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Understanding processes that generate and maintain large disjunctions within plant species can provide valuable insights into plant diversity and speciation. The butterfly bush Buddleja alternifolia has an unusual disjunct distribution, occurring in the Himalaya, Hengduan Mountains (HDM) and the Loess Plateau (LP) in China. We generated a high-quality, chromosome-level genome assembly of B. alternifolia, the first within the family Scrophulariaceae. Whole-genome re-sequencing data from 48 populations plus morphological and petal colour reflectance data covering its full distribution range were collected. Three distinct genetic lineages of B. alternifolia were uncovered, corresponding to Himalayan, HDM and LP populations, with the last also differentiated morphologically and phenologically, indicating occurrence of allopatric speciation likely to be facilitated by geographic isolation and divergent adaptation to distinct ecological niches. Moreover, speciation with gene flow between populations from either side of a mountain barrier could be under way within LP. The current disjunctions within B. alternifolia might result from vicariance of a once widespread distribution, followed by several past contraction and expansion events, possibly linked to climate fluctuations promoted by the Kunlun-Yellow river tectonic movement. Several adaptive genes are likely to be either uniformly or diversely selected among regions, providing a footprint of local adaptations. These findings provide new insights into plant biogeography, adaptation and different processes of allopatric speciation.

Impatiens wutaishanensis R.L. Liao & Lei Cai, a new species from Southeast Yunnan, China, is here described and illustrated. This new species is most similar to Impatiens parvisepala S.X. Yu & Y.T. Hou in its racemose inflorescences, its four lateral sepals, the leaf arrangement, and in having yellow flowers. However, it differs in the height of the plants, the length of the petiole, the size and shape of the leaf blade, the shape of the spur, and the number of flowers in each inflorescence. A detailed description, color photographs, and a provisional IUCN red list assessment are provided, and its geographical distribution, ecology, and morphological relationship with relevant similar species are discussed.

Acer yangbiense Y.S. Chen & Q.E. Yang (Aceraceae) is a threatened tree species endemic to China, formerly presumed to have declined to only five extant individuals, restricted to Yangbi County, Yunnan Province. Our surveys in 2016, however, located 577 individuals in 12 localities, but only three localities (with a total of 62 individuals) are protected. Nine localities are on private forest land. The population's size structure is an inverse J-curve, but there is a scarcity of trees of the smallest size class and of seedlings. Our surveys also showed that the habitat of A. yangbiense is degraded as a result of the negative effects of agriculture, logging and wood harvesting. Assessment with the IUCN Red List categories and criteria indicates that A. yangbiense should be recategorized from Critically Endangered to Endangered.

Both in situ and ex situ conservation are important strategies for protecting threatened plant species. Nevertheless, the success of conservation depends on whether the plant species can naturally regenerate and accomplish its life cycle over a long-term. Here we studied the pollination biology of the threatened species Hibiscus aridicola, Amorphophallus albus, Stemona parviflora and S. japonica aiming to get data about pollination strategies, pollinators as well as mating systems. These experiments were performed at Kunming Botanical Garden (KBG) for ex situ and the plant species’ natural habitat for in situ conservation. The results indicated that H. aridicola is self-compatible and had pollinators under both ex situ and in situ conditions. The other three species are all self-incompatible and a limited number of pollinators for S. parviflora and S. japonica were observed at cultivated and natural habitats. Amorphophallus albus had no pollinators at KBG but a large number of rove beetles (Atheta sp.) could be observed in the plant species’ natural habitat. This resulted in a high fruit set under natural conditions (73.3%, n = 30). The results showed clearly, that appropriate pollinators for the four plant species are not present all the time and all localities, which further influences the reproduction success of a plant species. Hence, for a successful conservation, it is vital to assess the species reproduction strategy prior deciding whether in situ and/or ex situ conservation should be carried out.

Magnolia ovoidea is a narrowly endemic, Critically Endangered tree with a fragmented distribution in south-west Yunnan, China. We examined the size structure of this species, documented threats and assessed its extinction risk. We found the species in six locations, all in unprotected areas, in Maguan County. The largest subpopulations are in Donggua Lin and Youfang Po, with 17 and 50 living individuals, respectively. The distribution of the diameter at breast height of the M. ovoidea population has an inverse J-shape, indicating a stable size structure. However, the distribution of the height of seedlings is L-shaped, suggesting inhibited regeneration. The natural vegetation is severely fragmented in all six locations, surrounded by roads, farmlands, and Alnus nepalensis or Cunninghamia lanceolata plantations. We recommend that M. ovoidea should be categorized on the IUCN Red List as Critically Endangered based on criteria B2ab(iii) + C2a(i).

Background It has been recognized that a certain amount of habitat disturbance is a facilitating factor for the occurrence of natural hybridization, yet to date we are unaware of any studies exploring hybridization and reproductive barriers in those plants preferentially occupying disturbed habitats. Buddleja plants (also called butterfly bush) generally do grow in disturbed habitats, and several species with hybrid origin have been proposed, based solely on morphological evidence. Results In the present study, we test the hypothesis that B. × wardii is of natural hybridization origin in two sympatric populations of three taxa including B. × wardii and its parents (B. alternifolia and B. crispa) plus 4 referenced parental populations, using four nuclear genes and three chloroplast intergenic spacers, as well as with 10 morphological characters. Our results suggest that at both sites B. × wardii is likely to be a hybrid between B. alternifolia and B. crispa, and moreover, we confirm that most of the hybrids examined are F1s. That these plants are F1s is further supported by morphology, as no transgressive characters were detected. B. crispa was found to be the maternal parent in the Bahe (BH) population, from cpDNA evidence. However, in the Taji (TJ) population, the direction of hybridization was difficult to establish due to the shared cpDNA haplotypes between B. alternifolia and B. crispa, however we still predicted a similar unidirectional hybridization pattern due to results from cross-specific pollination treatments which supported the “SI × SC rule”. Conclusions The presence of mainly F1 hybrids can successfully impede gene flow and thus maintain species boundaries in parental species in a typical distribution of Buddleja, i.e. in disturbed habitats.

Nyssa yunnanensis is a deciduous tree species in family Nyssaceae within the order Cornales. As owning only eight individuals in two sites recorded in Yunnan province of China, this species was listed as the China’s national grade-I protection species in 1999, and also as one of 120 PSESP (Plant Species with Extremely Small Populations) in Implementation Plan of Rescuing and Conserving China’s Plant Species with extremely Small Populations(PSESP) (2011-2-15). N. yunnanensis was also been evaluated as Critically Endangered in IUCN red list and Threatened Species List of China's Higher Plants. Hence understanding the genomic characteristics of this highly endangered Tertiary relict tree species is essential, especially for developing conservation strategies. Here we present the draft genome assembly of N. yunnanensis. Using 10X genomics linked-reads sequencing data, we carried out the de novo assembly and annotation analysis. The N. yunnanensis genome assembly is 1475 Mb in length containing 288,519 scaffolds with a scaffold N50 length of 985.59 kb. 799.51 Mb of the assembled genome was identified as repetitive elements, accounting for 54.24% of the sequenced genome. And a total of 39,803 protein-coding genes were annotated. The genomic data of N. yunnanensis provided in this study will provide basic information for future genomic and evolutionary studies. With the genomic characteristics of N. yunnanensis available, our study might also facilitate in future conservation biology studies to help protecting this extremely threatened tree species.

The habitats of Craigia yunnanensis (Tiliaceae), an endangered tree with extremely small populations, have been mostly occupied by human settlements and farmlands, and thus population restoration in these areas might not be a practical option. Therefore, we have attempted an innovative reintroduction practice since 2017. Local volunteers, who were also landowners, adopted and planted seedlings dispersively to their private idle plots around the countryside, which helped to avoid the interference of seedling growth with their crops or houses and also increased the guarantees to the reintroduction success.

Based on the comprehensive studies on the conservation biology of Trigonobalanus doichangensis, a globally threatened plant species with extremely small populations (PSESP), some 247 2-year-old saplings propagated from seeds were planted into its originally distributed site (99°44′57.5″E, 22°28′43.0″N and altitude of 1352 m) in south Yunnan province of China in September 2007. In cooperation with the local Forestry Bureau of Lancang County and a local family near the site, the necessary aftercares, monitoring, and data collecting have all been continuously carried out, around 50% of the survived plants bloomed and fruited in 2015. Twelve years after planting in September 2019, a survival rate of 46.56%, an average height increase of 7.89 m, and an average base diameter increase of 5.40 cm of T. doichangensis were all recorded and documented. Certainly, this successful reintroduction practice can be a template for accelerating the multiple-scale population restoration of T. doichangensis in China.

Flowers or inflorescences often deploy various signals, including visual, olfactory, and gustatory cues, that can be detected by their pollinators. In many plants, these cues and their functions are poorly understood. Deciphering the interactions between floral cues and pollinators is crucial for analyzing the reproductive success of flowering plants. In this study, we examined the composition of the fetid floral scents produced by several Stemona species, including nine S. tuberosa populations from across China, using dynamic headspace adsorption, gas chromatography, and mass spectrometry techniques. We compared variations in floral phenotype, including floral longevity, nectar rewards, pollinator behavior, and flower length and color among the Stemona species. Of the 54 scent compounds identified, the major compounds include fetid dimethyl disulfide, dimethyl trisulfide, 1-pyrroline, butyric acid, p-cresol, isoamyl alcohol, and indole. We detected striking differentiation in floral scent at both the species and population level, and even within a population of plants with different colored flowers. Floral characteristics related to sapromyophily and deceptive pollination, including flower color mimicking livor mortis and a lack of nectar, were found in five Stemona species, indicating that Stemona is a typical sapromyophilous taxon. Species of this monocot genus might employ evolutionary tactics to exploit saprophilous flies for pollination.

Buddlejeae comprise c. 108 species in five commonly accepted genera: Buddleja, Chilianthus, Emorya, Gomphostigma and Nicodemia. Conflicting generic and infrageneric level classifications based on morphology attest to a need to evaluate relationships and trait evolution in a molecular phylogenetic framework. We use multiple independent loci from the nuclear and plastid genomes and representative taxonomic sampling to infer phylogenetic relationships using maximum likelihood and Bayesian analyses with single-locus and concatenated data and Bayesian multispecies coalescent analyses. Nicodemia and Gomphostigma are resolved as monophyletic. Chilianthus is not monophyletic, with three species in one clade and Buddleja glomerata (=Chilianthus lobulatus) possibly separate. Buddleja is paraphyletic with respect to Chilianthus, Emorya, Nicodemia and, probably, Gomphostigma. We propose a new classification to reflect phylogenetic relationships in Buddlejeae. Only Buddleja is retained at the generic level. Chilianthus, Nicodemia, Gomphostigma and Emorya are combined with Buddleja, with a new name and new combination erected for the two Emorya spp., Buddleja normaniae and B. rinconensis. Sectional classification of Buddleja is revised, with two new monotypic sections being proposed, Salviifoliae and Pulchellae, and Gomphostigma being lowered to sectional rank. Reproductive morphological traits traditionally used to define genera, including stamen exsertion, corolla shape and inflorescence type, were reconstructed on the phylogenetic tree and are inferred to have converged on similar states multiple times. Plesiomorphic trait states in Buddlejeae include capsular fruits, included stamens, white and tube-shaped corollas and paniculate inflorescences. Fil: Chau, J.ohm. University of Washington; Estados Unidos Fil: O'Leary, Nataly Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion. Academia Nacional de Ciencias Exactas, Físicas y Naturales. Instituto de Botánica Darwinion; Argentina Fil: Wei-Bang, Sun. Chinese Academy of Sciences; República de China Fil: Olmstead, Richard. University of Washington; Estados Unidos

Corybas taliensis is an endangered terrestrial orchid, endemic to China. In this study, we assembled the complete chloroplast (cp) genome using Illumina pair-end sequencing data. The whole cp genom...

The rare and threatened fern Christensenia aesculifolia of South-east Asia is listed in China as a second-ranked plant for national protection and is also categorized as one of 62 plant species with extremely small populations by the Yunnan provincial government. Field investigations during 2014–2017 failed to relocate one previously known population, and revealed that the single known extant population of C. aesculifolia contains only 10 individual plants. The most urgent conservation requirement for this species is to conserve the threatened habitat of the remnant population. Further field surveys and research are also required for an improved understanding of the species’ status.

Background Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”. Findings We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ∼666 Mb, with 13 chromosomes covering ∼97% of the genome and scaffold N50 sizes of 45 Mb. Further, BUSCO analysis recovered 95.5% complete BUSCO genes. The total number of repetitive elements account for 68.0% of the A. yangbiense genome. Genome annotation generated 28,320 protein-coding genes, assisted by a combination of prediction and transcriptome sequencing. In addition, a nearly 1:1 orthology ratio of dot plots of longer syntenic blocks revealed a similar evolutionary history between A. yangbiense and grape, indicating that the genome has not undergone a whole-genome duplication event after the core eudicot common hexaploidization. Conclusion Here, we report a high-quality de novo genome assembly of A. yangbiense, the first genome for the genus Acer and the family Aceraceae. This will provide fundamental conservation genomics resources, as well as representing a new high-quality reference genome for the economically important Acer lineage and the wider order of Sapindales.

BackgroundAcer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”.FindingsWe generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ∼666 Mb, with 13 chromosomes covering ∼97% of the genome and scaffold N50 sizes of 45 Mb. Further, BUSCO analysis recovered 95.5% complete BUSCO genes. The total number of repetitive elements account for 68.0% of the A. yangbiense genome. Genome annotation generated 28,320 protein-coding genes, assisted by a combination of prediction and transcriptome sequencing. In addition, a nearly 1:1 orthology ratio of dot plots of longer syntenic blocks revealed a similar evolutionary history between A. yangbiense and grape, indicating that the genome has not undergone a whole-genome duplication event after the core eudicot common hexaploidization.ConclusionHere, we report a high-quality de novo genome assembly of A. yangbiense, the first genome for the genus Acer and the family Aceraceae. This will provide fundamental conservation genomics resources, as well as representing a new high-quality reference genome for the economically important Acer lineage and the wider order of Sapindales.

Background Primula is a large genus of flowering herbs well known for their heterostyly. Currently few natural hybrids are known and reproductive barriers in this genus in the wild have received little attention. However, there is instance of hybridization between rare and widely-spread species, and conservation implications of such situation is poorly understood. In the present study, we investigated hybridization patterns and reproductive barriers between a wide spread species, Primula poissonii and a rare species P. anisodora, of which only three populations are currently known. Results Pollinator-mediated reproductive isolation was strong between parental species but not significant between hybrids and parental species. Hand pollination experiments showed significant reduction of both fruit- and seed-set for heterospecific pollination as compared with conspecific pollination for both parental species. Furthermore, hybrids had higher fruit- and seed-set when pollinated with P. anisodora pollen as opposed to P. poissonii pollen. Microsatellites identified backcrosses to P. anisodora in two of the three populations of P. anisodora, and additionally more individuals of P. anisodora showed introgression from P. poissonii than vice versa. Conclusions These results provide evidence for potential genetic swamping of the P. anisodora populations, which could pose a serious threat for this locally endemic species. Electronic supplementary material The online version of this article (10.1186/s12870-019-1881-0) contains supplementary material, which is available to authorized users.

Pollination and seed dispersal in angiosperms have long been investigated in order to understand the coevolution of plants and animals. However, the signals from flowers and/or seeds to attract pollinators and/or seed dispersers have received comparatively little attention. In this study, the pollination biology and seed dispersal of the vulnerable agarwood plant Aquilaria sinensis (Lour.) Gilg, a traditional medicinal plant in China, was studied in its natural distribution range. The reproductive tactics of A. sinensis were studied in detail by employing various tests dealing with fruit set and also seed dispersal. Dynamic headspace extraction followed by GC-MS analysis was also performed in order to reveal the composition of floral scent. The results showed that noctuids and pyralids are the most effective pollinators of pollinator-dependent A. sinensis. The main compounds of the floral scent were (E, E)-α-Farnesene (61.9 ± 3.2%), trans-Ocimene (16.6 ± 1.2%), and Benzyl salicylate (4.6 ± 1.1%). The results obtained from seed dispersal experiments indicate that hornets are effective seed dispersers and they may play an important role in long-distance seed dispersal of A. sinensis. Based on our findings, we recommend several protection methods for this threatened agarwood plant in China.

Of the genus Craigia, widespread in the Tertiary, only two relict species survived to modern times. One species is now possibly extinct and the other one, Craigia yunnanensis, is severely endangered. Extensive surveys have located six C. yunnanensis populations in Yunnan province, southwest China. Using fluorescent amplified fragment length polymorphism (AFLP), the genetic diversity and population structure of these populations were examined. It was found that genetic diversity of C. yunnanensis was moderate at the species level, but low at regional and population levels. Analysis of population structure showed significant genetic differentiation between Wenshan and Dehong regions, apparently representing two geographically isolated for long time refuges. There are also clear indications of isolation between populations, which, together with anthropogenically caused decline of population size, will lead to general loss of the species genetic variation with subsequent loss of adaptive potential. To conserve the genetic integrity of C. yunnanensis, we recommend that ex-situ conservation should include representative samples from every population of the two differentiated regions (e.g. Wenshan and Dehong). The crosses between individuals originated from different regions should be avoided because of a high risk of outbreeding depression. As all the extant populations of C. yunnanensis are in unprotected areas with strong anthropogenic impact, there is no alternative to reintroduction of C. yunnanensis into suitable protected locations.

Several conservation programs have been started for the critically endangered Sichou oak ( Quercus sichourensis ) since 2007. These programs include detailed field investigations, seedling cultivation and research on the fruit biology of the species. In this study, we first report on the five mature individual trees found in our 9-year field investigation. Thus far, a total of 10 mature individuals have been recorded. All Q. sichourensis trees are healthy and most produce healthy acorns. Acorns of Q. sichourensis are large with dry masses of 8.0–14.0 g. These acorns had high moisture contents at collection and died shortly after (7–28 d) when dried with silica gel. Characteristics of Q. sichourensis acorns varied between populations. Compared with the acorns from Funing, the acorns collected from Ceheng were bigger, more viable (germination percentage was up to 96%), less sensitive to desiccation, and germinated faster. Q. sichourensis occurs in regions with a distinct 5–6 month dry season. Habitat degradation is largely responsible for the rareness of Quercus sichorensis , but desiccation sensitivity of the acorns may also limit the regeneration of the species and potentially lead to its continued rareness. As a species with extremely small populations (PSESP), Q. sichourensis is facing high risk of extinction and should be defined as a Critically Endangered species in the global IUCN Red List.

Diaspores of myrmecochorous plants often include nutrient rich elaiosomes, which are known as appendages of seeds to attract ants for seed dispersion. The benefits of myrmecochory were investigated thoroughly in previous studies, such as, the diaspores may be carried to the ant nests where the elaiosomes are consumed and the seeds are discarded unharmed. However, it remained unclear whether the secondary dispersers could shape the traits of diaspores in myrmecochory. In this study, we analyzed the nutritional quality of seeds and their corresponding elaiosomes of the myrmecochorous plant Stemona tuberosa (Stemonaceae), whose diaspores are dispersed by ants and/or hornets. Our results show significant differences in the quantitative pattern of nutrients between seeds and elaiosomes, respectively. The elaiosomes of S. tuberosa contain significantly higher concentration of proteins, free fatty acids, free amino acids, soluble carbohydrates, and micronutrients than the seeds. The low mass ratio of seed/elaiosomes in S. tuberosa compared to other myrmecochorous species imply that seed dispersal by hornets might be discriminated from seed dispersal by ants. In conclusion, we suggest that hornets and ants obtain the essential nutrients from the elaiosomes of S. tuberosa and these rewards contribute to shape the hornet/ant-plant mutualism.

Buddleja colvilei Hook.f. & Thomson (Scrophulariaceae) is a threatened alpine plant with a distribution throughout the Himalayas, also used as an ornamental plant. The name Buddleja sessilifolia B.S. Sun ex S.Y. Pao was assigned in 1983 to a plant distributed throughout the Gaoligong Mountains, but the name was later placed in synonymy with B. colvilei in the Flora of China. In this study we sequenced the complete chloroplast (cp) genomes of two individuals of B. colvilei and three individuals of B. sessilifolia from across the range. Both molecular and morphological analysis support the revision of B. sessilifolia. The phylogenetic analysis constructed with the whole cp genomes, the large single-copy regions (LSC), small single-copy regions (SSC), inverted repeat (IR) and the nuclear genes 18S/ITS1/5.8S/ITS2/28S all supported B. sessilifolia as a distinct species. Additionally, coalescence-based species delimitation methods (bGMYC, bPTP) using the whole chloroplast datasets also supported B. sessilifolia as a distinct species. The results suggest that the B. sessilifolia lineage was early diverging among the Asian Buddleja species. Overall gene contents were similar and gene arrangements were found to be highly conserved in the two species, however, fixed differences were found between the two species. A total of 474 single nucleotide polymorphisms (SNPs) were identified between the two species. The Principal Coordinate Analysis of the morphological characters resolved two groups and supported B. sessilifolia as a distinct species. Discrimination of B. colvilei and B. sessilifolia using morphological characters and the redescription of B. sessilifolia are detailed here.

Magnolia sinica, distributed in Yunnan province of China, is a critically endangered (CR) species based on the IUCN Red List. Here we provide the complete chloroplast (cp) genome sequence of M. sin...

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 secondarymetabolites 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.

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.

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.

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.

Manglietia ventii is a highly endangered plant species endemic to Yunnan province in China, where there are only five known small populations. Despite abundant flowering there is very low fruit and seed set, and very few seedlings in natural populations, indicating problems with reproduction. The causes of low fecundity in M. ventii are not known, largely because of insufficient knowledge of the species pollination ecology and breeding system. We conducted observations and pollination experiments, and analyzed floral scents to understand the pollinator–plant interactions and the role of floral scent in this relationship, as well as the species breeding system. Like the majority of Magnoliaceae, M. ventii has protogynous and nocturnal flowers that emit a strong fragrance over two consecutive evenings. There is a closing period (the pre-staminate stage) during the process of anthesis of a flower, and we characterize the key flowering process as an “open-close-reopen” flowering rhythm with five distinct floral stages observed throughout the floral period of this species: pre-pistillate, pistillate, pre-staminate, staminate, and post-staminate. Flowers are in the pistillate stage during the first night of anthesis and enter the staminate stage the next night. During anthesis, floral scent emission occurs in the pistillate and staminate stages. The effective pollinators were weevils (Sitophilus sp.) and beetles (Anomala sp.), while the role of Rove beetles (Aleochara sp.) and thrips (Thrips sp.) in pollination of M. ventii appears to be minor or absent. The major chemical compounds of the floral scents were Limonene, β-Pinene, α-Pinene, 1,8-Cineole, Methyl-2-methylbutyrate, p-Cymene, Methyl-3-methyl-2-butenoate and 2-Methoxy-2-methyl-3-buten, and the relative proportions of these compounds varied between the pistillate and staminate stages. Production of these chemicals coincided with flower visitation by weevils and beetles. The results of pollination experiments suggest that M. ventii is pollinator-dependent, and low seed set in natural populations is a result of insufficient pollen deposition. Thus, conservation of the species should focus on improving pollination service through the introduction of genetically variable individuals and increase in density of reproducing trees.

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 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.

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.

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.