Paphiopedilum Species Research Articles

Fungal community characteristics of the last remaining habitat of three paphiopedilum species in China.

Paphiopedilum armeniacum, Paphiopedilum wenshanense and Paphiopedilum emersonii are critically endangered wild orchids. Their populations are under severe threat, with a dramatic decline in the number of their natural distribution sites. Ex situ conservation and artificial breeding are the keys to maintaining the population to ensure the success of ex situ conservation and field return in the future. The habitat characteristics and soil nutrient information of the last remaining wild distribution sites of the three species were studied. ITS high-throughput sequencing was used to reveal the composition and structure of the soil fungal community, analyze its diversity and functional characteristics, and reveal its relationship with soil nutrients. The three species preferred to grow on low-lying, ventilated and shaded declivities with good water drainage. There were significant differences in soil alkali-hydrolyzed nitrogen and available phosphorus among the three species. There were 336 fungal species detected in the samples. On average, there were different dominant groups in the soil fungal communities of the three species. The functional groups of soil fungi within their habitats were dominated by saprophytic fungi and ectomycorrhizae, with significant differences in diversity and structure. The co-occurrence network of habitat soil fungi was mainly positive. Soil pH significantly affected soil fungal diversity within their habitats of the three paphiopedilum species. The study confirmed that the dominant groups of soil fungi were significantly correlated with soil nutrients. The three species exhibit comparable habitat inclinations, yet they display substantial variations in the composition, structure, and diversity of soil fungi. The fungal functional group is characterized by a rich presence of saprophytic fungi, a proliferation of ectomycorrhizae, and a modest occurrence of orchid mycorrhizae. The symbiotic interactions among the soil fungi associated with these three species are well-coordinated, enhancing their resilience against challenging environmental conditions. There is a significant correlation between soil environmental factors and the composition of soil fungal communities, with pH emerging as a pivotal factor regulating fungal diversity. Our research into the habitat traits and soil fungal ecosystems of the three wild Paphiopedilum species has established a cornerstone for prospective ex situ conservation measures and the eventual reestablishment of these species in their native landscapes.

Additions to orchid flora of Bhutan III: <i>Paphiopedilum × pradhanii</i>

Paphiopedilum × pradhanii, a natural hybrid between P. fairrieanum (subgenus Paphiopedilum, section Ceratopetalum) and P. venustum (subgenus Paphiopedilum, section Barbata), is reported for the first time from Bhutan. Detailed geographical distribution, ecology, and conservation status, and dichotomous keys to all Paphiopedilum species found in Bhutan are provided. A brief description of the parents of this hybrid is also provided.

Floral longevity is related to flower nutrient stoichiometry in endangered orchids, Paphiopedilum species

The relationship between floral longevity and the chemical elements that support plant survival, growth and reproduction remains unclear. Here, we used 12 Paphiopedilum (Cypripedioideae, Orchidaceae) species with long-lived flowers to investigate the possible association of chemical elements with floral longevity. We found that floral longevity was significant and positive related to the contents of phosphorus, calcium, magnesium, and silicon in flowers, but was not significantly related to carbon, nitrogen, potassium or boron contents. In addition, the contents of certain chemical elements in flowers were correlated. In our principal component analysis of eight floral trace elements and floral longevity, the first two principal components explained 50.5% and 20.5% of the total variation, respectively. The second major axis of calcium variation in flowers was independent of the first major axis. Our findings highlight a relationship between floral longevity and nutrient stoichiometry that needs to be understood when developing conservation plans for endangered orchids of Paphiopedilum species.

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.

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.

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.

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.

Comparative Analyses of Chloroplast Genomes Provide Comprehensive Insights into the Adaptive Evolution of Paphiopedilum (Orchidaceae)

An elucidation of how the selection pressures caused by habitat environments affect plant plastid genomes and lead to the adaptive evolution of plants, is a very intense area of research in evolutionary biology. The genus Paphiopedilum is a predominant group of orchids that includes over 66 species with high horticultural and ornamental value. However, owing to the destructive exploitation and habitat deterioration of wild germplasm resources of Paphiopedilum, it needs more molecular genetic resources and studies on this genus. The chloroplast is cytoplasmically inherited and often used in evolutionary studies. Thus, for this study, we newly sequenced, assembled and annotated five chloroplast genomes of the Paphiopedilum species. The size of these genomes ranged from 155,886 bp (P. henryanum) to 160,503 bp (P. ‘GZSLKY’ Youyou) and they contained 121–122 genes, which consisted of 76 protein coding genes, eight ribosomal RNAs, and 37–38 transfer RNAs. Combined with the other 14 Paphiopedilum species, the characteristics of the repeat sequences, divergent hotspot regions, and the condo usage bias were evaluated and identified, respectively. The gene transfer analysis showed that some fragments of the ndh and ycf gene families were shared by both the chloroplast and nucleus. Although the genomic structure and gene content was conserved, there was a significant boundary shift caused by the inverted repeat (IR) expansion and small single copy (SSC) contraction. The lower GC content and loss of ndh genes could be the result of adaptive evolutionary responses to its unique habitats. The genes under positive selection, including accD, matK, psbM, rpl20, rps12, ycf1, and ycf2 might be regarded as potential candidate genes for further study, which significantly contribute to the adaptive evolution of Paphiopedilum.

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.

Genome Size and Labellum Epidermal Cell Size Are Evolutionarily Correlated With Floral Longevity in Paphiopedilum Species.

Genome size is known to influence phenotypic traits in leaves and seeds. Although genome size is closely related to cellular and developmental traits across biological kingdoms, floral longevity is a floral trait with important fitness consequence, but less is known about the link between floral longevity and sizes of genomes and cells. In this study, we examined evolutionary coordination between genome size, floral longevity, and epidermal cell size in flowers and leaves in 13 Paphiopedilum species. We found that, across all the study species, the genome size was positively correlated with floral longevity but negatively associated with labellum epidermal cell size, and a negative relationship was found between floral longevity and labellum epidermal cell size. This suggested that genome size is potentially correlated with floral longevity, and genome size has an important impact on life-history trait. In addition, genome size was positively correlated with leaf epidermal cell size, which was different from the relationship in flower due to different selective pressures they experienced or different functions they performed. Therefore, genome size constraints floral longevity, and it is a strong predictor of cell size. The impact of genome size on reproduction might have more implications for the evolution of flowering plants and pollination ecology.

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.

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.

Is seasonal flowering time of Paphiopedilum species caused by differences in initial time of floral bud differentiation?

Members of the genus Paphiopedilum are world-famous for their large, colourful flowers, unique floral morphology and long floral lifespan. Most Paphiopedilum species bloom in spring or autumn. The control of flowering time is of great significance to the commercial production of floral crops, because it affects the sales and prices of flowers. However, the mechanism that regulates when Paphiopedilum species bloom is unclear. In the present study, floral bud initiation and development of P. micranthum (spring-flowering species with one flower per stalk), P. dianthum (autumn-flowering species with multiple flowers per stalk) and P. henryanum (autumn-flowering species with one flower per stalk) were investigated by morphological and anatomical methods. We divided Paphiopedilum floral bud differentiation into six phases: the initiation of differentiation, inflorescence primordium differentiation, flower primordium differentiation, sepal primordium differentiation, petal primordium differentiation and column primordium differentiation. We found that the timing of floral bud differentiation for the three species was synchronized when experiencing the same environment, while the period from initiation to flowering largely differed. In addition, initiation of floral bud differentiation in P. dianthum was earlier at a warmer environment. The difference in flowering time of three species was mainly caused by the duration of floral bud development, rather than the initiation time. The findings were of great significance for the cultivation and flowering regulation of Paphiopedilum species.

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.

Leaf physiological and anatomical responses of two sympatric Paphiopedilum species to temperature

Paphiopedilum dianthum and P. micranthum are two endangered orchid species, with high ornamental and conservation values. They are sympatric species, but their leaf anatomical traits and flowering period have significant differences. However, it is unclear whether the differences in leaf structure of the two species will affect their adaptabilities to temperature. Here, we investigated the leaf photosynthetic, anatomical, and flowering traits of these two species at three sites with different temperatures (Kunming, 16.7 ± 0.2 °C; Puer, 17.7 ± 0.2 °C; Menglun, 23.3 ± 0.2 °C) in southwest China. Compared with those at Puer and Kunming, the values of light-saturated photosynthetic rate (Pmax), stomatal conductance (gs), leaf thickness (LT), and stomatal density (SD) in both species were lower at Menglun. The values of Pmax, gs, LT, adaxial cuticle thickness (CTad) and SD in P. dianthum were higher than those of P. micranthum at the three sites. Compared with P. dianthum, there were no flowering plants of P. micranthum at Menglun. These results indicated that both species were less resistance to high temperature, and P. dianthum had a stronger adaptability to high-temperature than P. micranthum. Our findings can provide valuable information for the conservation and cultivation of Paphiopedilum species.

Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection.

In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50–54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.

Transcriptome analysis provides insights into the non-methylated lignin synthesis in Paphiopedilum armeniacum seed

BackgroundsPaphiopedilum is an important genus of the orchid family Orchidaceae and has high horticultural value. The wild populations are under threat of extinction because of overcollection and habitat destruction. Mature seeds of most Paphiopedilum species are difficult to germinate, which severely restricts their germplasm conservation and commercial production. The factors inhibiting germination are largely unknown.ResultsIn this study, large amounts of non-methylated lignin accumulated during seed maturation of Paphiopedilum armeniacum (P. armeniacum), which negatively correlates with the germination rate. The transcriptome profiles of P. armeniacum seed at different development stages were compared to explore the molecular clues for non-methylated lignin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that a large number of genes associated with phenylpropanoid biosynthesis and phenylalanine metabolism during seed maturation were differentially expressed. Several key genes in the lignin biosynthetic pathway displayed different expression patterns during the lignification process. PAL, 4CL, HCT, and CSE upregulation was associated with C and H lignin accumulation. The expression of CCoAOMT, F5H, and COMT were maintained at a low level or down-regulated to inhibit the conversion to the typical G and S lignin. Quantitative real-time RT-PCR analysis confirmed the altered expression levels of these genes in seeds and vegetative tissues.ConclusionsThis work demonstrated the plasticity of natural lignin polymer assembly in seed and provided a better understanding of the molecular mechanism of seed-specific lignification process.

Phylogeny and Historical Biogeography of Paphiopedilum Pfitzer (Orchidaceae) Based on Nuclear and Plastid DNA.

The phylogeny and biogeography of the genus Paphiopedilum were evaluated by using phylogenetic trees derived from analysis of nuclear ribosomal internal transcribed spacer (ITS) sequences, the plastid trnL intron, the trnL-F spacer, and the atpB-rbcL spacer. This genus was divided into three subgenera: Parvisepalum, Brachypetalum, and Paphiopedilum. Each of them is monophyletic with high bootstrap supports according to the highly resolved phylogenetic tree reconstructed by combined sequences. There are five sections within the subgenus Paphiopedilum, including Coryopedilum, Pardalopetalum, Cochlopetalum, Paphiopedilum, and Barbata. The subgenus Parvisepalum is phylogenetic basal, which suggesting that Parvisepalum is comprising more ancestral characters than other subgenera. The evolutionary trend of genus Paphiopedilum was deduced based on the maximum likelihood (ML) tree and Bayesian Evolutionary Analysis Sampling Trees (BEAST). Reconstruct Ancestral State in Phylogenies (RASP) analyses based on the combined sequence data. The biogeographic analysis indicates that Paphiopedilum species were firstly derived in Southern China and Southeast Asia, subsequently dispersed into the Southeast Asian archipelagoes. The subgenera Paphiopedilum was likely derived after these historical dispersals and vicariance events. Our research reveals the relevance of the differentiation of Paphiopedilum in Southeast Asia and geological history. Moreover, the biogeographic analysis explains that the significant evolutionary hotspots of these orchids in the Sundaland and Wallacea might be attributed to repeated migration and isolation events between the south-eastern Asia mainland and the Sunda Super Islands.