Circulating microbiome DNA as biomarkers for early diagnosis and recurrence of lung cancer.

Lung cancer mortality is exacerbated by late-stage diagnosis. Emerging evidence indicates the potential clinical significance of distinct microbial signatures as diagnostic and prognostic biomarkers across various cancers. However, circulating microbiome DNA (cmDNA) profiles are underexplored in lung cancer (LC). Here, whole-genome sequencing is performed on plasma of LC patients and healthy controls (HCs). Differentially enriched microbial species are identified between LC and HC. A diagnostic model is developed, which has a high sensitivity of 87.7% and achieves an AUC of 93.2% in the independent validation dataset. Crucially, this model demonstrates the capability to detect early-stage LC, achieving a sensitivity of 86.5% for stage I and 87.1% for tumors <1 cm. In addition, we construct a cmDNA model for recurrence, which precisely predicts LC recurrence after surgery. Overall, this study highlights the significant alterations of cmDNA profiles in LC, indicating its potential as biomarkers for early diagnosis and recurrence.

Characterization of the distinct immune microenvironments between hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

As two major types of primary liver cancers, the tumor immune microenvironment (TIME) of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) have been well studied separately. However, a systemic assessment of the similarities and differences between the TIME of HCC and ICC is still lacking. In this study, we pictured a landscape of combined TIME of HCC and ICC by sequencing and integrating 41 single-cell RNA-seq samples from four different tissue types of both malignancies. We found that T cells in HCC tumors generally exhibit higher levels of immunosuppression and exhaustion than those in ICC tumors. Myeloid cells in HCC and ICC tumors also exhibit distinct phenotypes and may serve as a key factor driving the differences between their TIMEs. Besides, we identified a cluster of EGR1+ macrophages specifically enriched in HCC tumors. Together, our study provides new insights into cellular composition, states and interactions in the TIMEs of HCC and ICC, which could pave the way for the development of future therapeutic targets for liver cancers.

Genomes, fossils, and the concurrent rise of modern birds and flowering plants in the Late Cretaceous.

The phylogeny and divergence timing of the Neoavian radiation remain controversial despite recent progress. We analyzed the genomes of 124 species across all Neoavian orders, using data from 25,460 loci spanning four DNA classes, including 5,756 coding sequences, 12,449 conserved nonexonic elements, 4,871 introns, and 2,384 intergenic segments. We conducted a comprehensive sensitivity analysis to account for the heterogeneity across different DNA classes, leading to an optimal tree of Neoaves with high resolution. This phylogeny features a novel Neoavian dichotomy comprising two monophyletic clades: a previously recognized Telluraves (land birds) and a newly circumscribed Aquaterraves (waterbirds and relatives). Molecular dating analyses with 20 fossil calibrations indicate that the diversification of modern birds began in the Late Cretaceous and underwent a constant and steady radiation across the KPg boundary, concurrent with the rise of angiosperms as well as other major Cenozoic animal groups including placental and multituberculate mammals. The KPg catastrophe had a limited impact on avian evolution compared to the Paleocene-Eocene Thermal Maximum, which triggered a rapid diversification of seabirds. Our findings suggest that the evolution of modern birds followed a slow process of gradualism rather than a rapid process of punctuated equilibrium, with limited interruption by the KPg catastrophe. This study places bird evolution into a new context within vertebrates, with ramifications for the evolution of the Earth's biota.

Comparison of a novel hand-held retractor-assisted transforaminal lumbar interbody fusion by the wiltse approach and posterior TLIF: a one-year prospective controlled study.

BACKGROUND: This study aims to compare the clinical outcomes and safety of a novel hand-held retractor system-assisted Wiltse TLIF with that P-TLIF and assess whether this hand-held retractor system assisted Wiltse TLIF can yield less paraspinal muscle injury. METHODS: 56 patients (P-TLIF: 26, Wiltse TLIF: 30) were included in this one year prospective controlled study. The operation time, intraoperative blood loss, postoperative drainage, mobilization time, and discharge time were recorded. The clinical outcomes were evaluated by ODI, VAS, JOA, and SF-36 scores (7 days, 3, 6, and 12 months after surgery). Paraspinal muscle injury was assessed by postoperative MRI (6 months after surgery). CK and C-reaction protein were measured pre and postoperatively, and CT or X-ray (one year postoperatively) was used to assess bony union/non-union. RESULTS: The Wiltse (study) group was associated with significantly less estimated blood loss (79.67 ± 28.59 ml vs 192.31 ± 59.48 ml, P = 0.000*), postoperative drainage (43.33 ± 27.89 ml vs 285.57 ± 123.05 ml, P = 0.000*), and shorter mobilization (4.1 ± 1.2 d vs. 3.0 ± 0.9 d, P < 0.05) and discharge times (7.7 ± 1.9 d vs. 6.1 ± 1.2 d, P = 0.002*) than the P-TLIF (control) group. Serum CK activity at 24 h postoperatively in the study group was significantly lower than in the control group (384.10 ± 141.99 U/L vs 532.76 ± 225.76 U/L, P = 0.018*). At 7 days after surgery, VAS (2.3 ± 0.6 vs 3.2 ± 0.7, P = 0.000*)and ODI scores (43.9 ± 11.9 vs 55.2 ± 12.9, P = 0.001*) were lower, while the JOA scores (18.4 ± 3.4 vs 16.3 ± 4.2, P = 0.041*) was higher in the control group than in the study group. Results observed at 3 months of follow-up were consistent with those at 7 days. After six months postoperatively, paraspinal muscle degeneration in the control group was more significant than in the study group (P = 0.008*). CONCLUSION: Our study showed that this novel hand-held retractor system assisted Wiltse approach TLIF can significantly reduce paraspinal muscle injury, postoperative drainage, and intraoperative blood loss, mobilization and discharge time, as well as yield better short-term outcomes compared to P-TLIF. TRIAL REGISTRATION: 25/09/2023 NCT06052579.

Serine/threonine kinase 36 induced epithelial-mesenchymal transition promotes docetaxel resistance in prostate cancer.

To investigate the role and potential mechanism of serine/threonine kinase 36 (STK36) in docetaxel resistance-prostate cancer (PCa). The expression of STK36 in PCa and the correlation with clinicopathological characteristics of PCa patients were analyzed using the data from different databases and tissue microarrays. To investigate the role of STK36 on cell proliferation, invasion, and migration, STK36 was overexpressed and silenced in DU-145 and PC-3 cell lines. Cell counting kit-8 (CCK8) was used to test cell proliferation. Cell invasion and migration were detected by cell wound scratch assay and trans well, respectively. The expression profile of STK36, E-Cadherin, and Vimentin was analyzed by Western blot. Cell apoptosis was detected by the TUNEL assay. STK36 expression was upregulated in PCa tissue compared with adjacent benign PCa tissue; it was higher in patients with advanced stages compared with lower stages and was significantly correlated with decreased overall survival. Up-regulation of STK36 significantly promoted the proliferation, invasion, and migration of DU-145 and PC-3 cells and compensated for the suppression caused by docetaxel treatment in vitro. A striking apoptosis inhibition could be observed when dealing with docetaxel, although the apoptosis of DU-145 and PC-3 cells was not affected by the STK36 exclusive overexpression. Besides, E-Cadherin expression was restrained while the expression levels of vimentin were all enhanced. The knockdown of STK36 reversed the above process. STK36 up-regulation could accelerate the biological behavior and docetaxel resistance of PCa by epithelial-mesenchymal transition (EMT) activation. STK36 may be potentially used as a target in PCa resolvent with docetaxel.

RNA N6-methyladenosine modification-based biomarkers for absorbed ionizing radiation dose estimation.

Radiation triage and biological dosimetry are critical for the medical management of massive potentially exposed individuals following radiological accidents. Here, we performed a genome-wide screening of radiation-responding mRNAs, whose N6-methyladenosine (m6A) levels showed significant alteration after acute irradiation. The m6A levels of three genes, Ncoa4, Ate1 and Fgf22, in peripheral blood mononuclear cells (PBMCs) of mice showed excellent dose-response relationships and could serve as biomarkers of radiation exposure. Especially, the RNA m6A of Ncoa4 maintained a high level as long as 28 days after irradiation. We demonstrated its responsive specificity to radiation, conservation across the mice, monkeys and humans, and the dose-response relationship in PBMCs from cancer patients receiving radiation therapy. Finally, NOCA4 m6A-based biodosimetric models were constructed for estimating absorbed radiation doses in mice or humans. Collectively, this study demonstrated the potential feasibility of RNA m6A in radiation accidents management and clinical applications.

Responses of root-associated fungal community structure of mycorrhizal plants to nitrogen and/or phosphorus addition in a subtropical forest.

Root-associated fungi play a vital role in maintaining nutrient absorption and health of host plants. To compare the responses of root-associated fungal community structures to nitrogen (N) and/or phosphorus (P) additions across differential mycorrhizal types, we collected roots of nine plant species belonging to three mycorrhizal types (arbuscular mycorrhiza, ectomycorrhiza, and ericoid mycorrhiza) under control and N and/or P addition treatments from a subtropical forest, and detected the diversity and community composition of fungi inhabiting roots through the high-throughput sequencing technique. The results showed that root-associated fungal communities of all nine plant species were mainly composed of Basidiomycota and Ascomycota. The relative abundance of Ascomycota and Basidiomycota was significantly lower and higher under the P addition than that under control, respectively. The relative abundance of Ascomycota of ericoid mycorrhizal trees was significantly higher than those of arbuscular mycorrhizal and ectomycorrhizal trees, while the relative abundance of Basidiomycota was significantly lower than the other two mycorrhizal types. Compared with the control, P addition significantly reduced the α-diversity and changed community composition of root-associated fungi across different mycorrhizal plant types, while no effect of N addition or mycorrhizal type was observed. Compared with the control and N addition treatments, NP addition caused root-associated fungal communities of all plants becoming integrally divergent. In addition, the fungal communities of ectomycorrhizal mycorrhizal trees became apparently convergent in comparison with those of arbuscular and ericoid mycorrhizal trees under the NP addition. Collectively, our results highlighted that P was a critical factor influencing community structures of tree root-associated fungi in subtropical forest soils. This study would enhance our understanding of the responses and maintenance mechanisms of plant root-associated fungal diversity under global environmental changes in the subtropical region.

Immune-related gene IL17RA as a diagnostic marker in osteoporosis.

Objectives: Bone immune disorders are major contributors to osteoporosis development. This study aims to identify potential diagnostic markers and molecular targets for osteoporosis treatment from an immunological perspective. Method: We downloaded dataset GSE56116 from the Gene Expression Omnibus database, and identified differentially expressed genes (DEGs) between normal and osteoporosis groups. Subsequently, differentially expressed immune-related genes (DEIRGs) were identified, and a functional enrichment analysis was performed. A protein-protein interaction network was also constructed based on data from STRING database to identify hub genes. Following external validation using an additional dataset (GSE35959), effective biomarkers were confirmed using RT-qPCR and immunohistochemical (IHC) staining. ROC curves were constructed to validate the diagnostic values of the identified biomarkers. Finally, a ceRNA and a transcription factor network was constructed, and a Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to explore the biological functions of these diagnostic markers. Results: In total, 307 and 31 DEGs and DEIRGs were identified, respectively. The enrichment analysis revealed that the DEIRGs are mainly associated with Gene Ontology terms of positive regulation of MAPK cascade, granulocyte chemotaxis, and cytokine receptor. protein-protein interaction network analysis revealed 10 hub genes: FGF8, KL, CCL3, FGF4, IL9, FGF9, BMP7, IL17RA, IL12RB2, CD40LG. The expression level of IL17RA was also found to be significantly high. RT-qPCR and immunohistochemical results showed that the expression of IL17RA was significantly higher in osteoporosis patients compared to the normal group, as evidenced by the area under the curve Area Under Curve of 0.802. Then, we constructed NEAT1-hsa-miR-128-3p-IL17RA, and SNHG1-hsa-miR-128-3p-IL17RA ceRNA networks in addition to ERF-IL17RA, IRF8-IL17RA, POLR2A-IL17RA and ERG-IL17RA transcriptional networks. Finally, functional enrichment analysis revealed that IL17RA was involved in the development and progression of osteoporosis by regulating local immune and inflammatory processes in bone tissue. Conclusion: This study identifies the immune-related gene IL17RA as a diagnostic marker of osteoporosis from an immunological perspective, and provides insight into its biological function.

Late Oligocene fossil acorns and nuts of Quercus section Cyclobalanopsis from the Nanning Basin, Guangxi, South China.

Quercus is the largest genus within the Fagaceae and has a rich fossil record. Most of the fossil material is attributed to the subgenus Quercus based on leaves, pollen or rarely acorns and nuts. Fossil records of Q. section Cyclobalanopsis characterized by ring-cupped acorns are relatively few and especially those described based on nuts are scant. In this study, we described four new species of Quercus section Cyclobalanopsis based on mummified acorns and nuts: Q. paleodisciformis X.Y. Liu et J.H. Jin sp. nov., Q. paleohui X.Y. Liu et J.H. Jin sp. nov., Q. nanningensis X.Y. Liu et J.H. Jin sp. nov. and Q. yongningensis X.Y. Liu et J.H. Jin sp. nov. These species closely resemble the extant species Q. disciformis, Q. hui, Q. kerrii, and Q. dinghuensis. The occurrence of Q. section Cyclobalanopsis in the Oligocene stratum of Guangxi, South China, suggests that the section has diversified within its extant distribution center since the Oligocene. By combining records from other areas, we propose that the section first appeared in the middle Eocene of East Asia (Sino-Japan), has diversified in situ with a few elements scattering into West Asia and southern Europe since the Oligocene and Pliocene, respectively, and finally became restricted in East Asia since the Pleistocene. This indicates that the section originated and diversified in East Asia, before spreading into West Asia no later than the Oligocene and into southern Europe by the Pliocene. Subsequently it disappeared from South Europe and West Asia due to the appearance of the (summer dry) Mediterranean climate and widespread cooling during the Pleistocene.

Enhanced Mitophagy in Cholesteatoma Epithelial Cells.

HYPOTHESIS: Mitophagy may have a potential role in the pathogenesis of acquired cholesteatoma. BACKGROUND: Enhanced mitophagy has been proven to be involved in various cancers. However, its role in the pathogenesis of cholesteatoma, which shares some common features with cancer, is controversial. This study investigated mitophagy in cholesteatoma epithelial cells. METHODS: The autophagy protein markers LC3-II and p62 and mitophagy proteins BNIP3, Parkin, and PINK1 were analyzed in cholesteatoma epithelial cells and external auditory canal epithelium cells by immunoblotting. The results were confirmed by immunohistochemistry. Adenovirus Ad-mCherry-GFP-LC3B and Ad-GFP-LC3B were used to evaluate autophagic activity. Transmission electron microscopy was used to observe and analyze autophagosomes. RESULTS: LC3-II expression was increased in cholesteatoma cells, whereas soluble and insoluble p62 levels were decreased. The expressions of BNIP3, Parkin, and PINK1 were higher in total protein and mitochondrial protein of cholesteatoma cells compared with normal external auditory canal epithelium cells. Autophagic activity was increased in cholesteatoma cells compared with normal external auditory canal epithelium cells. CONCLUSION: Mitophagy was enhanced in cholesteatoma epithelial cells and may have a potential role in the pathogenesis of acquired cholesteatoma.

First reliable Miocene fossil winged fruits record of Engelhardia in Asia through anatomical investigation.

Fossil genera with similar features to the winged fruits of the living Engelhardia Lesch. ex Blume (e.g., Palaeocarya G. Saporta) have been widely reported in Cenozoic fossil floras of the Northern Hemisphere. However, fossil winged fruits of Engelhardia with detailed anatomical structures have only been found in the upper Eocene of North America. This study reports the first Engelhardia fossil winged fruits with detailed anatomical structures in East Asia from the Miocene Erzitang Formation of Guangxi, South China. The anatomical and morphological features of the new fossils, including the unique structure of secondary septa, clearly distinguish them from other fossil genera and show unambiguously their attribution to the genus Engelhardia. This discovery suggests that Engelhardia had reached its modern distribution during the Miocene and the climate of the Guiping Basin in Guangxi during the Miocene was similar to that of present-day tropical and subtropical regions in Asia.

Metformin Protects Radiation-Induced Early Brain Injury by Reducing Inflammation and DNA Damage.

Radiation-induced brain injury (RIBI) is one of the most common and fatal complications of cranial radiation therapy (CRT); however, no effective intervention is available currently. Metformin has been reported to have anti-RIBI activity as a first-line anti-diabetic drug. However, the mechanism of action is unclear. An RIBI mice model and an in vitro cell model under 30 and 10 Gy 60Co γ-rays exposure were established to investigate the mechanism of metformin in RIBI. The results showed that pre-treatment with metformin protects hippocampal neurogenesis in the brain of mice and improves learning and memory ability after irradiation. Further investigations revealed that metformin pretreatment reduces inflammation and decreases DNA damage in the in vitro BV2 cell line. In addition, we observed that metformin inhibits the activation of IκB and IRF-3, which are downstream components of the cGAS-STING pathway. These findings suggest that metformin might protect the brain from RIBI, at least partly, through the cGAS pathway, making it a potential therapeutic drug for RIBI.

Bacterial communities in the phyllosphere are distinct from those in root and soil, and sensitive to plant species changes in subtropical tree plantations.

Deciphering the local diversity and community composition of plant-associated microorganisms is crucial to predict their ecological functions in forest ecosystems. The differences in microbial diversity and community composition between the aboveground and belowground tree compartments remain largely unknown. Here, we examined bacterial communities in the leaf surface (phyllosphere) and root-associated (root and rhizospheric soil) habitats of 13 tree species. Bacterial richness substantially differed across the three compartments, with the highest value observed in rhizospheric soil. Tree species exerted a significant effect on α-diversity of leaf- and soil- but not root-inhabiting bacteria. Bacterial communities were distinct across habitats and were significantly more divergent in leaf- than in root-associated habitats. Leaf nutrients and soil pH and NH4+-N were the main factors regulating leaf- and root-related community composition, respectively. This study highlights that host selection effects on bacterial community structure were more prominent in aboveground than in belowground habitats. Our findings contribute to a better understanding of the effect of compartments and subtropical tree species on microbial diversity, with crucial implications for sustainable forest plantation management.

RegVar: Tissue-specific Prioritization of Non-coding Regulatory Variants.

Non-coding genomic variants constitute the majority of trait-associated genome variations; however, the identification of functional non-coding variants is still a challenge in human genetics, and a method for systematically assessing the impact of regulatory variants on gene expression and linking these regulatory variants to potential target genes is still lacking. Here, we introduce a deep neural network (DNN)-based computational framework, RegVar, which can accurately predict the tissue-specific impact of non-coding regulatory variants on target genes. We show that by robustly learning the genomic characteristics of massive variant-gene expression associations in a variety of human tissues, RegVar vastly surpasses all current non-coding variant prioritization methods in predicting regulatory variants under different circumstances. The unique features of RegVar make it an excellent framework for assessing the regulatory impact of any variant on its putative target genes in a variety of tissues. RegVar is available as a web server at https://regvar.omic.tech/.

Comprehensive analysis of senescence-related genes and immune infiltration in intervertebral disc degeneration: a meta-data approach utilizing bulk and single-cell RNA sequencing data.

Objectives: This study aims to identify the key senescence genes and potential regulatory mechanisms that contribute to the etiology of intervertebral disc degeneration (IDD). Method: We analyzed GSE34095 and GSE70362 datasets, identifying key senescence-related differentially expressed genes (DEGs) in IDD using lasso regression. Risk scores classified patients into high- and low-risk groups. We compared pathways, functions, and immune infiltration between these groups. Diagnostic ability was assessed using ROC curves and a nomogram predicted IDD incidence. In single-cell dataset GSE165722, we evaluated expression of key senescence-related DEGs. Results: We identified 12 key senescence-related DEGs distinguishing high- and low-risk IDD patients. Enrichment analysis revealed cellular stress response, apoptotic signaling pathway, and protein kinase activation differences. Immune cell analysis showed elevated eosinophils in low-risk group and increased effector memory CD8 T, central memory CD4 T, myeloid-derived suppressor, natural killer, monocyte, Type 1 T helper, plasmacytoid dendritic, and natural killer T cells in high-risk group. A nomogram using AUC >0.75 genes (CXCL8, MAP4K4, MINK1, and TNIK) predicted IDD incidence with good diagnostic power. High senescence scores were observed in neutrophils. Conclusion: Our diagnostic model, based on key senescence-related DEGs and immune cell infiltration, offers new insights into IDD pathogenesis and immunotherapy strategies.

CommPath: An R package for inference and analysis of pathway-mediated cell-cell communication chain from single-cell transcriptomics.

Single-cell transcriptomics offers opportunities to investigate ligand-receptor (LR) interactions between heterogeneous cell populations within tissues. However, most existing tools for the inference of intercellular communication do not allow prioritization of functional LR associations that provoke certain biological responses in the receiver cells. In addition, current tools do not enable the identification of the impact on the downstream cell types of the receiver cells. We present CommPath, an open-source R package and webserver, to analyze and visualize the LR interactions and pathway-mediated intercellular communication chain with single-cell transcriptomic data. CommPath curates a comprehensive signaling pathway database to interpret the consequences of LR associations and therefore infers functional LR interactions. Furthermore, CommPath determines cell-cell communication chain by considering both the upstream and downstream cells of user-defined cell populations. Applying CommPath to human hepatocellular carcinoma dataset shows its ability to decipher complex LR interaction patterns and the associated intercellular communication chain, as well as their changes in disease versus homeostasis.

Use of plant growth regulators to reduce 2-methyl-4-chlorophenoxy acetic acid-Na (MPCA-Na) damage in cotton (Gossypium hirsutum).

BACKGROUND: 2-methyl-4-chlorophenoxy acetic acid-Na (MPCA-Na) is a phenoxy carboxylic acid selective hormone herbicide that is widely used in the crop fields. However, drift of MPCA-Na during application is highly damaging to cotton (Gossypium hirsutum) and other crop plants. This study was carried out from 2019 to 2020 to determine the effects of different concentrations of MPCA-Na on physiological and metabolic activities besides growth and yield of cotton plants at seedling, budding, flowering and boll stages. Moreover, we evaluated the different combinations of 24-epibrassinolide, gibberellin (GA3), phthalanilic acid and seaweed fertilizer to ameliorate herbicide damage. RESULTS: 2-methyl-4-chlorophenoxy acetic acid-Na (MPCA-Na) exposure caused a decrease in the chlorophyll content, and an increase in the soluble protein content, Malondialdehyde (MDA) content and protective enzyme activity. It also caused significant reductions in plant height, boll number and the single boll weight at the seedling and budding stages, but had little effects on plant height and the single boll weight at flowering and boll stage. Under the maximum recommended dose of MPCA-Na (130 g/L), the number of cotton bolls at seedling and budding stages decreased by 75.33 and 79.50%, respectively, and the single boll weight decreased by 46.42 and 36.31%, respectively. Nevertheless, the number of G. hirsutum bolls and single boll weight at flowering and boll stage decreased by 48.15 and 5.38%, respectively. Application of plant growth regulators decreased the MDA content, and increased chlorophyll, soluble protein content and protective enzyme activity, and alleviated MCPA-Na toxicity. Positive effects in case of growth regulators treated plants were also observed in terms of G. hirsutum yield. Phthalanilic acid + seaweed fertilizer, 24-epibrassinolide + seaweed fertilizer, and GA3 + seaweed fertilizer should be used at the seedling, budding, and flowering and boll stages, respectively. CONCLUSIONS: The results of current study suggest that certain plant growth regulators could be used to alleviate MPCA-Na damage and maintain G. hirsutum yield. When the cotton exposed to MCPA-Na at the seedling stage, it should be treated with phthalanilic acid + seaweed fertilizer, while plants exposed at the budding stage should be treated with 24-epibrassinolide + seaweed fertilizer, and those exposed at the flowering and boll stages should be treated with GA3 + seaweed fertilizer to mitigate stress.

Fossil Fruits of Ceratophyllum from the Upper Eocene and Miocene of South China.

Ceratophyllum L. is a cosmopolitan genus of perennial aquatic herbs that occur in quiet freshwaters. Fossils of this genus have been widely reported from the Northern Hemisphere, most of them occurring in the temperate zone. Here, we describe two species of fossil fruits discovered from subtropical areas of China. The fossil fruit discovered from the upper Eocene Huangniuling Formation of the Maoming Basin is designated as C. cf. muricatum Chamisso, and fruits discovered from the Miocene Erzitang Formation of the Guiping Basin are assigned to the extant species C. demersum L. The discovery of these two fossil species indicates that Ceratophyllum had spread to South China by the late Eocene and their distribution expanded in subtropical China during the Miocene.