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For most species, a precise understanding of how climatic parameters determine the timing of seasonal life cycle stages is constrained by limited long‐term data. Further, most long‐term studies of plant phenology that have examined relationships between phenological timing and climate have been local in scale or have focused on single climatic parameters. Herbarium specimens, however, can expand the temporal and spatial coverage of phenological datasets. Using Trillium ovatum specimens collected over > 100 yr across its native range, we analyzed how seasonal climatic conditions (mean minimum temperature (Tₘᵢₙ), mean maximum temperature and total precipitation (PPT)) affect flowering phenology. We then examined long‐term changes in climatic conditions and in the timing of flowering across T. ovatum's range. Warmer Tₘᵢₙ advanced flowering, whereas higher PPT delayed flowering. However, Tₘᵢₙ and PPT were shown to interact: the advancing effect of warmer Tₘᵢₙ was strongest where PPT was highest, and the delaying effect of higher PPT was strongest where Tₘᵢₙ was coldest. The direction of temporal change in climatic parameters and in the timing of flowering was dependent on geographic location. Tₘᵢₙ, for example, decreased across the observation period in coastal regions, but increased in inland areas. Our results highlight the complex effects of climate and geographic location on phenology.

Trillium govanianum is traditionally used to treat innumerable alignments like sexual disorders, cancer, inflammation etc. Mainly rhizomes of T. govanianum have been explored for phytochemical profiling but comprehensive metabolomics of other parts has not been yet deeply investigated. Thus, current study was aimed for organs-specific (roots, rhizomes, rhizomatous buds, stems, leaves, and fruits) phytochemical profiling of T. govanianum via metabolomics approach. Targeted (steroidal saponins and free sugars) and non-targeted metabolomics were performed by UPLC-PDA/ELSD & UHPLC-Q-TOF-IMS. Among steroidal compounds, 20-hydroxyecdysone, pennogenin-3-O- β -chacotrioside, dioscin were found predominantly in all samples while diosgenin was identified only in rhizomes. Further, four free sugars viz. 2-deoxyribose (116.24 ± 1.26 mg/g: leaves), fructose (454.76 ± 12.14 mg/g: rhizomes), glucose (243.21 ± 7.53 mg/g: fruits), and galactose (69.06 ± 2.14 mg/g: fruits) were found significant in respective parts of T. govanianum . Elemental analysis of targeted samples was determined by atomic absorption spectrophotometer. Heavy metals (Cd, Hg, Pd, As) were absent while micro- (Mn, Na, Zn, Cu) and macro- (Ca, Fe, Mg, K) elements were found in all samples. Furthermore, UHPLC-Q-TOF-IMS had identified 103 metabolites based on their mass fragmentation patterns and 839 were tentatively predicted using METLIN database. The multivariate statistical analysis showed organs specific clustering and variance of metabolites. Apart from this, extracts were evaluated for in vitro anticholinesterase activity, and found potentials inhibitors with IC 50 values 2.02 ± 0.15 to 27.65 ± 0.89 mg/mL and 3.58 ± 0.12 to 16.81 ± 2.48 mg/mL of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme, respectively. Thus, comprehensive metabolomics and anti-cholinesterase activity of different parts of T. govanianum would lay the foundation for improving medicinal importance and health benefits of T. govanianum.

Background and aimsBacterial ventriculitis is common in children with temporary external ventricular drains and diagnosis is challenging due to frequent reoperations, blood contamination of cerebrospinal fluid (CSF), presence of chemical ventriculitis and elevation of blood laboratory markers by concomitant bacterial infection.MethodsProspective, observational study enrolling children with external ventricular drainage at surgical ward and paediatric intensive care unit. CD64in in CSF together with CSF leukocyte count, glucose, proteins and blood leukocyte and differential count, CRP, PCT were studied at the time of suspected ventriculitis. CD64in was measured by flow cytometry (Trillium Diagnostics, LLC, Brewer, ME). Wilcoxon-test was used for comparison between groups and diagnostic accuracy determined by the area under the ROC curves (AUC) was defined for each marker.ResultsThirty-three episodes of clinically suspected ventriculitis in twenty-one children (male 14, female 7, median age: 9 months, range: 8 days-167 months) were observed during a 26-month period. Episodes were classified into those with microbiologically proven ventriculitis (13 episodes: 9 Gram-positive and 4 Gram-negative) and those with microbiologically negative CSF (20 episodes). CD64in was the only CSF marker that could differentiate between groups (p = 0.0003); its diagnostic accuracy was 0.875 (95% CI: 0.713-0.963). Among blood markers only CRP and band neutrophils differentiated between groups (p = 0.0032 and p = 0.0463) with their diagnostic accuracy of 0.808 (0.633-0.923) and 0.721 (0.524-0.870); respectively.ConclusionsCD64in in CSF is a promising diagnostic marker of bacterial ventriculitis in children with external ventricular drainage before microbiological confirmation.

For many organisms, stage is a better predictor of demographic rates than age. Yet no general theoretical framework exists for understanding or predicting evolution in stage-structured populations. Here, we provide a general modeling approach that can be used to predict evolution and demography of stage-structured populations. This advances our ability to understand evolution in stage-structured populations to a level previously available only for populations structured by age. We use this framework to provide the first rigorous proof that Lande’s theorem, which relates adaptive evolution to population growth, applies to stage-classified populations, assuming only normality and that evolution is slow relative to population dynamics. We extend this theorem to allow for different means or variances among stages. Our next major result is the formulation of Price’s theorem, a fundamental law of evolution, for stage-structured populations. In addition, we use data fromTrillium grandiflorumto demonstrate how our models can be applied to a real-world population and thereby show their practical potential to generate accurate projections of evolutionary and population dynamics. Finally, we use our framework to compare rates of evolution in age- versus stage-structured populations, which shows how our methods can yield biological insights about evolution in stage-structured populations.

A major goal in ecology is to understand mechanisms that increase invasion success of exotic species. A recent hypothesis implicates altered species interactions resulting from ungulate herbivore overabundance as a key cause of exotic plant domination. To test this hypothesis, we maintained an experimental demography deer exclusion study for 6 y in a forest where the native ungulate Odocoileus virginianus (white-tailed deer) is overabundant and Alliaria petiolata (garlic mustard) is aggressively invading. Because population growth is multiplicative across time, we introduce metrics that correctly integrate experimental effects across treatment years, the cumulative population growth rate, λc, and its geometric mean, λper-year, the time-averaged annual population growth rate. We determined λc and λper-year of the invader and of a common native, Trillium erectum. Our results conclusively demonstrate that deer are required for the success of Alliaria; its projected population trajectory shifted from explosive growth in the presence of deer (λper-year = 1.33) to decline toward extinction where deer are excluded (λper-year = 0.88). In contrast, Trillium's λper-year was suppressed in the presence of deer relative to deer exclusion (λper-year = 1.04 vs. 1.20, respectively). Retrospective sensitivity analyses revealed that the largest negative effect of deer exclusion on Alliaria came from rosette transitions, whereas the largest positive effect on Trillium came from reproductive transitions. Deer exclusion lowered Alliaria density while increasing Trillium density. Our results provide definitive experimental support that interactions with overabundant ungulates enhance demographic success of invaders and depress natives' success, with broad implications for biodiversity and ecosystem function worldwide.

BACKGROUND AND AIMS: The light availability on a temperate, deciduous-forest floor varies greatly, reflecting the seasonal leaf dynamics of the canopy trees. The growth and/or reproductive activity of understorey plants should be influenced by the length of the high-irradiance period from snowmelt to canopy closure. The aim of the present study was to clarify how spring-blooming species regulate the translocation of photosynthetic products to current reproduction and storage organs during a growing season in accordance with the changing light conditions. METHODS: Growth pattern, net photosynthetic rate, seed production, and shoot and flower production in the next year of Trillium apetalon were compared between natural and experimentally shaded conditions. Furthermore, translocation of current photosynthetic products within plants was assessed by a labelled carbon-chase experiment. KEY RESULTS: During the high-irradiance period, plants showed high photosynthetic ability, in which current products were initially used for shoot growth, then reserved in the rhizome. Carbon translocation to developing fruit occurred after canopy closure, but this was very small due to low photosynthetic rates under the darker conditions. The shading treatment in the early season advanced the time of carbon translocation to fruit, but reduced seed production in the current year and flower production of the next year. CONCLUSIONS: Carbon translocation to the storage organ had priority over seed production under high-irradiance conditions. A shortened bright period due to early canopy closure effectively restricts carbon assimilation, which greatly reduces subsequent reproductive output owing to low photosynthetic products for fruit development and small carbon storage for future reproduction. As populations of this species are maintained by seedling recruitment, acceleration of canopy closure timing may influence the maintenance and dynamics of populations.

1. Spring ephemeral herb species in temperate deciduous forests are active above-ground only briefly each year. This study tested experimentally how two countervailing constraints – cold and darkness – influence the phenology of six spring herb species. 2. Dormancy of underground structures, maintained by cold temperatures in a growth chamber, was broken at six 25-day intervals from January or February to June in two consecutive years. Upon emergence, survival and flowering were measured on cohorts grown outdoors. Shade cloth was added at the time of normal canopy closure. 3. Cardamine concatenata, Dicentra cucullaria, Erythronium albidum and Trillium recurvatum had no or low 2-year survival in the two or three earliest cohorts and no or low survival in the latest cohort, relative to their natural cohort. Allium canadense and Claytonia virginica had survival in all cohorts. Flowering never occurred in the first two or three cohorts for three species and never occurred or declined in later cohorts in all species. 4. Despite widely differing emergence dates, senescence was completed within a 40-day period soon after shade was imposed for all cohorts for all species. Consequently, leaf life span became shorter as date of emergence was delayed among cohorts. 5. In general, the brief growth period of spring herb species is an adaptation to avoid winter cold and late-spring canopy shade. These constraints are species-specific and differ for survival and flowering for some species. Claytonia virginica is the most tolerant among the species to a wider range of conditions. 6. Synthesis. Knowing that cold and shade constrain a plant's non-dormant period is important because of the significant role plant phenology plays in responses to climate change.

Trillium govanianum , an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum .

The present study is designed to provide a comprehensive insight into the ex situ conservation of the medicinally important endangered herb Trillium govanianum Wall. ex D. Don (Himalayan Trillium) through biotechnological tools. To prioritize ex-situ conservation, it is imperative to first understand the status of existing genetic diversity, followed by phytochemical screening to identify elite individuals or locations. Therefore, the genetic diversity of T. govanianum individuals from different locations was assessed using SSR markers. The individuals grouped into diversified clusters were further analyzed by HPLC to identify elite chemotype(s), followed by the standardization of an efficient in vitro propagation method for conservation purposes. The neighbor-joining tree and dendrogram revealed a low level of genetic diversity, thereby indicating more similarity among individuals from the same topographical environment. Based on HPLC analysis, it was revealed that at a higher elevation, the content of diosgenin was higher, which may have an impact on the accumulation of the bioactive compound (diosgenin) in T. govanianum. The rhizome bud explants harvested from the individuals of identified elite locations exhibited the maximum survival rate and shoot regeneration on MS medium enriched with BAP, Kn, and GA3. For multiplication, a substantial increase in the production of lateral buds was noticed on MS medium fortified with GA3, BAP, Kn, and casein hydrolysate. Meanwhile, higher concentrations of sucrose, growth regulators (BAP and TDZ), and additives (casein hydrolysate and L-glutamine) showed a significant effect on mini rhizome development from in vitro derived lateral buds. The in vitro regenerants were further rooted and hardened successfully. Besides, SSR and ISSR markers confirmed the genetic uniformity of in vitro raised regenerants. Thus, the study demonstrates an efficient and competent way for propagating the valuable endangered medicinal herb T. govanianum by developing in vitro mini rhizomes using rhizome bud explants. The findings will be of immense potential for its large-scale ex-situ conservation due to its narrow genetic base, along with fostering knowledge about conservation strategies.Key messageAn efficient and competent way to propagate the valuable endangered medicinal herb Trillium govanianum (Himalayan Trillium) was demonstrated by in vitro mini rhizomes production using rhizome bud explants.

Background Robust phylogenies for species with giant genomes and closely related taxa can build evolutionary frameworks for investigating the origin and evolution of these genomic gigantisms. Paris japonica (Melanthiaceae) has the largest genome that has been confirmed in eukaryotes to date; however, its phylogenetic position remains unresolved. As a result, the evolutionary history of the genomic gigantisms in P. japonica remains poorly understood. Results We used next-generation sequencing to generate complete plastomes of P. japonica , P. verticillata , Trillium govanianum , Ypsilandra thibetica and Y. yunnanensis . Together with published plastomes, the infra-familial relationships in Melanthiaceae and infra-generic phylogeny in Paris were investigated, and their divergence times were calculated. The results indicated that the expansion of the ancestral genome of extant Paris and Trillium occurred approximately from 59.16 Mya to 38.21 Mya. The sister relationship between P. japonica and the section Euthyra was recovered, and they diverged around the transition of the Oligocene/Miocene (20 Mya), when the Japan Islands were separated from the continent of Asia. Conclusions The genome size expansion in the most recent common ancestor for Paris and Trillium was most possibly a gradual process that lasted for approximately 20 million years. The divergence of P. japonica (section Kinugasa ) and other taxa with thick rhizome may have been triggered by the isolation of the Japan Islands from the continent of Asia. This long-term separation, since the Oligocene/Miocene boundary, would have played an important role in the formation and evolution of the genomic gigantism in P. japonica. Moreover, our results support the taxonomic treatment of Paris as a genus rather than dividing it into three genera, but do not support the recognition of T. govanianum as the separate genus Trillidium .