Fritillaria Species Research Articles

Biochemometric-guided isolation of new Isosteroidal alkaloids from Fritillaria cirrhosa D.Don (Liliaceae, syn. Fritillaria roylei Hook) as acetylcholinesterase inhibitors

Globally, Alzheimer's disease is an urgent public health concern with the ageing population in developing nations. Recent studies have identified isosteroidal alkaloids as promising therapeutic agents for Alzheimer's treatment. Fritillaria species are well-known rich sources of steroidal and isosteroidal alkaloids. In this context, the current study focuses on the biochemometric-guided isolation of three previously undescribed and two known isosteroidal alkaloids as acetylcholinesterase (AChE) inhibitors from the bulbs of Fritillaria cirrhosa D.Don. The isolated molecules were characterized by NMR, HR-ESI-MS, FT-IR, and DP4+ analysis. Subsequently, all isolates were evaluated for AChE inhibitory activity using Ellman's method. Among the evaluated molecules, 1 (IC50: 33.0 ± 4.4 μM) and 5 (IC50: 24.7 ± 4.5 μM) showed promising AChE inhibition in vitro. Enzyme kinetic studies of isolated molecules revealed mixed inhibition kinetics with Ki varying from 1.3 to 24.4 μM. Moreover, the in silico studies showed excellent binding affinities of isolated molecules with the target protein and good drug-like ADMET properties. The present study identified new isosteroidal alkaloids as promising AChE inhibitors from F. cirrhosa bulbs via a biochemometric approach and advocated their further exploration for treating neurodegenerative disorders.

An automatic LC-MS/MS data analysis workflow for herbal compound annotation with AutoAnnotatoR: A case study of ten botanical origins of Fritillaria species

BackgroundDespite the widespread implementation of analytical hardware capable of recording large-scale datasets for botanical natural products, the data processing procedures for compound annotation remain a bothersome obstacle that demand a tremendous amount of time and expert knowledge. MethodsHerein, an automatic LC-MS/MS data analysis workflow with AutoAnnotatoR was introduced for the compound annotation of plant derived natural products, which has the merits of great efficiency, high accuracy, saving time and simplified process. This procedure enabled automatic matching of MS2 data with characteristic fragment ions, as well as MS1 data with compound libraries, which improves the accuracy of structural elucidation. Notably, the optimization of collision energy for each target ion was successfully performed for the first time, facilitating the acquisition of comprehensive fragmentation information. ResultsThe automatic analysis workflow with AutoAnnotatoR was successfully applied for the annotation of alkaloids from 10 botanical origins of Fritillaria species. Consequently, a total of 2684 chemical constituents were tentatively characterized, with 23 components being unambiguously validated by reference standards and 2434 being probable novel chemicals. ConclusionThe entire data analysis procedure takes only a few hours, vastly improving analysis speed while assuring high accuracy. This method provides a powerful tool for the rapid and precise annotation of complex natural products. The workflow is publicly accessible on Github as an open-source R package called AutoAnnotatoR (https://github.com/anyaling2022/AutoAnnotatoR).

First Report of Clonostachys rosea Causing Bulb Rot on Fritillaria taipaiensis in China.

Taibai Beimu (Fritillaria taipaiensis) is a species of Fritillaria commonly used in traditional Chinese medicine for its antitussive, expectorant, and antihypertensive properties. In April of 2021 and 2022, an incidence 10-30% of yellowing or purpling, wilting, and dying symptoms was observed on Taibai Beimu in Wanyuan, Sichuan province. Infected roots and bulbs displayed spots ranging from brown to black, along with necrotic rot. In severe cases, the entire bulbs rotted. Fifteen symptomatic bulbs were cut into 0.5 × 0.5 cm pieces, surface sterilized in 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min under aseptic conditions, rinsed with sterile water 3 times, and air-dried. The segments were placed on potato dextrose agar (PDA) and incubated at 25℃ for 7 days in the dark. Six Clonostachys-like monospore isolates were obtained. Colonies on PDA reached 32 to 43 mm in diameter in 7 days at 25℃ in the dark, felty to tomentose to granulose aerial mycelia with a white or light yellow appearance, and reverse colors matching. On cornmeal-dextrose agar, primary conidiophores had a Verticillium-like structure with 1 to 3 levels. Stipes were 36.1 to 236.3μm long. Phialides formed in whorls of 2 to 5, 15.3 to 45.7μm long, 1.1 to 3.4μm wide at the base, and 1.03 to 2.41μm wide near opening (n=95). Each producing a small hyaline drop of conidia. Conidia were 3.7 to 11.3μm × 2.1 to 4.1μm (n=110). Secondary conidiophores displayed Penicillium-like structures, and stipes were 23.1 to 142.3μm long. Phialides formed in compressed whorls of 4 to 8 per metula, 7.0 to 16.0μm in length, 1.3 to 3.1μm in width at the base, 1.8 to 3.6μm at the widest point, and 0.8 to 1.8μm near opening (n=50). Conidia were 3.0 to 6.4μm ×1.6 to 3.4μm (n=65). The morphology was consistent with the previous description of Clonostachys rosea (Hans-Josef et al. 1999). The ATP citrate lyase (ACL1), β-tubulin (TUB2), translation elongation factor 1-α (tef1α), and the nuclear ribosomal internal transcribed spacer (ITS) of three strains were amplified and sequenced using primers acl1-230up/acl1-1220low (Gräfenhan et al. 2011), T1/CYLTUB1R (Crous et al. 2004; O'Donnell and Cigelnik 1997), EF1-728F/EF2 (Carbone and Kohn 1999; O'Donnell et al. 1998), and ITS1/ITS4 (White et al. 1990), respectively. Blastn homology search showed a > 97% similarity to the ex-type strains of C. rosea (CBS710.86). All sequences have been deposited in GenBank (PP394342 to PP394350, and PP396901 to PP396903). A phylogenetic tree was constructed using Bayesian analysis based on the alignment of the combined ACL1, TUB2, tef1α, and ITS sequences through IQ-TREE. The tree displayed clustering with known strains of C. rosea. Pathogenicity was confirmed by inoculating five healthy five-year-old Taibai beimu plants with a spore suspension (1.0 × 106 spores mL-1) of the strain WYEB1101, while sterilized water was used as a control. The inoculation process involved pouring the spore suspension over the wounded bulbs and covering with them sterile soil. Subsequently, all plants were cultivated in sterile soil indoors under natural conditions suitable for Taibai beimu. The pathogenicity assays were repeated twice. After 20 days of cultivation, the infected plants displayed symptoms similar to those observed in the field, while all control plants remained asymptomatic. Sequencing confirmed the re-isolation of C. rosea from the inoculated plants, satisfying Koch's hypothesis. Clonostachys rosea has been previously reported to cause root rot of Chinese medicine herb, such as Astragalus membranaceus and Gastrodia elata (Lee et al. 2020; Qi et al. 2022). To our knowledge, this is the first report of C. rosea infecting Taibai Beimu in China, highlighting a potential risk to this crop.

Determination of ITS1 haplotypes of Fritillariae Cirrhosae Bulbus by amplicon sequencing

BackgroundFritillariae Cirrhosae Bulbus is an antitussive and expectorant Chinese medicinal material derived from the dried bulbs of six Fritillaria species. In the 2015 edition of the Chinese Pharmacopoeia, the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) is the officially listed method for their authenfication. Specifically, the ~ 300-bp ITS1 amplicon of only Fritillariae Cirrhosae Bulbus but not other Fritillaria species can be cleaved into two smaller fragments with restriction enzyme SmaI. Considering repeated reported cases of incomplete digestion of ITS1 amplicon, this study aims to investigate the possibility of heterogeneous ITS1 sequences contained in the Fritillariae Cirrhosae Bulbus.MethodsIn this study, ITS1 amplicons of Fritillaria Cirrhosae Bulbus and four other Fritillaria species were sequenced on Illumina platform. We utilised high-throughout amplicon sequencing to determine ITS1 haplotypes and their frequencies in Fritillaria genomes.ResultsOur results showed that all six botanical sources of Fritillariae Cirrhosae Bulbus indeed possess ITS1 haplotypes with no SmaI restriction site, and the average percentages of ITS1 reads containing SmaI restriction site ranged from 63.60% to 91.81%.ConclusionOur findings suggest that the incomplete digestion in PCR–RFLP analysis of Fritillariae Cirrhosae Bulbus is caused by the presence of ITS1 haplotypes without SmaI restriction site due to intragenomic heterogeneity.

Taxonomic relevance of stem and leaf anatomy in 10 endemic Fritillaria species from the Mediterranean Region

Fritillaria has important medicinal and horticultural values. This research aims to comprehensively assess the taxonomic relevance of micro-anatomical features within ten species belonging to Fritillaria subgenus Fritillaria, namely F. aurea, F. bithynica, F. crassifolia, F. enginiana, F. glaucoviridis, F. kittaniae, F. serpenticola, F. sibthorpiana, F. wendelboi, and F. whittallii, utilizing light microscopy. The majority of these species exhibit endemism within the Turkish Mediterranean Region, with F. aurea and F. crassifolia representing endemic Irano-Turanian elements, while the rest are native to the Eastern Mediterranean Region. In this anatomical investigation, we described and compare the cross-sections of the stem and leaves. Results revealed substantial variation in anatomical traits among the studied species. The presence or absence of eglandular trichomes, crystal granules in the leaf surface and cross section and in stem pith region, and the arrangement of parenchymal and sclerenchymal layers in the stem offered useful taxonomic markers. Similarly, leaf characteristics such as epidermal cell sizes, stomatal indices, mesophyll types, and guard cell shapes provided distinguishing features. A dichotomous key was developed based on these anatomical traits to aid in the accurate identification of Fritillaria species, highlighting the practical utility of these features in taxonomic studies. The findings underscore the importance of micro-anatomical traits as valuable tools for distinguishing and classifying plant species, especially in cases where morphological traits alone may not provide sufficient differentiation.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics

BackgroundFritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. MethodsThis paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. ResultsFive species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. ConclusionThe investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

The Identification of Fritillaria Species Using Hyperspectral Imaging with Enhanced One-Dimensional Convolutional Neural Networks via Attention Mechanism.

Combining deep learning and hyperspectral imaging (HSI) has proven to be an effective approach in the quality control of medicinal and edible plants. Nonetheless, hyperspectral data contains redundant information and highly correlated characteristic bands, which can adversely impact sample identification. To address this issue, we proposed an enhanced one-dimensional convolutional neural network (1DCNN) with an attention mechanism. Given an intermediate feature map, two attention modules are constructed along two separate dimensions, channel and spectral, and then combined to enhance relevant features and to suppress irrelevant ones. Validated by Fritillaria datasets, the results demonstrate that an attention-enhanced 1DCNN model outperforms several machine learning algorithms and shows consistent improvements over a vanilla 1DCNN. Notably under VNIR and SWIR lenses, the model obtained 98.97% and 99.35% for binary classification between Fritillariae Cirrhosae Bulbus (FCB) and other non-FCB species, respectively. Additionally, it still achieved an extraordinary accuracy of 97.64% and 98.39% for eight-category classification among Fritillaria species. This study demonstrated the application of HSI with artificial intelligence can serve as a reliable, efficient, and non-destructive quality control method for authenticating Fritillaria species. Moreover, our findings also illustrated the great potential of the attention mechanism in enhancing the performance of the vanilla 1DCNN method, providing reference for other HSI-related quality controls of plants with medicinal and edible uses.

Biomod2 modeling for predicting the potential ecological distribution of three Fritillaria species under climate change.

The Fritillaria species ranked as a well-known traditional medicine in China and has become rare due to excessive harvesting. To find reasonable strategy for conservation and cultivation, identification of new ecological distribution of Fritillaria species together with prediction of those responses to climate change are necessary. In terms of current occurrence records and bioclimatic variables, the suitable habitats for Fritillaria delavayi, Fritillaria taipaiensis, and Fritillaria wabuensis were predicted. In comparison with Maxent and GARP, Biomod2 obtained the best AUC, KAPPA and TSS values of larger than 0.926 and was chosen to construct model. Temperature seasonality was indicated to put the greatest influence on Fritillaria taipaiensis and Fritillaria wabuensis, while isothermality was of most importance for Fritillaria delavayi. The current suitable areas for three Fritillaria species were distributed in south-west China, accounting for approximately 17.72%, 23.06% and 20.60% of China's total area, respectively. During 2021-2100 period, the suitable habitats of F. delavayi and F. wabuensis reached the maximum under SSP585 scenario, while that of F. taipaiensis reached the maximum under SSP126 scenario. The high niche overlap among three Fritillaria species showed correlation with the chemical composition (P ≤ 0.05), while no correlation was observed between niche overlap and DNA barcodes, indicating that spatial distribution had a major influence on chemical composition in the Fritillaria species. Finally, the acquisition of species-specific habitats would contribute to decrease in habitat competition, and future conservation and cultivation of Fritillaria species.

Investigation of biochemical profiles derived from different Fritillaria species in Kurdistan Region using GC-MS

Fritillaria species belongs to Liliaceae family, it has been used as medicinal plants in traditional Chinese medicine for more than 2000 years. These plants are known with significant variations in their chemical profiles. Consequently, the characterization of the profile of the major bioactive constituents in various regions are important for pharmaceutical purposes. Despite the identification of numerous species of Fritillaria in Kurdistan, there is no previous study examining phytochemical components of Fritillaria in this region. This study aims to investigate the distribution of major bioactive compounds in wild bulbs of Fritillaria spp. in Kurdistan. 40 compounds were totally detected using Gas Chromatography-Mass Spectrometry (GC-MS). Among the detected compounds, 15 of them were previously found to have effective biological activities. Results have also shown that amongst the underlying Fritillaria species, variations of the types and quantities of the 15 bioactive compounds were significant. This result is of importance for the classification of different Fritillaria. spp. with distinct geographic distributions and medicinal applications.

Fingerprint profiling and gut microbiota regulation of polysaccharides from Fritillaria species

Few studies reported the quality evaluation and gut microbiota regulation effect of polysaccharides from Fritillaria species. In this study, polysaccharides extracted from ten Fritillaria species were compared and distinguished through multi-levels evaluation strategy and data fusion. Furthermore, the gut microbiota regulation effect of polysaccharides among different species was analyzed and evaluated. The fingerprint profiling of IR, molecular weight distribution of polysaccharides, chromatogram of partially hydrolyzed polysaccharides (oligosaccharides) and completely hydrolyzed polysaccharides (monosaccharides) were similar, and no exclusive signals were observed. However, the signal strength of functional group, oligosaccharides abundance and monosaccharides proportion showed obvious differences in inter- and intra-species. Glucan may be the main component of polysaccharides in Fritillaria species, CIRR derived from CIR, PRZ, DEL, TAI, UNI possessed higher total polysaccharides content, polymerization degree, oligosaccharides abundance (DP 2–4), and glucose content than the others. Meanwhile, data fusion model was established for identification of affinis and multi-original species, the accuracy of which proved to be 100 %. In addition, Fritillaria polysaccharides could increase the bacterial community richness and diversity, regulate the gut microbiota composition and possessed potential therapeutic effects on gastrointestinal diseases and nervous system diseases.

Nucleolar number variation in Fritillaria (liliaceae) taxa from Greece

The current study aims to estimate the most frequent number of nucleoli in taxonomically interesting Fritillaria species. Silver nitrate staining is applied using a modified protocol in 14 taxa and one hybrid from 17 populations from Greece. This is the first report of the number of nucleoli for all the Fritillaria taxa studied here. In general, the number of nucleoli ranges from 0-8, with Fritillaria pontica and F. theophrasti characterised by the greatest number of observed nucleoli. The results reinforce the classification of taxonomically intriguing taxa, such as Fritillaria sporadum and F. theophrasti, as distinct species, even though they have recently been considered synonyms of F. ehrhartii and F. pontica, respectively.

Taxonomical relations of tribe Lilieae (Liliaceae) based on palynological issues

Pollen micromorphology and ultrastructure of fifteen species from the genera Cardiocrinum, Fritillaria, Lilium, and Notholirion were examined by Light Microscopy (LM), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). Our results showed that all quantitative characters of pollen micromorphology (LM and SEM observation) varied in the studied taxa. Pollen grains in all studied species were oblate except Notholirion bulbuliferum with oblate-spheroidal. The studied species of Fritillaria had medium pollen type and the rest showed large. Although, two major exine ornamentation in central region of equatorial surface and sulcus margin region were seen such as reticulate (taxa of Cardiocrinum) and reticulum-cristatum (taxa of genera of Lilium, and Notholirion). These characters were varied in taxa of Fritillaria in both regions (foveolate-rugulate, reticulate and foveolate). Also, there were three sulcus membrane ornamentation including striate-tuberculate (Fritillaria gibbosa), tuberculate (Lilium lophophorum and with the rest studied taxa of Fritillaria), and macrotuberculate (the genera Cardiocrinum, Lilium, and Notholirion). Moreover, three kinds of lumina types and two kinds of muri types were detected among these taxa. The qualitative and quantitative characters of the pollen wall were varied such as kind of endexine situation, number of intine layers in the sulcus membrane region, and thickness of elements from the pollen wall. Dendrogram of all qualitative and quantitative palynological characters constructed two pollen groups, five pollen types, and four pollen subtypes. The palynological dendrogram verified that Nomocharis is part of genus Lilium. Palynological types supported the heterogeneous phylogenetic situation of Cardiocrinum. Palynological subtypes were partly valuable in delimitation of sectional levels of the genus Lilium.

Reproductive Biology of Fritillaria aurea Schott (Liliaceae), a Rare Species Endemic to Turkey

Fritillaria aurea, unlike other Fritillaria species, is a narrowly endemic species. It has a brown checkered speckled color on each tepal and also has regular yellow intense colors on the tepals. Its large bell-shaped appearance when the flower is open is one of its most striking ornamental features. However, F. aurea plant numbers are limited in their natural habitats, and their numbers are decreasing day by day; they are even confronted with the risk of extinction owing to the damage resulting from human activities. This detailed investigation of reproductive biology plays a vital role in determining the evolutionary success and survival of F. aurea, largely in determining the effectiveness of their reproductive performance. Our results indicate that the species exhibits a very low partial dichogamy and herkogamy structure, as well as self-incompatible pollination. The flowering period ranges from March to April. Average seed viability, average seed number, and seed germination were determined as 71%, 255, and 45%, respectively. The pollen viability, pollen grains, and stigma receptivity were found as 96%, 392.000, and 85%, respectively. The pollen/ovule ratio (P/O) and self-incompatibility index (SII) in the spontaneous cross-pollination were detected as 1537 and 0, respectively. Flowers of F. aurea were visited by several insect species, mostly honeybees (Apis mellifera); Bombus sp. and Vespa sp. also played a minor role in pollination. To sum up, these results not only lay a solid foundation for further reproductive biology investigations to more broadly reveal the mechanisms of F. aurea endangerment in the future but also provide a reliable theoretical basis for hybridization breeding of parents.

The complete chloroplast genome sequences of three lilies: genome structure, comparative genomic and phylogenetic analyses.

We sequenced and analyzed the complete chloroplast genomes of Lilium amoenum, Lilium souliei, and Nomocharis forrestii in detail, including the first sequence and structural comparison of Nomocharis forrestii. We found that the lengths and nucleotide composition of the three chloroplast genes showed little variation. The chloroplast genomes of the three Lilium species contain 87 protein coding genes (PCGs), 38 tRNAs, and 8 rRNA genes. The only difference is that Nomocharis forrestii had an additional infA pseudogene. In the sequence analysis of the Lilium chloroplast genomes, 216 SSRs, 143 pairs of long repeats, 571 SNPs, and 202 indels were detected. In addition, we identified seven hypervariable regions that can be used as potential molecular markers and DNA barcodes of Lilium through complete sequence alignment. The phylogenetic tree was constructed from the three chloroplast genome sequences of Lilium obtained here and 40 chloroplast genome sequences from the NCBI database (including 35 Lilium species, 4 Fritillaria species, and one species of Smilax). The analysis showed that the species clustering of the genus Lilium essentially conformed to the classical morphological classification system of Comber, but differences in the classification of individual species remained. In our report, we support the reclassification of Lilium henryi and Lilium rosthorniiy in the genus Lilium. In general, this study not only provides genome data for three Lilium species, but also provides a comparative analysis of the Lilium chloroplast genomes. These advances will help to identify Lilium species, clarify the phylogenetic analysis of the Lilium genus, and help to solve and improve the disputes and deficiencies in the traditional morphological classification.

A Review on the Composition and Biosynthesis of Alkaloids and on the Taxonomy, Domestication, and Cultivation of Medicinal Fritillaria Species

Fritillaria is a perennial herb with medicinal properties. There are 158 Fritillaria species worldwide, 33 of which have reported therapeutic efficacy. Alkaloids are the principal constituents in Fritillaria. Fritillaria species growing at 2700–4000 m are the sources of extract namely Chuan Beimu (the Pharmacopoeia of the People’s Republic of China, 2020 Edition), with low biomass, mainly containing more 5α-cevanine isosteroidal alkaloids with cis-configuration. In contrast, species growing below 1500 m are usually taller than 50 cm, and they mainly contain more trans-configuration isosteroidal alkaloids. There are two schemes of the biosynthetic pathways of steroidal alkaloids with different frameworks and catalytic reactions and combined high-throughput omics data. Based on the distributed elevations, Fritillaria species were divided into three major categories, which met classification features based on phylogenetic analysis or morphological features. Artificial or in vitro cultivations are effective strategies for balancing economical requirements and ecological protection. Fritillaria species growing at lower altitudes can be cultivated by bulb reproduction, but species growing at higher altitudes still rely mainly on gathering a large number of wild resources. Integration of asexual tissue culture and bulb reproduction with sexual artificial or imitated wild cultivation may create a very promising and effective way to maintain sustainable industrial development of Fritillaria.

Integrative analysis of the steroidal alkaloids distribution and biosynthesis of bulbs Fritillariae Cirrhosae through metabolome and transcriptome analyses

BackgroundBulbus Fritillariae Cirrhosae (BFC) is an endangered high-altitude medicine and food homology plant with anti-tumor, anti-asthmatic, and antitussive activities as it contains a variety of active ingredients, especially steroidal alkaloids. Bulbus Fritillariae Thunbergia (BFT) is another species of Fritillaria that grows at lower altitude areas. Production of plant-derived active ingredients through a synthetic biology strategy is one of the current hot topics in biological research, which requires a complete understanding of the related molecular pathways. Our knowledge of the steroidal alkaloid biosynthesis in Fritillaria species is still very limited.ResultsTo promote our understanding of these pathways, we performed non-target metabolomics and transcriptome analysis of BFC and BFT. Metabolomics analysis identified 1288 metabolites in BFC and BFT in total. Steroidal alkaloids, including the proposed active ingredients of Fritillaria species peimine, peimisine, peiminine, etc., were the most abundant alkaloids detected. Our metabolomics data also showed that the contents of the majority of the steroidal alkaloids in BFC were higher than in BFT. Further, our comparative transcriptome analyses between BFC and BFT identified differentially expressed gene sets among these species, which are potentially involved in the alkaloids biosynthesis of BFC.ConclusionThese findings promote our understanding of the mechanism of steroidal alkaloids biosynthesis in Fritillaria species.

Transcriptome and metabolite profiling analyses reveal the molecular mechanism underlying the characteristic accumulation of anthocyanins and flavonols in Fritillaria unibracteata and F. delavayi.

As an important medicinal herb with high profit, Sichuan Fritillaria is on the verge of extinction because of unreasonable exploitation, but the information on flavonoids which possess diverse medicinal activities was almost unknown. Here, two representative Sichuan Fritillaria species (F. unibracteata and F. delavayi) were studied with UHPLC-Q-Orbitrap HRMS (ultra-high performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry). Anthocyanins mainly accumulate in flowers especially in F. unibracteata (8.22 mg/g; mainly consisting of cyanidin 3-O-rutinoside), with trace amounts in leaves and stems, but to the lowest levels in bulbs. Flavonols accumulates considerably in all tissues of the two Fritillaria species except bulbs, to the highest level in the flower of F. delavayi (28.94 mg/g; mainly consisting of quercetin glycosides). Further transcriptomic and RT-qPCR (real-time quantitative Polymerase Chain Reaction) analysis revealed the anthocyanin-specific biosynthetic unigenes displayed the highest expression levels in the flower of F. unibracteata while most of flavonol-specific biosynthetic unigenes showed the highest transcript abundance in the flower of F. delavayi, but to the lowest expression levels in the bulbs of the two species, accounting for the tissue- and species-specific accumulation of anthocyanins and flavonols. Moreover, the functions of FcMYB90, FcTT8 and FcMYB4 was carefully studied in tobacco. These results prove that several MYB and bHLH transcription factors coordinately controlled the direction of the phenylpropanoid metabolic flux which finally resulted in the characteristic accumulation pattern of anthocyanins and flavonols in Sichuan Fritillaria. This work provides a good foundation and novel insights for the further industrial development, resource utilization and breeding of Sichuan Fritillaria.

DNA Barcoding and Phylogenomic Analysis of the Genus Fritillaria in China Based on Complete Chloroplast Genomes.

The Fritillaria is an extremely complicated genus in taxonomy and phylogeny, which contains numerous medicinal species in China. Both traditional characteristic-based taxonomy and universal DNA barcodes (ITS, trnH-psbA, and rbcL) are difficult to effectively identify the species. Here, we generated a large dataset of chloroplast genomes from multiple accessions per species of Fritillaria to evaluate their effectiveness in species discrimination. Moreover, phylogeny of species in China was explored based on the complete chloroplast genomes, and then divergence times of each node were estimated. The results showed that all 21 species in Fritillaria here (including two suspicious species) could be correctly discriminated using cpDNA genomes except F. cirrhosa, which suggested that DNA super-barcode could greatly enhance species discriminatory resolution for complicated genera. Furthermore, four regions (ycf1, matK-trnG-GCC, rpoC1, and matK) gained remarkably higher resolution than that of other plastid regions, but only matK might be suitable to identify Fritillaria species in consideration of its lengths. Phylogenomic analysis showed that the subgenus Fritillaria in China was divided into four major clades with obvious geographic structure. Among them, Clade I, mainly distributed in southwest China, was a young and complicated group. Moreover, according to the analysis, taxonomic treatments of the two suspicious species, namely “F. omeiensis” and “F. hupehensis” in Flora of China (2000) are questionable and might need further revision. Molecular dating revealed that both origin and divergence of subgenus Fritillaria, as well as its four major clades, were significantly associated with geological and climatic fluctuations during the Middle to Late Miocene. This study would enrich case studies of DNA super-barcode and provide new insights on speciation, lineage diversification, and biogeography of the Fritillaria in China.

Modeling of the Potential Geographical Distribution of Three Fritillaria Species Under Climate Change.

Fritillaria species, a well-known Chinese traditional medicine for more than 2,000 years, have become rare resources due to excessive harvesting. In order to balance the economical requirement and ecological protection of Fritillaria species, it is necessary to determine (1) the important environmental variables that were responsible for the spatial distribution, (2) distribution change in response to climate change in the future, (3) ecological niche overlap between various Fritillaria species, and (4) the correlation between spatial distribution and phylogenies as well. In this study, the areas with potential ecological suitability for Fritillaria cirrhosa, Fritillaria unibracteata, and Fritillaria przewalskii were predicted using MaxEnt based on the current occurrence records and bioclimatic variables. The result indicated that precipitation and elevation were the most important environmental variables for the three species. Moreover, the current suitable habitats of F. cirrhosa, F. unibracteata, and F. przewalskii encompassed 681,951, 481,607, and 349,199 km2, respectively. Under the scenario of the highest concentration of greenhouse gas emission (SSP585), the whole suitable habitats of F. cirrhosa and F. przewalskii reach the maximum from 2021 to 2100, while those of F. unibracteata reach the maximum from 2021 to 2100 under the scenario of moderate emission (SSP370) from 2021 to 2100. The MaxEnt data were also used to predict the ecological niche overlap, and thus high overlap occurring among three Fritillaria species was observed. The niche overlap of three Fritillaria species was related to the phylogenetic analysis despite the non-significance (P > 0.05), indicating that spatial distribution was one of the factors that contributed to the speciation diversification. Additionally, we predicted species-specific habitats to decrease habitat competition. Overall, the information obtained in this study provided new insight into the potential distribution and ecological niche of three species for the conservation and management in the future.

Seasonal variations in the plant diet of the Chinese Monal revealed by fecal DNA metabarcoding analysis

The Chinese Monal (Lophophorus lhuysii) is an alpine-obligate galliform species of global conservation priority. It has been listed as a first class protected wildlife species in China, requiring conservation actions during the 14th Five-Year Plan period. However, the diet composition of Chinese Monal and its seasonal variations have rarely been studied, constraining the effective conservation of the species. Here, we investigated the plant diet composition of the Chinese Monal and its seasonal variations using a DNA metabarcoding approach on fecal samples. We collected 190 fecal samples of the Chinese Monals from the central Qionglai Mountains located in China, and analyzed the plant diet of this species using a DNA metabarcoding approach. Taxonomic profiling of higher plants in the fecal samples was performed using the second internal transcribed spacer (ITS2) amplicon. Downstream analyses, including rarefaction curves, nonmetric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PERMANOVA), were used to explore the seasonal variations in diet composition. The Chinese Monal foraged a wide range of plant recipes composed of 35 families and 83 genera throughout the year, with Brassicaceae, Apiaceae, and Poaceae as the dominant families, and Cardamine as the dominant genus. The species consumed plants from 62 genera from 28 families during the breeding season (n ​= ​81) and 66 genera from 31 families during the non-breeding season (n ​= ​109). Further, the plant diet composition significantly varied between the breeding and non-breeding seasons, especially for the frequency of occurrence and relative read abundances at genus level. Our study analyzed the plant diet of the Chinese Monal at a high resolution for the first time, and the results revealed that the seasonal variations in its plant diet composition was adapted to plant phenology and foraging strategy. Fritillaria species, a previously confirmed important food resource for the Chinese Monal, were not detected in any fecal samples, potentially due to overharvesting of Fritillaria bulbs for Traditional Chinese Medicine. Therefore, we highly recommend further restriction of herb gathering in Chinese Monal habitats to facilitate the conservation of this endangered species. Altogether, our study enriches essential ecological information for the Chinese Monal and also provides insights into conservation management for this endangered species.