2011; Wang et al., 2009), this study investigates adaptive changes in AQP genes across rodent species distributed along a habitat humidity gradient. Due to the limited research on AQP0 and AQP12 in rodents, we focus on the evolutionary patterns of AQP1 to AQP11 in arid-adapted species, followed by functional validation of positively selected AQP genes.
           
    Since the Tertiary Period, the orogenic uplift of the Tibetan Plateau and the formation of the Hengduan Mountains have transformed the regional paleoclimate from warm and humid to arid and cold (Geng and Zhu, 2024; Li et al., 2014). This region is now characterized by mild annual temperature variation, high diurnal temperature range and pronounced vertical climatic zonation (Ren et al., 2023). These heterogeneous topographic and climatic conditions have likely acted as key drivers of genetic and phenotypic differentiation in local species (Luo et al., 2024). In rodents, different functional categories of AQPs may face distinct selection pressures and their evolutionary patterns may correlate with habitat humidity gradients. To address this research gap, we therefore conducted water-deprivation experiments on Eothenomys miletus, a species endemic to the Hengduan Mountains, to elucidate how AQP expression underpins its adaptability to varying rainfall regimes. The complex paleoclimatic changes in this region provide the context for studying adaptive evolution, while research on this specific species can directly uncover mechanisms of local adaptation, addressing a gap left by previous studies that often focused on widely distributed or model species. The findings of this study furnish valuable understanding of the potential mechanisms by which rodents may cope with growing water scarcity under global warming conditions.

    MATERIALS AND METHODS

    Data acquisition for AQP genes
     
    The coding sequence (CDS) regions of AQP1 to AQP11 genes from 15 rodent species were obtained from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov). Ecological metadata, including species habitat characteristics, were obtained from the Animal Diversity Web (https://animaldiversity.org) (Table 1).

    Table 1: NCBI numbers for AQP1-11 genes in 15 rodent species.


     
    Phylogenetic analysis of AQP genes
     
    The amino acid sequence information of the AQP genes were aligned using MUSCLE software embedded in MEGA X (Kulikov et al., 2024). The most appropriate amino acid substitution model was selected using ModelGenerator (Keane et al., 2004). Maximum likelihood (ML) phylogenetic analysis was performed in MEGA X, with 1000 bootstrap replicates employed to estimate node support (Kumar et al., 2016). Ultimately, the resulting tree was graphically presented and annotated through the Interactive Tree of Life (iTOL) online resource (https://itol.embl.de).
     
    Selection pressure analysis of AQP genes
     
    Position-specific selection pressures acting on AQP genes were assessed using the site models (M7 and M8) implemented in PAML 4.7. To enhance the reliability of positively selected site identification across the 15 rodent species, two additional models-the Fixed Effects Likelihood (FEL) and Single-Likelihood Ancestor Counting (SLAC) approaches-accessible via the Datamonkey web server (http://www.datamonkey.org), were also employed. The strength of natural selection was quantified by calculating the ratio of nonsynonymous (dN) to synonymous (dS) substitution rates (ω = dN/dS). Selective pressures were interpreted as follows: ω<1 indicates purifying selection; ω>1 indicates positive selection and ω = 1 suggests neutral evolution. Branch-specific models in PAML 4.7 were applied to test for positive selection acting along specific lineages of rodent AQP genes (Zhang et al., 2025). Finally, branch-site models were used to examine whether AQP genes in rodent species inhabiting arid environments have undergone positive selection.
     
    Water deprivation experiment design
     
    Wild-caught E. miletus individuals were collected from farmland and shrubland habitats in Xiaguan Town, Dali City, Yunnan Province, China (GPS coordinates: 25°34′54″ N, 100°14′6″E; altitude: 2061 m). Following capture, all animals were disinfected and cleared of ectoparasites by local health authorities prior to transportation. Thereafter, the animals were transferred to the laboratory animal facility of Yunnan Normal University. They were individually housed in standard cages (260 mm × 160 mm × 150 mm) under a 12-hour light:12-hour dark photoperiod and a consistent surrounding temperature held at 25±1°C. All animals were provided with a standard laboratory diet (produced by Kunming Medical University) and were acclimatized for four weeks with food and water available ad libitum. Only healthy, non-reproductive adult individuals were selected for the experiment. Post-acclimation, the animals were randomly assigned to one of two experimental groups: a water-deprived treatment group (n = 6), which received standard feed but no water access and a control group (n = 6), which maintained ad libitum access to both food and water. Pre-experiment body weights did not differ significantly between the two groups (p>0.05). At the conclusion of the experimental period, all animals were humanely euthanized via CO‚  asphyxiation. Target tissues were promptly dissected, forthwith flash-frozen in liquid nitrogen and preserved at -80°C for subsequent molecular assays. As AQP1, AQP5 and AQP7 have been extensively characterized in previous literature and because AQP10 deficiency has been reported to produce minimal phenotypic consequences in murine models (Morinaga et al., 2002; Rebez and Jacob, 2024), the present study focused on the functional validation of AQP11.
     
    ELISA for AQP11 protein in E. miletus
     
    AQP11 expression levels were quantified in seven tissue types-kidney, brain, liver, heart, small intestine, white adipose tissue (WAT) and brown adipose tissue (BAT)-using ELISA. Frozen tissue samples of E. miletus, stored at -80°C, were analyzed. The measurements were performed using a commercially available rat AQP11 ELISA Kit (Baika Biological Technology Co., Kunming, China), strictly following the manufacturer’s protocol.
     
    Data processing and analysis
     
    SPSS Statistics version 27.0 was employed for all statistical analyses. First, the distribution of the data was assessed using normality tests. With the exception of the AQP11 expression data in BAT, which deviated from a normal distribution, all other datasets satisfied the assumption of normality. Based on the distribution characteristics of the data, appropriate statistical methods were selected for between-group comparisons: For data meeting the normality assumption, Variations in the expression levels of AQP11 between the control and treatment groups within the same tissue type were assessed via an independent-samples t-test. In the case of BAT data with a non-normal distribution, the non-parametric Mann Whitney U test was employed. To compare AQP11 expression levels among different tissues within the same experimental group, one-way analysis of variance (one-way ANOVA) was used. Prior to conducting the ANOVA, the assumption of homogeneity of variance was verified. To identify specific group differences after a significant ANOVA, we applied Tukey’s HSD post hoc test for pairwise comparisons.
           
    All boxplot figures were generated using OriginPro 2024 software.

    RESULTS AND DISCUSSION

    Phylogenetic analysis of AQP genes
     
    AQPs are critical membrane proteins that regulate water homeostasis in organisms and are classified into three major categories: classical water-selective AQPs (AQP0, 1, 2, 4, 5 and 6). Among these, AQP2, 5 and 6 are functionally important in the skin, bladder, salt glands and kidneys across amphibians, sauropsids and mammals. (Morinaga et al., 2002; Nishimura and Yang, 2013). Deficiencies in these AQPs can result in severe physiological defects (Ikeda et al., 2002; Noda and Sasaki, 2006; Oshio et al., 2006). Classical aquaglyceroporins (AQP3, 7, 9 and 10) are involved in osmoregulation and energy metabolism through glycerol transport (Hara-Chikuma, 2005; Sohara et al., 2006) and play crucial roles in metalloid homeostasis (Bienert et al., 2008). Unorthodox AQPs (AQP11 and AQP12) are considered “superaquaporins” within the AQP subfamily (Ishibashi, 2006). Additionally, AQP8 is capable of transporting water, urea, ammonia and free radicals (Bienert et al., 2007).
           
    A total of 164 AQP genes were phylogenetically clustered into three major groups: AQP1, 2, 4, 5, 6 and 8 formed one cluster; AQP3, 7, 9 and 10 grouped together; while AQP11 formed a distinct separate branch. This phylogenetic structure corresponds exactly to the three recognized AQP subfamilies (Fig 1). Furthermore, AQP genes within each subfamily exhibited clearly resolved, species-specific phylogenetic relationships. Notably, rodent species inhabiting humid environments consistently clustered together within the phylogenetic tree.

    Fig 1: Phylogenetic tree of AQP1-11 genes.


           
    Within this research, phylogenetic trees were reconstructed based on AQP genes from 15 rodent species. The resulting clustering patterns revealed a correlation with species ecotypes. Specifically, lineages from humid habitats frequently formed independent clades, suggesting that adaptation to arid environments may have driven unique evolutionary changes in rodent AQP genes to fulfill physiological requirements under water stress. Interestingly, O. torridus-which inhabits arid regions including deserts, sand dunes and savannas in the western and southwestern United States and northern Mexico, where rainfall is relatively low-was found to group within a clade comprising species from more humid environments across the AQP1-11 gene tree (Harold and Egoscue, 1960). This anomalous positioning suggests that O. torridus may have evolved alternative physiological mechanisms for coping with water scarcity, potentially involving regulatory changes in AQP expression or function rather than rapid gene evolution. This contrasts with the rapid evolutionary pattern observed in AQP genes among other drought-adapted species, such as the genomic data from the comparatively arid and humid groups of Liangzhou donkeys, where the CYP4A11 gene was identified as a key factor in their adaptation to arid environments (Wang et al., 2022). This suggests that rodents may employ diverse molecular evolutionary pathways in responding to drought stress. However, the specific reasons still require subsequent experimental verification.
     
    Positively selected sites in rodent AQP genes
     
    To explore the molecular basis of adaptation in arid-adapted rodents, we identified sites under positive selection. Analysis of positively selected sites across the AQP gene family in the 15 rodent species identified a single positively selected site in each of three genes: site 131 in AQP1, site 111 in AQP5 and site 8 in AQP7. No significant signals of positive selection were detected in the remaining AQP genes analyzed (Table 2).

    Table 2: Site model testing of positive selection sites in reptile AQP genes.


           
    Signatures of positive selection were detected in AQP1, AQP5 and AQP7. The selection on AQP1, AQP5 and AQP7 aligns with their established roles in mediating adaptive responses to environmental stress in other mammals, such as enhanced renal water reabsorption in camels (Wang et al., 2014) and thermoregulation in goats (Kaushik et al., 2024). Notably, AQP7-the sole glycerol channel in adipose tissue-is closely linked to adipocyte morphology and physiology, obesity development and the maintenance of energy balance and glucose homeostasis (Miranda et al., 2010). Therefore, the positively selected sites identified in AQP1, AQP5 and AQP7 in this study may therefore represent adaptive modifications that enhance responses to environmental water variability by regulating water metabolism, thermoregulatory capacity and energy balance.
     
    Positive selection in AQP genes of rodents from arid habitats
     
    Various ω ratio models were employed to assess whether positive selection has acted on AQP genes in rodents. Significant heterogeneity in ω ratios among branches was identified for AQP2 (p = 0.0002*), AQP3 (p = 0.0074*), AQP5 (p = 0.0203*), AQP6 (p = 0.0034*), AQP9 (p< 0.0001), AQP10 (p<0.0001) and AQP11 (p = 0.0001*). In contrast, the remaining AQP genes exhibited no significant branch-specific variation in ω ratios (Table 3).

    Table 3: Phylogenetic analysis of positive selection in the AQP gene branch.


           
    Branch-site model A revealed positive selection acting on rodent AQP genes. Specifically, sites under significant positive selection were identified in AQP10 (site 294, p = 0.0020*) and AQP11 (site 184, p = 0.0037*) in species inhabiting arid environments. No significant signals of positive selection were detected in the other AQP genes (Table 4). Consequently, in environments with lower water content, AQP10 and AQP11 assume a more significant role in rodents. However, the specific reasons for this phenomenon require further investigation.

    Table 4: Selection pressure analysis of AQP gene orthologous sites in rodents.


     
    AQP11 expression in tissues of E. miletus
     
    AQP11 is an unconventional AQP that localises to the endoplasmic reticulum (ER) and is implicated in regulating subcellular water distribution among organelles, particularly in maintaining osmotic balance in critical organs such as the kidneys (Morishita et al., 2004). It functions as a peroxiporin, facilitating the transport of H2O2 across the ER membrane. Expressed in multiple tissues-including kidney, liver, intestine and brain-AQP11 contributes to organellar redox homeostasis and participates broadly in cellular stress responses (Bestetti et al., 2020; Markou et al., 2022; Yakata et al., 2007).
           
    Compared with the control group, water-deprived E. miletus exhibited significantly higher AQP11 expression (p<0.05) in the kidney, brain, liver, heart, small intestine, WAT and BAT (Fig 2). Post-hoc analysis further revealed that expression in the small intestine was significantly lower than in the other six tissues (p<0.05), indicating the most pronounced differential expression among all tissues examined.

    Fig 2: Box plot showing AQP11 expression levels in seven tissues of E. miletus.


           
    We quantified AQP11 expression in E. miletus under water-restricted conditions and observed significant upregulation across all seven tissues examined, with the most pronounced increase in the kidney. In mammals, AQP11 deficiency is associated with ER-derived vacuolation in proximal tubules, polycystic kidney disease and early renal failure, highlighting its essential role in renal tubule development and ER homeostasis (Michałek and Grabowska, 2019; Morishita et al., 2005; Schwartz and Johnson, 1971). The marked renal upregulation suggests a protective mechanism against osmotic stress, potentially through enhanced H2O2 efflux from the ER, thereby mitigating redox imbalance and preventing ER stress-induced pathology.
           
    Notable upregulation was also observed in the brain and heart, indicating a previously underappreciated role for AQP11 in supporting neural and cardiovascular function during dehydration. In the brain, AQP11 is localised to the choroid plexus epithelium and brain capillary endothelium, suggesting a possible role in water regulation at the blood-brain barrier (Benga and Huber, 2012).  Its activity may involve interplay with other AQPs, such as AQP4, in maintaining cerebral water balance (Zhang et al., 2022). Although the cardiac mechanism remains unclear, our data imply AQP11 involvement in systemic adaptation to osmotic stress.
           
    In metabolic tissues, strong upregulation in BAT points to a role in alleviating ER stress via H2O2 transport, which may be critical for sustaining thermogenesis and energy balance under metabolic challenge (Calamita and Delporte, 2021). Similarly, hepatic upregulation likely supports the clearance of H2O2 generated during oxidative protein folding, thereby promoting hepatocyte function under high ER load (Ishibashi et al., 2021). By contrast, AQP11 expression in the small intestine showed only a mild response to water stress, suggesting that other mechanisms may predominately regulate hydric balance in this organ under drought conditions.

    CONCLUSION

    AQPs are phylogenetically widespread across most species and investigations of AQP genes in rodents are likely to provide valuable insights for related human studies. Focusing on rodents with adaptations to arid habitats, this research identified a positively selected site within AQP11, suggesting that the gene may be critical for adaptive responses to xeric conditions. In E. miletus, AQP11 expression was significantly up-regulated across all seven tissues examined, supporting the hypothesis that AQP11 acts as a key physiological regulator of endoplasmic reticulum redox homeostasis-mediating H2O2 efflux and facilitating adaptive responses to water deprivation and other stressors in multiple organ systems. This study has certain limitations. Specifically, as the research focused on particular rodent species, the generalizability of the conclusions to a broader range of species requires further verification. Additionally, the relatively limited sample size may affect the statistical power of some analyses. Future studies could employ gain- and loss-of-function experiments while expanding both sample sizes and the range of species examined to more directly validate the specific role of AQP11 in adaptation to arid environments.

    ACKNOWLEDGEMENT

    This work was supported by the National Natural Scientific Foundation of China (No. 32560262; 32500436), the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities’ Association (NO. 202301BA070001-076).
     
    Ethical approval
     
    All animal procedures were within the rules of Animals Care and Use Committee of School of Life Sciences, Yunnan Normal University. This study was approved by the committee (13-0901-011).

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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