Salvia guidongensis sp. nov.: unraveling a critical evolutionary link in East Asian Salvia from Central China integrating morphology, phylogeny, and plastid genomics.

Introduction: Salvia L., representing the largest genus within the mint family, is noted for its global distribution of approximately 1000 species, with East Asia, and particularly China, recognized as a critical center of diversity for the genus. Methods: Our research was conducted through extensive fieldwork in Guidong County, Hunan Province, China, where we identified a previously undescribed species of Salvia. The identification process involved detailed morphological observations, phylogenetic analyses, and plastid genomics. Results: The newly discovered species, Salvia guidongensis, exhibits unique characteristics not commonly observed in the East Asian lineage of Salvia, including dual floral colors within natural populations-either pale purple or pale yellow. Morphologically, while it shares similarities with members of sect. Glutinaria, S. guidongensis is distinct in its floral morphology, stature, and specific foliar traits. Phylogenetic analysis places S. guidongensis in a unique clade within the East Asian lineage of Salvia, suggesting it may serve as an important evolutionary link. Additionally, we explored the plastome features of S. guidongensis, comparing them with those of closely related species. Discussion: The discovery of S. guidongensis not only entriches the taxonomic tapestry of Salvia but also provides critical insights into the biogeography and evolutionary pathways of the genus in East Asia. By integrating morphological and molecular data, we validate the novel status of S. guidongensis and highlight its significance in bridging taxonomic and evolutionary gaps within Sect. Glutinaria of Salvia.

Evolution and chemical diversity of the volatile compounds in Salvia species.

INTRODUCTION: The plant essential oils are composed of volatile compounds and have significant value in preventing and treating neurological diseases, anxiety, depression, among others. The genus Salvia has been shown to be an important medicinal resource, especially the aerial parts of genus Salvia, which are rich in volatile compounds; however, the chemical diversity and distribution patterns of volatile compounds in Salvia species are still unknown. OBJECTIVE: The work is performed to analyse the chemical diversity and distribution patterns of volatile compounds in genus Salvia. METHODS: The genomic single nucleotide polymorphisms (SNPs) combined with gas chromatography-mass spectrometry (GC-MS) were used to explore the evolution and chemical diversity of Salvia volatile compounds. Initially, the genetic relationship of genus Salvia was revealed by phylogenetic tree that was constructed based on SNPs. And then, GC-MS was applied to explore the chemical diversity of volatile compounds. RESULTS: The results indicated that the volatile compounds were mainly monoterpenoids, sesquiterpenoids, and aliphatic compounds. The genomic SNPs divided species involved in this work into four branches. The volatile compounds in the first and second branches were mainly sesquiterpenoids and monoterpenoids, respectively. Species in the third branch contained more aliphatic compounds and sesquiterpenoids. And those in the fourth branch were also rich in monoterpenoids but had relatively simple chemical compositions. CONCLUSION: This study offered new insights into the phylogenetic relationships besides chemistry diversity and distribution pattern of volatile compounds of genus Salvia, providing theoretical guidance for the investigations and development of secondary metabolites.

Metabolomic and antioxidant analyses of Salvia miltiorrhiza Bunge and Salvia prattii Hemsl. seeds.

Salvia miltiorrhiza and Salvia prattii seeds are rich in metabolites that are beneficial to human health and can be utilised as nutritional supplements. In this study, UPLC-MS and GC-MS based on extensively focused metabolomics were used to compare the seed metabolomics of the two species. LC-MS detected 118 metabolites, primarily Lipids and phenylpropanoids. GC- MS detected a total of 188 metabolites, mainly organic acids and their derivatives, of which Salvia prattii seeds contain high levels of nutrients. In addition, we experimentally determined antioxidant activity of two Salvia species, and the results showed that the antioxidant activity of Salvia prattii seeds was about twice as high as that of Salvia miltiorrhiza seeds. We used WGCNA to group the metabolites, and found the central metabolites in the focal modules including flavonoids and terpenoids. Our study contributes valuable knowledge for future research on the chemical makeup of Salvia prattii seeds.

Unveiling the spatial distribution and molecular mechanisms of terpenoid biosynthesis in Salvia miltiorrhiza and S. grandifolia using multi-omics and DESI-MSI.

Salvia miltiorrhiza and S. grandifolia are rich in diterpenoids and have therapeutic effects on cardiovascular diseases. In this study, the spatial distribution of diterpenoids in both species was analyzed by a combination of metabolomics and mass spectrometry imaging techniques. The results indicated that diterpenoids in S. miltiorrhiza were mainly abietane-type norditerpenoid quinones with a furan or dihydrofuran D-ring and were mainly distributed in the periderm of the roots, e.g. cryptotanshinone and tanshinone IIA. The compounds in S. grandifolia were mainly phenolic abietane-type tricyclic diterpenoids with six- or seven-membered C-rings, and were widely distributed in the periderm, phloem, and xylem of the roots, e.g. 11-hydroxy-sugiol, 11,20-dihydroxy-sugiol, and 11,20-dihydroxy-ferruginol. In addition, the leaves of S. grandifolia were rich in tanshinone biosynthesis precursors, such as 11-hydroxy-sugiol, while those of S. miltiorrhiza were rich in phenolic acids. Genes in the upstream pathway of tanshinone biosynthesis were highly expressed in the root of S. grandifolia, and genes in the downstream pathway were highly expressed in the root of S. miltiorrhiza. Here, we describe the specific tissue distributions and mechanisms of diterpenoids in two Salvia species, which will facilitate further investigations of the biosynthesis of diterpenoids in plant synthetic biology.

The complete chloroplast genome sequence of Salvia chienii E.Peter, 1936 (Lamiaceae).

Salvia chienii E.Peter is a medicinal herb mainly distributed in Huangshan Mountain of Anhui province, China. In this study, the first complete chloroplast genome of S. chienii was sequenced and assembled. The genome length was 151,530 bp and encoded 143 genes (91 protein-coding genes, eight rRNA genes, and 37 tRNA genes). The phylogenomic analysis showed that S. chienii was closely related to S. miltiorrhiza. Further evolutionary studies of the genus Salvia could benefit from the complete chloroplast genome of S. chienii present in this study.

Comparison of potent odorants in raw and cooked mildly salted large yellow croaker using odor-active value calculation and omission test: Understanding the role of cooking method.

Cooking improves food aroma, but few studies have explored how cooking affects food aromas. Here, aroma changes in mildly salted large yellow croaker (Larimichthys crocea, MSLYC) after steaming, baking, frying, and deep frying was investigated. The raw fish was dominated by fishy notes but after cooking, the aroma became dominated by fatty notes. Nine volatiles, including hexanal, nonanal, (E, Z)-2, 6-nonadienal, (E, E)-2, 4-decadienal, 1-octen-3-ol, linalool, ethyl hexanoate, acetic acid and anethole, were identified as key odor-active compounds using GC-MS, OAV, and omission tests analyses. Changes in the concentrations of key odor-active compounds were mainly due to evaporation, oxidation of linolenic acids, and thermal catalyzed reactions. Interestingly, anethole was the key odor-active compound, providing new insight into the underlying reactions of cooked fish aroma.

Unusual patterns of hybridization involving two alpine Salvia species: Absence of both F1 and backcrossed hybrids.

Natural hybridization plays an important role in speciation; however, we still know little about the mechanisms underlying the early stages of hybrid speciation. Hybrid zones are commonly dominated by F1s, or backcrosses, which impedes further speciation. In the present study, morphological traits and double digest restriction-site associated DNA sequencing (ddRAD-seq) data have been used to confirm natural hybridization between Salvia flava and S. castanea, the first case of identification of natural hybridization using combined phenotypic and molecular evidence in the East Asian clade of Salvia. We further examined several reproductive barriers in both pre-zygotic and post-zygotic reproductive stages to clarify the causes and consequences of the hybridization pattern. Our results revealed that reproductive isolation between the two species was strong despite the occurrence of hybridization. Interestingly, we found that most of the hybrids were likely to be F2s. This is a very unusual pattern of hybridization, and has rarely been reported before. The prevalence of geitonogamy within these self-compatible hybrids due to short distance foraging by pollinators might explain the origin of this unusual pattern. F2s can self-breed and develop further, therefore, we might be witnessing the early stages of hybrid speciation. Our study provides a new case for understanding the diversification of plants on the Qinghai-Tibet Plateau.

Preparation and Tribological Properties of a Multilayer Graphene-Reinforced TiO2 Composite Nanolubricant Additive.

The unique structure and physical properties of graphene and anatase TiO2 make them suitable for use as additives for engine lubricants. This study describes the use of dielectric barrier discharge plasma-assisted ball milling to synthesize a multilayer graphene-reinforced TiO2 composite nanolubricant additive (MGTC). A variety of physical and chemical tests were performed to characterize the resulting experimental materials, including X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Raman, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). Four-ball friction and wear testing machines were used to study the tribological properties and extreme pressure anti-wear properties of a base oil containing 0.1, 0.5, 1.0, and 1.5 wt % of the modified TiO2. Raman spectroscopy, XPS, SEM, and energy-dispersive spectrometry (EDS) analyses were used to examine and analyze the microstructure of the friction pairs. As a result of the plasma-assisted ball milling process, expanded graphite was successfully separated into multilayer graphene nanosheets, and spherical TiO2 was successfully bonded to the nanosheets of the multilayer graphene. The 1.0 wt % composite oil was found to provide good friction reduction and wear resistance. It had a film thickness of 27.5 nm, which was 167% thicker than base oil. Due to its excellent dispersion stability, the MGTC nanocomposite exhibited excellent lubrication performance, which was attributed to the formation of carbon protective films, titanium dioxide deposition films, transfer films, and the occurrence of nano ball effects on the surface of friction pairs.

Characterization of complete chloroplast genome of Salvia honania L. H. Bailey (Lamiaceae).

Salvia honania L. H. Bailey is an endemic species, mainly distributed in Henan and Hubei provinces in China. The first complete chloroplast genome of Salvia honania was sequenced and assembled in this study. The genome is 151,559 bp in length and contains 132 encoded genes in total, including 87 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The phylogenomic analysis showed that Salvia honania was closely related to Salvia meiliensis according the current sampling extent.

The sage genome provides insight into the evolutionary dynamics of diterpene biosynthesis gene cluster in plants.

The widely cultivated medicinal and ornamental plant sage (Salvia officinalis L.) is an evergreen shrub of the Lamiaceae family, native to the Mediterranean. We assembled a high-quality sage genome of 480 Mb on seven chromosomes, and identified a biosynthetic gene cluster (BGC) encoding two pairs of diterpene synthases (diTPSs) that, together with the cytochromes P450 (CYPs) genes located inside and outside the cluster, form two expression cascades responsible for the shoot and root diterpenoids, respectively, thus extending BGC functionality from co-regulation to orchestrating metabolite production in different organs. Phylogenomic analysis indicates that the Salvia clades diverged in the early Miocene. In East Asia, most Salvia species are herbaceous and accumulate diterpenoids in storage roots. Notably, in Chinese sage S. miltiorrhiza, the diterpene BGC has contracted and the shoot cascade has been lost. Our data provide genomic insights of micro-evolution of growth type-associated patterning of specialized metabolite production in plants.

The complete chloroplast genome of Salvia liguliloba Y. Z. Sun (Lamiaceae).

Salvia liguliloba Y. Z. Sun is a plant species endemic to the Tianmu Mountains. In this study, we assembled the complete chloroplast genome of S. liguliloba. The chloroplast genome of S. liguliloba was 151,490 bp with quadripartite structure in length, which contained 124 encoded genes, including 79 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. Our phylogenetic analysis result based on 54 chloroplast genomes revealed that S. liguliloba was closely related to S. miltiorrhiza according to the current sampling extent in Lamiaceae.

Specific Flavonoids and Their Biosynthetic Pathway in Scutellaria baicalensis.

Scutellaria baicalensis, is one of the most traditional medicinal plants in the Lamiaceae family, and has been widely used to treat liver and lung complaints and as a complementary cancer treatment in traditional Chinese medicine. The preparation from its roots, called "Huang Qin," is rich in specialized flavones such as baicalein, wogonin, and their glycosides which lack a 4'-hydroxyl group on the B ring (4'-deoxyflavones), with anti-tumor, antioxidant, and antiviral activities. Baicalein has recently been reported to inhibit the replication of the COVID-19 virus. These 4'-deoxyflavones are found only in the order Lamiales and were discovered in the genus Scutellaria, suggesting that a new metabolic pathway synthesizing 4'-deoxyflavones evolved recently in this genus. In this review, we focus on the class of 4'-deoxyflavones in S. baicalensis and their pharmacological properties. We also describe the apparent evolutionary route taken by the genes encoding enzymes involved in the novel, root-specific, biosynthetic pathway for baicalein and wogonin, which provides insights into the evolution of specific flavone biosynthetic pathways in the mint family.

Two types of O-methyltransferase are involved in biosynthesis of anticancer methoxylated 4'-deoxyflavones in Scutellaria baicalensis Georgi.

The medicinal plant Scutellaria baicalensis Georgi is rich in specialized 4'-deoxyflavones, which are reported to have many health-promoting properties. We assayed Scutellaria flavones with different methoxyl groups on human cancer cell lines and found that polymethoxylated 4'-deoxyflavones, like skullcapflavone I and tenaxin I have stronger ability to induce apoptosis compared to unmethylated baicalein, showing that methoxylation enhances bioactivity as well as the physical properties of specialized flavones, while having no side-effects on healthy cells. We investigated the formation of methoxylated flavones and found that two O-methyltransferase (OMT) families are active in the roots of S. baicalensis. The Type II OMTs, SbPFOMT2 and SbPFOMT5, decorate one of two adjacent hydroxyl groups on flavones and are responsible for methylation on the C6, 8 and 3'-hydroxyl positions, to form oroxylin A, tenaxin II and chrysoeriol respectively. The Type I OMTs, SbFOMT3, SbFOMT5 and SbFOMT6 account mainly for C7-methoxylation of flavones, but SbFOMT5 can also methylate baicalein on its C5 and C6-hydroxyl positions. The dimethoxylated flavone, skullcapflavone I (found naturally in roots of S. baicalensis) can be produced in yeast by co-expressing SbPFOMT5 plus SbFOMT6 when the appropriately hydroxylated 4'-deoxyflavone substrates are supplied in the medium. Co-expression of SbPFOMT5 plus SbFOMT5 in yeast produced tenaxin I, also found in Scutellaria roots. This work showed that both type I and type II OMT enzymes are involved in biosynthesis of methoxylated flavones in S. baicalensis.

A 2-oxoglutarate-dependent dioxygenase converts dihydrofuran to furan in Salvia diterpenoids.

Tanshinone ⅡA (TⅡA), a diterpene quinone with a furan ring, is a bioactive compound found in the medicinal herb redroot sage (Salvia miltiorrhiza Bunge), in which both furan and dihydrofuran analogs are present in abundance. Progress has been made recently in elucidating the tanshinone biosynthetic pathway, including heterocyclization of the dihydrofuran D-ring by cytochrome P450s; however, dehydrogenation of dihydrofuran to furan, a key step of furan ring formation, remains uncharacterized. Here, by differential transcriptome mining, we identified six 2-oxoglutarate-dependent dioxygenase (2-ODD) genes whose expressions corresponded to tanshinone biosynthesis. We showed that Sm2-ODD14 acts as a dehydrogenase catalyzing the furan ring aromatization. In vitro Sm2-ODD14 converted cryptotanshinone to TⅡA and thus was designated TⅡA synthase (SmTⅡAS). Furthermore, SmTⅡAS showed a strict substrate specificity, and repression of SmTⅡAS expression in hairy root by RNAi led to increased accumulation of total dihydrofuran-tanshinones and decreased production of furan-tanshinones. We conclude that SmTⅡAS controls the metabolite flux from dihydrofuran- to furan-tanshinones, which influences medicinal properties of S. miltiorrhiza.

Elsholtziazhongyangii (Lamiaceae), a new species from Sichuan, China.

Elsholtziazhongyangii (Lamiaceae), a new species from Sichuan Province, China, is described and illustrated. The new species is morphologically similar to E.feddeif.feddei, but it can be easily distinguished from E.feddeif.feddei by smaller corolla (3.2-3.5 mm vs. 4.5-5.3 mm), bract indumentum (glabrous, except margin ciliate vs. villous, especially on veins abaxially, glabrous adaxially) and bract stalked (ca. 1.2 mm vs. sessile). Phylogenetic analyses, based on two nuclear ribosomal (ETS, ITS) and five plastid (rbcL, matK, trnL-F, ycf1, ycf1-rps15) regions, confirmed that the new species formed a monophyletic clade with robust support. The new species is currently known from western Sichuan.

Occurrence and Prevention of Delayed Autonomous Selfing in Salvia umbratica (Lamiaceae).

Delayed autonomous selfing (DAS) provides reproductive assurance under conditions of pollinator and/or pollen-limitation. Few plant species have been investigated to determine if DAS is terminated when a flower is sufficiently pollinated by a pollen vector, thereby saving plant resources for other purposes. We examined this possibility in bumblebee-pollinated Salvia umbratica. We first showed that DAS resulting in high fruit set (100%) and seed set (>80%) per flower occurred in the absence of insect pollinators by means of style recurvature and was completed in 94% of flowers 72 h after they opened. In contrast, in flowers pollinated immediately after opening, DAS was prevented by corollas dropping away before styles recurve toward the upper thecae. We next showed that hand-pollination of flowers immediately after they opened resulted in high fruit set (100%) and seed set (>80%) when 5-10 pollen grains or more were deposited on their stigmas, whereas fruit set and seed set were reduced to 45.00 and 22.50%, respectively, when pollen loads were reduced to 1-3 pollen grains. Finally, we showed that on average single pollinator visits deposited 26 pollen grains on stigmas of flowers that had just opened, which is more than enough to ensure high fruit and seed set. Our results indicate that flower longevity is highly correlated with the pollinator environment and female fitness of S. umbratica, with extended flower longevity allowing DAS to occur being advantageous when pollinators are absent, while reduced floral longevity and prevention of DAS being favored when flowers are pollinated by pollinators. Thus, flower longevity in S. umbratica varies so as to optimize reproductive output and resource efforts, and is dependent on the availability and effectiveness of pollinators to pollinate flowers.

Conservation Genomics of Wild Red Sage (Salvia miltiorrhiza) and Its Endangered Relatives in China: Population Structure and Interspecific Relationships Revealed From 2b-RAD Data.

Red sage (Salvia miltiorrhiza) is a widely used medicinal plant for treatment of cardiovascular and cerebrovascular diseases. Because of excessive excavation by huge market demand and habitat loss by human activities, the wild population resources of S. miltiorrhiza have reduced drastically in recent years. Meanwhile, population status of two closely related species S. bowleyana and S. paramiltiorrhiza were in a trend of decreasing due to their potential replacement of S. miltiorrhiza. Particularly, S. paramiltiorrhiza was threatened and endemic to a small region in eastern China. However, to date there has been no conservation genetic research reported for wild S. miltiorrhiza population and its endangered relatives. Assess the wild germplasm diversity for S. miltiorrhiza and its related species would provide fundamental genetic background for cultivation and molecular breeding of this medicinally important species. In the present study, we investigated the genetic diversity, population structure, and intra/inter-specific differentiation of S. miltiorrhiza and above two relatives using 2b-RAD genome-wide genotyping method. By investigating 81 individuals of S. miltiorrhiza, 55 individuals of S. bowleyana and 15 individuals of S. paramiltiorrhiza from 23 locations in China, we obtained 23,928 SNPs in total. A comparatively high genetic diversity was observed in S. miltiorrhiza (π = 0.0788, H e = 0.0783 ± 0.0007). The observed and expected heterozygosity in populations of these three species ranged from 0.0297 to 0.1481 and 0.0251 to 0.831, respectively. Two major lineage groups were detected in the examined S. miltiorrhiza populations. The results indicated that Dabie Mountain as a genetic diversity center of S. miltiorrhiza and possible complex inter-specific genetic exchange/hybridization occurred between S. miltiorrhiza and the two relatives. We suggest that strategic conservation and germplasm preservation should be considered not only for wild populations of S. miltiorrhiza, but also for its related S. bowleyana and S. paramiltiorrhiza.

Integrated Transcriptomics and Proteomics to Reveal Regulation Mechanism and Evolution of SmWRKY61 on Tanshinone Biosynthesis in Salvia miltiorrhiza and Salvia castanea.

Tanshinones found in Salvia species are the main active compounds for the treatment of cardiovascular and cerebrovascular diseases, but their contents are hugely different in different species. For example, tanshinone IIA content in Salvia castanea Diels f. tomentosa Stib. is about 49 times higher than that in Salvia miltiorrhiza Bunge. The molecular mechanism responsible for this phenomenon remains largely unknown. To address this, we performed comparative transcriptomic and proteomic analyses of S. miltiorrhiza and S. castanea. A total of 296 genes in S. castanea and 125 genes in S. miltiorrhiza were highly expressed at both the transcriptional and proteome levels, including hormone signal regulation, fungus response genes, transcription factors, and CYP450. Among these differentially expressed genes, the expression of SmWRKY61 was particularly high in S. castanea. Overexpression of SmWRKY61 in S. miltiorrhiza could significantly increase the content of tanshinone I and tanshinone IIA, which were 11.09 and 33.37 times of the control, respectively. Moreover, SmWRKY61 had a strong regulatory effect, elevating the expression levels of tanshinone pathway genes such as DXS2, CMK, HMGS2, 1, KSL1, KSL2, CYP76AH1, and CYP76AK3. For the WRKY family, 79 SmWRKYs were originally obtained and classified into three main groups. Collinearity analysis indicated a more specific extension of WRKY gene family in Salvia genus. In 55 Salvia species, only 37 species contained the WRKY61 sequence, and high SmWRKY61 expression in some Salvia L. species was often accompanied by high tanshinone accumulation. The above results suggest that SmWRKY61 is a highly effective regulator of tanshinone accumulation and may be a key factor resulting in high tanshinone accumulation in S. castanea.

[Effects of different carbon sources on growth and active component contents in Salvia miltiorrhiza and S. castanea f. tomentosa hairy roots].

Salvia miltiorrhiza(Sm) and Salvia castanea f. tomentosa(Sc) hairy roots were used as experimental materials to study the effects of six different carbon sources, galactose, fructose, lactose, glucose, arabinose and sucrose(control), on fresh weight, dry weight, contents and yields of salvianolic acids and tanshinones. The results showed that galactose was most beneficial to the growth of two kinds of hairy roots, while lactose and arabinose were not conducive to their growth. As for Sm hairy roots, fructose significantly promoted the accumulation of salvianolic acid B, and the content increased by 5.801 times and 10.151 times compared with the control group, respectively. Glucose significantly promoted the accumulation of salvianolic acids. The content and yield of rosmarinic acid were 7.674 times and 9.260 times of that of the control group, and the content and yield of salvianolic acid B were 5.532 times and 6.675 times of the control group. For the hairy roots of Sc, galactose significantly increased the content and yield of rosmarinic acid, reaching 7.820 times and 9.944 times of the control group, respectively. Fructose promoted the increase of the content and yield of cryptotanshinone, reaching 9.242 times and 6.609 times of the control group, respectively. The study confirmed the optimal carbon source for the hairy root culture of Sm and Sc, and provided theoretical guidance for large-scale production of Sm drug-derived components and the utilization of Sc.

Development of 19 novel microsatellite markers of lily-of-the-valley (Convallaria, Asparagaceae) from transcriptome sequencing.

The lily-of-the-valley Convallaria (Asparagaceae) consists of three herbaceous perennial species. The plants are commonly found in northern hemisphere, and are best-known for their ornamental and pharmaceutical value. In order to assess the genetic structure, diversity and demographic history of Convallaria species, 19 novel microsatellite markers were developed based on transcriptome data of C. keiskei. Polymorphism and cross-amplification of the markers were tested in three populations of C. keiskei and one population each of C. majalis and C. montana. The transferability rate in two species was both 89.5%. The average number of alleles detected per locus was 7.7, 3.3 and 2.7 in C. keiskei, C. majalis and C. montana, respectively, and the polymorphism information content correspondingly varied from 0.067 to 0.730, from 0.071 to 0.637 and from 0.195 to 0.680 at the population level. The observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.833, respectively. Seven of the 19 loci showed significant deviation from Hardy-Weinberg equilibrium. The availability of these markers will provide a useful molecular tool for further population genetics, phylogeographic and breeding studies of Convallaria species.