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Two previously undescribed 3(4), 8(9), 9(10)-trisecocycloartane triterpene dilactones with a featured eleven-membered ring, pseudoladilactones A and B (1-2), together with three known ones (4-5), were isolated from the 80 % ethanol extract of the cones of Pseudolarix amabilis. The structures of compounds 1 and 2 were determined on the basis of analysis of 1D- and 2D-NMR spectroscopic data. The absolute configuration of pseudoladilactones A (1) was elucidated by single crystal X-ray diffraction (CuKα). All isolates were tested for cytototoxicity against four human tumor cell lines A549, HCT116, SK-BR-3, and HepG2.

It has been well reported in many studies globally that the increased atmospheric carbon dioxide (CO2) concentration has positively influenced plant growth in the past century. However, it is crucial to understand how long the tree growth trend will remain alongside the rising CO2 concentration before it is tipped over. In this study, we focused on studying the long-term tree growth responses to the rising CO2 concentration, in combination with other environmental factors, and its feedback to the changing climate. This study has reported two tree species, Pseudolarix amabilis (P. amabilis) and Cryptomeria japonica (C. japonica), sampled from a subtropical monsoon forest located in eastern China, and established the long-term tree ring chronologies by applying tree ring width measurement and cross dating, and then, basal area increment (BAI) was calculated. Meanwhile, intrinsic water-use efficiency (iWUE) was calculated through the measurement of carbon isotope composition (δ13C) in tree ring samples, and its relationships with BAI and atmospheric CO2 concentration were also quantified. BAI values continuously increased in both P. amabilis and C. japonica with the rising of CO2 concentration until the atmospheric CO2 concentration tipping points were reached, after which tree growth started to decline with the rising CO2, while iWUE exerted a continuous increase trend with the increasing CO2 concentration. Tree growth of both species was more photosynthesis driven before reaching the maximum BAI points, once the tipping points of atmospheric CO2 were passed, leading to the positive feed of forest carbon cycling to climate change and global warming. Therefore, tree growth was more water limitation driven in the last 20 years.

Abstract Four new 3,4-secocycloartane triterpenoids, pseudolactones A–D (1–4), were isolated from the ethanol extract of the cones of Pseudol arixamabilis. Their structures were established by extensive 1D- and 2D-NMR experiments. The cones of P. arixamabilis are enriched in the ring-expanded or cleaved cycloartane triterpenoids. This work provides new insight into cycloartane triterpenoids from the cones of P. arixamabilis. Graphic Abstract

A formal synthesis of pseudolaric acid B, a diterpene isolated from the root bark of Pseudolarix kaempferi Gordon (Pinaceae), to Trost’s synthetic intermediate was achieved in 17 steps from a known ketone. Key features of this synthesis include a Claisen rearrangement and iodoetherification to construct quaternary stereocenters and ring-closing metathesis to form the seven-membered ring.

Pseudolarix amabilis (Nelson) Rehd, is a monotypic genus plant belonging to the family Pinaceae. The root bark , known as “Tu-Jin-Pi” has been used for the treatment of skin diseases. During our activity screening, the water-soluble part of the 70% EtOH extract of P. amabilis bark showed excellent inhibitory bioactivities on PTP1B enzyme, leading to the phytochemical isolation of the root bark of P. amabilis. Three oleanane-type compounds (1-3) and seven phenolic compounds (4-10) were isolated, in which oleanolic acid 3-O-β-D-glucuronyl-6′-ethyl ester (1) was identified as a new saponin. The chemical structures of these compounds were elucidated by 1D/2D nuclear magnetic resonance and high resolution mass spectra. In addition, their pharmacological inhibitory bioactivities on PTP1B enzyme were evaluated, and the three oleanane-type compounds 1-3 exhibited inhibitory bioactivities with IC50 values of 1.90 ± 0.37, 19.15 ± 0.10 and 10.44 ± 0.59 μM, respectively.

The root bark of Pseudolarix kaempferi, a famous traditional Chinese medicine, has been commonly used in China to treat skin diseases caused by fungal infections. To evaluate its pharmatherapeutic quality, a sensitive, simple and accurate high-performance liquid chromatography (HPLC) method was developed for the quantitative analysis of three major bioactive diterpenoids, pseudolaric acids A-C in P. kaempferi. A Zorbax XDB RP-C18 (4.6 mm×250 mm, 5 μm) was used in this HPLC system, with UV detection at 260 nm. A gradient elution was effected by a mobile phase composed of acetonitrile and 1% aqueous acetic acid, where acetonitrile concentration was changed from 30% to 60% in 30 min. These three constituents were well resolved by this method with good linear relationships in the range of 0.982-98.2 μg/mL for pseudolaric acid A, 0.618-61.8 μg/mL for pseudolaric acid B, and 1.052-105.2 μg/mL for pseudolaric acid C, respectively. The correlation coefficients of the calibration curves were all over 0.999. In addition, these three compounds in six batches of the root barks of P. kaempferi were quantitatively determined to evaluate the effectiveness of this method.

Pseudolaric acids A (1) and B (2) are diterpene acids isolated from the root bark of Pseudolarix kaempferi as the main biologically active constituents of tujinpi, a traditional Chinese medicinal herb. Studies toward the total synthesis of 2 are described. The unusual trans-fused [5–7] ring system in the core structure of 2 was successfully synthesized via Dieckmann condensation.

Seven undescribed cycloartane triterpenoids, pseudolarnoids A-G, together with ten known ones, were isolated from the seeds of Pseudolarix amabilis (J. Nelson) Rehder. Their structures were elucidated on the basis of spectroscopic analysis, X-ray crystallography, and ECD data. Pseudolarnoids A-C are cycloartane triterpenoids with a unique 16S, 23R-spirolactone moiety. Pseudolarnoids F, G, and pseudolarolide C demonstrated potent antiviral effects on HSV-1 in vitro.

A series of sesquiterpene polyol esters, including two new β-dihydroagarofuran sesquiterpenes ( 1 - 2 ) and 14 known compounds ( 3 - 16 ), were isolated from the root bark of Pseudolarix kaempferi. The esters⿿ structures were elucidated by employing extensive spectroscopic analyses and by comparison to literature compounds. The absolute configurations of the two new compounds were determined by the CD exciton chirality method. The known compounds 3 - 16 were isolated from the Pseudolarix genus for the first time.

The Quilchena fossil locality is dated (51.5 ± 0.4 Ma) to the Early Eocene Climatic Optimum, and this locality is reconstructed as the warmest and wettest of the Early Eocene upland sites from the Okanagan Highlands of British Columbia and northern Washington State. Mean annual temperature (MAT) is estimated from leaf margin analysis, using 55 dicot morphotypes, as 16.2 ± 2.1 °C/14.6 ± 4.8 °C. Using bioclimatic analysis of 45 nearest living relatives, a moist mesothermal climate is indicated (MAT 12.7–16.6 °C; cold month mean temperature (CMMT) 3.5–7.9 °C; mean annual precipitation (MAP) 103–157 cm/year. Leaf size analysis estimates MAP at 121 ± 39 cm/year. Estimates from the climate leaf analysis multivariate program corroborate these results, although with a slightly cooler MAT (13.3 ± 2.1 °C). Plants that support an interpretation of warm winters with minimal or no frost include Azolla, Glyptostrobus, Taxodium, Keteleeria, Pseudolarix, Eucommia, Dipteronia, Hovenia, Ternstroemia, and others. These thermophilous elements occur together with temperate genera such as Alnus, Betula, Ulmus, Calocedrus, and Fraxinus. Palynological assemblages at Quilchena are dominated by bisaccate conifers and Cupressaceae. Common angiosperms include Ulmus type, triporates, Pterocarya, and Alnus. Insect fossils at Quilchena that today inhabit tropical and subtropical regions also support warm and equable climate without significant frost, and include obligate palm-feeding beetles (Pachymerina), which indicate CMMT perhaps as high as 8 °C. These are found together with temperate aphids, wasps, giant lacewings, brown lacewings, and a panorpoid scorpionfly, supporting an interpretation of equable climatic conditions during the Early Eocene Climatic Optimum.

Phylogeny of the ten Pinaceous genera has long been contentious. Plastid genomes (plastomes) provide an opportunity to resolve this problem because they contain rich evolutionary information. To comprehend the plastid phylogenomics of all ten Pinaceous genera, we sequenced the plastomes of two previously unavailable genera, Pseudolarix amabilis (122,234 bp) and Tsuga chinensis (120,859 bp). Both plastomes share similar gene repertoire and order. Here for the first time we report a unique insertion of tandem repeats in accD of T. chinensis. From the 65 plastid protein-coding genes common to all Pinaceous genera, we re-examined the phylogenetic relationship among all Pinaceous genera. Our two phylogenetic trees are congruent in an identical tree topology, with the five genera of the Abietoideae subfamily constituting a monophyletic clade separate from the other three subfamilies: Pinoideae, Piceoideae, and Laricoideae. The five genera of Abietoideae were grouped into two sister clades consisting of (1) Cedrus alone and (2) two sister subclades of Pseudolarix—Tsuga and Abies—Keteleeria, with the former uniquely losing the gene psaM and the latter specifically excluding the 3 psbA from the residual inverted repeat.

Pinaceae comprises 11 genera, and represents the largest family of conifers with an extensive wild distribution in the Northern Hemisphere. Intergeneric relationships of Pinaceae have been investigated using many morphological characters and molecular markers, but phylogenetic positions of four genera, including Cathaya, Cedrus, Nothotsuga and Pseudolarix, remain controversial or have not been completely resolved. To completely resolve the intergeneric relationships of Pinaceae, we conducted a comparative transcriptomic study of 14 species representing all Pinaceae genera. Multiple data sets, containing up to 6,369,681 sites across 4676 loci, were analyzed using concatenation and coalescent methods. Our study generated a robust topology, which divides Pinaceae into two clades, one (pinoid) including Cathaya, Laric, Picea, Pinus, and Pseudotsuga, and the other (abietoid) including Abies, Cedrus, Keteleeria, Nothotsuga, Pseudolarix, and Tsuga. Cathaya and Pinus form a Glade sister to Picea; Cedrus is sister to the remaining abietoid genera, and the two genera Nothotsuga and Tsuga form a Glade sister to Pseudolarix. The discordant positions of Cathaya, Cedrus and Pseudolarix in different gene trees could be explained by ancient radiation and/or molecular homoplastic evolution. The hybrid origin hypothesis of Nothotsuga is not supported. Based on molecular dating, extant Pinaceae genera diverged since about 206 Mya, earlier than the break-up of Pangea, and the divergence among the pinoid genera occurred earlier than the split among the abietoid genera. Moreover, our study indicates that two radiation events occurred in the evolution of Pinaceae genera, and some important morphological characters evolved multiple times based on ancestral state reconstruction.

Pseudolaric acid B (PAB), which is the main biologically active diterpene acid of Pseudolarix kaempferi, has presented anti-fungal, anti-tumor, anti-fertility, and anti-tubulin activities. In this study, a sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method with multiple-reaction monitoring mode was employed for quantification of PAB in rat plasma. The calibration curve was linear over the range of 0.86-288 ng/mL with correlation coefficient (r) greater than 0.995 for PAB, and the lower limit of quantification was 0.86 ng/mL in rat plasma. The accuracy of PAB was between -9.1% and 7.0% relative error, and precision ranged from 1.2 to 10.6% relative standard deviation. This method was successfully applied to the pharmacokinetic studies of PAB in Sprague-Dawley rats. After single intravenous administration of 2.0, 4.0, and 8.0mg/kg PAB to rats, the t1/2 were (16.1 ± 5.6), (30.0 ± 13.7), and (27.4 ± 5.3)min, respectively. The pharmacokinetics (C(2min), AUC) of PAB within the used dosage range were in accordance with linear pharmacokinetic characteristics.

Pseudolarix (Pinaceae) is a vulnerable (sensu IUCN) monotypic genus restricted to southeastern China. To better understand levels of genetic diversity, population structure and gene flow among populations of P. amabilis, we developed five compound SSR markers and ten novel polymorphic expressed sequence tags (EST) derived microsatellites. The results showed that all 15 loci were polymorphic with the number of alleles per locus ranging from two to seven. The expected and observed heterozygosities varied from 0.169 to 0.752, and 0.000 to 1.000, respectively. The inbreeding coefficient ranged from −0.833 to 1.000. These markers will contribute to research on genetic diversity and population genetic structure of P. amabilis, which in turn will contribute to the species conservation.

Pseudolarix amabilis belongs to one of three monotypic genera in Pinaceae. This species had 2n=44 chromosomes in somatic cells and its karyotype was composed of four long submetacentric chromosomes and 40 short telocentric chromosomes that varied gradually in length, supporting previous reports by conventional staining. The chromosomes were stained sequentially with the fluorochromes, chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). CMAbands appeared on 12 chromosomes at near terminal region and proximal region. DAPI-bands appeared at centromeric terminal regions of all 40 telocentric chromosomes. The fluorescent-banded karyotype of this species was compared with those of other Pinaceae genera considering taxonomical treatment and molecular phylogenetic analyses reported. On the basis of the fluorescent-banded karyotype, the relationship between Pseudolarix amabilis and other Pinaceae genera was discussed.

One novel and eight known oxidatively rearranged cycloartane triterpenoids were isolated from the seeds of Pseudolarix amabilis. The structure of the new isolate was elucidated on extensive spectroscopic analyses. Results indicated that pseudolarolide Q (4) exhibited strong antimicrobial activity against Gram-positive Staphylococcus aureus and Candida albicans at the MICs of 6.08 and 24.32 μM, respectively. Pseudolarolide I (2) showed the 11β-HSD1 inhibitory property at the IC50 value of 34.5 nM.

Six new triterpenoids, pseudolarins A-F (1-6), were isolated from the twigs of Pseudolarix amabilis, together with four known triterpenoids (7-10) and five known diterpenoids (11-15). Their structures were determined by extensive spectroscopic analysis, and the absolute configuration of 1 was assigned by single-crystal X-ray diffraction. Compound 1 is a 3,4:9,10-diseco-cycloartane triterpenoid possessing an unprecedented 5/5/7/6/5/6/5 ring system. Compounds 1, 5, 7, 9-11, and 13 showed inhibition against protein tyrosine phosphatase 1B (PTP1B) in vitro.

A flora from Thomas Ranch near Princeton, British Columbia, Canada, is assessed for biodiversity and paleoclimate. This latest Early to early Middle Eocene flora occurs in the Allenby Formation. Seventy-six megafossil morphotypes have been recognized, representing at least 62 species, with 29 identified to genus or species. Common taxa include Ginkgo L., Metasequoia Miki, Sequoia Endl., Abies Mill., Pinus L., Pseudolarix Gordon, Acer L., Alnus Mill., Betula L., Fagus L., Sassafras J Presl, Macginitiea Wolfe & Wehr, Prunus L., and Ulmus L. More than 70 pollen and spore types are recognized, 32 of which are assignable to family or genus. The microflora is dominated by conifers (85%–97% abundance), with Betulaceae accounting for most of the angiosperms. The Climate Leaf Analysis Multivariate Program (CLAMP) calculates a mean annual temperature (MAT) of 9.0 ± 1.7 °C and bioclimatic analysis (BA) calculates a MAT of 12.8 ± 2.5 °C. Coldest month mean temperature (CMMT) was >0 °C. Mean annual precipitation (MAP) was >70 cm/year but is estimated with high uncertainty. Both the CLAMP and BA estimates are at the low end of the MAT range previously published for other Okanagan Highland localities, indicating a temperate climate consistent with a mixed conifer–deciduous forest.

The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch ( Pseudolarix amabilis , Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined “pseudolaratriene.” Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P . amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7- trans -fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene.

Previously, the identification of fossil Pseudolarix at the species level has been based on the morphology of the bract-scale complex of the seed cone. The morphological consistence of fossils through most of the Cenozoic with extant P. amabilis has led them to be considered conspecific, suggesting that P. amabilis is an extraordinary example of morphological stasis. However, the lack of cuticular evidence, especially for the leaf-homologous bract, reduces the accuracy of fossil identification based on morphology, thus weakening the evidence for morphological stasis in P. amabilis. For the first time, we provide cuticular evidence of the bract-scale of fossil P. amabilis based on the bract-scale complex from the late Miocene Shengxian Formation, Zhejiang, East China, which improves the identification accuracy and reinforces the concept of morphological stasis in this species. Second, we preliminarily reveal the niche stability of P. amabilis, which corresponds to its morphological stasis. Finally, we infer that the late Miocene forest containing P. amabilis in Zhejiang was an evergreen sclerophyllous broad-leaved or mixed mesophytic forest, which combined with the evergreen broad-leaved forest suggested by previous megafossil studies, indicates the occurrence of vertical vegetation zonation.

Pseudolaridimers A (1) and B (2), two unprecedented heterodimers formed via a [4 + 2] Diels - Alder cycloaddition between a cycloartane triterpenoid unit and a labdane diterpenoid unit, were isolated from the cones of Pseudolarix amabilis. Their structures were established by extensive analysis of HRESIMS and NMR spectra. The absolute configuration of 1 was determined by single crystal X-ray diffraction (Cu K-alpha) of its methyl esterified derivative. Pseudolaridimer A (1) showed strong cytotoxicity against HCT116, ZR-75-30, and HL-60 human tumor cell lines, with IC50 values 9.62, 7.84, 8.29 mu g/mL, respectively. Pseudolaridimer B (2) only exhibited potent inhibition against the HL-60 cell line with an IC50 value of 7.50 mu g/mL.

Pseudolarix amabilis is a rare and endangered tree species in China. With the method of spatial sere substituting for time sere, by the size structure in place of age structure, using life stable, survival analysis, the structure and dynamics of P. amabilis population at Yaoluoping Natural reserve of Anhui Province was studied. Based on the life table of population and the theory of survival analysis, we worked out the life table of P. amabilis population drew the curves of mortality density function, hazard rate function and survival, and analyzed the population dynamics. The resulted showed that, along the altitude, the population density increased, distribution range of size class decreased. It indicated that the survival curve of the population trended to the type of Deevey Ⅱ.

Flora composition and species diversity features of Pseudolarix amabilis community at the Yaoluoping natural reserve in Anhui Province were analyzed. According to the statistics from 4 sample plots, there are 88 species (including varieties) of 77 genus belonging to 48 families of vascular plants. There are various plants in the community of Pseudolarix amabilis, and its family and genus are of scattered composition. Their floral composition is complicated. In terms of geographical composition of seed plant genus, most of them are distributed in the temperate zone. The species diversity of shrub layer is the highest, the tree layer is the second, and the herb layer is the lowest in the vertical structure of the community. The species diversity index of different layers shows: shrub layer>tree layer>herb layer.

In general, simultaneously separation and purification of components with a broad polarity range from traditional Chinese medicine (TCM) is a challenge by an ordinary high-speed counter-current chromatography (HSCCC) method. In this paper, we describes a rapid and efficient separation method of combining three-step gradient elution and two-step flow-rate gradient elution using high-performance counter-current chromatography (HPCCC) to separate 8 diterpene compounds simultaneously within 80 min in a single run from the alcohol extract of Pseudolarix kaempferi Gordon. This separation process produced 166 mg pseudolaric acid B O-β- d -glucopyranoside (PABGly), 152 mg pseudolaric acid C (PAC), 8 mg deacetylpseudolaric acid A (deacetylPAA), 5 mg pseudolaric acid A O-β- d -glucopyranoside (PAAGly), 484 mg pseudolaric acid B (PAB), 33 mg pseudolaric acid B methyl ester (PAB methyl ester), 10 mg pseudolaric acid A (PAA) and 18 mg pseudolaric acid H (PAH) from 1.0 g crude sample with purities of 98.6%, 99.6%, 92.3%, 92.2%, 99.2%, 99.4%, 98.3%, 91.0%, respectively. Our study indicates that the suitable combination of step-wise gradient elution and flow-rate gradient elution using HPCCC is an effective strategy to separate complex components from natural products.

Field data were sampled by the method of contiguous grid quadrate. The spatial patterns of Pseudolarix amabilis populations were studied by the following methods: DispItalic textersal index (C), David moores index (I), Lloyd index (m*/m), Morisita index (Iδ), Parameter of negative index (K) and Cassie index (1/K). The quadrate variance analysis, Greig-Smith and Kershaw methods were used to study the spatial distribution pattern, pattern scale and pattern intensity of Pinus armandi population at different sizes. The result showed that P. amabilis population is most clumped. The result indicated the pattern intensity was decreasing with the size increasing and a single peak value appeared in the curve with the area increasing. There was a slow change in the pattern intensity of population, which means a smaller variation in environment and a bigger difference in the relative density of population patches during diffusion process.

Three novel triterpenoids, pseudoferic acids A and B (1 and 2) possessed a unique 16,24-cyclo-26-norlanostane skeleton, and pseudoferic acid C (3) featured a cis-fused D/E-ring system, were isolated from the root bark of Pseudolarix kaempferi. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compounds 2 and 3 exhibited weak inhibitory activities against 110-HSD1 (110-hydroxysteroid dehydrogenase 1) with IC50 values of 0.44 (mouse 11 beta-HSD1) and 0.75 mu M (human 11 beta-HSD1), respectively. (C) 2011 Elsevier Ltd. All rights reserved.

Three new 3(4),9(10)-disecocycloartane peroxy triterpene lactones, named as pseudolarolides Q2, T1, and T2 (1–3, resp.), along with five known triterpenoids, were isolated from the seeds of Pseudolarix kaempferiGord. Their structures were elucidated to be (9R,10R,16R,23R,25R)-16,23-epoxy-9,10-epidioxy-3,4:9,10-disecocycloart-1(2)-ene-3,4:26,23-diolide (1), (1R,9R,10S,16R,23S,25R)-1,4:16,23-diepoxy-9,10-epidioxy-3,4;9,10-disecocycloartan-26(23)-olid-3-oic acid methyl ester (2), and (1R,9R,10S,16R,23R,25R)-1,4:16,23-diepoxy-9,10-epidioxy-3,4;9,10-disecocycloartan-26(23)-olid-3-oic acid methyl ester (3) on the basis of spectroscopic analysis.

A rapid and reliable method based on ultra-performance liquid chromatography (UPLC) coupled with photodiode-array detection (PDA) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS) has been developed for separation and identification of major constituents in extracts of root bark of Pseudolarix kaempferi Gordon (PKG). Identification of the constituents was carried out by interpretation of their retention times, UV absorption spectra, MS and MS/MS spectra, as well as the data provided by authentic standards and literatures. A total of 20 components were separated in only 8.0 min on a small particle size C18 column (1.7 microm). These components included nine diterpene acids, seven glycosides and four triterpenoids, among which pseudolaric acid C-O-beta-D-glucopyranoside and pseudolaric acid C2-O-beta-D-glucopyranoside were separated and identified for the first time in this study. Furthermore, the fragmentation patterns of the three types of compounds were elucidated for the first time. This established UPLC-PDA/Q-TOF-MS/MS method is reliable and effective for the separation and identification of the 20 compounds and will be useful for quality control of the crude materials of Pseudolarix kaempferi Gordon and their related preparations.

Three species of Pseudolarix including P. japonica, P. sp. A, and P. sp. B, all belonging to Pinaceae, are systematically described from the Middle Miocene Duho Formation of the Yeonil Group based on the detached seminiferous scales. The presence of Pseudolarix in the Yeonil flora indicates that various kinds of Pinaceae did flourish during the Middle Miocene. The occurrence of Pseudolarix is the first record in the Korean Tertiary flora.

This study, which includes 51 species and six genera of subfamily Abietoideae (Pinaceae), assesses the systematic significance of the wood structure in this group. In particular, the presence of normal and traumatic resin canals, the ray structure and the axial parenchyma constitute phylogenetically informative features. Comparative wood anatomy of Abietoideae clearly supports the monophyly of the genera Abies–Cedrus–Keteleeria–Nothotsuga–Pseudolarix–Tsuga, all of which have axial parenchyma with nodular transverse end walls in the regions of growth ring boundaries, crystals in the ray parenchyma and pitted horizontal and nodular end walls of ray parenchyma cells. Axial resin canals support a subdivision of the subfamily into two groups: Abies, Cedrus, Pseudolarix and Tsuga, without axial resin canals, and Keteleeria and Nothotsuga, with axial resin canals and a specific arrangement of traumatic axial resin canals. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 184–196.

We review the fossil history of seed plant genera that are now endemic to eastern Asia. Although the majority of eastern Asian endemic genera have no known fossil record at all, 54 genera, or about 9%, are reliably known from the fossil record. Most of these are woody (with two exceptions), and most are today either broadly East Asian, or more specifically confined to Sino-Japanese subcategory rather than being endemic to the Sino-Himalayan area. Of the “eastern Asian endemic” genera so far known from the fossil record, the majority formerly occurred in Europe and/or North America, indicating that eastern Asia served as a late Tertiary or Quaternary refugium for taxa. Hence, many of these genera may have originated in other parts of the Northern Hemisphere and expanded their ranges across continents and former sea barriers when tectonic and climatic conditions allowed, leading to their arrival in eastern Asia. Although clear evidence for paleoendemism is provided by the gymnosperms Amentotaxus, Cathaya, Cephalotaxus, Cunninghamia, Cryptomeria, Glyptostrobus, Ginkgo, Keteleeria, Metasequoia, Nothotsuga, Pseudolarix, Sciadopitys, and Taiwania, and the angiosperms Cercidiphyllum, Choerospondias, Corylopsis, Craigia, Cyclocarya, Davidia, Dipelta, Decaisnea, Diplopanax, Dipteronia, Emmenopterys, Eucommia, Euscaphis, Hemiptelea, Hovenia, Koelreuteria, Paulownia, Phellodendron, Platycarya, Pteroceltis, Rehderodendron, Sargentodoxa, Schizophragma, Sinomenium, Tapiscia, Tetracentron, Toricellia, Trapella, and Trochodendron, we cannot rule out the possibility that neoendemism plays an important role especially for herbaceous taxa in the present-day flora of Asia, particularly in the Sino-Himalayan region. In addition to reviewing paleobotanical occurrences from the literature, we document newly recognized fossil occurrences that expand the geographic and stratigraphic ranges previously known for Dipelta, Pteroceltis, and Toricellia.

In order to provide the chemical markers for the quality control of herbal medicines, four diterpenoids, pseudolaric acids A and B (PAA and PAB), and their glucosides were isolated from the methanol extract of the Chinese herb Pseudolarix kaempferi using high-speed counter-current chromatography (HSCCC). The diphase solvent system was n-hexane/EtOAc/MeOH/H(2)O which was used at two ratios (5:5:5:5 and 1:9:4:6 by volume) in the separation of pseudolaric acids and their glycosides, respectively. As a result, PAA (14 mg), PAB (129 mg), PAA-O-beta-D-glucopyranoside (8 mg, PAAG), and PAB-O-beta-D-glucopyranoside (42 mg, PABG) were obtained from 0.5 g of the crude extract. Their purities were determined to be above 97% by HPLC analysis. Their chemical structures were confirmed by( 1)H and( 13)C NMR analysis or HPLC comparison with the reference compounds.

Uncertainties in the age and phylogenetic position of Pinaceae fossils present significant obstacles to our understanding of the timing of diversification in the family. We demonstrate that simultaneous phylogenetic analyses of chloroplast DNA (matK and rbcL) and nonmolecular characters that include both extant genera and a limited number of fossil taxa provide useful hypotheses for calibrating molecular trees. Root placements varied for Pinaceae, with Bayesian analyses recovering mutually monophyletic subfamilies Pinoideae and Abietoideae and parsimony analyses recovering Abietoideae as paraphyletic by placing the root between Cedrus and the remaining genera. The inferred phylogenetic positions of fossil taxa Pityostrobus bernissartensis as the sister group to Pinus and Pseudolarix erensis as the sister group to extant Pseudolarix were used to guide divergence‐time calibrations; these calibrations yielded an Early Cretaceous and an Early Jurassic age for crown‐group Pinaceae, respectively. The older age ...

The plants of the genus Abies are dominant and key species in the dark coniferous forest in the Northern Hemisphere. There are 52 species, one subspecies and 12 varieties in the genus Abies in the world. The history and modern distribution of Abies were discussed. The genus has three modern distribution centers: South Europe, North America and East Asia. These areas are also rich in fossil records. The vertical distribution regions of Abies are from sea level to an elevation of 4,700 m, concentrated in 1,000–2,000 m (15 species). In China, the genus distributes in 20 provinces, especially abundant in the Hengduan Mountains. Meanwhile, endemic and relic phenomena are obvious in this genus. There are seven relic species with both limited individuals and limited distributed regions. Based on the fossil records and the latest phylogenetic data, the following hypothesis was proposed: Abies originated from the middle and high altitudes of the Northern Hemisphere in the middle Cretaceous and it was dispersed southward in the Eocene due to global climate cooling down. The distribution of Abies was deeply influenced by geological events such as the uplift of Himalaya, the Alps, the Rocky Mountains, the occurrence of the Asian Monsoon as well as the Quaternary glaciers. Finally, the current distribution pattern appeared in the Quaternary. The genus Abies has a fossil history and modern distribution pattern similar to that of Cathaya and Pseudolarix.

A reversed phase high-performance liquid chromatography method was established for the first time to simultaneously qualify the seven major diterpenoids in Pseudolarix kaempferi, namely pseudolaric acid B O-beta-D-glucopyranoside (1), pseudolaric acid C2 (2), pseudolaric acid C1 (3), deacetylpseudolaric acid A (4), pseudolaric acid A O-beta-D-glucopyranoside (5), pseudolaric acid B (6) and pseudolaric acid A (7). The optimal conditions of separation and detection were achieved on an Inertsil ODS-3 column with gradient elution of methanol and 0.5% aqueous acetic acid (v/v) at the flow rate of 0.6 ml min(-1) within 40 min and detection wavelength set at 262 nm. All calibration curves showed good linear regression (r2>0.9999) within test ranges. This method provided good accuracy with recoveries in the range of 94.3-106.1% and good precision with R.S.D.s of repeatability and intermediate precision less than 0.57% and 4.67%, respectively. The method was successfully applied to qualitative and quantitative determination of 20 P. kaempferi among the 54 samples collected from different areas. The results revealed that the commercial crude drugs were seriously confused and the developed HPLC assay could be used as a suitable qualitative and quantitative determination method for P. kaempferi.

The differentiation of Pseudolarix from other bisaccate pollen is considered as a tool to investigate the fossil history of this genus. Using Light Microscope (LM) and Scanning Electron Microscopy (SEM) we performed a descriptive and morphometric analysis of Pseudolarix amabilis (Pinaceae) bisaccate fresh pollen grains. Descriptive differentiation characters from Pinus and from other bisaccates have been established. A morphometric differentiation from pine pollen belonging to Pinus sylvestris type was obtained through a study of twelve living species of Pinus . A new identification key for bisaccate pollen grains of living conifers has been elaborated. Although it is addressed to identification of fresh pollen, the proposed descriptive criteria may also be applicable to well-preserved fossil material. A Pliocene site from N-Italy, rich in Pseudolarix cone scales and needles, was used to test the reliability of the proposed criteria on fossil pollen. We also identified Pseudolarix pollen from the lacustrine sediments from Massif Central (France), about 3 My. We carried out a complementary evaluation of the ecological and climate ranges both of the living species and of the fossil assemblages that included Pseudolarix. This tree was a component of the warm-temperate mixed conifers rainforests of the Cenozoic, a biome yielding, after considerable reduction of floristic diversity, overall conifer decimation, to the modern “mixed mesophytic” and “mixed hardwood” forest types. Although its fossil record reveals a wide distribution in the Northern Hemisphere during the Cenozoic, macrofossils are only scattered. On the other hand, information about the pollen production, aerodynamics and taphonomy of Pseudolarix pollen suggests that this tree is among the properly or moderately over-represented genera in pollen images from its natural habitat. Therefore, its pollen record may add clarifications to palaeoclimate reconstructions of Cenozoic environments.

The genus Liquidambar (Hamamelidaceae) is one of a number of taxa associated with the Daijima-type floras of late Early to early Middle Miocene age, which also include many warm-temperate to subtropical elements. However, Liquidambar is very rare or absent in the Aniai-type floras of the early Early Miocene. We describe fossil leaves, fruits and pollen of this genus found in the Lower Miocene Nakamura Formation in the southern part of Gifu Prefecture. The flora of the Nakamura Formation (Hiyoshi flora) shows features common to those of the conformably underlying Hachiya Formation (earliest Early Miocene) but differs from the unconformably overlying Hiramaki Formation (late Early Miocene) in the same region. This fact combined with the occurrence of Liquidambar in the Hiyoshi flora, associated with warm-temperate elements such as Pseudolarix, Castanea, Celtis and Lindera, clearly supports one of two former views: the flora is of a southern type within the Aniai-type floras.

Palynofloras from the middle Early to early Middle Eocene Okanagan Highlands (northern Washington State and southern British Columbia) are used to reconstruct vegetation across a broad upland Eocene landscape. In this preliminary report, forest floristic composition is reconstructed using palynological analysis of sediments from Republic, Washington; localities of the Allenby Formation in the Princeton region (Hospital Hill, One Mile Creek and Summers Creek Road), Hat Creek, McAbee, Falkland, Horsefly, and Driftwood Canyon, British Columbia. Wind-dispersed taxa were dominant in all samples, consistent with floras preserved in lacustrine and paludal depositional environments. Pseudolarix was dominant in five of the floras, but Abies (Falkland) or Ulmus (Republic Corner Lot site) were dominant in individual samples for some floras. Betulaceae were dominant for McAbee (Alnus) and Allenby Formation (Betula), matching megafloral data for these sites. Some taxa common to most sites suggest cool conditions (e.g., Abies, other Pinaceae; Alnus, other Betulaceae). However, all floras contained a substantive broad-leaved deciduous element (e.g., Fagaceae, Juglandaceae) and conifers (e.g., Metasequoia) indicative of mesothermal conditions. Palms were only abundant in the Hat Creek coal flora, with very low counts recorded for the Falkland, McAbee, and Allenby Formation sites, suggesting that they were rare in much of the landscape and likely restricted to specialized habitats. Thermophilic (principally mesothermal) taxa, including palms (five sites) and "taxodiaceous" conifers, may have occurred at their climatic limits. The limiting factor controlling the regional distribution of thermophilic flora, which include primarily wetlands taxa, may be either climatic or edaphic.

Angiogenesis is controlled by a number of growth factors, including vascular endothelial growth factor (VEGF). In this study, pseudolarix acid B, isolated from the traditional Chinese medicinal plant Pseudolarix kaempferi and originally identified as an early pregnancy-terminating agent, was evaluated for its potential as an angiogenesis inhibitor, using in vitro and in vivo models. After exposure to pseudolarix acid B 0.625–5 μM for 72 h, the proliferation of human umbilical vein endothelial cells was significantly inhibited. Pseudolarix acid B 0.313–2.5 μM for 24 h potently blocked the VEGF-induced tube formation of human umbilical vein endothelial cells in a dose-dependent manner. Matrigel plug assays disclosed that pseudolarix acid B reduced angiogenesis induced by VEGF in vivo. In addition, pseudolarix acid B antagonized VEGF-mediated anti-apoptotic effects on serum-deprived human umbilical vein endothelial cells and increased apoptosis of endothelial cells induced by VEGF in Matrigel plug assays. Moreover, pseudolarix acid B significantly inhibited VEGF-induced tyrosine phosphorylation of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1), in correlation with a marked decrease in the phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings collectively suggest that pseudolarix acid B possesses anti-angiogenic activity. One of the main anti-angiogenesis mechanisms of pseudolarix acid B may involve antagonism of the VEGF-mediated anti-apoptosis effect via inhibition of KDR/flk-1, ERK1/2, and Akt phosphorylation in endothelial cells.

Pseudolarix amabilis Rehd. extract was examined in vitro for antibacterial effects, anti-inflammatory effects, and inhibitory effects on histamine release. Pseudolarix amabilis Rehd. extract was also examined for efficacy on dermatitis in atopic dermatitis model mice (NC mice) and effects on keratinous moisture level and transepidermal water loss in miniature pigs. Pseudolarix amabilis Rehd. extract had antibacterial effects on Staphylococcus aureus, Candida albicans, and Streptococcus pyogenes; however this antibacterial effect varied with the temperature at which and conditions under which Pseudolarix amabilis Rehd. was extracted. Pseudolarix amabilis Rehd. extract at the final concentration of 2 mg/mL significantly inhibited the hyaluronidase activity; and at 0.005, 0.05, and 0.5 mg/mL, it also significantly inhibited the histamine release. In the mice in which atopic dermatitis had been induced, 28-day administration of Pseudolarix amabilis Rehd. extract at 4 and 400 mg/mL significantly inhibited aggravation of dermatitis without having effects on body weight. In the dorsal skin of miniature pigs, Pseudolarix amabilis Rehd. extract at 4 and 400 mg/mL significantly increased keratinous moisture level with the increase in the number of dosing days, and caused no changes in transepidermal water loss. From the above results, it is clear that Pseudolarix amabilis Rehd. extract inhibits both proliferation of bacteria and inflammation caused by antigens. Furthermore, it is suggested that Pseudolarix amabilis Rehd. extract will serve as a medicinal drug which effectively moistens the skin and prevents and heals dermatitis.

Molecular phylogenetic reconstructions of Pinaceae based on immunological approaches plus nuclear and organellar DNA sequences consistently resolve the generic pairs Larix – Pseudotsuga and Abies – Keteleeria. In most analyses Pseudolarix and Tsuga (including Nothotsuga) form a clade that is sister to the Abies Keteleeria clade, and Cathaya, Picea, and Pinus make up an unresolved trichotomy. The position of Cedrus remains problematic; molecular evidence supports a basal position in the family. Within Pinaceae genera, molecular phylogenetic reconstructions have markedly improved our understanding of species relationships and biogeography in Pinus, Larix, Pseudotsuga, and Tsuga. Molecular phylogenetic approaches have become routine in plant systematics, and many researchers have applied these methods in conifers. This paper reviews molecular phylogenetic studies of Pinaceae and its constituent genera, with a focus on Pinus, the largest genus in the family. PINACEAE Pinaceae comprises eleven recognized genera and 225 species (Table 1), making it the largest family of non-flowering seed plants. Pinaceae often form the dominant component of boreal, coastal, and montane forests over large regions of the Northern Hemisphere, and several species are now widely planted for reforestation and wood production in both hemispheres. Thus the family has tremendous ecological and economic importance throughout the temperate and in many subtropical regions of the world. There is considerable morphological and molecular evidence for the monophyly of Pinaceae. Hart’s (1987) morphological cladistic analysis of all conifer genera recorded ten synapomorphies, including ovulate cone scales distinct from subtending bracts, two inverted ovules per scale, and a terminal seed wing. Additional synapomorphies come from embryology, chemistry, and phloem characters (see Price, 1989 for details), rbcL (Chase et al. 1993), and nuclear 18S rRNA (Chaw et al. 1997) sequences. Price (1989) reviewed the history of Pinaceae subfamilial classification. He concluded that either two subfamilies (Table 1) or three subfamilies (Abietoideae, Laricoideae and a monogeneric Pinoideae) were “reasonable alternatives.” Frankis (1989) recognized four subfamilies: Abietoideae, Laricoideae (containing Cathaya, Larix and Pseudotsuga) and monogeneric Piceoideae and Pinoideae. We prefer a more broadly circumscribed Pinoideae (Table 1). Proc 4 IS on Conifer

Pseudolarolides O (1) and P (2), two novel triterpenoids with a cycloartane-type framework, were isolated from the seeds of Pseudolarix kaempferi Gord. (Pinaceae). Their structures were elucidated as (16R,23S,25R)-16,23-epoxy-3,4 : 9,10-disecocycloartan-1(10),9(11)-diene-3(4),26(23)-diolide (1), and (9S,16R,23S,25R)-1,9 : 16,23-diepoxy-8,9 : 9,10-disecocycloartan-1(29),5(6),10(19)-triene-3(4),26(23)-diolide (2), respectively, on the basis of spectroscopic techniques and X-ray-diffraction studies.

The phytochemical investigation of the root bark of Pseudolarix kaempferi yielded eight eudesmane-type sesquiterpenoids, including three new ones, 1α-hydroxyl-4(14)-en-β-dihydroagarafuran (1), 1α, 2α-diacetoxy-8β-isobutanoyloxy-9α-benzoyl oxy-15-β-(β-furancarbonyloxy)-4β, 6β-dihydroxy-β-dihydroagarofuran (7), and 1α-acetoxy-2 α-furancarbonyloxy-8β-isobutanoyloxy-9α-benzoyloxy-15β-(β-acetoxy)-4β, 6β-dihydroxy-β-dihydroagarofuran (8). Herein the new compounds 7 and 8 were reported as a mixture. The molecular structures of the isolated compounds were elucidated on the basis of extensive spectroscopic analysis, including UV, IR, NMR, and MS, and comparison with the literature data.

Five new diterpenoids, pseudolaric acids F (1), G (2), and H (3), 2',3'-dihydroxy-1'-propoxypseudolarate B (4), and 6'-O-acetylpseudolaric acid B O-beta-D-glucopyranoside (5), along with nine known diterpenoids, pseudolaric acids A, B, and C(1), deacetylpseudolaric acid C(2), deacetylpseudolaric acid A, methyl pseudolarate A, methyl pseudolarate B, pseudolaric acid A-O-beta-D-glucopyranoside, and pseudolaric acid B-O-beta-D-glucopyranoside, were isolated from the root bark of Pseudolarix kaempferi. Their structures and stereochemistry were elucidated mainly by spectral data, especially 2D NMR techniques.

The seed fatty acid (FA) compositions of Abietoids (Abies, Cedrus, Hesperopeuce, Keteleeria, Pseudolarix, and Tsuga) are reviewed in the present study in conclusion to our survey of Pinaceae seed FA compositions. Many unpublished data are given. Abietoids and Pinoids (Pinus, Larix, Picea, and Pseudotsuga)-constituting the family Pinaceae-are united by the presence of several delta5-olefinic acids, taxoleic (5,9-18:2), pinolenic (5,9,12-18:3), coniferonic (5,9,12,15-1 8:4), keteleeronic (5,11-20:2), and sciadonic (5,11,14-20:3) acids, and of 14-methyl hexadecanoic (anteiso-17:0) acid. These acids seldom occur in angiosperm seeds. The proportions of individual delta5-olefinic acids, however, differ between Pinoids and Abietoids. In the first group, pinolenic acid is much greater than taxoleic acid, whereas in the second group, pinolenic acid is greater than or equal to taxoleic acid. Moreover, taxoleic acid in Abietoids is much greater than taxoleic acid in Pinoids, an apparent limit between the two subfamilies being about 4.5% of that acid relative to total FA. Tsuga spp. appear to be a major exception, as their seed FA compositions are much like those of species from the Pinoid group. In this respect, Hesperopeuce mertensiana, also known as Tsuga mertensiana, has little in common with Abietoids and fits the general FA pattern of Pinoids well. Tsuga spp. and H. mertensiana, from their seed FA compositions, should perhaps be separated from the Abietoid group and their taxonomic position revised. It is suggested that a "Tsugoid" subfamily be created, with seed FA in compliance with the Pinoid pattern and other botanical and immunological criteria of the Abietoid type. All Pinaceae genera, with the exception of Pinus, are quite homogeneous when considering their overall seed FA compositions, including delta5-olefinic acids. In all cases but one (Pinus), variations from one species to another inside a given genus are of small amplitude. Pinus spp., on the other hand, have highly variable levels of delta5-olefinic acids in their FA compositions, particularly when sections (e.g., Cembroides vs. Pinus sections) or subsections (e.g., Flexiles and Cembrae subsections from the section Strobus) are compared, although they show qualitatively the same FA patterns characteristic of Pinoids. Multicomponent analysis of Abietoid seed FA allowed grouping of individual species into genera that coincide with the same genera otherwise characterized by more classical botanical criteria. Our studies exemplify how seed FA compositions, particularly owing to the presence of delta5-olefinic acids, may be useful in sustaining and adding some precision to existing taxonomy of the major family of gymnosperms, Pinaceae.