•Nutritional quality of diaspores of Stemona tuberosa was analyzed.•Significant differences of nutrients were revealed between seeds and elaiosomes.•Seed dispersal by hornets might be discriminated from seed dispersal by ants.•Elaiosomes contribute to shape the hornet/ant-plant mutualism.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 crispa is a species with high genetic diversity.•The populations of B. crispa were divided into two genetic groups.•Most of higher diversity populations were located in the center of the distribution areas.•Population differentiations of B. crispa are more influenced by barriers of rivers and mountains than geographic distance.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.

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.

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 scent profiles of two Buddleja species were chemically characterized.•Intra-specific variation of scent compositions was investigated.•High intra-specific variations of scent compositions were found in both species.•Both species showed distinct patterns of intra-specific variation.•Introgression and founder effect account for the high intra-specific variation.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.

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.

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.

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-β-ocimene (53.4%), lilac alcohol (9.6%) and lilac aldehyde (5.6%). Main volatile compounds in B. lindleyana were β-caryophyllene (17.6%), α-farnesene (16.0%) and 3-octanone (9.8%). Main volatile compounds in B. loricata were 4-oxoisophorone (27.1%), α-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.

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.

By using a modified biotin–streptavidin capture method, a total of 12 microsatellite loci were developed and characterized for Michelia coriacea (Magnoliaceae), a critically endangered endemic to Southeast Yunnan, China. The number of alleles (A) ranged from two to six in 30 samples of this species. The ranges of observed (HO) and expected (HE) heterozygosities were 0.033–0.8000 and 0.033–0.7910, respectively. Cross-species amplification in M. yunnanensis showed that a subset of these markers holds promise for congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on M. coriacea and other congeneric species.

The extent of genetic variation in seed and seedling traits of Trigonobalanus doichangensis, a rare and endangered tree in southwest China, was evaluated at the population level. Seeds were collected from four natural populations in the south and southwest Yunnan province, China. Significant differences (P < 0.05) between populations were observed in seed length, seed weight and seedling morphological characters. The analyses revealed that none of the seed and seedling traits had significant correlations with the geo-climatic variables of the populations. Among seed and seedling traits, significant correlations (P < 0.05) were discovered among seed length, seed weight and height of 30-day-old seedlings. Significant correlation (r = 1.00, P < 0.01) occurred also between the height and root collar diameter of 4-year-old seedlings. With the exception of seed width and seed germination percentage, 51–96% of total variation in other seed and seedling related traits was attributed to population effect, as determined by the broad sense heritability. In conclusion, these observations on variation in seed and seedling traits should be a reference point, when considering seed collection of this species for ex situ conservation and species restoration.

Trigonobalanus doichangensis is recognized as a rare and endangered plant of China. The morphological and physiological traits of fruits were investigated in one population over 2 years (2006–2007) and in four populations during 2007 in order to facilitate ex-situ conservation and eventual reforestation through planting of propagules. The results indicated that morphological traits including fruit length, fruit width and 1,000-fruit weight showed significant variation among individual trees within populations (P < 0.01), however, not among populations. And each of them had significant positive correlation with each other (P < 0.01). Seed abortion occurred, and there was significant variation in percentage of fruit fill among populations and among individual trees within populations, whereas, percentage of fruit fill showed no correlation with the morphological traits. As for fruit germination, germination percentage, germination index and vigor index presented significant variation among populations and among individual trees within populations. The three germination-related indices had significant positive correlations with each other (r ≥ 0.9, P < 0.01) and with percentage of fruit fill (r > 0.86, P < 0.01), however, possessing weak correlations with morphological traits. It was also found that each of the traits mentioned above showed significant variation among individual trees within years, however, not between years. In addition, more than 48% of the variation occurred among individual trees both within populations and within years in all the fruit characters. We suggest that collecting fruits from various individuals at each of populations will be the preferred strategy to conserve the most genetic diversity of the species.

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.

Magnolia sinica is one of the most threatened trees endemic to Southeast Yunnan. Based on our investigations, only 52 individuals and eight populations are found in the wild. M. sinica has been categorized as Critically Endangered on the IUCN Red List and identified as a “Plant Species with Extremely Small Populations (PSESP)”. Its fruit/seed set is very low and seedlings are rarely found in the wild. It is hypothesized that it may encounter obstacles to reproductive success. This study, therefore, focuses on its reproductive biology, knowledge of which is essential for effective conservation. Flowers of this species are protogynous and nocturnal, and possess a two-day rhythm of sexual presentation. For the first night of anthesis, the flowers are in the pistillate stage during which tepals open at dusk and close approx. 1 h later (except for the open outer ones). They remain closed until the next afternoon, when flowers, now in the staminate stage, re-open and remain so until the tepals drop. Nocturnal beetles enter into the flowers and remain trapped throughout the night as the flower closes, during which time they feed on tepals. Pollen-gathering bees are found to visit the re-opened flowers and the beetles are released during this stage. Two species of Pleocomidae and Curculionidae beetles appear to be effective pollinators.M. sinica is a self-compatible, pollinator-dependent species, and its fruit/seed set can be significantly increased by hand-pollination. No functional seed dispersers have been found in its extant natural habitats. These findings suggest that it may face both pollination and seed disperser insufficiencies in its current fragmented habitats, which may account for its low regeneration. Here we propose conservation strategies based on our findings.

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.

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.

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 μl to 0.64 μl 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-α-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.

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.

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 (F4,25=35.139, P<0.0001) and seed set per fruit (F4,25=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 Andrenidae 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.