Phylotranscriptomic analyses reveal deep gene tree discordance in Camellia (Theaceae).

Gene tree discordance is a significant legacy of biological evolution. Multiple factors can result in incongruence among genes, such as introgression, incomplete lineage sorting (ILS), gene duplication or loss. Resolving the background of gene tree discordance is a critical way to uncover the process of species diversification. Camellia, the largest genus in Theaceae, has controversial taxonomy and systematics due in part to a complex evolutionary history. We used 60 transcriptomes of 55 species, which represented 15 sections of Camellia to investigate its phylogeny and the possible causes of gene tree discordance. We conducted gene tree discordance analysis based on 1,617 orthologous low-copy nuclear genes, primarily using coalescent species trees and polytomy tests to distinguish hard and soft conflict. A selective pressure analysis was also performed to assess the impact of selection on phylogenetic topology reconstruction. Our results detected different levels of gene tree discordance in the backbone of Camellia, and recovered rapid diversification as one of the possible causes of gene tree discordance. Furthermore, we confirmed that none of the currently proposed sections of Camellia was monophyletic. Comparisons among datasets partitioned under different selective pressure regimes showed that integrating all orthologous genes provided the best phylogenetic resolution of the species tree of Camellia. The findings of this study reveal rapid diversification as a major source of gene tree discordance in Camellia and will facilitate future investigation of reticulate relationships at the species level in this important plant genus.

[Genetic Subtypes and Pretreatment Drug Resistance in the Newly Reported Human Immunodeficiency Virus-Infected Men Aged≥50 Years Old in Guangxi].

Objective To analyze the genetic subtypes of human immunodeficiency virus (HIV) and the prevalence of pretreatment drug resistance in the newly reported HIV-infected men in Guangxi. Methods The stratified random sampling method was employed to select the newly reported HIV-infected men aged≥50 years old in 14 cities of Guangxi from January to June in 2020.The pol gene of HIV-1 was amplified by nested reverse transcription polymerase chain reaction and then sequenced.The mutation sites associated with drug resistance and the degree of drug resistance were then analyzed. Results A total of 615 HIV-infected men were included in the study.The genetic subtypes of CRF01_AE,CRF07_BC,and CRF08_BC accounted for 57.4% (353/615),17.1% (105/615),and 22.4% (138/615),respectively.The mutations associated with the resistance to nucleoside reverse transcriptase inhibitors (NRTI),non-nucleoside reverse transcriptase inhibitors (NNRTI),and protease inhibitors occurred in 8 (1.3%),18 (2.9%),and 0 patients,respectively.M184V (0.7%) and K103N (1.8%) were the mutations with the highest occurrence rates for the resistance to NRTIs and NNRTIs,respectively.Twenty-two (3.6%) patients were resistant to at least one type of inhibitors.Specifically,4 (0.7%),14 (2.3%),4 (0.7%),and 0 patients were resistant to NRTIs,NNRTIs,both NRTIs and NNRTIs,and protease inhibitors,respectively.The pretreatment resistance to NNRTIs had much higher frequency than that to NRTIs (2.9% vs.1.3%;χ2=3.929,P=0.047).The prevalence of pretreatment resistance to lamivudine,zidovudine,tenofovir,abacavir,rilpivirine,efavirenz,nevirapine,and lopinavir/ritonavir was 0.8%, 0.3%, 0.7%, 1.0%, 1.3%, 2.8%, 2.9%, and 0, respectively. Conclusions CRF01_AE,CRF07_BC,and CRF08_BC are the three major strains of HIV-infected men≥50 years old newly reported in Guangxi,2020,and the pretreatment drug resistance demonstrates low prevalence.

HIV-1 Transmission among Injecting Drug Users is Principally Derived from Local Circulating Strains in Guangxi, China.

Objective: The mode of human immunodeficiency virus (HIV) transmission via injection drug use (IDU) still exists, and the recent shift in IDU-related transmission of HIV infection is largely unknown. The purpose of this study was to analyze the spatiotemporal sources and dynamics of HIV-1 transmission through IDU in Guangxi. Methods: We performed a molecular epidemiological investigation of infections across Guangxi from 2009 to 2019. Phylogenetic and Bayesian time-geographic analyses of HIV-1 sequences were performed to confirm the characteristics of transmission between IDUs in combination with epidemiological data. Results: Among the 535 subjects, CRF08_BC (57.4%), CRF01_AE (28.4%), and CRF07_BC (10.7%) were the top 3 HIV strains; 72.6% of infections were linked to other provinces in the transmission network; 93.6% of sequence-transmitted strains were locally endemic, with the rest coming from other provinces, predominantly Guangdong and Yunnan; 92.1% of the HIV transmission among people who inject drugs tended to be transmitted between HIV-positive IDUs. Conclusion: HIV recombinants were high diversity, and circulating local strains were the transmission sources among IDUs in Guangxi. However, there were still cases of IDUs linked to other provinces. Coverage of traditional prevention strategies should be expanded, and inter-provincial collaboration between Guangxi, Yunnan, and Guangdong provinces should be strengthened.

Species discrimination in Schima (Theaceae): Next-generation super-barcodes meet evolutionary complexity.

Plastid genome and nuclear ribosomal DNA (nrDNA) arrays, proposed recently as "super-barcodes," might provide additional discriminatory power and overcome the limitations of traditional barcoding loci, yet super-barcodes need to be tested for their effectiveness in more plant groups. Morphological homoplasy among Schima species makes the genus a model for testing the efficacy of super-barcodes. In this study, we generated multiple data sets comprising standard DNA barcodes (matK, rbcL, trnH-psbA, nrITS) and super-barcodes (plastid genome, nrDNA arrays) across 58 individuals from 12 out of 13 species of Schima from China. No samples were correctly assigned to species using standard DNA barcodes and nrDNA arrays, while only 27.27% of species with multiple accessions were distinguished using the plastid genome and its partitioned data sets-the lowest estimated rate of super-barcode success in the literature so far. For Schima and other taxa with similarly recently divergence and low levels of genetic variation, incomplete lineage sorting, hybridization or taxonomic oversplitting are all possible causes of the failure. Taken together, our study suggests that by no means are super-barcodes immune to the challenges imposed by evolutionary complexity. We therefore call for developing multilocus nuclear markers for species discrimination in plant groups.

Phylotranscriptomics of Theaceae: generic-level relationships, reticulation and whole-genome duplication.

BACKGROUND AND AIMS: Theaceae, with three tribes, nine genera and more than 200 species, are of great economic and ecological importance. Recent phylogenetic analyses based on plastomic data resolved the relationships among the three tribes and the intergeneric relationships within two of those tribes. However, generic-level relationships within the largest tribe, Theeae, were not fully resolved. The role of putative whole-genome duplication (WGD) events in the family and possible hybridization events among genera within Theeae also remain to be tested further. METHODS: Transcriptomes or low-depth whole-genome sequencing of 57 species of Theaceae, as well as additional plastome sequence data, were generated. Using a dataset of low-copy nuclear genes, we reconstructed phylogenetic relationships using concatenated, species tree and phylogenetic network approaches. We further conducted molecular dating analyses and inferred possible WGD events by examining the distribution of the number of synonymous substitutions per synonymous site (Ks) for paralogues in each species. For plastid protein-coding sequences , phylogenies were reconstructed for comparison with the results obtained from analysis of the nuclear dataset. RESULTS: Based on the 610 low-copy nuclear genes (858 606 bp in length) investigated, Stewartieae was resolved as sister to the other two tribes. Within Theeae, the Apterosperma-Laplacea clade grouped with Pyrenaria, leaving Camellia and Polyspora as sister. The estimated ages within Theaceae were largely consistent with previous studies based mainly on plastome data. Two reticulation events within Camellia and one between the common ancestor of Gordonia and Schima were found. All members of the tea family shared two WGD events, an older At-γ and a recent Ad-ß; both events were also shared with the outgroups (Diapensiaceae, Pentaphylacaceae, Styracaceae and Symplocaceae). CONCLUSIONS: Our analyses using low-copy nuclear genes improved understanding of phylogenetic relationships at the tribal and generic levels previously proposed based on plastome data, but the phylogenetic position of the Apterosperma-Laplacea clade needs more attention. There is no evidence for extensive intergeneric hybridization within Theeae or for a Theaceae-specific WGD event. Land bridges (e.g. the Bering land bridge) during the Late Oligocene may have permitted the intercontinental plant movements that facilitated the putative ancient introgression between the common ancestor of Gordonia and Schima.

The complete chloroplast genome sequence of Laplacea alpestris and its phylogenetic position.

Laplacea alpestris is a member of the genus Laplacea, which distributes in Central and South America. Genetic information of L. alpestris would provide guidance for the phylogenetic position of this species. Here, we reported and characterized its complete chloroplast (cp) genome using Illumina pair-end sequencing data. The total chloroplast genome size of this species was 157,211 bp, including inverted repeats (IRs) of 26,103 bp, separated by a large single copy (LSC) and a small single copy (SSC) of 86,749 and 18,256 bp, respectively. A total of 132 genes, including 37 tRNA, 8 rRNA, and 87 protein-coding genes were identified. Phylogenetic analysis showed that L. alpestris formed a monophyletic clade with Laplacea fruticosa, and then grouped with Apterosperma oblata. The systematic position of Southeast Asian Laplacea species needs further studies.

Antioxidative Flavan-3-ol Dimers from the Leaves of Camellia fangchengensis.

Camellia fangchengensis Liang et Zhong, belonging to the genus Camellia sect. Thea (Theaceae), is an endemic tea species to the south and southwest areas of Guangxi province, People's Republic of China. Known as a wild tea plant, the leaves have been used for producing green tea or black tea by the local people of its growing area. HPLC and LC-MS analysis showed the leaves contain oligomeric catechins as major phenolic components. Further detailed phytochemical study led to the identification of five flavan-3-ol dimers (1-5) including two new ones, fangchengbisflavans A (1) and B (2) from the leaves of C. fangchengensis, together with six known monomers (6-11) and one glucoside (12), in addition to gallic acid (13). Their structures were determined by extensive spectroscopic analysis. Most of the isolates displayed significant antioxidant activities in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical scavenging assays. The results suggested that the leaves of C. fangchengensis, rich in flavan-3-ol oligomers and monomers as potent antioxidants, could be a valuable plant resource for the production of tea and natural beverages.

Insights into the historical assembly of East Asian subtropical evergreen broadleaved forests revealed by the temporal history of the tea family.

Subtropical evergreen broadleaved forests (EBLFs) inhabit large areas of East Asia. Although paleovegetation reconstructions have revealed that the subtropical EBLFs existed in Southwest China during the Miocene, the historical construction of these forests remains poorly known. Here, we used the tea family (Theaceae), a characteristic component of the subtropical EBLFs, to gain new insights into the assembly of this important biome. Using a robust phylogenetic framework of Theaceae based on plastome and nuclear ribosomal DNA sequence data, the temporal history of the family was reconstructed. Data from other characteristic components of subtropical EBLFs, including Fagaceae, Lauraceae and Magnoliaceae, were also integrated. Most of the essential elements of the subtropical EBLFs appear to have originated around the Oligocene-Miocene (O-M) boundary. However, small woody lineages (e.g. Camellia, Hartia) from Theaceae were dated to the late Miocene. Accelerated net diversification rates within Theaceae were also detected near the O-M transition period and the late Miocene. Our results suggest that two independent intensifications of the East Asian summer monsoon (EASM) around the O-M boundary and the late Miocene may have facilitated the historical assembly of the subtropical EBLFs in East Asia.

Clinical application of a novel computer-aided detection system based on three-dimensional CT images on pulmonary nodule.

The aim of this study was to investigate the clinical application effects of a novel computer-aided detection (CAD) system based on three-dimensional computed tomography (CT) images on pulmonary nodule. 98 cases with pulmonary nodule (PN) in our hospital from Jun, 2009 to Jun, 2013 were analysed in this study. All cases underwent PN detection both by the simple spiral CT scan and by the computer-aided system based on 3D CT images, respectively. Postoperative pathological results were considered as the "gold standard", for both two checking methods, the diagnostic accuracies for determining benign and malignant PN were calculated. Under simple spiral CT scan method, 63 cases is malignant, including 50 true positive cases and 13 false positive cases from the "gold standard"; 35 cases is benign, 16 true negative case and 19 false negative cases, the Sensitivity 1 (Se1)=0.725, Specificity1 (Sp1)=0.448, Agreement rate1 (Kappa 1)=0.673, J1 (Youden's index 1)=0.173, LR(+)1=1.616, LR(-)1=0.499. Kappa 1=0.673 between the 0.4 and 0.75, has a moderate consistency. Underwent computer-aided detection (CAD) based on 3D CT method, 67cases is malignant, including 62 true positive cases and 7 false positive cases; 31 cases is benign, 24 true negative case and 7 false negative cases, Sensitivity 2 (Se2)=0.899, Specificity2 (Sp2)=0.828, Agreement rate (Kappa 2)=0.877, J2 (Youden's index 2)=0.727, LR(+)2=5.212, LR(-)2=0.123. Kappa 2=0.877 >0.75, has a good consistency. Computer-aided PN detecting system based on 3D CT images has better clinical application value, and can help doctor carry out early diagnosis of lung disease (such as cancer, etc.) through CT images.

Genetic diversity and domestication origin of tea plant Camellia taliensis (Theaceae) as revealed by microsatellite markers.

BACKGROUND: Tea is one of the most popular beverages in the world. Many species in the Thea section of the Camellia genus can be processed for drinking and have been domesticated. However, few investigations have focused on the genetic consequence of domestication and geographic origin of landraces on tea plants using credible wild and planted populations of a single species. Here, C. taliensis provides us with a unique opportunity to explore these issues. RESULTS: Fourteen nuclear microsatellite loci were employed to determine the genetic diversity and domestication origin of C. taliensis, which were represented by 587 individuals from 25 wild, planted and recently domesticated populations. C. taliensis showed a moderate high level of overall genetic diversity. The greater reduction of genetic diversity and stronger genetic drift were detected in the wild group than in the recently domesticated group, indicating the loss of genetic diversity of wild populations due to overexploitation and habitat fragmentation. Instead of the endangered wild trees, recently domesticated individuals were used to compare with the planted trees for detecting the genetic consequence of domestication. A little and non-significant reduction in genetic diversity was found during domestication. The long life cycle, selection for leaf traits and gene flow between populations will delay the emergence of bottleneck in planted trees. Both phylogenetic and assignment analyses suggested that planted trees may have been domesticated from the adjacent central forest of western Yunnan and dispersed artificially to distant places. CONCLUSIONS: This study contributes to the knowledge about levels and distribution of genetic diversity of C. taliensis and provides new insights into genetic consequence of domestication and geographic origin of planted trees of this species. As an endemic tea source plant, wild, planted and recently domesticated C. taliensis trees should all be protected for their unique genetic characteristics, which are valuable for tea breeding.

Comparative chloroplast genomes of camellia species.

BACKGROUND: Camellia, comprising more than 200 species, is a valuable economic commodity due to its enormously popular commercial products: tea leaves, flowers, and high-quality edible oils. It is the largest and most important genus in the family Theaceae. However, phylogenetic resolution of the species has proven to be difficult. Consequently, the interspecies relationships of the genus Camellia are still hotly debated. Phylogenomics is an attractive avenue that can be used to reconstruct the tree of life, especially at low taxonomic levels. METHODOLOGY/PRINCIPAL FINDINGS: Seven complete chloroplast (cp) genomes were sequenced from six species representing different subdivisions of the genus Camellia using Illumina sequencing technology. Four junctions between the single-copy segments and the inverted repeats were confirmed and genome assemblies were validated by PCR-based product sequencing using 123 pairs of primers covering preliminary cp genome assemblies. The length of the Camellia cp genome was found to be about 157kb, which contained 123 unique genes and 23 were duplicated in the IR regions. We determined that the complete Camellia cp genome was relatively well conserved, but contained enough genetic differences to provide useful phylogenetic information. Phylogenetic relationships were analyzed using seven complete cp genomes of six Camellia species. We also identified rapidly evolving regions of the cp genome that have the potential to be used for further species identification and phylogenetic resolution. CONCLUSIONS/SIGNIFICANCE: In this study, we wanted to determine if analyzing completely sequenced cp genomes could help settle these controversies of interspecies relationships in Camellia. The results demonstrate that cp genome data are beneficial in resolving species definition because they indicate that organelle-based "barcodes", can be established for a species and then used to unmask interspecies phylogenetic relationships. It reveals that phylogenomics based on cp genomes is an effective approach for achieving phylogenetic resolution between Camellia species.

New flavan-3-ol dimer from green tea produced from Camellia taliensis in the Ai-Lao mountains of Southwest China.

Camellia taliensis (W. W. Smith) Melchior, belonging to the genus Camellia sect. Thea (Theaceae), is an endemic species distributed from the west and southwest of Yunnan province, China, to the north of Myanmar. Known as a wild tea tree, its leaves have been used commonly for producing tea beverages by the local people of its growing area. One new flavan-3-ol dimer, talienbisflavan A (1), was isolated from green tea prepared from the leaves of C. taliensis collected from the east side of the Ai-Lao mountains, Yuanjiang county of Yunnan province, China. In addition, five hydrolyzable tannins (2-6), five flavonols and flavonol glycosides (9-13), three flavan-3-ols (14-16), nine simple phenolic compounds and glycosides (7, 8, and 17-23), and caffeine (24) were identified. Their structures were determined by detailed spectroscopic analysis. All of the isolated phenolic compounds were tested for their antioxidant activities by DPPH and ABTS(+) radical scavenging assays. The contents of its main chemical compositions were also compared with those collected from the Lincang area of Yunnan province by high-performance liquid chromatography analysis.

Phylogeography of Camellia taliensis (Theaceae) inferred from chloroplast and nuclear DNA: insights into evolutionary history and conservation.

BACKGROUND: As one of the most important but seriously endangered wild relatives of the cultivated tea, Camellia taliensis harbors valuable gene resources for tea tree improvement in the future. The knowledge of genetic variation and population structure may provide insights into evolutionary history and germplasm conservation of the species. RESULTS: Here, we sampled 21 natural populations from the species' range in China and performed the phylogeography of C. taliensis by using the nuclear PAL gene fragment and chloroplast rpl32-trnL intergenic spacer. Levels of haplotype diversity and nucleotide diversity detected at rpl32-trnL (h = 0.841; π = 0.00314) were almost as high as at PAL (h = 0.836; π = 0.00417). Significant chloroplast DNA population subdivision was detected (GST = 0.988; NST = 0.989), suggesting fairly high genetic differentiation and low levels of recurrent gene flow through seeds among populations. Nested clade phylogeographic analysis of chlorotypes suggests that population genetic structure in C. taliensis has been affected by habitat fragmentation in the past. However, the detection of a moderate nrDNA population subdivision (GST = 0.222; NST = 0.301) provided the evidence of efficient pollen-mediated gene flow among populations and significant phylogeographical structure (NST > GST; P < 0.01). The analysis of PAL haplotypes indicates that phylogeographical pattern of nrDNA haplotypes might be caused by restricted gene flow with isolation by distance, which was also supported by Mantel's test of nrDNA haplotypes (r = 0.234, P < 0.001). We found that chlorotype C1 was fixed in seven populations of Lancang River Region, implying that the Lancang River might have provided a corridor for the long-distance dispersal of the species. CONCLUSIONS: We found that C. taliensis showed fairly high genetic differentiation resulting from restricted gene flow and habitat fragmentation. This phylogeographical study gives us deep insights into population structure of the species and conservation strategies for germplasm sampling and developing in situ conservation of natural populations.