The day-night differences in cognitive and anxiety-like behaviors of mice after chronic sleep restriction.

Animal behavioral tests are often conducted during the day. However, rodents are nocturnal animals and are primarily active at night. The aim of this study was to determine whether there are diurnal changes in cognitive and anxiety-like performance of mice following chronic sleep restriction (SR). We also investigated whether this phenotypic difference is related to the diurnal variation of glymphatic clearance of metabolic wastes. Mice received 9-day SR by the use of the modified rotating rod method, followed by the open field, elevated plus maze, and Y-maze tests conducted during the day and at night, respectively. Brain ß-amyloid (Aß) and tau protein levels, the polarity of aquaporin4 (AQP4), a functional marker of the glymphatic system, and glymphatic transport ability were also analyzed. SR mice exhibited cognitive impairment and anxiety-like behaviors during the day, but not at night. AQP4 polarity and glymphatic transport ability were higher during the day, with lower Aß1-42 , Aß1-40 , and P-Tau levels in the frontal cortex. These day-night differences were totally disrupted after SR. These results reveal the diurnal changes in behavioral performance after chronic SR, which may be related to circadian control of AQP4-mediated glymphatic clearance of toxic macromolecules from the brain.

A novel dual-signal output strategy for POCT of CEA based on a smartphone electrochemical aptasensing platform.

A smartphone-based electrochemical aptasensing platform was developed for the point-of-care testing (POCT) of carcinoembryonic antigen (CEA) based on the ferrocene (Fc) and PdPt@PCN-224 dual-signal labeled strategy. The prepared PdPt@PCN-224 nanocomposite showed a strong catalytic property for the reduction of H2O2. Phosphate group-labeled aptamer could capture PdPt@PCN-224 by Zr-O-P bonds to form PdPt@PCN-224-P-Apt. Therefore, a dual signal labeled probe was formed by the hybridization between Fc-DNA and PdPt@PCN-224-P-Apt. The presence of CEA forced PdPt@PCN-224-P-Apt to leave the electrode surface due to the specific affinity, leading to the decrease of the reduction current of H2O2. At the same time, the Fc-DNA strand changed to hairpin structure, which made Fc closer to the electrode and resulted in the increase of the oxidation current of Fc. Thus, CEA can be accurately determined through both signals: the decrease of H2O2 reduction current and the increase of Fc oxidation current, which could avoid the false positive signal. Under the optimal conditions, the prepared aptasensor exhibited a wide linear range from 1 pg·mL-1 to 100 ng·mL-1 and low detection limits of 0.98 pg·mL-1 and 0.27 pg·mL-1 with Fc and PdPt@PCN-224 as signal labels, respectively. The aptasensor developed in this study has successfully demonstrated its capability to detect CEA in real human serum samples. These findings suggest that the proposed sensing platform will hold great potential for clinical tumor diagnosis and monitoring.

Mucilage secretion by aerial roots in sorghum (Sorghum bicolor): sugar profile, genetic diversity, GWAS and transcriptomic analysis.

Aerial root mucilage can enhance nitrogen fixation by providing sugar and low oxygen environment to the rhizosphere microbiome in Sierra Mixe maize. Aerial root mucilage has long been documented in sorghum (Sorghum bicolor), but little is known about the biological significance, genotypic variation, and genetic regulation of this biological process. In the present study, we found that a large variation of mucilage secretion capacity existed in a sorghum panel consisting of 146 accessions. Mucilage secretion occurred primarily in young aerial roots under adequately humid conditions but decreased or stopped in mature long aerial roots or under dry conditions. The main components of the mucilage-soluble were glucose and fructose, as revealed by sugar profiling of cultivated and wild sorghum. The mucilage secretion capacity of landrace grain sorghum was significantly higher than that of wild sorghum. Transcriptome analysis revealed that 1844 genes were upregulated and 2617 genes were downregulated in mucilage secreting roots. Amongst these 4461 differentially expressed genes, 82 genes belonged to glycosyltransferases and glucuronidation pathways. Sobic.010G120200, encoding a UDP-glycosyltransferase, was identified by both GWAS and transcriptome analysis as a candidate gene, which may be involved in the regulation of mucilage secretion in sorghum through a negative regulatory mechanism.

A label-free electrochemical aptasensor based on Bi-Sb alloy materials for potential POCT of HER-2.

As a prognostic biomarker for breast cancer, human epidermal growth factor receptor 2 (HER-2) is of crucial diagnostic value. Here, a label-free electrochemical aptasensor was established for the ultrasensitive detection of HER-2 using a modified electrode of Bi-Sb alloy materials (Bi-Sb AMs). The performance of the aptasensor was enhanced greatly due to the introduction of Bi-Sb alloy materials (Bi-Sb AMs) with high conductivity. Furthermore, by integrating the aptasensor with the Sensit Smart U-disk electrochemical analyzer, the point-of-care testing (POCT) for HER-2 was realized. Under the optimal experimental parameters, the POCT analyzer showed a wide linear response from 0.01 pg mL-1 to 100 ng mL-1, with a low detection limit (LOD) of 5.96 fg mL-1 for the detection of HER-2. The presented POCT analyzer exhibited good specificity, stability, and reproducibility. Benefiting from the simple operation and rapid testing, the developed analyzer will have potential application in the prognostic diagnosis and treatment of breast cancer.

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

Dimethyl itaconate inhibits LPS-induced inflammatory release and apoptosis in alveolar type II epithelial and bronchial epithelial cells by activating pulmonary surfactant proteins A and D.

BACKGROUND: Injury to the lung is a common, clinically serious inflammatory disease. However, its pathogenesis remains unclear, and the existing treatments, including cytokine therapy, stem cell therapy, and hormone therapy, are not completely effective in treating this disease. Dimethyl itaconate (DMI) is a surfactant with important anti-inflammatory effects. OBJECTIVE: The present study used alveolar type II (AT II) and bronchial epithelial cells as models to determine the role of DMI in lung injury. MATERIAL AND METHODS: First, the effects of DMI were established on the survival, inflammatory release, and apoptosis in lipopolysaccharide (LPS)-induced AT II and bronchial epithelial cells. The association between DMI and Sirtuin1 (SIRT1) was assessed using molecular docking. Next, by constructing interference plasmids to inhibit surfactant protein (SP)-A and SP-D expressions, the effect of DMI was observed on inflammatory release and apoptosis. RESULTS: The results revealed that DMI increased the survival rate and expression levels of SP-A, SP-D, and SIRT1, and inhibited inflammatory factors as well as apoptosis in LPS-induced cells. Furthermore, DMI could bind to SIRT1 to regulate SP-A and SP-D expressions. After SP-A and SP-D expressions were inhibited, the inhibitory effect of DMI was reversed on inflammatory release and apoptosis. CONCLUSION: The findings of the present study revealed that DMI inhibited LPS-induced inflammatory release and apoptosis in cells by targeting SIRT1 and then activating SP-A and SP-D. This novel insight into the pharmacological mechanism of DMI lays the foundation for its later use for alleviating lung injury.

[Efficacy and safety of levetiracetam versus phenytoin as second-line drugs for the treatment of children with convulsive status epilepticus: a Meta analysis].

OBJECTIVE: To systematically evaluate the efficacy and safety of levetiracetam (LEV) versus phenytoin (PHT) as second-line drugs for the treatment of convulsive status epilepticus (CSE) in children. METHODS: English and Chinese electronic databases were searched for the randomized controlled trials comparing the efficacy and safety of LEV and PHT as second-line drugs for the treatment of childhood CSE. RevMan 5.3 software was used for data analysis. RESULTS: Seven studies with 1 434 children were included. The Meta analysis showed that compared with the PHT group, the LEV group achieved a significantly higher control rate of CSE (RR=1.12, 95%CI:1.00-1.24, P=0.05), but there was no significant difference between the two groups in the recurrence rate of epilepsy within 24 hours (RR=0.82, 95%CI:0.22-3.11, P=0.77) and the rate of further antiepileptic drug therapy (RR=0.97, 95%CI:0.64-1.45, P=0.87). There was no significant difference in the incidence rate of adverse events between the two groups (RR=0.77, 95%CI:0.55-1.09, P=0.15). CONCLUSIONS: LEV has a better clinical effect than PHT in the treatment of children with CSE and does not increase the incidence rate of adverse events.

Genome-wide association study reveals that different pathways contribute to grain quality variation in sorghum (Sorghum bicolor).

BACKGROUND: In sorghum (Sorghum bicolor), one paramount breeding objective is to increase grain quality. The nutritional quality and end use value of sorghum grains are primarily influenced by the proportions of tannins, starch and proteins, but the genetic basis of these grain quality traits remains largely unknown. This study aimed to dissect the natural variation of sorghum grain quality traits and identify the underpinning genetic loci by genome-wide association study. RESULTS: Levels of starch, tannins and 17 amino acids were quantified in 196 diverse sorghum inbred lines, and 44 traits based on known metabolic pathways and biochemical interactions amongst the 17 amino acids calculated. A Genome-wide association study (GWAS) with 3,512,517 SNPs from re-sequencing data identified 14, 15 and 711 significant SNPs which represented 14, 14, 492 genetic loci associated with levels of tannins, starch and amino acids in sorghum grains, respectively. Amongst these significant SNPs, two SNPs were associated with tannin content on chromosome 4 and colocalized with three previously identified loci for Tannin1, and orthologs of Zm1 and TT16 genes. One SNP associated with starch content colocalized with sucrose phosphate synthase gene. Furthermore, homologues of opaque1 and opaque2 genes associated with amino acid content were identified. Using the KEGG pathway database, six and three candidate genes of tannins and starch were mapped into 12 and 3 metabolism pathways, respectively. Thirty-four candidate genes were mapped into 16 biosynthetic and catabolic pathways of amino acids. We finally reconstructed the biosynthetic pathways for aspartate and branched-chain amino acids based on 15 candidate genes identified in this study. CONCLUSION: Promising candidate genes associated with grain quality traits have been identified in the present study. Some of them colocalized with previously identified genetic regions, but novel candidate genes involved in various metabolic pathways which influence grain quality traits have been dissected. Our study acts as an entry point for further validation studies to elucidate the complex mechanisms controlling grain quality traits such as tannins, starch and amino acids in sorghum.

NF-κB-Regulated miR-99a Modulates Endothelial Cell Inflammation.

Objective. The present study was performed to investigate the effects and mechanisms of miR-99a on LPS-induced endothelial cell inflammation, as well as the regulation of NF-κB on miR-99a production. Methods and Results. ELISA showed that LPS treatment significantly promoted the secretion of inflammatory factors (TNF-α, IL-6, IL-1ß, and MCP-1). LPS treatment also inhibited miR-99a production and promoted mTOR expression and NF-κB nuclear translocation. Overexpression of miR-99a suppressed the LPS-induced TNF-α, IL-6, IL-1ß, and MCP-1 overproduction, mTOR upregulation, and NF-κB nuclear translocation. The PROMO software analysis indicated NF-κB binding site in the -1643 to -1652 region of miR-99a promoter. Dual luciferase reporter analysis, electrophoretic mobility shift assays (EMSA), and chromosome immunoprecipitation (ChIP) assays demonstrated that NF-κB promoted the transcription of miR-99a by binding to the -1643 to -1652 region of miR-99a promoter. Further studies on HUVECs verified the regulatory effects of NF-κB on miR-99a production. Conclusion. MiR-99a inhibited the LPS-induced HUVECs inflammation via inhibition of the mTOR/NF-κB signal. NF-κB promoted miR-99a production by binding to the -1643 to -1652 region of miR-99a promoter. Considering the importance of endothelial inflammation on cardiovascular diseases, such as atherosclerosis, our results may provide a new insight into the pathogenesis and therapy of atherosclerosis.

Meta-Analysis of miR-146a Polymorphisms Association with Coronary Artery Diseases and Ischemic Stroke.

Coronary artery disease (CAD) and ischemic stroke (IS) are manifestations of atherosclerosis, with a high death rate. miR-146a is a microRNA that participates in the progress of CAD and IS. A single nucleotide polymorphism (SNP) in the precursor of miR-146a, rs2910164, was found to be associated with the risks of CAD and IS. However, the results were inconsistent and inconclusive. A meta-analysis was performed to assess the relationship of rs2910164 and CAD as well as IS susceptibility. The database Pubmed, Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Chinese National Knowledge Infrastructure (CNKI), and Chinese Biomedical Literature Database (CBM) were searched for related studies. Crude odds ratios with 95% confidence intervals were used to investigate the strength of the association by random- or fixed-effect model. A total of eight studies, with 3138 cases and 3097 controls were identified for the meta-analysis. The results shows that rs2910164 is associated with the risk of CAD significantly in allelic model (OR = 0.86), homozygous model (OR = 0.70), heterozygous model (OR = 0.80) and dominant model (OR = 0.76). The subjects carrying the GG genotype, GG + GC genotype or G allele are at lower risks of CAD. For the susceptibility of IS, there are no significant associations between rs2910164 and total studies. However, in subgroup analysis by sample size and ethnicity, the GG, GG + GC and G allele of rs2910164 are found to be associated with higher risks of IS in large sample size group and in Koreans, under homozygous and dominant models. In conclusion, the current meta-analysis suggests lower risks of CAD for GG, GG + GC genotype and G allele of rs2910164, while rs2910164 is not associated with the risk of IS. Thus rs2910164 might be recommended as a predictor for susceptibility of CAD, but not IS.

[Hydrochemical Characteristics and Formation Mechanism of Groundwater in the Western Region of Hepu Basin, Beihai City].

Beihai City is a typical coastal city where groundwater provides a strong support for social and economic development. Studies on the hydrochemical characteristics and formation mechanism in Beihai City play an important role in the scientific management of water resources and coastal ecological environment protection. In this study, we revealed the main hydrogeochemical processes controlling groundwater quality by means of groundwater survey and water sample collection in the western region of Hepu Basin, Beihai City, combined with hydrochemistry and isotope theories and methods. The results showed that groundwater had the remarkable features of low pH value and low mineralization degree. For pore water, hydrochemistry type by primarily NO3 type water and concentrations of Na+ and Cl- were modestly increased along the flow path. Ca-HCO3, Ca-Cl·HCO3, Ca·Na-HCO3, and Na-Cl·HCO3 types were predominant in fissure water. The groundwater was of meteoric origin, hydrogeochemical evolutions were mainly affected by water-rock interactions, cation exchange, and anthropogenic activities. Na+, K+, and Cl- were mainly derived from evaporite and silicate rocks; Ca2+, Mg2+, HCO3-, and SO42- were from carbonatite and evaporite; and NO3- principally arose from anthropogenic activities. This study suggests that the groundwater pollution prevention and control should be carried out as soon as possible in the area where the NO3 type water occurs to avoid the further deterioration of water quality.

Isolation and characterisation of 17 microsatellite DNA loci from RAD reduced-representation genomes for Asian warty newts, genus Paramesotriton (Caudata: Salamandridae).

Asian warty newts, genus Paramesotriton, are endemic to southern China and northern Vietnam. Despite the achievements in biodiversity, molecular systematics and biogeography of species in this genus, population genetic diversity studies are lacking due to the lack of economical and available genetic markers. In this study, we developed 17 highly polymorphic microsatellite loci from RAD simplified genomic data for the Asian warty newts, genus Paramesotriton and successfully completed cross-species amplification tests on 20 samples of four species of Paramesotriton. These microsatellite markers can be used as important tools to study population genetic structure, levels of gene flow, population differentiation, mating systems and landscape genetics within the genus Paramesotriton and, thus, to make scientific conservation decisions and actions for the conservation of these rare and endangered amphibians.

A smartphone-based electrochemical POCT for CEA based on signal amplification of Zr6MOFs.

Carcinoembryonic antigen (CEA) is a biomarker of high expression in cancer cells. Highly sensitive and selective detection of CEA holds significant clinical value in the diagnosis, monitoring and efficacy evaluation of malignant tumors. In this work, a smartphone-based electrochemical point-of-care testing (POCT) platform for the detection of CEA was developed based on a Zr6MOF signal amplification strategy. Ferrocene labeled DNA strands (Fc-DNA) were immobilized on Zr6MOFs to form a Fc-DNA/Zr6MOF signal probe. Double-stranded DNA (dsDNA) formed by complementary DNA (cDNA) and CEA aptamer was assembled on a screen-printed electrode via an Au-S bond. When CEA was added, the aptamer specifically bound with CEA, resulting in the exposure of cDNA. Then, Fc-DNA/Zr6MOF signal probes were introduced on the electrode surface through hybridization between Fc-DNA and cDNA. The detection of CEA was realized by measuring the electrochemical response of Fc. The POCT device was made by connecting a modified electrode with a smartphone through a Sensit Smart USB flash disk. Due to the signal amplification of Zr6MOFs, this POCT platform exhibited high sensitivity, wide linear range, and low detection limit for CEA detection. The developed POCT platform has been used for the detection of CEA in actual human serum samples with satisfactory results.

Oreonectesdamingshanensis (Cypriniformes, Nemacheilidae), a new species of stream fish from Guangxi, Southwest China.

In this work, a new species of the genus Oreonectes is described, named Oreonectesdamingshanensis Yu, Luo, Lan, Xiao & Zhou, sp. nov., collected from the Damingshan Mountains of the Guangxi Zhuang Autonomous Region, China. Phylogenetic trees constructed based on the mitochondrial Cyt b showed that the new species represents an independent evolutionary lineage, with uncorrected genetic distances (p-distance) from congeners ranging from 6.1% to 8.9%. Morphologically, the new species can be distinguished from five other species of the genus by a combination of characters. The discovery of this new species raises the number of known species of Oreonectes from five to six. Our study suggests that O.platycephalus may be a complex containing multiple species and that previously recorded areas need to be further delimited and reevaluated.

Integrated phylogenetic analyses reveal the evolutionary, biogeographic, and diversification history of Asian warty treefrog genus Theloderma (Anura, Rhacophoridae).

Asian warty treefrogs, genus Theloderma, are morphologically variable arboreal frogs endemic to Southeast Asia and Southern China. However, integrated systematic studies are lacking, and knowledge of the genus in terms of diversity, origin, and historical diversification remains limited. To address these knowledge gaps, we used three mitochondrial and five nuclear gene fragments to reconstruct the Theloderma phylogeny, estimate divergence times, and examine the biogeography of the genus. Phylogenetic and species delimitation analyses suggest that the genus Theloderma comprises three major clades corresponding to two subgenera and seven species groups, and mPTP identified at least 12 putative cryptic species, suggesting that species diversity has been underestimated. Biogeographic analyses indicated that most recent common ancestor of Theloderma originated in the Indochina Peninsula during the Middle Oligocene (ca. 27.77 Ma) and the splitting of Clade A to C occurred in the Late Oligocene (ca. 23.55-25.57 Ma). Current biogeographic patterns result from two distinct processes: in situ diversification in the Indochina Peninsula and dispersal in multiple areas, namely southward dispersal to the Malay Peninsula and Borneo, northeastward dispersal to Southern China, northward dispersal to the Himalayas, and dispersal from Southern China to the Indochina Peninsula. Ancestral character reconstruction suggests that the ancestor of Theloderma may have possessed a small body size, rough dorsal skin, and absence of vomerine teeth and hand webbing, and that these four characters have undergone multiple evolutions. Principal component analysis based on eight bioclimatic variables did not clearly distinguish the three major clades of Theloderma, suggesting that species in these clades may occupy similar climatic ecological niches. Our research highlights the importance of orogeny and paleoclimatic changes, in shaping amphibian biodiversity in mountain ecosystems.

AP-1 and NF-κB transcriptionally regulate interleukin-8 in EA.Hy926 cells under shear stress.

Cardiovascular and cerebrovascular diseases remain the leading cause of death in the world. AS (atherosclerosis) is not only an inflammatory disease in which chemokines play the main role but also a disorder that is related to blood SS (shear stress). We have investigated the action of IL-8 (interleukin-8) mRNA expression in human endothelial cells line-EA.Hy926 under SS at different intensities and duration. Expression increases with time in an intensity dependent manner. With regard to the transcriptional mechanism involved, transient transfection of the human wild-type IL-8 promoter (-162/+44)/luciferase reporter plasmid, or site mutation of one of the binding sites [AP-1 (activator protein 1) or NF-κB (nuclear factor κB)] in the IL-8 promoter region was investigated. Both AP-1 and NF-κB were essential for SS-activated transcription, with the cells responding to NF-κB activation within minutes. After stimulated at low SS (4.20 dyne/cm2) for 30 min, the P65 subunit was translocated from the cytoplasm to nucleus for at least 60 min, while the cytoplasmic level of IκB (inhibitory κB) gradually decreased. The combined activation of NF-κB and AP-1 are the upstream regulators of low SS-induced IL-8 production in EA.Hy926 cells, which subsequently trigger an inflammatory reaction in endothelium.

[Susceptibility to prostate cancer in Han Chinese: single nucleotide polymorphism analysis of 1 667 cases].

OBJECTIVE: Prostate cancer (PCa) has the highest incidence among male malignancies in Western industrialized countries and, as a most common malignant disease in urology, its incidence has been increasing in recent years in Chinese men. This study was to investigate the risk loci associated with PCa susceptibility in Han Chinese by analyzing single nucleotide polymorphisms (SNP). METHODS: We collected peripheral blood samples from 1 667 PCa patients and 1 525 healthy men, and detected 40 loci associated with PCa susceptibility by analyzing SNPs using Sequenom technology. RESULTS: Of the 40 known loci, 16 were confirmed to be significantly associated with PCa susceptibility (P < 0.05). The loci 1, 2 and 5 at 8q24, 10q11 and 22q13.2 also contributed to PCa susceptibility in different ethnic groups. CONCLUSION: PCa susceptibility is obviously associated with the risk loci rs1465618, rs721048, rs12621278, rs7679673, rs12653946, rs339331, rs1512268, rs10086908, rs16901979, rs1447295, rs10993994, rs10896449, rs902774, rs9600079, rs11649743 and rs5759167 in Chinese Han population.

[Biosynthesis of CdS quantum dots in Saccharomyces cerevisiae and spectroscopic characterization].

In the present work, CdS quantum dots (QDs) were successfully biosynthesized at room temperature by using saccharomyces cerevisiae yeast as a carrier. Fluorescence emission spectra, ultraviolet-visible (UV/Vis) absorption spectra, and inverted fluorescence microscope images confirmed that saccharomyces cerevisiae can be used to biosynthesize CdS QDs. The as-prepared CdS QDs show the fluorescence emission peak at 443 nm and emit blue-green fluorescence under UV light (with excitation at 365 nm). Transmission electron microscopy (TEM) was applied to characterize the as-prepared CdS QDs and the TEM results showed that the as-prepared CdS QDs had the structure of hexagonal wurtzite. Fluorescence emission spectrum and UV/Vis absorption spectrum were used as the performance indicatiors to study the effects of saccharomyces cerevisiae yeast incubation times, reactant Cd2+ concentrations and reaction times on CdS QDs synthesis. Saccharomyces cerevisiae yeast grown in early stable phase can get the highest fluorescence intensity of CdS QDs when they were co-cultured with 0.5 mmol x L(-1) of Cd2+ with 24 h incubation time. Furthermore, much more CdS QDs can be obtained by changing the culture medium during the synthesis process.

Phylogeography of the endangered "eyed" turtles (genus Sacalia) and the discovery of a lineage derived from natural interspecific hybridization.

The herpetofauna of the Indomalayan bioregion of Asia suffers from severe habitat loss, unsustainable harvesting, and lack of research and conservation. Here, we investigated the range-wide phylogeography of the endangered "eyed" turtles (genus Sacalia, including the Beale's Eyed Turtle S. bealei and the Four-eyed Turtle S. quadriocellata) and discovered a natural interspecific hybrid turtle population in China. Based on phylogeny of the mitochondrial Cytochrome b gene of 101 samples in this study and public data, three major clades and six subclades were identified: S. bealei (SBE) in eastern-southern China, east S. quadriocellata in South China (northern east [SQUen] and southern east [SQUes] subclades), and west S. quadriocellata mainly in Vietnam (northern west [SQUwn], central west [SQUwc], and southern west [SQUws] subclades). We sequenced 16 nuclear DNA loci of 87 samples from SBE, SQUen, SQUes, and SQUwn subclades. Population genetic clustering analysis suggested a structure similar to the mitochondrial phylogeny, where most samples were classified into four genetic clusters corresponding to the four mtDNA subclades. However, a proportion of samples carrying SQUen mtDNA haplotypes formed an additional distinct cluster SHY. Those samples are found in the contact zone of the two species bearing mosaic and intermediate morphological characteristics. We detected an admixed ancestry in SHY from SBE and SQUen that conformed to an intrapopulation breeding scenario for at least hundreds of generations after the initial hybrid event, leading to a conclusion that SHY is a distinct and near-panmictic population derived from natural interspecific hybridization. In addition, SQUes (Hainan Island endemic) is of special concern due to significant isolation and low genetic diversity. We suggest that seven evolutionarily significant units should be recognized to facilitate appropriate conservation actions. These findings also highlight the urgent need for further herpetological research and conservation in this region.