Phylogenomic analyses using a new 1013-gene Vitaceae bait-set support major groups of North American Vitis.

A set of newly designed Vitaceae baits targeting 1013 genes was employed to explore phylogenetic relationships among North American Vitis. Eurasian Vitis taxa including Vitis vinifera were found to be nested within North American Vitis subgenus Vitis. North American Vitis subgenus Vitis can be placed into nine main groups: the Monticola group, the Occidentales group, the Californica group, the Vinifera group (introduced from Eurasia), the Mustangensis group, the Palmata group, the Aestivalis group, the Labrusca group, and the Cinerea group. Strong cytonuclear discordances were detected in North American Vitis, with many species non-monophyletic in the plastid phylogeny, while monophyletic in the nuclear phylogeny. The phylogenomic analyses support recognizing four distinct species in the Vitis cinerea complex in North America: V. cinerea, V. baileyana, V. berlandieri, and V. simpsonii. Such treatment will better serve the conservation of wild Vitis diversity in North America. Yet the evolutionary history of Vitis is highly complex, with the concordance analyses indicating conflicting signals across the phylogeny. Cytonuclear discordances and Analyses using the Species Networks applying Quartets (SNaQ) method support extensive hybridizations in North American Vitis. The results further indicate that plastid genomes alone are insufficient for resolving the evolutionary history of plant groups that have undergone rampant hybridization, like the case in North American Vitis. Nuclear gene data are essential for species delimitation, identification and reconstructing evolutionary relationships; therefore, they are imperative for plant phylogenomic studies.

Climate-influenced boreotropical survival and rampant introgressions explain the thriving of New World grapes in the north temperate zone.

The north temperate region was characterized by a warm climate and a rich thermophilic flora before the Eocene, but early diversifications of the temperate biome under global climate change and biome shift remain uncertain. Moreover, it is becoming clear that hybridization/introgression is an important driving force of speciation in plant diversity. Here, we applied analyses from biogeography and phylogenetic networks to account for both introgression and incomplete lineage sorting based on genomic data from the New World Vitis, a charismatic component of the temperate North American flora with known and suspected gene flow among species. Biogeographic inference and fossil evidence suggest that the grapes were widely distributed from North America to Europe during the Paleocene to the Eocene, followed by widespread extinction and survival of relicts in the tropical New World. During the climate warming in the early Miocene, a Vitis ancestor migrated northward from the refugia with subsequent diversification in the North American region. We found strong evidence for widespread incongruence and reticulate evolution among nuclear genes within both recent and ancient lineages of the New World Vitis. Furthermore, the organellar genomes showed strong conflicts with the inferred species tree from the nuclear genomes. Our phylogenomic analyses provided an important assessment of the wide occurrence of reticulate introgression in the New World Vitis, which potentially represents one of the most important mechanisms for the diversification of Vitis species in temperate North America and even the entire temperate Northern Hemisphere. The scenario we report here may be a common model of temperate diversification of flowering plants adapted to the global climate cooling and fluctuation in the Neogene.

Comparative Chloroplast Genome Analyses of the Winter-Blooming Eastern Asian Endemic Genus Chimonanthus (Calycanthaceae) With Implications For Its Phylogeny and Diversification.

Chimonanthus of Calycanthaceae is a small endemic genus in China, with unusual winter-blooming sweet flowers widely cultivated for ornamentals and medicinal uses. The evolution of Chimonanthus plastomes and its phylogenetic relationships remain unresolved due to limited availability of genetic resources. Here, we report fully assembled and annotated chloroplast genomes of five Chimonanthus species. The chloroplast genomes of the genus (size range 153,010 - 153,299 bp) reveal high similarities in gene content, gene order, GC content, codon usage, amino acid frequency, simple sequence repeats, oligonucleotide repeats, synonymous and non-synonymous substitutions, and transition and transversion substitutions. Signatures of positive selection are detected in atpF and rpoB genes in C. campanulatus. The correlations among substitutions, InDels, and oligonucleotide repeats reveal weak to strong correlations in distantly related species at the intergeneric levels, and very weak to weak correlations among closely related Chimonanthus species. Chloroplast genomes are used to reconstruct a well-resolved phylogenetic tree, which supports the monophyly of Chimonanthus. Within Chimonanthus, C. praecox and C. campanulatus form one clade, while C. grammatus, C. salicifolius, C. zhejiangensis, and C. nitens constitute another clade. Chimonanthus nitens appears paraphyletic and is closely related to C. salicifolius and C. zhejiangensis, suggesting the need to reevaluate the species delimitation of C. nitens. Chimonanthus and Calycanthus diverged in mid-Oligocene; the radiation of extant Chimonanthus species was dated to the mid-Miocene, while C. grammatus diverged from other Chimonanthus species in the late Miocene. C. salicifolius, C. nitens (a), and C. zhejiangensis are inferred to have diverged in the Pleistocene of the Quaternary period, suggesting recent speciation of a relict lineage in the subtropical forest regions in eastern China. This study provides important insights into the chloroplast genome features and evolutionary history of Chimonanthus and family Calycanthaceae.

Application of droplet digital polymerase chain reaction of plasma methylated septin 9 on detection and early monitoring of colorectal cancer.

Methylated septin 9 (SEPT9) has been approved for non-invasive screening of colorectal cancer (CRC), but data on monitoring of CRC is sparse. Droplet digital polymerase chain reaction (ddPCR), with higher detection precision and simpler quantification than conventional PCR, has not been applied in SEPT9 detection. We explored the role of SEPT9 ddPCR for CRC detection and to measure serial SEPT9 levels in blood samples of CRC patients before and 3-month after surgery. SEPT9 methylated ratio, methylated abundance, and CEA levels were all higher in CRC patients than normal controls (all P < 0.05). The area under the curve (AUC) for methylated ratio and abundance to detect CRC was 0.707 and 0.710, respectively. There was an increasing trend for SEPT9 methylated abundance from proximal to distal cancers (P = 0.017). At 3-month after surgery, both methylated abundance and ratio decreased (P = 0.005 and 0.053, respectively), especially methylated abundance in stage III and distal cancer (both P < 0.01). We have developed a ddPCR platform for the quantitative detection of plasma SEPT9 in CRC patients. SEPT9 methylated abundance had an early post-operative decline, which may be useful in monitoring of treatment response.

Vitisshizishanensis, a new species of the grape genus from Hubei province, China.

Vitisshizishanensis (Vitaceae), a new species from Hubei, China, is described and illustrated. It is morphologically similar to V.flexuosa and V.bryoniifolia, but differs in leaf lobing and pubescence. It can be easily distinguished from the two species based on its glabrous or with very sparse arachnoid tomentum on the abaxial mature leaf surface, and its unlobed to 3-7 lobed leaves. A detailed description, along with photographs for the new species, and a table for morphological comparisons with similar Vitis species, are also provided.

On the recognition of the long neglected Vitis adenoclada Hand.-Mazz. (Vitaceae) from southern China.

This study reports the recognition of Vitis adenoclada Hand.-Mazz. from southern China. The species was not recognized in the Flora Reipublicae Ropularis Sinicicae and Flora of China treatments. Recent field studies and examination of herbarium collections including the type material suggest that Vitis adenoclada is morphologically similar to V. heyneana, in their densely arachnoid tomentose abaxial leaves, yet it can be easily distinguished from the latter by its red-purple glandular hairs on the young branches (vs. glandular hairs absent in V. heyneana) and inflorescences usually subtended by a tendril at the base (vs. only occasionally with a tendril in V. heyneana). Vitis adenoclada may be a species of hybrid origin, with the highly tomentose Vitis heyneana as one of the parental species, and likely the glandular-hair bearing V. davidii as the other parental species. Vitis adenoclada is recorded from southern China in Guangdong, Guangxi, Guizhou, Hunan and Zhejiang provinces.

Phylogenomic relationships and character evolution of the grape family (Vitaceae).

The grape family consists of 16 genera and ca. 950 species. It is best known for the economically important fruit crop - the grape Vitis vinifera. The deep phylogenetic relationships and character evolution of the grape family have attracted the attention of researchers in recent years. We herein reconstruct the phylogenomic relationships within Vitaceae using nuclear and plastid genes based on the Hyb-Seq approach and test the newly proposed classification system of the family. The five tribes of the grape family, including Ampelopsideae, Cayratieae, Cisseae, Parthenocisseae, and Viteae, are each robustly supported by both nuclear and chloroplast genomic data and the backbone relationships are congruent with previous reports. The cupular floral disc (raised above and free from ovary at the upper part) is an ancestral state of Vitaceae, with the inconspicuous floral disc as derived in the tribe Parthenocisseae, and the state of adnate to the ovary as derived in the tribe Viteae. The 5-merous floral pattern was inferred to be the ancestral in Vitaceae, with the 4-merous flowers evolved at least two times in the family. The compound dichasial cyme (cymose with two secondary axes) is ancestral in Vitaceae and the thyrse inflorescence (a combination of racemose and cymose branching) in tribe Viteae is derived. The ribbon-like trichome only evolved once in Vitaceae, as a synapomorphy for the tribe Viteae.

Testing morphological trait evolution and assessing species delimitations in the grape genus using a phylogenomic framework.

The grape genus (Vitis L.) is of great agronomic importance and represents an economically valuable resource. Researchers have explored the phylogenetic relationships of subgenus Vitis for decades. However, the evolutionary patterns of many morphological characters of subgenus Vitis have not yet been explored in the context of a robust phylogenetic framework. Within the East Asian clade, V. bryoniifolia and its closely related taxa form the V. bryoniifolia clade, which is taxonomically complex. The phylogenetic relationships and species delimitation within this clade remain poorly resolved, due to the limited sampling in prior studies. We tested morphological trait evolution based on ancestral character state reconstruction using a phylogenomic framework. With 89 accessions from the East Asian subgenus Vitis sampled, a robust phylogenetic relationship of the V. bryoniifolia clade is reconstructed using the restriction-site associated DNA sequencing (RAD-seq) data, which support the monophyly of most taxa of the V. bryoniifolia clade. Ancestral character state reconstructions suggest that the weak climbing ability and simplified tendrils of Vitis each evolved multiple times. This study provides a reliable phylogenomic framework for the V. bryoniifolia clade. Coupled with morphological analyses, we discuss the taxonomic status of some taxa in the V. bryoniifolia clade and untangle a taxonomic dilemma in the grape genus.

Methylated Septin 9 and Carcinoembryonic Antigen for Serological Diagnosis and Monitoring of Patients with Colorectal Cancer After Surgery.

With the increasing incidence and mortality of colorectal cancer (CRC), early and accurate diagnosis is of paramount priority to combat this cancer. Epigenetic alterations such as DNA methylation are innovative biomarkers for CRC, due to their stability, frequency, and accessibility in bodily fluids. In this study, blood samples were prospectively collected from patients before and after operation for CRC for determination of methylated septin 9 (mSEPT9) and compared to carcinoembryonic antigen (CEA). The sensitivity of using mSEPT9 methylation status for diagnosing CRC was significantly higher than using elevated CEA levels (73.2% vs 48.2%; p value < 0.001). The sensitivities of both tests increased with higher tumor staging (P = 0.004 and 0.04 respectively). Combined mSEPT9 and CEA had higher accuracy than single CEA or mSEPT9 (P = 0.009 and 0.532 separately). An increase in the methylation level of mSEPT9 detected in the post-operative samples was associated with a higher mortality rate (15.2% vs 1.8%; P = 0.024) and the presence of metastasis (27.3% vs 7.0%; P = 0.013). mSEPT9 was more sensitive than CEA for diagnosing CRC, and combined mSEPT9 and CEA was more accurate. After curative resection, detection of increased mSEPT9 methylation level may indicate adverse outcomes.

Transcriptomic and proteomic approaches to explore the differences in monoterpene and benzenoid biosynthesis between scented and unscented genotypes of wintersweet.

Wintersweet (Chimonanthus praecox L.) is an important ornamental plant in China with a pleasant floral scent. To explore the potential mechanisms underlying differences in the fragrances among genotypes of this plant, we analyzed floral volatile organic compounds (VOCs) from two different genotypes: SW001, which has little to no fragrance, and the scented genotype H29. The major VOCs in H29 were linalool, trans-ß-ocimene, benzyl acetate, methyl salicylate, benzyl alcohol (BAlc) and methyl benzoate. The most important aroma-active compound in H29, linalool, was emitted at a low concentration in SW001, which had markedly higher levels of trans-ß-ocimene than H29. Next, to investigate scent biosynthesis, we analyzed the transcriptome and proteome of fully open flowers of the two genotypes. A total of 14 443 differentially expressed unigenes and 196 differentially expressed proteins were identified. Further analyses indicated that 56 differentially expressed genes involved in the terpenoid and benzenoid biosynthesis pathways might play critical roles in regulating floral fragrance difference. Disequilibrium expression of four terpene synthase genes resulted in diverse emission of linalool and trans-ß-ocimene in both genotypes. In addition, the expressions of two CpMYC2 transcription factors were both upregulated in H29, implying that they may regulate linalool production. Notably, 16 of 20 genes in the benzenoid biosynthesis pathway were downregulated, corresponding to the relatively low level of benzenoid production in SW001. The lack of benzyl acetate might indicate that SW001 may lack substrate BAlc or functional acetyl-CoA:benzylalcohol acetyltransferase.

Phylogenomics, biogeography, and adaptive radiation of grapes.

The application of whole-genome resequencing based on next-generation sequencing technologies provides an unprecedented opportunity for researchers to resolve long-standing evolutionary problems. Taxa belonging to the grape genus (Vitis L.) represent important genetic resources for the improvement of cultivated grapes. However, it has been challenging to resolve the deep phylogenetic relationships within Vitis, limiting the current understanding of the evolutionary history of Vitis and preventing the use of valuable wild grape resources. In this study, we obtained whole-genome sequence data from 41 accessions representing most taxa within subgenus Vitis and aligned these sequences to the Vitis vinifera L. reference genome. We reconstructed deep phylogenetic relationships within subgenus Vitis based on 2068 single-copy orthologous genes, which led to a robust topology with bootstrap support values of 100% for almost all branches. Three main clades are recovered within subgenus Vitis reflecting their continental distribution through North America, Europe, and East Asia, respectively. Our results suggest that the most possible migration route of the East Asian Vitis is from northeastern Asia southward to South Asia and Southeast Asia. The East Asian Vitis seems to have experienced adaptive radiation during the Miocene. This study provides novel insights into the diversification history of the grape genus Vitis.

Morphology, Structure, and Ontogeny of Trichomes of the Grape Genus (Vitis, Vitaceae).

Trichomes are widely distributed on surfaces of different organs in the grape genus Vitis and are of taxonomic utility. To explore the morphology, structure and ontogeny of Vitis trichomes, we investigated the diversity and distribution of trichomes in 34 species of Vitis. Two main types of trichomes in Vitis are documented: non-glandular and glandular. Within non-glandular trichomes, ribbon and simple trichomes are found on different vegetative plant organs. The morphology and ontogeny of these types of trichomes are further examined with light microscopy and scanning electron microscopy. The ultrastructure of the glandular trichomes is explored with transmission electron microscopy. The ribbon trichomes are twisted, greatly elongated and unicellular, and this trichome type may be a morphological synapomorphy of Vitis and its closest tropical relative Ampelocissus and Pterisanthes in Vitaceae. The simple trichomes are documented in most species sampled in the genus. The glandular trichomes are multicellular, non-vascularized and composed of both epidermis and subjacent layers. We show that prickles occurring along the stems and petioles of Vitis davidii are modified glandular trichomes. We observed that glandular trichomes of V. romanetii secrete mucilage and volatile substances which trap insectes on the glands. Transmission electron microscopy indicates that metabolic products accumulate in vacuoles, the cytoplasm and intercellular spaces. We infer that glandular trichomes and young prickles are involved in the secretion of these metabolic products and the intercellular spaces may be the places of temporary storage of these secretions.