Bile acids (BAs) are vital for liver health, and their dysregulation causes hepatobiliary disorders. Microcystins (MCs) are pervasive hepatotoxins in freshwaters worldwide that threaten public health; yet, their effects on BA homeostasis─particularly regulation mechanisms─remain poorly defined. Here, we show that chronic exposure to environmentally relevant doses of microcystin-LR (MC-LR) induces not only cholestatic liver injury but also gallstone formation in mice. Targeted metabolomics revealed that MC-LR strikingly altered BA profiles across the liver, gallbladder bile, and serum, characterized by increased hydrophobicity and excessive deconjugation. Crucially, this compositional distortion overrides the quantitative expansion of the gallbladder BA pool, creating a prolithogenic environment. Gene and protein expression analyses further demonstrated dysregulation of BA synthetic enzymes, transporters, and nuclear receptors FXR and SHP, indicative of a disrupted FXR-SHP signaling axis. Microscale thermophoresis and molecular docking confirmed the high-affinity binding of MC-LR to human FXR and SHP. Rescue experiments further showed that pharmacological activation of FXR using GW4064 or obeticholic acid (OCA) effectively reversed MC-LR-induced intracellular BA accumulation in human HepG2 cell line. Collectively, our findings establish MC-LR as an overlooked environmental driver of gallstone disease and highlight direct FXR-SHP antagonism as a key mechanism with therapeutic potential.

Spawning diversity plays an essential role in fish survival and reproduction, which contributes to the exceptional diversity of teleosts among vertebrates. Different zona radiata structures reflect the adaptability of fish to the environment of spawning and early embryonic development. The morphological and transcriptional characteristics of fish follicle development between different spawning habits, particularly the zona radiata variations, have been poorly documented. In this study, we integrated histology and transcriptomics to investigate the differences in the zona radiata structure and gene expression profiles among follicles from different spawning habits of Culter alburnus. Our results revealed that stage Ⅲ was the crucial period for zona radiata thickening and structure differentiation. Transcriptomic analyses of adhesive and semi-buoyant eggs at stage Ⅲ revealed a significant upregulation of genes involved in glycoprotein synthesis, extracellular matrix formation, and regulation of protease activity in adhesive eggs, such as the wfdc and a2ml gene family. This upregulation likely underpins the thicker zona radiata in adhesive eggs, facilitating their attachment to substrates. This study represents the first elucidation of the ultrastructure of the zona radiata and gene expression patterns in different developmental stages of adhesive and semi-buoyant eggs of Culter alburnus, offering new perspectives for aquaculture research in understanding fish reproductive adaptations.

The processes of fertilization and egg activation are vital for early embryogenesis. However, while the mechanisms associated with key events during these processes differ among species and modes of spawning, the signal pathways underlying these processes are opaque for many fishes, including economically important species. We investigated phenotypic traits, ultrastructure and protein expression levels in the eggs of the topmouth culter (Culter alburnus), a protected and economically important freshwater fish that exhibits two spawning modes, producing semi-buoyant eggs and adhesive eggs. Unfertilized eggs of C. alburnus were examined, as well as eggs at fertilization and 30 min post fertilization. Our results showed that in semi-buoyant eggs, energy metabolism was activated at fertilization, followed by elevated protein expression of cytoskeleton and extracellular matrix (ECM)-receptor interactions that resulted in rapid egg swelling; a recognized adaptation for lotic habitats. In contrast, in adhesive eggs fertilization initiated the process of sperm-egg fusion and blocking of polyspermy, followed by enhanced protein expression of lipid metabolism and the formation of egg envelope adhesion and hardening, which are adaptive in lentic habitats. Our findings indicate that alternative signal pathways differ between modes of spawning and timing during the key processes of fertilization and egg activation, providing new insights into the molecular mechanisms involved in adaptive early embryonic development in teleost fishes.

In years of blended teaching of Microbiology Experiment, we found that students’ initiative is the key to the learning effect. In the past, some students were passive in spite of diverse teaching methods applied. In recent years, we have adopted the task-driven teaching method: responsibility system of team leader rotation, which has remarkably incentivized all the students. As a team leader, each student has played an important role in pre-class tasks, the implementation in class, and summarization after class. In addition, they actively cooperated with the team leader when not serving as the team leader. Finally, the proportion of students achieving excellent results in the course and the marks on the quality and ability were significantly improved. Moreover, this method has been recognized by the students.

Microcystins (MCs) produced by some cyanobacteria can cause toxicity in animals and humans. In recent years, growing evidence suggests that MCs can act as endocrine disruptors. This research systematically investigated effects of microcystin-LR (MC-LR) on endocrine organs, biosynthesis of hormones and positive/negative feedback of the endocrine system in rats. Male, Sprague-Dawley rats were acutely administrated MC-LR by a single intraperitoneal injection at doses of 45, 67.5 or 90 μg MC-LR/kg body mass (bm), and then euthanized 24 h after exposure. In exposed rats, histological damage of hypothalamus, pituitary, adrenal, testis and thyroid were observed. Serum concentrations of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT), expressions of genes and proteins for biosynthesis of hormones were lesser, which indicated an overall suppression of the hypothalamus-pituitary-adrenal (HPA) axis. Along the hypothalamus-pituitary-gonadal (HPG) axis, lesser concentrations of gonadotropin-releasing hormone (GnRH) and testosterone (T), but greater concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2) were observed. Except for greater transcription of cyp19a1 in testes, transcriptions of genes and proteins for T and E2 biosynthesis along the HPG axis were lesser. As for the hypothalamus-pituitary-thyroid (HPT) axis, after MCs treatment, greater concentrations of thyroid-stimulating hormone (TSH), but lesser concentrations of free tri-iodothyronine (fT3) were observed in serum. Concentrations of free tetra-iodothyronine (fT4) were greater in rats dosed with 45 μg MCs/kg, bm, but lesser in rats dosed with 67.5 or 90 μg MCs/kg, bm. Transcripts of genes for biosynthesis of hormones and receptors along the HPT axis and expressions of proteins for biosynthesis of tetra-iodothyronine (T4) and tri-iodothyronine (T3) in thyroid were significantly altered. Cross-talk among the HPA, HPG and HPT axes probably occurred. It was concluded that MCs caused an imbalance of positive and negative feedback of hormonal regulatory axes, blocked biosynthesis of key hormones and exhibited endocrine-disrupting effects.

Microcystins (MCs) can cause reproductive and developmental toxicity and disrupt endocrine homeostasis in mammals. In the present study, male, Sprague-Dawley (SD) rats were administrated 3 or 30 μg MC-LR/kg, body mass (bm) per day via intraperitoneal (i.p.) injections for 6 weeks. Effects of MC-LR on histology, hormone concentrations, gene transcriptional profiles and protein expressions along the hypothalamic-pituitary-adrenal (HPA), -gonad (HPG) and -thyroid (HPT) axes were assessed. Sub-chronic administration with MC-LR caused histological damage to hypothalamus, pituitary, adrenal, testes and thyroid and affected relative masses of pituitary, adrenal and testes. The HPA axis was activated and serum concentrations of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were significantly augmented. Along the HPG axis, serum concentrations of gonadotropin-releasing hormone (GnRH) and dihydrotestosterone (DHT) were diminished, while concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T) and estradiol (E2) were augmented. For the HPT axis, only concentrations of free tetra-iodothyronine (fT4) were significantly diminished, while concentrations of thyrotropin-releasing hormone (TRH), thyroid-stimulating hormone (TSH) or free tri-iodothyronine (fT3) were not significantly changed. Also, several genes and proteins related to synthesis of steroid hormones were significantly altered. Findings of the present study illustrate that MC-LR can cause endocrine-disrupting effects through the disruption of synthesis and secretion of hormones along the HPA, HPG and HPT axes and negative feedback regulation. Also, there could be crosstalk among HPA, HPG and HPT axes. These findings elucidate mechanisms of endocrine-disrupting effects of MCs.

Microcystins (MCs), a group of most widespread freshwater cyanotoxins that possess strong neurotoxicity, can adversely affect brain structures and functions and are linked to neurodegenerative diseases. Despite the essential role of lipids in brain structures and functions, the brain lipidome profile of mammals exposed to MCs remains unexplored, hindering a clear understanding of the neurotoxic effects of MCs and underlying mechanisms. In this study, we performed untargeted lipidomic profiling using ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) on the prefrontal cortex and hippocampus of mice orally exposed to 30 and 300 μg/kg body mass/day of microcystin-leucine arginine (MC-LR) for 180 days to evaluate the impacts of MC-LR on the brain lipidome profile and functions. Our results show that MC-LR resulted in a decline in cognitive parameters, as assessed by the Morris water maze test. Interestingly, apparent neurodegenerative changes were observed in the prefrontal cortex, but not in the hippocampus. Comprehensive lipidomic analyses uncovered profound, region-specific changes in the phospholipid and sphingolipid profile at the levels of lipid subclasses, lipid species, and fatty acyl composition. These changes showed overall decrease trends of lipid content in the prefrontal cortex yet increasing trends in the hippocampus. We identified distinct transcriptional regulations of lipid metabolism and apoptosis by MC-LR in the two regions, which appeared to underlie the neurodegenerative changes. Collectively, this study uncovers region-specific changes in the brain lipidome profile and functions induced by MCs, shedding light on the role of lipid dysfunction in neurotoxicity mechanism of MCs.

Documenting the origins of megadiverse (sub)tropical aquatic ecosystems is an important goal for studies of evolution and ecology. Nonetheless, the geological and ecological establishment of the modern Yangtze River remains poorly understood. Here, we reconstruct the geographic and ecological history of an endemic clade of East Asian fishes based on the mitochondrial phylogenomics analysis of Cyprinidae using 15 fossil calibrations. We estimate an ancestral condition of benthic spawning with demersal or adhesive eggs in southern East Asia before ∼23 Ma and a derived condition of riverine spawning with semibuoyant eggs in the Yangtze by ∼18 Ma. These results imply the formation of Yangtze riverine ecosystems around the Oligocene-Miocene boundary in response to plateau uplift and monsoon strengthening. Some of these cyprinids reverted to benthic spawning with adhesive eggs by ∼15 Ma, a time of rising to peak net diversification rates, indicating the formation of potamo-lacustrine ecosystems by the mid-Miocene during a strong East Asian summer monsoon. Our study provides increased spatiotemporal resolution for the co-evolutionary histories of the Yangtze River and its biodiversity and highlights biological evidence concerning the geomorphological dynamics of the Yangtze River.

Progressive morphological and cytochemical processes occur in the egg after fertilization in which the egg envelope changes dramatically. However, the biochemical and structural alterations of egg envelope between common carp adhesive eggs and grass carp semi-buoyant eggs remain poorly understand. Here, this study found that compared with grass carp semi-buoyant eggs, common carp adhesive eggs possess a smaller egg diameter and a thicker egg envelope, and the difference was most significant at 1 h postfertilization. After fertilization, an adhesive layer was found and gradually thickened in common carp eggs, especially at 1 h postfertilization. The surface of fertilized adhesive egg of common carp was covered by a dense adhesive layer which gradually grew to be thickened after fertilization. While grass carp semi-buoyant eggs did not possess an adhesive layer after fertilization and the surface of grass carp semi-buoyant egg was smoother. Moreover, proteomic changes of egg envelopes between common carp adhesive eggs and grass carp semi-buoyant eggs at 1 h postfertilization showed that a rapid fold change of cytoskeleton-associated proteins (including actin, filamin-A, alpha-actinin, cytoskeletonassociated protein 5, and ezrin) was observed in common carp egg envelope after fertilization, which might be conductive to maintaining cell morphology and fish egg morphology. Significant upregulation of proteins such as a coagulation factor XIII A protein, zona pellucida proteins and cystatin in the fertilized egg of common carp might be responsible for inducing the process of egg envelope hardening in order to the protection of embryonic development. Obviously up-regulated level of alpha-2-macroglobulin-like protein in the fertilized egg of common carp could reflect the potentials of the effective immune defense in fertilized adhesive eggs of common carp. In conclusion, this study is the first to present the ultrastructure and protein composition in the egg envelope of common carp adhesive eggs and grass carp semi-buoyant eggs. Extensive changes in the abundance of proteins of egg envelope would throw some new lights on the biochemical processes underlying the early developmental processes after fertilization.

Health risks of chronic exposure to microcystins (MCs), a family of aquatic contaminants produced mainly by cyanobacteria, are critical yet unsolved problems. Despite a few epidemiological studies, the metabolic profiles of humans exposed to MCs remain unknown, hindering the deep understanding of the molecular toxicity mechanisms. Here, sensitive nuclear magnetic resonance (NMR)- and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics were applied to investigate the serum metabolic profiles of humans living near Lake Chao, where toxic cyanobacterial blooms occur annually. MCs were positively detected in 92 of 144 sera by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a median concentration of 0.016 μg/L. The estimated daily intake (0.15-0.27 μg MC-LReq/day) was less than the tolerable daily intake (TDI, 2.4 μg MC-LR for 60 kg adults) recommended by the World Health Organization (WHO). Obvious disruptions of the amino acid metabolism were confirmed and played important roles in renal impairments associated with serum MC burdens. Chronic oral exposure of mice to 30 μg MC-LR/kg body mass, which is less than the no observed adverse effect level, also led to obvious renal lesions and metabolic dysfunction. These observations provide the first evidence of metabolic disturbance of humans exposed to MCs and indicate that the WHO's TDI value determined traditionally should be lessened to protect human health effectively.

ETHNOPHARMACOLOGICAL RELEVANCE:Glycyrrhiza and sargassum are among the 18 incompatible medicaments according to traditional Chinese medicine (TCM) theory. Although it contains glycyrrhiza and sargassum, Haizao Yuhu decoction (HYD) is a classic prescription widely used as TCM to treat goiter. According to the Chinese Pharmacopoeia, glycyrrhiza is divided into three varieties: Glycyrrhiza uralensis Fish., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat. Whether the three varieties of glycyrrhiza have different efficacy or toxicity when applied in the HYD is unknown.AIM OF THE STUDY:To explore whether the HYDs comprising three varieties of glycyrrhiza have different efficacy or toxicity when used to treat goiter in rats and the underlying mechanisms of these HYDs.MATERIALS AND METHODS:For two weeks, the goiter model was replicated by intragastric propylthiouracil (PTU) administration. Samples were divided into the control group, model group, euthyrox group, HYD with glycyrrhiza uralensis (HYD-U) group, HYD with glycyrrhiza glabra (HYD-G) group, and HYD with glycyrrhiza inflata (HYD-I) group. After four weeks of treatment, body weight, rectal temperature, thyroid/liver/kidney coefficient, thyroid/liver/kidney function, thyroid/liver/kidney histomorphology, and thyroid ultrastructure were evaluated. Then, real-time quantitative reverse-transcription polymerase chain reaction (RTqPCR), Western blot, and immunofluorescence analyses were performed to detect genes and proteins affecting autophagy and apoptosis in thyroid cells in the AMP-activated Protein Kinases (AMPK)/Mammalian target of rapamycin (mTOR) pathway.RESULTS:All three HYDs increased thyroid hormones (THs) levels, relieved thyroid pathological tissue and ultrastructure, and activated vital proteins and genes in the AMPK/mTOR pathway. Comparisons among the efficacy of the three HYDs indicated that HYD-U restored the THs most effectively; however, no difference in the anti-goiter effect was observed. Moreover, the three HYDs resulted in no toxicity and promoted the recovery of impaired liver and kidney function caused by PTU. Comparisons among the recovery effects of the three HYDs on the liver and kidney were the same.CONCLUSION:Our experiments demonstrated that the three HYDs had outstanding anti-goiter effects and protected liver and kidney function. Their anti-goiter effects were attributed to AMPK/mTOR pathway-induced autophagy and apoptosis. HYD-U resulted in the best THs recovery. It was further indicated that in our present study, glycyrrhiza and sargassum were compatible in the three HYDs, thereby suggesting their safety of compounding in HYD and providing a basis for the research of the 18 incompatible medicaments.

The proteasome is a highly conserved polycatalytic enzyme that is required for cellular processes and is widely present in the nucleus and cytoplasm of archaea, as well as all eukaryotes. A total of 22 members of the proteasome subunit (CiPS) gene family were identified and characterized by scanning the grass carp (Ctenopharyngodon idella) genome. These genes were classified into two subfamilies, CiPSA and CiPSB, based on phylogenetic analysis, which was consistent with the results from other species. We examined the response of this gene family to high density and saline-alkali stresses in aquaculture using publicly available transcriptome data resources. In grass carp, CiPS member transcripts were detected in all tested tissues, with the highest expression level in the head kidney and the lowest in the liver. According to transcriptome-based expression analysis, CiPS genes play a role in response to environmental stresses in grass carp, mainly in the form of negative regulation. Interestingly, a cluster of members belonging to the CiPSB subfamily on a 15 kb region on chromosome segment CI01000319, including CiPSB8, 9, 9b, and 10, showed marked responses to high density and saline-alkali stress. It appears that CiPS genes confer stress tolerance through the regulation of common genes, as well as specific genes. In summary, our genome-wide characterization, evolutionary, and transcriptomic analysis of CiPS genes in grass carp provides valuable information for characterizing the molecular functions of these genes and utilizing them to improve stress tolerance in aquaculture.

Despite the widespread recognition of adaptive radiation as a driver of speciation, the mechanisms by which natural selection generates new species are incompletely understood. The evolutionary radiation of endemic East Asian cyprinids has been proposed as evolving through a change in spawning habits, involving a transition from semibuoyant eggs to adhesive eggs in response to crosslinked river-lake system formation. Here, we investigated the molecular mechanisms that underpin this radiation, associated with egg hydration and adhesiveness. We demonstrated that semibuoyant eggs enhance hydration by increasing the degradation of yolk protein and accumulation of Ca2+ and Mg2+ ions, while adhesive eggs improve adhesiveness and hardness of the egg envelope by producing an adhesive layer and a unique 4th layer to the egg envelope. Based on multiomics analyses and verification tests, we showed that during the process of adaptive radiation, adhesive eggs downregulated the "vitellogenin degradation pathway," "zinc metalloprotease pathway," and "ubiquitin-proteasome pathway" and the pathways of Ca2+ and Mg2+ active transport to reduce their hydration. At the same time, adhesive eggs upregulated the crosslinks of microfilament-associated proteins and adhesive-related proteins, the hardening-related proteins of the egg envelope, and the biosynthesis of glycosaminoglycan in the ovary to generate adhesiveness. These findings illustrate the novel molecular mechanisms associated with hydration and adhesiveness of freshwater fish eggs and identify critical molecular mechanisms involved in the adaptive radiation of endemic East Asian cyprinids. We propose that these key egg attributes may function as "magic traits" in this adaptive radiation.

本文研究滚装码头混合泊位分配和劳动力分配的联合调度优化问题.首先,考虑潮汐时间窗约束、装卸劳动力约束、泊位缆桩分布约束以及泊位不规则布局因素,建立以最小化船舶总服务时间为目标的混合整数规划模型.其次,采用内外嵌套算法设计策略,提出求解该类问题的组合算法.其中,外层是多种群并行进化的遗传算法,生成多种船舶计划顺序,内层为基于规则的启发式算法,用于计算给定计划顺序的目标函数值.然后,基于实际运营数据,生成多组不同规模的算例进行全面数值实验,结果表明所提出的算法可在10分钟内求解包含50艘船、100个泊段的算例.最后,开展基于真实滚装码头运营实例的案例分析,对所提模型和算法在实际码头调度问题中的适用性与高效性进行验证.

The Yangtze River is cross-linked with numerous lakes within its floodplain and is a worldwide biodiversity hotspot. There is no evidence indicating when this unique river-lake system developed. The endemic East Asian cyprinid clade has evolved diverse spawning adaptations to different flow conditions. Our ancestral egg-type reconstruction showed an ancestral state of adhesive eggs and later demersal eggs origination (both stream adaptations). Semi-buoyant eggs emerged ~18 Mya as a fast-flowing river adaptation, with increased hydration via three yolk protein degradation pathways, ion transport pathways and egg envelope permeability transition pores. Adhesive eggs evolved secondarily ~14 Mya with the egg envelope increasing to four layers and an adhesive layer, along with an increase in adhesiveness via microfilament/adhesive-related protein crosslinking and enhanced glycosaminoglycan biosynthesis, improving adherence to submerged lake plants, indicating that the cross-linked river-lake system formed in the mid-Miocene. This study provides a unique biological evidence for large-scale water system evolution.

本文基于整车物流智能调度决策支持系统的研发、实施与运维的成功应用,论述运筹学在智能化上的应用路径以及实践驱动的学术路径.该系统是国内较早在汽车物流企业实现落地的智能化调度系统,其所形成的思想理论与方法技术揭示了运筹学在智能化应用上的核心价值,以及实践驱动的学术价值,对解决"卡脖子"难题提供示范性思路.本文提出运筹学在智能化研发上"三环七步"的整体研发框架.首先,分析智能化需求的运筹学特征,详细介绍汽车整车物流的发展趋势、瓶颈及智能调度需求;其次,论述运筹学系统模型的作用与建模方法,分析汽车整车物流系统模型的决策要素、目标及约束,提出汽车整车物流智能调度的运筹学应用问题.然后,提出"模式装箱"的新装箱理论问题,明确问题的计算难解性、可解性及核心科学特征.进一步,建立汽车整车物流调度应用问题与科学问题的混合整数线性规划模型;提出求解汽车整车物流调度问题的分支定界算法,以及大规模问题求解的时空分解及滚动求解方法与技术;提出面向运筹应用的生产测试及压力测试方法,给出汽车整车物流调度的测试分析的流程与结果.此外,提出深度集成整车运输管理系统与仓库管理系统、优化算法引擎驱动的分布式、多视图、多系统融合的智能调度决策支持系统.最后,论述该系统在实施过程中的推广使用和运维情况,并对运筹学应用及实践驱动的科学研究进行总结与展望.

Alternative reproductive tactics (ARTs) are behavioural, morphological, and physiological traits associated with alternative reproductive phenotypes within a population or species. ARTs are widespread in nature, and are a particular feature of teleost fishes. However, few studies have examined egg buoyancy mechanisms in the context of the evolution of ARTs in freshwater fishes. In marine fishes, egg buoyancy is achieved chiefly through hydration. While the buoyancy of freshwater fish eggs has been suggested to be determined primarily through the presence of oil droplets, the majority (60%) of freshwater pelagic eggs do not possess an oil droplet. We applied a physical model of buoyancy to understand the contributions of oil droplets and hydration to the buoyancy of pelagic freshwater fish eggs. We further used phylogenetic regression to estimate the effect of the relative size of the perivitelline space, habitat and parental care on the occurrence of oil droplets, while controlling for non-independence among species due to phylogenetic relatedness. Our analysis demonstrates that the probability of oil droplets in freshwater pelagic eggs exhibits a significant negative relationship with the size of perivitelline space, which may reflect a trade-off relating to energy allocation in contrasting habitats. We also demonstrate a positive association between the probability of oil droplets and the provision of parental care and occupancy of lentic habitats. These findings indicates the evolution of contrasting buoyancy mechanisms as novel ARTs in freshwater fishes. A theoretical model in combination with empirical analysis indicate the evolution of novel ARTs in freshwater fishes as adaptive responses to flow conditions.

目的 检测叉头状转录因子3(Foxp3)和程序性死亡受体1(PD1)在结直肠癌组织中的表达,探讨两者表达与患者临床病理特征及预后的关系.方法 应用免疫组化(IHC)技术检测Foxp3及PD1在结直肠癌组织中和癌旁组织中的表达,Chi-square统计分析Foxp3及PD1表达强度的相关性及与患者临床病理特征,Kaplan-Meiere统计分析Foxp3及PD1表达强度和预后的关系.结果 结直肠癌组织中Foxp3及PD1表达均明显高于癌旁组织中的表达,其表达分别与浸润深度、TNM分期以及淋巴结转移相关.结直肠癌组织中Foxp3表达与PD1表达呈正相关.与Foxp3阴性表达及PD1阴性表达患者相比较,Foxp3阳性表达及PD1阳性表达患者生存率低.结论 结直肠癌组织中Foxp3及PD1的表达显著升高,其高表达可能促进了结直肠癌的发生发展.

The Yangtze River is the largest river in Asia, but its evolutionary history has long been debated. Diverse groups of endemic freshwater fishes have evolved in this river. Here we represent the historical spatiotemporal pattern of the endemic East Asian cyprinid clade based on the largest molecular phylogeny of Cyprinidae, including 1420 species and fossil records. Based on the evolution of egg types adapting to different hydrological conditions, we show that the ancestors of this endemic clade (laying adhesive eggs) were distributed in southern East Asia before ~24 Ma and subsequently dispersed to the Yangtze River basin to spawn semi-buoyant eggs at ~19 Ma. These results indicate that the Yangtze River reversed its flow direction from southward to eastward to form the present river system within the Oligocene-Miocene boundary (~24-19 Ma). Some East Asian cyprinids evolved into fishes producing adhesive eggs again at ~13 Ma, together with an increased net diversification rate, indicating that the river began to form a potamo-lacustrine system during the Mid-Miocene. The new sketch of the formation history of the Yangtze River system through Cyprinidae phylogeny, together with the evolution of egg types in endemic East Asia cyprinids, has better spatiotemporal integrity than traditional geological studies.

The Yangtze River is the longest river in Asia, but its evolutionary history has long been debated. So far no robust biological evidences can be found to crack this mystery. Here we reconstruct spatiotemporal and diversification dynamics of endemic East Asian cyprinids based on a largest molecular phylogeny of Cyprinidae, including 1420 species, and show that their ancestors laying adhesive eggs were distributed in southern East Asia before ~24 Ma, subsequently dispersed to the Yangtze River to spawn semi-buoyant eggs at ~19 Ma. This indicates that the Yangtze River diverted eastward around the Oligocene-Miocene boundary. Some of these cyprinids evolved again into fishes producing adhesive eggs at ~13 Ma, together with a peaked net diversification rate, indicating that the river formed a potamo-lacustrine ecosystem during the Mid-Miocene. Our reconstruction of the history of the Yangtze River has higher time resolution and much better continuity than those deriving from geological studies.