The role of botanical gardens in scientific research, conservation, and citizen science

Plant diversity is currently being lost at an unprecedented rate, resulting in an associated decrease in ecosystem services. About a third of the world＇s vascular plant species face the threat of extinction due to a variety of devastating activities, including, over-harvesting and over exploitation, destructive agricul- tural and forestry practices, urbanization, environmental pollution, land-use changes, exotic invasive species, global climate change, and more. We therefore need to increase our efforts to develop integrative conservation approaches for plant species conservation. Botanical gardens devote their resources to the study and conservation of plants, as well as making the world＇s plant species diversity known to the public. These gardens also play a central role in meeting human needs and providing well-being. In this minireview, a framework for the integrated missions of botanical gardens, including scientific research, inJex situ conservation, plant resource utilization, and citizen science are cataloged. By reviewing the history of the development of Kunming Botanical Garden, we illustrate successful species conservation approaches （among others, projects involving Camellia, Rhododendron, Magnolia, Begonia, Alliurn, Ne- penthes, medicinal plants, ornamental plants, and Plant Species with Extreme Small Populations）, as well as citizen science, and scientific research at Kunming Botanical Garden over the past 80 years. We emphasize that Kunming Botanical Garden focuses largely on the ex situ conservation of plants from Southwest China, especially those endangered, endemic, and economically important plant species native to the Yunnan Plateau and the southern Hengduan Mountains. We also discuss the future chal- lenges and responsibilities of botanical gardens in a changing world, including： the negative effects of outbreeding and/or inbreeding depression; promoting awareness, study, and conservation of plant species diversity; accelerating global access to information about plant diversity; increasing capacity building and training activities. We hope this minireview can promote understanding of the role of botanical gardens.

Floral characteristics and pollination ecology of Manglietia ventii(Magnoliaceae),a plant species with extremely small populations(PSESP)endemic to South Yunnan of China

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 pollinatoreplant 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, b-Pinene, a-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.

Plant species with extremely small populations(PSESP) in China:A seed and spore biology perspective

Approximately one fifth of the world's plants are at risk of extinction.Of these,a significant number exist as populations of few individuals,with limited distribution ranges and under enormous pressure due to habitat destruction.In China,these most-at-risk species are described as 'plant species with extremely small populations'(PSESP).Implementing conservation action for such listed species is urgent.Storing seeds is one of the main means of ex situ conservation for flowering plants.Spore storage could provide a simple and economical method for fern ex situ conservation.Seed and spore germination in nature is a critical step in species regeneration and thus in situ conservation.But what is known about the seed and spore biology(storage and germination) of at-risk species? We have used China's PSESP(the first group listing) as a case study to understand the gaps in knowledge on propagule biology of threatened plant species.We found that whilst germination information is available for 28 species(23%of PSESP),storage characteristics are only known for 8%of PSESP(10 species).Moreover,we estimate that 60%of the listed species may require cryopreservation for long-term storage.We conclude that comparative biology studies are urgently needed on the world's most threatened taxa so that conservation action can progress beyond species listing.

Reproductive biology of Magnolia sinica(Magnoliaecea),a threatened species with extremely small populations in Yunnan,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.

High regional genetic differentiation of an endangered relict plant Craigia yunnanensis and implications for its conservation

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.

Conservation and fruit biology of Sichou oak {Quercus sichourensis,Fagaceae)- A critically endangered species in China

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.

Morphological characteristics of leaf epidermis and size variation of leaf,flower and fruit in different ploidy levels in Buddleja macrostachya (Buddlejaceae)

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 orB. macrostachya. In general, flower and fruit length, absolute leaf size, and stomatal length in,eased with an increase at ploidy level （P〈0.01）, whereas adaxial cell and stomatal density decreased with an increase at ploidy level （P〈0.01）. We found no conspicuous differences in relative leaf size （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 hexaptoid from dodecaploid cytotypes either in the field or in herbarium specimens.

The genome of Magnolia biondii Pamp. provides insights into the evolution of Magnoliales and biosynthesis of terpenoids

Magnolia biondii Pamp.(Magnoliaceae,magnoliids)is a phylogenetically,economically,and medicinally important ornamental tree species widely grown and cultivated in the north-temperate regions of China.Determining the genome sequence of M.biondii would help resolve the phylogenetic uncertainty of magnoliids and improve the understanding of individual trait evolution within the Magnolia genus.We assembled a chromosome-level reference genome of M.biondii using~67,~175,and~154Gb of raw DNA sequences generated via Pacific Biosciences single-molecule real-time sequencing,10X Genomics Chromium,and Hi-C scaffolding strategies,respectively.The final genome assembly was~2.22Gb,with a contig N50 value of 269.11 kb and a BUSCO complete gene percentage of 91.90%.Approximately 89.17%of the genome was organized into 19 chromosomes,resulting in a scaffold N50 of 92.86Mb.The genome contained 47,547 protein-coding genes,accounting for 23.47%of the genome length,whereas 66.48%of the genome length consisted of repetitive elements.We confirmed a WGD event that occurred very close to the time of the split between the Magnoliales and Laurales.Functional enrichment of the Magnolia-specific and expanded gene families highlighted genes involved in the biosynthesis of secondary metabolites,plant–pathogen interactions,and responses to stimuli,which may improve the ecological fitness and biological adaptability of the lineage.Phylogenomic analyses revealed a sister relationship of magnoliids and Chloranthaceae,which are sister to a clade comprising monocots and eudicots.The genome sequence of M.biondii could lead to trait improvement,germplasm conservation,and evolutionary studies on the rapid radiation of early angiosperms.

Karyomorphology of three species in Dipentodon(Dipentodontaceae),Perrottetia(Celastraceae),and Tapiscia(Tapisciaceae) of the order Huerteales and their phylogenetic implications

Abstract The karyomorphology of three species in Dipentodon （Dipentodontaceae）, Perrottetia （Celastraceae）, and Tapiscia （Tapisciaceae）, namely Dipentodon sinicus, Perrottetia racemosa, and Tapiscia sinensis, was investigated in the present study. Recent molecular research has discovered close relationships among these three genera, which has led to the establishment of the order Huerteales with Perrottetia being placed in Dipentodontaceae. Herein we report the chromosome numbers of D. sinicus and P. racemosa for the first time, and present their karyotype formulas as 2n = 34 = 22 sm ＋ 12 st （D. sinicus）, 2n = 20 = 11 m ＋ 9 sm （P. racemosa）, and 2n = 30 = 22 m（2SAT） ＋ 8sm （T. sinensis）. Asymmetry of their karyotypes is categorized to be Type 3B in D. sinicus, Type 2A in P. racemosa, and Type 2A in T. sinensis. Each of the species shows special cytological features. Compared with Perrottetia, Dipentodon has a different basic chromosome number, a higher karyotype asymmetry, and different karyomorphology of its interphase nuclei, mitotic prophase, and metaphase. Thus, on the basis of these results, we have reservations regarding the suggestion of placing Dipentodon and Perrottetia together in the family Dipentodontaceae.

Words from the Guest Editor-in-Chief

PSESP(Plant Species with Extremely Small Populations)is a new concept developed to rescue the most globally threatened plant species.It first appeared in'Proposal for the Extremely Small Population protection project for the endemic plants and animals of Yunnan',a report submitted to China's State Forestry Administration(SFA)through the Yunnan Forestry Department(YFD)in 2005.In September 2009,four years after the launch of the project。

Applying image clustering to phylogenetic analysis:A trial

Phylogenetic studies have increased in recent years,largely due to rapid developments in sequencing techniques (Tucker et al.,2017).However,molecular phylogenetic studies rely on collecting biomaterials,which limits their applicability to many,especially small,rare plants,or inaccessible plants.Recently.

Pollinator attraction in Cornus capitata (Cornaceae): the relative role of visual and olfactory cues

Aims it is generally accepted that visual displays and floral scent play important roles in communication between flowering plants and their pollinators.However,the relative role of visual and olfactory cues in pollinator attraction is largely unknown.in this study,we determined the roles of both types of cue in attracting pollinators to Cornus capitata,a medium sized tree with each capitulum sur-rounded by four large,white,petaloid bracts.Methods Pollinator observations and pollination experiments were con-ducted in a natural population;the inflorescences’visual and olfactory signals were characterized by spectral and chemical analyses;the responses of pollinators to visual and olfactory cues were tested using dual-choice behavioural bioassays;the relative roles of visual and olfactory cues in pollinator attrac-tion were tested by comparing the responses of pollinators to inflorescences subjected to three experimental treatments(intact,all bracts removed,and capitulum removed)within the natural population.Important Findings For fruit set,C.capitata is entirely dependent on pollinators,with a bee,Anthophora sp.,being the main pollinator.bracts present high colour distance and green contrast against the leaves.Twelve volatile compounds in the floral scent were detected,most of which have previously been reported to be attractive to a broad spectrum of bee species.behavioural bioassays showed that both,visual cues alone and olfactory cues alone,are attractive to pollinating bees.However,vis-ual cues alone attracted significantly more approaches than olfactory cues alone,while olfactory cues alone elicited a significantly higher landing percentage than visual cues alone.The finding suggests that,in the C.capitata-Anthophora sp.interaction,visual cues are mainly used for location from long distances,while olfactory cues mainly aid landing from short distances.our results indicate that different modalities of floral cues should be considered together to understand fully the communication between flowering plant and pollinators.

Seed dispersal by hornets:An unusual insect-plant mutualism

Vespicochory, seed dispersal by hornets, is a rare seed dispersal mechanism in angiosperms and, to date, there are few records of this phenomenon.Through field investigations and behavioral assays conducted in four populations of Stemona tuberosa from 2011–2016, we demonstrate that hornets are the primary seed dispersers of S. tuberosa and play an important role in "long-distance" seed dispersal in this species. Furthermore, some ant species act as secondary dispersers and may transport the seeds to safe sites.Hornets and ants provide complementary seed dispersal at different spatial scales. This unique example of insect-plant mutualism may be an underestimated but important strategy to ensure "long-distance" seed dispersal in other myrmecochorous plants.

Variation in floral characters,particularly floral scent,in sapromyophilous Stemona species

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.

Natural Hybridization between Rhododendron delavayi and R.cyanocarpum(Ericaceae),from Morphological,Molecular and Reproductive Evidence

The natural hybridization that occurs between two sympatric species of Rhododendron subgenus Hymenanthes in Yunnan, China, was investigated. In field observations, it was noted that the putative hybrids between R. delavayi Franch. and R. cyanocarpum （Franch.） Franch. ex W.W. Sm. had intermediate morphologies. On the basis of morphology, chloroplast DNA （trnL-rp132） and nuclear DNA （waxy）, hybrids and parental species were identified. Hybridization occurred in both directions, but was asymmetrical, with R. delavayi as the major maternal parent. Reciprocal hand pollination treatments showed that either species, as pollen donor or pollen receiver, could produce fruits. It was noted that fruit set varied among treatments. The same pollinators （bumblebees） were shared in both parental species. From these results, we conclude that individuals with intermediate morphologies are indeed of hybrid origin from natural hybridization between R. cyanocarpum and R. delavayi. Furthermore, we presume the hybridization at the study site could have been initiated by habitat disturbance in the 1950s, and we may hence witness the early stages of hybrid swarm formation.

Characterization of 24 microsatellite markers in Primula chungensis(Primulaceae),a distylous-homostylous species,using MiSeq sequencing

Primula chungensis is a species with considerable floral and mating-system variation,including distylous(outcrossing),homostylous(selfing) and mixed populations that contain both outcrossing and selfing forms.We isolated 24 microsatellite markers from P.chungensis using Illumina Mi Seq sequencing.Polymorphism and genetic diversity were then measured based on a sample of 24 individuals from a natural population in southern Tibet.All loci were polymorphic with the number of alleles per locus ranging from 2 to 4.The observed and expected heterozygosity ranged from 0 to 1 and 0.219 to 0.708,respectively.The microsatellite markers we have identified will serve as valuable tools for the investigation of the population genetic structure and phylogeography of P.chungensis and will inform models of the evolutionary history of mating systems in the species.