Genus Cypripedium Research Articles

Phylogenomic Analysis of Target Enrichment and Transcriptome Data Uncovers Rapid Radiation and Extensive Hybridization in Slipper Orchid Genus Cypripedium L.

Cypripedium is the most widespread and morphologically diverse genus of slipper orchids. Despite several published phylogenies, the topology and monophyly of its infrageneric taxa remained uncertain. Here, we aimed to reconstruct a robust section-level phylogeny of Cypripedium and explore its evolutionary history using target capture data for the first time. We used the orchid-specific bait set Orchidaceae963 in combination with transcriptomic data to reconstruct the phylogeny of Cypripedium based on 913 nuclear loci, covering all 13 sections. Subsequently, we investigated discordance among nuclear and chloroplast trees, estimated divergence times and ancestral ranges, searched for anomaly zones, polytomies, and diversification rate shifts, and identified potential gene (genome) duplication and hybridization events. All sections were recovered as monophyletic, contrary to the two subsections within sect. Cypripedium. The two subclades within this section did not correspond to its subsections but matched the geographic distribution of their species. Additionally, we discovered high levels of discordance in the short backbone branches of the genus and within sect. Cypripedium, which can be attributed to hybridization events detected based on phylogenetic network analyses, and incomplete lineage sorting caused by rapid radiation. Our biogeographic analysis suggested a Neotropical origin of the genus during the Oligocene (~30 Ma), with a lineage of potentially hybrid origin spreading to the Old World in the Early Miocene (~22 Ma). The rapid radiation at the backbone likely occurred in Southeast Asia around the Middle Miocene Climatic Transition (~15-13 Ma), followed by several independent dispersals back to the New World. Moreover, the Pliocene-Quaternary glacial cycles may have contributed to further speciation and reticulate evolution within Cypripedium. Our study provided novel insights into the evolutionary history of Cypripedium based on high-throughput molecular data, shedding light on the dynamics of its distribution and diversity patterns from its origin to the present.

Habitats and population structure of rare orchids from the genus Cypripedium in Siberia

Cypripedium calceolus L., C. macranthos Sw. and C. ventricosum Sw. are rare Eurasian orchid species, whose habitats are poorly studied in Siberia. In this paper, we describe 55 sympatric and 10 separated populations of the three species, located in Russia within Republic of Buryatia, Irkutsk Region, Krasnoyarsk Territory, and Republic of Altai. Cypripedium macranthos plants dominated the most of the sympatric populations. Vegetation surveys were performed in all localities, and the collected data were analyzed using the IBIS software. We applied the phytoindication scales elaborated by Tsatsenkin and his colleagues for ecological indicator value analysis. The Cypripedium populations were characterized by high floristic diversity of their habitats; however, all of them were found in areas where both canopy density and ground cover were low. In the most of the separated populations, ontogenetic spectra of Cypripedium species corresponded to the base spectrum with a predominance of the adult vegetative and reproductive shoots.

Species Distribution Modeling Reveals Recent Shifts in Suitable Habitat for Six North American Cypripedium spp. (Orchidaceae)

Accelerating climate change is expected to cause range shifts of numerous taxa worldwide. While climatic projections and predicted consequences typically focus on the future (2050 or later), a measurable change in climatic conditions has occurred over recent decades. We investigate whether recent climate change has caused measurable shifts in suitable habitat for six North American species in the highly threatened genus Cypripedium (Orchidaceae). We constructed species distribution models using a maximum entropy approach from species occurrence records, 19 bioclimatic variables, land cover data, and soil data for two decadal time intervals (1980–1989 and 2010–2019). Models were compared between time intervals to assess shifts in locality, size, fragmentation, and mean elevation of suitable habitat. For all six congeners, the centroids of suitable habitat shifted between time intervals, although the directionality varied. There was, however, consistency among species within geographic regions. Consistent with our expectations, the optimal habitat for most species shifted to a higher elevation and for western species it shifted northwards. However, the habitat for one northwestern species shifted southwards and the habitat for eastern species converged on the Great Lakes region from different directions. This work illustrates the somewhat idiosyncratic responses of congeneric species to changing climatic conditions and how the geographic region occupied by a species may be more important for predicting shifts in habitat than is the response of a closely related taxon.

Comparative Chloroplast Genomics of Seven Endangered Cypripedium Species and Phylogenetic Relationships of Orchidaceae.

The species in the genus Cypripedium (Orchidaceae) are considered endangered, mainly distributed in the temperate regions of the Northern Hemisphere, with high ornamental and economic value. Despite previous extensive studies based on both morphology and molecular data, species and sections relationships within Cypripedium remain controversial. Here, we employed two newly generated Cypripedium chloroplast genomes with five other published genomes to elucidate their genomic characteristics. The two genomes were 162,773–207,142 bp in length and contained 128–130 genes, including 82–84 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. We identified 2,192 simple sequence repeats, 786 large repeat sequences, and 7,929 variable loci. The increase of repeat sequences (simple sequence repeats and large repeat sequences) causes a significant amplification in the chloroplast genome size of Cypripedium. The expansion of the IR region led to the pseudogenization or loss of genes in the SSC region. In addition, we identified 12 highly polymorphic loci (Pi > 0.09) suitable for inferring the phylogeny of Cypripedium species. Based on data sets of whole chloroplast genomes (IRa excluded) and protein-coding sequences, a well-supported phylogenetic tree was reconstructed, strongly supporting the five subfamilies of Orchidaceae and the genus Cypripedium as monophyletic taxa. Our findings also supported that C. palangshanense belonged to sect. Palangshanensia rather than sect. Retinervia. This study also enriched the genomic resources of Cypripedium, which may help to promote the conservation efforts of these endangered species.

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.

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.

Cypripedium lichiangense (Orchidaceae) mimics a humus-rich oviposition site to attract its female pollinator, Ferdinandea cuprea (Syrphidae).

Most species in the genus Cypripedium (Cypripedioideae) produce trap flowers, making it a model lineage to study deceptive pollination. Floral attractants in most species studied appear to target bee species of different sizes. However, more recent publications report fly pollination in some subalpine species, suggesting novel suites of adaptive floral traits. Cypripedium lichiangense (section Trigonopedia) is an endangered subalpine species endemic to the Hengduan Mountains, China. We observed and analysed its floral traits, pollinators and breeding systems over 2 years insitu and in the lab. Cypripedium lichiangense was visited by females of Ferdinandea cuprea (Syrphidae). The pollinia were carried dorsally on the fly thoraces. The eggs of this fly were frequently found in the saccate labellum and on other floral organs, suggesting brood-site mimesis. The orchid is self-compatible, but cross-pollination produces more viable embryos. We propose a new mode of floral mimesis, humus-rich oviposition site mimicry, for C. lichiangense. Compared with the mimesis of aphid colonies attracting syrphid pollinators (subfamily Syrphinae), whose larvae are entomophagic, as reported in some Paphiopedilum species (Cypripedioideae), pollination by deceit in C. lichiangense represents a distinct and separate mode of exploitation of another saprophagic (or phytophagic) larvae syrphid lineage in the subfamily Eristalinae and appears to indicate diversity of pollination strategies in SectionTrigonopedia of Cypripedium. However, this new brood-site mimesis seems to be less attractive to pollinators. As a possible adaptation to the weak attracted pollination strategy, this plant species has a long flowering period and extended lifespan of individual flowers to ensure reproductive success.

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.

The Impact of Human Pressure and Climate Change on the Habitat Availability and Protection of Cypripedium (Orchidaceae) in Northeast China.

Human pressure on the environment and climate change are two important factors contributing to species decline and overall loss of biodiversity. Orchids may be particularly vulnerable to human-induced losses of habitat and the pervasive impact of global climate change. In this study, we simulated the extent of the suitable habitat of three species of the terrestrial orchid genus Cypripedium in northeast China and assessed the impact of human pressure and climate change on the future distribution of these species. Cypripedium represents a genus of long-lived terrestrial orchids that contains several species with great ornamental value. Severe habitat destruction and overcollection have led to major population declines in recent decades. Our results showed that at present the most suitable habitats of the three species can be found in Da Xing’an Ling, Xiao Xing’an Ling and in the Changbai Mountains. Human activity was predicted to have the largest impact on species distributions in the Changbai Mountains. In addition, climate change was predicted to lead to a shift in distribution towards higher elevations and to an increased fragmentation of suitable habitats of the three investigated Cypripedium species in the study area. These results will be valuable for decision makers to identify areas that are likely to maintain viable Cypripedium populations in the future and to develop conservation strategies to protect the remaining populations of these enigmatic orchid species.

Морфометрическая изменчивость видов рода Cypripedium в коллекции Центрального сибирского ботанического сада СО РАН

The article is devoted to the study of the genus Cypripedium L., three of the studied species of this genus are included in the Red Book of the Russian Federation. Biological material was taken from natural phytocenoses of the Novosibirsk region and the Altai Republic and introduced on the collection site of the Central Siberian Botanical Garden. The adaptive response of individuals was studied at the morphological level depending on environmental changes in ex situ conditions. The presence of all main stages in the phenological cycle in the adapting representatives of the three species (except for fruiting in Cypripedium × ventricosum) is an indicator of optimal ecological conditions for ex situ. A comparative analysis of morphometric characters in generative individuals was carried out and significant differences between in situ and ex situ were revealed. The adaptive changes associated with the response to natural abiotic ecological conditions were also identified. The revealed differences in morphometric data for the three species allow us to note the presence of a significant adaptive reaction in C. calceolus and C. macranthon and relative adaptive inertia in C. × ventricosum. The ecological optimum in abiotic factors for the three studied species from the genus Cypripedium is the indicators for 2018.The factors for 2018 became optimal for the developmentt of generative buds, which led to 100 % flowering of individuals in C. macranthon in 2019. We also performed a dental analysis of the lower surface of the bracts in three species and studied the morphology of the stomatal apparatus. We have noted significant differences between the species, under the same eco-conditions, in the size of stomata and their number per 1 cm2.

Cultivation of Cypripedium L. species in vitro and growing them ex vitro

Asymbiotic seed germination in vitro of 10 species of the genus Cypripedium L. is described. Several methods of seed sterilization and some modifications of Harvais, Chu and Mudge, Norstog, Malmgren growth media weretested. Adaptation ability in the open ground of the seedlings obtained in vitro was investigated. In addition, the results ofintroduction of 17 cypripedium species in Moscow region are listed.

Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady\u2019s slipper orchid endemic to East Asia

BackgroundThe genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady’s slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List.ResultsWe investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid–late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage.ConclusionOur overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

The natural history of the genus Cypripedium (Orchidaceae)

The genus Cypripedium is one of five monophyletic genera within slipper orchids. Nuclear ribosomal ITS, nuclear low copy gene (ACO) and plastid data were used for molecular clock analyses. According to these analyses, the most recent common ancestor of Cypripedium appeared in the early Eocene. The Irapeana section is the oldest phylogenetic line within Cypripedium. The analyses indicate that after the Middle Eocene Climatic Optimum (c. 41 Ma) three main evolutionary lineages were split. Other important evolutionary processes took place in the late Oligocene-early Miocene. In this period, there were numerous divergence events within species groups characterized by transcontinental disjunction.

Does evolutionary history determine specificity in broad ecological interactions?

AbstractMost species engage in broad interactions, in which they interact with multiple partner species. The evolutionary processes that generate such generalized interactions remain unknown, partly due to the difficulty in comparing their breadth.We argue that the interaction specificity of species involved in broad interactions evolves in three ways: (a)assemblage specialization, in which a species specializes on particular host species that contribute unique resources, yielding specialization on the entire host assemblage; (b)apparent generalism, in which a species specializes on one or few host species that contribute unique resources, but also associates with other host species that contribute functionally redundant resources; and (c)true generalism, in which a species associates with multiple hosts that overlap functionally, and that are geographically interchangeable based on opportunity for encounter, leading to frequent host switching.We performed a phylogenetically controlled analysis of data on mycorrhizal fungal associations for approximately 25% of the orchid subfamily Cypripedioideae to determine whether these plants have specialized on their mycorrhizal fungal communities, or whether they are true generalists. We also assessed the impact of environmental factors on these associations.Our results suggested strong support for apparent generalism, suggesting strong specialization on particular, dominant fungi and weak specialization on others. Large orchid clades associated with dominant fungal species, notablyTulasnella cystidiophorafor genusCypripedium, andT. cystidiophoraandT. calosporafor genusPaphiopedilum. Significant phylogenetic signal in fungal species richness per plant species, but not in the fungal phylogenetic diversity per plant species nor in the composition of fungal assemblages across orchid species suggested that plant phylogeny is an important determinant of fungal association. Mixed linear models showed that environment influenced specificity across plant species, and that observed differences were strongly driven by differences in sampling effort.Synthesis.We found evidence of specialization of plant species on dominant fungal species, and to a lesser extent on their close relatives. The strong dominance of particular fungal species in these associations suggests important ecological roles for them, while environmental gradients in specificity suggest strong environmental filtering of these interactions.

Comparative transcriptomics provides insight into the molecular basis of species diversification of section Trigonopedia (Cypripedium) on the Qinghai-Tibetan Plateau

Deceptive pollination is key to the species richness of Orchidaceae. However, the genetic basis of species diversification is still under study. Section Trigonopedia is a monophyletic clade of genus Cypripedium distributed in the southwest of China. The species of this section are pollinated by different flies. Pollinator differentiation makes section Trigonopedia an ideal group for studying the genetic basis underlying species diversification. Here, we sequenced the transcriptomes of eight species of the genus Cypripedium, including six co-flowering species of section Trigonopedia and two species outside this section as an outgroup. We reconstructed the phylogeny of the section with the combined 1572 single-copy genes extracted from the eight species and produced a highly resolved tree of the section. Furthermore, we combined substitution rate estimation and differential expression analysis to identify candidate genes, including genes related to floral scent synthesis and environmental adaptation, involved in species differentiation. Field investigations showed that these species have adapted to different habitats. We propose that the species diversification in this section is initiated by floral scent differentiation, followed by habitat differentiation, finally leading to speciation. This study sheds novel light on the diversification of closely related orchid species in the Qinghai-Tibetan region.

REPORT OF A FLORAL VISITOR OF Cypripedium irapeanum (ORCHIDACEAE) IN TEPOZTLAN, MORELOS, MEXICO

The process and participating actors of pollination have fascinated many scholars since the time of Darwin. Nevertheless, for many species of orchids, the identity of the pollinator or the purpose of floral visits remains unknown. Cypripedium (Orchidaceae) is a genus of worldwide distribution that presents a broad range of colors and sizes. Three species are reported in Mexico: C. dickinsonianum, C. irapeanum and C. molle . However, there is very little information regarding the ecological interactions of these species. Firstly, a preliminary bibliographic review was conducted in order to compile information pertaining to the pollinators and floral visitors of the genus Cypripedium. Secondly, a population of C. irapeanum was monitored in a pine-oak forest at 1680 m.a.s.l. in the municipality of Tepoztlan, in Morelos, Mexico. The municipality of Tepoztlan in Morelos, Mexico, hosts a population of approximately 250 individuals of the species C. irapeanum . During a visit to this site in July 2015, two specimens of bees, belonging to the genus Lasioglossum subgenus Eickwortia , were found exiting from the apertures of the rear part of the C. irapeanum labellum. This event was photographed and one bee specimen was collected and identified to species, thus contributing to the first record of a floral visitor to this species of orchid. Lasioglossum nyctere is a floral visitor and possibly a pollinator of C. irapeanum. Future studies should confirm whether or not this bee is indeed a pollinator of the orchid.

Effects of altering medium strength and sucrose concentration onin vitrogermination and seedling growth ofCypripedium macranthosSw.

The genus Cypripedium, known as lady's slipper orchid, has been considered to have a considerable marketability as a potted and garden plant with beautiful flowers; however, this species is becoming endangered and, in some places extinct, due to habitat destruction and illegal collections. As such, an optimized artificial propagation system is necessary for its conservation and horticultural cultivation. For the establishment of the in vitro proliferation of the endangered Cypripedium macranthos Sw., native to Korea, the effects of medium strength and sucrose concentrations on germination, protocorm formation and seedling growth were investigated through asymbiotic seed culture. The highest germination rate and protocorm formation rate were obtained with a 1/4 MS medium; higher MS medium strengths did not generate the favorable conditions required for germination and protocorm growth. The overall growth characteristics of roots and buds developed from protocorms were best in cultures of 1/4 MS medium. On this medium containing 10 g·L -1 sucrose, both the germination rate and protocorm formation rate increased significantly. The general growth properties of seedlings developed on the medium with 10 g·L -1 sucrose were best, showing the highest bud formation rate and root number. Our results demonstrate that the 1/4 MS medium, supplemented with 10 g·L -1 sucrose, could improve in vitro germination, and facilitate the growth of seedlings developed from the protocorms of C. macranthos Sw.

Fly pollination in Cypripedium: a case study of sympatric C. sichuanense and C. micranthum

Most Cypripedium spp. are known to be pollinated by bees. However, myiophilous traits are found in some species, especially in sections Trigonopedia and Sinopedilum. Here we chose C. micranthum and C. sichuanense, two sympatric species endemic to Sichuan, China, to test whether these orchids are fly pollinated. Artificial pollination showed that both flowers are self-compatible but need pollen vectors for successful reproduction. Field observation showed that C. micranthum was pollinated by fruit flies and C. sichuanense by dung flies, both novel pollinators of Cypripedium orchids. These sympatric Cypripedium spp. are also cross-compatible, but hybrids were not found in nature. The pollination syndromes of C. sichuanense and C. micranthum fit into the complex sapromyiophily pattern. It appears that pollinator specificity is responsible for their reproductive isolation. The discovery of fly pollination in C. sichuanense and C. micranthum, which belong to the related sections Trigonopedia and Sinopedilum, suggests a shift from bee to fly pollination in the genus Cypripedium. Unlike most Cypripedium spp., the anthers of C. micranthum release discrete pollinia with narrow stalks instead of the usual amorphous pollen smears. This ‘proto-pollinarium’ is described, probably for the first time. These pollinia are most likely an adaptation for pollination by microdiptera, so the fly can carry the contents of both chambers in the same anther. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 50‐58.

Evolution and biogeography of the slipper orchids: Eocene vicariance of the conduplicate genera in the Old and New World Tropics.

Intercontinental disjunctions between tropical regions, which harbor two-thirds of the flowering plants, have drawn great interest from biologists and biogeographers. Most previous studies on these distribution patterns focused on woody plants, and paid little attention to herbs. The Orchidaceae is one of the largest families of angiosperms, with a herbaceous habit and a high species diversity in the Tropics. Here we investigate the evolutionary and biogeographical history of the slipper orchids, which represents a monophyletic subfamily (Cypripedioideae) of the orchid family and comprises five genera that are disjunctly distributed in tropical to temperate regions. A relatively well-resolved and highly supported phylogeny of slipper orchids was reconstructed based on sequence analyses of six maternally inherited chloroplast and two low-copy nuclear genes (LFY and ACO). We found that the genus Cypripedium with a wide distribution in the northern temperate and subtropical zones diverged first, followed by Selenipedium endemic to South America, and finally conduplicate-leaved genera in the Tropics. Mexipedium and Phragmipedium from the neotropics are most closely related, and form a clade sister to Paphiopedilum from tropical Asia. According to molecular clock estimates, the genus Selenipedium originated in Palaeocene, while the most recent common ancestor of conduplicate-leaved slipper orchids could be dated back to the Eocene. Ancestral area reconstruction indicates that vicariance is responsible for the disjunct distribution of conduplicate slipper orchids in palaeotropical and neotropical regions. Our study sheds some light on mechanisms underlying generic and species diversification in the orchid family and tropical disjunctions of herbaceous plant groups. In addition, we suggest that the biogeographical study should sample both regional endemics and their widespread relatives.

Molecular phylogeny of Cypripedium (Orchidaceae: Cypripedioideae) inferred from multiple nuclear and chloroplast regions

A molecular analysis was performed on 56 taxa in the orchid genus Cypripedium using nrDNA ITS and five chloroplast regions ( trnH- psbA, atpI- atpH, trnS- trnfM, trnL- F spacer, and the trnL intron). The genus Cypripedium was confirmed as monophyletic. Our data provided strong support for monophyletic grouping of eight infrageneric sections ( Subtropica, Obtusipetala, Trigonopedia, Sinopedilum, Bifolia, Flabelinervia, Arietinum, and Cypripedium) defined in earlier taxonomic treatments, and paraphyletic grouping of two sections ( Irapeana and Retinervi). Within the genus Cypripedium, the first divergent lineage consisted of two Mesomaerican species, and subsequently the Cypripedium debile lineage from eastern Asia was split. Our study did not support the notion that two Asian species ( Cypripedium subtropicum and Cypripedium singchii) were closely related to either Mesoamerican Cypripedium irapeanum or North American Cypripedium californicum, as indicated by previous interpretations based on morphological evidences. In addition, one pair of vicariant species, Cypripedium plectrochilum (eastern Asia) and Cypripedium arietinum (North America), unique to section Arietinum, was confirmed. Furthermore, within the monophyletic section Cypripedium two previously recognized subsections, Cypripedium and Macrantha, were shown to be paraphyletic. Our results suggested that this section split into two groups based on distribution (North America vs. Eurasia) instead of such previously used, morphological traits as flower color, and the shape of the lips (labellum) and lateral petals.