Inbreeding in Chinese fir: Insights into the adaptive growth traits of selfed progeny from mRNA, miRNA, and copy number variation.

PREMISE: The impact of inbreeding on biological processes is well documented in individuals with severe inbreeding depression. However, the biological processes influencing the adaptive growth of normal selfed individuals are unknown. Here, we aimed to investigate how inbreeding affects gene expression for adaptive growth of normal selfed seedlings from a self-fertilizing parent in Chinese fir (Cunninghamia lanceolata). METHODS: Using RNA-seq data from above- and underground tissues of abnormal and normal selfed seedlings, we analyzed GO biological processes network. We also sequenced small RNAs in the aboveground tissues and measured the copy number variations (CNV) of the hub genes. RESULTS: Phenotypic fitness analysis revealed that the normal seedlings were better adapted than their abnormal counterparts. Upregulated differentially expressed genes (DEGs) were associated with development processes, and downregulated DEGs were mainly enriched in fundamental metabolism and stress response. Results of mRNA-miRNA parallel sequencing revealed that upregulated target genes were predominantly associated with development, highlighting their crucial role in phosphorylation in signal transduction networks. We also discovered a moderate correlation (0.1328 < R2 < 0.6257) between CNV and gene expression levels for three hub genes (TMKL1, GT2, and RHY1A). CONCLUSIONS: We uncovered the key biological processes underpinning the growth of normal selfed seedlings and established the relationship between CNV and the expression levels of hub genes in selfed seedlings. Understanding the candidate genes involved in the growth of selfed seedlings will help us comprehend the genetic mechanisms behind inbreeding depression in the evolutionary biology of plants.

Pollen and viruses contribute to spatio-temporal variation in asthma-related emergency department visits.

BACKGROUND: Asthma exacerbations are an important cause of emergency department visits but much remains unknown about the role of environmental triggers including viruses and allergenic pollen. A better understanding of spatio-temporal variation in exposure and risk posed by viruses and pollen types could help prioritize public health interventions. OBJECTIVE: Here we quantify the effects of regionally important Cupressaceae pollen, tree pollen, other pollen types, rhinovirus, seasonal coronavirus, respiratory syncytial virus, and influenza on asthma-related emergency department visits for people living near eight pollen monitoring stations in Texas. METHODS: We used age stratified Poisson regression analyses to quantify the effects of allergenic pollen and viruses on asthma-related emergency department visits. RESULTS: Young children (<5 years of age) had high asthma-related emergency department rates (24.1 visits/1,000,000 person-days), which were mainly attributed to viruses (51.2%). School-aged children also had high rates (20.7 visits/1,000,000 person-days), which were attributed to viruses (57.0%), Cupressaceae pollen (0.7%), and tree pollen (2.8%). Adults had lower rates (8.1 visits/1,000,000 person-days) which were attributed to viruses (25.4%), Cupressaceae pollen (0.8%), and tree pollen (2.3%). This risk was spread unevenly across space and time; for example, during peak Cuppressaceae season, this pollen accounted for 8.2% of adult emergency department visits near Austin where these plants are abundant, but 0.4% in cities like Houston where they are not; results for other age groups were similar. CONCLUSIONS: Although viruses are a major contributor to asthma-related emergency department visits, airborne pollen can explain a meaningful portion of visits during peak pollen season and this risk varies over both time and space because of differences in plant composition.

Pattern of sensitization to Juniperus oxycedrus 4EF-hand polcalcin, Jun o 4, compared with the 2EF-hand grass homolog Phl p 7 in a general Italian population of subjects suffering from pollinosis.

Cupressaceae pollen causes allergic reactions worldwide with long-lasting symptomatic periods. Currently, no cypress polcalcin is available for diagnostic purposes. With the aim to investigate the pattern of sensitization to a cypress polcalcin, a synthetic gene of Jun o 4, the Juniperus oxycedrus 4EF-hand polcalcin, was cloned and expressed in Escherichia coli. Its features were investigated in comparison with the grass 2EF-hand Phl p 7. Rhinitis was the symptom most frequently reported in a cohort of Italian patients sensitized to rJun o 4 and/or rPhl p 7. The detection of many pollen allergic patients sensitized to the cypress polcalcin, but negative to Phl p 7, indicates that Phl p 7 cannot be further considered a marker of sensitization towards all the polcalcins. A 4EF-hand cypress polcalcin claims the inclusion in allergy diagnostic tests. In addition, the sensitivity of polcalcins to gastrointestinal digestion is reported and discussed for the first time.

Phylogeny and evolution of Cupressaceae: Updates on intergeneric relationships and new insights on ancient intergeneric hybridization.

After the merger of the former Taxodiaceae and Cupressaceae s.s., currently the conifer family Cupressaceae (sensu lato) comprises seven subfamilies and 32 genera, most of which are important components of temperate and mountainous forests. With the exception of a recently published genus-level phylogeny of gymnosperms inferred from sequence analysis of 790 orthologs, previous phylogenetic studies of Cupressaceae were based mainly on morphological characters or a few molecular markers, and did not completely resolve the intergeneric relationships. In this study, we reconstructed a robust and well-resolved phylogeny of Cupressaceae represented by all 32 genera, using 1944 genes (Orthogroups) generated from transcriptome sequencing. Reticulate evolution analyses detected a possible ancient hybridization that occurred between ancestors of two subclades of Cupressoideae, including Microbiota-Platycladus-Tetraclinis (MPT) and Juniperus-Cupressus-Hesperocyparis-Callitropsis-Xanthocyparis (JCHCX), although both concatenation and coalescent trees are highly supported. Moreover, divergence time estimation and ancestral area reconstruction indicate that Cupressaceae very likely originated in Asia in the Triassic, and geographic isolation caused by continental separation drove the vicariant evolution of the two subfamilies Cupressoideae and Callitroideae in the northern and southern hemispheres, respectively. Evolutionary analyses of some morphological characters suggest that helically arranged linear-acicular leaves and imbricate bract-scale complexes represent ancestral states, and the shift from linear-acicular leaves to scale-like leaves was associated with the shift from helical to decussate arrangement. Our study sheds new light on phylogeny and evolutionary history of Cupressaceae, and strongly suggests that both dichotomous phylogenetic and reticulate evolution analyses be conducted in phylogenomic studies.

Shoot dimorphism enables Sequoia sempervirens to separate requirements for foliar water uptake and photosynthesis.

PREMISE: Trees in wet forests often have features that prevent water films from covering stomata and inhibiting gas exchange, while many trees in drier environments use foliar water uptake to reduce water stress. In forests with both wet and dry seasons, evergreen trees would benefit from producing leaves capable of balancing rainy-season photosynthesis with summertime water absorption. METHODS: Using samples collected from across the vertical gradient in tall redwood (Sequoia sempervirens) crowns, we estimated tree-level foliar water uptake and employed physics-based causative modeling to identify key functional traits that determine uptake potential by setting hydraulic resistance. RESULTS: We showed that Sequoia has two functionally distinct shoot morphotypes. While most shoots specialize in photosynthesis, the axial shoot type is capable of much greater foliar water uptake, and its within-crown distribution varies with latitude. A suite of leaf surface traits cause hydraulic resistance, leading to variation in uptake capacity among samples. CONCLUSIONS: Shoot dimorphism gives tall Sequoia trees the capacity to absorb up to 48 kg H2 O h-1 during the first hour of leaf wetting, ameliorating water stress while presumably maintaining high photosynthetic capacity year round. Geographic variation in shoot dimorphism suggests that plasticity in shoot-type distribution and leaf surface traits helps Sequoia maintain a dominate presence in both wet and dry forests.

Persistence of airborne tree pollen from the Cupressaceae family during the last decade in the city of Bahia Blanca.

Airborne pollen is a major contributor to allergic respiratory diseases. However, the literature on patterns and seasonality of specific pollen types mainly comes from northern hemisphere and developed regions, limiting our ability to extrapolate these findings to other geographical locations. With this research letter, our aim is to contribute to the current knowledge of aerobiology by analyzing the decade-long patterns of airborne tree pollen in the city of Bahia Blanca, Argentina. In general, we found late winter to early spring as the period with the highest Cupressaceae pollen counts. In 2020, the average pollen count during July through September was 78.81 (SD 79.70), the highest recorded during the last 10 years. These months had varying pollen concentrations from moderate (15-89 grains/m3 of air) to high (90-1499 grains/m3 of air). Regardless of such variation, August had consistently been the month with the highest peak concentrations from 2010 to 2020 (61.2, SD 57.3), followed by September (27.1, SD 20.3). We did not find any month with very high (˃1500 grains/m3 of air) peak concentration. Further studies need to be done to expand our knowledge on aerobiology to characterize specific pollen sub-types and determine the exact allergenic potential of airborne pollen in different regions.

Chemical Constituents of the Leaves of Thujopsis dolabrata and Their Cytotoxicity.

Five podophyllotoxin derivatives (1-5), two diterpenoids (6 and 7), three diterpenoid xylosides (8-10), a flavanonol glycoside (11), flavonol (12), and biflavonoid (13) were isolated from the leaves of Thujopsis dolabrata (Cupressaceae). Compounds 1, 6, and 8 were named (-)-ß-isopeltatin, epi-nootkastatin 2, and acetoxyanticopalol 15-O-ß-D-xylopyranoside, respectively. The structures of the isolated compounds were determined based on a detailed analysis of NMR spectroscopic data and through chromatographic and spectroscopic analyses following specific chemical transformations. The isolated compounds (1-5 and 7-11) were evaluated for their cytotoxicity toward HL-60 human promyelocytic leukemia cells and Caki-1 human kidney carcinoma cells. The podophyllotoxin derivatives (1-5) exhibited cytotoxicity against both HL-60 and Caki-1 cells with IC50 values ranging from 0.00069 to 5.4 µM, and the diterpenoid derivatives (7-10) demonstrated cytotoxicity against HL-60 cells with IC50 values ranging from 4.5 to 11 µM. HL-60 cells treated with 8 exhibited apoptosis characteristics, such as accumulation of sub-G1 cells and nuclear chromatin condensation.

Analysis of the annual pollen integral in Albuquerque, New Mexico, shows a negative trend with temperatures for Juniper, Cottonwood, Elm, and Mulberry.

The goal of this study is to determine if the annual pollen integral (APIn) for the top tree allergens in the City of Albuquerque is correlated with meteorological variables. This analysis would be the first of its kind for this area. We used 17 consecutive years from 2004 to 2020 and data collected by the city of Albuquerque using a Spore Trap (Burkard) volumetric air sampler in a location designed to represent a typical desert environment. The pollen studied include Juniper, Elm, Ash, Cottonwood, and Mulberry. We found a negative linear correlation with early summer temperatures of the previous year and APIn for Elm, Cottonwood, and Mulberry, and early fall temperatures for Juniper. Linear regression models developed for Elm, Cottonwood, and Mulberry used the monthly mean maximum temperature for the month of June of the prior year as the independent variable to yield a R squared statistic (R 2) of 0.88, 0.91 and 0.78, respectively. For Juniper, the average monthly mean minimum temperature for the previous September and October served as the independent variable and yielded the R 2 value of 0.80. We also observed a positive trend for the annual maximum temperature over time and a negative trend for the total APIn. Summers in New Mexico are hot and dry, and they may be getting hotter and drier because of climate change. Our analysis predicts that climate change in this area may lead to reduced allergies if temperatures continue to increase and if precipitation patterns remain the same.

Secondary Metabolites with Antimicrobial Activities from Chamaecyparis obtusa var. formosana.

Seven new compounds, including one dimer novel skeleton, chamaecyformosanin A (1); three diterpenes, chamaecyformosanins B-D (2-4); one sesquiterpene, chamaecyformosanin E (5); and two monoterpenes, chamaecyformosanins F and G (6 and 7) were isolated from the methanol extract of the bark of Chamaecyparis obtusa var. formosana. Their structures were established by the mean of spectroscopic analysis and the comparison of NMR data with those of known analogues. Their structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis, and high-resolution mass spectrometry. Furthermore, the isolated compounds were subjected to an evaluation of their antimicrobial activity. Metabolites 1, 3, and 4 present antibacterial activities. It is worth mentioning that the chemical composition of the bark of C. obtusa var. formosana has never been studied in the past. This is the first time the barks from C. obtusa var. formosana were studied and two new skeleton compounds, 1 and 7, were obtained.

Chemical Composition of Essential Oils from Leaves and Fruits of Juniperus foetidissima and Their Attractancy and Toxicity to Two Economically Important Tephritid Fruit Fly Species, Ceratitis capitata and Anastrepha suspensa.

The present study analyzed the chemical composition of Juniperus foetidissima Willd. essential oils (EOs) and evaluated their attractancy and toxicity to two agriculturally important tephritid fruit flies. The composition of hydrodistilled EOs obtained from leaves (JFLEO) and fruits (JFFEO) of J. foetidissima was analyzed by GC-FID and GC-MS. The main compounds were α-pinene (45%) and cedrol (18%) in the JFLEO and α-pinene (42%), α-thujone (12%), and ß-thujone (25%) in the JFFEO. In behavioral bioassays of the male Mediterranean fruit fly, Ceratitis capitata (Wiedemann), both JFLEO and JFFEO showed strong attraction comparable to that observed with two positive controls, Melaleuca alternifolia and Tetradenia riparia EOs. In topical bioassays of the female Caribbean fruit fly, Anastrepha suspensa (Loew), the toxicity of JFFEO was two-fold higher than that of JFLEO, with the LD50 values being 10.46 and 22.07 µg/µL, respectively. This could be due to differences in chemical components between JFLEO and JFFEO. The JFFEO was dominated by 48% monoterpene hydrocarbons (MH) and 46% oxygenated monoterpenes (OM), while JFLEO consisted of 57% MH, 18% OM, and 20% oxygenated sesquiterpenes (OS). This is the first study to evaluate the attractancy and toxicity of J. foetidissima EOs to tephritid fruit flies. Our results indicate that JFFEO has the potential for application to the management of pest tephritid species, and further investigation is warranted.

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms.

Taiwania cryptomerioides is a monotypic gymnosperm species, valued for the high decay resistance of its wood. This durability has been attributed to the abundance of terpenoids, especially the major diterpenoid metabolite ferruginol, with antifungal and antitermite activity. Specialized diterpenoid metabolism in gymnosperms primarily recruits bifunctional class-I/II diterpene synthases (diTPSs), whereas monofunctional class-II and class-I enzymes operate in angiosperms. In this study, we identified a previously unrecognized group of monofunctional diTPSs in T. cryptomerioides, which suggests a distinct evolutionary divergence of the diTPS family in this species. Specifically, five monofunctional diTPS functions not previously observed in gymnosperms were characterized, including monofunctional class-II enzymes forming labda-13-en-8-ol diphosphate (LPP, TcCPS2) and (+)-copalyl diphosphate (CPP, TcCPS4), and three class-I diTPSs producing biformene (TcKSL1), levopimaradiene (TcKSL3) and phyllocladanol (TcKSL5), respectively. Methyl jasmonate (MeJA) elicited the accumulation of levopimaradiene and the corresponding biosynthetic diTPS genes, TcCPS4 and TcKSL3, is consistent with a possible role in plant defense. Furthermore, TcCPS4 and TcKSL3 are likely to contribute to abietatriene biosynthesis via levopimaradiene as an intermediate in ferruginol biosynthesis in Taiwania. In conclusion, this study provides deeper insight into the functional landscape and molecular evolution of specialized diterpenoid metabolism in gymnosperms as a basis to better understand the role of these metabolites in tree chemical defense.

Characterization of a 7 kDa pollen allergen belonging to the gibberellin-regulated protein family from three Cupressaceae species.

BACKGROUND: Severe allergy to fruits mediated by a 7 kDa allergen belonging to the gibberellin-regulated protein (GRP) family is known to be associated with Cupressaceae pollinosis. OBJECTIVE: To identify and characterize Cupressaceae pollen allergens involved in GRP-related fruit allergy. METHODS: Pru p 7-related proteins from pollen of Cupressus sempervirens, Juniperus ashei and Cryptomeria japonica were identified using a rabbit anti-Pru p 7 antiserum, purified chromatographically and sequenced by mass spectrometry and bioinformatic comparisons. The C sempervirens protein was produced as a recombinant allergen in Pichia pastoris. IgE antibody binding to pollen GRP proteins was analysed in a peach allergic (n = 54) and a cypress pollen allergic (n = 88) patient population from southern France using ImmunoCAP. RESULTS: In each of the three Cupressaceae species studied, a 7 kDa pollen protein related to Pru p 7 was identified and found to comprise an amino acid sequence of 63 residues in length, 92%-98% identical to each other and 67%-68% identical to Pru p 7. The C sempervirens, J ashei and C japonica GRP allergens have been officially recognized by the WHO/IUIS Allergen Nomenclature Sub-Committee and named Cup s 7, Jun a 7 and Cry j 7, respectively. Recombinant Cup s 7 showed IgE antibody binding capacity comparable to that of the purified natural allergen. Among 51 peach allergic subjects sensitized to Pru p 7, substantially higher levels of IgE to Cup s 7 than to Pru p 7 were found. Further, the pollen protein was able to completely outcompete IgE binding to Pru p 7, while the reverse competition effect was modest, consistent with primary sensitization by the pollen allergen. CONCLUSION AND CLINICAL RELEVANCE: Pru p 7-related pollen allergens from three Cupressaceae species have been characterized and may become useful for the identification of pollinosis patients at risk of developing severe fruit allergy.

Fragmented and isolated: limited gene flow coupled with weak isolation by environment in the paleoendemic giant sequoia (Sequoiadendron giganteum).

PREMISE: Patterns of genetic structure across a species' range reflect the long-term interplay between genetic drift, gene flow, and selection. Given the importance of gene flow in preventing the loss of diversity through genetic drift among spatially isolated populations, understanding the dynamics of gene flow and the factors that influence connectivity across a species' range is a major goal for conservation of genetic diversity. Here we present a detailed look at gene flow dynamics of Sequoiadendron giganteum, a paleoendemic tree species that will likely face numerous threats due to climate change. METHODS: We used microsatellite markers to examine nineteen populations of S. giganteum for patterns of genetic structure and to estimate admixture and rates of gene flow between eight population pairs. Also, we used Generalized Dissimilarity Models to elucidate landscape factors that shape genetic differentiation among populations. RESULTS: We found minimal gene flow between adjacent groves in the northern disjunct range. In most of the southern portion of the range, groves showed a signal of connectivity which degrades to isolation in the extreme south. Geographic distance was the most important predictor of genetic dissimilarity across the range, with environmental conditions related to precipitation and temperature explaining a small, but significant, portion of the genetic variance. CONCLUSIONS: Due to their isolation and unique genetic composition, northern populations of S. giganteum should be considered a high conservation priority. In this region, we suggest germplasm conservation as well as restoration planting to enhance genetic diversity.

Spirobiflavonoid stereoisomers from the endangered conifer Glyptostrobus pensilis and their protein tyrosine phosphatase 1B inhibitory activity.

Six spirobiflavonoid stereoisomers including two new ones, spiropensilisols A (1) and B (2), were isolated from a mass-limited trunk barks of Glyptostrobus pensilis, an endangered conifer endemic to China. The new structures featuring a benzofuran-containing spirolactone and their absolute configurations were determined by extensive spectroscopic methods. All the isolates showed significant inhibitory activities against the human protein tyrosine phosphatase 1B (PTP1B) enzyme, a potential therapeutic target for diabetes and obesity, with IC50 values ranging from 3.3 to 17.1 µM. A preliminary SAR analysis with assistance of the molecular modeling approach was performed for the most potent compound (i.e., 1), to understand the nature of interactions governing the binding mode of spirobiflavonids within the active site of the PTP1B enzyme.

Novel 28 microsatellite loci using high-throughput sequencing for an endangered species on Metasequoia glyptostroboides (Cupressaceae).

Metasequoia glyptostroboides is a living fossil and an endangered species listed in the International Union for Conservation of Nature (IUCN). Distinguishing the genotypes of all wild individuals of M. glyptostroboides is important to delimit management units and key germplasm resources. We characterized 28 novel polymorphic microsatellite loci using a streptavidin-biotin microsatellite-enriched library and Illumina high-throughput sequencing. Characteristics of each locus were tested using 140 individuals collected from five natural populations of M. glyptostroboides. The number of alleles per locus ranged from 3 to 20, with a mean number of about 8 alleles. The observed and expected heterozygosities in each population ranged from 0.0000 to 1.0000 and from 0.0000 to 0.8958, respectively. Four to nine loci were cross-amplified successfully in seven species of Cupressaceae. The novel SSR markers will provide a toolkit for DNA identification of all of the extant wild individuals guiding further conservation efforts of M. glyptostroboides.

Evolutionary diversification of taiwanioid conifers: evidence from a new Upper Cretaceous seed cone from Hokkaido, Japan.

A single cylindrical seed cone 2 cm long, 1.1 cm wide has been found preserved in a calcium carbonate marine concretion from the Hakobuchi Formation (late Campanian-early Maastrichtian) of Hobetsu, Hokkaido, Japan. The cone, attached to a bent peduncle lacking leaves, has helically arranged bract/scale complexes that arise at right angles from the cone axis in the middle of the cone. The cone axis, ca. 1 mm wide, has a broad cylinder of secondary vascular tissue, and lacks a continuous resin canal system. Bract-scale complexes are laminar, cordate-orbiculate, and upturned distally, consisting primarily of bract tissue with no visible scale tip. The vascular trace to the bract/scale complex originates as a rod that divides laterally into several traces at the level of seed attachment. A single resin canal originates at the base of the bract-scale complex abaxial to the vascular strand, but more distally there are up to ca. 15 large resin canals that form a single row. Two to three inverted winged seeds are attached adaxially near the cone periphery. Cone structure and vascularization are most similar to those in the Cupressaceae, Subfamily Taiwanioideae, differing from living Taiwania cryptomerioides by having up to three seeds/scale rather than two, an abruptly upturned bract tip, in details of bract/scale vasculature, and a cone peduncle lacking leaves. This cone is described as Mukawastrobus satoi Stockey, Nishida and Rothwell. Together with previously described Early to Late Cretaceous taiwanioid seed cones from Mongolia and Hokkaido the new species demonstrates that the taxonomically diagnostic characters of such conifers are as subtle as those of Cretaceous and Cenozoic sequoioid Cupressaceae. This realization emphasizes that evolutionary diversification and turnover among taiwanioid conifers during the Cretaceous and Paleogene are probably far greater than currently recognized.

Molecular phylogenetic analyses reveal a deep dichotomy in the conifer-inhabiting genus Trisetacus (Eriophyoidea: Nalepellidae), with the two lineages differing in their female genital morphology and host associations.

We analyzed the phylogenetic relationships of the genus Trisetacus using two genes [cytochrome c oxidase subunit I (COI) and D1-D2 region of 28S rDNA (D1-D2 28S)], a representive taxon sampling (nearly 40% of known diversity), and a large set of close and distant outgroups. Our analyses suggest the presence of a dichotomy between Trisetacus associated with Cupressaceae and Pinaceae. The following smaller molecular clades were found: Pin-1 (bud mites, twig sheath mites, bark gall mites, and endoparasitic mites from pinaceans), Pin-2 (needle sheath mites from pines), Pin-2a (putative Nearctic group of needle sheath mites), Pin-2b (putative Palearctic group of needle sheath mites), Cup-1 and 2 (bud, cone, seed mites and mites living under bark scales from cupressaceans). The monophyly of the recently proposed subgenus Brevithecus nested within clade Cup-2 was confirmed. Ancestral character reconstruction analyses recovered: (1) Pinaceae as the ancestral hosts of Nalepellidae and Trisetacus, (2) repetitive reductions of the spermathecal tube independently occurred in two lineages of Trisetacus from Cupressaceae, and (3) several mite habitats on host (galls, cones, twig sheaths, seeds, inside leaves, and under scales) are evolutionarily derived states, whereas living in buds or needle sheaths are ancestral states for Trisetacus clades Cup and Pin. Using confocal microscopy, we identified six basic types of the female internal genitalia of Trisetacus based on shapes of the spermatheca and spermathecal tube. These genitalic types are strongly correlated with lineages recovered by molecular phylogenetic analyses, suggesting that the female genital morphology is both evolutionarily conserved and is a factor influencing macroevolutionary patterns in this group of mites.

Strong dispersal limitation in postfire regeneration of Baker cypress, a rare serotinous conifer.

PREMISE: Dispersal capacity primarily determines the spatial establishment patterns that drive range expansions and contractions in tree species. Seedling establishment in Baker cypress (Hesperocyparis bakeri [(Jeps.) Bartel]) relies predominantly on fire events, due to its cone serotiny, shade intolerance, and small seeds that require the optimal conditions of fire-exposed, mineral soil seedbeds. METHODS: We quantified the density and spatial distribution of post-disturbance seedlings following the 2014 Eiler Fire in northern California and compared the observed recruitment to predictions from a mechanistic seed dispersal model. RESULTS: Postfire recruitment was dense, averaging 11 seedlings/m2 , and occurred primarily in the first year after fire. We estimated the mean descent velocity of the wingless seeds as ~4 m/s, the highest value reported for any putatively wind-dispersed tree species. The rapid seed descent contributed to markedly spatially constrained recruitment. Most seedlings (~81%) established within 5 m of the parent tree, and 94% established within 10 m. The maximum observed dispersal distance was 48.5 m; dispersal distance scaled linearly with canopy height. Distributions of modeled seed dispersal distance and observed seedling establishment in Baker cypress did not differ, demonstrating that wind disperses seeds a short distance because of the lack of a wing, and secondary dispersal appeared to be minimal at this recently burned site. CONCLUSIONS: If seed dispersal is solely reliant on wind, migration in response to rapid climate change will be impeded and potentially present difficulties in sustaining populations of this and other obligate seeder species with equally constrained dispersal.

Within-crown plasticity in leaf traits among the tallest conifers.

PREMISE OF THE STUDY: Leaves are the sites of greatest water stress in trees and a key means of acclimation to the environment. We considered phenotypic plasticity of Pseudotsuga menziesii leaves in their ecological context, exploring responsiveness to natural gradients in water stress (indicated by sample height) and light availability (measured from hemispherical photos) to understand how leaf structure is controlled by abiotic factors in tall tree crowns. METHODS: After measuring anatomy, morphology, and carbon isotope composition (δ13 C) of leaves throughout crowns of P. menziesii >90 m tall, we compared structural plasticity of leaves among the three tallest conifer species using equivalent data from past work on Sequoia sempervirens and Picea sitchensis. KEY RESULTS: Leaf mass per projected area (LMA) and δ13 C increased and mesoporosity (airspace/area) decreased along the water-stress gradient, while light did not play a detectable role in leaf development. Overall, leaves of P. menziesii were far less phenotypically responsive to within-crown abiotic gradients than either P. sitchensis, whose leaves responded strongly to light availability, or S. sempervirens, whose leaves responded equally strongly to water stress. CONCLUSIONS: P. menziesii maintain remarkably consistent leaf structure despite pronounced vertical gradients in abiotic factors. Contrasting patterns of leaf structural plasticity underlie divergent ecological strategies of the three tallest conifer species, which coexist in Californian rainforests.

Growth inhibitory activity of biflavonoids and diterpenoids from the leaves of the Libyan Juniperus phoenicea against human cancer cells.

Three biflavonoids [cupressuflavone (1), amentoflavone (2), and sumaflavone (3)], four diterpenoids [13-epi-cupressic acid (4), imbricatholic acid (5), 3-hydroxy-sandaracopimaric acid (6), and dehydroabietic acid (7)], and one lignan [ß-peltatin methyl ether (8)] were isolated from the cytotoxic fractions of the extracts of the leaves of the Libyan Juniperus phoenicea L. The structures of these compounds were elucidated by spectroscopic means. Cytotoxicity of compounds 1-6 were assessed against the human lung cancer cell line A549 using the MTT assay. Compounds 1 and 3 showed cytotoxicity against the A549 cells (IC50  = 65 and 77 µM, respectively), whereas compound 2 did not show any activity. Diterpenes 4-6 exhibited weak cytotoxicity against the A549 cells with the IC50 values of 159, 263, and 223 µM, respectively. The cytotoxicity of each compound was compared with the anticancer drug, etoposide (IC50  = 61 µM). Cupressuflavone (1) was evaluated also for cytotoxicity against both the human PC3 cancer cell line and the normal prostate cell line (PNT2), and this compound revealed a high degree of cytotoxic selectivity towards the prostate cancer cells (PC3), with IC50 value of 19.9 µM, without any evidence of cytotoxicity towards the normal prostate cell line (PNT2).