Molecular and cellular mechanisms of teneurin signaling in synaptic partner matching.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.

Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

Archaeogenomic distinctiveness of the Isthmo-Colombian area.

The recently enriched genomic history of Indigenous groups in the Americas is still meager concerning continental Central America. Here, we report ten pre-Hispanic (plus two early colonial) genomes and 84 genome-wide profiles from seven groups presently living in Panama. Our analyses reveal that pre-Hispanic demographic events contributed to the extensive genetic structure currently seen in the area, which is also characterized by a distinctive Isthmo-Colombian Indigenous component. This component drives these populations on a specific variability axis and derives from the local admixture of different ancestries of northern North American origin(s). Two of these ancestries were differentially associated to Pleistocene Indigenous groups that also moved into South America, leaving heterogenous genetic footprints. An additional Pleistocene ancestry was brought by a still unsampled population of the Isthmus (UPopI) that remained restricted to the Isthmian area, expanded locally during the early Holocene, and left genomic traces up to the present day.

Cell-Surface Proteomic Profiling in the Fly Brain Uncovers Wiring Regulators.

Molecular interactions at the cellular interface mediate organized assembly of single cells into tissues and, thus, govern the development and physiology of multicellular organisms. Here, we developed a cell-type-specific, spatiotemporally resolved approach to profile cell-surface proteomes in intact tissues. Quantitative profiling of cell-surface proteomes of Drosophila olfactory projection neurons (PNs) in pupae and adults revealed global downregulation of wiring molecules and upregulation of synaptic molecules in the transition from developing to mature PNs. A proteome-instructed in vivo screen identified 20 cell-surface molecules regulating neural circuit assembly, many of which belong to evolutionarily conserved protein families not previously linked to neural development. Genetic analysis further revealed that the lipoprotein receptor LRP1 cell-autonomously controls PN dendrite targeting, contributing to the formation of a precise olfactory map. These findings highlight the power of temporally resolved in situ cell-surface proteomic profiling in discovering regulators of brain wiring.

Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing.

The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

Oxygen-evolving photosystem II structures during S1-S2-S3 transitions.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Gut cytokines modulate olfaction through metabolic reprogramming of glia.

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival1,2. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms3-5. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. Here we show that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. Our findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies.

Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing.

Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.

Alleles on locus chromosome 4B from different parents confer tiller number and the yield-associated traits in wheat.

Pleiotropy is frequently detected in agronomic traits of wheat (Triticum aestivum). A locus on chromosome 4B, QTn/Ptn/Sl/Sns/Al/Tgw/Gl/Gw.caas-4B, proved to show pleiotropic effects on tiller, spike, and grain traits using a recombinant inbred line (RIL) population of Qingxinmai × 041133. The allele from Qingxinmai increased tiller numbers, and the allele from line 041133 produced better performances of spike traits and grain traits. Another 52 QTL for the eight traits investigated were detected on 18 chromosomes, except for chromosomes 5D, 6D, and 7B. Several genes in the genomic interval of the locus on chromosome 4B were differentially expressed in crown and inflorescence samples between Qingxinmai and line 041133. The development of the KASP marker specific for the locus on chromosome 4B is useful for molecular marker-assisted selection in wheat breeding.

Allelic variation of terpene synthases drives terpene diversity in the wild species of the Freesia genus.

Terpene synthases (TPSs) play pivotal roles in conferring the structural diversity of terpenoids, which are mainly emitted from flowers, whereas the genetic basis of the release of floral volatile terpenes remains largely elusive. Though quite similar in sequence, TPS allelic variants still function divergently, and how they drive floral terpene diversity in closely related species remains unknown. Here, TPSs responsible for the floral scent of wild Freesia species were characterized, and the functions of their natural allelic variants, as well as the causal amino acid residues, were investigated in depth. Besides the 8 TPSs previously reported in modern cultivars, 7 additional TPSs were functionally evaluated to contribute to the major volatiles emitted from wild Freesia species. Functional characterization of allelic natural variants demonstrated that allelic TPS2 and TPS10 variants changed the enzymatic capacity while allelic TPS6 variants drove the diversity of floral terpene products. Further residue substitution analysis revealed the minor residues determining the enzyme catalytic activity and product specificity. The clarification of TPSs in wild Freesia species reveals that allelic TPS variants evolved differently to determine the interspecific floral volatile terpenes in the genus and might be used for modern cultivar improvement.

Glycine-Ti3C2Tx Hybrid Material Improves the Electrochemical Corrosion Resistance of a Water-Borne Epoxy Coating.

Improving the dispersibility and compatibility of nanomaterials in water-borne epoxy resins is an important means to improve the protection ability and corrosion resistance of coatings. In this study, glycine-functionalized Ti3C2Tx (GT) was used to prepare an epoxy composite coating. The results of Fourier transform infrared spectroscopy and X-ray diffraction showed that glycine was successfully modified. The scanning electron microscopy and transmission electron microscopy results showed that the aggregation of Ti3C2Tx was alleviated. Electrochemical impedance spectroscopy test results show that, after 60 days of immersion, GT coating still shows the best protection performance, and the composite coating |Z|f = 0.01 Hz is 3 orders of magnitude higher than that of the pure epoxy coating. This is mainly because, after adding glycine, the -COOH group on the surface of glycine binds to the -OH group on the surface of Ti3C2Tx, improving the aggregation of Ti3C2Tx itself. At the same time, the -NH group of glycine can also participate in the curing reaction of epoxy resin to strengthen the bonding strength between the coating and the metal. The good dispersion of GT in epoxy resin makes it fill the pores and holes left by epoxy resin curing and strengthen the corrosion resistance. The easy availability and green properties of glycine provide a simple and environmentally friendly method for the modification of Ti3C2Tx.

A prediction nomogram for hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: The present study aimed to develop and validate a new nomogram for predicting the incidence of hepatocellular carcinoma (HCC) among chronic hepatitis B (CHB) patients receiving antiviral therapy from real-world data. METHODS: The nomogram was established based on a real-world retrospective study of 764 patients with HBV from October 2008 to July 2020. A predictive model for the incidence of HCC was developed by multivariable Cox regression, and a nomogram was constructed. The predictive accuracy and discriminative ability of the nomogram were assessed by the concordance index (C-index), calibration curves, and decision curve analysis (DCA). Risk group stratification was performed to assess the predictive capacity of the nomogram. The nomogram was compared to three current commonly used predictive models. RESULTS: A total of 764 patients with HBV were recruited for this study. Age, family history of HCC, alcohol consumption, and Aspartate aminotransferase-to-Platelet Ratio Index (APRI) were all independent risk predictors of HCC in CHB patients. The constructed nomogram had good discrimination with a C-index of 0.811. The calibration curve and DCA also proved the reliability and accuracy of the nomogram. Three risk groups (low, moderate, and high) with significantly different prognoses were identified (p < 0.001). The model's performance was significantly better than that of other risk models. CONCLUSIONS: The nomogram was superior in predicting HCC risk among CHB patients who received antiviral treatment. The model can be utilized in clinical practice to aid decision-making on the strategy of long-term HCC surveillance, especially for moderate- and high-risk patients.

Key processes in tumor metastasis and therapeutic strategies with nanocarriers: a review.

Cancer metastasis is the leading cause of cancer-related death. Metastasis occurs at all stages of tumor development, with unexplored changes occurring at the primary site and distant colonization sites. The growing understanding of the metastatic process of tumor cells has contributed to the emergence of better treatment options and strategies. This review summarizes a range of features related to tumor cell metastasis and nanobased drug delivery systems for inhibiting tumor metastasis. The mechanisms of tumor metastasis in the ideal order of metastatic progression were summarized. We focus on the prominent role of nanocarriers in the treatment of tumor metastasis, summarizing the latest applications of nanocarriers in combination with drugs to target important components and processes of tumor metastasis and providing ideas for more effective nanodrug delivery systems.

Transistor-based immunosensor using AuNPs-Ab2-HRP enzyme nanoprobe for the detection of antigen biomarker in human blood.

Alpha-fetoprotein (AFP) is inextricably linked to various diseases, including liver cancer. Thus, detecting the content of AFP in biology has great significance in diagnosis, treatment, and intervention. Motivated by the urgent need for affordable and convenient electronic sensors in the analysis and detection of aqueous biological samples, we combined the solution-gated graphene transistor (SGGT) with the catalytic reaction of enzyme nanoprobes (HRP-AuNPs-Ab2) to accurately sense AFP. The SGGT immunosensor demonstrated high specificity and stability, excellent selectivity, and excessive linearity over a range of 4 ng/mL to 500 ng/mL, with the lower detection limit down to 1.03 ng/mL. Finally, clinical samples were successfully detected by the SGGT immunosensor, and the results were consistent with chemiluminescence methods that are popular in hospitals for detecting AFP. Notably, the SGGT immunosensor is also recyclable, so it has excellent potential for use in high-throughput detection.

Evaluation and Identification of Powdery Mildew Resistance Genes in Aegilops tauschii and Emmer Wheat Accessions.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a serious threat to wheat (Triticum aestivum L.) production. Narrow genetic basis of common wheat boosted the demand for diversified donors against powdery mildew. Aegilops tauschii Coss (2n = 2x = DD) and emmer wheat (2n = 4x = AABB), as the ancestor species of common wheat, are important gene donors for genetic improvement of common wheat. In this study, a total of 71 Ae. tauschii and 161 emmer wheat accessions were first evaluated for their powdery mildew resistance using the Bgt isolate E09. Thirty-three Ae. tauschii (46.5%) and 108 emmer wheat accessions (67.1%) were resistant. Then, all these accessions were tested by the diagnostic markers for 21 known Pm genes. The results showed that Pm2 alleles were detected in all the 71 Ae. tauschii and only Pm4 alleles were detected in 20 of 161 emmer wheat accessions. After haplotype analysis, we identified four Pm4 alleles (Pm4a, Pm4b, Pm4d, and Pm4f) in the emmer wheat accessions and three Pm2 alleles (Pm2d, Pm2e, and Pm2g) in the Ae. tauschii. Further resistance spectrum analysis indicated that these resistance accessions displayed different resistance reactions to different Bgt isolates, implying they may have other Pm genes apart from Pm2 and/or Pm4 alleles. Notably, a new Pm2 allele, Pm2S, was identified in Ae. tauschii, which contained a 64-bp deletion in the first exon and formed a new termination site at the 513th triplet of the shifted reading frame compared with reported Pm2 alleles. The phylogenetic tree of Pm2S showed that the kinship of Pm2S was close to Pm2h. To efficiently and accurately detect Pm2S and distinguish with other Pm2 alleles in Ae. tauschii background, a diagnostic marker, YTU-QS-3, was developed, and its effectiveness was verified. This study provided valuable Pm alleles and enriched the genetic diversity of the powdery mildew resistance in wheat improvement.

Metabolic and Transcriptomic Profile Revealing the Differential Accumulating Mechanism in Different Parts of Dendrobium nobile.

Dendrobium nobile is an important orchid plant that has been used as a traditional herb for many years. For the further pharmaceutical development of this resource, a combined transcriptome and metabolome analysis was performed in different parts of D. nobile. First, saccharides, organic acids, amino acids and their derivatives, and alkaloids were the main substances identified in D. nobile. Amino acids and their derivatives and flavonoids accumulated strongly in flowers; saccharides and phenols accumulated strongly in flowers and fruits; alkaloids accumulated strongly in leaves and flowers; and a nucleotide and its derivatives and organic acids accumulated strongly in leaves, flowers, and fruits. Simultaneously, genes for lipid metabolism, terpenoid biosynthesis, and alkaloid biosynthesis were highly expressed in the flowers; genes for phenylpropanoids biosynthesis and flavonoid biosynthesis were highly expressed in the roots; and genes for other metabolisms were highly expressed in the leaves. Furthermore, different members of metabolic enzyme families like cytochrome P450 and 4-coumarate-coA ligase showed differential effects on tissue-specific metabolic accumulation. Members of transcription factor families like AP2-EREBP, bHLH, NAC, MADS, and MYB participated widely in differential accumulation. ATP-binding cassette transporters and some other transporters also showed positive effects on tissue-specific metabolic accumulation. These results systematically elucidated the molecular mechanism of differential accumulation in different parts of D. nobile and enriched the library of specialized metabolic products and promising candidate genes.

Transcriptome analysis of mesenteric arterioles changes and its mechanisms in cirrhotic rats with portal hypertension.

Portal hypertension (PHT) is a major cause of liver cirrhosis. The formation of portosystemic collateral vessels and splanchnic vasodilation contribute to the development of hyperdynamic circulation, which in turn aggravates PHT and increases the risk of complications. To investigate the changes in mesenteric arterioles in PHT, cirrhotic rat models were established by ligating the common bile ducts. After 4 weeks, the cirrhotic rats suffered from severe PHT and splanchnic hyperdynamic circulation, characterized by increased portal pressure (PP), cardiac output (CO), cardiac index (CI), and superior mesenteric artery (SMA) flow. Mesenteric arterioles in cirrhotic rats displayed remarkable vasodilation, vascular remodeling, and hypocontractility. RNA sequencing was performed based on these findings. A total of 1,637 differentially expressed genes (DEGs) were detected, with 889 up-regulated and 748 down-regulated genes. Signaling pathways related to vascular changes were enriched, including the vascular endothelial growth factor (VEGF), phosphatidylinositol-3-kinase-AKT (PI3K-AKT), and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) signaling pathway, among others. Moreover, the top ten hub genes were screened according to the degree nodes in the protein-protein interaction (PPI) network. Functional enrichment analyses indicated that the hub genes were involved in cell cycle regulation, mitosis, and cellular response to oxidative stress and nitric oxide (NO). In addition, promising candidate drugs for ameliorating PHT, such as resveratrol, were predicted based on hub genes. Taken together, our study highlighted remarkable changes in the mesenteric arterioles of cirrhotic rats with PHT. Transcriptome analyses revealed the potential molecular mechanisms of vascular changes in splanchnic hyperdynamic circulation.

XFab-α4-1BB/CD40L fusion protein activates dendritic cells, improves expansion of antigen-specific T cells, and exhibits antitumour efficacy in multiple solid tumour models.

BACKGROUND: Additional immunotherapies are still warranted for non-responders to checkpoint inhibitors with refractory or relapsing cancers, especially for patients with "cold" tumours lacking significant immune infiltration at treatment onset. We developed XFab-α4-1BB/CD40L, a bispecific antibody targeting 4-1BB and CD40 for dendritic cell activation and priming of tumour-reactive T cells to inhibit tumours. METHODS: XFab-α4-1BB/CD40L was developed by engineering an anti-4-1BB Fab arm into a CD40L trimer based on XFab® platform. Characterisation of the bispecific antibody was performed by cell-based reporter assays, maturation of dendritic cell assays, and mixed lymphocyte reactions. The abilities of antigen-specific T-cell expansion and antitumour efficacy were assessed in syngeneic mouse tumour models. Toxicological and pharmacodynamic profiles were investigated in non-human primates. RESULTS: XFab-α4-1BB/CD40L demonstrated independent CD40 agonistic activity and conditional 4-1BB activity mediated by CD40 crosslinking, leading to dendritic cell maturation and T-cell proliferation in vitro. We confirmed the expansion of antigen-specific T cells in the vaccination model and potent tumour regression induced by the bispecific antibody alone or in combination with gemcitabine in vivo, concomitant with improved tumour-reactive T-cell infiltration. XFab-α4-1BB/CD40L showed no signs of liver toxicity at doses up to 51 mg/kg in a repeated-dose regimen in non-human primates. CONCLUSIONS: XFab-α4-1BB/CD40L is capable of enhancing antitumour immunity by modulating dendritic cell and T-cell functions via targeting 4-1BB agonism to areas of CD40 expression. The focused, potent, and safe immune response induced by the bispecific antibody supports further clinical investigations for the treatment of solid tumours.

Molecular phylogenetics of the fresh water sleepers Odontobutis (Gobiiformes: Odontobutidae) and its implications on biogeography of freshwater ichthyofauna of East Asia.

The genus Odontobutis is a group of freshwater fishes endemic to East Asia. Phylogenetic relationships among the Odontobutis species have never been fully tested due to incomplete taxon sampling and that molecular data have not been collected in many Odontobutis species. In the present study, we sampled 51 specimens from all known eight Odontobutis species with two outgroups (Perccottus glenii and Neodontobutis hainanensis). We collected sequence data of 4434 single-copy nuclear coding loci using gene capture and Illumina sequencing. A robust phylogeny of the Odontobutis with many individuals for each species was built, supporting the current taxonomy that all extant Odontobutis species are valid. The two species from Japan (O. hikimius + O. obscurus) formed an independent clade sister to the "continental odontobutids", whereas the species from southern China (O. sinensis + O. haifengensis) separated from the rest species of the genus. Surprisingly species from the lower reaches of the Yangtze River (O. potamophilus) was more closely related to species from the Korean Peninsula and northeastern China than to the middle reaches of the Yangtze River, such that their relationship was ((O. sinensis + O. haifengensis)(O. platycephala + (O. yaluensis + (O. potamophilus + O. interruptus)))). Divergence time among the Odontobutis was estimated using 100 most clock-like loci and three fossil calibration points. The crown group of the Odontobutis was estimated at 9.0 Ma during the late Miocene (5.6-12.7 Ma, 95% HPDs). Ancestral range of the genus was reconstructed using Reconstruct Ancestral States in Phylogenies (RASP) and BioGeoBEARS. The result suggested that the common ancestor of modern Odontobutis probably was distributed in Japan, southern China or the Korean Peninsula. A series of geographical events in East Asia since the late Miocene, such as the opening of the Japan/East Sea, rapid uplift of the Tibetan Plateau and climate change in the northern region of the Yellow River might account for diversification and current distribution pattern of the Odontobutis.