Slipper Orchid Research Articles

Detecting preservation and reintroduction sites for endangered plant species using a two‐step modeling and field approach

AbstractTo withstand the surge of species loss worldwide, (re)introduction of endangered plant species has become an increasingly common technique in conservation biology. Successful (re)introduction plans, however, require identifying sites that provide the optimal ecological conditions for the target species to thrive. In this study, we propose a two‐step approach to identify appropriate (re)introduction sites. The first step involves modeling the niche and distribution of the species with bioclimatic and topographical predictors, both at continental and at national scales. The second step consists of refining these bioclimatic predictions by analyzing stationary ecological parameters, such as soil conditions, and relating them to population‐level fitness values. We demonstrate this methodology using Swiss populations of the lady's slipper orchid (Cypripedium calceolus L., Orchidaceae), for which conservation plans have existed for years but have generally been unfruitful. Our workflow identified sites for future (re)introductions based on the species requirements for mid‐to‐sunny light conditions and specific soil physico‐chemical properties, such as basic to neutral pH and low soil organic matter content. Our findings show that by combining wide‐scale bioclimatic modeling with fine scale field measurements it is possible to carefully identify the ecological requirements of a target species for successful (re)introductions.

Home, the Farm

Home, the Farm

Characterization of the complete chloroplast genome of orchid family species Paphiopedilum bellatulum

The genus Paphiopedilum is well known as the lady’s slipper orchid in Orchidaceae family. Paphiopedilum bellatulum (Rchb.f.) Stein 1892, has important medicinal and ornamental value, which occurs in the tropical Asia. However, in recent decades, it was threatened with extinction by significantly reduced small population size. In this study, we sequenced and characterized the complete chloroplast genome of P. bellatulum based on the Illumina Hiseq platform. The size of P. bellatulum chloroplast genome was 156,567 bp, including a large single-copy (LSC) region of 88,243 bp, a small single-copy (SSC) region of 3652 bp, and two inverted repeat regions (IRs) of 32,336 bp. The overall GC contents of the chloroplast genome were 35.71%. A total of 122 genes were annotated, including 76 protein-coding genes, 38 transfer RNAs (tRNAs), and eight ribosomal RNAs (rRNAs). The phylogenetic analysis indicated that P. bellatulum formed a close relationship with another Paphiopedilum species P. wenshanense. The results will provide helpful genetic resource for further phylogenetic studies of the genus Paphiopedilum.

Insight into Paphiopedilum x Dalatense Aver. (Orchidaceae) origin based on morphological and molecular markers

Paphiopedilum callosum, Paphiopedilum villosum and Paphiopedilum ´ dalatense are endangered species and are currently listed as Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES, 2019) Appendix I species. Thus, the international trade for these naturally occurring species is forbidden. However, the in vitro propagated materials and their inter-species hybrids forced by breeders can be traded for commercial purposes. P. ´ dalatense is an endemic slipper orchid species of Vietnam with high aesthetic value. Currently, the natural reserves of this species have been greatly reduced by overexploitation. In this study, P. ´ dalatense was demonstrated to be the reversible hybrid species between P. callosum and P. villosum based on both morphological and molecular markers. P. ´ dalatense possesses intermediate morphological characteristics between P. callosum and P. villosum which could be recorded in leaf, in flower and its components such as lip, petal, and dorsal sepal. There were 23 sites in ITS1-5.8S rRNA-ITS2 region sequence of P. ´ dalatense which possess the nucleotide characteristic of both P. callosum and P. villosum. There were two out of three P. ´ dalatense investigated samples possessed partial matK gene which was identical to the corresponding sequence in P. villosum and the last sample possessed identical partial matK gene to P. callosum. Proving P. ´ dalatense to be the reversible hybrid species between P. callosum and P. villosum is the essentially scientific base for commercial breeding of P. ´ dalatense by artificial hybridization which contributes to avoid overexploiting the natural P. ´ dalatense resources.

Mesoamerican Cypripedium: Mycorrhizal Contributions to Promote Their Conservation as Critically Endangered Species

In the valuable orchid genus Cypripedium, the section Irapeana consists of a distinctive group of Mesoamerican species that is formed by Cypripedium dickinsonianum Hágsater, C. irapeanum Lex., and C. molle Lindl. All lady slipper orchids exhibit different distributions and abundances. Data analysis that used herbarium accessions and field investigations indicated that the habitats of these three species have been dramatically reduced. Prospecting for suitable habitats based on climatic, vegetation, and soil parameters allows us to predict potential distributions. Conservation strategies, such as ex situ propagation by asymbiotic and symbiotic approaches, have indicated that the culture media used are a determining factor for seedling development. Mycorrhizal isolates play a main role in the compatibility and further development of germinated seeds. The fungi isolated from adult plants belong to two different families, which makes it possible that widely distributed C. irapeanum populations will be fungal-specific as well as restricted for C. molle. Root mycorrhization patterns occur high on the secondary roots. In contrast with other species of the genus, in situ germination can occur over a short period of two months, but we have documented periods as long as ten years. Cypripedium is a highly problematic genus for ex situ conservation because the germination requirements and cultures are poorly documented, and there is great urgency for in situ conservation to develop strategies for identifying hotspot habitats and actualize the protection status to avoid extinction of this genus.

Multiple Fungi May Connect the Roots of an Orchid (Cypripedium reginae) and Ash (Fraxinus nigra) in Western Newfoundland.

Showy lady's slipper (Cypripedium reginae Walter, Orchidaceae) and black ash (Fraxinus nigra Marshall, Oleaceae) often co-occur in close proximity in fens in western Newfoundland, Canada. Metabarcoding of DNA extracted from root samples of both species following surface sterilization, and others without surface sterilization was used to determine if there were shared fungal endophytes in the roots of both species that could form a common mycorrhizal network between them. A wide variety of fungi were recovered from primers amplifying the nuclear ribosomal internal transcribed spacer region (ITS2). Sixty-six fungal sequences were shared by surface-sterilized roots of both orchid and ash, among them arbuscular mycorrhizal fungi (Claroideoglomus, Dominikia, Glomus and Rhizophagus), ectomycorrhizal fungi (Inocybe and Tomentella), the broad-host root endophyte Cadophora orchidicola, along with root pathogens (Dactylonectria, Ilyonectria, Pyricularia, and Xylomyces) and fungi of unknown function. There appear to be multiple fungi that could form a common mycorrhizal network between C. reginae and F. nigra, which might explain their frequent co-occurrence. Transfer of nutrients or carbon between the orchid and ash via one or more of the shared fungal endophytes remains to be demonstrated.

Comparative Analysis of the Complete Chloroplast Genomes of Nine Paphiopedilum Species.

Paphiopedilum is known as “lady’s or Venus” slipper orchids due to its prominent shoe-shaped labellum, with high ornamental value. Phylogenetic relationships among some species in Paphiopedilum genus cannot be effectively determined by morphological features alone or through the analysis of nuclear or chloroplast DNA fragments. In order to provide aid in understanding the evolutionary and phylogenetic relationship in Paphiopedilum at chloroplast (cp) genome-scale level, the complete cp genomes of six Paphiopedilum species were newly sequenced in this study, and three other published cp genome sequences of Paphiopedilum were included in the comparative analyses. The cp genomes of the six Paphiopedilum species ranged from 154,908 bp (P. hirsutissimum) to 161,300 bp (P. victoria-mariae) in size, all constituting four-part annular structures. Analyses of the nucleotide substitutions, insertions/deletions, and simple sequence repeats in the cp genomes were conducted. Ten highly variable regions that could serve as potential DNA barcodes or phylogenetic markers for this diverse genus were identified. Sequence variations in the non-coding regions were greater than that in the conserved protein-coding regions, as well as in the large single copy (LSC) and small single copy (SSC) regions than in the inverted repeat (IR) regions. Phylogenetic analysis revealed that all Paphiopedilum species clustered in one monophyletic clade in the Cypripedioideae subfamily and then subdivided into seven smaller branches corresponding to different subgenus or sections of the genus, with high bootstrap supports, indicate that cp genome sequencing can be an effective means in resolving the complex relationship in Paphiopedilum.

Paphiopedilum charlesworthii var. lannaense, a new slipper orchid from Northern Thailand identified by morphological and AFLP analyses

A new endemic slipper orchid in the family Orchidaceae from Northern Thailand was described according to morphological and molecular analyses. Morphological information specifies the new slipper orchid Paphiopedilum charlesworthii var. lannaense to be similar to Paphiopedilum charlesworthii (Rolfe) Pfitzer 1895, with the exception of its staminode being obovate-obcordate with yellow color, glittery and rough surface. Molecular analysis by AFLP technique indicates that Paphiopedilum charlesworthii var. lannaense can be included as a member of section Paphiopedilum and is closely related to Paphiopedilum coccineum Perner, H. & Herrmann, R. (2000) (syn. Paphiopedilum barbigerum var. coccineum), from which it differs morphologically by dorsal sepal and petal characters. The morphological and molecular evidence supported that Paphiopedilum charlesworthii var. lannaense is a new slipper orchid in the genus Paphiopedilum (Cypripedioideae, Orchidaceae).

FIRST DESCRIPTION OF Cypripedium AND Cypripedium calceolus var. citrina B. HERGT IN ROMANIA: MORPHOLOGY, POLLINATION AND CONSERVATION STATUS

Cypripedium genus is represented by 59 species and nothospecies (species of hybrid origin), mainly distributed across North, Central and South America, Europe and Asia, but absent from Madagascar and Australia. The most important characteristic of this genus is their showy and large labellum, which transformed into the specific slipper-shaped, vividly coloured, inflated pouch. In Romania, the genus is represented by one species only, Cypripedium calceolus. The article contains the first description of a newly discovered population in Prahova County, as well as a complete illustration of a Cypripedium calceolus flower pollinated by a female bee of the Lasioglossum genus. The flowers are passive traps pollinated by bees of particular type and size. The article describes in detail different mimicry (deceit) pollination strategies employed by the orchid in order to exploit its insect pollinators (generalized food deception mimicry, shelter mimicry, nest/brood-site mimicry, rendezvous attraction). Using advanced techniques of ultra-macro photography, details of floral organs are illustrated in order to describe in great detail the intriguing deceptive pollination mechanisms and mimicry strategies employed by the Slipper Orchids to attract their highly specific and rather scarce pollinating insects. Along with the detailed description and illustration of the common single-flowered plants, we also describe rare Cypripedium calceolus f. biflorum plants in Prahova County and present for the first time in Romania, a unique Cypripedium calceolus var. citrina individual, found in 2019, in Suceava County.

Genetic Analysis of Lady's Slippers

Genetic Analysis of Lady's Slippers

Characterization of phytohormone and transcriptome profiles during protocorm-like bodies development of Paphiopedilum

BackgroundPaphiopedilum, commonly known as slipper orchid, is an important genus of orchid family with prominent horticultural value. Compared with conventional methods such as tillers and in vitro shoots multiplication, induction and regeneration of protocorm-like bodies (PLBs) is an effective micropropagation method in Paphiopedilum. The PLB initiation efficiency varies among species, hybrids and varieties, which leads to only a few Paphiopedilum species can be large-scale propagated through PLBs. So far, little is known about the mechanisms behind the initiation and maintenance of PLB in Paphiopedilum.ResultsA protocol to induce PLB development from seed-derived protocorms of Paphiopedilum SCBG Huihuang90 (P. SCBG Prince × P. SCBG Miracle) was established. The morphological characterization of four key PLB developmental stages showed that significant polarity and cell size gradients were observed within each PLB. The endogenous hormone level was evaluated. The increase in the levels of indoleacetic acid (IAA) and jasmonic acid (JA) accompanying the PLBs differentiation, suggesting auxin and JA levels were correlated with PLB development. Gibberellic acid (GA) decreased to a very low level, indicated that GA inactivation may be necessary for shoot apical meristem (SAM) development.Comparative transcriptomic profiles of four different developmental stages of P. SCBG Huihuang90 PLBs explore key genes involved in PLB development. The numbers of differentially expressed genes (DEGs) in three pairwise comparisons (A vs B, B vs C, C vs D) were 1455, 349, and 3529, respectively. KEGG enrichment analysis revealed that DEGs were implicated in secondary metabolite metabolism and photosynthesis. DEGs related to hormone metabolism and signaling, somatic embryogenesis, shoot development and photosynthesis were discussed in detail.ConclusionThis study is the first report on PLB development in Paphiopedilum using transcriptome sequencing, which provides useful information to understand the mechanisms of PLB development.

Effects of forest stand structure on population of endangered orchid species Cypripedium calceolus L.

Lady’s slipper orchid (Cypripedium calceolus L.) has suffered a dramatic decline and is one of the most endangered orchid species in Europe. It grows mainly on calcareous soils in deciduous and mixed forests. Although numerous studies have contributed to our understanding of the causes of C. calceolus decline, surprisingly, little is known about the effects of the forest stand structure on the spatial distribution and morphometric characteristics of C. calceolus ramets. In this study, we analyzed how the spatial distribution of tree species and several structural characteristics of the forest stand affect the distribution and morphology of ramets of C. calceolus. For this, we used a remnant population of this species located in extensively managed forests in the Krowiarki Range (the Sudetes, SW Poland). The alarming decline in C. calceolus in this region over the last century is commonly attributed to forest management and land-use changes (abandonment of pastoralism in forests). We analyzed the morphometric characteristics and spatial distribution of C. calceolus ramets in this population in relation to the spatial distributions of trees, canopy gaps, saplings and structural characteristics of the forest stand. For this, we combined precise measurements of the forest stand structure, spatial analyzes in GIS and multivariate logistic regression modeling.The probability of the occurrence of C. calceolus was best predicted by its proximity to silver fir trees. We attributed this mainly to a positive effect by silver fir on the topsoil moisture, which can be especially beneficial to C. calceolus as this species is sensitive to drought. The occurrence of C. calceolus ramets was negatively affected by the basal area and positively affected by the diameter at the breast height of trees growing in a 5 m buffer zone. The structural attributes of the forest stand structure also affected the orchid’s morphology. Ramets growing close to beech or sycamore had lower leaf dimensions than ramets close to fir, and a pattern of decreasing leaf size with proximity to beech or sycamore was visible.This study spotlights the relationship between forest stand structure and the population of the endangered orchid C. calceolus. Based on the results of this and previous research, we have formulated several recommendations for the protection of the remnant population of this species in managed forests. These include maintaining mixed beech-fir forests rather than pure beech forest stands, supporting forest regeneration by species other than European beech, cutting young beech trees to prevent overgrowing the orchid ramets, fencing remnant populations of C. calceolus to exclude browsing pressure and maintaining spatial heterogeneity to support wild pollinators.

Niche evolution and historical biogeography of lady slipper orchids in North America and Eurasia

AbstractAimDispersal and subsequent adaptation to local environmental and climatic conditions play major roles in the evolution and current‐day distribution of plants and animals, but their relative importance is not always easy to disentangle. The aim of this study was to provide a comprehensive phylogenetic analysis of the terrestrial orchid genus Cypripedium to better understand the role of niche evolution and historical dispersal events in driving the radiation of the genus in North America and Eurasia.LocationNorthern Hemisphere.TaxonCypripedium (Orchidaceae).MethodsWe used a relaxed molecular clock analysis to infer phylogenetic relationships among 45 Cypripedium species and to estimate divergence times. Niche modelling was used to assess the potential distribution of each species in both continents, whereas ancestral area reconstructions using BioGeoBEARS were used to estimate changes in historical biogeography. Subsequently, the ancestral state of niche conditions was assessed for each species using maximum likelihood methods and we tested for phylogenetic signal in the evolution of niche conditions using Blomberg's K and Pagel's λ. A mantel test was used to test for a correlation between phylogenetic distances and niche overlap in Cypripedium.ResultsWe found that Cypripedium originated in Central America and underwent multiple dispersal events between North America and Eurasia after long‐distance seed dispersal or dispersal via the Bering land bridge. The mountainous area of Southwest China provided refuge areas for Cypripedium orchids during episodes of increased glaciation. Our results further showed that Cypripedium has gradually increased its tolerance to seasonal temperature changes, providing evidence for significant niche evolution through time. No significant correlation between current niches and phylogenetic relatedness was observed.Main conclusionsOur study provides clear evidence that repeated dispersal and adaptation to increased cooling after the Eocene−Oligocene Transition has contributed to the radiation of Cypripedium in the Northern Hemisphere.

Materials to the creation of the “Lady slipper grove” nature monument of regional importance (Altai Krai)

Materials on the flora and vegetation of the projected nature monument of regional importance “Ladyslipper grove” are given. Flora comprises 150 species of higher vascular plants, including 4 rare species: Cypripediumcalceolus L., C. macranthon Sw., C. ventricosum Sw., Orchis militaris L. The vegetation is typical for left-bank forest-steppe:birch, aspen and birch-aspen communities in combination with steppe meadows along the edges, and steppes with Festucavalesiaca.

Ресурсы лекарственных растений Национального парка «Башкирия» Республики Башкортостан

The vegetation of the Bashkiria National Park is one of the basic components for studying the flora and vegetation of the Republic of Bashkortostan. Accounting and protection of such plant biodiversity is important for their conservation and study. Based on the results of geobotanical surveys in the Bashkiria National Park in the spring-summer period of 2020, 270 species of medicinal plants belonging to 61 families were identified, of which the majority in terms of the number of species are represented by the families Asteraceae, Lamiaceae, Rosaceae, Ranunculaceae, Fabaceae and Apiaceae. Despite the fact that in geomorphological terms the national park is included in the region of low-mountainous relief of the eastern region of the mountains of the Southern Urals, medicinal plants belonging to meadow and forest coenotic groups prevail in the Bashkiria National Park. The presence of previously discovered species such as Rhodiola iremelica Boriss Rhodiola Iremel, Helichrysum arenarium (L.) Moench Sandy cumin, Cypripedium guttatum Sw. spotted ladys slipper, included in the Red Book of Bashkiria. In addition, Stipa pennata L. was found feather grass included in both the Red Book of Bashkiria and the Red Book of Russia.

Genetic History of the Remnant Population of the Rare Orchid Cypripedium calceolus Based on Plastid and Nuclear rDNA.

The lady’s slipper orchid (Cypripedium calceolus), which inhabits shady deciduous and mixed forests and meadows, is now threatened with extinction in many European countries, and its natural populations have been dramatically declining in recent years. Knowledge of its evolutionary history, genetic variability, and processes in small populations are therefore crucial for the species’ protection. Nowadays, in south-west Poland, it is only distributed in seven small remnant and isolated populations, which we examined. One nuclear (ITS rDNA) and two plastid (accD-psa1, trnL-F) markers were analyzed and compared globally in this study. Based on the nuclear marker, the most common ancestor of C. calceolus and Cypripedium shanxiense existed about 2 million years ago (95% HPD: 5.33–0.44) in Asia. The division of the C. calceolus population into the European and Asian lineages indicated by C/T polymorphism started about 0.5 million years ago (95% HPD: 1.8–0.01). The observed variation of plastid DNA, which arose during the Pleistocene glacial–interglacial cycles, is still diffuse in Poland. Its distribution is explained by the result of fragmentation or habitat loss due to human impact on the environment.

Peduncle vulnerability to embolism is related to conduit dimensions of the critically endangered slipper orchids in Southwest China

Members of the family Orchidaceae are amongst the most ornamental and protected plants in the world. The understanding of physiological and ecological characteristics of orchids is of great significance for predicting their response to environmental changes and establishing orchid conservation strategies. The physiological characteristics of flowers affect their relationship with pollinators and successful plant reproduction, but research on water physiology of flowers is still lacking. Cypripedium and Paphiopedilum are closely related phylogenetically in Orchidaceae, but they are different in floral longevity, life history, and habitat. In this study, we used the optical vulnerability method to investigate differences in flower peduncle drought resistance in the four slipper orchids of Cypripedium and Paphiopedilum, which are critically endangered orchids in Southwest China, and to test whether peduncle conduit dimensions determine embolism resistance. We found that the peduncles of Paphiopedilum species had greater xylem embolism resistance than that of Cypripedium. Across species, the time of 50% embolism of the peduncles was strongly correlated with the mean ratio (t/b)3 in peduncles. Our results are in line with habitat-based expectations that flower peduncle embolism resistance and conduit traits in Paphiopedilum species has helped them adapt to seasonal drought stress in karst habitats. However, the peduncle traits of Cypripedium reflect adaptations to an environment characterized by abundant soil water during the flowering period. Moreover, drought resistance of the flower peduncle was related to conduit dimensions. The results of this study contribute to our understanding of the divergent adaptation of flower traits in slipper orchids, which is beneficial for the conservation of endangered orchids.

Floral Longevity of Paphiopedilum and Cypripedium Is Associated With Floral Morphology.

Floral longevity (FL) is an important trait influencing plant reproductive success by affecting the chance of insect pollination. However, it is still unclear which factors affect FL, and whether FL is evolutionarily associated with structural traits. Since construction costs and water loss by transpiration play a role in leaf longevity, we speculated that floral structures may affect the maintenance and loss of water in flowers and, therefore, FL. Here, we investigated the slipper orchid Paphiopedilum and Cypripedium, which are closely related, but strongly differ in their FL. To understand the evolutionary association of floral anatomical traits with FL, we used a phylogenetic independent comparative method to examine the relationships between 30 floral anatomical traits and FL in 18 species of Paphiopedilum and Cypripedium. Compared with Paphiopedilum species, Cypripedium species have lower values for floral traits related to drought tolerance and water retention capacity. Long FL was basically accompanied by the thicker epidermal and endodermal tissues of the floral stem, the thicker adaxial and abaxial epidermis of the flower, and low floral vein and stomatal densities. Vein density of the dorsal sepals and synsepals was negatively correlated with stomatal density. Our results supported the hypothesis that there was a correlation between FL and floral anatomical traits in slipper orchids. The ability to retain water in the flowers was associated with FL. These findings provide a new insight into the evolutionary association of floral traits with transpirational water loss for orchids under natural selection.

The chloroplast genome evolution of Venus slipper (Paphiopedilum): IR expansion, SSC contraction, and highly rearranged SSC regions

BackgroundPaphiopedilum is the largest genus of slipper orchids. Previous studies showed that the phylogenetic relationships of this genus are not well resolved, and sparse taxon sampling documented inverted repeat (IR) expansion and small single copy (SSC) contraction of the chloroplast genomes of Paphiopedilum.ResultsHere, we sequenced, assembled, and annotated 77 plastomes of Paphiopedilum species (size range of 152,130 – 164,092 bp). The phylogeny based on the plastome resolved the relationships of the genus except for the phylogenetic position of two unstable species. We used phylogenetic and comparative genomic approaches to elucidate the plastome evolution of Paphiopedilum. The plastomes of Paphiopedilum have a conserved genome structure and gene content except in the SSC region. The large single copy/inverted repeat (LSC/IR) boundaries are relatively stable, while the boundaries of the inverted repeat and small single copy region (IR/SSC) varied among species. Corresponding to the IR/SSC boundary shifts, the chloroplast genomes of the genus experienced IR expansion and SSC contraction. The IR region incorporated one to six genes of the SSC region. Unexpectedly, great variation in the size, gene order, and gene content of the SSC regions was found, especially in the subg. Parvisepalum. Furthermore, Paphiopedilum provides evidence for the ongoing degradation of the ndh genes in the photoautotrophic plants. The estimated substitution rates of the protein coding genes show accelerated rates of evolution in clpP, psbH, and psbZ. Genes transferred to the IR region due to the boundary shift also have higher substitution rates.ConclusionsWe found IR expansion and SSC contraction in the chloroplast genomes of Paphiopedilum with dense sampling, and the genus shows variation in the size, gene order, and gene content of the SSC region. This genus provides an ideal system to investigate the dynamics of plastome evolution.

Vegetative anatomy and photosynthetic performance of the only known winter-greenCypripediumspecies: implications for divergent and convergent evolution of slipper orchids

AbstractCypripedium subtropicum is the only known winter-green species in the genus Cypripedium, whereas the other nearly 50 species keep their leaves for less than half the year. Life form has an important effect on carbon acquisition and adaptation of plants. However, the physiological mechanism behind it remains unclear. In this study, we investigated vegetative anatomy and photosynthetic performance of C. subtropicum across with its leaf ages. These anatomical and photosynthetic traits were also compared with typical Cypripedium spp. and other members of subfamily Cypripedioideae. The obtained results confirmed that this species exhibited many characters of shade plants, such as thin leaves, extremely low photosynthetic rate and light saturation point and high chlorophyll content. Unlike the strategy adopted by typical Cypripedium spp. that quickly achieve annual carbon gain with a high assimilation rate in a short growing season, C. subtropicum obtains its carbon through a low assimilation rate but a much longer leaf lifespan. The local climate and favourable light condition guaranteed the comparable carbon income in winter to compensate for its low photosynthetic capacity. The long-lived, thin leaves of C. subtropicum, differing from the long-lived, thick leathery leaves in conduplicate-leaved genera, represent a distinct adaptive strategy in subfamily Cypripedioideae. Our findings shed light on the divergent and convergent evolution in slipper orchids, and we hope these findings will contribute to the conservation of such an endangered orchid.