Orchid Cypripedium Research Articles

Monitoring of a <i>Cypripedium guttatum</i> (Orchidaceae, Liliopsida) population on the northern border of its distribution range (Komi Republic, European Russia)

The results of our 12-year study of a population of the rare orchid Cypripedium guttatum Sw. on the northern border of its range, on the territory of the Komi Republic (north-east of European Russia) are presented. Our research has shown that weather factors (temperature and precipitation) have an impact on the seasonal development of shoots and plant size. The number of shoots is influenced by the weather conditions of the previous growing season, namely: the temperature of the first decade of August influences positively while early autumn frosts and the amount of precipitation of the entire period do negatively. A negative correlation was found between the number of flowering shoots and the temperature of the second decade of July of the previous growing season and a positive relationship between the number of juvenile plants and the precipitation of August of the previous growing season. Fruit set was quite low (17.1%), which is typical for this species. The pollination efficiency of C. guttatum was negatively affected by the high temperature during its flowering (the second decade of June). One box contained an average of 4065 seeds. The real seed productivity per shoot varied from 1989 to 7064 pcs., it is associated with precipitation in the second decade of July of the current growing season. The high number of the population studied (25 thousand shoots), the correspondence of the ontogenetic spectrum to the basic one of the species, the annual presence of juvenile plants of seed origin indicates its stable condition.

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.

Effective population size in a partially clonal plant is not predicted by the number of genetic individuals.

Estimating effective population size (N e) is important for theoretical and practical applications in evolutionary biology and conservation. Nevertheless, estimates of N e in organisms with complex life-history traits remain scarce because of the challenges associated with estimation methods. Partially clonal plants capable of both vegetative (clonal) growth and sexual reproduction are a common group of organisms for which the discrepancy between the apparent number of individuals (ramets) and the number of genetic individuals (genets) can be striking, and it is unclear how this discrepancy relates to N e. In this study, we analysed two populations of the orchid Cypripedium calceolus to understand how the rate of clonal versus sexual reproduction affected N e. We genotyped >1000 ramets at microsatellite and SNP loci, and estimated contemporary N e with the linkage disequilibrium method, starting from the theoretical expectation that variance in reproductive success among individuals caused by clonal reproduction and by constraints on sexual reproduction would lower N e. We considered factors potentially affecting our estimates, including different marker types and sampling strategies, and the influence of pseudoreplication in genomic data sets on N e confidence intervals. The magnitude of N e/N ramets and N e/N genets ratios we provide may be used as reference points for other species with similar life-history traits. Our findings demonstrate that N e in partially clonal plants cannot be predicted based on the number of genets generated by sexual reproduction, because demographic changes over time can strongly influence N e. This is especially relevant in species of conservation concern in which population declines may not be detected by only ascertaining the number of genets.

Soil properties and plant species can predict population size and potential introduction sites of the endangered orchid Cypripedium calceolus

Background and AimsTo counteract the ongoing worldwide biodiversity loss, conservation actions are required to re-establish populations of threatened species. Two key factors predominantly involved in finding the most suitable habitats for endangered plant species are the surrounding plant community composition and the physicochemical parameters of the soil rooting zone. However, such factors are likely to be context- and species-dependent, so it remains unclear to what extent they influence the performance of target species.MethodsWe studied large and small Swiss populations of the endangered orchid Cypripedium calceolus. We measured functional traits related to C. calceolus plant and population performance (clonal patch area, plant height, number, of leaf, stems, flowers and fruits), realized vegetation surveys, soil profile analyses, and tested for relationships between plant traits and the surrounding vegetation structure or soil physicochemical parameters.ResultsLarge populations contained bigger patches with more stems and leaves, and produced more flower per individual than small populations. Neither vegetation alliances nor soil classes per se could predict C. calceolus functional traits and population size. However, functional traits explaining population performance and size were related to specific soil parameters (soil organic matter content, pH and phosphorus), in addition to a combination of presence-absence of plant indicator species, relating to ecotones between forests and clearings.ConclusionWe show that even for species that can grow across a wide range of vegetation groups both indicator species and specific soil parameters can be used to assess the most favourable sites to implement (re)-introduction actions.

Cytological Study of Cypripedium japonicum Thunb. (Orchidaceae Juss.): An Endangered Species from Korea.

Changes in chromosome number and karyotype evolution are important to plant diversification, as they are both major drivers of speciation processes. Herein, chromosome number, karyotype, and genome size of the Korean lady’s slipper orchid Cypripedium japonicum Thunb., an endangered species, were investigated in natural populations. Furthermore, all cytological data from this species are reported herein for the first time. The chromosome number of all investigated C. japonicum plants was diploid (2n = 2x = 22), with x = 11 as base chromosome number, whereby the species can now be clearly distinguished from the Japanese lady’s slipper orchid. The karyotypes of all studied individuals were of similar length, symmetrical, and rather unimodal. Flow cytometry of the C. japonicum revealed that the genome size ranged from 28.38 to 30.14 pg/1C. Data on chromosome number and karyotypes were largely consistent with previous results indicating that Korean (x = 11) populations of C. japonicum are more closely related to Chinese populations (x = 11) compared to Japanese (x = 10) populations. These comprehensive cytological results will benefit the efforts to discriminate the geographically isolated and endangered Eastern Asian (China, Japan, and Korea) lady’s slipper orchid species.

Pollination ecology and breeding systems of Cypripedium kentuckiense (Orchidaceae) in Tennessee1

The orchid Cypripedium kentuckiense is listed as imperiled in Tennessee and Oklahoma and critically imperiled in seven other states. We followed individuals' vegetative and floral traits over two seasons. Patterns of floral senescence were found to vary over two seasons and might be related to April temperatures when buds develop. Mechanical self-pollination was uncommon but perhaps “leaky.” Frequencies of insect-mediated pollination were surprisingly high (62%) for a Cypripedium species with large flowers but conversion of flowers into fruit was low (7% and 14%), suggesting that cryptic factors lower reproductive success, not inadequate pollinator visits. Hand-pollinations suggest flowers are self-compatible. As in other Cypripedium species, floral architecture relates to pollinator dimensions but at this site, only two bee species, Anthophora abrupta and A. bomboides, were primary pollinators. Male A. abrupta were common visitors although male bees are uncommon pollen vectors of Cypripedium species. For the first time, exit patterns of bees inside Cypripedium flowers were recorded based on bee species and gender. This behavior varied and was somewhat atypical of previous studies on other bee-pollinated species in the clade and genus. Specifically, Anthophora species within labella chewed on the epidermis lingering within the interior and exit canals. Consequently, C. kentuckiense might not be a trap.

Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile.

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

Does the Rarity of a Flower's Scent Phenotype in a Deceptive Orchid Explain Its Pollination Success?

Floral scent, a key mediator in plant–pollinator interactions, varies not only among plant species, but also within species. In deceptive plants, it is assumed that variation in floral scents and other traits involved in pollinator attraction is maintained by negative frequency-dependent selection, i.e., rare phenotypes are more attractive to pollinators and hence, have a higher fitness than common phenotypes. So far, it is unknown whether the rarity of multivariate and/or continuous floral scent traits influences the pollination success of flowers. Here, we tested in the deceptive orchid Cypripedium calceolus, whether flowers with rarer scent bouquets within a population have a higher chance to getting pollinated than flowers with more common scents. We collected the scent of more than 100 flowers in two populations by dynamic headspace and analyzed the samples by gas chromatography coupled to mass spectrometry (GC/MS). From the same flowers we also recorded whether they set a fruit or not. We introduced rarity measures of uni- and multivariate floral scent traits for single flowers, which allowed us to finally test for frequency-dependent pollination, a prerequisite for negative frequency-dependent selection. Our results do not show rarity has an effect on the likelihood to set fruits in neither of the two populations and in none of the scent characteristics analyzed. Hence, there is no evidence of negative frequency-dependent pollination mediated by the floral scent of C. calceolus. We discuss that our approach to determine rarity of a scent is applicable to any univariate or multivariate (semi)quantitative trait.

The rare and endangered orchid Cypripedium calceolus L. in Croatia - refound in Gorski Kotar (West Croatia) after 126 years

The rare and endangered orchid Cypripedium calceolus L. in Croatia - refound in Gorski Kotar (West Croatia) after 126 years

Consistent pollination services to Cypripedium macranthos var. rebunense (Orchidaceae) by Bombus pseudobaicalensis

AbstractBombus pseudobaicalensis queens are known pollinators of the threatened orchid Cypripedium macranthos var. rebunense. However, whether other bumblebee species also pollinate this orchid has not yet been ascertained. A 10‐year observation provided convincing evidence that B. pseudobaicalensis is the only consistent pollinator. B. beaticola moshkarareppus queens were newly found to rarely carry pollen smears of the orchid, whereas B. hypocrita sapporoensis and B. diversus queens were practically mere flower visitors. Why these three species do not equal B. pseudobaicalensis in pollination effectiveness remains unclear but differences in body size and individual abundance between B. pseudobaicalensis and the other species are considered as possible primary causes. Given the almost complete dependence of C. macranthos var. rebunense on B. pseudobaicalensis for pollination and its potential risk, conservation managers should take care not to reduce the bumblebee colonies.

Genetic diversity in the endangered terrestrial orchid Cypripedium japonicum in East Asia: Insights into population history and implications for conservation

Little is known about levels and patterns of genetic diversity for the entire range of endangered orchids native to China, Korea, and Japan. In this study, we focus on Cypripedium japonicum and suggest three hypotheses: 1) that genetic drift has been a primary evolutionary force; 2) that populations in central and western China harbor higher levels of genetic variation relative to those from eastern China; and 3) that C. japonicum in China maintains the highest genetic variation among the three countries. Using ISSR and SCoT markers, we investigated genetic diversity in 17 populations to test the three hypotheses. As anticipated, we found low levels of genetic diversity at the species level with substantially high degree of genetic divergence, which can be mainly attributed to random genetic drift. Chinese populations harbor the highest within-population genetic variation, which tends to increase from east to west. We also found a close relationship between Korean populations and central/western Chinese populations. Historical rarity coupled with limited gene flow seems to be important factors for shaping genetic diversity and structure of C. japonicum. Our results indicate that the mountain areas in central and western China were likely refugia at the Last Glacial Maximum.

Hormonal and morphological changes during seed development of Cypripedium japonicum.

With the goal of evaluating the factors that may be influencing the reproductive success of an endangered, terrestrial orchid Cypripedium japonicum Thunb., the anatomical and morphological characteristics of seed development were assessed, as were the levels of endogenous hormones to investigate the possible relationship between hormones and seed development. The results showed that morphological characteristics of seeds were changing from milky white and filiform to brown and fusiform. The development of ovule that initiated on the fertile valves only began after pollination, and a mature embryo sac was produced at 40days after pollination (DAP). Fertilization occurred 50 to 60DAP, and the newly formed zygote divided into two unequal cells that became the embryo proper and the suspensor. Seeds reached maturity at about 115DAP, at which point they consisted only of a globular embryo and two layers of seed coat, without endosperm. The suspensor was lost, and only lipid particles and a large number of starch grains distributed in the cytoplasm. The peak of gibberellic acid (GA3), zeatin riboside (ZR), and indole acetic acid (IAA) occurred in a time sequence, as 20, 30, and 40DAP. There was a highly significant positive correlation among them (P<0.01). Abscisic acid (ABA) content increased at 60DAP and reached the maximum at 115DAP. ABA contents were significantly negatively correlated with the three hormones above (P<0.05). It indicated that the lack of endosperm and suspensor tissues, delayed differentiation of embryos, and high levels of ABA may lead to the difficulty of germination.

Isolation and characterization of novel microsatellite loci for the endangered orchid Cypripedium japonicum (Orchidaceae).

Premise of the study:Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum (Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan.Methods and Results:Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in at least one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged from 0.00 to 0.92 and 0.00 to 0.71, respectively.Conclusions:The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.

The Largest Plastid Genome of Monocots: a Novel Genome Type Containing AT Residue Repeats in the Slipper Orchid Cypripedium japonicum

Plastid genome studies on Orchidaceae, one of the largest families of flowering plants, have been limited to the subfamily Epidendroideae. To increase understanding of the plastid genome evolution in Orchidaceae, we performed a complete plastid genome analysis of the slipper orchid Cypripedium japonicum positioned in the basal clade of the family. No significant gene deletions were observed in the plastid genome of the slipper orchid composed of 85 coding genes including members of the ndh gene family, which are mostly deleted or show pseudogenisation in orchids, although matK was identified as a pseudogene due to a frameshift mutation. Results also revealed that C. japonicum contains the largest plastid genome (174,417 bp) within monocots and the third largest one in Magnoliophyta. This is a new type of plastid genome extended due to abnormally frequent AT residue repeats within noncoding regions without inverted repeat (IR) expansion. In addition, we detected 25 plastid microsatellites and compared them among seven populations from Korea and Japan. These microsatellites may be applicable to population genetics and conservation biology studies on slipper orchids, which have become rare and are nearing extinction. AT-rich regions in the introns may also play a role in effective splicing of the genome after matK lost its function. As AT-rich regions are difficult to amplify and sequence using normal sequencing technologies, we propose a revised methodology for sequencing these regions.

Pollination biology of the endangered orchid Cypripedium japonicum in a fragmented forest of Japan

AbstractPollination biology studies of the endangered orchid Cypripedium japonicum were conducted in its natural habitat using pollinator observation and hand‐pollination experiments. The observed fruit set was as follows: artificial outcross‐pollinated, 100%; artificial self‐pollinated, 100%; pollinator‐excluded, 0%; and emasculated flowers, 0%. These results show that this species, although self‐compatible, is neither autogamous nor agamospermous. The fruit set for open‐pollinated flowers was 14.9%, which suggests that the study population was subject to pollinator limitation. The nectarless flowers of C. japonicum were exclusively visited and pollinated by the queens of two bumblebee species (Bombus ardens and B. diversus diversus). It is probable that the nectarless flowers of C. japonicum attract pollinators through a generalized food deceptive system.

Contributions of Covariance: Decomposing the Components of Stochastic Population Growth in Cypripedium calceolus

Although correlations between vital rates can have important effects on evolution and demography, few studies have investigated their effects on population dynamics. Here, we extend life-table response experiments (LTREs) to variable environments, showing how to quantify contributions made by (1) mean vital rates, (2) variability driven by environmental fluctuations, (3) correlations implying demographic trade-offs and reflecting stage transition synchrony, and (4) elasticities reflecting local selection pressures. Applying our methods to the lady's slipper orchid Cypripedium calceolus, we found that mean rates accounted for 77.1% of all effects on the stochastic growth rate, variability accounted for 12.6%, elasticities accounted for 6.6%, and correlations accounted for 3.7%. Stochastic effects accounted for 17.6%, 15.3%, and 35.9% of the total in our three populations. Larger elasticities to transitions between dormancy states and stronger correlations between emergence and survival suggest that one population was under greater pressure to remain active while the other two showed survival payoffs for dormancy in poor years. Strong negative correlations between dormancy, emergence, and stasis balanced opposing contributions, resulting in near stationarity in two populations. These new methods provide an additional tool for researchers investigating stochastic population dynamics and should be useful for a broad range of applications in basic ecology and conservation biology.

The root colonizing fungi of the terrestrial orchid Cypripedium irapeanum

Este estudio investigó el status micorrízico y la identificación de los hongos que colonizan la raíz de la orquídea terrestre Cypripedium irapeanum por medio de los polimorfismos de los fragmentos de la restricción y la secuenciación del espacio tránscrito interno del rDNA. Esta orquídea es endémica de diferentes regiones de México, Guatemala y Honduras localizándose entre los 1400 y 2250 m de altitud. Crece principalmente en los remanentes de los bosques de encino del altiplano y está en la lista roja de México de las plantas amenazadas. Los bosques de encino en este país están constantemente amenazados. El examen al microscopio de los segmentos de raíces teñidas de las orquídeas nos mostró la presencia de estructuras fúngicas tanto de hongos de micorriza orquidoide (pelotones e hifas enrrolladas) como de los llamados endófitos obscuros septados (DSE) (hifas mielinizadas y microesclerosios). El análisis de la secuenciación de la región ITS1-5.6-ITS2 sugiere que el tejido micorrizado está dominado por Tulasneaceae: Sistotrema sp., Rhizoctonia solani, and Epulorhiza sp. Entre los DSE, uno de los 10 aislados mostró 100% de similaridad con Phomopsis sp XJ-05, y otro 99% con el endofito MUT 885, ambos reportados como endófitos obscuros septados. El hipotético endófito septado obscuro Phomopsis sp XJ-05 fue aislado tanto de las raíces como de las semillas que germinaron de C. irapeanum restriction fragment length polymorphisms and by rDNA internal transcribed spacer sequencing.

Asymbiotic germination of the White Mountain Orchid (Pseudorchis albida) from immature seed on media enriched with complex organics or phytohormones

The arctic-alpine White Mountain Orchid (Pseudorchis albida) has so-far resisted attempts at in vitro reproduction from seed, but a practical method is required for its conservation. We hypothesised that, having similar seeds to the notoriously intractable orchid Cypripedium calceolus, immature seeds can germinate in vitro. We collected mature and immature seeds from the Italian Alps, and sowed these in sterile conditions on Malmgren’s basal nutrient medium modified with either phytohormones or complex organic media (COM). Different COM treatments included coconut milk, banana powder, malt extract and yeast extract, all at a concentration of 5%. Phytohormones, all at 20 mg L, included 6-benzylaminopurine (BA), gibberellic acid (GA3), kinetin and thidiazuron (TDZ), added to autoclaved growth media via filter sterilization. After six months we found absolutely no germination of mature seeds for any treatment. Only 3.9% of immature seeds germinated in the control treatment and germination in all COM treatments was not significantly greater than this. However, most phytohormones significantly increased germination of immature seed and promoted protocorm development, BA being most effective (50.5% germination) – this method can therefore be used to propagate the species.

Pollination of the Lady's slipper orchid (Cypripedium calceolus) in Scandinavia – taxonomic and conservational aspects

The Lady's slipper orchid Cypripedium calceolus L. is considered one of the most beautiful orchids of Europe. Consequently, the species has suffered from over‐collecting and is now critically endangered in many countries. Although pollination success is suspected to influence the long‐term survival of Cypripedium calceolus, relatively little is known about the identity of its pollinators in mainland Sweden – a region that comprises the largest European populations. In order to identify which species pollinate eight representative populations in mainland Sweden, we observed and sampled visitors to flowers using a standardized protocol. Specimens were identified and any pollen smear found on their body was examined for the presence of Cypripedium pollen. Nine species were recognized as effective pollen vectors (Andrena cineraria, A. carantonica, A. haemorrohoa, A. helvola, A. nigroaenea, A. praecox, Colletes cunicularius, Lasioglossum fratellum and L. fulvicorne), four of them for the first time in Scandinavia. This is the first time that a species of Colletes is reported to carry pollen of Cypripedium in this region. All but one specimens were females. Our results suggest a taxonomically heterogeneous pollinator fauna for Cypripedium calceolus and are discussed in light of the management of this species.

Characterization of mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two phylogenetically distinct fungi associated with the orchid

We isolated Rhizoctonia-like fungi from populations of the threatened orchid Cypripedium macranthos. In ultrastructural observations of the septa, the isolates had a flattened imperforate parenthesome consisting of two electron-dense membranes bordered by an internal electron-lucent zone, identical to the septal ultrastructure of Rhizoctonia repens (teleomorph Tulasnella), a mycorrhizal fungus of many orchid species. However, hyphae of the isolates did not fuse with those of known tester strains of R. repens and grew less than half as fast as those of R. repens. In phylogenetic analyses, sequences for rDNA and internal transcribed spacer (ITS) regions of the isolates were distinct from those of the taxonomically identified species of Tulasnella. On the basis of the ITS sequences, the isolates clustered into two groups that corresponded exactly with the clades demonstrated for other Cypripedium spp. from Eurasia and North America despite the geographical separation, suggesting high specificity in the Cypripedium-fungus association. In addition, the two phylogenetic groups corresponded to two different plant clones at different developmental stages. The fungi from one clone constituted one group and did not belong to the other fungal group isolated from the other clone. The possibility of switching to a new mycorrhizal partner during the orchid's lifetime is discussed.