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Big tree rhododendron, Rhododendron protistum var. giganteum (Ericaceae) is an endangered and important germplasm source with high ornamental value in China. To design appropriate germination and seedling establishment protocols for a species conservation and restoration program, we studied the effects of temperature, photoperiod, storage, and gibberellic acid (GA(3)) treatment on seed germination in a laboratory experiment. The effects of soil substrate and sowing depth on seedling emergence were studied in a greenhouse experiment. We hypothesized that photoperiod is a limiting factor for seed germination of big tree rhododendron, and soil type and sowing depth would affect seedling emergence. Results showed that seeds of big tree rhododendron were non-dormant, and temperature significantly affected germination percentages (GP) and mean germination time (MGT). The optimal temperatures for seed germination were relatively low (15 degrees C and 20 degrees C). High temperature negatively affected seed germination. The seeds had less than 5% of GP when incubated in complete darkness, suggesting that darkness significantly inhibits seed germination. Storage period and temperature had significant effect on GP and MGT of big tree rhododendron seeds, and the effect of their interaction on MGT was also significant. GA(3) treatment increased GP and germination speed. Soil substrates did not significantly affect seedling emergence, but native soil is beneficial to seedling growth. When the seeds were sown on the soil surface, seedling emergence was higher than 80%, but it decreased with increasing sowing depth, and no seedling emerged at 3 cm soil depth. We present some suggestions for the ex situ conservation and restoration program, including seed germination and seedling artificial propagation of big tree rhododendron based on the research results. (C) 2015 Elsevier GmbH. All rights reserved.

By emitting strong scents resembling rotting organic materials suitable for oviposition and/or foraging of flies, sapromyiophilous flowers mimic the substrates that attract flies as pollinators. It has been suggested that the wide range of volatile organic compounds emitted by this deceptive pollination system reflects the trophic preferences of flies to different types of substrate, including herbivore and carnivore feces, carrion, and fruiting bodies of fungi. Previous studies suggest that floral scents play a particularly important role in sapromyiophily. However, few studies on the relative importance of floral color or synergy between visual and olfactory cues in sapromyiophily have been substantiated. In this study, we analyzed fetid floral odor, floral pigment composition, and reflectance of an Amorphophallus konjac C. Koch inflorescence, and we conducted bioassays with different visual and/or olfactory cues to explore an unsubstantiated color profile in sapromyiophily: mimicking livor mortis. Our analysis showed A. konjac can emit oligosulphide-dominated volatile blends similar to those emitted by carrion. Necrophagous flies cannot discriminate between the color of an inflorescence, livor mortis, and floral pigments. We concluded that mimicking livor mortis may represent a common tactic of pollinator attraction in "carrion flower" systems within angiosperms.

The newly discovered endemic maple Acer yangbiense of China has only five individuals left in the wild, and thus has been classified as a plant species with extremely small populations (PSESP). PSESP species call for emergency protection procedures, such as ex-situ conservation and reintroduction. Our objectives were to examine the genetic diversity of A. yangbiense and to evaluate former conservation strategies from a genetic point of view. Our results suggested that A. yangbiense was not genetically depauperate, but its genetic loss at a species level was obvious. A parentage analysis indicated a high selfingrate in A. yangbiense and suggested the existence of a previously unknown wild individual. Former conservation strategies did not include all genetic variations of the wild population, and gene diversity of the ex-situ conserved seedlings is lower than that of the wild population. From our findings, we make suggestions to guide the subsequent protection of this species. (C) 2015 Elsevier Ltd. All rights reserved.

Magnolia sinica, a Critically Endangered tree endemic to Yunnan, China, is one of the 20 plant species with extremely small populations approved by the Yunnan government for urgent rescue action before 2015. Information on the geographical distribution and population size of this species had not previously been reported, hindering effective conservation. We therefore carried out a survey of the literature and of herbarium specimens, followed by a detailed field survey and morphological measurements and observations of surviving individuals. We located 52 individuals in the wild, in eight localities. Two distinguishing morphological characters (tepal colour and tepal number) were revised based on observations of all remaining wild individuals that produced flowers and on one 30-year-old flowering plant in Kunming Botanical Garden. The survival rate of individuals propagated from seed for ex situ conservation at the Garden was 100% over 5 years; of 100 individuals transplanted to each of two reinforcement sites, 20 and 18, respectively, were alive after 6 years. We propose two groups of measures to protect M. sinica: (1) in situ conservation, population monitoring, and public engagement, and (2) ex situ conservation with reinforcement or reintroduction.

By emitting scent resembling that of organic material suitable for oviposition and/or consumption by flies, sapromyophilous flowers use these flies as pollinators. To date, intensive scent analyses of such flowers have been restricted to Apocynaceae, Annonaceae, and Araceae. Recent studies have suggested that the wide range of volatile organic compounds (VOCs) from sapromyophilous flowers play an important role in attracting saprophagous flies by mimicking different types of decomposing substrates (herbivore and carnivore feces, carrion, and the fruiting bodies of fungi, etc.). In this study, we report the flower visitors and the floral VOCs of Stemona japonica (Blume) Miquel, a species native to China. The flowers do not produce rewards, and pollinators were not observed consuming pollen, thus suggesting a deceptive pollination system. Headspace samples of the floral scent were collected via solid-phase micro-extraction and analysed by gas chromatography coupled with mass spectrometry. Main floral scent compounds were 1-pyrroline (59.2 %), 2-methyl-1-butanol (27.2 %), and 3-methyl-1-butanol (8.8 %), and resulted in a semen-like odor of blooming flowers. The floral constituents of S. japonica were significantly different from those found in previous sapromyophilous plants. An olfaction test indicated that 1-pyrroline is responsible for the semen-like odor in S. japonica flowers. Main flower visitors were shoot flies of the genus Atherigona (Muscidae). Bioassays using a mixture of all identified floral volatiles revealed that the synthetic volatiles can attract Atherigona flies in natural habitats. Our results suggest that the foul-smelling flowers of S. japonica may represent a new type of sapromyophily through scent mimicry.

Buddleja crispa Benth. is one of the most morphologically variable species in genus Buddleja, and it is widely distributed in the Himalaya-Hengduan Mountains (HHM) region. This study used AFLPs as a tool to examine the genetic variation among and within 25 populations of B. crispa. Analysis of population genetics of the species aimed to clarify morphological variation, current distribution patterns, strong adaptability to habitats, and the effects of geological factors in the HHM region. The genetic structure results, based on PCoA and NJ cluster analyses, revealed that the populations of B. crispa were divided into two genetic groups. Furthermore, the peripheral populations had lower genetic diversity than the populations in the center of the distribution areas (Three Parallel Rivers). We conclude that the gene flow (predominantly seed and pollen flow) and the population differentiation of B. crispa might be more affected by the barriers formed by rivers and mountains than by geographic distance. (C) 2014 Elsevier Ltd. All rights reserved.

• Floral color is a key reproductive character, often associates with environmental adaptation, and undergoes human intervention. A large number of Rhododendron species spread widely with various flower colors, which makes a good model for flower coloration. The chromatic features and anthocyanin compositions of 30 species from seven subgenera were systematically analyzed. • The Royal Horticultural Society Color Chart and CIE L*a*b* system were employed to describe and investigate flower colors. The UPLC-PDA/ESI-MSn system was used to identify and quantify anthocyanins in petal extracts. • The flower colors of 30 Rhododendron species were categorized into four groups—red, purplish pink, purple and white. Seven anthocyanins were identified and quantified in petals, including delphinidin, cyanidin and malvidin 3-O-arabinoside-5-O-glucosides, cyanidin 3,5-di-O-glucoside, 3-O-galactoside and 3-O-arabinoside, and delphinidin 3-O-glucoside. The red-flowered species mainly contained cyanidin monoglycosides and had much higher total anthocyanin contents than purplish pink- and purple-flowered species. Purplish pink- and purple-flowered species had similar anthocyanin types and contents. The chromatic differences were significant among groups, except the purplish pink and purple groups. Statistical analysis showed that Cy3Gal and Cy3Arb are characteristic for red-flowered species, and Mv3Arb5G and Dp3Arb5G play important roles in purple coloration; their contents were major components that greatly affected the chromatic parameters. In total, 21 flavonols derivates were identified. However, total flavonols content and copigmentation index showed no significant difference or correlation among /with color groups, suggesting that flavonols might not play a major role in coloration. • These results enhance our knowledge of the biochemical basis of flower coloration in Rhododendron species, and will lay a foundation for genetic variation study and aid the breeding of cultivars with novel flower colors. This article is protected by copyright. All rights reserved.

Nine new indole alkaloids, vinmajines A-I (1-9), and 43 known indole alkaloids were isolated from cultivated Vinca major in Kunming. The new structures were elucidated by extensive spectroscopic and quantum theory analysis. In addition, the results also supported that types of indole alkaloids from V. major might be influenced significantly by the ecological environment. (C) 2014 Elsevier Ltd. All rights reserved.

In this study, floral color, scent composition and emission rate, nectar property, pollinators, and breeding system of dimorphic Buddleja delavayi Gagnep. were investigated. Flower color of B. delavayi was determined using a standard color chart and spectrophotometer, and two distinct color polymorphisms were observed having purple or white flowers. Floral scents of B. delavayi were collected using dynamic headspace adsorption and identified with coupled gas chromatography and mass spectrometry. In total, 28 compounds were identified from the flowers of B. delavayi. The identified scents were divided into three chemical classes based on their biosynthetic origin: terpenes, fatty acid derivatives, and benzenoids. The scent profiles in all individuals were dominated by a few components, such as lilac aldehyde and alcohol, 4-oxoisophorone, benaldehyde, and oxoisophorone oxide. Floral scent composition (benzenoids and terpenes) showed a significant difference between white and purple flower morphs. Flower color-flower scent associations in B. delavayi were identified with two distinct scent profiles in the two color phenotypes. The studies of other floral characteristics (nectar, floral visitors, breeding system, and fruit set) indicated that floral scent emission rate, nectar volume, visitor visitation frequency, and natural fruit set were not significantly different between the two flower color morphs. Bagging experiments revealed that seed production of B. delavayi is dependent mainly on honeybee Apis cerana. Lastly, this study implies that dimorphic floral color in B. delavayi may have been maintained by floral visitors and nectar guide color.

The pollination biology of Rhododendron cyanocarpum (Franch.) W. W. Smith, an alpine species endemic to NW Yunnan, China, was investigated between 2007 and 2008. Floral traits including flowering time, floral morphology, petal color, and floral scents were assessed, and the associated pollinator assemblage and their foraging behavior were recorded. The flowering period of R. cyanocarpum ranges from late March to middle May and a single flower can last 8-10 days in its natural habitat. Flowers generally show marked herkogamy and the protruding style encourages pollinator foraging behavior that counteracts self-pollination. Floral scents comprise mostly aliphatics (64.37%) and terpenoids (29.88%), which make the flowers attractive for several insect groups, but a peak at 430nm in the reflectance spectrum of petals suggest selection for the attraction of bumblebees. The two Bombus species, B. festivus and B. richardsiellus, representing 90% of total recorded visits, were the only insect species that can be considered to effectively pollinate R. cyanocarpum. Pollination treatments indicated the general self-compatibility of R. cyanocarpum; however, outcrossing seemed to be the dominant strategy, with low rates of self-fertilization providing a certain level of reproductive assurance in its alpine habitat.

Floral scents are important olfactory signals for communication between plants and pollinators. Several studies have focused on inter-specific variation of floral scents, but little is known about the intra-specific variation, especially in some polychromic species. In this study, we investigated the floral scent compositions of Buddleja fallowiana and Buddleja officinalis in situ by dynamic headspace collection and coupled GC-MS. Variations of scent compositions within and between populations as well as among species were compared. In spite of substantial intra- and inter-population variability, B. fallowiana and B. officinalis were clearly differentiated in their scent profiles. In B. fallowiana, obvious differentiation was found between studied populations, while all investigated populations in B. officinalis are part of a metapopulation. These high intra-specific variations are discussed in relation to the introgression through hybridization and founder effects from different populations. (C) 2014 Elsevier Ltd. All rights reserved.

The Rhododendron of China exhibit great diversity, however no conservation evaluation of the genus had been performed, as a result of no governmental financial support. Based on our recent field investigations covering a total of seven provinces and 95 days from 2011 to 2013 in China, we herein provide an updated conservation status on eight species plus two further species that are listed as endangered and vulnerable in the Red List of Rhododendrons in which the evaluation was mainly based on old records. Our surveys indicated that urgent conservation actions to the two critically endangered species R. magniflorum (less than 10 extant plants) and R. roxieoides (only 22 mature individuals) should be performed. Moreover, we proposed that species represented by few plants, and that grow at low altitude, in disturbed habitats, should be given priority for ex- and in-situ conservation. Despite current distributions of all listed endangered Rhododendrons were not fully covered, our report aimed to arouse conservation attention and actions on Rhododendrons preferably by governmental financial support in China.

The ecological roles and biological mechanisms of zoochory in plants have long been foci in studies of co-evolutionary processes between plants and animals. However, the dispersal of fungal spores by animals has received comparatively little attention. In this study, the dispersal of spores of a selected fetid fungus, Lysurus mokusin, via feces of mycophagous insects was explored by: collecting volatiles emitted by the fungus using dynamic headspace extraction and analyzing them by GC-MS; testing the capacity of mycophagous insects to disperse its spores by counting spores in their feces; comparing the germinability of L. mokusin spores extracted from feces of nocturnal earwigs and natural gleba of the fungus; and assessing the ability of L. mokusin volatiles to attract insects in bioassays with synthetic scent mixtures. Numerous spores were detected in insects' feces, the bioassays indicated that L. mokusin odor (similar to that of decaying substances) attracts diverse generalist mycophagous insects, and passage through the gut of Anisolabis maritima earwigs significantly enhanced the germination rate of L. mokusin spores. Therefore, nocturnal earwigs and diurnal flies probably play important roles in dispersal of L. mokusin spores, and dispersal via feces may be an important common dispersal mechanism for fungal reproductive tissue.

Natural hybridization was assumed to play an essential role for the diversification of Primula; however, only one study of hybridization in the region of the Himalayas has been undertaken. In the present study, we examined another natural hybrid zone where morphologically putative hybrids as well as P. beesiana Forrest, P. bulleyana Forrest, and P. poissonii Franch. co-occurred. We used molecular data to confirm the parental species of putative hybrids and the unidirectional hybridization pattern between P. beesiana and P. bulleyana. Moreover, with reference to synthetic F(1)s, most hybrids examined are possibly advanced generations, although the possibility of F-1 hybrids currently examined could not be completely excluded. In addition, pollinator observations on experimental arrays of transplanted parental species showed interspecific pollen flows during visitations of shared pollinators, indicating an incomplete pre-zygotic barrier between P. beesiana and P. bulleyana. Seed productions from both flower morphs of putative hybrids were significantly lower than parental species, suggesting lower reproductive success in these hybrids. Combined with the evidence of recent habitat disturbance in the study area, we might witness the early process of hybridization between P. beesiana and P. bulleyana.

In this study, flower color, nectar properties, and inflorescence scent composition of eight natural and one introduced Buddleja davidii populations were investigated. Flower color of B. davidii was determined using the Royal Horticultural Society Color Chart and ranged from purple to white. Volume of nectar produced by a single flower ranged from 0.36 mu l to 0.64 mu l and total sugar concentration produced by inflorescence ranged from 17.0% to 33.5% in all populations. Floral nectar volume and sugar concentration were not significantly different between two flower color morphs in the B. davidii populations. Floral scents of B. davidii were collected using dynamic headspace adsorption and identified with coupled gas chromatography and mass spectrometry. In total, 33 compounds were identified from the inflorescences of B. davidii. The identified scents were divided into five chemical classes based on their biosynthetic origin: irregular terpenes, monoterpenoids, sesquiterpenoids, fatty acid derivatives, and benzenoids. The scent profiles in all populations were dominated by few components, such as: 4-oxoisophorone, E,E-alpha-farnesene, and 1-octen-3-ol. Given that inflorescence scents from natural and introduced individuals coming from the same population have discrepant chemical composition, we infer that phenotype plasticity may mediate floral scent composition. Based on the comparison of present and other data available on floral scent in B. davidii, we conclude that inflorescence scent may serve as a specific signal helping to attract pollinating butterflies to locate flowers as nectar sources, and may have evolved in conjunction with the sensory capabilities of butterflies and moths as a specific group of pollinators. (C) 2014 Elsevier GmbH. All rights reserved.

RNA extraction of plant tissues (in our case Bauhinia leaves) using a pBIOZOL/CTAB lysis buffer

Over the past decades, biodiversity conservation in China has achieved a number of successes. However, due to inadequate conservation policies, poor implementation and lack of financial support, wild plant species that are extremely small in population size and therefore seriously threatened have not had the attention they require. But the new concept of plant species with extremely small populations (PSESP), first promulgated in Yunnan Province, is becoming more widely accepted in China. Several national and regional-level conservation strategies and actions for conserving China’s PSESP are being implemented over the next 5 years. With this new policy framework leading the way, plant conservation in China is set to make important new advances.

Mexican sunflower is a native species of North and Central America that was introduced into China early last century, but it has widely naturalized and become a harmful invasive plant in tropical and subtropical regions in South China. Inter-simple sequence repeat (ISSR) markers were employed to assess genetic diversity and variation in Mexican sunflower populations from China and neighboring regions. The karyotypes of populations were also studied. Our research showed high levels of genetic diversity in all populations. The lowest genetic diversity estimates were represented in two populations in Laos, suggesting prevention of new introductions into Laos is critical. Partitioning of genetic variance revealed that genetic variation was mostly found within populations, and unweighted pair group method with arithmetic means (UPGMA) analysis showed that the introductions into China and Laos were independent. There were no obvious correlations between genetic relationships and geographic distance of populations in China, consistent with the human associated dispersal history of Mexican sunflower. Previous cytological data and our chromosome count (2n = 34) and karyotype analysis showed chromosome stability among populations. The high levels of genetic diversity within invasive Mexican sunflower populations could be challenging for its management in China, and further expansion and potential negative effects on ecological systems of this plant should be monitored.

Long-term ecological and genetic studies in natural populations of tree species require marking techniques so that individuals can be re-visited over time, even in difficult terrain. Both GPS coordinates and physical labels have disadvantages that can make re-finding trees difficult. We tested passive and semi-active radio frequency identification (RFID) tags and readers as a means to relocate individual trees. Passive RFID tags do not provide a good solution because of low transmission power of hand-held readers and strong directionality. Semi-active RFID tags provide detection over longer distances, but also suffer from strong directionality. Active RFID tags promise an improvement over semi-passive tags, and could be evaluated in a future study. We conclude that RFID technology has the potential to improve the ability of researchers to locate individual trees repeatedly under natural conditions, and can be used in conjunction with other marking techniques such as physical tags and GPS coordinates.

Reproductive isolation (RI) plays an important role for speciation, but assessing reproductive barriers at all life-cycle stages remains challenging. In plants, most studies addressing the topic have been focusing on herbs with short generation times. The present study attempted to quantify several reproductive barriers between a hybridizing species pair of long-lived woody rhododendrons. Consistent with findings of previous studies, pre-zygotic reproductive barriers contributed more to total RI than post-zygotic reproductive barriers. Especially in the more widespread species geographic isolation was an important barrier and pollinator constancy contributed exceptionally to RI in both species. Additionally to strong pre-zygotic reproductive barriers, post-zygotic reproductive barriers were considerable and had asymmetric tendencies favoring one of the species as maternal parent. Overall, despite occasional hybridization, the present study provides evidence for strong RI between R. cyanocarpum and R. delavayi.

Floral scents from Buddleja lindleyana, Buddleja loricata, Buddleja cordata and Buddleja tubiflora were evaluated using the dynamic headspace adsorption method and identified with coupled gas chromatography and mass spectrometry. In total, 13 compounds were identified from the flowers of B. cordata, 19 from B. lindleyana and 29 from B. loricata, representing 95.5%, 93.1% and 96.2% of the total floral scents in the three species, respectively. No floral scents were recorded from B. tubiflora. Main volatile compounds in B. cordata were trans-beta-ocimene (53.4%), lilac alcohol (9.6%) and lilac aldehyde (5.6%). Main volatile compounds in B. lindleyana were beta-caryophyllene (17.6%), alpha-farnesene (16.0%) and 3-octanone (9.8%). Main volatile compounds in B. loricata were 4-oxoisophorone (27.1%), alpha-farnesene (17.9%) and linalool (10.6%). The study suggested that the floral scents may have evolved in conjunction with the sensory capabilities of different visitors as a specific group of pollinators in representative Buddleja species. (C) 2012 Elsevier Ltd. All rights reserved.

Background and aims - Reproductive success through autonomous self-fertilization is thought to be one of the main advantages in plants when effective pollinators are limited. The present study examined an autonomous self-fertilization mechanism in a perennial ginger, Hedychium yunnanense.

Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei's G(st) = 0.187 for ISSR and Wright's F(st) = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions.

Wild grapes are woody climbers, found mostly in temperate regions of the northern hemisphere, comprising the genus Vitis. Despite its importance, the evolutionary history of Vitis is still contentious. Past studies have led to conflicting hypotheses about the phylogeny, speciation events, and biogeographic history of the genus. Here we investigate the evolutionary history of Vitis using data from four chloroplast spacers (trnH-psbA, trnK-rps16, trnF-nahJ, and rp132-trnL) and the nuclear gene RPB2-I, and we explore mechanisms that could have shaped the observed distribution of current species. Maximum likelihood and Bayesian analyses provided similar results, strongly supporting the presence of two subgenera and suggesting a species clustering within subgenus Vitis that mainly mirrors the disjunction between the Old and New World. Vitis vinifera subsp. syivestris was found to be sister to the Asian species while three major clades were found in the American species. A network approach confirmed the main geographic groups and highlighted different chloroplast haplotype patterns between Asian and American species. Molecular dating analysis provided the time boundaries to discuss our results. Our study shows wild grape diversification to be a continuous and complex process that concerned the Tertiary as well as the Quaternary, most likely involving both geographical and climatic forces. Local variations in extent and timing of these forces were discussed based on observed differences between groups. In the context of the Tertiary-Quaternary debate, we provide evidence in favor of the "continuous hypothesis" to explain present diversity. Finally, two directions for future research are highlighted: (i) was the earliest grape American or Asian? and (ii) are all modern grape species real? (C) 2011 Elsevier Inc. All rights reserved.

Buddleja davidii is a widespread shrub in Asia while B. yunnanensis is a narrowly endemic species limited to Yunnan Province, China. To explore whether floral volatiles, morphological characters of flower and seed and breeding system are correlated with their distributions, we measured length and width of corolla, trichome density at corolla throat, level of stigma/anthers relationship, seed size and weight. The results indicated that these characteristics were significantly different between the two species (P < 0.01). Bagging experiments revealed that B. davidii is a self-incompatible plant while B. yunnanensis is self-compatible. Thick trichome density at the corolla throat may reduce out-crossing in B. yunnanensis. Autogamy plays an important role in fruit production of this species while B. davidii requires pollinators for fruiting. Scents were collected using dynamic headspace adsorption method and identified with coupled gas chromatography and mass spectrometry. In total, 27 floral scent compounds were identified. The volatile composition in the two species was very different. We attempted to determine if these features, associated with commonness and with rarity found in these two taxa, could also help to explain the distribution pattern of other species of the genus Buddleja. (C) 2011 Elsevier GmbH. All rights reserved.

Hibiscus aridicola (Malvaceae) is an endangered ornamental shrub endemic to the dry-hot valleys of Jinsha River in southwest China. Only four natural populations of H. aridicola exist in the wild according to our field investigation. It can be inferred that H. aridicola is facing a very high risk of extinction in the wild and an urgent conservation strategy is required. By using a modified biotin-streptavidin capture method, a total of 40 microsatellite markers were developed and characterized in H. aridicola for the first time. Polymorphisms were evaluated in 39 individuals from four natural populations. Fifteen of the markers showed polymorphisms with two to six alleles per locus; the observed heterozygosity ranged from 0.19 to 0.72. These microsatellite loci would be useful tools for population genetics studies on H. aridicola and other con-generic species which are important to the conservation and development of endangered species.

Three new clerodane diterpenoids, splendidins A - C (1 - 3, resp.), were isolated from Salvia splendens, together with six known ones. Their structures were elucidated by extensive spectroscopic analysis. Splendidin C (3) was the first diterpenoid glucoside reported from this plant. These compounds were evaluated for their cytotoxic activity; however, none of them were cytotoxic.

The leaf epidermis of all 23 species from Asian Buddleja was investigated with scanning electron microscopy (SEM). Characteristics of trichome type, stomatal shape, cuticular membrane on adaxial and abaxial epidermis, inner margin of outer stomatal ledge and ornamentation of outer stomatal ledge are presented in this study. Among the Buddleja species, some distinct epidermal patterns were observed. These can be used to characterize and identify different sections among the Asian Buddleja species, viz. Alternifoliae. Curviflorae and Rectiflorae. In general, stomatal length of Buddleja increases with ploidy level. This character can be used to distinguish diploids from polyploids without preparation of caryograms. Based on this correlation Buddleja microstachya, B. subcapitata and B. bhutanica are characterized to be diploids, as concluded from their stomatal dimensions. Leaf epidermis characters can also be used to differentiate species pairs that are easily confused with each other, viz., B. asiatica and B. bhutanica. B. albiflora and B. nivea, B. candida and B. fallowiana, B. delavayi and B. forrestii, B. japonica and B. lindleyana, B. officinalis and B. paniculata. (C) 2010 Elsevier GmbH. All rights reserved.

The giant tree rhododendron (R. protistum var. giganteum) is one of the most famous plants in China, and is critically endangered. As previous conservation efforts only focused on the two recorded populations of this variety, key issues regarding relatedness to R. protistum var. protistum still remain. Our field investigations and SSR analysis of the two varieties in China and Nepal suggest that the two varieties are not significantly different, and we suggest that limited conservation resources could be better used elsewhere given the numerous endangered plants urgently needing protection in China. We suspect that the problems we identified in this study will also apply to many other taxa in developing countries identified as endangered on the basis of incomplete information. Keywords: Rhododendron protistum var. giganteum, Conservation priority, Genetic structure

Cytological studies were carried out on two populations of Trigonobalanus verticillata in Yinggeling, Hainan Province, China (YGL), and Fraser’s Hill, Malaysia (FH). In the two populations, the pattern of interphase nuclei was of the simple chromocentre type, the mitotic prophases were of the proximal interstitial type and chromosome numbers were 2n = 2x = 14 (YGL) and 2n = 6x = 42 (FH), representing diploid and hexaploid respectively. The basic chromosome number of the species was x = 7; this is a unique number in the Fagaceae and in the Fagales as well. The characteristic karyotype of the diploid population in China was 2n = 14 = 8m + 4sm + 2st. Based on the new finding of the chromosome number in this study, we clarify the previous dispute about the basic chromosome number and propose that the two Asian Trigonobalanus species have closer affinity.

Buddleja macrostachya (Buddlejaceae) is a widespread shrub native to the Sino-Himalayan mountains and beyond. It has been found to occur at two ploidy levels, hexaploid, 2n=6x=114 and dodecaploid, 2n=12x=228. To determine if morphological characters might be used as indicators of ploidy levels, we measured floral and fruit length, relative and absolute leaf size, trichome density on both leaf surfaces, and stomatal density and length in different populations of B. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length increased with an increase at ploidy level (P 0.05) in different populations. Other characters studied such as trichome type, cuticular membrane and ornamentation of stomata, cell and stomatal shape, and anticlinal wall pattern were quite constant in this species. Thus it appears that flower and fruit length, absolute leaf size, and stomatal frequency and length can be used to distinguish hexaploid from dodecaploid cytotypes either in the field or in herbarium specimens.

As a critically endangered tree in the Magnoliaceae family, huagaimu (Manglietiastrum sinicum) is represented by only 10 mature individuals in evergreen broadleaved montane forests of southeastern Yunnan Province, China. Our previous work revealed the existence of a seed dormancy period for this species. The current study was performed to evaluate the effects of plant growth regulators (PGRs) and moist chilling on breaking seed dormancy in this species. Germination of seeds pretreated for 24 h with gibberellic acid (GA3), α-naphthaleneacetic acid, 6-benzyladenine, and 2,4-dichlorophenoxyacetic acid indicated that only GA3, at concentrations of 300 and 500 mg·L−1, can significantly break the seed dormancy of huagaimu after 50 days of incubation, with about 66% germination under 500 mg·L−1 GA3. Moist chilling at 4 °C for 3 weeks can also effectively break the seed dormancy of the species, with 56% of seeds treated in this way germinating after 30 days of incubation. The combined treatments of PGRs followed by moist chilling were also conducted. Based on germination results after 30 days of incubation, the seed germination of combined treatments was significantly higher than that of PGR treatments. However, the seeds treated only with moist chilling presented the highest germination percentage among all the treatments.

Michelia coriacea (Magnoliacae) is a critically endangered tree, endemic to Southeast Yunnan province, China. Most of the individuals in the extant populations normally bear flowers, but fruit set and fertile seed production rates were only 6.7% and 0.2%, respectively. To identify possible causes of reproductive barriers, the following studies were carried out: examination of stamen and pistil development; experiments using controlled pollination; observations of behavior and frequency of floral visitors in both cultivated and natural populations. The results revealed that about 60% of pollen was abnormal and approx. 70% of ovules had delayed development. Hand pollination could effectively enhance the fruit set (F(4.25) = 35.139, P < 0.0001) and seed set per fruit (F(4.25) = 85.022, P < 0.0001). Both cultivated and wild M. coriacea had an extremely low frequency of floral visitors. Some beetles, a few species of Andremdae and some Bombus sp. are likely to be the effective pollinators. The fruit set and seed set per fruit from controlled self-pollination and cross-pollination were significantly different (P < 0.05), and thus it is inferred, that inbreeding depression may be a contributing factor in the very low seed production. It appears that low seed set in M. coriacea is due to a combination of factors: abnormalities in pollen and ovules, low number of effective pollinators, and inbreeding depression. (C) 2009 Elsevier GmbH. All rights reserved.

Cytological studies were carried out for the first time on five populations of Trigonobalanus doichangensis in China and Thailand. In all populations, the pattern of interphase nuclei was of the simple chromocentre type, the mitotic prophases were of the proximal interstitial type and chromosome numbers were 2n = 2 chi = 14. Two B chromosomes were commonly observed at prophase and prophase-metaphase, but rarely at metaphase. Karyotype variation among the populations at the diploid level was limited, but there were some distinguishing cytological characters. Based on the comparison of all the available data on cytology, taxonomy, phytogeography and molecular systematics related to the genus Trigonobalanus, we recommend that the three species of Trigonobalanus comprise the subfamily Trigonobalanoideae and that the genus should not be segregated into three monotypic genera. (c) 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 154, 321-330.

The chromosome numbers of 27 populations of Buddleja, comprising 14 species, were counted. The basic chromosome number of all species was x = 19, confirming previous reports. Different ploidy levels (2n = 38, 76, 114, 228) were observed in these taxa, representing diploids, tetraploids, hexaploids, and dodecaploids, respectively. The chromosome numbers of B. yunnanensis, B. brachystachya, and B. macrostachya are reported for the first time. The tetraploid 2n = 76 is a new ploidy level for B. myriantha. Particular attention was given to B. macrostachya, because of the variation in morphology and ploidy level between isolated populations of this species. Two types of interphase nuclei were recognized: the complex chromocentre type in B. macrostachya and the simple chromocentre type in the other species. Biogeographically, most of the polyploidy in the Asiatic species occurs in the Sino-Himalayan region. It seems to be associated with the uplift of the Himalayan Mountains, the orogeny of this region playing an important role in the evolution of polyploidy in these taxa. (c) 2007 The Linnean Society of London. Botanical Journal of the Linnean Society, 2007, 154, 305-312.

Despite acceptance in the scientific community that population information and suites of characters are crucial for circumscription of taxonomic groups, new taxa continue to be published on the basis of few herbarium specimens. Given that there is increasing evidence that hybridisation plays an integral part in evolution, it is desirable to identify groups in which it occurs. In this study, we showcase how population variation can be used to identify distinguishing characters and why closely related species that are growing in sympatry should be considered when describing new taxonomic entities., Hybridization has become a focal topic in evolutionary biology, and many taxonomists are aware that the process occurs more frequently than previously assumed. Nonetheless many species and varieties are still described without explicitly considering the possibility of hybridization, especially in countries that have relatively short scientific histories, but which often possess the highest species diversities. Furthermore, new taxa are often described based only on herbarium specimens, not taking into account information from wild populations, significantly decreasing the potential to detect morphologies arising from hybridization at this crucial descriptive stage. We used morphological data from a hybrid swarm involving two Rhododendron species to showcase possible character combinations in intermediates. Certain characters used to distinguish taxa were more variable within the same individual than between species, emphasizing the importance of population information for an adequate choice of characters. Most described varieties of the two species fell within the spectrum of hybrid morphology, suggesting that these taxa would be unlikely to have merited formal description if contemporary standards had been employed. In all investigated cases the hybrid nature of described varieties seems to have been detectable with adequate morphological data alone, if populations had been assessed. A post hoc assessment of taxa is often complicated, especially if certain types of information are not provided. To avoid accumulation of such invalid taxa, careful scrutiny should be employed for new descriptions. Hybrids (not hybrid species) described as taxa obscure valuable information about natural processes and impact negatively on further research that depends on taxonomic data.

Trigonobalanus doichangensis is a national rare and endangered plant of China. It is restricted to 4 sites in southwest Yunnan, China and 1 site in Chiang-Rai, northern Thailand. Investigations revealed that 4 community types are currently extant in Yunnan: isolated individuals, sprouting woods, mono-dominant forest and co-dominant forest. The habitats have been severely damaged and the populations there are facing a high risk of extinction. The adult phase of T. doichangensis is reached when a tree attains a height of about 4 m. The flowering and fruiting time varies slightly among populations and/or across the micro-habitats. In comparison with other fagaceous plants in the community, T. doichangensis has an inverse flowering and fruiting period from October through to May. Although microspore genesis and the development of male gametes are normal, the pollen does not germinate until at least 8 h culture. The highest germination rate was 37.8%. In addition, only 9.8% of the nuts contain well-developed seeds. Genetic variation analyzed with random amplified polymorphic DNA indicated that its total genetic diversity was 0.1600 and genetic diversity within populations was 0.0749, the coefficient of gene differentiation was 0.5320. Therefore, T. doichangensis has high genetic differentiation, a low level of genetic diversity and a poor gene flow compared to the other fagaceous species. It seems that habitat degradation, over exploitation and reproductive barriers, are most likely to be the factors threatening the species. However, it may be a combination of geographical catastrophic events, habitat deterioration, declining genetic diversity and physiological stress. Therefore, a practical conservation strategy for T. doichangensis is urgently needed.

This is the first study of the genetic diversity and structure of the big tree rhododendron, Rhododendron protistum var. giganteum, which is a highly endangered species with only two known endemic populations in a small area in the southern part of Yunnan Province in China. We detected moderate to high genetic diversity at the species level, but low genetic differentiation between the two extant populations. These results suggest that some rare and endangered species are able to maintain high levels of genetic diversity even at small population sizes., Comprehensive studies on the genetic diversity and structure of endangered species are urgently needed to promote effective conservation and management activities. The big tree rhododendron, Rhododendron protistum var. giganteum, is a highly endangered species with only two known endemic populations in a small area in the southern part of Yunnan Province in China. Unfortunately, limited information is available regarding the population genetics of this species. Therefore, we conducted amplified fragment length polymorphism (AFLP) analysis to characterize the genetic diversity and variation of this species within and between remaining populations. Twelve primer combinations of AFLP produced 447 unambiguous and repetitious bands. Among these bands, 298 (66.67 %) were polymorphic. We found high genetic diversity at the species level (percentage of polymorphic loci = 66.67 %, h = 0.240, I = 0.358) and low genetic differentiation (Gst = 0.110) between the two populations. Gene flow between populations (Nm) was relatively high at 4.065. Analysis of molecular variance results revealed that 22 % of the genetic variation was partitioned between populations and 78 % of the genetic variation was within populations. The presence of moderate to high genetic diversity and low genetic differentiation in the two populations can be explained by life history traits, pollen dispersal and high gene flow (Nm = 4.065). Bayesian structure and principal coordinate analysis revealed that 56 sampled trees were clustered into two groups. Our results suggest that some rare and endangered species are able to maintain high levels of genetic diversity even at small population sizes. These results will assist with the design of conservation and management programmes, such as in situ and ex situ conservation, seed collection for germplasm conservation and reintroduction.

In this study, we report that the phoretic flower mite Neocypholaelaps indica can infest flowers of Pachysandra axillaris and Apis cerana in Yunnan, China. We suggest that beekeepers, researchers and regulators should be aware of this species to regulate its potential harm to beekeeping and output of crops in China.

Microsatellite primers were developed to assess genetic diversity and population structure in Acer yangbiense, a critically endangered endemic species that occurs in northwestern Yunnan Province, China. •Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol, 34 microsatellite loci were isolated and characterized in A. yangbiense. Polymorphisms were evaluated in 39 individuals from two distinct populations, one of which was naturally occurring and the other an ex situ grouping. Nine of the markers showed polymorphisms with two to five alleles per locus; observed (H(o)) and expected (H(e)) heterozygosities ranged from 0.0000 to 0.8000 and from 0.0000 to 0.6889, respectively. •These microsatellite primers will prove useful in the further investigation of population genetics studies in A. yangbiense and, in addition, will assist related research of other congeneric species.

We engaged experts in various fields of study (pollination ecology, chemical ecology, and ethnobotany), invited community participation, and provided environmental education in an effort to conserve an endangered birthwort (Aristolochia delavayi) and a vulnerable pipevine swallowtail (Byasa daemonius). Scientists studied the uptake and sequestration of the secondary metabolites aristolochic acids from A. delavayi leaves by different stages of pipevine swallowtail as a defense mechanism; low fruit set of the myophilous A. delavayi due to pollinator limitation; and the emission of chemical signals that attract parasitic wasps by the prepupae of B. daemonius. The results of these studies were part of an education program delivered by personnel of non-governmental organizations. The program was devised to deliver information to the public about the health risks of consuming A. delavayi individuals (aristolochic-acid-associated cancers) and to establish a bridge between the public and scientific research. Following delivery of the program, the behavior of residents changed considerably. Community residents were involved in management activities, including participation in a program to promote understanding of ecological interactions between A. delavayi and B. daemonius; designing an in situ conservation site; monitoring A. delavayi and B. daemonius individuals; and promoting the natural fruit set of A. delavayi by scattering animal excrement to attract fly pollinators. The integration of scientific information and community participation appears to have resulted in an increase in abundance of threatened A. delavayi and B. daemonius populations. We believe the involvement of local people in conservation is necessary for successful species conservation.

Buddleja caryopteridifolia was treated as a synonym of B. crispa in Flora of China . However, it was found that the two entities had different phenologies as well as morphology in the sympatric habitat investigated. Bud dleja crispa flowers from mid-March to mid-May while B. caryopteridifolia blooms from early June to mid-September. In this study, the comprehensive comparative analyses of morphological traits, floral scent composition and amplified fragment length polymorphism (AFLP) data were utilized to determine whether B. caryopteridifolia should be recognized as a separate species. Among 13 morphological characters, all but calyx length were significantly different (all p

Pulse tube refrigerator (PTR) is a new type of mechanical cryocooler with the potential of long-term operation in space. Theoretical and experimental studies are currently on the way in the Cryogenic Laboratory of Chinese Academy of Sciences (CL/CAS) in order to develop a 85 K/250 mW class pulse tube cryocooler to be used to cool space-borne infrared devices. A theoretical model is established based on the analyses of the thermodynamic behavior of gas parcels in oscillating flow regenerators. It helps us to understand the cooler and can be used to study the influence of DC flow on the refrigeration performance. The flow resistance of the regenerator is an important factor for the cooler performance. A test bench, including a hot-wire anemometer has been set up to investigate the flow resistance characteristics of regenerators with oscillating flow. The results of measurement are correlated and served for practical design. The Oxford type linear pressure wave generator with flexure bearings is also under development in CL/CAS. The prototype miniature pulse tube cryocooler, driven by a linear pressure wave generator of 1.06 cm 3 maximum swept volume, provides at present 200 mW net cooling power at 81 K with 28.4 W input power. Improvements are being made to further increase the cooling power and reduce the input power.