Population genetic structure and demography of Magnolia kobus: variety borealis is not supported genetically.

Species delimitations by morphological and by genetic markers are not always congruent. Magnolia kobus consists of two morphologically different varieties, kobus and borealis. The latter variety is characterized by larger leaves than the former. For the conservation of M. kobus genetic resources in natural forests, the relationships between morphological and genetic variation should be clarified. We investigated variations in nuclear microsatellites, chloroplast DNA (cpDNA) sequences and leaf morphological traits in 23 populations of M. kobus over the range of species. Two genetically divergent lineages, northern and southern were detected and their geographical boundary was estimated to be at 39°N. The northern lineage consisted of two genetic clusters and a single cpDNA haplotype, while the southern one had multiple genetic clusters and cpDNA haplotypes. The northern lineage showed significantly lower genetic diversity than the southern. Approximate Bayesian computation indicated that the northern and southern lineages had experienced, respectively, population expansion and long-term stable population size. The divergence time between the two lineages was estimated to be 565,000 years ago and no signature of migration between the two lineages after divergence was detected. Ecological niche modeling showed that the potential distribution area in northern Japan at the last glacial maximum was very small. It is thus considered that the two lineages have experienced different population histories over several glacial-inter-glacial cycles. Individuals of populations in the central to northern part of Honshu on the Sea of Japan side and in Hokkaido had large leaf width and area. These leaf characteristics corresponded with those of variety borealis. However, the delimitation of the northern and southern lineages detected by genetic markers (39°N) was not congruent with that detected by leaf morphologies (36°N). It is therefore suggested that variety borealis is not supported genetically and the northern and southern lineages should be considered separately when identifying conservation units based not on morphology but on genetic markers.

Genetic patterns in Neotropical Magnolias (Magnoliaceae) using de novo developed microsatellite markers.

Conserving tree populations safeguards forests since they represent key elements of the ecosystem. The genetic characteristics underlying the evolutionary success of the tree growth form: high genetic diversity, extensive gene flow and strong species integrity, contribute to their survival in terms of adaptability. However, different biological and landscape contexts challenge these characteristics. This study employs 63 de novo developed microsatellite or SSR (Single Sequence Repeat) markers in different datasets of nine Neotropical Magnolia species. The genetic patterns of these protogynous, insect-pollinated tree species occurring in fragmented, highly-disturbed landscapes were investigated. Datasets containing a total of 340 individuals were tested for their genetic structure and degree of inbreeding. Analyses for genetic structure depicted structuring between species, i.e. strong species integrity. Within the species, all but one population pair were considered moderate to highly differentiated, i.e. no indication of extensive gene flow between populations. No overall correlation was observed between genetic and geographic distance of the pairwise species' populations. In contrast to the pronounced genetic structure, there was no evidence of inbreeding within the populations, suggesting mechanisms favouring cross pollination and/or selection for more genetically diverse, heterozygous offspring. In conclusion, the data illustrate that the Neotropical Magnolias in the context of a fragmented landscape still have ample gene flow within populations, yet little gene flow between populations.

Evolutionary Comparisons of the Chloroplast Genome in Lauraceae and Insights into Loss Events in the Magnoliids.

Available plastomes of the Lauraceae show similar structure and varied size, but there has been no systematic comparison across the family. In order to understand the variation in plastome size and structure in the Lauraceae and related families of magnoliids, we here compare 47 plastomes, 15 newly sequenced, from 27 representative genera. We reveal that the two shortest plastomes are in the parasitic Lauraceae genus Cassytha, with lengths of 114,623 (C. filiformis) and 114,963 bp (C. capillaris), and that they have lost NADH dehydrogenase (ndh) genes in the large single-copy region and one entire copy of the inverted repeat (IR) region. The plastomes of the core Lauraceae group, with lengths from 150,749 bp (Nectandra angustifolia) to 152,739 bp (Actinodaphne trichocarpa), have lost trnI-CAU, rpl23, rpl2, a fragment of ycf2, and their intergenic regions in IRb region, whereas the plastomes of the basal Lauraceae group, with lengths from 157,577 bp (Eusideroxylon zwageri) to 158,530 bp (Beilschmiedia tungfangensis), have lost rpl2 in IRa region. The plastomes of Calycanthus (Calycanthaceae, Laurales) have lost rpl2 in IRb region, but the plastome of Caryodaphnopsis henryi (Lauraceae) remain intact, as do those of the nonLaurales magnoliid genera Piper, Liriodendron, and Magnolia. On the basis of our phylogenetic analysis and structural comparisons, different loss events occurred in different lineages of the Laurales, and fragment loss events in the IR regions have largely driven the contraction of the plastome in the Lauraceae. These results provide new insights into the evolution of the Lauraceae as well as the magnoliids as a whole.

Identification of Magnolia wufengensis (Magnoliaceae) cultivars using phenotypic traits, SSR and SRAP markers: insights into breeding and conservation.

A combination of phenotypic characterization and molecular markers may provide reliable information on new plant varieties and elucidate the conservation status of rare species. Five newly developed Magnolia wufengensis cultivars, an endangered plant species endemic to Hubei Province, China, possess more distinctive phenotypes than common Magnolia cultivars. With reference to a wild species population of M. wufengensis and a population of Magnolia denudata, morphological traits of flower organs, simple sequence repeat (SSR), and sequence-related amplified polymorphism (SRAP) markers were used. In the morphological study, six traits of floral organs were investigated and their relationships were analyzed between cultivars. In the genetic study, 9 SSR primer pairs and 10 SRAP primer combinations were screened. The five cultivars maintained a high level of genetic diversity. Genetic diversity of each M. wufengensis cultivar was much lower than that of the wild population, but was slightly higher than that of the M. denudata population. Analysis of molecular variance (AMOVA) revealed that genetic variation among populations was 20% (SRAP) and 30% (SSR), which showed a high degree of genetic differentiation among populations of the five cultivars. The dendrograms illustrated a clear separation between M. wufengensis populations and outer species, and identified two major groups among cultivars. Correlation analysis indicated a good fit between the two marker systems, but a relatively low fit between morphological and genetic traits (SRAP: r = 0.60, SSR: r = 0.52). These findings provide reliable references for the application of these molecular markers in the breeding and conservation of M. wufengensis.

Development of microsatellite markers in Parakmeria nitida (Magnoliaceae).

PREMISE OF THE STUDY: We developed microsatellite makers for Parakmeria nitida to investigate its population structure and conservation genetics. METHODS AND RESULTS: A total of 25 microsatellite primer pairs were developed using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, and polymorphism was assessed in three natural populations of P. nitida. Among these markers, 11 were monomorphic and 14 showed polymorphism. CONCLUSIONS: These markers are potentially useful for future population genetic analyses of P. nitida and will serve as an important tool for conservation efforts.

[High-throughput pyrosequencing of the complete chloroplast genome of Magnolia officinalis and its application in species identification].

Chloroplast genome sequences have comprehensive application prospects in DNA barcoding and chloroplast engineering in traditional Chinese medicine. The complete chloroplast genome of Magnolia officinalis sequenced by high-throughput pyrosequencing and a sequencing procedure was established. Fourteen contigs were obtained after de nove assembly. The sequencing percent of coverage was 99.99%. The chloroplast genome is 160 183 bp in size, and has a typical quadripartite structure with the large (LSC, 88 210 bp) and small copy (SSC, 18 843 bp) regions separated by two copies of an inverted repeat (IRs, 26 565 bp each). chloroplast genes were successfully annotated, of which 17 genes located in each IR region. The chloroplast genome features in Magnolia officinalis are nearly identical to those from other Magnoliid chloroplast genomes. Phylogenetic analyses were performed based on 81 shared coding-genes for a total of 9 Magnolia samples of 5 closely related species. Results showed that distinguishing among species was generally straightforward at the species and population level. This study confirmed the effectiveness of our chloroplast genome sequencing procedure. The chloroplast genome can provide distinguishing differences to help identify Magnolia officinalis and its closely related plants.

[Comparative study of effect of Magnolia officinalis and M. officinalis var. biloba on gastrointestinal motility dysfunction].

OBJECTIVE: To compare the effect of Magnolia officinalis and M. officinalis var. biloba on gastrointestinal motility dysfunction. METHOD: The gastrointestinal motility dysfunction induced by atropine and L-Arg were used in this study. The effect of M. officinalis on the gastrointestinal function was evaluated by propulsive motility of the total gastrointestinal tract test by recording the time of excreting carbo medicinalis, the gastric residualrate, the intestinal propulsion rate, the serum levels of D-xylose, motilin (MTL) and gastrin (GAS). ED50 were calculated with Bliss's method. RESULT: M. officinalis and M. officinalis var. biloba improved the gastrointestinal motility, decreased the gastric residual rate, enhanced the intestinal propulsion rate and increased serum levels of D-xylose, motilin (MTL) and gastrin(GAS). The ED50 value of M. officinalis var. biloba was much higher than that of M. officinalis. CONCLUSION: The effect of improving gastrointestinal motility of M. officinalis was proved to be superior to that of M. officinalis var. biloba.

[Population genetic study of Magnolia officinalis and Magnolia officinalis var. biloba].

OBJECTIVE: population genetic study of Magnolia officinalis and M. officinalis var. biloba was performed in this paper. The objectives of this study are to provide baseline data of molecular pharmaceutics for quality control of Cortex Magnoliae Officinalis. METHOD: A total of 15 populations of M. officinalis and M. officinalis var. biloba were collected. PCR amplification and sequencing were performed with two chloroplast intergenic spacers psbA-trnH and trnL-trnF. Chloroplast haplotype frequencies were calculated, genetic diversity and genetic structure were estimated by using the program HAPLONST, and a haplotype network depicting the mutational relationships among distinct haplotypes was drawn following the principle of parsimony by TCS version 1.13. RESULT: The differentiation of haplotype frequencies between M. officinalis and M. officiunalis var. biloba was significant although there was no private haplotypes of themselves. CONCLUSION: There was certain genetic divergence between M. officinalis and M. offcinalis var. biloba although the monophyletic clade of themselves was not still formed.

Honokiol and magnolol production by in vitro micropropagated plants of Magnolia dealbata, an endangered endemic Mexican species.

An efficient protocol for the in vitro propagation of Magnolia dealbata Zucc., an important medicinal plant that is the source of the anxiolytic and anticancer compounds honokiol and magnolol, was established. This plant is wild-crafted, and conservationists have expressed concerns with regard to the sustainability of production. In the present work, two factors were found to be of importance for the regeneration of M. dealbata and the production of honokiol and magnolol. These factors were the type of explants and the combination and concentration of plant-growth regulators. Green, compact, nodular organogenic callus was obtained from leaf explants in a medium fortified with Murashige and Skoog salts and supplemented with 1.5 mg/L 2,4-dicholorophenoxyacetic acid and 1.5 mg/L kinetin. Shoots multiplication from callus cultures was achieved in the Murashige and Skoog (MS) medium with 1.5 mg/L thidiazuron (TDZ). Phenol secretion was controlled by the addition of 250 mg/L of activated charcoal. For rooting, shoots were transferred to MS medium supplemented with several auxins. After root induction, the plants were hardened in earthen pots containing sand, soil, and vermiculite. The contents of honokiol (HK) and magnolol (MG) were determined in different plant materials by high-performance liquid chromatography-diode-array detection techniques. This analysis revealed that the honokiol and magnolol content in aerial and underground parts of micropropagated M. dealbata were higher than that observed in wild plants (both 6 months old). Our results suggest that conservation of M. dealbata is possible by means of in vitro multiplication of leaf-derived callus. The usefulness of M. dealbata regeneration and production of HK and MG may be attributed to the proper selection of explant sourcing and identification of the correct growth medium to support adequate growth. This careful selection of explants and growth medium leads to a very useful source of plant material for pharmacological and phytomedicinal screening applications and, above all, would safeguard this plant species from the threat of extinction.

[Identification of the different cultivated Magnolia biondii].

OBJECTIVE: To distinguish Magnolia biondii of ten different cultivated. METHODS: The original species can be identified by the morphological characteristics and microscopic characteristics. The volatile oil is determinated by the method of Chinese pharmacopoeia. RESULTS: The ten cultivated Mangolia biondii have obviously differents in morphological characteristics, microscopic characteristics and volatile oil. CONCLUSION: It is important for development application of Magnolia biondii and variety improvement.

[Construction on ISSR fingerprint of Magnolia officinalis from Enshi in Hubei province].

OBJECTIVE: To establish the ISSR fingerprint of the cortex of Magnolia officinalis produced from Enshi. METHODS: Cultivar and habit identification was based on Inter-simple sequence repeats (ISSR). RESULTS: Two ISSR primes could be used to construct the ISSR fingerprint of M. officinalis from Enshi. CONCLUSION: The ISSR marker is an effective method for the cultivar and habit identification of the cortex of M. officinralis.

Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae).

Magnolia officinalis subsp. biloba, a traditional Chinese medicinal plant, experienced severe declines in the number of populations and the number of individuals in the late 20th century due to the widespread harvest of the subspecies. A large-scale cultivation program was initiated and cultivated populations rapidly recovered the loss in individual plant numbers, but wild populations remained small as a consequence of cutting. In this study, the levels of genetic variation and genetic structure of seven wild populations and five domestic populations of M. officinalis subsp. biloba were estimated employing an AFLP methodology. The plant exhibited a relatively high level of intra-population genetic diversity (h = 0.208 and H(j) = 0.268). The cultivated populations maintained approximately 95% of the variation exhibited in wild populations, indicating a slight genetic bottleneck in the cultivated populations. The analysis of genetic differentiation revealed that most of the AFLP diversity resided within populations both for the wild group (78.22%) and the cultivated group (85.92%). Genetic differentiation among populations in the wild group was significant (F(ST) = 0.1092, P < 0.005), suggesting wild population level genetic structure. Principal coordinates analysis (PCO) did not discern among wild and cultivated populations, indicating that alleles from the wild population were maintained in the cultivated gene pool. Results from the present study provide important baseline data for effectively conserving the genetic resources of this medicinal subspecies.

[Phenols in seedling cortex of Magnolia officinalis from Enshi].

OBJECTIVE: To study the phenols in Cortex of Magnolia officinalis of biernial seedings produced in Enshi, Hubei. METHODS: The content of magnolol and honokiol in cortexes were determined by HPLC. The chromatograms of 10 samples seedling cortexes were recorded and compared. RESULTS: The content of magnolol and honokiol in Cortex of Magnolia officinalis of the seedlings from Enshi was higher than other samples. There were ten characteristic absorption bands in the HPLC chromatograms, which differed from the cortex of adult trees. CONCLUSION: The results can be used to identify the quality of the seedlings for the breeding.

Inhibitions of mast cell-derived histamine release by different Flos Magnoliae species in rat peritoneal mast cells.

Flos Magnoliae (FM) is a commonly used Chinese medicinal herb for symptomatic relief of allergic rhinitis, sinusitis and headache. A number of FM species have been used as substitutes or adulterants for clinical application, although the differences in their pharmacological actions have not been reported. The present study investigated the effects of six identified FM species M. biondii, M. denudata, M. kobus, M. liliflora, M. sargentiana and M. sprengeri, as well as the marker compounds magnolin and fargesin on compound 48/80-induced histamine release in rat peritoneal mast cells (RPMC) in vitro. Ethanolic extracts of all FM species produced a concentration-dependent inhibition of compound 48/80-induced histamine release in RPMC. The rank order of the IC(50)s was M. biondii

[Photosynthesis of five magnolia species in Nanjing City in winter and spring].

The photosynthesis and fluorescence characteristics of five magnolia ornamental species naturally distributed in various subtropical areas were studied in Nanjing City in winter and spring. The results showed that the diurnal changes of net photosynthetic rate (P(n)) and water use efficiency (WUE) of test species in winter were different from those in spring. The diurnal integral values of P(n), apparent quantum yield (AQY) and carboxylation efficiency (CE) were lower in winter than in spring, and the F(o) was higher, while the F(v)/F(m) F(v)/F(o) phi PS II, F(v)'/F(m)', ETR, qP and NPQ were lower in winter than in spring. The differences among the five species were remarkable, among which, Parakmeria lotungensis and Michelia platypetala mainly distributed in middle-subtropical area had higher diurnal integral values of P(n), AQY, CE and LSP, but lower value of LCP than other species in winter and spring. Their F(v)/F(m), F(v)/F(o),phi PS II, F(v)'/F(m)', ETR, qP and NPQ were also higher, indicating that they had higher photosynthetic capacity and wider ecological ranges of light adaptability. Manglietia insignis and Michelia wilsonii mainly distributed in southern subtropical area had lower photosynthetic capacity, and their fluorescence parameters were also lower in winter. Grey correlation analysis showed the main factors affecting the P(n) of test magnolia species in winter were T(a) and PAR.

[Identification of Magnolia biondii Pamp by X-Ray diffraction Fourier pattern method].

OBJECTIVE: To establish a new identification and analysis method of Magnolia biondii Pamp. METHODS: Powder X-Ray diffraction fourier fingerprint pattern method. RESULT: Experiment and analysis were carried out on five samples. The standard X-Ray diffraction Fourier fingerprint pattern method and characteristic diffraction peaks of Magnolia biondii Pamp were obtained. CONCLUSION: The method can be used for the identification of Magnolia biondii Pamp.

Nuclear ribosomal DNA sequence variation and evolution of spotted marsh-orchids (Dactylorhiza maculata group).

Sequences of both internal and external transcribed spacers of nuclear ribosomal DNA were sequenced for four species belonging to the Dactylorhiza maculata group or "spotted marsh-Orchids". These four species are D. fuchsii, D. saccifera, D. foliosa, and D. maculata. Extensive nuclear ribosomal DNA polymorphism was uncovered within the diploid D. fuchsii and the putative autotetraploid D. maculata. Within the phylogenetic trees reconstructed using parsimony and Bayesian analyses, four main lineages (A, B, C, and D) were well supported. While D. saccifera, D. maculata, and D. foliosa were confined to clades B, C, and D, respectively, D. fuchsii accessions were spread over three clades (A, B, and C). Lineage C, which included accessions of the diploid D. fuchsii and the tetraploid D. maculata, was closely related to the lineage of D. foliosa (lineage D), an endemic diploid species from Madeira. Moreover, intra-individual polymorphism was found within accessions of D. maculata, D. fuchsii, and D. saccifera. It is shown that in some instances two lineages, contributed to the observed intra-individual polymorphism (C and A in D. maculata, A and B in D. fuchsii and D. saccifera). Evolutionary scenarios leading to this extensive nuclear ribosomal DNA polymorphism are discussed in the light of results from maternally inherited chloroplast DNA markers and an autopolyploid origin of D. maculata from a D. foliosa-like ancestor is postulated.

[Studies on random amplified polymorphic DNA fingerprinting of Cortex Magnoliae Officinalis].

OBJECTIVE: To identify Chinese traditional medicine (CTM) "Hou-pu" (Cortex Magnoliae Officinalis), its counterfeits and substitues. METHODS: Total genomic DNA samples of ten plant species were amplified by randomly amplified polymorphic DNA (RAPD). RESULTS: Ten samples were able to be distinguished through their amplified DNA banding patterns on the agarose gels after electrophoresis. CONCLUSION: RAPD is able to identify "Hou-pu", its counterfeits and substitutes quickly and truly, which is also quite valuable for correctly introducing plant.

[Research on DNA molecular marker of Magnolia officinalis Rehd. et Wils.--RAPD study on certified species].

AIM: To discuss the intraspecific relationship in Magnolia officinalis and the genuineness of Cortex Magnoliae officinalis, and to find some DNA characters of certified "Houpo". METHODS: Thirty-three samples from eleven locations, which can represent most of the distribution of M. officinalis, were selected. The total DNA was extracted. Severty-four random primers were tried to get good amplification. RESULTS: One hundred and sixteen bands amplified from seventeen primers, were clustered by NTSYS-pc software. Three branches were obtained. Some distinctive primers and bands, which represent certified species or fine breed, were obtained also. CONCLUSION: 1) M. officinalis should be divided into three geographic clans instead of two subspecies or varieties, they are, a) typical officinalis, b) typical biloba and c) Middle type. This conclusion agrees with the leaf form and other characters. 2) The genetic difference between "Chuanpo" and "Wenpo" is evident and the difference is in correspondence with the quantities of their chemical constituents. So, the genetic difference is the main reason of the genuineness of Cortex Magnoliae officinalis. 3) These results may be used to establish DNA database for identification of Cortex Magnoliae officinalis.

[Study on genuineness of traditional Chinese medicine cortex Magnoliae officinalis].

It's found that a significant correlation between the samples of Magnoliae officinalis from the provenance in phenols content and varieties or forms. The total content of magnolol and honokiol in its bark is usually used as an index to measure the quality. Therefore, Cortex Magnoliae officinalis produced in Sichuan and Hubei being considered genuinenss and good in quality, should be actually referred to local varieties. This conclusion can also reveals the nature and scientific connotation of genuineness on Cortex Magnoliae Officinalis.