Sperm-Specific CatSper is Not Conserved in All Vertebrates and May Not be the Only Progesterone-Responsive Ion Channel Present in Sperm.

Progesterone (P4) acts as a key conserved signalling molecule in vertebrate reproduction. P4 is especially important for mature sperm physiology and subsequent reproductive success. "CatSpermasome", a multi-unit molecular complex, has been suggested to be the main if not the only P4-responsive atypical Ca2+-ion channel present in mature sperm. Altogether, here we analyse the protein sequences of CatSper1-4 from more than 500 vertebrates ranging from early fishes to humans. CatSper1 becomes longer in mammals due to sequence gain mainly at the N-terminus. Overall the conservation of full-length CatSper1-4 as well as the individual TM regions remain low. The lipid-water-interface residues (i.e. a 5 amino acid stretch sequence present on both sides of each TM region) also remain highly diverged. No specific patterns of amino acid distributions were observed. The total frequency of positively charged, negatively charged or their ratios do not follow in any specific pattern. Similarly, the frequency of total hydrophobic, total hydrophilic residues or even their ratios remain random and do not follow any specific pattern. We noted that the CatSper1-4 genes are missing in amphibians and the CatSper1 gene is missing in birds. The high variability of CatSper1-4 and gene-loss in certain clades indicate that the "CatSpermasome" is not the only P4-responsive ion channel. Data indicate that the molecular evolution of CatSper is mostly guided by diverse hydrophobic ligands rather than only P4. The comparative data also suggest possibilities of other Ca2+-channel/s in vertebrate sperm that can also respond to P4.

The evolution of mammalian Rem2: unraveling the impact of purifying selection and coevolution on protein function, and implications for human disorders.

Rad And Gem-Like GTP-Binding Protein 2 (Rem2), a member of the RGK family of Ras-like GTPases, is implicated in Huntington's disease and Long QT Syndrome and is highly expressed in the brain and endocrine cells. We examine the evolutionary history of Rem2 identified in various mammalian species, focusing on the role of purifying selection and coevolution in shaping its sequence and protein structural constraints. Our analysis of Rem2 sequences across 175 mammalian species found evidence for strong purifying selection in 70% of non-invariant codon sites which is characteristic of essential proteins that play critical roles in biological processes and is consistent with Rem2's role in the regulation of neuronal development and function. We inferred epistatic effects in 50 pairs of codon sites in Rem2, some of which are predicted to have deleterious effects on human health. Additionally, we reconstructed the ancestral evolutionary history of mammalian Rem2 using protein structure prediction of extinct and extant sequences which revealed the dynamics of how substitutions that change the gene sequence of Rem2 can impact protein structure in variable regions while maintaining core functional mechanisms. By understanding the selective pressures, protein- and gene - interactions that have shaped the sequence and structure of the Rem2 protein, we gain a stronger understanding of its biological and functional constraints.

Positive selection analysis of Cyt proteins from Bacillus thuringiensis: a conservative trend driven by negative (purifying) selection.

Bacillus thuringiensis is a Gram-positive entomopathogenic bacterium that produces different pesticidal proteins: vegetative insecticidal proteins (Vpb1/Vpa2, Vip3, and Vpb4) during vegetative growth and δ-endotoxins (Cry and Cyt) during sporulation, which accumulate into parasporal crystals. Cyt proteins are the smaller subset of δ-endotoxins targeting Diptera species. While Cry and Vip3 proteins undergo positive selection, our analysis suggests that Cyt proteins evolve following a conservative trend driven negative (purifying) selection.

The genome-wide signature of short-term temporal selection.

Despite evolutionary biology's obsession with natural selection, few studies have evaluated multigenerational series of patterns of selection on a genome-wide scale in natural populations. Here, we report on a 10-y population-genomic survey of the microcrustacean Daphnia pulex. The genome sequences of [Formula: see text]800 isolates provide insights into patterns of selection that cannot be obtained from long-term molecular-evolution studies, including the following: the pervasiveness of near quasi-neutrality across the genome (mean net selection coefficients near zero, but with significant temporal variance about the mean, and little evidence of positive covariance of selection across time intervals); the preponderance of weak positive selection operating on minor alleles; and a genome-wide distribution of numerous small linkage islands of observable selection influencing levels of nucleotide diversity. These results suggest that interannual fluctuating selection is a major determinant of standing levels of variation in natural populations, challenge the conventional paradigm for interpreting patterns of nucleotide diversity and divergence, and motivate the need for the further development of theoretical expressions for the interpretation of population-genomic data.

Toward a semi-supervised learning approach to phylogenetic estimation.

Models have always been central to inferring molecular evolution and to reconstructing phylogenetic trees. Their use typically involves the development of a mechanistic framework reflecting our understanding of the underlying biological processes, such as nucleotide substitu- tions, and the estimation of model parameters by maximum likelihood or Bayesian inference. However, deriving and optimizing the likelihood of the data is not always possible under complex evolutionary scenarios or even tractable for large datasets, often leading to unrealistic simplifying assumptions in the fitted models. To overcome this issue, we coupled stochastic simulations of genome evolution with a new supervised deep learning model to infer key parameters of molecular evolution. Our model is designed to directly analyze multiple sequence alignments and estimate per-site evolutionary rates and divergence, without requiring a known phylogenetic tree. The accuracy of our predictions matched that of likelihood-based phylogenetic inference, when rate heterogeneity followed a simple gamma distribution, but it strongly exceeded it under more complex patterns of rate variation, such as codon models. Our approach is highly scalable and can be efficiently applied to genomic data, as we showed on a dataset of 26 million nucleotides from the clownfish clade. Our simulations also showed that the integration of per-site rates obtained by deep learning within a Bayesian framework led to significantly more accu- rate phylogenetic inference, particularly with respect to the estimated branch lengths. We thus propose that future advancements in phylogenetic analysis will benefit from a semi-supervised learning approach that combines deep-learning estimation of substitution rates, which allows for more flexible models of rate variation, and probabilistic inference of the phylogenetic tree, which guarantees interpretability and a rigorous assessment of statistical support.

Crystal structure of L-2-keto-3-deoxyfuconate 4-dehydrogenase reveals a unique binding mode as a α-furanosyl hemiketal of substrates.

L-2-Keto-3-deoxyfuconate 4-dehydrogenase (L-KDFDH) catalyzes the NAD+-dependent oxidization of L-2-keto-3-deoxyfuconate (L-KDF) to L-2,4-diketo-3-deoxyfuconate (L-2,4-DKDF) in the non-phosphorylating L-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of L-KDF. On the other hand, BDH2, a mammalian homolog with L-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-L-proline (C4LHyp) with the cyclic structure. We found that L-KDFDH and BDH2 utilize C4LHyp and L-KDF, respectively. Therefore, to elucidate unique substrate specificity at the atomic level, we herein investigated for the first time the crystal structures of L-KDFDH from Herbaspirillum huttiense in the ligand-free, L-KDF and L-2,4-DKDF, D-KDP (D-2-keto-3-deoxypentonate; additional substrate), or L-2,4-DKDF and NADH bound forms. In complexed structures, L-KDF, L-2,4-DKDF, and D-KDP commonly bound as a α-furanosyl hemiketal. Furthermore, L-KDFDH showed no activity for L-KDF and D-KDP analogs without the C5 hydroxyl group, which form only the acyclic α-keto form. The C1 carboxyl and α-anomeric C2 hydroxyl groups and O5 oxygen atom of the substrate (and product) were specifically recognized by Arg148, Arg192, and Arg214. The side chain of Trp252 was important for hydrophobically recognizing the C6 methyl group of L-KDF. This is the first example showing the physiological role of the hemiketal of 2-keto-3-deoxysugar acid.

A new strategy to generate nanobodies for the coumaphos based on the synthesized nanobody libraries.

To break through the bottleneck in preparation of nanobody (Nb) for chemical contaminants induced by the difficulties in the synthesis of immunogen, complexity and unexpectable efficiency of immunization, a novel strategy to generate Nbs based on the designed synthetic Nb libraries with final size up to 109 cfu/mL was adopted and succeeded in selection of anti-coumaphos Nb A4. Furthermore, an affinity-matured mutant Nb 3G was obtained from the secondary library. Finally, an ic-ELISA was established with the limit of detection for coumaphos low to 1.90 ng/mL, 6.4-fold improved than the parent Nb A4, and the detection range from 3.06 to 15.77 ng/mL. Meanwhile, the recovery rate of vegetable samples was from 89.9% to 98.5%. Finally, the accuracy was testified by the standard UPLC-MS/MS method with R2 up to 0.99. Overall, fully synthetic Nb libraries constructed in this work provided an alternative possibility to generate the specific Nbs for chemical contaminants.

Selection Shapes the Genomic Landscape of Introgressed Ancestry in a Pair of Sympatric Sea Urchin Species.

A growing number of recent studies have demonstrated that introgression is common across the tree of life. However, we still have a limited understanding of the fate and fitness consequence of introgressed variation at the whole-genome scale across diverse taxonomic groups. Here, we implemented a phylogenetic hidden Markov model to identify and characterize introgressed genomic regions in a pair of well-diverged, nonsister sea urchin species: Strongylocentrotus pallidus and Strongylocentrotus droebachiensis. Despite the old age of introgression, a sizable fraction of the genome (1% to 5%) exhibited introgressed ancestry, including numerous genes showing signals of historical positive selection that may represent cases of adaptive introgression. One striking result was the overrepresentation of hyalin genes in the identified introgressed regions despite observing considerable overall evidence of selection against introgression. There was a negative correlation between introgression and chromosome gene density, and two chromosomes were observed with considerably reduced introgression. Relative to the nonintrogressed genome-wide background, introgressed regions had significantly reduced nucleotide divergence (dXY) and overlapped fewer protein-coding genes, coding bases, and genes with a history of positive selection. Additionally, genes residing within introgressed regions showed slower rates of evolution (dN, dS, dN/dS) than random samples of genes without introgressed ancestry. Overall, our findings are consistent with widespread selection against introgressed ancestry across the genome and suggest that slowly evolving, low-divergence genomic regions are more likely to move between species and avoid negative selection following hybridization and introgression.

Designing monomeric IFNγ: The significance of domain-swapped dimer structure in IFNγ immune responses.

Interferon (IFN) γ can initiate immune responses by inducing the expression of major histocompatibility complex molecules, suggesting its potential for cancer immunotherapy. However, it also has an immunosuppressive function that limits its application as a therapeutic agent. IFNγ has a characteristic domain-swapped dimer structure with two of the six α-helices exchanged with each other. As we hypothesized that the contrasting functions of IFNγ could be attributed to its unique domain-swapped structure, we designed monomeric IFNγ by transforming the domain-swapped dimer structure of WT IFNγ. We conjectured the evolution of this domain-swapped dimer and hypothesized that the current IFNγ structure emerged through shortening of the loop structure at the base of the swapped domain and the accumulation of hydrophobic amino acids at the newly generated interface during domain-swapping. We then designed and generated a stable monomeric IFNγ by retracing this evolutionary process, complementing the lost loop structure with a linker, and replacing the accumulated hydrophobic amino acids with hydrophilic ones. We determined that the designed variant was a monomer based on molecular size and number of epitopes and exhibited activity in cell-based assays. Notably, the monomeric IFNγ showed a qualitatively similar balance between immunostimulatory and immunosuppressive gene expression as WT IFNγ. This study demonstrates that the structural format of IFNγ affects the strength of its activity rather than regulating the fate of downstream gene expression.

Identification, molecular evolution, codon bias, and expansion analysis of NLP transcription factor family in foxtail millet (Setaria italica L.) and closely related crops.

The NODULE-INCEPTION-like protein (NLP) family is a plant-specific transcription factor (TF) family involved in nitrate transport and assimilation in plants, which are essential for improving plant nitrogen use efficiency. Currently, the molecular nature and evolutionary trajectory of NLP genes in the C4 model crop foxtail millet are unknown. Therefore, we performed a comprehensive analysis of NLP and molecular evolution in foxtail millet by scanning the genomes of foxtail millet and representative species of the plant kingdom. We identified seven NLP genes in the foxtail millet genome, all of which are individually and separately distributed on different chromosomes. They were not structurally identical to each other and were mainly expressed on root tissues. We unearthed two key genes (Si5G004100.1 and Si6G248300.1) with a variety of excellent characteristics. Regarding its molecular evolution, we found that NLP genes in Gramineae mainly underwent dispersed duplication, but maize NLP genes were mainly generated via WGD events. Other factors such as base mutations and natural selection have combined to promote the evolution of NLP genes. Intriguingly, the family in plants showed a gradual expansion during evolution with more duplications than losses, contrary to most gene families. In conclusion, this study advances the use of NLP genetic resources and the understanding of molecular evolution in cereals.

Evolution of rhodopsin in flatfishes (Pleuronectiformes) is associated with depth and migratory behavior.

Visual signals are involved in many fitness-related tasks and are therefore essential for survival in many species. Aquatic organisms are ideal systems to study visual evolution, as the high diversity of spectral properties in aquatic environments generates great potential for adaptation to different light conditions. Flatfishes are an economically important group, with over 800 described species distributed globally, including halibut, flounder, sole, and turbot. The diversity of flatfish species and wide array of environments they occupy provides an excellent opportunity to understand how this variation translates to molecular adaptation of vision genes. Using models of molecular evolution, we investigated how the light environments inhabited by different flatfish lineages have shaped evolution in the rhodopsin gene, which is responsible for mediating dim-light visual transduction. We found strong evidence for positive selection in rhodopsin, and this was correlated with both migratory behavior and several fundamental aspects of habitat, including depth and freshwater/marine evolutionary transitions. We also identified several mutations that likely affect the wavelength of peak absorbance of rhodopsin, and outline how these shifts in absorbance correlate with the response to the light spectrum present in different habitats. This is the first study of rhodopsin evolution in flatfishes that considers their extensive diversity, and our results highlight how ecologically-driven molecular adaptation has occurred across this group in response to transitions to novel light environments.

Comparative analysis the chloroplast genomes of Celastrus (Celastraceae) species: Provide insights into molecular evolution, species identification and phylogenetic relationships.

BACKGROUND: The genus Celastrus is an important medicinal plant resource. The similarity of morphology and the lack of complete chloroplast genome analysis have significantly impeded the exploration of species identification, molecular evolution and phylogeny of Celastrus. PURPOSE: In order to resolve the phylogenic controversy of Celastrus species, the chloroplast genome comparative analysis was performed to provide genetic evidence. METHODS: In this study, we collected and sequenced ten chloroplast genomes of Celastrus species from China and downloaded three chloroplast genomes from the databases. The chloroplast genomes were compared and analyzed to explore their characteristics and evolution. Furthermore, the phylogenetic relationships of Celastrus species were inferred based on the whole chloroplast genomes and protein-coding genes. RESULTS: All the 13 Celastrus species chloroplast genomes showed a typical quadripartite structure with genome sizes ranging from 155,113 to 157,366 bp. The intron loss of the rps16 gene occurred in all the 13 Celastrus species. The GC content, gene sequence, repeat types and codon bias pattern were highly conserved. Ten highly variation regions were identified, which can be used as potential DNA markers in molecular identification of Celastrus species. Eight genes, including accD, atp4, ndhB, rpoC1, rbcL, rpl2, rpl20 and ycf1, were detected to experience positive selection. Phylogenetic analysis showed that Celastrus was a monophyletic group and Tripterygium was the closest sister-group. Noteworthy, C. gemmatus Loes. and C. orbiculatus Thunb. can be discriminated using the chloroplast genome as a super barcode. The comparative and phylogenetic analysis results proposed that C. tonkinensis Pitard. was the synonym of C. hindsii Benth. CONCLUSION: The comparative analysis of the Celastrus chloroplast genomes can provide comprehensive genetic evidence for molecular evolution, species identification and phylogenetic relationships.

The substrate quality of CK2 target sites has a determinant role on their function and evolution.

Most biological processes are regulated by signaling modules that bind to short linear motifs. For protein kinases, substrates may have full or only partial matches to the kinase recognition motif, a property known as "substrate quality." However, it is not clear whether differences in substrate quality represent neutral variation or if they have functional consequences. We examine this question for the kinase CK2, which has many fundamental functions. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions, whereas minimal substrates are more weakly phosphorylated and have regulatory functions. Optimal CK2 sites tend to be more conserved, and substrate quality is often tuned by selection. For intermediate sites, increases or decreases in substrate quality may be deleterious, as we demonstrate for a CK2 substrate at the kinetochore. The results together suggest a strong role for substrate quality in phosphosite function and evolution. A record of this paper's transparent peer review process is included in the supplemental information.

Epistasis arises from shifting the rate-limiting step during enzyme evolution of a ß-lactamase.

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a ß-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme's conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

Genetic drift drives faster-Z evolution in the salmon louse Lepeophtheirus salmonis.

Sex chromosome evolution is a complex sub-field of population genetics with unresolved questions about how quickly and adaptively these chromosomes should evolve compared to autosomes. One key limitation to existing knowledge is an intense focus on only a handful of taxa, resulting in uncertainty about whether observed patterns reflect general processes or are idiosyncratic to the more widely-studied clades. In particular, the Z chromosomes of female heterogametic (ZW) systems tend to be quickly but not adaptively evolving in birds, while in butterflies and moths Z chromosomes tend to be evolving adaptively, but not always faster than autosomes. To understand how these two observations fit into broader evolutionary patterns, we explore patterns of Z chromosome evolution outside of these two well-studied clades. We utilize a publicly available high-quality genome, gene expression, population, and outgroup data for the salmon louse Lepeophtheirus salmonis, an important aquacultural pest copepod. We find that the Z chromosome is faster evolving than the autosomes, but that this effect is driven by increased drift rather than adaptive evolution. Due to high rates of female reproductive failure, the Z chromosome exhibits only a slightly lower effective population size than the autosomes which is nonetheless sufficient to decrease efficiency of hemizygous selection acting on the Z. These results highlight the usefulness of organismal life history in calibrating population genetic expectations and demonstrate the value of the ever-expanding wealth of modern publicly available genomic data to help resolve outstanding evolutionary questions.

Escalation by duplication: Milkweed bug trumps Monarch butterfly.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit. Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity. A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha- and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

Evolutionary trajectory of TRPM2 channel activation by adenosine diphosphate ribose and calcium.

Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca2+-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins.

Comparative analysis of the molecular starvation response of Southern Ocean copepods.

Large lipid-storing copepods dominate mesozooplankton biomass in the polar oceans and form a critical link between primary production and higher trophic levels. The ecological success of these species depends on their ability to survive periods of food deprivation in a highly seasonal environment, but the molecular changes that mediate starvation tolerance in these taxa are unknown. We conducted starvation experiments for two dominant Southern Ocean copepods, Calanoides acutus and Calanus propinquus, allowing us to compare the molecular starvation response between species. These species differ in life history, diet and metabolic traits, and expressed overlapping but distinct transcriptomic responses to starvation. Most starvation-response genes were species-specific, but we identified a conserved core set of starvation-response genes related to RNA and protein metabolism. We used phylotranscriptomics to place these results in the context of copepod evolution and found that starvation-response genes are under strong purifying selection at the sequence level and stabilizing selection at the expression level, consistent with their role in mediating essential biological functions. Selection on starvation-response genes was especially strong in our focal lipid-storing lineage relative to other copepod taxa, underscoring the significance of starvation tolerance for these species. We also found that certain key lipid enzymes (elongases and desaturases) have experienced diversification and positive selection in lipid-storing lineages, reflecting the unique lipid storage needs of these animals. Our results shed light on the molecular adaptations of high-latitude zooplankton to variable food conditions and suggest that starvation-response genes are under particularly strong sequence and expression constraints.

Evolutionary trends of respiratory syncytial viruses: Insights from large-scale surveillance and molecular dynamics of G glycoprotein.

Human respiratory syncytial virus (RSV) is an underlying cause of lower respiratory illnesses in children, elderly and immunocompromised adults. RSV contains multiple structural and non-structural proteins with two major glycoproteins that control the initial phase of infection, fusion glycoprotein and the attachment (G) glycoprotein. G protein attaches to the ciliated cells of airways initiating the infection. The hypervariable G protein plays a vital role in evolution of RSV strains. We employed multiple bioinformatics tools on systematically accessed large-scale data to evaluate mutations, evolutionary history, and phylodynamics of RSV. Mutational analysis of central conserved region (CCR) on G protein-coding sequences between 163 and 189 positions revealed frequent mutations at site 178 in human RSV (hRSV) A while arginine to glutamine substitutions at site 180 positions in hRSV B, remained prevalent from 2009 to 2014. Phylogenetic analysis indicates multiple signature mutations within G protein responsible for diversification of clades. The USA and China have highest number of surveillance records, followed by Kenya. Markov Chain Monte Carlo Bayesian skyline plot revealed that RSV A evolved steadily from 1990 to 2000, and rapidly between 2003 and 2005. Evolution of RSV B continued from 2003 to 2022, with a high evolution stage from 2016 to 2020. Throughout evolution, cysteine residues maintained their strict conserved states while CCR has an entropy value of 0.0039(±0.0005). This study concludes the notion that RSV G glycoprotein is continuously evolving while the CCR region of G protein maintains its conserved state providing an opportunity for CCR-specific monoclonal antibodys (mAbs) and inhibitors as potential candidates for immunoprophylaxis.

Characterization of the genetic variation and evolutionary divergence of the CLEC18 family.

BACKGROUND: The C-type lectin family 18 (CLEC18) with lipid and glycan binding capabilities is important to metabolic regulation and innate immune responses against viral infection. However, human CLEC18 comprises three paralogous genes with highly similar sequences, making it challenging to distinguish genetic variations, expression patterns, and biological functions of individual CLEC18 paralogs. Additionally, the evolutionary relationship between human CLEC18 and its counterparts in other species remains unclear. METHODS: To identify the sequence variation and evolutionary divergence of human CLEC18 paralogs, we conducted a comprehensive analysis using various resources, including human and non-human primate reference genome assemblies, human pangenome assemblies, and long-read-based whole-genome and -transcriptome sequencing datasets. RESULTS: We uncovered paralogous sequence variants (PSVs) and polymorphic variants (PVs) of human CLEC18 proteins, and identified distinct signatures specific to each CLEC18 paralog. Furthermore, we unveiled a novel segmental duplication for human CLEC18A gene. By comparing CLEC18 across human and non-human primates, our research showed that the CLEC18 paralogy probably occurred in the common ancestor of human and closely related non-human primates, and the lipid-binding CAP/SCP/TAPS domain of CLEC18 is more diverse than its glycan-binding CTLD. Moreover, we found that certain amino acids alterations at variant positions are exclusive to human CLEC18 paralogs. CONCLUSIONS: Our findings offer a comprehensive profiling of the intricate variations and evolutionary characteristics of human CLEC18.