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Having a comprehensive understanding of genetic differentiation, responses to environmental change and demographic history is critical for genetic improvement and conservation efforts. Forest trees are an excellent resource for understanding population differentiation and adaptive genetic variation due to their ability to adapt to different climates and environments. Cephalotaxus oliveri is a relict conifer endemic to China. In this study, we generated transcriptome data and identified 17 728 high‐quality single‐nucleotide polymorphisms (SNPs) from 18 populations. We found significant negative correlations between expression diversity and nucleotide diversity within and among populations, suggesting that gene expression and nucleotide diversity have a reciprocal relationship when the species adapts to the environment. The analyses of population structure showed that C. oliveri displayed a striking genetic structure with four groups. BayeScEnv and RDA methods detected the signatures of local adaptation, and identified that 738 outlier SNPs were associated with precipitation, temperature and soil conditions across heterogeneous environmental conditions. Approximate Bayesian computation analyses showed that the first and second divergence occurred in the late Miocene (c. 10.075 million years ago [Ma]) and the middle Pleistocene transition (c. 0.815 Ma), respectively. Ecological niche modeling of C. oliveri revealed signs of westward expansion after the last glacial maximum, while it was predicted to experience significant range contractions in future climate change scenarios. Geographical factors and environmental factors in southern China have played a critical role in establishing the current genetic diversity and population structure of C. oliveri. This study provides an important reference for forest resource management and conservation for C. oliveri. [ABSTRACT FROM AUTHOR]

Abstract Five undescribed alkaloids were isolated from the seeds of Cephalotaxus oliveri along with 27 known ones. The new structures were elucidated based on spectroscopic data including 1D and 2D NMR, MS and calculated ECD spectra. Among them, (+)-acetylcephalofortine C was an enantiomeric Cephalotaxine alkaloids. The performed bioassay revealed that those alkaloids were not cytotoxic against cancer cells and had no neuroprotective properties in the HEI-OC-1 cells model.

Evaluating the contributions of geographic distance and environmental heterogeneity to the genetic divergence can inform the demographic history and responses to environmental change of natural populations. The isolation‐by‐distance (IBD) reveals that genetic differentiation among populations increases with geographic distance, while the isolation‐by‐environment (IBE) assumes a linear relationship between genetic variation and environmental differences among populations. Here, we sampled and genotyped 330 individuals from 18 natural populations of Cephalotaxus oliveri throughout the species' distribution. Twenty‐eight EST‐SSR markers were applied to analyze population genetics, for the investigation of the driving factors that shaped spatial structure. In addition, we identified the outlier loci under positive selection and tested their association with environmental factors. The results showed a moderate genetic diversity in C. oliveri and high genetic differentiation among populations. Population structure analyses indicated that 18 populations were clustered into two major groups. We observed that the genetic diversity of central populations decreased and the genetic differentiation increased towards the marginal populations. Additionally, the signatures of IBD and IBE were detected in C. oliveri, and IBE provided a better contribution to genetic differentiation. Six outlier loci under positive selection were demonstrated to be closely correlated with environmental variables, among which bio8 was associated with the greatest number of loci. Genetic evidence suggests the consistency of the central‐marginal hypothesis (CMH) for C. oliveri. Furthermore, our results suggest that temperature‐related variables played an important role in shaping genetic differentiation. [ABSTRACT FROM AUTHOR]

Five undescribed alkaloids were isolated from the seeds of Cephalotaxus oliveri along with 27 known ones. The new structures were elucidated based on spectroscopic data including 1D and 2D NMR, MS and calculated ECD spectra. Among them, (+)-acetylcephalofortine C was an enantiomeric Cephalotaxine alkaloids. The performed bioassay revealed that those alkaloids were not cytotoxic against cancer cells and had no neuroprotective properties in the HEI-OC-1 cells model. [ABSTRACT FROM AUTHOR]

Three new biflavonoids, umcephabiovins C − E (1 − 3), along with fourteen known compounds were isolated from the twigs and leaves of Cephalotaxus oliveri. Their structures and configurations were elucidated by UV, IR, NMR, ECD, and HR-ESI-MS spectra. Compounds 1 − 3 exhibited significant α-glucosidase inhibitory activity with IC50 values of 7.05 ± 2.66, 24.45 ± 4.73, and 1.84 ± 1.14 μM, respectively. Compound 11 showed moderate cytotoxicity against the BaF3/T315I cell line. [ABSTRACT FROM AUTHOR]

Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3′UTR, and 5′UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune, Ametotaxus argotaenia, and Pseudotaxus chienii, respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species.

Cephalotaxus oliveri is an endemic conifer of China, which has medicinal and ornamental value. However, the limited molecular markers and genetic information are insufficient for further genetic studies of this species. In this study, we characterized and developed the EST-SSRs from transcriptome sequences for the first time. The results showed that a total of 5089 SSRs were identified from 36446 unigenes with a density of one SSR per 11.1 kb. The most common type was trinucleotide repeats, excluding mononucleotide repeats, followed by dinucleotide repeats. AAG/CTT and AT/AT exhibited the highest frequency in the trinucleotide and dinucleotide repeats, respectively. Of the identified SSRs, 671, 1125, and 1958 SSRs were located in CDS, 3′UTR, and 5′UTR, respectively. Functional annotation showed that the SSR-containing unigenes were involved in growth and development with various biological functions. Among successfully designed primer pairs, 238 primer pairs were randomly selected for amplification and validation of EST-SSR markers and 47 primer pairs were identified as polymorphic. Finally, 28 high-polymorphic primers were used for genetic analysis and revealed a moderate level of genetic diversity. Seven natural C. oliveri sampling sites were divided into two genetic groups. Furthermore, the 28 EST-SSRs had 96.43, 71.43, and 78.57% of transferability rate in Cephalotaxus fortune , Ametotaxus argotaenia, and Pseudotaxus chienii , respectively. These markers developed in this study lay the foundation for further genetic and adaptive evolution studies in C. oliveri and related species. [ABSTRACT FROM AUTHOR]

Three new biflavonoids, named oliveriflavones A-C (1–3), together with two known flavonoids (quercetin (4) and rutin (5)), were isolated from the endangered plant Cephalotaxus oliveri. The chemical structures of these compounds were elucidated by comprehensive spectroscopic methods including NMR, HRESIMS, IR, UV, and CD spectra. Compounds 1–5 were first isolated from the genus Cephalotaxus. All the compounds were tested for their antioxidant activity. Compounds 4 and 5 showed excellent activity with IC50 values of 0.03 ± 0.06 μM and 0.02 ± 0.10 μM, respectively. [ABSTRACT FROM AUTHOR]

Three new biflavonoids, umcephabiflovin A ( 1 ), umcephabiflovin B ( 2 ), and S -taiwanhomoflavone-B ( 3 ), together with seven known flavonoids ( 4–10 ) and seven known alkaloids ( 11 – 17 ), were isolated from the twigs of Cephalotaxus oliveri Mast. The flavonoids were found to inhibit α-glucosidase activity. [ABSTRACT FROM AUTHOR]

Two new Cephalotaxus alkaloids, (±)-cephaloliverine A ( 1 ) and cephalolancine A ( 2 ) were isolated from Cephalotaxus oliveri and Cephalotaxus lanceolata respectively. Their structures were determined on the basis of spectroscopic techniques including NMR, IR, UV, mass spectrometry, and X-ray diffraction. Cephalolancine A ( 2 ) features a new skeleton-type. The discovery of these alkaloids provides biogenetic evidences of the formation of Cephalotaxus alkaloids. [ABSTRACT FROM AUTHOR]

Habitat heterogeneity, physical barriers, and the uplift of the Yungui Plateau were found to deeply affect the phylogeographic pattern and evolutionary history of Cephalotaxus oliveri , a perennial conifer endemic to China. In this study, we explored the phylogeography using three chloroplast sequences ( trn L– trn F, trn T– trn D and atp B– rbc L) in 22 natural populations of C. oliveri distributed throughout its range. The Yungui Plateau populations of C. oliveri were revealed to origin ca. 9.15 Ma by molecular clock estimation, which is consistent with rapid uplift of the Qinghai–Tibetan Plateau (QTP) ca. 8–10 Ma. Additionally, geological effects of the Yungui Plateau were suggested to promote the rapid intra-specific differentiation of C. oliveri in the Pliocene and Early Pleistocene. The relatively low level of genetic diversity ( h = 0.719, θ = 1.17 × 10 −3 ) and high population differentiation ( N ST = 0.771 and G ST = 0.642) implied restricted gene flow among populations, which was confirmed by the Nested Clade Analysis (NCA). Mismatch distribution and haplotypes network provided evidences of recent demographic population expansion. Furthermore, the statistical dispersal–vicariance analysis indicated that the center of origin was in Central China. The comparison of haplotype distribution patterns indicated that the regions of HNHPS and HBLD were the potential refugia during the Pleistocene ice ages. Our results highlighted that habitat heterogeneity and physical barriers presenting in a species range can predict genetic patterns. [ABSTRACT FROM AUTHOR]

Six undescribed isoquinoline alkaloids, named as cephaloliverines A−F, were isolated from the seeds of Cephalotaxus oliveri. They were identified by NMR and MS spectroscopic data analyses, combined with the time-dependent density functional theory ECD calculation for cephaloliverines A and B and also by X-ray crystal diffraction for cephaloliverine E. Biosynthetic considerations suggest that cephaloliverines A−D are homologous of cephalotaxine-, homoerythrina- and Erythrina -type alkaloids. The performed bioassay revealed no cytotoxic activity against cancer cells and no neuroprotective properties on HEI–OC–1 cells model. Six undescribed isoquinoline alkaloids, named as cephaloliverines A−F, were isolated from the seeds of Cephalotaxus oliveri. Biosynthesis analysis disclosed cephaloliverines A−D were homologous of cephalotaxine-, homoerythrina- and erythrina-type alkaloids. Cephaloliverines E−F were contracted ring derivatives of cephalotaxine. [Display omitted] • Six undescribed isoquinoline alkaloids, named as cephaloliverines A−F, were isolated from Cephalotaxus oliveri. • Cephaloliverines A−D were parallel alkaloids to cephalotaxine-, homoerythrina- and erythrina-type. • Cephaloliverines E−F were contracted ring derivatives of cephalotaxine. [ABSTRACT FROM AUTHOR]

We have determined the complete chloroplast (cp) genome sequence of Cephalotaxus oliveri. The genome is 134,337 bp in length, encodes 113 genes, and lacks inverted repeat (IR) regions. Genome-wide mutational dynamics have been investigated through comparative analysis of the cp genomes of C. oliveri and C. wilsoniana. Gene order transformation analyses indicate that when distinct isomers are considered as alternative structures for the ancestral cp genome of cupressophyte and Pinaceae lineages, it is not possible to distinguish between hypotheses favoring retention of the same IR region in cupressophyte and Pinaceae cp genomes from a hypothesis proposing independent loss of IRA and IRB. Furthermore, in cupressophyte cp genomes, the highly reduced IRs are replaced by short repeats that have the potential to mediate homologous recombination, analogous to the situation in Pinaceae. The importance of repeats in the mutational dynamics of cupressophyte cp genomes is also illustrated by the accD reading frame, which has undergone extreme length expansion in cupressophytes. This has been caused by a large insertion comprising multiple repeat sequences. Overall, we find that the distribution of repeats, indels, and substitutions is significantly correlated in Cephalotaxus cp genomes, consistent with a hypothesis that repeats play a role in inducing substitutions and indels in conifer cp genomes. [ABSTRACT FROM AUTHOR]

Cephalotaxus oliveri is a scarce medicinal conifer endemic to the south central region of China and Vietnam. A small fragmented population presently exists due to anthropogenic disturbance. C. oliveri has been used for its alkaloids harringtonine and homoharringtonine, which are effective against leucocythemia and lymphadenosarcoma. Monoecious plants have been detected in nature, although they were understood to be dioecious. In order to study the mating system, population genetics and the genetic effects of habitat fragmentation on C. oliveri, 15 polymorphic and 12 monomorphic microsatellite loci were developed for C. oliveri by using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol. The polymorphisms were assessed in 96 individuals from three natural populations (32 individuals per population). The number of alleles per locus ranged from two to 33, the observed and expected heterozygosity per locus ranged from 0.000 to 1.000 and from 0.000 to 0.923, respectively. These loci would facilitate a comprehensive understanding of the genetic dynamics on C. oliveri, which will be useful for establishing effective conservation strategies for this species. [ABSTRACT FROM AUTHOR]

* Premise of the study: Microsatellite loci were developed for Cephalotaxus oliveri, an endemic and endangered conifer in China, which will allow assessment of the levels of genetic diversity and a means to understand the genetic consequences of habitat fragmentation. * Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining (FIASCO) Repeats protocol, 19 microsatellite loci were identified in C. oliveri, 13 of which were polymorphic within a sample of 52 individuals representing five natural populations. The actual number of alleles per locus ranged from one to five. Twelve polymorphic loci were also successfully amplified in C. fortunei. * Conclusions: These microsatellite loci will provide a useful tool for further investigating genetic variation in natural populations of C. oliveri, which will inform future conservation and management strategies. Additionally, cross-amplification in C. fortunei suggested the potential utility of these loci in this and other congeneric species. [ABSTRACT FROM AUTHOR]

Cephalotaxus oliveri is a tertiary relict conifer endemic to China, regarded as a national second-level protected plant in China. This species has experienced severe changes in temperature and precipitation in the past millions of years, adapting well to harsh environments. In view of global climate change and its endangered conditions, it is crucial to study how it responds to changes in temperature and precipitation for its conservation work. In this study, single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing were combined to generate the complete transcriptome of C. oliveri. Using the RNA-seq data to correct the SMRT sequencing data, the four tissues obtained 63,831 (root), 58,108 (stem), 33,013 (leaf) and 62,436 (male cone) full-length unigenes, with a N50 length of 2523, 3480, 3181, and 3267 bp, respectively. Additionally, 35,887, 11,306, 36,422, and 25,439 SSRs were detected for the male cone, leaf, root, and stem, respectively. The number of long non-coding RNAs predicted from the root was the largest (11,113), and the other tissues were 3408 (stem), 3193 (leaf), and 3107 (male cone), respectively. Functional annotation and enrichment analysis of tissue-specific expressed genes revealed the special roles in response to environmental stress and adaptability in the different four tissues. We also characterized the gene families and pathways related to abiotic factors. This work provides a comprehensive transcriptome resource for C. oliveri, and this resource will facilitate further studies on the functional genomics and adaptive evolution of C. oliveri. [ABSTRACT FROM AUTHOR]

• A comprehensive phytochemical investigation on the endangered Cephalotaxus oliveri. • Six cephalotaxine-type alkaloids and three C 31 -biflavonoids were newly obtained. • Alkaloids 1 – 4 are the first natural cephalotaxine esters in the N -oxide form. • Some isolates showed cytotoxicities with IC 50 values ranging from 3 nM to 0.1 μM. • 21 polyketides were isolated from an endophyte associated with the title plant. Rare and endangered plants (REPs) and their associated endophytes survived in unique habitats are promising sources for natural product-derived drug discovery. In this study, six new (cephaloverines A–F, 1 – 6 , resp.) and 16 known (11 – 26) cephalotaxine-type alkaloids, together with three new (oliverbiflavones A–C, 7 – 9 , resp.) and 11 known (27 – 37) biflavonoids were isolated and characterized from the twigs and leaves of Cephalotaxus oliveri , an endangered plant endemic to China. Meanwhile, a preliminary investigation on the secondary metabolites from a selected fungal endophyte (i.e., Alternaria alternate Y-4–2) associated with the title plant led to the isolation of 21 structurally distinct polyketides including one new dimeric xanthone (10). The new structures (1 – 10) with the absolute configurations were determined by detailed spectroscopic analyses, electronic circular dichroism (ECD) or Na 2 MoO 4 -induced ECD, the modified Mosher's method, and some chemical transformations. Compounds 1 – 4 are the first representatives of naturally occurring N -oxides of cephalotaxine esters, while compounds 7 – 9 have a special structural feature of having a C -methylated biflavonoid skeleton. The Cephalotaxus alkaloids with ester side-chains at C-3 (1 – 6 , 13 – 22 , and 26) and four biflavonoids (27 – 29 and 34) were found to show pronounced cytotoxicities against a small panel of human cancer cell lines (A549, NCI-H460, HL60, NCI-H929, and RPMI-8226), with IC 50 values mainly ranging from 0.003 to 9.34 μM. The most potent compound, deoxyharringtonine (16), generally exhibited IC 50 values less than 10 nM. The structure–activity relationship (SAR) of the aforementioned Cephalotaxus alkaloids was briefly discussed. [ABSTRACT FROM AUTHOR]