Slipper Orchid Research Articles

Flowers meet Newton: testing the role of gravitational pull in resupination of orchid flowers.

Resupination refers to the developmental orientation changes of flowers through ≈180º, leaving them effectively upside-down. It is a widespread trait present in 14 angiosperm families, including the Orchidaceae, where it is a gravitropic phenomenon actively controlled by auxins. Here, we demonstrate that the passive gravitational pull on flower parts can have an additional influence on resupination. We studied a lady's slipper orchid in which some flowers naturally fail to resupinate. We conducted a manipulative experiment removing floral parts and showed that both the probability of complete resupination and the degree of flower vertical movement (from 0º - 180º) are related to the mass of floral organs. During flower development, the tip of the ovary slightly curves actively (14.75º) due to gravitropism. This promotes a lever arm effect so that the gravitational pull acting on flower mass creates a torque that bends the ovary, orienting the flower into a resupinate position that is accessible to pollinators. The role of the mass of floral organs in resupination provides new insights into flower development and its role in pollination mechanisms.

Improvement of Growth Rate in In Vitro Culture of Paphiopedilum primulinum M. W. Wood & P. Taylor and Paphiopedilum glaucophyllum J. J. Smith using Banana Enrichment Media.

Paphiopedilum primulinum and Paphiopedilum glaucophyllum have unique labellum colour and shaped like lady's slippers. These orchids are from the Cochlopetalum section, which is exclusively found in Sumatra and Java. There are so many people that desire to collect these plants illegally. Due to extensive commercial exploitation, Paphiopedilum is in danger of going extinct. Tissue culture techniques are utilised to conserve threatened orchid germplasm in a short time. The success of the in vitro culture depends on the accuracy of the basic media composition used. The Ambon Lumut banana (ALB) can accelerate plant growth and cell division. Banana added to the culture medium was prepared by mashing the ripe flesh (3.5 months old) using a mortar. This research aims to investigate the effect of banana homogenate supplemented media for the orchids P. primulinum and P. glaucophyllum based on the parameters of difference of plant height (calculated from the base of the stem to the tip of the plant stem), number of leaves, and number of roots. The measurement method was carried out using a ruler with a centimetre scale. Observations and documentation were carried out once a week for 7 weeks after planting (WAP) for P. primulinum and P. glaucophyllum. The results showed that ½ Murashige and Skoog (MS) + ALB homogenate is a better medium for P. primulinum and P. glaucophyllum growth than media without banana homogenate. The highest values of plant height, leaf growth and root growth of P. primulinum with banana homogenate were 0.44 cm, 0.63 leaves, and 0.50 roots, respectively. The highest values of plant height and leaf growth of P. glaucophyllum were 0.75 cm and 1.90 leaves, respectively. Culture medium added banana homogenate was able to support the propagation of plants, some of which are returned to nature and others used for industrial purposes (conventionally cultivated by the community).

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.

Phylogenomic insights into the historical biogeography, character-state evolution, and species diversification rates of Cypripedioideae (Orchidaceae)

Cypripedioideae (slipper orchids; Orchidaceae) currently consist of ∼200 herbaceous species with a strikingly disjunctive distribution in tropical and temperate regions of both hemispheres. In this study, an updated phylogeny with representatives from all five cypripedioid genera was presented based on maximum likelihood and Bayesian inference of plastome and low-copy nuclear genes. Phylogenomic analyses indicated that each genus is monophyletic, but some relationships (e.g., those among Cypripedium sects. Acaulia, Arietinum, Bifolia, Flabellinervia, Obtusipetala and Palangshanensia) conflict with those in previous studies based on Sanger data. Cypripedioideae appeared to have arisen in South America and/or the adjacent Qinghai-Tibet Plateau and Hengduan Mountains ∼35 Mya. We inferred multiple dispersal events between East Asia and North America in Cypripedium, and between mainland Southeast Asia and the Malay Archipelago in Paphiopedilum. In the Americas, divergences among four genera (except Cypripedium) occurred around 31–20 Mya, long before the closure of the Isthmus of Panama, indicating the importance of long-distance dispersal. Evolutionary patterns between morphological and plastome character evolution suggested several traits, genome size and NDH genes, which are likely to have contributed to the success of slipper orchids in alpine floras and low-elevation forests. Species diversification rates were notably higher in epiphytic clades of Paphiopedilum than in other, terrestrial cypripedioids, paralleling similar accelerations associated with epiphytism in other groups. This study also suggested that sea-level fluctuations and mountain-building processes promoted the diversification of the largest genera, Paphiopedilum and Cypripedium.

Saramsa Orchid Sanctuary, Ranipool, Sikkim

An erstwhile experimental garden at Saramsa is now a Sanctuary of Tropical Orchids of Sikkim with about 200 species under cultivation. Slipper orchids like Paphiopedilum fairrieanum, the elegant Cymbidium eburneum and Dendrobium chrysotoxum are conseved here.

Fixed non-random orientation to the Sun (conversotropism) in two window-flowered greenhood orchids, Diplodium spp. (Orchidaceae: Pterostylidinae); implications for other window flowers and pollinator behaviour

Abstract Many plant species orient their flowers to the Sun. Such orientations may be dynamic (heliotropism or solar tracking) or static, adopting fixed non-random azimuths to points on the daily arc of the Sun (conversotropism as defined herein). Two cauline greenhood orchid species, Diplodium reflexum and D. ampliatum (Pterostylidinae), are shown to be negatively conversotropic and non-heliotropic. Cauline greenhoods are trap and window flowers having large translucent light panels that glow when backlit flowers are viewed from the front. Conversotropism in greenhoods is thought to maximize light transmission through the window panels to manipulate the behaviour of fungus gnat pollinators. Conversotropism is so far documented in only seven species in five plant families, but is likely to be more widespread. The general characteristics of conversotropism are summarized and a new two stage model of window function in greenhoods is proposed. Window flowers in the Lady Slipper orchids (Cypripedium) and in Arums of the genus Arisaema are also predicted to be conversotropic. The evolution of conversotropism in cauline greenhoods highlights the importance of floral windows in these orchids. The highly unusual trap flowers may use light both as a pollinator attractant and repellent at different stages of the pollination process. Greenhoods appear to exploit the instinct of flies to escape confinement and the behaviours enabling it. This probably also applies to other trap flowers and adds insect escape behaviours to the many other drives, instincts, and behaviours exploited by plants for pollination.

Incomplete resupination during floral development leads to pollination failure in a slipper orchid.

In many families, plants undergo floral resupination by twisting through approximately 180° during floral development so that the flower is effectively positioned upside down. In most orchids, resupination results in the median petal (i.e., the labellum) becoming lowermost, which plays a crucial role in pollination by serving as a landing platform or as a trapping device, or both. Incomplete resupination is predicted to lead to reduced pollination, although tests of this assumption are still lacking. We investigated the effect of resupination using Phragmipedium vittatum, a rare lady's slipper orchid whose specialized labellum forms a trapping device. First, we surveyed the natural occurrence of incomplete resupination. Then we manipulated flowers into non- (≈0°), half- (≈90°), and fully resupinate (≈180°) positions to test the effect of orientation on pollen smear removal and deposition by pollinators (female hoverflies). We found that ca. 10% of flowers in the natural population were not fully resupinate, being either non- (upward, 0-60°) or half-resupinate (sideward, 60-120°). The change in orientation prevented the effectiveness of pollination by hoverflies since no pollen smear removal or deposition were found in flowers from non- and half-resupinate treatments. Although these flowers still attracted hoverflies, they were not trapped effectively. As this orchid is incapable of autonomous self-pollination, flowers that do not resupinate fail to set fruits. These results highlight the importance of correct floral orientation provided by resupination to ensure pollination in orchids and other resupinate flowers.

<i>Cypripedium himalaicum</i> (Orchidaceae), a new distributional record for the flora of Arunachal Pradesh, India

Cypripedium himalaicum Rolfe belonging to the family Orchidaceae is reported here as an addition to the flora of Arunachal Pradesh, India. The species commonly known as ‘The Himalayan Slipper Orchid’ was earlier known from Himachal Pradesh, Jammu & Kashmir, Sikkim and Uttarakhand in India and four other countries (Bhutan, China, Myanmar and Nepal). The species is categorized as Endangered by the IUCN.

Unveiling Limestone Orchid Hotspots in the Karst Hills of Northern Peninsular Malaysia

To date, documentation of the diversity of orchids from the limestone hills in the northern part of Peninsular Malaysia, particularly in the states of Kedah and Perak, is still lacking, with limited literature available. There is indeed an urgent need to fill this knowledge gap, so that proper documentation of the diversity of orchids from this unique karst habitat can be prepared. In this study, a series of 12 months of diversity assessments on five limestone hills in Kedah and Perak has resulted in the discovery of 56 orchid species from 37 genera. From this account, 12 species are new records within Kedah and 2 species are new records within Malaysia, namely Bulbophyllum meson J.J.Verm., Schuit. & de Vogel and Luisia brachystachys (Lindl.) Blume. Three species are endemic to Peninsular Malaysia which are Anoectochilus sanguineus P.T.Ong & P.O’Byrne, Cheirostylis goldschmidtiana Schltr. and Phalaenopsis appendiculata Carr. Findings of two keystone species, the long-lost Cheirostylis goldschmidtiana and the endangered snow-white slipper orchid, Paphiopedilum niveum (Rchb.f.) Stein, have catalyzed the need for a more comprehensive study to unveil the species richness and endemism within a limestone habitat. Heat maps using geographical data produced from ArcGIS software have enabled precise determination of the areas with the highest concentration of orchid diversity. Results from this study have shown that Gunung Fakir Terbang houses the greatest number of orchid species, followed by Gunung Batu Putih and Gunung Baling. Meanwhile, lower species occurrences were recorded from Gunung Pulai and Gunung Pong. However, lack of attention and delayed conservation action on this unique karst habitat would only lead to more habitat loss, leading to the decline of limestone orchid populations. Orchids as well as other plants are threatened by anthropogenic activity such as quarrying and forest clearing for agriculture. Thus, the results of this study will serve as baseline data for future work in documenting and conserving limestone orchids and their karst habitat in northern Peninsular Malaysia.

Construction of a de novo assembly pipeline using multiple transcriptome data sets from Cypripedium macranthos (Orchidaceae).

The family Orchidaceae comprises the most species of any monocotyledonous family and has interesting characteristics such as seed germination induced by mycorrhizal fungi and flower morphology that co-adapted with pollinators. In orchid species, genomes have been decoded for only a few horticultural species, and there is little genetic information available. Generally, for species lacking sequenced genomes, gene sequences are predicted by de novo assembly of transcriptome data. Here, we devised a de novo assembly pipeline for transcriptome data from the wild orchid Cypripedium (lady slipper orchid) in Japan by mixing multiple data sets and integrating assemblies to create a more complete and less redundant contig set. Among the assemblies generated by combining various assemblers, Trinity and IDBA-Tran yielded good assembly with higher mapping rates and percentages of BLAST hit contigs and complete BUSCO. Using this contig set as a reference, we analyzed differential gene expression between protocorms grown aseptically or with mycorrhizal fungi to detect gene expressions required for mycorrhizal interaction. A pipeline proposed in this study can construct a highly reliable contig set with little redundancy even when multiple transcriptome data are mixed, and can provide a reference that is adaptable to DEG analysis and other downstream analysis in RNA-seq.

Morphology and DNA marker for distinguishing Paphiopedilum hangianum and Paphiopedilum emersonii from Vietnam

Genus Paphiopedilum has species having lovely flowers which are incredibly attractive to everyone. Their ornamental and commercial value caused over-collection and illegal poaching and trade. Due to these reasons, nowadays, the Venus slipper orchids are facing to deplete in nature. Therefore, it is important to consider these species conservation. Mainly, it is necessary to prioritize the identification and phylogenetic analysis methods of the genus Paphiopedilum which includes many species with similar morphological characteristics. Consequently, it isn't easy to distinguish the identical species of this genus when the plants are young or not yet fully flowering. Therefore, this study aimed to distinguish two Paphiopedilum species, i.e. P. hangianum and P. emersonii, which have similar morphological characteristics, through comparative morphological analysis and differences in DNA barcoding sequences. To solve the problem associated with species identifications, a morphological comparison table was created with the four DNA sequence markers matK, rbcL, rpoC1 and trnH-psbA. The results of the morphological analysis showed that P. hangianum and P. emersonii are significantly different from each other in the flower's characteristics. While the difference in leaf morphology of both selected species is found very little, it is also distinguishable upon careful comparison. Moreover, the DNA barcoding indicator gave accurate and rapid distinctions between the two species, even when the plants are young or without flowers. Furthermore, this DNA barcoding can establish an evolutionary relationship between the two selected species and the other species of the genus Paphiopedilum. The results of this study also suggested that the indicator trnH-psbA is a suitable marker for distinguishing these two species and can be applied for the phylogenetic analysis of the genus Paphiopedilum in Vietnam.

Illegal Trade in Exotic Animals and Its Impacts in Slovenia-A Case Study.

Currently, the illegal wildlife trade is one of the most profitable illegal enterprises in the world. The aim of our study was to determine the situation with respect to wildlife trade in Slovenia, which is mainly a transit country, before changes to the Schengen borders came into effect. The volume of trade is significant but not extensive. The most common endangered species involved in illegal trade in Slovenia are the brown bear, the peregrine falcon, the date mussel, the lady's slipper orchid, the common snowdrop, the cyclamen, the sea turtle, the otter, and various reptile species. The smuggling of shells (date shells), ivory (ivory products), certain plants, and various hunting trophies (bears, big cats) has decreased in recent years. Nevertheless, counteracting crimes continues to be important for the conservation of some species in Slovenia, notably the lynx, and for the reduction of poaching. Improvements are needed in the detection and prevention of wildlife crime, especially in light of changes made to the Schengen borders and the consequent inclusion of new trading partners for Slovenia. The lack of people properly trained to identify, detect, and investigate wildlife crime is especially acute.

Predicting Distribution and Range Dynamics of Three Threatened Cypripedium Species under Climate Change Scenario in Western Himalaya

Climate change and anthropogenic pressure have significantly contributed to the decline of biodiversity worldwide, particularly in mountain ecosystems such as the Himalaya. In addition to being relatively sensitive to disturbances, orchids may also respond more quickly to climate change impacts than other plant species. Because of their complex biology and anthropogenic pressures on their habitat in the Himalayan region, lady’s slipper orchids are considered to be a highly vulnerable group of orchids. In the present study, we examine the effect of climate change on the distribution of three threatened Cypripedium species (Cypripedium cordigerum, Cypripedium elegans, and Cypripedium himalaicum), utilizing ecological niche modeling for present and future climatic scenarios to identify key environmental determinants and population parameters. A community climate system model (CCSM ver. 4) was used to identify suitable distribution areas for future scenarios. Based on the least correlated characteristics of the species bioclimatic, topographical, and physiological characteristics, the species’ climatic niche was determined. According to the results, the true skill statistic (TSS), area under the receiver operating characteristic curve (AUC), and Cohen’s kappa provide more reliable predictions. Precipitation during the wettest month and precipitation during the coldest quarter are the primary climatic variables that influence the distribution of suitable areas. A total of 192 km2 of the area was estimated to be suitable for all three species under current climate conditions. Under future climate conditions, the model predicts a trivial increase in suitable habitat areas with a shift toward the northwest. However, highly suitable habitat areas will be severely diminished. There are currently highly suitable habitats in Tungnath and the Valley of Flowers, but due to climatic factors, the habitats will become unsuitable in the future. Additionally, under future climatic scenarios, viable habitats will be identified for priority conservation to cope with the effects of climate change and anthropogenic activities. In light of these findings, conservation methods for the target species may be designed that will be successful and have the potential to prevent local extinctions.

Forest Disturbances Threatening Cypripedium calceolus Populations Can Improve Its Habitat Conditions

Inappropriate forest management activities, above all clear-cutting, are recognized as the major threats to an iconic orchid species—the lady’s slipper orchid (Cypripedium calceolus), the flagship species of nature protection in Europe. Although clear-cutting in protected species’ localities is strongly regulated in many European countries, salvage logging is allowed in some circumstances (e.g., following windstorms, and insect outbreaks) and can threaten C. calceolus populations. In this paper, we review a database of recently registered populations of this species in the Czech Republic and study historical maps, to better understand the history of local habitat conditions and assess threats to C. calceolus populations by bark beetle outbreaks. We found that about one-third of the C. calceolus populations have suffered in coniferous or mixed tree plantations with a high proportion of spruce trees, which are forests with a high risk of bark beetle infestation. We review bark beetle management measures and distinguish management efforts in areas with known C. calceolus populations that result in no damage to the population and, at times, improve habitat conditions for the species. Thus, the bark beetle—usually understood as the enemy—can be transformed into a savior if smart management measures will replace the panic salvage logging.

The ecological adaptation of the unparalleled plastome character evolution in slipper orchids.

Plastomes may have undergone adaptive evolution in the process of plant adaptation to diverse environments, whereby species may differ in plastome characters. Cypripedioideae successfully colonized distinct environments and could be an ideal group for studying the interspecific variation and adaptive evolution of plastomes. Comparative study of plastomes, ancestral state reconstruction, phylogenetic-based analysis, ecological niche modelling, and selective pressure analysis were conducted to reveal the evolutionary patterns of plastomes in Cypripedioideae and their relationship with environmental factors. The plastomes of the three evolved genera had reduced plastome size, increased GC content, and compacted gene content compared to the basal group. Variations in plastome size and GC content are proved to have clear relationships with climate regions. Furthermore, ecological niche modelling revealed that temperature and water factors are important climatic factors contributing to the distributional difference which is directly correlated with the climate regions. The temperature-sensitive genes ndh genes, infA, and rpl20 were found to be either lost/pseudogenized or under positive selection in the evolved groups. Unparalleled plastome character variations were discovered in slipper orchids. Our study indicates that variations in plastome characters have adaptive consequences and that temperature and water factors are important climatic factors that affect plastome evolution. This research highlights the expectation that plants can facilitate adaptation to different environmental conditions with the changes in plastome and has added critical insight for understanding the process of plastome evolution in plants.

The lady's 'slippery' orchid: functions of the floral trap and aphid mimicry in a hoverfly-pollinated Phragmipedium species in Brazil.

Trap flowers are fascinating cases of adaptation, often linked to oviposition-site mimicry systems. Some trap flowers do not imprison pollinators for a pre-determined period, but rather force them to move through a specific path, manipulating their movements in a way that culminates in pollen transfer, often as they leave through a secondary opening. We investigated the previously unknown pollination system of the lady's slipper orchid Phragmipedium vittatum and assessed the function of micro-morphological traits of its trap flowers. Our observations revealed that P. vittatum is pollinated by females of two hoverfly species (Syrphidae). Eggs laid by flies on or near raised black spots on the flowers indicate that the orchid mimics aphids which serve as food for their aphidophagous larvae. Dark, elevated aphid-like spots appear to attract the attention of hoverflies to a slipping zone. This region has downward projecting papillate cells and mucilage secretion that promote slipperiness, causing potential pollinators to fall into the labellum. They then follow a specific upward route towards inner aphid-like spots by holding onto upward oriented hairs that aid their grip. As hoverflies are funnelled by the lateral constriction of the labellum, they pass the stigma, depositing pollen they may be carrying. Later, they squeeze under one of the articulated anthers which places pollen smears onto their upper thorax. Then, they depart through one of the narrow lateral holes by holding onto hairs projecting from the petals. This study confirms the system of aphid mimicry in Phragmipedium and highlights the sophisticated micro-morphological traits used by trap flowers in pollinator attraction, trapping, guidance and release, thus promoting precise pollen transfer.

In vitro seed germination of Paphiopedilum lowii, an endangered slipper orchid in North Borneo

Abstract. Ragu VS, Ombokou R, Repin R, Molidin D, Miadin R, Aziz ZA. 2022. In vitro seed germination of Paphiopedilum lowii, an endangered slipper orchid in North Borneo. Biodiversitas 23: 5687-5694. Paphiopedilum lowii (Lindl.) Stein is alarmingly decreasing in numbers due to habitat destruction and over-collection. Propagation through seed is one effort to conserve the species. In vitro method offers better results for seed germination in orchids, but the success of germination is species-specific. Hence, this research aimed to evaluate different types of media [(½ strength Murashige & Skoog and Orchimax Medium (Duchefa Biochemie)], and to determine the effects of sucrose [0% 1%, 2% and 3% (w/v)], peptone [0%, 0.1% and 0.2% (w/v)], coconut water [0%, 10%, 20% and 30% (v/v)] and fertilizer [Orchid Focus - 0%, 0.1% and 0.2% (v/v)] on seed germination. The protocorm size was also determined after 12 weeks of observation. Half-strength MS media consistently gave higher seed germination, ½ MS supplemented with 1% (w/v) sucrose, 0.1% (w/v) peptone, 0% (v/v) coconut water and 0.1 % (v/v) fertilizer resulted in the highest seed germination at 5.32% ± 5.9, 19.27% ± 9.34, 11.33% ± 3.80 and 19.31% ± 9.03 respectively. Despite being 12 weeks in culture, the sizes of the protocorms are small (0.222±0.089 mm diameter, 0.703±0.280 mm circumference, and 0.045±0.043 mm2 area); this requires future investigation. These findings can serve as base information for further enhancement of seed germination and development of propagation methods of P. lowii for use in a conservation program.

Trait-linked phylogenetic framework of Paphiopedilum distributed in India revealed species passport trait to prevent unethical trade through in-silico study

• In-silico trait-linked phylogenetic framework of 9 Paphiopedilum species distributed in India based on combined nrDNA and cpDNA ( ITS2 + matK + rbcL ) dataset by MP and Bayesian analysis. • Ancestral trait tracing of 47 characters viz., morphological and chromosomal using Mesquite 3.61. • Sharing of passport traits to public may augment to prevent unethical trade of Paphiopedilum species throughout the distributional range. Paphiopedilum Pfitzer is one of the largest horticulturally utilized genera of slipper orchids. Similar characters of slipper orchids within Paphiopedilum may camouflage identity and mislead a nonprofessional to species substitution. All the species of Paphiopedilum are enlisted in CITES Appendix I and barred for unethical trading from wild sources. An in-silico study on the trait-linked phylogenetic framework of the Paphiopedilum species distributed in India aimed to identify the species passport trait which may prevent unethical trade. The phylogenetic framework was reconstructed using downloaded DNA sequences of ITS2, matK, and rbcL genes from GenBank for 22 slipper orchids of the subfamily Cypripedioideae including 9 Paphiopedilum species of India. Parsimonious and Bayesian interpretations were carried out using PAUP version 4.0a and MrBayes with ancestral trait reconstruction of 47 characters by Mesquite 3.61. The combined DNA ( ITS2 + matK + rbcL ) dataset supported the monophyly of the genus Paphiopedilum with a distinct clade and section Barbata (Krzl.) V.A. Albert & Borge Pett. with a subclade. The monophyly of the conduplicate-leaved genera Paphiopedilum, Phragmipedium, and Mexipedium was congruent to recent taxonomic classifications whereas plicate-leaved Selenipedium and Cypripedium showed polytomy in parsimonious tree. Ancestral reconstruction exhibited basal leaves, conduplicate leaves and deciduous perianth as symplesiomorphic traits. Undulate-revolute, rounded synsepal, ciliate lip; pubescent dorsal sepal, glabrous, glossy lip; coriaceous rough leaves , shortly hirsute peduncle; hairy lip; glandular pubescent peduncle; and brownish dorsal sepal with greenish yellow margin can be considered as passport traits for P. fairrieanum, P. spicerianum, P. venustum, P. charlesworthii, P. druryi and P. hirsutissimum respectively.

Cypripedium subtropicum embryo development and cytokinin requirements for asymbiotic germination

BackgroundCypripedium subtropicum is a unique, endangered lady’s slipper orchid with evergreen leaves on non-dormant shoots that is native to southwestern China. This study documents the major developmental events in C. subtropicum seed development from fertilization to seed maturity, determines the optimum period for seed collection, and examines the cytokinin requirements for asymbiotic germination and protocorm survival.ResultsStructural studies revealed that embryo development proceeded after successful fertilization at 60 days after pollination (DAP). At 105 DAP, a globular embryo with the shrinking inner seed coat was observed, and seeds collected at this time point exhibited optimal germination. After 120 DAP, most seeds had a mature embryo within the capsule, and within the cells of the embryo proper, numerous proteins/lipid bodies were present as the main storage products. In addition, the inner seed coat had compressed into a thin layer that tightly enclosed the embryo, while the outer seed coat had progressively elongated, resulting in a hair-like appearance of the mature seed. Histochemical staining using Nile red and toluidine blue O (TBO) indicated that the lignified inner and outer seed coats may lead to coat-imposed dormancy. Seeds collected at this stage germinated poorly. Analyses of cytokinin preferences and optimal concentrations for germination and protocorm survival showed that both 6-(γ,γ-dimethylallylamino) purine (2iP) and 6-benzylaminopurine (BA) enhanced germination compared with the control, although higher concentrations of BA (4 and 8 μM) suppressed germination. The protocorm survival rate improved with increasing 2iP concentration.ConclusionsThis study provides a reproducible procedure for culturing immature seeds of C. subtropicum based on a defined time schedule of seed development. In addition, the cytokinin 2iP was shown to improve germination and protocorm survival. This study provides a scientific basis for seedling establishment through asymbiotic seed culture for further reintroduction efforts.

High-Density Genetic Linkage Map Construction and QTLs Identification Associated with Four Leaf-Related Traits in Lady’s Slipper Orchids (Paphiopedilum concolor × Paphiopedilum hirsutissimum)

Lady’s slipper orchids (Paphiopedilum spp.) are highly valuable within the flower industry. Recently, both Paphiopedilum concolor and Paphiopedilum hirsutissimum (2n = 2x = 26) have been widely used for hybrid parents, ornamental, and economic purposes. However, high-density genetic maps and leaf traits related to quantitative trait loci (QTLs) in these two Paphiopedilum species have been poorly studied. Herein, an interspecific F1 population of 95 individuals was developed from the cross between P. concolor and P. hirsutissimum with contrasting leaf length (LL), leaf width (LW), leaf thickness (LT), and leaf number (LN). RNA extracted from the F1 population and their parents was subjected to high-throughput RNA sequencing. Approximately 745.59 Gb of clean data were generated, and were assembled into 349,730 transcripts and 185,089 unigenes. In total, 165,196 high-resolution polymorphic single nucleotide polymorphism (SNP) markers were initially identified. Finally, 8410 SNP markers satisfied the requirements and were used to construct a genetic map. The integrated map contained 13 linkage groups (LGs) and spanned 1616.18 cM, with an average distance of 0.19 cM between adjacent markers. QTL analysis in the F1 population identified 12 QTLs linked to four leaf-related traits, including LL, LW, LT, and LN. These QTLs by composition interval mapping, explained 11.86% to 21.58% of the phenotypic variance, and were distributed on eight LGs, but not on LGs 4, 6, 8, 12, and 13. Furthermore, 25 unigenes were identified via BLAST searches between the SNP markers in the QTL regions and our assembled transcriptome, of which 11 unigenes were enriched with 59 gene ontology (GO) terms. The information generated in this study will be useful for candidate genes for further molecular regulation studies on leaf traits, future marker-assisted selection of leaf ornamental improvement breeding, genome assembly, and comparative genome analyses.