Independent Expansion of SLC7A3 in Mammals to Meet the Challenge of Nitrogen Metabolism

Independent Expansion of SLC7A3 in Mammals to Meet the Challenge of Nitrogen Metabolism | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Independent Expansion of SLC7A3 in Mammals to Meet the Challenge of Nitrogen Metabolism Jianghong Wu, Sile Hu, Jiahui Shi, Wei Li, Yan Zhu, Binhong Wen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8632196/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Adequate nitrogen metabolism is crucial for survival, yet carnivores and herbivores face distinct challenges: carnivores must efficiently detoxify ammonia, while herbivorous ruminants need to conserve and recycle nitrogen. Gene family expansion through duplication is a key evolutionary mechanism for adaptation. Here we show that the Cationic Amino Acid Transporter gene SLC7A3 has independently expanded via duplication in both ruminants (e.g., sheep, cattle) and carnivores to address their specific nitrogen balance pressures. These parallel expansions occurred at different, lineage-specific genomic locations. Following duplication, the SLC7A3 copies in carnivores underwent relaxed selection, whereas those in ruminants were under strong purifying selection. Expression analyses indicate high expression of these gene copies in intestines, blood, lungs, and spleen, suggesting a role in enhancing arginine transport to support urea cycle function. This study demonstrates convergent evolution through copy number variation, linking specific dietary challenges to large-scale genomic adaptation. Biological sciences/Evolution/Molecular evolution Biological sciences/Biochemistry/Ion channels Parallel Evolution SLC7A3 gene family Nitrogen balance Comparative Genomics Expression pattern Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction How species rapidly adapt to new ecological niches and physiological challenges is a central question in evolutionary biology[ 1 ]. The expansion of gene families is considered a key mechanism for providing new genetic material and facilitating functional innovation[ 2 ]. Typically, the expansion of functionally important genes occurs through post-speciation gene duplication, resulting in different species possessing varying copy numbers of a gene in homologous genomic regions[ 3 , 4 ]. This model predicts a clear history of duplication events in phylogenetic trees that corresponds with species divergence[ 5 ]. However, when distantly related species face remarkably similar environmental or physiological pressures, do they independently converge on expanding the same gene? If so, does this expansion stem from a single, ancient, shared ancestral event, or does it occur through independent and repeated events within their respective genomes? Investigating this can reveal the power of natural selection and the plasticity of genome evolution[ 6 ]. Both carnivores[ 7 ] and ruminants[ 8 ] derive the majority of their required glucose from gluconeogenesis. Ammonia is a highly toxic waste product generated when amino acids serve as substrates for gluconeogenesis[ 7 ] in carnivores. It is crucial for carnivores to rapidly remove excess ammonia to prevent ammonia toxicity[ 9 ]. For ruminants, nitrogen conservation and recycling are essential to cope with the persistent low-quality nitrogen in their diet and periodic nitrogen scarcity[ 10 ]. Consequently, the different challenges in nitrogen metabolism between carnivores and ruminants have shaped their respective paths of adaptive natural selection. Carnivores detoxify ammonia by rapidly converting it to urea for excretion[ 11 ]. In contrast, ruminants have evolved the urea cycle to recycle ammonia nitrogen back to the rumen for microbial reuse[ 12 ]. Both require that ammonia be converted into urea as completely as possible through the urea cycle. This scenario constitutes a strong natural selection pressure under the nitrogen metabolism for the specific mammalian lineage. The Solute Carrier family 7 (SLC7) is crucial for nitrogen metabolism because its transporters[ 13 ], particularly its cationic amino acid transporter (CAT) subfamily[ 14 ], plays a central role in amino acid transport and in supplying precursors for the urea cycle (such as L-arginine and L-ornithine)[ 14 , 15 ]. Although SLC7A3 (CAT-3) is known to be expressed in specific tissues[ 15 ], its evolutionary patterns in relation to ecological adaptation across the entire class of Mammalia, as well as its genomic evolutionary history, remain entirely unexplored. Existing research has predominantly focused on functional characterization within single species, lacking a macro-scale, comparative genomics perspective. In this study, we used sheep as a ruminant model to identify genes of the solute carrier (SLC) superfamily. Comparative genome analysis with humans revealed significant expansion of the SLC7 subfamily genes in sheep. Further investigation into SLC7 expansion patterns across mammals uncovered parallel evolution of SLC7A3 in both ruminants and carnivores, with expansion sites exhibiting lineage-specific distributions. This study presents a classic case of parallel evolution, but one that occurs at the relatively unstable genomic structural level of copy number variation rather than through the typically recognized convergent evolution of protein-coding sequences. This significantly expands our understanding of the molecular mechanisms underlying "convergent evolution." By directly linking a specific physiological challenge (ammonia metabolism) to macro-scale genomic evolutionary patterns, it establishes a paradigm for multi-level research bridging diet intaking, physiology, and genomics. Insights into the SLC7A3 expansion model will help explain interspecies differences (including livestock and humans) in protein metabolic efficiency, susceptibility to nitrogen imbalance diseases, and related traits. Results Identification of members of the os-SLC gene superfamily in Ovis aries We identified 438 SLC members for the genome of Ovis aries . Compare with Homo sapiens , the number of os-SLC has gained 75 members and lost 30 members. Table S1 provides basic information on the subcellular localization, PI, Molecular weight, lengths, instability index, Aliphatic index, GRAVY of the 438 os-SLC. The proteins produced by these genes have different lengths, with amino acids ranging from 130 AA (os-SLC51B) to 1239 AA (os-SLC4A2). The instability coefficients of these proteins range from 15.76 (os-SLC25A6) to 97.93 (os-SLC17A5). The predicted isoelectric points of the proteins range from 4.47 (os-SLC51B) to 10.09 (os-SLC25A17). Of these, 271 proteins have isoelectric points higher than 7, making them positively charged in acidic solutions. The proteins have different levels of hydrophilicity, ranging from − 0.585 (os-SLC39A10) to 0.917 (os-SLC22A18). Furthermore, 297 proteins are located on the cell membrane, 48 proteins are located on the endosome, 69 proteins are found in the endomembrane system. Phylogenetic analysis and classification of os-SLC proteins To investigate the evolutionary relationship of os-SLC proteins, a phylogenetic tree was constructed from 393 solute carrier gene superfamily from Homo sapiens (hs-SLC) proteins and 438 os-SLC proteins by the ML method (Fig. 1 ). A comparison of the SLC proteins in Homo sapiens and Ovis aries reveals homology among them. This phylogenetic analysis categorizes the 831 proteins into thirteen distinct clans, with the 438 os-SLC proteins from Ovis aries being distributed across various families. The clan 1 constitutes the second-largest branch within this evolutionary tree and it is primarily constituted by SCL2 , SLC8 , SLC12 , SLC25 , SLC27 , SLC28 , SLC35 , SLC39 and SLC41 gene family. The clan 2 is primarily constituted by SLC17 , SLC22 , SLC29 , SLC37 , SLC39 and SLC52 gene family. The clan 3 is primarily constituted by the SLC1 gene family. The clan 4 only includes 7 genes, which belong to SLC18 and SLC30 gene family. The clan 5 encompasses a wide variety of gene families, mainly consisting of SLC4 , SLC22 , SLC24 , SLC30 , SLC35 and SLC43 gene family. The clan 6 only includes 15 genes, which belong to SLC11 , SLC13 and SLC41 gene family. Clan 7 mainly encompasses SLC6 and six other minor members of the gene family. The clan 8 is the largest one among the branches of this tree, encompassing 248 genes. It is mainly constituted by 30 distinct gene families, among which are SLC7 , SLC9 , SLC16 , SLC26 , SLC35 and SLC44 gene family. Interestingly, family os-SLC7 may have undergone stronger expansions. From the evolutionary tree, the os-SLC7 gene family consists of 49 members, of which 37 are newly expanding genes (from os-LOC105605834 to os-LOC101115153 , os-LOC132657146 , os-LOC1114368 , os-LOC105610833 , os-LOC101107086 , red branches in Fig. 1 ) comparing with 12 members in human hs-SLC7 gene family. Gene duplication and collinearity analysis of os-SLC7A3 gene family During species evolution, gene duplication and retention of existing genes allow species genomes to expand. The distribution of the 49 os-SLC7 genes across chromosomes 1–26, X and Y exhibited non-uniformity (Fig. 2 ). Among these chromosomes, Chr14 have the highest number of os-SLC7 genes (38 genes, 77.55%), followed by Chr7, Chr9, and Chr17 (2 genes, 4.08%). Chromosomes 1, 3, 10, 26 and X contained 1 (2.04%) os-SLC7 genes, respectively. As can be seen from Fig. 3 , The segmental duplication of the os-SLC7 gene were not observed in Ovis aries , but a large number of tandem duplications were found. Chromosomes 14 contained the highest number of tandem duplication genes (34 genes), which belong to subfamily os-CAT3. These findings suggest that numerous os-SLC7 members might have originated from tandem duplication events. Next, we counted the members of the SLC gene family in six other species and found that, apart from sheep, the SLC7 subfamily members underwent significant expansion in horses, cattle, tigers, and domestic dogs (Table S2). Structure and motif composition of SLC7 expanded genes To determine the phylogeny relationship and structure of SLC7 expanded genes, a maximum likelihood tree for the os-SLC7 gene family was constructed and divided into two sub-branches (CATs clan and LATs clan) (Fig. 2 A), and there are significant differences in conserved motifs and domains between them (Fig. 3 -B, C). 34 expanding genes ( os-LOC101111827 to os-LOC101111313 ) and os-SLC7A1 ~ 4 , os-SLC7A14 were belong to the os-CATs clan, which have closest relationship with the os-SLC7A3 , encoding the CAT-3 cationic amino acid transporter. However, the os-SLC7A3 gene and its expanded genes are located on different chromosomes: the os-SLC7A3 gene is located on the X chromosome, while these expanded genes are situated on chromosome 14. The os-LATs clan contains os-SLC7A5 ~ 11 and 3 expanding genes (os- LOC101114368 , os-LOC105610833 and os-LOC101107086 ). To determine the composition and number of these conserved motifs in the os-SLC7 gene family, we utilized online MEME software. This analysis identified a total of 14 conserved motifs, labeled motif 1 to motif 14 (Fig. 3 -B). The presence of motif 2, 4, 7, 11, 13 and 14 is a common feature among the members of os-CATs clan, whereas the members of os-LATs clan only encompass a segment of motifs, specifically motifs 4, 6, 11, and 13 among others. The os-CATs clan protein matches with 2A0303 superfamily domain, and the os-LATs clan protein matches with 2A0308 superfamily domain (Fig. 3 -C). All os-SLC7 family members contain multiple exons and introns (Fig. 3 -D). The number of exons in the gene’s ranges from 13 to 3, the number and length of exons of genes situated on the same evolutionary tree branch are approximately similar. They contain introns, and are expressed in multiple tissues, which is inconsistent with the typical characteristics of retrogenes. Therefore, the expansion of SLC7A3 may result from genomic breakage and recombination. Expansion and loss of CAT during the evolution of mammalian Given substantial interspecies variation in CAT3 gene copy number, we further investigated the evolutionary dynamics of CAT gene family expansion and loss across mammals. By analyzing 27 mammalian species, lineage-specific expansion patterns of CAT1-4 genes were characterized (Table S4-S7). Our findings demonstrate that CAT3 and CAT4 underwent significant expansion in some clades. CAT3 genes expanded in Artiodactyla (Suina, Tylopoda and Ruminantia) and Carnivora , with particularly notable diversification within Artiodactyla : ruminants exhibited the highest copy numbers (sheep 38, goat 42, cattle 28, Zebu 35), while non-ruminants showed reduced counts (pig 16, hippopotamus 12, camel 5) (Fig. 4 ). Strikingly, cetaceans retained only 2 copies that is the lowest among Cetartiodactylas and comparable to non-cetartiodactyl species. The copy number of expanded SLC7A3 genes in each species may be influenced by the completeness of genome assembly and annotation. This ruminant-specific amplification suggests recent evolutionary innovation in the CAT3 subfamily. CAT3 exhibits significant copy number divergence even between closely related species Bos indicus (zebu cattle) and Bos taurus (taurine cattle) with 35 copies in zebu versus 28 in taurine cattle. Furthermore, it is unexpected that there are 41 copies CAT3 paralog genes in hybrid cattle ( Bos indicus x Bos taurus ). This disparity indicates post-speciation gene duplication events within the CAT3 family, suggesting that extensive duplicate of CAT3-like novel genes has occurred. Conversely, CAT4 expansion occurred exclusively in Perissodactyla, with all other lineages maintaining a single-copy state. To elucidate the evolutionary origins of the CAT3 and CAT4 gene families, we reconstructed the phylogeny of CAT3 and CAT4 paralogs individually. The expansion mechanisms of SLC7A3 and SLC7A4 differ significantly. In SLC7A3 , orthologous genes cluster together, with expansion members forming distinct clades, one cluster in Artiodactyla (even-toed ungulates), and another cluster in Carnivora (carnivores)(Fig. 5 A). These findings demonstrate lineage-specific convergent expansions occurring independently in each group following speciation for SLC7A3 . In contrast, the phylogenetic tree of SLC7A4 shows disorganized clustering among its expansion members, exhibiting no clear species-specific grouping pattern (Fig. 5 B). Analysis of SLC7A3 paralog exon numbers reveals that herbivores have exon counts concentrated between 8 and 12, whereas carnivores have exon counts concentrated between 0 and 4(Fig. 5 C-D). This may be attributed to relaxed selection allowing for the retention of gene mutations, which subsequently affects the recognition of gene structures such as introns and exons. Expansion locus of CAT3 between ruminants and Carnivora The results demonstrate that although evolutionary expansions of the SLC7A3 gene (CAT3) in Artiodactyla and Carnivora both adhere to deterministic patterns, they are driven by clade-specific genomic architectures. The localization of the original SLC7A3 gene to the X chromosome is a conserved feature across mammalian species (Table S6). Crucially, the chromosomal localization of expansion events differs fundamentally between these lineages. In Artiodactyla, the CAT3 expansion locus in the ZNF331-DPRX-NLRP12-MYADM-PRKCG-CACNG7 ( ZDNMPC ) cluster occupies an ancestral syntenic block conserved across most mammals (excluding Platypus, Kangaroo) in this study (Fig. 6 A). Within this region, humans retain two CAT3 pseudogenes, suggesting functional decay post-expansion in primates. The genome recombination happened in this block of hybrid cattle (Bos indicus × Bos taurus) compare with the Bos indicus and Bos taurus (Fig S1 ). A syntenic block was identified on chromosome 18, with its specific coordinates being 4.2–5.1 Mb in hybrid cattle, 60.8–61.6 Mb in Bos taurus , and 61-61.9 Mb in Bos indicus . Strikingly, the order of the gene cluster ZDNMPC was inverted in hybrid cattle relative to the other two species. Additionally, we observed that four specific genes ( LOC109572792 , LOC109571833 , LOC109571835 , LOC109572110 ) were present in two copies in the hybrid cattle, indicating a hybridization-induced gene duplication event at this CAT3 expansion locus (Fig. 6 C). Further research has revealed that genomic inversions and rearrangements also occur within the ZDNMPC region across different sheep breeds, and the copy number variation of the newly expanded SLC7A3 gene in this region is substantial. However, this region remains stable without undergoing genomic structural changes among Bactrian camels, dromedaries, and alpacas (Fig S2). In Carnivora, the expansion occurs within a derived syntenic region ( SLCO1A2-IAPP-PYROXD1-RECQL-GOLT1B-SPX-GYS2, SIPRGSG ), indicative of lineage-specific genomic reorganization (Fig. 6 B). Notably, no CAT3 pseudogenes are present in the homologous human locus ( Homo sapiens as an outgroup), underscoring Carnivora's unique evolutionary trajectory (Fig S3). Furthermore, a substantial fraction of the expanded CAT3 gene members has evolved into pseudogenes in the gray wolf and other carnivores (Table S6). Further research has revealed that genomic inversions and rearrangements also occur within the SIPRGSG region across different dog breeds, and the copy number variation of the newly expanded SLC7A3 gene in this region is substantial (Fig S4). The expansion event of the SLC7A3 gene may be associated with recombination hotspots within the region, disrupting genomic stability in this area and thereby making it prone to subsequent structural variations (such as inversions) and copy number changes. Expression pattern of the expansion of SLC7A3 genes family To better understand the basis for functional divergence, we conducted gene expression analysis of CAT3s in tissues of four representative species (sheep, cattle, goat, dog) (Fig. 7 A-D). Comparative analysis of the top 10 tissues with the highest expression across four species identified blood, spleen, intestines, and lungs as commonly expressed tissues, while the parathyroid gland, pituitary gland, tendon, cervix, bone marrow, and eyeball exhibited specific expression in domestic dogs (Fig. 7 E). This suggests that CAT3 expanded genes may function differently between ruminants and carnivores. Specifically, sheep showed highest expression in the thymus, goats in the intestine, cattle in the thyroid gland, and domestic dogs in the intestines. Expression levels of the expanded CAT3 members varied significantly among the species analyzed. Notably, the gene with the highest expression was species-specific: LOC101108819 in sheep, LOC108633279 in goats, LOC101904151 in cattle, and LOC119877631 in domestic dogs. Consistent with this pattern, no collinear relationship was detected among these genes, demonstrating strong evolutionary lineage-specificity. The results indicate that the pineal gland exhibits the highest expression levels among the examined organs in both cattle and goats. Specifically, transcript XM_013971618.1 ( LOC102190544 ) and transcript XM_024979355.2 ( LOC532048 ) show the highest specific expression in the pineal gland of goats and cattle, respectively. Although the goat LOC102190544 and cattle LOC532048 genes are both highly and specifically expressed in the pineal gland, the lack of collinearity between them suggests they are not orthologs derived from a common ancestor (Fig. 7 ). Their similar expression patterns likely result from convergent evolution, where distinct genes independently evolve to fulfill analogous roles in the pineal gland. Variation in the selective pressure over SLC7A3 gene family The results obtained from the one-ratio model indicated that the SLC7A3 gene was under purifying selection ( \(\:\omega\:\) = 0.65). This model gave a log maximum-likelihood value of -8477.11. The free‐ratio model assumes an independent \(\:\omega\:\) ratio for each branch in the tree. In the species tree, 453 branches were assumed to have their own \(\:\omega\:\) ratios under the free‐ratio estimation, which leads to a log maximum‐likelihood value of -8238.92. In comparison with the one‐ratio model, the free‐ratio model involved 452 extra parameters (Fig. 8 .). The branch-specific \(\:\omega\:\) values of Carnivora SLC7A3 paralog members show a significant increase, further confirming the relaxation of selective constraints, which has led to an increase in the number of pseudogenes within Carnivora SLC7A3 copies. The SLC7A3 genes and expanded SLC7A3 gene members of ruminants and carnivores branch was specified as a foreground branch to test for adaptive selection. Results of all two-ratio model tests for three branches are shown in Table 1 . The results showed that the carnivores branch had significantly higher \(\:\omega\:\) value compared with other branches, suggesting the potential action of positive selection. However, the original SLC7A3 gene and ruminants branch had significantly lower \(\:\omega\:\) value compared with other branches, suggesting the potential action of negative selection. Furthermore, we estimated \(\:\omega\:\) value utilized branch models with heterogeneous \(\:\omega\:\) cross three branches. The results showed that the selective pressure was variation in three branches (0.10 for SLC7A3 , 0.95 for carnivores, and 0.55 for ruminants in Table 1 ). Table 1 Parameter estimates under models of variable \(\:\omega\:\) ratios among lineages and LRTs of their fit to the SLC7A3 gene family between carnivora and ruminants. Models \(\:{\varvec{\omega\:}}_{0}\) \(\:{\varvec{\omega\:}}_{\varvec{S}\varvec{L}\varvec{C}7\varvec{A}3}\) \(\:{\varvec{\omega\:}}_{\varvec{C}}\) \(\:{\varvec{\omega\:}}_{\varvec{R}}\) ℓ P-value H 0 : \(\:\:{{\varvec{\omega\:}}_{0}=\varvec{\omega\:}}_{\varvec{S}\varvec{L}\varvec{C}7\varvec{A}3}={\varvec{\omega\:}}_{\varvec{C}}={\varvec{\omega\:}}_{\varvec{R}}\) 0.65 0.65 0.65 0.65 -8477.11 not allowed H 1 : \(\:\:{\varvec{\omega\:}}_{0}={\varvec{\omega\:}}_{\varvec{C}}={\varvec{\omega\:}}_{\varvec{R}}{\ne\:\varvec{\omega\:}}_{\varvec{S}\varvec{L}\varvec{C}7\varvec{A}3}\) 0.67 0.10 0.67 0.67 -8467.76 1.53×10 − 5 H 2 : \(\:\:{{\varvec{\omega\:}}_{0}=\varvec{\omega\:}}_{\varvec{S}\varvec{L}\varvec{C}7\varvec{A}3}={\varvec{\omega\:}}_{\varvec{C}}\ne\:{\varvec{\omega\:}}_{\varvec{R}}\) 0.78 0.78 0.78 0.55 -8471.31 6.60×10 − 4 H 3 : \(\:\:{{\varvec{\omega\:}}_{0}=\varvec{\omega\:}}_{\varvec{S}\varvec{L}\varvec{C}7\varvec{A}3}={\varvec{\omega\:}}_{\varvec{R}}\ne\:{\varvec{\omega\:}}_{\varvec{C}}\) 0.50 0.50 0.50 0.91 -8462.30 5.26×10 − 8 H 4 : \(\:\:{\varvec{\omega\:}}_{0}\ne\:{\varvec{\omega\:}}_{\varvec{S}\varvec{L}\varvec{C}7\varvec{A}3}\ne\:{\varvec{\omega\:}}_{\varvec{C}}\ne\:{\varvec{\omega\:}}_{\varvec{R}}\) 0.13 0.10 0.95 0.55 -8443.64 6.60×10 − 4 The topology and branch specific \(\:\varvec{\omega\:}\) ratios are presented in Fig. 8 . Discussion Changes in Earth's ecological environment (e.g., climatic shifts) can drive shifts in vegetation types[ 20 ], which directly trigger adaptive evolution in herbivorous ungulates and alter population (functional) diversity[ 23 , 24 ] and indirectly drive the evolution of carnivora[ 25 ]. Within this context, arginine serves as a key nitrogen storage form in plants for assimilating excess environmental ammonia. arginine is the primary amino acid form for nitrogen redistribution during autumn leaf senescence and winter storage. The stored nitrogen (in the form of arginine) is mobilized in spring to support the growth of new shoots[ 26 ]. Consequently, understanding how herbivores utilize plant-derived arginine is of critical importance. SLC transporters serve as ‘metabolic gate’ of cells and mediate the transport of a wide range of essential nutrients and metabolites such as glucose, amino acids, vitamins, neurotransmitters, and inorganic/metal ions. Interestingly, an additional 37 genes were identified in herbivores (sheep), which are homologous to the SLC7 gene family comparing with Omnivores (human). These results indicated that the expansion of SLC7 genes could have contributed to formation of unique traits of herbivores and meeting the demand of the lower nitrogen diets. Furthermore, ruminants can secrete urea back into the rumen through saliva, which is utilized by microorganisms for protein synthesis, significantly enhancing the utilization efficiency of low-quality plant resources [ 27 ]. The CATs members of SLC7 family serves as key proteins mediating the transmembrane transport of cationic amino acids such as arginine and lysine[ 14 ]. Furthermore, we investigated the evolutionary dynamics of CAT gene family expansion and loss across mammals. Expansions of the CAT gene family were observed in both herbivores and carnivores. Specifically, expansion of CAT3 subfamily genes occurred in ruminants and carnivores, while expansion of CAT4 was found in Perissodactyla. The expansion of CAT family members in these lineages may have been accompanied by changes in grassland vegetation. With the formation of grassland ecosystems during the Late Cretaceous around 70 million years ago [ 28 ], the ancestors of Perissodactyla diverged from other mammals around 56 million years ago, near or before the Paleocene-Eocene boundary[ 29 ]. Approximately 40 million years ago (late Eocene to early Oligocene), global climate underwent dramatic changes, with a sharp drop in temperature and increased aridity. This led to a contraction of global forests[ 30 , 31 ], while more drought-tolerant C3 herbaceous plants began to expand [ 30 ]. It is possible that perissodactyls collectively expanded CAT4 during this period to adapt to C3 grasses as a food source, enhancing nitrogen absorption capacity. This may explain why perissodactyls reached their peak diversity during the subsequent Oligocene to early Miocene[ 17 ]. Following the rise of C 4 plants to ecological dominance globally during the Late Miocene to Pliocene[ 18 , 31 ], the functional diversity of global ungulates began to decline after peaking around 10 million years ago, a trend synchronous with global aridification driven by the expansion of C 4 vegetation[ 23 ]. It was precisely during this period that artiodactyls (especially ruminants) underwent a crucial adaptive radiation and functional reorganization. This was facilitated by two key adaptations: first, the expansion of the CAT3 gene family, which maximized the absorption of arginine from C₄ plants to supplement nitrogen sources[ 26 ]; and second, the emergence of the rumen, which utilizes urea nitrogen. Together, these adaptations ensured that ruminants could meet their nitrogen demands even when feeding exclusively on C 4 plants, ultimately allowing them to become the dominant herbivores in grassland ecosystems by the Late Eocene[ 32 ]. In contrast, camels, whose primary food source is C 3 plants[ 33 , 34 ], and cetaceans, which inhabit aquatic environments, were not subjected to selection pressures from C 4 plants, and thus their CAT3 copy numbers almost unchanged. The large-scale turnover and adaptive radiation of herbivorous ungulates during the Late Miocene may have led to instability in prey sources for the ancestors of modern Carnivora[ 35 ], thereby preserving their omnivorous potential[ 36 , 37 ], This instability is likely a primary reason for the concurrent expansion of CAT3 within the order Carnivora. As carnivoran diets later stabilized towards specialization in meat consumption, the newly duplicated CAT3 copies gradually lost their function, becoming pseudogenes (as seen in gray wolves, tigers, etc.). Conversely, when species like the giant panda shifted their diet to become herbivorous, CAT3 underwent a secondary expansion. However, the underlying selective mechanisms remain to be fully elucidated: why did perissodactyls choose to expand CAT4 when C 3 vegetation was dominant, while artiodactyls and carnivorans opted for CAT3 expansion during the era of C 4 vegetation dominance. These question warrants further future investigation. The parallel expansion of SLC7A3 occurred at distinct genomic locations in different lineages. In ruminants, the expansion is fixed in an ancestral syntenic block ZDNMPC, whereas in carnivores, it is independently acquired in a derived region SIPRGSG . All placental mammals contain a single copy of the SLC7A3 gene within the ZDNMPC genomic structure of Boreoeutheria, while humans possess two pseudogene copies of SLC7A3 at this locus. Expansion of SLC7A3 through duplication appears to have initiated specifically within the Artiodactyla (even-toed ungulates). Notably, cetaceans within the Cetancodonta (which includes dolphins), having diverged and adapted to an aquatic environment, did not undergo this expansion. This difference may be directly related to dietary intake and could be associated with the expansion of C4 vegetation during the Artiodactyla radiation[ 24 ]. Following the divergence of Artiodactyla, the mechanisms by which environmental factors drove divergent species to select the same gene for duplication at a specific genomic locus warrant in-depth investigation. Multiple members of the SIPRGSG gene cluster (particularly SLCO1A2, IAPP, SPX, and GYS2) are directly involved in or regulate the absorption, metabolism, and homeostasis of nutrients, encompassing lipid absorption, glucose metabolism, and energy storage[ 38 – 41 ]. The fact that this cluster serves as the site for SLC7A3 expansion in carnivores suggests that, during evolution, genes related to nutrient absorption and metabolism may be functionally co-arranged in the genome to cope with specific dietary selection pressures. Members of the ZDNMPC gene cluster (particularly NLRP12 and the MYADM region) are directly or indirectly associated with nutrient absorption and metabolic processes. NLRP12 influences the absorption environment by regulating intestinal immunity[ 42 ], while the PRKCG participates in intracellular signaling triggered by insulin, affecting downstream pathways that control glucose uptake, utilization, and storage in cells[ 43 ]. As the site of expansion for a key metabolic gene (SLC7A3) in ruminants, this cluster suggests that during evolution, genes related to nutrient absorption, metabolism, and immune regulation may be functionally co-arranged in the genome to cope with specific dietary and digestive ecological pressures. This pattern may be associated with differences in the nutritional status of food intake between herbivores and carnivores. The nutritional status of an organism acts as a powerful selective pressure, leaving characteristic signatures within its genome[ 44 ]. There is a significant difference in nitrogen intake between carnivores and ruminants[ 36 ]. Through large-scale comparative genomics and evolutionary analysis across species, we revealed a phenomenon that defies the traditional paradigm of single-origin inheritance. The SLC7A3 gene has undergone significant, independent expansions in multiple, distantly related mammalian lineages, such as Carnivora and Artiodactyla. Critical evidence lies in the fact that these expansions occurred at distinct genomic loci and contradict the expected species phylogeny. This indicates that these are not conserved remnants of a single ancient duplication event inherited across lineages. Instead, repeated, independent selective sweeps favoring increased SLC7A3 copy number occurred in each lineage in response to divergent nitrogen metabolic pressures (high ammonia load from protein-rich diets in carnivores[ 9 ] versus heightened efficiency of nitrogen utilization on protein-scarce diets in ruminants[ 45 , 46 ]). Further functional evolutionary analysis demonstrated that these independently expanded SLC7A3 copies are under different selection pressure, which indicated different lineage may experience different selection pressures based on their unique food intake, leading to divergent evolutionary paths. The expression pattern of SLC7A3 gene was difference between carnivores and ruminants, resulting in genes with different functions[ 47 ]. In ruminants, arginine contributes to urea production, while urea supports microbial protein synthesis—ensuring a symbiotic relationship that optimizes nitrogen use and reduces the host’s dependency on dietary amino acids[ 46 ]. This system is central to the ruminant’s ability to convert low-quality forage into high-quality protein[ 48 ]. Unlike monogastric animals, ruminants recycle urea back to the rumen. Urea produced in the liver enters the bloodstream and is excreted (via urine) or transported to the rumen via saliva or direct diffusion[ 49 ]. Mammals repeatedly adapted to specialized nitrogen balance of diets intake, including plant-based diets for herbivores and meat-based diets for carnivores. The function of the CAT family (including SLC7A3 ) is to transport arginine, ornithine, and lysine[ 14 ], which particularly critical in nitrogen metabolic pathways and urea cycle[ 50 ]. Our extensive cross-species comparative genomic and evolutionary analyses reveal a paradigm-shifting finding: SLC7A3 has repeatedly expanded independently and significantly in distantly related mammals like Carnivora (carnivores) and Artiodactyla (ungulates/ruminants). Carnivores must continuously detoxify and excrete the large amounts of ammonia generated from nitrogen metabolism[ 9 ], whereas ruminants require the retention of ingested nitrogen to achieve high nitrogen utilization efficiency[ 48 ]. Although carnivores and ruminants face distinctly different nitrogen metabolic demands, both processes likely necessitate enhanced efficiency of the urea cycle. This is because the urea cycle plays a pivotal role both in the detoxification of ammonia to urea for excretion in carnivores and in the reutilization of urea as a non-protein nitrogen source in ruminants. Our study shows that SLC7A3 gene families have been repeatedly recruited in convergently evolved urea cycle, even across vast evolutionary timescales. Gene duplication plays a crucial role in the formation of novel traits throughout animal evolution, from lower to higher organisms[ 2 , 51 , 52 ]. Key evidence includes the expansions occurring at specific, non-conserved genomic locations mismatched with phylogenetic relationships. This demonstrates these expansions are not passive inheritance of an ancient duplication event, but rather the result of repeated, parallel natural selection for increased SLC7A3 copy number-likely driven by contrasting nitrogen metabolic demands (ammonia stress vs. nitrogen conservation). These results indicate that animals from different lineages have converged on duplicating the same gene to cope with nitrogen metabolism challenges, but the locations of these expansions in the genome vary across lineages, while they are identical within the same lineage. Additionally, we found that after hybridization between zebu cattle and taurine cattle, the copy number of SLC7A3 in the hybrid offspring differed from that of the parental lines, and this chromosomal region is prone to rearrangement and duplication during hybridization. This phenomenon suggests that the copy number of the SLC7A3 gene may exhibit copy number variation among different populations within the same animal species[ 53 ]. The rearrangement and combination of exons allow rapid creation of SLC7A3 genes with different functions, a key driver in gene family expansion. Comparison of the expression levels of SLC7A3 expanded members across different tissues in three ruminants and one carnivore revealed that tissues with consistently high expression in all four species include blood, intestine, spleen, and lung. The overlap in highly expressed organs (such as intestine, blood, lung, and spleen) suggests that both animal groups face common ammonia-nitrogen pressure and have evolved similar adaptive strategies: enhancing arginine transport to optimize urea cycle function and immune responses. Despite the conventional notion that the urea cycle takes place exclusively in the liver, here we demonstrate that these tissues also have the potential to maintain nitrogen balance. High expression in blood ensures efficient systemic circulation of arginine, supporting urea synthesis and effecting the nitrogen metabolism[ 54 ]. The intestine, as the frontline site for arginine absorption from plant[ 26 ], expresses expanded SLC7A3 members to improve arginine uptake efficiency[ 55 ]. Increased expression in the lungs can elevate arginine levels in pulmonary cells. Arginine serves as a substrate for nitric oxide synthase (NOS) in the lungs, participating in the production of nitric oxide (NO), thereby playing multiple key physiological and pathophysiological roles in the lungs and airways[ 56 , 57 ]. The finding that the other six highly expressed tissues are entirely distinct between carnivores and ruminants indicates that the function of SLC7A3 diverged, following distinct evolutionary paths in the two lineages. Even the most highly expressed SLC7A3 gene copy in each species shows no synteny across the ruminants. This indicates that the copy number expansion of SLC7A3 and gain of function has followed independent, species-specific evolutionary trajectories. Functional evolutionary analysis demonstrated that these independently expanded SLC7A3 copies are under different selection pressure between carnivores and ruminants. Following the duplication of the SLC7A3 gene, the new copies in ruminants and carnivores likely evolved toward different functions[ 58 ]. As the environment changed, the new copies in carnivores gradually became redundant, with reduced environmental constraints leading to increased genetic variation[ 59 ]. This ultimately resulted in these copies becoming pseudogenes within the carnivore lineage. Only a subset of SLC7A3 gene members was retained to meet the demands of arginine absorption and ammonia detoxification. This has enabled carnivoran animals to retain evolutionary plasticity, allowing them to evolve toward omnivory or herbivory (as seen in the giant panda and the dog), as well as toward strict carnivory (as seen pseudogenes in the gray wolf and dingo). This strongly implicates enhanced transport capacity for arginine in ammonia detoxification pathways as the adaptive driver behind these convergent evolutionary events. In contrast, ruminants, due to the low nitrogen content in their ingested food, evolved the ability to utilize non-protein nitrogen (urea) via rumen microbes to maintain nitrogen balance. This adaptation allows for the maximal utilization of dietary nitrogen, particularly under conditions of nutritional scarcity such as on cold-season grasslands[ 48 ], in order to sustain vital functions. This may also explain why the newly duplicated members of SLC7A3 in ruminants faced greater selective pressure than carnivores. Conclusion The study reveals that the SLC7A3 gene, part of the Cationic Amino Acid Transporter (CAT) family, has undergone independent expansion via gene duplication in two distinct mammalian lineages—ruminants (herbivores) and carnivores—to address challenges related to nitrogen metabolism and balance. Following duplication, the SLC7A3 copies in carnivores and ruminants experienced different evolutionary pressures. Genes in the carnivore branch underwent relaxed selection, while those in the ruminant branch were under strong purifying selection. The genomic locations of these independent expansion events differ between the two lineages but are conserved within each lineage. In ruminants, expansion occurred in an ancestral syntenic block (ZDNMPC cluster), while in carnivores, it occurred in a derived region (SIPRGSG cluster). The SLC7A3 paralogs are highly expressed in tissues like intestines, blood, lungs, and spleen. This suggests their role is to optimize arginine absorption and urea cycle function, enhancing the transport of arginine—a key precursor for the urea cycle. Despite facing opposite nitrogen balance challenges (carnivores need to efficiently detoxify and excrete ammonia as urea, while ruminants need to recycle nitrogen via urea for microbial protein synthesis), both lineages convergently evolved SLC7A3 gene expansion as a strategy to enhance urea cycle and arginine transport capabilities. This represents a classic case of parallel evolution, but occurring at the level of genomic structural variation (copy number variation) rather than through convergent evolution of protein-coding sequences. This expands the understanding of molecular mechanisms underlying convergent evolution. The study directly links a specific physiological challenge (ammonia metabolism under different dietary nitrogen regimes) to large-scale genomic evolutionary patterns, establishing a research paradigm bridging diet, physiology, and genomics. Materials and methods Identification of SLC gene superfamily members We use the sheep genome as the focal reference to identify SLC gene during ruminant evolution. The genomic data of Ovis aries and Homo sapiens were obtained from the genome database of NCBI website ( https://www.ncbi.nlm.nih.gov/datasets/genome (GCF_016772045.2, GCA_ 000001405.29). Then we obtained the hidden Markov model (HMM) profile of the SLC superfamily from Pfam protein family database (Table S3). We found 438 os-SLC genes from Ovis aries genome. Specifically, According to the study about human SLC gene superfamily[ 60 ], we downloaded 392 SLC superfamily members of Homo sapiens (hs-SLC) protein sequences from UniProt database. We identified all os-SLC genes from the Ovis aries genome that had these hs-SLC protein sequences as targets. The conserved domains of these os-SLC genes were analyzed, and the genes without the SLC domain were removed. The identified Ovis aries SLC genes were then queried via BLASTp in the NCBI database to determine whether they belonged to the os-SLC gene family. Finally, 438 genes with SLC domains were identified at the genome-wide scale. Sequences of CATs of 27 mammalian species representative of Primates, Rodentia, Lagomorpha, Artiodactyla, Perissodactyla, Carnivora, Chiroptera, Soricomorpha, Proboscidea, Kangaroo order and Monotremes were obtained from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ ). Phylogenetic analysis and gene structure The SLC proteins of Homo sapiens for the construction of Maximum Likelihood (ML) systematic evolutionary trees were obtained from the UniProt database ( https://www.uniprot.org ). The sequences of the SLC proteins in Homo sapiens and Ovis aries were compared with Clustal Omega. The systematic evolutionary tree included Homo sapiens and Ovis aries and was constructed using IQtree with the ML method[ 61 ], and the Best-fit model was: PMB + F + R5 chosen according to bayesian information criterion[ 62 ]. The DNA and cDNA sequences of the os-SLC genes were used to predict intron structure via the online Gene Structure Display Service ( http://gsds.cbi.pku.edu.cn ). The conserved motifs of os-SLC proteins in Ovis aries were determined with MEME software ( http:/meme.nbcr.net/meme/intro.html ) and in accordance with the corresponding parameters set by Liu et al.[ 63 ]. Intraspecific and Interspecific collinearity analysis The locations of 51 os-SLC7 genes on chromosomes were obtained based on the information annotated for the Ovis aries genome and analyzed through the Gene Location Visualization of TBtools[ 64 ]. The genome sequence and GTF file of Homo sapiens (GCF_000001405.40), Capra hircus (GCF_001704415.2), Bos taurus (GCA_002263795.4), Camelus dromedarius (GCF_036321535.1), Tursiops truncatus (GCF_011762595.1) downloaded from NCBI. Analysis of genome collinearity was performed using MCScanX software[ 65 ]. Circos[ 66 ] and Dual Synteny Plot were used Comparative Genome Viewer online tools ( https://www.ncbi.nlm.nih.gov/cgv/15545/9940 ) for visualized mapping of the collinear gene pairs. Search and filter for functionally characterized CATs gene in mammalians Data for the 666 functionally characterized CATs in 27 mammalians (including 6 Carnivoras , and 5 Perissodactylas , and 8 Artiodactylas , and 1 Cetaceas , and 1 Chiroptera , and 1 Insectivora , and 1 Rodentia , and 1 Lagomorpha , and 1 Primates , and 1 Proboscidea , and 1 Marsupialia , and 1 Monotreme ) (Tables S4-S7) were sourced from GenBank. In addition, the distribution of exon counts of SLC7A3 expanded genes were analyzed between carnivores and herbivores based on the genome annotation file. Variation in Selective Pressure between Ruminants and Carnivores in CAT3s Expansion members of SLC7A3 originated from tandem duplication events with lineage-specific convergent expansions. We therefore examined the selection pressures on the candidate branch as foreground by comparing their sequences with the background branch and calculating the ratio of non-synonymous substitution rate ( dN ) to synonymous substitution rate ( dS ) between the paralogs. We first aligned the codon sequences of the SLC7A3 and their paralogous copies using ClustalW codon in MEGA12[ 67 ], and then transformed the results into codon-level alignment using the DAMBE tool[ 68 ]. Then, we calculated the ω values (dN/dS) and performed the likelihood ratio test (LRT) using CODEML in the PAML4.10 package [ 32 ]. We also execute CODEML under the branch model when assuming different selective pressures (i.e., different ω ratios) for ruminants and carnivores linages on the tree and conduct an LRT for positive selection affecting prespecified branches using M0 as the null model. The expression analysis of CAT3s from RNA‑Seq Data To study the expression patterns of CAT3s gene in different organ between ruminant and carnivora, the RNA-seq data of 3 ruminant animals[ 69 ] and 1 carnivores’ animal[ 70 ] were downloaded from the NCBI (Accession number: PRJNA1017964, PRJNA867700 and PRJNA 906635). The gene expression level (transcripts per million) TPM was calculated using the Salmon tool [ 71 ]. Salmon estimated transcript abundances for each sample based on the FASTQ files. After running salmon quant for all samples, the individual TPM results were merged into a single table for easier downstream analysis using the salmon quantmerge command. The average expression value from the biological replication represents the organ expression level for each species. Finally, the CAT3s expansion gene member’s expression values were extracted for downstream analysis (Table S8). Declarations Competing interests The authors declare that there is no conflict of interests. Author Contributions Conceptualization, J.W. and S.H.; methodology, B.W. and W.L.; software, Y.Z.; validation, J.S., Y.Z. and J.W.; formal analysis, D.W.; investigation, J.S. and B.W.; resources, J.S.; data curation, J.S. and Y.Z.; writing—original draft preparation, S.H.; writing—review and editing, J.W. and J.Y.; visualization, W.L.; supervision, J.W.; project administration, S.H.; funding acquisition, J.W. and S.H.. All authors have read and agreed to the published version of the manuscript. Acknowledgements The authors are grateful for the support by the National Natural Science Foundation of China (32260812), the Inner Mongolia Autonomous Region’s Key Technology Tackling Plan (2023YFDZ0027, 2025YFDZ0135), the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region (2023NJYT23015), the Youth Program for Grassland Elite in Inner Mongolia Autonomous Region for Dr. Wu and First-class Discipline Research Special Project in Inner Mongolia Autonomous Region Department of Education(YLXKZX-NMD-009), the Inner Mongolia Autonomous Region’s Key Technology Tackling Plan (2025KJHZ0001) for Dr. Hu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Bates, O.K., Bertelsmeier, C.: Climatic niche shifts in introduced species. Curr. Biol. 31 (19), R1252–R66 (2021). https://doi.org/10.1016/j.cub.2021.08.035 David, K.T., Schraiber, J.G., Crandall, J.G., Labella, A.L., Opulente, D.A., Harrison, M.-C., et al.: Convergent expansions of keystone gene families drive metabolic innovation in Saccharomycotina yeasts. Proceedings of the National Academy of Sciences. ;122(23):e2500165122. doi: (2025). 10.1073/pnas.2500165122 David, K.T., Oaks, J.R., Halanych, K.M.: Patterns of gene evolution following duplications and speciations in vertebrates. PeerJ. 8 , e8813 (2020). Epub 2020/04/09 10.7717/peerj.8813 Kondrashov, F.A.: Gene duplication as a mechanism of genomic adaptation to a changing environment. Proceedings Biological sciences. ;279(1749):5048-57. Epub 2012/09/15. (2012). 10.1098/rspb.2012.1108 Lallemand, T., Leduc, M., Landès, C., Rizzon, C., Lerat, E.: An Overview of Duplicated Gene Detection Methods: Why the Duplication Mechanism Has to Be Accounted for in Their Choice. Genes. 11 (9) (2020). Epub 2020/09/10 10.3390/genes11091046 Gabora, L.: Convergent Evolution. In: Maloy S, Hughes K, editors. Brenner's Encyclopedia of Genetics (Second Edition). San Diego: Academic Press; pp. 178 – 80. (2013) Schermerhorn, T.: Normal glucose metabolism in carnivores overlaps with diabetes pathology in non-carnivores. Front. Endocrinol. 4 , 188 (2013). Epub 2013/12/19 10.3389/fendo.2013.00188 Wang, G., Zhu, Y., Feng, D., Yao, J., Cao, Y., Deng, L.: Hepatic gluconeogenesis and regulatory mechanisms in lactating ruminants: A literature review. Anim. Res. One Health. 3 (3), 230–239 (2025) Morris, J.G.: Nutritional and Metabolic Responses to Arginine Deficiency in Carnivores. J. Nutr. 115 (4), 524–531 (1985). https://doi.org/10.1093/jn/115.4.524 Zhang, X.M., Chen, W.X., Yan, Q.X., Wang, C., Lin, B., Yi, S.Y., et al.: Low-protein diet promotes nitrogen retention efficiency via enhanced renal urea reabsorption and microbial hydrogen incorporation in the rumen of goats. Anim. Feed Sci. Technol. 305 , 115762 (2023). https://doi.org/10.1016/j.anifeedsci.2023.115762 Singer, M.A., Part, B.: Biochem. Mol. Biology. 134 (4), 543–558 (2003). https://doi.org/10.1016/S1096-4959(03)00027-7 Hristov, A.N., Bannink, A., Crompton, L.A., Huhtanen, P., Kreuzer, M., McGee, M., et al.: Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques. J. Dairy Sci. 102 (7), 5811–5852 (2019). https://doi.org/10.3168/jds.2018-15829 Closs, E., Boissel, J.-P., Habermeier, A., Rotmann, A.: Structure and function of cationic amino acid transporters (CATs). J. Membr. Biol. 213 (2), 67–77 (2006) Jungnickel, K.E., Parker, J.L., Newstead, S.: Structural basis for amino acid transport by the CAT family of SLC7 transporters. Nat. Commun. 9 (1), 550 (2018) Fotiadis, D., Kanai, Y., Palacín, M.: The SLC3 and SLC7 families of amino acid transporters. Mol. Aspects Med. 34 (2–3), 139–158 (2013) Álvarez-Carretero, S., Tamuri, A.U., Battini, M., Nascimento, F.F., Carlisle, E., Asher, R.J., et al.: A species-level timeline of mammal evolution integrating phylogenomic data. Nature. 602 (7896), 263–267 (2022). 10.1038/s41586-021-04341-1 Steinthorsdottir, M., Coxall, H.K., Boer, A.M., Huber, M., Barbolini, N., Bradshaw, C.D., et al.: The Miocene: The Future of the Past. Paleoceanography Paleoclimatology ; 36 . (2020) Edwards, E.J., Osborne, C.P., Strömberg, C.A., Smith, S.A., Bond, W.J., Christin, P.A., et al.: The origins of C4 grasslands: integrating evolutionary and ecosystem science. Sci. (New York NY). 328 (5978), 587–591 (2010). Epub 2010/05/01 10.1126/science.1177216 de Deus Vidal, J., le Roux, P.C., Johnson, S.D., te Beest, M., Clark, V.R. (eds.): Beyond the Tree-Line: The C3-C4 Grass-Line Can Track Global Change in the World’s Grassy Mountain Systems. Frontiers in Ecology and Evolution (2021) Sage, R.F., Christin, P.A., Edwards, E.J.: The C(4) plant lineages of planet Earth. J. Exp. Bot. 62 (9), 3155–3169 (2011). Epub 2011/03/19 10.1093/jxb/err048 Buckley, M.: Ancient collagen reveals evolutionary history of the endemic South American ‘ungulates’. Proceedings of the Royal Society B: Biological Sciences. ;282(1806):20142671. (2015) Ding, W., Silvestro, D., Onstein, R.E., Wu, M., Zhou, Z., Xing, Y.: The stepwise rise of angiosperm-dominated terrestrial ecosystems. Biol. Rev. (2025) Blanco, F., Lazagabaster, I.A., Sanisidro, Ó., Bibi, F., Heckeberg, N.S., Ríos, M., et al.: Two major ecological shifts shaped 60 million years of ungulate faunal evolution. Nat. Commun. 16 (1), 4648 (2025). Epub 2025/06/06 10.1038/s41467-025-59974-x Abraham, J.O., Upham, N.S., Damian-Serrano, A., Jesmer, B.R.: Evolutionary causes and consequences of ungulate migration. Nat. Ecol. Evol. 6 (7), 998–1006 (2022). 10.1038/s41559-022-01749-4 Wu, Y., Wang, H., Wang, H., Feng, J.: Arms race of temporal partitioning between carnivorous and herbivorous mammals. Sci. Rep. 8 (1), 1713 (2018). 10.1038/s41598-018-20098-6 Ávila, C., Llebrés, M.T., Cánovas, F.M., Castro-Rodríguez, V.: Arginine, a key amino acid for nitrogen nutrition and metabolism of forest trees. J. Exp. Bot. 76 (18), 5238–5251 (2025). Epub 2025/06/10 10.1093/jxb/eraf260 Hailemariam, S., Zhao, S., He, Y., Wang, J.: Urea transport and hydrolysis in the rumen: a review. Anim. Nutr. 7 (4), 989–996 (2021) Raven, J., Thomas, H., Grasses: Curr. Biol. 20 (19), R837–R9 (2010). 10.1016/j.cub.2010.08.031 Bai, B., Wang, Y.-Q., Meng, J.: The divergence and dispersal of early perissodactyls as evidenced by early Eocene equids from Asia. Commun. Biology. 1 (1), 115 (2018). 10.1038/s42003-018-0116-5 Wang, J., Zhou, X., Wang, S., Ye, J., Liu, J., Wu, W., et al.: Steppe development and mammalian adaptation in the middle Miocene, North Junggar Basin, Central Asia. Palaeogeography, Palaeoclimatology, Palaeoecology. ; 659 :112634. (2025). https://doi.org/10.1016/j.palaeo.2024.112634 Ding, W., Silvestro, D., Onstein, R.E., Wu, M., Zhou, Z., Xing, Y.: The stepwise rise of angiosperm-dominated terrestrial ecosystems. Biol. Rev. Camb. Philos. Soc. 100 (5), 2131–2149 (2025). Epub 2025/05/30 10.1111/brv.70039 Katona, K., Coetsee, C.: Impacts of Browsing and Grazing Ungulates on Faunal Biodiversity. In: Gordon, I.J., Prins, H.H.T. (eds.) The Ecology of Browsing and Grazing II, pp. 277–300. Springer International Publishing, Cham (2019) Saarinen, J.: The Palaeontology of Browsing and Grazing. In: Gordon, I.J., Prins, H.H.T. (eds.) The Ecology of Browsing and Grazing II, pp. 5–59. Springer International Publishing, Cham (2019) Colledge, S., Conolly, J., Dobney, K., Manning, K., Shennan, S.: The Origins and Spread of Domestic Animals in Southwest Asia and Europe. Eur. J. Archaeol. 18 (2), 356–360 (2015) Wang, S.-Q., Jiangzuo, Q., Bibi, F., Zhang, C., Li, C., Li, S., et al.: Central Asian radiation of modern large-mammal faunas in Miocene. Sci. Adv. 11 (43), eadt5079 (2025). 10.1126/sciadv.adt5079 Hecker, N., Sharma, V., Hiller, M.: Convergent gene losses illuminate metabolic and physiological changes in herbivores and carnivores. Proc. Natl. Acad. Sci. U.S.A. 116 (8), 3036–3041 (2019). Epub 2019/02/06 10.1073/pnas.1818504116 Morris, J.G.: Nutritional and metabolic responses to arginine deficiency in carnivores. J. Nutr. 115 (4), 524–531 (1985) Khan, A., Liu, Y., Gad, M., Kenny, T.C., Birsoy, K.: Solute carriers: The gatekeepers of metabolism. Cell. 188 (4), 869–884 (2025). 10.1016/j.cell.2025.01.015 Lai, Q., Ma, Y., Bai, J., Zhuang, M., Pei, S., He, N., et al.: Mechanisms for Bile Acids CDCA- and DCA-Stimulated Hepatic Spexin Expression. Cells. 11 (14) (2022). Epub 2022/07/28 10.3390/cells11142159 Karlsson, E.: IAPP as a regulator of glucose homeostasis and pancreatic hormone secretion (review). Int. J. Mol. Med. 3 (6), 577–584 (1999). Epub 1999/05/26 10.3892/ijmm.3.6.577 von Wilamowitz-Moellendorff, A., Hunter, R.W., García-Rocha, M., Kang, L., López-Soldado, I., Lantier, L., et al.: Glucose-6-phosphate-mediated activation of liver glycogen synthase plays a key role in hepatic glycogen synthesis. Diabetes. 62 (12), 4070–4082 (2013). Epub 2013/08/31 10.2337/db13-0880 Truax, A.D., Chen, L., Tam, J.W., Cheng, N., Guo, H., Koblansky, A.A., et al.: The Inhibitory Innate Immune Sensor NLRP12 Maintains a Threshold against Obesity by Regulating Gut Microbiota Homeostasis. Cell. Host Microbe. 24 (3), 364–78e6 (2018). https://doi.org/10.1016/j.chom.2018.08.009 Ross, R., Wang, P.Y., Chari, M., Lam, C.K., Caspi, L., Ono, H., et al.: Hypothalamic protein kinase C regulates glucose production. Diabetes. 57 (8), 2061–2065 (2008). Epub 2008/05/31 10.2337/db08-0206 Maharjan, R.P., Ferenci, T.: A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input–mutation output relationships. PLoS Biol. 15 (6), e2001477 (2017) Getahun, D., Alemneh, T., Akeberegn, D., Getabalew, M., Zewdie, D.: Urea metabolism and recycling in ruminants. Biomed. J. Sci. Tech. Res. 20 (1), 14790–14796 (2019) Huntington, G., Archibeque, S. (eds.): Practical aspects of urea and ammonia metabolism in ruminants. Proc Am Soc Anim Sci (1999) DuBose, J.G., de Roode, J.C.: The link between gene duplication and divergent patterns of gene expression across a complex life cycle. Evol. Lett. 8 (5), 726–734 (2024). 10.1093/evlett/qrae028 Poppi, D.P., McLennan, S.R.: Protein and energy utilization by ruminants at pasture. J. Anim. Sci. 73 (1), 278–290 (1995) Hailemariam, S., Zhao, S., He, Y., Wang, J.: Urea transport and hydrolysis in the rumen: A review. Animal nutrition (Zhongguo xu mu shou yi xue hui). ; 7 (4):989–996. Epub 2021/11/06. (2021). 10.1016/j.aninu.2021.07.002 Meireles, P., Mendes, A.M., Aroeira, R.I., Mounce, B.C., Vignuzzi, M., Staines, H.M., et al.: Uptake and metabolism of arginine impact Plasmodium development in the liver. Sci. Rep. 7 (1), 4072 (2017). 10.1038/s41598-017-04424-y Luzuriaga-Neira, A., Subramanian, K., Alvarez-Ponce, D.: Functional compensation of mouse duplicates by their paralogs expressed in the same tissues. Genome biology and evolution. ;14(8). Epub 2022/08/11. (2022). 10.1093/gbe/evac126 Conrad, B., Antonarakis, S.E.: Gene Duplication: A Drive for Phenotypic Diversity and Cause of Human Disease. Annual Review of Genomics and Human Genetics. ;8(Volume 8, 2007):17–35. (2007). https://doi.org/10.1146/annurev.genom.8.021307.110233 Schäfer, Y., Palitzsch, K., Leptin, M., Whiteley, A.R., Wiehe, T., Suurväli, J.: Copy number variation and population-specific immune genes in the model vertebrate zebrafish. Elife 2024;13. Epub 2024/06/04. 10.7554/eLife.98058 Reisz, J.A., Earley, E.J., Nemkov, T., Key, A., Stephenson, D., Keele, G.R., et al.: Arginine metabolism is a biomarker of red blood cell and human aging. Aging cell. 24 (2), e14388 (2025). Epub 2024/10/31 10.1111/acel.14388 Nüse, B., Holland, T., Rauh, M., Gerlach, R.G., Mattner, J.: L-arginine metabolism as pivotal interface of mutual host-microbe interactions in the gut. Gut microbes. 15 (1), 2222961 (2023). Epub 2023/06/26 10.1080/19490976.2023.2222961 Scott, J.A., Maarsingh, H., Holguin, F., Grasemann, H.: Arginine Therapy for Lung Diseases. Front. Pharmacol. 12 , 627503 (2021). Epub 2021/04/10 10.3389/fphar.2021.627503 King, N.E., Rothenberg, M.E., Zimmermann, N.: Arginine in Asthma and Lung Inflammation. J. Nutr. 134 (10), 2830S–6S (2004). 10.1093/jn/134.10.2830S Kuzmin, E., Taylor, J.S., Boone, C.: Retention of duplicated genes in evolution. Trends Genet. 38 (1), 59–72 (2022). Epub 2021/07/24 10.1016/j.tig.2021.06.016 Walter, G.M., Monro, K., Terranova, D., la Spina, E., Majorana, M., Pepe, G., et al.: Environmental effects on genetic variance are likely to constrain adaptation in novel environments. Evol. Lett. 8 (3), 374–386 (2024). 10.1093/evlett/qrad065 He, L., Vasiliou, K., Nebert, D.W.: Analysis and update of the human solute carrier (SLC) gene superfamily. Hum. Genomics. 3 (2), 195 (2009) Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., von Haeseler, A., et al.: IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 37 (5), 1530–1534 (2020). 10.1093/molbev/msaa015 Bouckaert, R.R., Drummond, A.J.: bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evol. Biol. 17 (1), 42 (2017). Epub 2017/02/09 10.1186/s12862-017-0890-6 Liu, M., Ma, Z., Wang, A., Zheng, T., Huang, L., Sun, W., et al.: Genome-wide investigation of the auxin response factor gene family in Tartary buckwheat (Fagopyrum tataricum). Int. J. Mol. Sci. 19 (11), 3526 (2018) Chen, C., Chen, H., Zhang, Y., Thomas, H.R., Frank, M.H., He, Y., et al.: TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant. 13 (8), 1194–1202 (2020) Wang, Y., Tang, H., DeBarry, J.D., Tan, X., Li, J., Wang, X., et al.: MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 40 (7), e49–e (2012) Krzywinski, M.I., Schein, J.E., Birol, I., Connors, J., Gascoyne, R., Horsman, D., et al.: Circos: An information aesthetic for comparative genomics. Genome Res. (2009). 10.1101/gr.092759.109 Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., Tamura, K.: MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol Biol Evol. ;41(12). Epub 2024/12/22. (2024). 10.1093/molbev/msae263 Xia, X.: DAMBE7: New and Improved Tools for Data Analysis in Molecular Biology and Evolution. Mol. Biol. Evol. 35 (6), 1550–1552 (2018). Epub 2018/04/19 10.1093/molbev/msy073 Ding, L., Wang, Y., Zhang, L., Luo, C., Wu, F., Huang, Y., et al.: The HTIRDB: A resource containing a transcriptional atlas for 105 different tissues from each of seven species of domestic herbivore. iMeta. 4 (1), e267 (2025). Epub 2025/03/03 10.1002/imt2.267 Krause, C., Suwada, K., Blomme, E.A.G., Kowalkowski, K., Liguori, M.J., Mahalingaiah, P.K., et al.: Preclinical species gene expression database: Development and meta-analysis. Front. Genet. 13 , 1078050 (2022). Epub 2023/02/04 10.3389/fgene.2022.1078050 Patro, R., Duggal, G., Love, M.I., Irizarry, R.A., Kingsford, C.: Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods. 14 (4), 417–419 (2017). 10.1038/nmeth.4197 Additional Declarations There is NO Competing Interest. Supplementary Files FigS14.docx Figure S1, Figure S2, Figure S3, Figure S4 SupportingInformationTableS18.rar Table S1-8 Supplementaryinformation.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8632196","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":595852112,"identity":"908112c0-b37f-4577-8f87-948c6db9a78a","order_by":0,"name":"Jianghong Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYHCChAMgAogZQQw5NvbmA0RrYQAxjPl4jiUQZxVMS+I8iRwFvEoNbiQ8PPBzR20ev3T7hQMfd9SmtzHkMDD8qNiGT0vCwd4zx4sl55wpODjzzPHcNoazBxh7ztzGqcUMqOUAb9uxxA03chIOAxm5bYx9CcyMbfi1HPyLpCWdjZnHgKAWoMoaoJb0AyBGAhsbAS32Zx4kHJZtO5A4c0YOw8GZbQcM23jYEg7i84tke07yx7dtdYn9EukPH3xsq5OXn//44IMfFbi1MDDwJACJwyCGAZQBiSA8gB0kXwdiPIAyRsEoGAWjYBSgAgCMsWqNiTwSrgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4027-2345","institution":"College of Animal Science and technology, Inner Mongolia Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Jianghong","middleName":"","lastName":"Wu","suffix":""},{"id":595852113,"identity":"97664430-26b3-46b5-9269-65518aa12d82","order_by":1,"name":"Sile Hu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sile","middleName":"","lastName":"Hu","suffix":""},{"id":595852114,"identity":"3a3c2c66-97d3-4068-8ee4-c1d1927f2bbd","order_by":2,"name":"Jiahui Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"Shi","suffix":""},{"id":595852115,"identity":"52a35b39-d658-496d-a685-fe972de84f5d","order_by":3,"name":"Wei Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""},{"id":595852116,"identity":"b838d4d1-8f96-486b-a34f-66e25b02ed9d","order_by":4,"name":"Yan Zhu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhu","suffix":""},{"id":595852117,"identity":"f1ab9401-2cb0-4e9d-924e-f80221592e31","order_by":5,"name":"Binhong Wen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Binhong","middleName":"","lastName":"Wen","suffix":""},{"id":595852118,"identity":"973741ff-a6af-4ea8-a91e-84d033fda2f0","order_by":6,"name":"Hua Mei","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Mei","suffix":""},{"id":595852119,"identity":"4624c954-30b7-4d27-9b3a-1c0480abecd5","order_by":7,"name":"Dubala Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dubala","middleName":"","lastName":"Wu","suffix":""},{"id":595852120,"identity":"bb0c3237-bcb5-47e0-bd01-630a30db3ae1","order_by":8,"name":"Chun Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Li","suffix":""},{"id":595852121,"identity":"17cb0cbd-6df6-4321-b41d-5f9cdfa161ed","order_by":9,"name":"Jun Yin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2026-01-18 15:15:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8632196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8632196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103487623,"identity":"5c4f0d81-2399-4296-9e27-5ec221c96454","added_by":"auto","created_at":"2026-02-26 09:18:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2936524,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaximum Likelihood phylogenetic tree representing relationships among 438 \u003c/strong\u003eo\u003cem\u003es-SLC\u003c/em\u003e\u003cstrong\u003eof\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Ovis aries\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and 393 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehs-SLC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHomo sapiens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe \u003cem\u003eos-SLC\u003c/em\u003eare marked in blue, while those hs-SLC are marked in red. The different color blocks represent different clans. Red branches represent the gene expansion in the \u003cem\u003eos-SLC7\u003c/em\u003e gene family.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/44b3f0e1eb735c30ca2ce64c.png"},{"id":103507517,"identity":"201bd5b6-1b75-4838-b10c-b88435144b05","added_by":"auto","created_at":"2026-02-26 13:41:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1783635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCollinearity analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSLC7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene family in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOvis aries\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe red and blue corresponding to high- and low-density value. The outer ring represents the chromosomes of \u003cem\u003eOvis aries\u003c/em\u003e, numbered from 1 to 26 and X Y. The lines connecting different chromosomal positions represent collinear blocks, indicating regions of conserved gene order (synteny) between different parts of the sheep genome. These connections highlight paralogous relationships resulting from gene duplication events. The colored rectangles (red to blue gradient) along the chromosomes represent the density of gene duplication events. According to the color bar, red indicates strong collinearity signal, while blue indicates lower values.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/f04a7c4d63777bac26cc1bca.png"},{"id":103507749,"identity":"3809b6d1-6a55-44fb-bf1b-7bd9da7c0346","added_by":"auto","created_at":"2026-02-26 13:44:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":891599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree, protein conserved motifs, gene structure, and conserved domain analysis of 49 os-SLC7 gene family.\u003c/strong\u003e (A) Phylogenetic tree constructed by maximum likelihood method. The red rectangle represents os-CATs clan, and the blue one represents os-LATs clan. (B) Motif analysis of protein encoded by \u003cem\u003eos-SLC7\u003c/em\u003egene family. (C) Conserved domain of \u003cem\u003eos-SLC7\u003c/em\u003e genes family. (D) \u003cem\u003eos-SLC7\u003c/em\u003egene family structure.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/72b36fa764aee57bd6122f78.png"},{"id":103487618,"identity":"1cbe631c-a2ab-463b-92dc-053ed5b8758b","added_by":"auto","created_at":"2026-02-26 09:18:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":801249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumbers of CAT1-4 genes in different clade of mammalian lineages based on species tree[16]and grasslands evolution.\u003c/strong\u003e In the diagram, carnivores are denoted by red branches, herbivores by green, and omnivores by black. Circles sizes are representative of gene copy numbers. Grasses (Poaceae) originated in the Late Cretaceous[17, 18] and continued Rarity and Early Diversification in Paleocene-Eocene (ca. 66-34 Ma)[19]. All early grasses used the C₃ photosynthetic pathway[20]. A significant drop in atmospheric CO₂ during the Oligocene created new selective pressures. The more efficient C₄ photosynthetic pathway evolved independently in grasses around 30-32 Ma[18]. Global C₄ Grassland Revolution in Late Miocene – Pliocene. Expansion of C₃ Grasslands and presence of C₄ Grasses in early-Middle Miocene (ca. 23-11 Ma). C₄ grasslands expanded dramatically and nearly synchronously across low- to mid-latitude regions globally, displacing previous forests and C₃ grasslands to become the dominant ecosystem in Late Miocene-Pliocene (ca. 11-2.6 Ma)[17]. Ungulates and grasses originated independently in time[21]. Their fates became closely intertwined during the middle to late Cenozoic (particularly the Neogene) due to global climatic changes. The expansion of grasslands provided new ecological niches for ungulates[22], driving their adaptive radiation from browsing to grazing (e.g., the evolution of hypsodont teeth and rumen).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/e9b05bac5362cbb8e4bdaa8f.png"},{"id":103507965,"identity":"81e1c13c-a7a7-430d-8b31-79f8d96bcc93","added_by":"auto","created_at":"2026-02-26 13:46:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1663962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ethe phylogeny tree of CAT3 and CAT4 expanded genes and the distribution of exon count for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSLC7A3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene copies. \u003c/strong\u003eA) the phylogeny tree of CAT3 expanded genes. B) the phylogeny tree of CAT4 expanded genes. C) the distribution of exon counts of \u003cem\u003eSLC7A3\u003c/em\u003egene copies for herbivores. D) the distribution of exon counts of \u003cem\u003eSLC7A3\u003c/em\u003e gene copies for carnivores.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/9aa4c49cfcfb9a6d4871bd3d.png"},{"id":103507864,"identity":"c7c7c980-ac19-47c8-a198-ab19347823c3","added_by":"auto","created_at":"2026-02-26 13:46:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10231721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCollinearity analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSLC7A3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expanded gene family in Ruminant(A), Carnivores(B) and Hybridization induced gene duplication in F1 hybrid of angus ×zebu(C). \u003c/strong\u003eThe reference species was \u003cem\u003eHomo sapiens \u003c/em\u003e(hs). \u003cem\u003eCapra hircus \u003c/em\u003e(ch), B\u003cem\u003eos taurus\u003c/em\u003e (bt), \u003cem\u003eBos indicus\u003c/em\u003e (bi), \u003cem\u003eOvis aries\u003c/em\u003e (oa), hybrid cattle (\u003cem\u003eBos indicus\u003c/em\u003e × \u003cem\u003eBos taurus\u003c/em\u003e) (bz), \u003cem\u003eAiluropoda melanoleuca\u003c/em\u003e(am) \u003cem\u003eCanis lupus familiaris \u003c/em\u003e(cf), \u003cem\u003eHyaena hyaena\u003c/em\u003e(cd), \u003cem\u003eCanis lupus baileyi\u003c/em\u003e (gf), \u003cem\u003eFelis catus \u003c/em\u003e(fc). The orange genes of out circle were the expanded \u003cem\u003eSLC7A3\u003c/em\u003e gene, and the black line represents the collinear block of non \u003cem\u003eSLC7A3\u003c/em\u003e gene. The blue arrow highlights the highest expression of \u003cem\u003eSLC7A3\u003c/em\u003e copy gene in \u003cem\u003eOvis aries, Bos taurus \u003c/em\u003eand\u003cem\u003e Canis lupus familiaris\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/6a414a45d4168dd8deaf945c.png"},{"id":103507634,"identity":"98d07c29-ba87-41fb-a5ec-4b4b27237100","added_by":"auto","created_at":"2026-02-26 13:42:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2293772,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression of CAT3 expanded genes in different tissue of four species. \u003c/strong\u003eA) Expression levels of different transcripts of the SLC7A3 gene ranked among the top ten tissues in sheep. B) Expression levels of different transcripts of the SLC7A3 gene ranked among the top ten tissues in goat. C) Expression levels of different transcripts of the SLC7A3 gene ranked among the top ten tissues in cattle. D) Expression levels of different transcripts of the SLC7A3 gene ranked among the top ten tissues in dog. E) Venn diagram showing overlap of highly expression SLC7A3 among four species.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/e2bb83445e500b5ec9ba2dcd.png"},{"id":103487624,"identity":"c93ab843-578e-4905-a1db-4a939cda8c34","added_by":"auto","created_at":"2026-02-26 09:18:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1343367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSLC7A3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene families in ruminants and carnivores marked with different colors. \u003c/strong\u003eThose red genes represent the top 10 transcripts of highly expressed in goat, sheep, cattle and dog. The number behind ’#’ on each branch represent the rate of evolution under the free-ratio model of CONDEML.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/b479883611fc04048ef54e3e.png"},{"id":104397405,"identity":"58709516-0418-403a-8bed-c90edea94830","added_by":"auto","created_at":"2026-03-11 11:47:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25248649,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/d843e46e-13fb-499f-aa9c-f679235e42ec.pdf"},{"id":103487625,"identity":"8e436bd4-7031-4e9d-8748-446c48dec600","added_by":"auto","created_at":"2026-02-26 09:18:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7571326,"visible":true,"origin":"","legend":"Figure S1, Figure S2, Figure S3, Figure S4","description":"","filename":"FigS14.docx","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/28a400b5384373ae223a132e.docx"},{"id":103487621,"identity":"ffed5163-f4bd-4226-a3a5-acf2b4e15ec9","added_by":"auto","created_at":"2026-02-26 09:18:17","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":253180,"visible":true,"origin":"","legend":"Table S1-8","description":"","filename":"SupportingInformationTableS18.rar","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/261baafa97ac1dcd2bcdf6f3.rar"},{"id":103507961,"identity":"36d774ec-0721-4704-b1ae-00995628f2d8","added_by":"auto","created_at":"2026-02-26 13:46:41","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14925,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8632196/v1/97d5fd9e0ca5b14b3f80fc23.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Independent Expansion of SLC7A3 in Mammals to Meet the Challenge of Nitrogen Metabolism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHow species rapidly adapt to new ecological niches and physiological challenges is a central question in evolutionary biology[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The expansion of gene families is considered a key mechanism for providing new genetic material and facilitating functional innovation[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Typically, the expansion of functionally important genes occurs through post-speciation gene duplication, resulting in different species possessing varying copy numbers of a gene in homologous genomic regions[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This model predicts a clear history of duplication events in phylogenetic trees that corresponds with species divergence[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, when distantly related species face remarkably similar environmental or physiological pressures, do they independently converge on expanding the \u003cem\u003esame\u003c/em\u003e gene? If so, does this expansion stem from a single, ancient, shared ancestral event, or does it occur through independent and repeated events within their respective genomes? Investigating this can reveal the power of natural selection and the plasticity of genome evolution[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoth carnivores[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and ruminants[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] derive the majority of their required glucose from gluconeogenesis. Ammonia is a highly toxic waste product generated when amino acids serve as substrates for gluconeogenesis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] in carnivores. It is crucial for carnivores to rapidly remove excess ammonia to prevent ammonia toxicity[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. For ruminants, nitrogen conservation and recycling are essential to cope with the persistent low-quality nitrogen in their diet and periodic nitrogen scarcity[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consequently, the different challenges in nitrogen metabolism between carnivores and ruminants have shaped their respective paths of adaptive natural selection. Carnivores detoxify ammonia by rapidly converting it to urea for excretion[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, ruminants have evolved the urea cycle to recycle ammonia nitrogen back to the rumen for microbial reuse[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Both require that ammonia be converted into urea as completely as possible through the urea cycle. This scenario constitutes a strong natural selection pressure under the nitrogen metabolism for the specific mammalian lineage.\u003c/p\u003e \u003cp\u003eThe Solute Carrier family 7 (SLC7) is crucial for nitrogen metabolism because its transporters[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], particularly its cationic amino acid transporter (CAT) subfamily[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], plays a central role in amino acid transport and in supplying precursors for the urea cycle (such as L-arginine and L-ornithine)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although \u003cem\u003eSLC7A3\u003c/em\u003e (CAT-3) is known to be expressed in specific tissues[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], its evolutionary patterns in relation to ecological adaptation across the entire class of Mammalia, as well as its genomic evolutionary history, remain entirely unexplored. Existing research has predominantly focused on functional characterization within single species, lacking a macro-scale, comparative genomics perspective.\u003c/p\u003e \u003cp\u003eIn this study, we used sheep as a ruminant model to identify genes of the solute carrier (SLC) superfamily. Comparative genome analysis with humans revealed significant expansion of the SLC7 subfamily genes in sheep. Further investigation into SLC7 expansion patterns across mammals uncovered parallel evolution of \u003cem\u003eSLC7A3\u003c/em\u003e in both ruminants and carnivores, with expansion sites exhibiting lineage-specific distributions. This study presents a classic case of parallel evolution, but one that occurs at the relatively unstable genomic structural level of copy number variation rather than through the typically recognized convergent evolution of protein-coding sequences. This significantly expands our understanding of the molecular mechanisms underlying \"convergent evolution.\" By directly linking a specific physiological challenge (ammonia metabolism) to macro-scale genomic evolutionary patterns, it establishes a paradigm for multi-level research bridging diet intaking, physiology, and genomics. Insights into the \u003cem\u003eSLC7A3\u003c/em\u003e expansion model will help explain interspecies differences (including livestock and humans) in protein metabolic efficiency, susceptibility to nitrogen imbalance diseases, and related traits.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of members of the os-SLC gene superfamily in Ovis aries\u003c/h2\u003e \u003cp\u003eWe identified 438 SLC members for the genome of \u003cem\u003eOvis aries\u003c/em\u003e. Compare with \u003cem\u003eHomo sapiens\u003c/em\u003e, the number of os-SLC has gained 75 members and lost 30 members. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e provides basic information on the subcellular localization, PI, Molecular weight, lengths, instability index, Aliphatic index, GRAVY of the 438 os-SLC. The proteins produced by these genes have different lengths, with amino acids ranging from 130 AA (os-SLC51B) to 1239 AA (os-SLC4A2). The instability coefficients of these proteins range from 15.76 (os-SLC25A6) to 97.93 (os-SLC17A5). The predicted isoelectric points of the proteins range from 4.47 (os-SLC51B) to 10.09 (os-SLC25A17). Of these, 271 proteins have isoelectric points higher than 7, making them positively charged in acidic solutions. The proteins have different levels of hydrophilicity, ranging from \u0026minus;\u0026thinsp;0.585 (os-SLC39A10) to 0.917 (os-SLC22A18). Furthermore, 297 proteins are located on the cell membrane, 48 proteins are located on the endosome, 69 proteins are found in the endomembrane system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhylogenetic analysis and classification of os-SLC proteins\u003c/h3\u003e\n\u003cp\u003eTo investigate the evolutionary relationship of os-SLC proteins, a phylogenetic tree was constructed from 393 solute carrier gene superfamily from \u003cem\u003eHomo sapiens\u003c/em\u003e (hs-SLC) proteins and 438 os-SLC proteins by the ML method (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A comparison of the SLC proteins in \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eOvis aries\u003c/em\u003e reveals homology among them. This phylogenetic analysis categorizes the 831 proteins into thirteen distinct clans, with the 438 os-SLC proteins from \u003cem\u003eOvis aries\u003c/em\u003e being distributed across various families. The clan 1 constitutes the second-largest branch within this evolutionary tree and it is primarily constituted by \u003cem\u003eSCL2\u003c/em\u003e, \u003cem\u003eSLC8\u003c/em\u003e, \u003cem\u003eSLC12\u003c/em\u003e, \u003cem\u003eSLC25\u003c/em\u003e, \u003cem\u003eSLC27\u003c/em\u003e, \u003cem\u003eSLC28\u003c/em\u003e, \u003cem\u003eSLC35\u003c/em\u003e, \u003cem\u003eSLC39\u003c/em\u003e and \u003cem\u003eSLC41\u003c/em\u003e gene family. The clan 2 is primarily constituted by \u003cem\u003eSLC17\u003c/em\u003e, \u003cem\u003eSLC22\u003c/em\u003e, \u003cem\u003eSLC29\u003c/em\u003e, \u003cem\u003eSLC37\u003c/em\u003e, \u003cem\u003eSLC39\u003c/em\u003e and \u003cem\u003eSLC52\u003c/em\u003e gene family. The clan 3 is primarily constituted by the \u003cem\u003eSLC1\u003c/em\u003e gene family. The clan 4 only includes 7 genes, which belong to \u003cem\u003eSLC18\u003c/em\u003e and \u003cem\u003eSLC30\u003c/em\u003e gene family. The clan 5 encompasses a wide variety of gene families, mainly consisting of \u003cem\u003eSLC4\u003c/em\u003e, \u003cem\u003eSLC22\u003c/em\u003e, \u003cem\u003eSLC24\u003c/em\u003e, \u003cem\u003eSLC30\u003c/em\u003e, \u003cem\u003eSLC35\u003c/em\u003e and \u003cem\u003eSLC43\u003c/em\u003e gene family. The clan 6 only includes 15 genes, which belong to \u003cem\u003eSLC11\u003c/em\u003e, \u003cem\u003eSLC13\u003c/em\u003e and \u003cem\u003eSLC41\u003c/em\u003e gene family. Clan 7 mainly encompasses SLC6 and six other minor members of the gene family. The clan 8 is the largest one among the branches of this tree, encompassing 248 genes. It is mainly constituted by 30 distinct gene families, among which are \u003cem\u003eSLC7\u003c/em\u003e, \u003cem\u003eSLC9\u003c/em\u003e, \u003cem\u003eSLC16\u003c/em\u003e, \u003cem\u003eSLC26\u003c/em\u003e, \u003cem\u003eSLC35\u003c/em\u003e and \u003cem\u003eSLC44\u003c/em\u003e gene family. Interestingly, family \u003cem\u003eos-SLC7\u003c/em\u003e may have undergone stronger expansions. From the evolutionary tree, the \u003cem\u003eos-SLC7\u003c/em\u003e gene family consists of 49 members, of which 37 are newly expanding genes (from \u003cem\u003eos-LOC105605834\u003c/em\u003e to \u003cem\u003eos-LOC101115153\u003c/em\u003e, \u003cem\u003eos-LOC132657146\u003c/em\u003e, \u003cem\u003eos-LOC1114368\u003c/em\u003e, \u003cem\u003eos-LOC105610833\u003c/em\u003e, \u003cem\u003eos-LOC101107086\u003c/em\u003e, red branches in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) comparing with 12 members in human \u003cem\u003ehs-SLC7\u003c/em\u003e gene family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGene duplication and collinearity analysis of os-SLC7A3 gene family\u003c/h3\u003e\n\u003cp\u003eDuring species evolution, gene duplication and retention of existing genes allow species genomes to expand. The distribution of the 49 \u003cem\u003eos-SLC7\u003c/em\u003e genes across chromosomes 1\u0026ndash;26, X and Y exhibited non-uniformity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Among these chromosomes, Chr14 have the highest number of \u003cem\u003eos-SLC7\u003c/em\u003e genes (38 genes, 77.55%), followed by Chr7, Chr9, and Chr17 (2 genes, 4.08%). Chromosomes 1, 3, 10, 26 and X contained 1 (2.04%) \u003cem\u003eos-SLC7\u003c/em\u003e genes, respectively. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, The segmental duplication of the \u003cem\u003eos-SLC7\u003c/em\u003e gene were not observed in \u003cem\u003eOvis aries\u003c/em\u003e, but a large number of tandem duplications were found. Chromosomes 14 contained the highest number of tandem duplication genes (34 genes), which belong to subfamily os-CAT3. These findings suggest that numerous os-SLC7 members might have originated from tandem duplication events. Next, we counted the members of the SLC gene family in six other species and found that, apart from sheep, the SLC7 subfamily members underwent significant expansion in horses, cattle, tigers, and domestic dogs (Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eStructure and motif composition of SLC7 expanded genes\u003c/h3\u003e\n\u003cp\u003eTo determine the phylogeny relationship and structure of SLC7 expanded genes, a maximum likelihood tree for the \u003cem\u003eos-SLC7\u003c/em\u003e gene family was constructed and divided into two sub-branches (CATs clan and LATs clan) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and there are significant differences in conserved motifs and domains between them (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-B, C). 34 expanding genes (\u003cem\u003eos-LOC101111827\u003c/em\u003e to \u003cem\u003eos-LOC101111313\u003c/em\u003e) and \u003cem\u003eos-SLC7A1\u0026thinsp;~\u0026thinsp;4\u003c/em\u003e, \u003cem\u003eos-SLC7A14\u003c/em\u003e were belong to the os-CATs clan, which have closest relationship with the \u003cem\u003eos-SLC7A3\u003c/em\u003e, encoding the CAT-3 cationic amino acid transporter. However, the \u003cem\u003eos-SLC7A3\u003c/em\u003e gene and its expanded genes are located on different chromosomes: the \u003cem\u003eos-SLC7A3\u003c/em\u003e gene is located on the X chromosome, while these expanded genes are situated on chromosome 14. The os-LATs clan contains \u003cem\u003eos-SLC7A5\u0026thinsp;~\u0026thinsp;11\u003c/em\u003e and 3 expanding genes (os-\u003cem\u003eLOC101114368\u003c/em\u003e, \u003cem\u003eos-LOC105610833\u003c/em\u003e and \u003cem\u003eos-LOC101107086\u003c/em\u003e). To determine the composition and number of these conserved motifs in the \u003cem\u003eos-SLC7\u003c/em\u003e gene family, we utilized online MEME software. This analysis identified a total of 14 conserved motifs, labeled motif 1 to motif 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-B). The presence of motif 2, 4, 7, 11, 13 and 14 is a common feature among the members of os-CATs clan, whereas the members of os-LATs clan only encompass a segment of motifs, specifically motifs 4, 6, 11, and 13 among others. The os-CATs clan protein matches with 2A0303 superfamily domain, and the os-LATs clan protein matches with 2A0308 superfamily domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-C). All \u003cem\u003eos-SLC7\u003c/em\u003e family members contain multiple exons and introns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e-D). The number of exons in the gene\u0026rsquo;s ranges from 13 to 3, the number and length of exons of genes situated on the same evolutionary tree branch are approximately similar. They contain introns, and are expressed in multiple tissues, which is inconsistent with the typical characteristics of retrogenes. Therefore, the expansion of \u003cem\u003eSLC7A3\u003c/em\u003e may result from genomic breakage and recombination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eExpansion and loss of CAT during the evolution of mammalian\u003c/h3\u003e\n\u003cp\u003eGiven substantial interspecies variation in CAT3 gene copy number, we further investigated the evolutionary dynamics of CAT gene family expansion and loss across mammals. By analyzing 27 mammalian species, lineage-specific expansion patterns of CAT1-4 genes were characterized (Table S4-S7). Our findings demonstrate that CAT3 and CAT4 underwent significant expansion in some clades. CAT3 genes expanded in \u003cem\u003eArtiodactyla\u003c/em\u003e (Suina, Tylopoda and Ruminantia) and \u003cem\u003eCarnivora\u003c/em\u003e, with particularly notable diversification within \u003cem\u003eArtiodactyla\u003c/em\u003e: ruminants exhibited the highest copy numbers (sheep 38, goat 42, cattle 28, Zebu 35), while non-ruminants showed reduced counts (pig 16, hippopotamus 12, camel 5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Strikingly, cetaceans retained only 2 copies that is the lowest among \u003cem\u003eCetartiodactylas\u003c/em\u003e and comparable to non-cetartiodactyl species. The copy number of expanded SLC7A3 genes in each species may be influenced by the completeness of genome assembly and annotation. This ruminant-specific amplification suggests recent evolutionary innovation in the CAT3 subfamily. CAT3 exhibits significant copy number divergence even between closely related species Bos indicus (zebu cattle) and Bos taurus (taurine cattle) with 35 copies in zebu versus 28 in taurine cattle. Furthermore, it is unexpected that there are 41 copies CAT3 paralog genes in hybrid cattle (\u003cem\u003eBos indicus\u003c/em\u003e x \u003cem\u003eBos taurus\u003c/em\u003e). This disparity indicates post-speciation gene duplication events within the CAT3 family, suggesting that extensive duplicate of CAT3-like novel genes has occurred. Conversely, CAT4 expansion occurred exclusively in Perissodactyla, with all other lineages maintaining a single-copy state.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the evolutionary origins of the CAT3 and CAT4 gene families, we reconstructed the phylogeny of CAT3 and CAT4 paralogs individually. The expansion mechanisms of \u003cem\u003eSLC7A3\u003c/em\u003e and \u003cem\u003eSLC7A4\u003c/em\u003e differ significantly. In \u003cem\u003eSLC7A3\u003c/em\u003e, orthologous genes cluster together, with expansion members forming distinct clades, one cluster in Artiodactyla (even-toed ungulates), and another cluster in Carnivora (carnivores)(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These findings demonstrate lineage-specific convergent expansions occurring independently in each group following speciation for \u003cem\u003eSLC7A3\u003c/em\u003e. In contrast, the phylogenetic tree of \u003cem\u003eSLC7A4\u003c/em\u003e shows disorganized clustering among its expansion members, exhibiting no clear species-specific grouping pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Analysis of SLC7A3 paralog exon numbers reveals that herbivores have exon counts concentrated between 8 and 12, whereas carnivores have exon counts concentrated between 0 and 4(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). This may be attributed to relaxed selection allowing for the retention of gene mutations, which subsequently affects the recognition of gene structures such as introns and exons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExpansion locus of CAT3 between ruminants and Carnivora\u003c/h2\u003e \u003cp\u003eThe results demonstrate that although evolutionary expansions of the SLC7A3 gene (CAT3) in Artiodactyla and Carnivora both adhere to deterministic patterns, they are driven by clade-specific genomic architectures. The localization of the original \u003cem\u003eSLC7A3\u003c/em\u003e gene to the X chromosome is a conserved feature across mammalian species (Table S6). Crucially, the chromosomal localization of expansion events differs fundamentally between these lineages. In Artiodactyla, the CAT3 expansion locus in the \u003cem\u003eZNF331-DPRX-NLRP12-MYADM-PRKCG-CACNG7\u003c/em\u003e (\u003cem\u003eZDNMPC\u003c/em\u003e) cluster occupies an ancestral syntenic block conserved across most mammals (excluding Platypus, Kangaroo) in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Within this region, humans retain two CAT3 pseudogenes, suggesting functional decay post-expansion in primates. The genome recombination happened in this block of hybrid cattle (Bos indicus \u0026times; Bos taurus) compare with the Bos indicus and Bos taurus (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A syntenic block was identified on chromosome 18, with its specific coordinates being 4.2\u0026ndash;5.1 Mb in hybrid cattle, 60.8\u0026ndash;61.6 Mb in \u003cem\u003eBos taurus\u003c/em\u003e, and 61-61.9 Mb in \u003cem\u003eBos indicus\u003c/em\u003e. Strikingly, the order of the gene cluster \u003cem\u003eZDNMPC\u003c/em\u003e was inverted in hybrid cattle relative to the other two species. Additionally, we observed that four specific genes (\u003cem\u003eLOC109572792\u003c/em\u003e, \u003cem\u003eLOC109571833\u003c/em\u003e, \u003cem\u003eLOC109571835\u003c/em\u003e, \u003cem\u003eLOC109572110\u003c/em\u003e) were present in two copies in the hybrid cattle, indicating a hybridization-induced gene duplication event at this CAT3 expansion locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Further research has revealed that genomic inversions and rearrangements also occur within the \u003cem\u003eZDNMPC\u003c/em\u003e region across different sheep breeds, and the copy number variation of the newly expanded \u003cem\u003eSLC7A3\u003c/em\u003e gene in this region is substantial. However, this region remains stable without undergoing genomic structural changes among Bactrian camels, dromedaries, and alpacas (Fig S2).\u003c/p\u003e \u003cp\u003eIn Carnivora, the expansion occurs within a derived syntenic region (\u003cem\u003eSLCO1A2-IAPP-PYROXD1-RECQL-GOLT1B-SPX-GYS2, SIPRGSG\u003c/em\u003e), indicative of lineage-specific genomic reorganization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Notably, no CAT3 pseudogenes are present in the homologous human locus (\u003cem\u003eHomo sapiens\u003c/em\u003e as an outgroup), underscoring Carnivora's unique evolutionary trajectory (Fig S3). Furthermore, a substantial fraction of the expanded CAT3 gene members has evolved into pseudogenes in the gray wolf and other carnivores (Table S6). Further research has revealed that genomic inversions and rearrangements also occur within the \u003cem\u003eSIPRGSG\u003c/em\u003e region across different dog breeds, and the copy number variation of the newly expanded \u003cem\u003eSLC7A3\u003c/em\u003e gene in this region is substantial (Fig S4). The expansion event of the SLC7A3 gene may be associated with recombination hotspots within the region, disrupting genomic stability in this area and thereby making it prone to subsequent structural variations (such as inversions) and copy number changes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExpression pattern of the expansion of SLC7A3 genes family\u003c/h3\u003e\n\u003cp\u003eTo better understand the basis for functional divergence, we conducted gene expression analysis of CAT3s in tissues of four representative species (sheep, cattle, goat, dog) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). Comparative analysis of the top 10 tissues with the highest expression across four species identified blood, spleen, intestines, and lungs as commonly expressed tissues, while the parathyroid gland, pituitary gland, tendon, cervix, bone marrow, and eyeball exhibited specific expression in domestic dogs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). This suggests that CAT3 expanded genes may function differently between ruminants and carnivores. Specifically, sheep showed highest expression in the thymus, goats in the intestine, cattle in the thyroid gland, and domestic dogs in the intestines. Expression levels of the expanded CAT3 members varied significantly among the species analyzed. Notably, the gene with the highest expression was species-specific: \u003cem\u003eLOC101108819\u003c/em\u003e in sheep, \u003cem\u003eLOC108633279\u003c/em\u003e in goats, \u003cem\u003eLOC101904151\u003c/em\u003e in cattle, and \u003cem\u003eLOC119877631\u003c/em\u003e in domestic dogs. Consistent with this pattern, no collinear relationship was detected among these genes, demonstrating strong evolutionary lineage-specificity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results indicate that the pineal gland exhibits the highest expression levels among the examined organs in both cattle and goats. Specifically, transcript \u003cem\u003eXM_013971618.1\u003c/em\u003e (\u003cem\u003eLOC102190544\u003c/em\u003e) and transcript \u003cem\u003eXM_024979355.2\u003c/em\u003e (\u003cem\u003eLOC532048\u003c/em\u003e) show the highest specific expression in the pineal gland of goats and cattle, respectively. Although the goat \u003cem\u003eLOC102190544\u003c/em\u003e and cattle \u003cem\u003eLOC532048\u003c/em\u003e genes are both highly and specifically expressed in the pineal gland, the lack of collinearity between them suggests they are not orthologs derived from a common ancestor (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Their similar expression patterns likely result from convergent evolution, where distinct genes independently evolve to fulfill analogous roles in the pineal gland.\u003c/p\u003e\n\u003ch3\u003eVariation in the selective pressure over SLC7A3 gene family\u003c/h3\u003e\n\u003cp\u003eThe results obtained from the one-ratio model indicated that the \u003cem\u003eSLC7A3\u003c/em\u003e gene was under purifying selection (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e = 0.65). This model gave a log maximum-likelihood value of -8477.11. The free‐ratio model assumes an independent \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e ratio for each branch in the tree. In the species tree, 453 branches were assumed to have their own \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e ratios under the free‐ratio estimation, which leads to a log maximum‐likelihood value of -8238.92. In comparison with the one‐ratio model, the free‐ratio model involved 452 extra parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.). The branch-specific \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e values of Carnivora SLC7A3 paralog members show a significant increase, further confirming the relaxation of selective constraints, which has led to an increase in the number of pseudogenes within Carnivora SLC7A3 copies.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eSLC7A3\u003c/em\u003e genes and expanded \u003cem\u003eSLC7A3\u003c/em\u003e gene members of ruminants and carnivores branch was specified as a foreground branch to test for adaptive selection. Results of all two-ratio model tests for three branches are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The results showed that the carnivores branch had significantly higher \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e value compared with other branches, suggesting the potential action of positive selection. However, the original \u003cem\u003eSLC7A3\u003c/em\u003e gene and ruminants branch had significantly lower \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e value compared with other branches, suggesting the potential action of negative selection. Furthermore, we estimated \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e value utilized branch models with heterogeneous \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e cross three branches. The results showed that the selective pressure was variation in three branches (0.10 for \u003cem\u003eSLC7A3\u003c/em\u003e, 0.95 for carnivores, and 0.55 for ruminants in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameter estimates under models of variable \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\omega\\:\\)\u003c/span\u003e\u003c/span\u003e ratios among lineages and LRTs of their fit to the \u003cem\u003eSLC7A3\u003c/em\u003e gene family between carnivora and ruminants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModels\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{\\omega\\:}}_{0}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{\\omega\\:}}_{\\varvec{S}\\varvec{L}\\varvec{C}7\\varvec{A}3}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{\\omega\\:}}_{\\varvec{C}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{\\omega\\:}}_{\\varvec{R}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eℓ\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e :\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{{\\varvec{\\omega\\:}}_{0}=\\varvec{\\omega\\:}}_{\\varvec{S}\\varvec{L}\\varvec{C}7\\varvec{A}3}={\\varvec{\\omega\\:}}_{\\varvec{C}}={\\varvec{\\omega\\:}}_{\\varvec{R}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-8477.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003enot allowed\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{\\varvec{\\omega\\:}}_{0}={\\varvec{\\omega\\:}}_{\\varvec{C}}={\\varvec{\\omega\\:}}_{\\varvec{R}}{\\ne\\:\\varvec{\\omega\\:}}_{\\varvec{S}\\varvec{L}\\varvec{C}7\\varvec{A}3}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-8467.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.53\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{{\\varvec{\\omega\\:}}_{0}=\\varvec{\\omega\\:}}_{\\varvec{S}\\varvec{L}\\varvec{C}7\\varvec{A}3}={\\varvec{\\omega\\:}}_{\\varvec{C}}\\ne\\:{\\varvec{\\omega\\:}}_{\\varvec{R}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-8471.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.60\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{{\\varvec{\\omega\\:}}_{0}=\\varvec{\\omega\\:}}_{\\varvec{S}\\varvec{L}\\varvec{C}7\\varvec{A}3}={\\varvec{\\omega\\:}}_{\\varvec{R}}\\ne\\:{\\varvec{\\omega\\:}}_{\\varvec{C}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-8462.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.26\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e4\u003c/em\u003e\u003c/sub\u003e:\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{\\varvec{\\omega\\:}}_{0}\\ne\\:{\\varvec{\\omega\\:}}_{\\varvec{S}\\varvec{L}\\varvec{C}7\\varvec{A}3}\\ne\\:{\\varvec{\\omega\\:}}_{\\varvec{C}}\\ne\\:{\\varvec{\\omega\\:}}_{\\varvec{R}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-8443.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.60\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe topology and branch specific \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{\\omega\\:}\\)\u003c/span\u003e\u003c/span\u003e ratios are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChanges in Earth's ecological environment (e.g., climatic shifts) can drive shifts in vegetation types[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which directly trigger adaptive evolution in herbivorous ungulates and alter population (functional) diversity[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and indirectly drive the evolution of carnivora[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Within this context, arginine serves as a key nitrogen storage form in plants for assimilating excess environmental ammonia. arginine is the primary amino acid form for nitrogen redistribution during autumn leaf senescence and winter storage. The stored nitrogen (in the form of arginine) is mobilized in spring to support the growth of new shoots[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, understanding how herbivores utilize plant-derived arginine is of critical importance. SLC transporters serve as \u0026lsquo;metabolic gate\u0026rsquo; of cells and mediate the transport of a wide range of essential nutrients and metabolites such as glucose, amino acids, vitamins, neurotransmitters, and inorganic/metal ions. Interestingly, an additional 37 genes were identified in herbivores (sheep), which are homologous to the SLC7 gene family comparing with Omnivores (human). These results indicated that the expansion of SLC7 genes could have contributed to formation of unique traits of herbivores and meeting the demand of the lower nitrogen diets. Furthermore, ruminants can secrete urea back into the rumen through saliva, which is utilized by microorganisms for protein synthesis, significantly enhancing the utilization efficiency of low-quality plant resources [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The CATs members of SLC7 family serves as key proteins mediating the transmembrane transport of cationic amino acids such as arginine and lysine[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, we investigated the evolutionary dynamics of CAT gene family expansion and loss across mammals. Expansions of the CAT gene family were observed in both herbivores and carnivores. Specifically, expansion of CAT3 subfamily genes occurred in ruminants and carnivores, while expansion of CAT4 was found in Perissodactyla. The expansion of CAT family members in these lineages may have been accompanied by changes in grassland vegetation. With the formation of grassland ecosystems during the Late Cretaceous around 70\u0026nbsp;million years ago [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the ancestors of Perissodactyla diverged from other mammals around 56\u0026nbsp;million years ago, near or before the Paleocene-Eocene boundary[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Approximately 40\u0026nbsp;million years ago (late Eocene to early Oligocene), global climate underwent dramatic changes, with a sharp drop in temperature and increased aridity. This led to a contraction of global forests[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while more drought-tolerant C3 herbaceous plants began to expand [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is possible that perissodactyls collectively expanded CAT4 during this period to adapt to C3 grasses as a food source, enhancing nitrogen absorption capacity. This may explain why perissodactyls reached their peak diversity during the subsequent Oligocene to early Miocene[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Following the rise of C\u003csub\u003e4\u003c/sub\u003e plants to ecological dominance globally during the Late Miocene to Pliocene[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], the functional diversity of global ungulates began to decline after peaking around 10\u0026nbsp;million years ago, a trend synchronous with global aridification driven by the expansion of C\u003csub\u003e4\u003c/sub\u003e vegetation[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It was precisely during this period that artiodactyls (especially ruminants) underwent a crucial adaptive radiation and functional reorganization. This was facilitated by two key adaptations: first, the expansion of the CAT3 gene family, which maximized the absorption of arginine from C₄ plants to supplement nitrogen sources[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]; and second, the emergence of the rumen, which utilizes urea nitrogen. Together, these adaptations ensured that ruminants could meet their nitrogen demands even when feeding exclusively on C\u003csub\u003e4\u003c/sub\u003e plants, ultimately allowing them to become the dominant herbivores in grassland ecosystems by the Late Eocene[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In contrast, camels, whose primary food source is C\u003csub\u003e3\u003c/sub\u003e plants[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and cetaceans, which inhabit aquatic environments, were not subjected to selection pressures from C\u003csub\u003e4\u003c/sub\u003e plants, and thus their CAT3 copy numbers almost unchanged. The large-scale turnover and adaptive radiation of herbivorous ungulates during the Late Miocene may have led to instability in prey sources for the ancestors of modern Carnivora[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], thereby preserving their omnivorous potential[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], This instability is likely a primary reason for the concurrent expansion of CAT3 within the order Carnivora. As carnivoran diets later stabilized towards specialization in meat consumption, the newly duplicated CAT3 copies gradually lost their function, becoming pseudogenes (as seen in gray wolves, tigers, etc.). Conversely, when species like the giant panda shifted their diet to become herbivorous, CAT3 underwent a secondary expansion. However, the underlying selective mechanisms remain to be fully elucidated: why did perissodactyls choose to expand CAT4 when C\u003csub\u003e3\u003c/sub\u003e vegetation was dominant, while artiodactyls and carnivorans opted for CAT3 expansion during the era of C\u003csub\u003e4\u003c/sub\u003e vegetation dominance. These question warrants further future investigation.\u003c/p\u003e \u003cp\u003eThe parallel expansion of \u003cem\u003eSLC7A3\u003c/em\u003e occurred at distinct genomic locations in different lineages. In ruminants, the expansion is fixed in an ancestral syntenic block ZDNMPC, whereas in carnivores, it is independently acquired in a derived region \u003cem\u003eSIPRGSG\u003c/em\u003e. All placental mammals contain a single copy of the \u003cem\u003eSLC7A3\u003c/em\u003e gene within the ZDNMPC genomic structure of Boreoeutheria, while humans possess two pseudogene copies of \u003cem\u003eSLC7A3\u003c/em\u003e at this locus. Expansion of \u003cem\u003eSLC7A3\u003c/em\u003e through duplication appears to have initiated specifically within the Artiodactyla (even-toed ungulates). Notably, cetaceans within the Cetancodonta (which includes dolphins), having diverged and adapted to an aquatic environment, did not undergo this expansion. This difference may be directly related to dietary intake and could be associated with the expansion of C4 vegetation during the Artiodactyla radiation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Following the divergence of Artiodactyla, the mechanisms by which environmental factors drove divergent species to select the same gene for duplication at a specific genomic locus warrant in-depth investigation. Multiple members of the \u003cem\u003eSIPRGSG\u003c/em\u003e gene cluster (particularly SLCO1A2, IAPP, SPX, and GYS2) are directly involved in or regulate the absorption, metabolism, and homeostasis of nutrients, encompassing lipid absorption, glucose metabolism, and energy storage[\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The fact that this cluster serves as the site for SLC7A3 expansion in carnivores suggests that, during evolution, genes related to nutrient absorption and metabolism may be functionally co-arranged in the genome to cope with specific dietary selection pressures. Members of the \u003cem\u003eZDNMPC\u003c/em\u003e gene cluster (particularly NLRP12 and the MYADM region) are directly or indirectly associated with nutrient absorption and metabolic processes. NLRP12 influences the absorption environment by regulating intestinal immunity[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while the PRKCG participates in intracellular signaling triggered by insulin, affecting downstream pathways that control glucose uptake, utilization, and storage in cells[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. As the site of expansion for a key metabolic gene (SLC7A3) in ruminants, this cluster suggests that during evolution, genes related to nutrient absorption, metabolism, and immune regulation may be functionally co-arranged in the genome to cope with specific dietary and digestive ecological pressures.\u003c/p\u003e \u003cp\u003eThis pattern may be associated with differences in the nutritional status of food intake between herbivores and carnivores. The nutritional status of an organism acts as a powerful selective pressure, leaving characteristic signatures within its genome[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. There is a significant difference in nitrogen intake between carnivores and ruminants[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Through large-scale comparative genomics and evolutionary analysis across species, we revealed a phenomenon that defies the traditional paradigm of single-origin inheritance. The \u003cem\u003eSLC7A3\u003c/em\u003e gene has undergone significant, independent expansions in multiple, distantly related mammalian lineages, such as Carnivora and Artiodactyla. Critical evidence lies in the fact that these expansions occurred at distinct genomic loci and contradict the expected species phylogeny. This indicates that these are not conserved remnants of a single ancient duplication event inherited across lineages. Instead, repeated, independent selective sweeps favoring increased \u003cem\u003eSLC7A3\u003c/em\u003e copy number occurred in each lineage in response to divergent nitrogen metabolic pressures (high ammonia load from protein-rich diets in carnivores[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] versus heightened efficiency of nitrogen utilization on protein-scarce diets in ruminants[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]). Further functional evolutionary analysis demonstrated that these independently expanded \u003cem\u003eSLC7A3\u003c/em\u003e copies are under different selection pressure, which indicated different lineage may experience different selection pressures based on their unique food intake, leading to divergent evolutionary paths. The expression pattern of \u003cem\u003eSLC7A3\u003c/em\u003e gene was difference between carnivores and ruminants, resulting in genes with different functions[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In ruminants, arginine contributes to urea production, while urea supports microbial protein synthesis\u0026mdash;ensuring a symbiotic relationship that optimizes nitrogen use and reduces the host\u0026rsquo;s dependency on dietary amino acids[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This system is central to the ruminant\u0026rsquo;s ability to convert low-quality forage into high-quality protein[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Unlike monogastric animals, ruminants recycle urea back to the rumen. Urea produced in the liver enters the bloodstream and is excreted (via urine) or transported to the rumen via saliva or direct diffusion[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMammals repeatedly adapted to specialized nitrogen balance of diets intake, including plant-based diets for herbivores and meat-based diets for carnivores. The function of the CAT family (including \u003cem\u003eSLC7A3\u003c/em\u003e) is to transport arginine, ornithine, and lysine[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which particularly critical in nitrogen metabolic pathways and urea cycle[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our extensive cross-species comparative genomic and evolutionary analyses reveal a paradigm-shifting finding: \u003cem\u003eSLC7A3\u003c/em\u003e has repeatedly expanded independently and significantly in distantly related mammals like Carnivora (carnivores) and Artiodactyla (ungulates/ruminants). Carnivores must continuously detoxify and excrete the large amounts of ammonia generated from nitrogen metabolism[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], whereas ruminants require the retention of ingested nitrogen to achieve high nitrogen utilization efficiency[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Although carnivores and ruminants face distinctly different nitrogen metabolic demands, both processes likely necessitate enhanced efficiency of the urea cycle. This is because the urea cycle plays a pivotal role both in the detoxification of ammonia to urea for excretion in carnivores and in the reutilization of urea as a non-protein nitrogen source in ruminants. Our study shows that \u003cem\u003eSLC7A3\u003c/em\u003e gene families have been repeatedly recruited in convergently evolved urea cycle, even across vast evolutionary timescales. Gene duplication plays a crucial role in the formation of novel traits throughout animal evolution, from lower to higher organisms[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Key evidence includes the expansions occurring at specific, non-conserved genomic locations mismatched with phylogenetic relationships. This demonstrates these expansions are not passive inheritance of an ancient duplication event, but rather the result of repeated, parallel natural selection for increased \u003cem\u003eSLC7A3\u003c/em\u003e copy number-likely driven by contrasting nitrogen metabolic demands (ammonia stress vs. nitrogen conservation).\u003c/p\u003e \u003cp\u003eThese results indicate that animals from different lineages have converged on duplicating the same gene to cope with nitrogen metabolism challenges, but the locations of these expansions in the genome vary across lineages, while they are identical within the same lineage. Additionally, we found that after hybridization between zebu cattle and taurine cattle, the copy number of \u003cem\u003eSLC7A3\u003c/em\u003e in the hybrid offspring differed from that of the parental lines, and this chromosomal region is prone to rearrangement and duplication during hybridization. This phenomenon suggests that the copy number of the \u003cem\u003eSLC7A3\u003c/em\u003e gene may exhibit copy number variation among different populations within the same animal species[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The rearrangement and combination of exons allow rapid creation of \u003cem\u003eSLC7A3\u003c/em\u003e genes with different functions, a key driver in gene family expansion. Comparison of the expression levels of \u003cem\u003eSLC7A3\u003c/em\u003e expanded members across different tissues in three ruminants and one carnivore revealed that tissues with consistently high expression in all four species include blood, intestine, spleen, and lung. The overlap in highly expressed organs (such as intestine, blood, lung, and spleen) suggests that both animal groups face common ammonia-nitrogen pressure and have evolved similar adaptive strategies: enhancing arginine transport to optimize urea cycle function and immune responses. Despite the conventional notion that the urea cycle takes place exclusively in the liver, here we demonstrate that these tissues also have the potential to maintain nitrogen balance. High expression in blood ensures efficient systemic circulation of arginine, supporting urea synthesis and effecting the nitrogen metabolism[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The intestine, as the frontline site for arginine absorption from plant[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], expresses expanded \u003cem\u003eSLC7A3\u003c/em\u003e members to improve arginine uptake efficiency[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Increased expression in the lungs can elevate arginine levels in pulmonary cells. Arginine serves as a substrate for nitric oxide synthase (NOS) in the lungs, participating in the production of nitric oxide (NO), thereby playing multiple key physiological and pathophysiological roles in the lungs and airways[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The finding that the other six highly expressed tissues are entirely distinct between carnivores and ruminants indicates that the function of \u003cem\u003eSLC7A3\u003c/em\u003e diverged, following distinct evolutionary paths in the two lineages. Even the most highly expressed \u003cem\u003eSLC7A3\u003c/em\u003e gene copy in each species shows no synteny across the ruminants. This indicates that the copy number expansion of \u003cem\u003eSLC7A3\u003c/em\u003e and gain of function has followed independent, species-specific evolutionary trajectories.\u003c/p\u003e \u003cp\u003eFunctional evolutionary analysis demonstrated that these independently expanded \u003cem\u003eSLC7A3\u003c/em\u003e copies are under different selection pressure between carnivores and ruminants. Following the duplication of the \u003cem\u003eSLC7A3\u003c/em\u003e gene, the new copies in ruminants and carnivores likely evolved toward different functions[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. As the environment changed, the new copies in carnivores gradually became redundant, with reduced environmental constraints leading to increased genetic variation[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This ultimately resulted in these copies becoming pseudogenes within the carnivore lineage. Only a subset of \u003cem\u003eSLC7A3\u003c/em\u003e gene members was retained to meet the demands of arginine absorption and ammonia detoxification. This has enabled carnivoran animals to retain evolutionary plasticity, allowing them to evolve toward omnivory or herbivory (as seen in the giant panda and the dog), as well as toward strict carnivory (as seen pseudogenes in the gray wolf and dingo). This strongly implicates enhanced transport capacity for arginine in ammonia detoxification pathways as the adaptive driver behind these convergent evolutionary events. In contrast, ruminants, due to the low nitrogen content in their ingested food, evolved the ability to utilize non-protein nitrogen (urea) via rumen microbes to maintain nitrogen balance. This adaptation allows for the maximal utilization of dietary nitrogen, particularly under conditions of nutritional scarcity such as on cold-season grasslands[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], in order to sustain vital functions. This may also explain why the newly duplicated members of \u003cem\u003eSLC7A3\u003c/em\u003e in ruminants faced greater selective pressure than carnivores.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study reveals that the SLC7A3 gene, part of the Cationic Amino Acid Transporter (CAT) family, has undergone independent expansion via gene duplication in two distinct mammalian lineages\u0026mdash;ruminants (herbivores) and carnivores\u0026mdash;to address challenges related to nitrogen metabolism and balance. Following duplication, the SLC7A3 copies in carnivores and ruminants experienced different evolutionary pressures. Genes in the carnivore branch underwent relaxed selection, while those in the ruminant branch were under strong purifying selection. The genomic locations of these independent expansion events differ between the two lineages but are conserved within each lineage. In ruminants, expansion occurred in an ancestral syntenic block (ZDNMPC cluster), while in carnivores, it occurred in a derived region (SIPRGSG cluster). The SLC7A3 paralogs are highly expressed in tissues like intestines, blood, lungs, and spleen. This suggests their role is to optimize arginine absorption and urea cycle function, enhancing the transport of arginine\u0026mdash;a key precursor for the urea cycle. Despite facing opposite nitrogen balance challenges (carnivores need to efficiently detoxify and excrete ammonia as urea, while ruminants need to recycle nitrogen via urea for microbial protein synthesis), both lineages convergently evolved SLC7A3 gene expansion as a strategy to enhance urea cycle and arginine transport capabilities. This represents a classic case of parallel evolution, but occurring at the level of genomic structural variation (copy number variation) rather than through convergent evolution of protein-coding sequences. This expands the understanding of molecular mechanisms underlying convergent evolution. The study directly links a specific physiological challenge (ammonia metabolism under different dietary nitrogen regimes) to large-scale genomic evolutionary patterns, establishing a research paradigm bridging diet, physiology, and genomics.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of SLC gene superfamily members\u003c/h2\u003e \u003cp\u003eWe use the sheep genome as the focal reference to identify SLC gene during ruminant evolution. The genomic data of \u003cem\u003eOvis aries\u003c/em\u003e and \u003cem\u003eHomo sapiens\u003c/em\u003e were obtained from the genome database of NCBI website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (GCF_016772045.2, GCA_ 000001405.29). Then we obtained the hidden Markov model (HMM) profile of the SLC superfamily from Pfam protein family database (Table S3). We found 438 os-SLC genes from Ovis aries genome. Specifically, According to the study about human SLC gene superfamily[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], we downloaded 392 SLC superfamily members of Homo sapiens (hs-SLC) protein sequences from UniProt database. We identified all os-SLC genes from the \u003cem\u003eOvis aries\u003c/em\u003e genome that had these hs-SLC protein sequences as targets. The conserved domains of these os-SLC genes were analyzed, and the genes without the SLC domain were removed. The identified \u003cem\u003eOvis aries\u003c/em\u003e SLC genes were then queried via BLASTp in the NCBI database to determine whether they belonged to the os-SLC gene family. Finally, 438 genes with SLC domains were identified at the genome-wide scale. Sequences of CATs of 27 mammalian species representative of Primates, Rodentia, Lagomorpha, Artiodactyla, Perissodactyla, Carnivora, Chiroptera, Soricomorpha, Proboscidea, Kangaroo order and Monotremes were obtained from the National Center for Biotechnology Information (NCBI, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis and gene structure\u003c/h2\u003e \u003cp\u003eThe SLC proteins of \u003cem\u003eHomo sapiens\u003c/em\u003e for the construction of Maximum Likelihood (ML) systematic evolutionary trees were obtained from the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The sequences of the SLC proteins in \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eOvis aries\u003c/em\u003e were compared with Clustal Omega. The systematic evolutionary tree included \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eOvis aries\u003c/em\u003e and was constructed using IQtree with the ML method[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and the Best-fit model was: PMB\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;R5 chosen according to bayesian information criterion[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The DNA and cDNA sequences of the \u003cem\u003eos-SLC\u003c/em\u003e genes were used to predict intron structure via the online Gene Structure Display Service (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conserved motifs of os-SLC proteins in \u003cem\u003eOvis aries\u003c/em\u003e were determined with MEME software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp:/meme.nbcr.net/meme/intro.html\u003c/span\u003e\u003cspan address=\"http://meme.nbcr.net/meme/intro.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and in accordance with the corresponding parameters set by Liu et al.[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIntraspecific and Interspecific collinearity analysis\u003c/h2\u003e \u003cp\u003eThe locations of 51 \u003cem\u003eos-SLC7\u003c/em\u003e genes on chromosomes were obtained based on the information annotated for the \u003cem\u003eOvis aries\u003c/em\u003e genome and analyzed through the Gene Location Visualization of TBtools[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The genome sequence and GTF file of \u003cem\u003eHomo sapiens\u003c/em\u003e (GCF_000001405.40), \u003cem\u003eCapra hircus\u003c/em\u003e (GCF_001704415.2), \u003cem\u003eBos taurus\u003c/em\u003e (GCA_002263795.4), \u003cem\u003eCamelus dromedarius\u003c/em\u003e (GCF_036321535.1), \u003cem\u003eTursiops truncatus\u003c/em\u003e (GCF_011762595.1) downloaded from NCBI. Analysis of genome collinearity was performed using MCScanX software[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Circos[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] and Dual Synteny Plot were used Comparative Genome Viewer online tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cgv/15545/9940\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cgv/15545/9940\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for visualized mapping of the collinear gene pairs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSearch and filter for functionally characterized CATs gene in mammalians\u003c/h2\u003e \u003cp\u003eData for the 666 functionally characterized CATs in 27 mammalians (including 6 \u003cem\u003eCarnivoras\u003c/em\u003e, and 5 \u003cem\u003ePerissodactylas\u003c/em\u003e, and 8 \u003cem\u003eArtiodactylas\u003c/em\u003e, and 1 \u003cem\u003eCetaceas\u003c/em\u003e, and 1 \u003cem\u003eChiroptera\u003c/em\u003e, and 1 \u003cem\u003eInsectivora\u003c/em\u003e, and 1 \u003cem\u003eRodentia\u003c/em\u003e, and 1 \u003cem\u003eLagomorpha\u003c/em\u003e, and 1 \u003cem\u003ePrimates\u003c/em\u003e, and 1 \u003cem\u003eProboscidea\u003c/em\u003e, and 1 \u003cem\u003eMarsupialia\u003c/em\u003e, and 1 \u003cem\u003eMonotreme\u003c/em\u003e) (Tables S4-S7) were sourced from GenBank. In addition, the distribution of exon counts of SLC7A3 expanded genes were analyzed between carnivores and herbivores based on the genome annotation file.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eVariation in Selective Pressure between Ruminants and Carnivores in CAT3s\u003c/h2\u003e \u003cp\u003eExpansion members of \u003cem\u003eSLC7A3\u003c/em\u003e originated from tandem duplication events with lineage-specific convergent expansions. We therefore examined the selection pressures on the candidate branch as foreground by comparing their sequences with the background branch and calculating the ratio of non-synonymous substitution rate (\u003cem\u003edN\u003c/em\u003e) to synonymous substitution rate (\u003cem\u003edS\u003c/em\u003e) between the paralogs. We first aligned the codon sequences of the \u003cem\u003eSLC7A3\u003c/em\u003e and their paralogous copies using ClustalW codon in MEGA12[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], and then transformed the results into codon-level alignment using the DAMBE tool[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Then, we calculated the \u003cem\u003eω\u003c/em\u003e values (dN/dS) and performed the likelihood ratio test (LRT) using CODEML in the PAML4.10 package [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. We also execute CODEML under the branch model when assuming different selective pressures (i.e., different \u003cem\u003eω\u003c/em\u003e ratios) for ruminants and carnivores linages on the tree and conduct an LRT for positive selection affecting prespecified branches using M0 as the null model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe expression analysis of CAT3s from RNA‑Seq Data\u003c/h2\u003e \u003cp\u003eTo study the expression patterns of CAT3s gene in different organ between ruminant and carnivora, the RNA-seq data of 3 ruminant animals[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] and 1 \u003cem\u003ecarnivores\u0026rsquo;\u003c/em\u003e animal[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] were downloaded from the NCBI (Accession number: PRJNA1017964, PRJNA867700 and PRJNA 906635). The gene expression level (transcripts per million) TPM was calculated using the Salmon tool [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Salmon estimated transcript abundances for each sample based on the FASTQ files. After running salmon quant for all samples, the individual TPM results were merged into a single table for easier downstream analysis using the salmon quantmerge command. The average expression value from the biological replication represents the organ expression level for each species. Finally, the CAT3s expansion gene member\u0026rsquo;s expression values were extracted for downstream analysis (Table S8).\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interests.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eConceptualization, J.W. and S.H.; methodology, B.W. and W.L.; software, Y.Z.; validation, J.S., Y.Z. and J.W.; formal analysis, D.W.; investigation, J.S. and B.W.; resources, J.S.; data curation, J.S. and Y.Z.; writing\u0026mdash;original draft preparation, S.H.; writing\u0026mdash;review and editing, J.W. and J.Y.; visualization, W.L.; supervision, J.W.; project administration, S.H.; funding acquisition, J.W. and S.H.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors are grateful for the support by the National Natural Science Foundation of China (32260812), the Inner Mongolia Autonomous Region\u0026rsquo;s Key Technology Tackling Plan (2023YFDZ0027, 2025YFDZ0135), the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region (2023NJYT23015), the Youth Program for Grassland Elite in Inner Mongolia Autonomous Region for Dr. Wu and First-class Discipline Research Special Project in Inner Mongolia Autonomous Region Department of Education(YLXKZX-NMD-009), the Inner Mongolia Autonomous Region\u0026rsquo;s Key Technology Tackling Plan (2025KJHZ0001) for Dr. Hu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBates, O.K., Bertelsmeier, C.: Climatic niche shifts in introduced species. Curr. Biol. \u003cb\u003e31\u003c/b\u003e(19), R1252\u0026ndash;R66 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cub.2021.08.035\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2021.08.035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid, K.T., Schraiber, J.G., Crandall, J.G., Labella, A.L., Opulente, D.A., Harrison, M.-C., et al.: Convergent expansions of keystone gene families drive metabolic innovation in Saccharomycotina yeasts. Proceedings of the National Academy of Sciences. ;122(23):e2500165122. doi: (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2500165122\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2500165122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid, K.T., Oaks, J.R., Halanych, K.M.: Patterns of gene evolution following duplications and speciations in vertebrates. PeerJ. \u003cb\u003e8\u003c/b\u003e, e8813 (2020). Epub 2020/04/09 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7717/peerj.8813\u003c/span\u003e\u003cspan address=\"10.7717/peerj.8813\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKondrashov, F.A.: Gene duplication as a mechanism of genomic adaptation to a changing environment. Proceedings Biological sciences. ;279(1749):5048-57. Epub 2012/09/15. (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1098/rspb.2012.1108\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2012.1108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLallemand, T., Leduc, M., Land\u0026egrave;s, C., Rizzon, C., Lerat, E.: An Overview of Duplicated Gene Detection Methods: Why the Duplication Mechanism Has to Be Accounted for in Their Choice. Genes. \u003cb\u003e11\u003c/b\u003e(9) (2020). Epub 2020/09/10 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/genes11091046\u003c/span\u003e\u003cspan address=\"10.3390/genes11091046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGabora, L.: Convergent Evolution. In: Maloy S, Hughes K, editors. Brenner's Encyclopedia of Genetics (Second Edition). San Diego: Academic Press; pp. 178\u0026thinsp;\u0026ndash;\u0026thinsp;80. (2013)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchermerhorn, T.: Normal glucose metabolism in carnivores overlaps with diabetes pathology in non-carnivores. Front. Endocrinol. \u003cb\u003e4\u003c/b\u003e, 188 (2013). Epub 2013/12/19 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2013.00188\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2013.00188\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, G., Zhu, Y., Feng, D., Yao, J., Cao, Y., Deng, L.: Hepatic gluconeogenesis and regulatory mechanisms in lactating ruminants: A literature review. Anim. Res. One Health. \u003cb\u003e3\u003c/b\u003e(3), 230\u0026ndash;239 (2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris, J.G.: Nutritional and Metabolic Responses to Arginine Deficiency in Carnivores. J. Nutr. \u003cb\u003e115\u003c/b\u003e(4), 524\u0026ndash;531 (1985). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jn/115.4.524\u003c/span\u003e\u003cspan address=\"10.1093/jn/115.4.524\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, X.M., Chen, W.X., Yan, Q.X., Wang, C., Lin, B., Yi, S.Y., et al.: Low-protein diet promotes nitrogen retention efficiency via enhanced renal urea reabsorption and microbial hydrogen incorporation in the rumen of goats. Anim. Feed Sci. Technol. \u003cb\u003e305\u003c/b\u003e, 115762 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anifeedsci.2023.115762\u003c/span\u003e\u003cspan address=\"10.1016/j.anifeedsci.2023.115762\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinger, M.A., Part, B.: Biochem. Mol. Biology. \u003cb\u003e134\u003c/b\u003e(4), 543\u0026ndash;558 (2003). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1096-4959(03)00027-7\u003c/span\u003e\u003cspan address=\"10.1016/S1096-4959(03)00027-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHristov, A.N., Bannink, A., Crompton, L.A., Huhtanen, P., Kreuzer, M., McGee, M., et al.: Invited review: Nitrogen in ruminant nutrition: A review of measurement techniques. J. Dairy Sci. \u003cb\u003e102\u003c/b\u003e(7), 5811\u0026ndash;5852 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3168/jds.2018-15829\u003c/span\u003e\u003cspan address=\"10.3168/jds.2018-15829\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCloss, E., Boissel, J.-P., Habermeier, A., Rotmann, A.: Structure and function of cationic amino acid transporters (CATs). J. Membr. Biol. \u003cb\u003e213\u003c/b\u003e(2), 67\u0026ndash;77 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJungnickel, K.E., Parker, J.L., Newstead, S.: Structural basis for amino acid transport by the CAT family of SLC7 transporters. Nat. Commun. \u003cb\u003e9\u003c/b\u003e(1), 550 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFotiadis, D., Kanai, Y., Palac\u0026iacute;n, M.: The SLC3 and SLC7 families of amino acid transporters. Mol. Aspects Med. \u003cb\u003e34\u003c/b\u003e(2\u0026ndash;3), 139\u0026ndash;158 (2013)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Aacute;lvarez-Carretero, S., Tamuri, A.U., Battini, M., Nascimento, F.F., Carlisle, E., Asher, R.J., et al.: A species-level timeline of mammal evolution integrating phylogenomic data. Nature. \u003cb\u003e602\u003c/b\u003e(7896), 263\u0026ndash;267 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41586-021-04341-1\u003c/span\u003e\u003cspan address=\"10.1038/s41586-021-04341-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinthorsdottir, M., Coxall, H.K., Boer, A.M., Huber, M., Barbolini, N., Bradshaw, C.D., et al.: The Miocene: The Future of the Past. Paleoceanography Paleoclimatology ;\u003cb\u003e36\u003c/b\u003e. (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdwards, E.J., Osborne, C.P., Str\u0026ouml;mberg, C.A., Smith, S.A., Bond, W.J., Christin, P.A., et al.: The origins of C4 grasslands: integrating evolutionary and ecosystem science. Sci. (New York NY). \u003cb\u003e328\u003c/b\u003e(5978), 587\u0026ndash;591 (2010). Epub 2010/05/01 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.1177216\u003c/span\u003e\u003cspan address=\"10.1126/science.1177216\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Deus Vidal, J., le Roux, P.C., Johnson, S.D., te Beest, M., Clark, V.R. (eds.): Beyond the Tree-Line: The C3-C4 Grass-Line Can Track Global Change in the World\u0026rsquo;s Grassy Mountain Systems. Frontiers in Ecology and Evolution (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSage, R.F., Christin, P.A., Edwards, E.J.: The C(4) plant lineages of planet Earth. J. Exp. Bot. \u003cb\u003e62\u003c/b\u003e(9), 3155\u0026ndash;3169 (2011). Epub 2011/03/19 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/err048\u003c/span\u003e\u003cspan address=\"10.1093/jxb/err048\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuckley, M.: Ancient collagen reveals evolutionary history of the endemic South American \u0026lsquo;ungulates\u0026rsquo;. Proceedings of the Royal Society B: Biological Sciences. ;282(1806):20142671. (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, W., Silvestro, D., Onstein, R.E., Wu, M., Zhou, Z., Xing, Y.: The stepwise rise of angiosperm-dominated terrestrial ecosystems. Biol. Rev. (2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanco, F., Lazagabaster, I.A., Sanisidro, \u0026Oacute;., Bibi, F., Heckeberg, N.S., R\u0026iacute;os, M., et al.: Two major ecological shifts shaped 60 million years of ungulate faunal evolution. Nat. Commun. \u003cb\u003e16\u003c/b\u003e(1), 4648 (2025). Epub 2025/06/06 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-025-59974-x\u003c/span\u003e\u003cspan address=\"10.1038/s41467-025-59974-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbraham, J.O., Upham, N.S., Damian-Serrano, A., Jesmer, B.R.: Evolutionary causes and consequences of ungulate migration. Nat. Ecol. Evol. \u003cb\u003e6\u003c/b\u003e(7), 998\u0026ndash;1006 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41559-022-01749-4\u003c/span\u003e\u003cspan address=\"10.1038/s41559-022-01749-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, Y., Wang, H., Wang, H., Feng, J.: Arms race of temporal partitioning between carnivorous and herbivorous mammals. Sci. Rep. \u003cb\u003e8\u003c/b\u003e(1), 1713 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-018-20098-6\u003c/span\u003e\u003cspan address=\"10.1038/s41598-018-20098-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Aacute;vila, C., Llebr\u0026eacute;s, M.T., C\u0026aacute;novas, F.M., Castro-Rodr\u0026iacute;guez, V.: Arginine, a key amino acid for nitrogen nutrition and metabolism of forest trees. J. Exp. Bot. \u003cb\u003e76\u003c/b\u003e(18), 5238\u0026ndash;5251 (2025). Epub 2025/06/10 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jxb/eraf260\u003c/span\u003e\u003cspan address=\"10.1093/jxb/eraf260\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHailemariam, S., Zhao, S., He, Y., Wang, J.: Urea transport and hydrolysis in the rumen: a review. Anim. Nutr. \u003cb\u003e7\u003c/b\u003e(4), 989\u0026ndash;996 (2021)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaven, J., Thomas, H., Grasses: Curr. Biol. \u003cb\u003e20\u003c/b\u003e(19), R837\u0026ndash;R9 (2010). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cub.2010.08.031\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2010.08.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai, B., Wang, Y.-Q., Meng, J.: The divergence and dispersal of early perissodactyls as evidenced by early Eocene equids from Asia. Commun. Biology. \u003cb\u003e1\u003c/b\u003e(1), 115 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s42003-018-0116-5\u003c/span\u003e\u003cspan address=\"10.1038/s42003-018-0116-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, J., Zhou, X., Wang, S., Ye, J., Liu, J., Wu, W., et al.: Steppe development and mammalian adaptation in the middle Miocene, North Junggar Basin, Central Asia. Palaeogeography, Palaeoclimatology, Palaeoecology. ;\u003cb\u003e659\u003c/b\u003e:112634. (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.palaeo.2024.112634\u003c/span\u003e\u003cspan address=\"10.1016/j.palaeo.2024.112634\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, W., Silvestro, D., Onstein, R.E., Wu, M., Zhou, Z., Xing, Y.: The stepwise rise of angiosperm-dominated terrestrial ecosystems. Biol. Rev. Camb. Philos. Soc. \u003cb\u003e100\u003c/b\u003e(5), 2131\u0026ndash;2149 (2025). Epub 2025/05/30 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/brv.70039\u003c/span\u003e\u003cspan address=\"10.1111/brv.70039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatona, K., Coetsee, C.: Impacts of Browsing and Grazing Ungulates on Faunal Biodiversity. In: Gordon, I.J., Prins, H.H.T. (eds.) The Ecology of Browsing and Grazing II, pp. 277\u0026ndash;300. Springer International Publishing, Cham (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaarinen, J.: The Palaeontology of Browsing and Grazing. In: Gordon, I.J., Prins, H.H.T. (eds.) The Ecology of Browsing and Grazing II, pp. 5\u0026ndash;59. Springer International Publishing, Cham (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColledge, S., Conolly, J., Dobney, K., Manning, K., Shennan, S.: The Origins and Spread of Domestic Animals in Southwest Asia and Europe. Eur. J. Archaeol. \u003cb\u003e18\u003c/b\u003e(2), 356\u0026ndash;360 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, S.-Q., Jiangzuo, Q., Bibi, F., Zhang, C., Li, C., Li, S., et al.: Central Asian radiation of modern large-mammal faunas in Miocene. Sci. Adv. \u003cb\u003e11\u003c/b\u003e(43), eadt5079 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/sciadv.adt5079\u003c/span\u003e\u003cspan address=\"10.1126/sciadv.adt5079\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHecker, N., Sharma, V., Hiller, M.: Convergent gene losses illuminate metabolic and physiological changes in herbivores and carnivores. Proc. Natl. Acad. Sci. U.S.A. \u003cb\u003e116\u003c/b\u003e(8), 3036\u0026ndash;3041 (2019). Epub 2019/02/06 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1818504116\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1818504116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris, J.G.: Nutritional and metabolic responses to arginine deficiency in carnivores. J. Nutr. \u003cb\u003e115\u003c/b\u003e(4), 524\u0026ndash;531 (1985)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan, A., Liu, Y., Gad, M., Kenny, T.C., Birsoy, K.: Solute carriers: The gatekeepers of metabolism. Cell. \u003cb\u003e188\u003c/b\u003e(4), 869\u0026ndash;884 (2025). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2025.01.015\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2025.01.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai, Q., Ma, Y., Bai, J., Zhuang, M., Pei, S., He, N., et al.: Mechanisms for Bile Acids CDCA- and DCA-Stimulated Hepatic Spexin Expression. Cells. \u003cb\u003e11\u003c/b\u003e(14) (2022). Epub 2022/07/28 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cells11142159\u003c/span\u003e\u003cspan address=\"10.3390/cells11142159\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarlsson, E.: IAPP as a regulator of glucose homeostasis and pancreatic hormone secretion (review). Int. J. Mol. Med. \u003cb\u003e3\u003c/b\u003e(6), 577\u0026ndash;584 (1999). Epub 1999/05/26 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/ijmm.3.6.577\u003c/span\u003e\u003cspan address=\"10.3892/ijmm.3.6.577\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evon Wilamowitz-Moellendorff, A., Hunter, R.W., Garc\u0026iacute;a-Rocha, M., Kang, L., L\u0026oacute;pez-Soldado, I., Lantier, L., et al.: Glucose-6-phosphate-mediated activation of liver glycogen synthase plays a key role in hepatic glycogen synthesis. Diabetes. \u003cb\u003e62\u003c/b\u003e(12), 4070\u0026ndash;4082 (2013). Epub 2013/08/31 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2337/db13-0880\u003c/span\u003e\u003cspan address=\"10.2337/db13-0880\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTruax, A.D., Chen, L., Tam, J.W., Cheng, N., Guo, H., Koblansky, A.A., et al.: The Inhibitory Innate Immune Sensor NLRP12 Maintains a Threshold against Obesity by Regulating Gut Microbiota Homeostasis. Cell. Host Microbe. \u003cb\u003e24\u003c/b\u003e(3), 364\u0026ndash;78e6 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chom.2018.08.009\u003c/span\u003e\u003cspan address=\"10.1016/j.chom.2018.08.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoss, R., Wang, P.Y., Chari, M., Lam, C.K., Caspi, L., Ono, H., et al.: Hypothalamic protein kinase C regulates glucose production. Diabetes. \u003cb\u003e57\u003c/b\u003e(8), 2061\u0026ndash;2065 (2008). Epub 2008/05/31 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2337/db08-0206\u003c/span\u003e\u003cspan address=\"10.2337/db08-0206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaharjan, R.P., Ferenci, T.: A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input\u0026ndash;mutation output relationships. PLoS Biol. \u003cb\u003e15\u003c/b\u003e(6), e2001477 (2017)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGetahun, D., Alemneh, T., Akeberegn, D., Getabalew, M., Zewdie, D.: Urea metabolism and recycling in ruminants. Biomed. J. Sci. Tech. Res. \u003cb\u003e20\u003c/b\u003e(1), 14790\u0026ndash;14796 (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuntington, G., Archibeque, S. (eds.): Practical aspects of urea and ammonia metabolism in ruminants. Proc Am Soc Anim Sci (1999)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuBose, J.G., de Roode, J.C.: The link between gene duplication and divergent patterns of gene expression across a complex life cycle. Evol. Lett. \u003cb\u003e8\u003c/b\u003e(5), 726\u0026ndash;734 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/evlett/qrae028\u003c/span\u003e\u003cspan address=\"10.1093/evlett/qrae028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoppi, D.P., McLennan, S.R.: Protein and energy utilization by ruminants at pasture. J. Anim. Sci. \u003cb\u003e73\u003c/b\u003e(1), 278\u0026ndash;290 (1995)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHailemariam, S., Zhao, S., He, Y., Wang, J.: Urea transport and hydrolysis in the rumen: A review. Animal nutrition (Zhongguo xu mu shou yi xue hui). ;\u003cb\u003e7\u003c/b\u003e(4):989\u0026ndash;996. Epub 2021/11/06. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.aninu.2021.07.002\u003c/span\u003e\u003cspan address=\"10.1016/j.aninu.2021.07.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeireles, P., Mendes, A.M., Aroeira, R.I., Mounce, B.C., Vignuzzi, M., Staines, H.M., et al.: Uptake and metabolism of arginine impact Plasmodium development in the liver. Sci. Rep. \u003cb\u003e7\u003c/b\u003e(1), 4072 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-017-04424-y\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-04424-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuzuriaga-Neira, A., Subramanian, K., Alvarez-Ponce, D.: Functional compensation of mouse duplicates by their paralogs expressed in the same tissues. Genome biology and evolution. ;14(8). Epub 2022/08/11. (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/gbe/evac126\u003c/span\u003e\u003cspan address=\"10.1093/gbe/evac126\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConrad, B., Antonarakis, S.E.: Gene Duplication: A Drive for Phenotypic Diversity and Cause of Human Disease. Annual Review of Genomics and Human Genetics. ;8(Volume 8, 2007):17\u0026ndash;35. (2007). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev.genom.8.021307.110233\u003c/span\u003e\u003cspan address=\"10.1146/annurev.genom.8.021307.110233\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026auml;fer, Y., Palitzsch, K., Leptin, M., Whiteley, A.R., Wiehe, T., Suurv\u0026auml;li, J.: Copy number variation and population-specific immune genes in the model vertebrate zebrafish. Elife 2024;13. Epub 2024/06/04. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.98058\u003c/span\u003e\u003cspan address=\"10.7554/eLife.98058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReisz, J.A., Earley, E.J., Nemkov, T., Key, A., Stephenson, D., Keele, G.R., et al.: Arginine metabolism is a biomarker of red blood cell and human aging. Aging cell. \u003cb\u003e24\u003c/b\u003e(2), e14388 (2025). Epub 2024/10/31 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/acel.14388\u003c/span\u003e\u003cspan address=\"10.1111/acel.14388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eN\u0026uuml;se, B., Holland, T., Rauh, M., Gerlach, R.G., Mattner, J.: L-arginine metabolism as pivotal interface of mutual host-microbe interactions in the gut. Gut microbes. \u003cb\u003e15\u003c/b\u003e(1), 2222961 (2023). Epub 2023/06/26 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/19490976.2023.2222961\u003c/span\u003e\u003cspan address=\"10.1080/19490976.2023.2222961\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott, J.A., Maarsingh, H., Holguin, F., Grasemann, H.: Arginine Therapy for Lung Diseases. Front. Pharmacol. \u003cb\u003e12\u003c/b\u003e, 627503 (2021). Epub 2021/04/10 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphar.2021.627503\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2021.627503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing, N.E., Rothenberg, M.E., Zimmermann, N.: Arginine in Asthma and Lung Inflammation. J. Nutr. \u003cb\u003e134\u003c/b\u003e(10), 2830S\u0026ndash;6S (2004). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/jn/134.10.2830S\u003c/span\u003e\u003cspan address=\"10.1093/jn/134.10.2830S\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuzmin, E., Taylor, J.S., Boone, C.: Retention of duplicated genes in evolution. Trends Genet. \u003cb\u003e38\u003c/b\u003e(1), 59\u0026ndash;72 (2022). Epub 2021/07/24 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tig.2021.06.016\u003c/span\u003e\u003cspan address=\"10.1016/j.tig.2021.06.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalter, G.M., Monro, K., Terranova, D., la Spina, E., Majorana, M., Pepe, G., et al.: Environmental effects on genetic variance are likely to constrain adaptation in novel environments. Evol. Lett. \u003cb\u003e8\u003c/b\u003e(3), 374\u0026ndash;386 (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/evlett/qrad065\u003c/span\u003e\u003cspan address=\"10.1093/evlett/qrad065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe, L., Vasiliou, K., Nebert, D.W.: Analysis and update of the human solute carrier (SLC) gene superfamily. Hum. Genomics. \u003cb\u003e3\u003c/b\u003e(2), 195 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D., von Haeseler, A., et al.: IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. \u003cb\u003e37\u003c/b\u003e(5), 1530\u0026ndash;1534 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/molbev/msaa015\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msaa015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouckaert, R.R., Drummond, A.J.: bModelTest: Bayesian phylogenetic site model averaging and model comparison. BMC Evol. Biol. \u003cb\u003e17\u003c/b\u003e(1), 42 (2017). Epub 2017/02/09 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12862-017-0890-6\u003c/span\u003e\u003cspan address=\"10.1186/s12862-017-0890-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, M., Ma, Z., Wang, A., Zheng, T., Huang, L., Sun, W., et al.: Genome-wide investigation of the auxin response factor gene family in Tartary buckwheat (Fagopyrum tataricum). Int. J. Mol. Sci. \u003cb\u003e19\u003c/b\u003e(11), 3526 (2018)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, C., Chen, H., Zhang, Y., Thomas, H.R., Frank, M.H., He, Y., et al.: TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant. \u003cb\u003e13\u003c/b\u003e(8), 1194\u0026ndash;1202 (2020)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., Tang, H., DeBarry, J.D., Tan, X., Li, J., Wang, X., et al.: MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. \u003cb\u003e40\u003c/b\u003e(7), e49\u0026ndash;e (2012)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrzywinski, M.I., Schein, J.E., Birol, I., Connors, J., Gascoyne, R., Horsman, D., et al.: Circos: An information aesthetic for comparative genomics. Genome Res. (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gr.092759.109\u003c/span\u003e\u003cspan address=\"10.1101/gr.092759.109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., Tamura, K.: MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol Biol Evol. ;41(12). Epub 2024/12/22. (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/molbev/msae263\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msae263\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, X.: DAMBE7: New and Improved Tools for Data Analysis in Molecular Biology and Evolution. Mol. Biol. Evol. \u003cb\u003e35\u003c/b\u003e(6), 1550\u0026ndash;1552 (2018). Epub 2018/04/19 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/molbev/msy073\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msy073\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, L., Wang, Y., Zhang, L., Luo, C., Wu, F., Huang, Y., et al.: The HTIRDB: A resource containing a transcriptional atlas for 105 different tissues from each of seven species of domestic herbivore. iMeta. \u003cb\u003e4\u003c/b\u003e(1), e267 (2025). Epub 2025/03/03 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/imt2.267\u003c/span\u003e\u003cspan address=\"10.1002/imt2.267\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrause, C., Suwada, K., Blomme, E.A.G., Kowalkowski, K., Liguori, M.J., Mahalingaiah, P.K., et al.: Preclinical species gene expression database: Development and meta-analysis. Front. Genet. \u003cb\u003e13\u003c/b\u003e, 1078050 (2022). Epub 2023/02/04 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fgene.2022.1078050\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2022.1078050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatro, R., Duggal, G., Love, M.I., Irizarry, R.A., Kingsford, C.: Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods. \u003cb\u003e14\u003c/b\u003e(4), 417\u0026ndash;419 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nmeth.4197\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.4197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Parallel Evolution, SLC7A3 gene family, Nitrogen balance, Comparative Genomics, Expression pattern","lastPublishedDoi":"10.21203/rs.3.rs-8632196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8632196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdequate nitrogen metabolism is crucial for survival, yet carnivores and herbivores face distinct challenges: carnivores must efficiently detoxify ammonia, while herbivorous ruminants need to conserve and recycle nitrogen. Gene family expansion through duplication is a key evolutionary mechanism for adaptation. Here we show that the Cationic Amino Acid Transporter gene \u003cem\u003eSLC7A3\u003c/em\u003e has independently expanded via duplication in both ruminants (e.g., sheep, cattle) and carnivores to address their specific nitrogen balance pressures. These parallel expansions occurred at different, lineage-specific genomic locations. Following duplication, the \u003cem\u003eSLC7A3\u003c/em\u003e copies in carnivores underwent relaxed selection, whereas those in ruminants were under strong purifying selection. Expression analyses indicate high expression of these gene copies in intestines, blood, lungs, and spleen, suggesting a role in enhancing arginine transport to support urea cycle function. This study demonstrates convergent evolution through copy number variation, linking specific dietary challenges to large-scale genomic adaptation.\u003c/p\u003e","manuscriptTitle":"Independent Expansion of SLC7A3 in Mammals to Meet the Challenge of Nitrogen Metabolism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-26 09:18:07","doi":"10.21203/rs.3.rs-8632196/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"195d79a0-da6a-49a7-9662-5a6b4bbc39e7","owner":[],"postedDate":"February 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":63403185,"name":"Biological sciences/Evolution/Molecular evolution"},{"id":63403186,"name":"Biological sciences/Biochemistry/Ion channels"}],"tags":[],"updatedAt":"2026-04-15T15:51:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-26 09:18:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8632196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8632196","identity":"rs-8632196","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}