Author Correction: The Apostasia genome and the evolution of orchids.

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The Apostasia genome and the evolution of orchids.

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

Phylogenetic analysis and character evolution of tribe Arethuseae (Orchidaceae) reveal a new genus Mengzia.

Delimitation of the tribe Arethuseae has varied considerably since it was first defined. The relationships within Arethuseae, particularly within the subtribe Arethusinae, remain poorly elucidated. In this study, we reconstructed the phylogeny of Arethuseae, using six plastid markers (matK, ycf1, rbcL rpoc1, rpl32-trnL and trnL-F) from 83 taxa. The ancestral state reconstruction of 11 selected morphological characters was also conducted to identify synapomorphies and assess potential evolutionary transitions. Morphological character comparision between the distinct species Bletilla foliosa and other species are conducted. Our results unequivocally supported the monophyly of Arethuseae, which included highly supported clades and a clear synapomorphy of non-trichome-like lamellae. Furthermore, B. foliosa formed a separate clade in the subtribe Arethusinae, instead of clustering with the other Bletilla species in the subtribe Coelogyninae. The morphological characters comparision further showed that the B. foliosa clade could be distinguished from other genera in Arethuseae by multiple characters, including presence of lateral inflorescence, three lamellae with trichome-like apex and four pollinia. In light of these molecular and morphological evidences, we propose Mengzia as a new genus to accommodate B. foliosa and accordingly provide descriptions of this new genus and combination.

[Research progress on molecular mechanism of Dendrobium officinale and its active components to metabolic syndrome].

Metabolic syndrome,a kind of clinical syndrome marked by the presence of symptoms such as hyperglycemia,dyslipidemia and hypertension,has an increasing incidence and comes to be present in younger people. More importantly,prolonged maintenance of this condition can significantly increase the incidence of chronic diseases such as diabetes,cardiovascular disease and cancer.However,the formation mechanism of metabolic syndrome is very complex and has not been fully studied and revealed. Dendrobium officinale is a traditional medicine and food substance with multiple physiological functions. In recent years,D. officinale has attracted much attention from the scholars both at home and abroad due to its functions such as improving blood lipid,lowering blood pressure and regulating blood sugar. However,there is no systematic review on the current studies about D. officinale in intervening metabolic syndrome and its underlying molecular mechanism. In this paper,the biological activity of the main active components,and the research or application status of D. officinale extract in the recent years were reviewed. Then,we analyzed the digestion,absorption and the safety and toxicity of D. officinale and its active components in the body. Finally,we summarized the effects of D. officinale and its active components on metabolic syndrome in animals and human bodies,and discussed its possible molecular mechanisms at the cellular level. This paper provides solid theoretical guidance and reliable molecular basis for further research and advanced development of D. officinale and its active components,especially for its oncoming clinical application.

Morphological Type Identification of Self-Incompatibility in Dendrobium and Its Phylogenetic Evolution Pattern.

Self-incompatibility (SI) is a type of reproductive barrier within plant species and is one of the mechanisms for the formation and maintenance of the high diversity and adaptation of angiosperm species. Approximately 40% of flowering plants are SI species, while only 10% of orchid species are self-incompatible. Intriguingly, as one of the largest genera in Orchidaceae, 72% of Dendrobium species are self-incompatible, accounting for nearly half of the reported SI species in orchids, suggesting that SI contributes to the high diversity of orchid species. However, few studies investigating SI in Dendrobium have been published. This study aimed to address the following questions: (1) How many SI phenotypes are in Dendrobium, and what are they? (2) What is their distribution pattern in the Dendrobium phylogenetic tree? We investigated the flowering time, the capsule set rate, and the pollen tube growth from the representative species of Dendrobium after artificial pollination and analysed their distribution in the Asian Dendrobium clade phylogenetic tree. The number of SI phenotypes exceeded our expectations. The SI type of Dendrobium chrysanthum was the primary type in the Dendrobium SI species. We speculate that there are many different SI determinants in Dendrobium that have evolved recently and might be specific to Dendrobium or Orchidaceae. Overall, this work provides new insights and a comprehensive understanding of Dendrobium SI.

[Original materials of traditional Chinese medicinal names of "Jinchai" and "Jinchai Shihu" based on vegetative morphology].

"Jinchai Shihu" were called Jinchai and recoded in "Taishang Zhouhou Yujingfang" of Tang Dynasty, which first clearly documented the name of Shihu in complex Dendrobium medicines and were condiered as superior medicinal articles. Morphological features are one of the naming principles for Chinese medicines. In this paper, botanical origin plants under the names of "Jinchai" and "Jinchai Shihu" were investigated. Based on documents from the local Chronicles and historical accounts, the Chinese characters of Jinchai have the distinctive features of gold color and two hair clasps. Moreover, the hair clasps are usually cylindrical in shape with uniform thickness in middle and upper part, and tapers off to the foot. And its bottom part style is simple and head part is complex. Thus we speculated the herbal "Jinchai" and "Jinchai Shihu" should have similar morphologic features as Chinese characters of Jinchai, including golden color and hairpin shape of stems without braches, short and solid sterm. After comparing the dried vegetative morphology of 10 common medicinal Dendrobium species, we suggested that of Dendrobium flexicaule matches well with the morphological features from historical herbal records.

[Breakthrough in key science and technologies in Dendrobium catenatum industry].

In view of the significant difficulties of propagation, planting and simple product in Dendrobium catenatum(D. officinale)industry development, a series of research were carried out. Genome study showed that D. catenatum is a specie of diploid with 38 chromosomes and 28 910 protein-coding genes. It was identified that specific genes accumulated in different organs at the transcriptome level. We got an insight into the gene regulation mechanism of the loss of the endospermous seed, the wide ecological adaptability and the synthesis of polysaccharides, which provided a theoretical basis for genetic engineering breeding and development and utilization of active pharmaceutical ingredients. The rapid propagation system was established for applying to industrialized production by overcoming breeding problems on seed setting and sprouting, which laid a foundation for artificial cultivation of D. catenatum. And in order to give a clear explanation of genetic variation of important economic traits, we built up the breeding system. Since special varieties of D. catenatum were bred, it helped solve the problem of trait segregation of seedling progeny and support the improvement of D. catenatum industry. The regulation of dynamic variation of target compounds, together with the mechanism of nutrient uptake, was revealed. The breakthrough of key technologies including culture substrates, light regulation and precisely collection was carried out. Several cultivation modes like facility cultivation, original ecological cultivation, cliff epiphytic cultivation, stereoscopic cultivation and potting cultivation were set up. Above all, the goal of cultivating D. catenatum as well as producing good D. catenatum will be achieved.

[Herbal textual research on relationship between Chinese medicine"Shihu" (Dendrobium spp.) and "Tiepi Shihu" (D. catenatum)].

Dendrobium species on the ancient Chinese herbal texts were investigated in this paper, including their dscriptions of original species, producing areas and quality. Our results indicated that the major producing areas were Lu'an, Anhui province and Wenzhou, Taizhou, Zhejiang province. In addition, the sweet flavor, short, thin and solid stems were standing for good quality. Based on the stable producing areas and quality descriptions, D. catenatum (D. officinale) ("Tiepi Shihu") and D. houshanense were high quality medicinal Dendrobium species ("Shihu" ) in ancient China. Besides, there were 3 scientific names for "Tiepi Shihu", including D. candidum, D. officinale, D. catenatum. After textual investigation, We suggest that D. catenatum should be its scientific name, and D. officinale was synonyms published later. However, the name "D. officinale" could be reserved as it is much more popular used in publication and commodities. Moreover, its Chinese name should be "Tiepi Shihu".

Distinct subcellular localization and potential role of LINE1-ORF1P in meiotic oocytes.

LINE-1 is an autonomous non-LTR retrotransposon in mammalian genomes and encodes ORF1P and ORF2P. ORF2P has been clearly identified as the enzyme supplier needed in LINE-1 retrotransposition. However, the role of ORF1P is not well explored. In this study, we employed loss/gain-of-function approach to investigate the role of LINE1-ORF1P in mouse oocyte meiotic maturation. During mouse oocyte development, ORF1P was observed in cytoplasm as well as in nucleus at germinal vesicle (GV) stage while was localized on the spindle after germinal vesicle breakdown (GVBD). Depletion of ORF1P caused oocyte arrest at the GV stage as well as down-regulation of CDC2 and CYCLIN B1, components of the maturation-promoting factor (MPF). Further analysis demonstrated ORF1P depletion triggered DNA damage response and most of the oocytes presented altered chromatin configuration. In addition, SMAD4 showed nuclear foci signal after Orf1p dsRNA injection. ORF1P overexpression held the oocyte development at MI stage and the chromosome alignment and spindle organization were severely affected. We also found that ORF1P could form DCP1A body-like foci structure in both cytoplasm and nucleus after heat shock. Taken together, accurate regulation of ORF1P plays an essential role in mouse oocyte meiotic maturation.

Seasonal variation in the mating system of a selfing annual with large floral displays.

BACKGROUND AND AIMS: Flowering plants display considerable variation in mating system, specifically the relative frequency of cross- and self-fertilization. The majority of estimates of outcrossing rate do not account for temporal variation, particularly during the flowering season. Here, we investigated seasonal variation in mating and fertility in Incarvillea sinensis (Bignoniaceae), an annual with showy, insect-pollinated, 'one-day' flowers capable of delayed selfing. We examined the influence of several biotic and abiotic environmental factors on day-to-day variation in fruit set, seed set and patterns of mating. METHODS: We recorded daily flower number and pollinator abundance in nine 3 × 3-m patches in a population at Mu Us Sand land, Inner Mongolia, China. From marked flowers we collected data on daily fruit and seed set and estimated outcrossing rate and biparental inbreeding using six microsatellite loci and 172 open-pollinated families throughout the flowering period. KEY RESULTS: Flower density increased significantly over most of the 50-d flowering season, but was associated with a decline in levels of pollinator service by bees, particularly on windy days. Fruit and seed set declined over time, especially during the latter third of the flowering period. Multilocus estimates of outcrossing rate were obtained using two methods (the programs MLTR and BORICE) and both indicated high selfing rates of ∼80 %. There was evidence for a significant increase in levels of selfing as the flowering season progressed and pollinator visitation declined. Biparental inbreeding also declined significantly as the flowering season progressed. CONCLUSIONS: Temporal variation in outcrossing rates may be a common feature of the mating biology of annual, insect-pollinated plants of harsh environments but our study is the first to examine seasonal mating-system dynamics in this context. Despite having large flowers and showy floral displays, I. sinensis attracted relatively few pollinators. Delayed selfing by corolla dragging largely explains the occurrence of mixed mating in I. sinensis, and this mode of self-fertilization probably functions to promote reproductive assurance when pollinator service is limited by windy environmental conditions and competition from co-occurring flowering plants.

[Investigation of original materials under traditonal Chinese medicine names of "Jinchai" and "Jinchai Shihu"].

To clear from botanical view the original materials under the traditional Chinese medicine names of "Jinchai", "Jinchai Shihu" within the genus Dendrobium of the family Orchidaceae. Combined of different methods including study of historical records from the local chronicles and historical accounts of past event in Hubei, Sichuan, Chongqing, Henan and Shaanxi provinces, interviewing face to face with the old traditional Chinese workers and folk doctors in 20 downtowns and countrysides, such as, Laohekou, Lichuan, Fangxian, Xixia, Neixiang, and Ankang, and collecting a few plants of "Jinchai" for taxonomic identification. The traditional Chinese medicine names of "Jinchai", "Jinchai Shihu" were widely used by the local people from the eastern Chongqing, western Hubei, northeasten Sichuan, southeastern Shaanxi, western Henan. Those two names were frequently found in the local Chronicles and historical accounts of past event, even in the local daily life such as folk songs and stories. The botanical identification results showed that a endemic species of D. flexicaule is the original materials of the traditional Chinese medicine names "Jinchai" and "Jinchai Shihu", and this species are also called "Longtoujin", "Renzijin", "Huanzijin" and "Longtoufengweijin" by the local people. The dried artifactitious specimens of D. flexicaule are traditionally named as "Jinerhuan". The botanic resource plants of the traditional Chinese medicine names of "Jinchai", "Jinchai Shihu" are the endemic species of D. flexicaule that is distributed mainly in central areas of China including eastern Chongqing, western Hubei, northeasten Sichuan, southeastern Shaanxi, and western Henan, rather than D. nobile as referring in both Chinese and English version of Flora of China, and in official recorded serious versions of The Chinese Pharmacopoeia since 1977. In order to avoid confusion in the traditional Chinese medicine dendrobiums industry, the Chinese name of D. nobile is suggested as "Biancao Shihu", which characterized one stem feature of this species, and the traditional Chinese medicine names "Jinchai" or "JinchaiShihu" is suggested to refer to the species D. flexicaule.

PLK4 is essential for meiotic resumption in mouse oocytes.

Polo-like kinase (PLK) 4 is a unique member of the PLK family that plays vital roles in centriole biogenesis during mitosis. The localization of PLK4 on centrioles must be precisely regulated during mitosis to ensure correct centriole duplication. However, little is known about the function of PLK4 in mammalian oocyte meiosis. We addressed this question by examining the expression and localization of PLK4 in mouse oocytes and using RNA interference and protein overexpression to investigate its function in meiosis. PLK4 expression peaked at the germinal vesicle breakdown (GVBD) stage, and the protein was localized in the cytoplasm throughout meiotic maturation. Depletion of PLK4 caused meiotic arrest at the GV stage and suppressed CYCLINB1 and CDC2 activities. Moreover, PLK4 depletion prevented the de-phosphorylation of CDC2-Tyr15 in nucleus and induced a decrease in the level of the CDC25C protein. PLK1 overexpression failed to rescue GV-stage arrest in PLK4-depleted oocytes, whereas overexpressing PLK4 resulted in normal GVBD in oocytes in which PLK1 activity was inhibited. In addition, PLK4 overexpression did not cause abnormal spindle formation or affect extrusion of the first polar body. These results illustrate the fact that PLK4 is essential for meiotic resumption but may not influence spindle formation in mouse oocytes during meiotic maturation.

Exogenous thymine DNA glycosylase regulates epigenetic modifications and meiotic cell cycle progression of mouse oocytes.

In mammalian cells, 5-methylcytosine (5-meC) can be transformed into 5-hydroxymethylcytosine (5-hmC) by the methylcytosine dioxygenase TET proteins (TET1, TET2 and TET3). Thymine DNA glycosylase (TDG), a downstream enzyme of TET proteins, not only functions in base excision repair, but also acts as a key enzyme that participates in active DNA demethylation. Here we microinjected exogenous TDG-mCherry mRNAs into germinal vesicle (GV) stage mouse oocytes, and found that initially TDG-mCherry localized in the nucleus. Just before GV breakdown (GVBD), TDG-mCherry was released from the nucleus into the cytoplasm. In contrast with TDG, another active DNA demethylation-associated enzyme, activation-induced cytidine deaminase (AID) became localized in the cytoplasm of GV oocytes, but entered the nucleus of oocytes just before GVBD. However, both TDG and AID could enter the G0 stage nuclei of cumulus cells injected into the ooplasm. To analyze the effects of TDG on oocyte maturation, we over-expressed TDG-mCherry in GV oocytes, and found that the rates of both GVBD and polar body extrusion rate were significantly decreased. When the TDG over-expressed oocytes were blocked at the GV stage, the oocyte chromatin became decondensed, and the histone 3 trimethyl lysine 9 (H3K9me3) and H3K9me2 levels were decreased. We also found that TDG could reduce the 5-meC level of oocyte genomic DNA. All these results indicate that aberrant TDG expression causes epigenetic modifications and meiotic cell cycle arrest of mouse oocytes.

Reticulate evolution and sea-level fluctuations together drove species diversification of slipper orchids (Paphiopedilum) in South-East Asia.

South-East Asia covers four of the world's biodiversity hotspots, showing high species diversity and endemism. Owing to the successive expansion and contraction of distribution and the fragmentation by geographical barriers, the tropical flora greatly diversified in this region during the Tertiary, but the evolutionary tempo and mode of species diversity remain poorly investigated. Paphiopedilum, the largest genus of slipper orchids comprising nearly 100 species, is mainly distributed in South-East Asia, providing an ideal system for exploring how plant species diversity was shaped in this region. Here, we investigated the evolutionary history of this genus with eight cpDNA regions and four low-copy nuclear genes. Discordance between gene trees and network analysis indicates that reticulate evolution occurred in the genus. Ancestral area reconstruction suggests that vicariance and long-distance dispersal together led to its current distribution. Diversification rate variation was detected and strongly correlated with the species diversity in subg. Paphiopedilum (~80 species). The shift of speciation rate in subg. Paphiopedilum was coincident with sea-level fluctuations in the late Cenozoic, which could have provided ecological opportunities for speciation and created bridges or barriers for gene flow. Moreover, some other factors (e.g. sympatric distribution, incomplete reproductive barriers and clonal propagation) might also be advantageous for the formation and reproduction of hybrid species. In conclusion, our study suggests that the interplay of reticulate evolution and sea-level fluctuations has promoted the diversification of the genus Paphiopedilum and sheds light into the evolution of Orchidaceae and the historical processes of plant species diversification in South-East Asia.

Cyclin O regulates germinal vesicle breakdown in mouse oocytes.

It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

Influence of asymmetrical mating patterns and male reproductive success on the maintenance of sexual polymorphism in Acer pictum subsp. mono (Aceraceae).

Populations of Acer species often contain more than three sex phenotypes with complex sexual polymorphism including duodichogamy, protandry and protogyny. We identified the mechanisms that maintain sexual polymorphism in Acer pictum subsp. mono, a temperate tree from northern China, by investigating maternal mating patterns and male reproductive success. We used paternity analyses to estimate rates of outcrossing and disassortative mating, as well as male outcrossed siring success, in a population of A. pictum subsp. mono with uneven sex phenotype ratios (duodichogamous 69.1%, protandrous 19.6%, protogynous 11.3%). We used a pollen-transfer model to investigate whether the unequal ratios of sex phenotypes could be explained by the observed patterns of mating. Most progeny resulted from outcrossing, particularly disassortative among the sex phenotypes. Although the duodichogamous phenotype showed a significant amount of intraphenotypic mating, the frequency did not exceed that of disassortative mating. We detected no significant differences in male outcrossed siring success among the sex phenotypes. The pollen-transfer model demonstrated that sex phenotype ratios could be maintained by the observed mating pattern in the population. Our results indicate that disassortative mating among the sex phenotypes can maintain sexual polymorphism in A. pictum subsp. mono and that ratios biased towards duodichogamy can result from frequent intraphenotypic mating in this phenotype.

Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy.

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

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

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

Reproductive isolation and pollination success of rewarding Galearis diantha and non-rewarding Ponerorchis chusua (Orchidaceae).

BACKGROUND AND AIMS: Increasing evidence challenges the conventional perception that orchids are the most distinct example of floral diversification due to floral or prezygotic isolation. Regarding the relationship between co-flowering plants, rewarding and non-rewarding orchids in particular, few studies have investigated whether non-rewarding plants affect the pollination success of rewarding plants. Here, floral isolation and mutual effects between the rewarding orchid Galearis diantha and the non-rewarding orchid Ponerorchis chusua were investigated. METHODS: Flowering phenological traits were monitored by noting the opening and wilting dates of the chosen individual plants. The pollinator pool and pollinator behaviour were assessed from field observations. Key morphological traits of the flowers and pollinators were measured directly in the field. Pollinator limitation and interspecific compatibility were evaluated by hand-pollination experiments. Fruit set was surveyed in monospecific and heterospecific plots. KEY RESULTS: The species had overlapping peak flowering periods. Pollinators of both species displayed a certain degree of constancy in visiting each species, but they also visited other flowers before landing on the focal orchids. A substantial difference in spur size between the species resulted in the deposition of pollen on different regions of the body of the shared pollinator. Hand-pollination experiments revealed that fruit set was strongly pollinator-limited in both species. No significant difference in fruit set was found between monospecific plots and heterospecific plots. CONCLUSIONS: A combination of mechanical isolation and incomplete ethological isolation eliminates the possibility of pollen transfer between the species. These results do not support either the facilitation or competition hypothesis regarding the effect of nearby rewarding flowers on non-rewarding plants. The absence of a significant effect of non-rewarding P. chusua on rewarding G. diantha can be ascribed to low levels of overlap between the pollinator pools of two species.

Development of polymorphic microsatellite markers for Incarvillea sinensis (Bignoniaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Incarvillea sinensis var. sinensis (Bignoniaceae), an annual herb endemic to Inner Mongolia, to study the degree to which delayed self-fertilization is favored. METHODS AND RESULTS: Eight polymorphic primer sets were isolated and characterized in two Inner Mongolia populations of I. sinensis var. sinensis with a relatively simple and fast subcloning method. Numbers of alleles per locus ranged from 2 to 7, with observed and expected heterozygosities ranging from 0 to 0.261 and from 0 to 0.778, respectively. CONCLUSIONS: These markers will be useful for future studies of self-fertilization adaptability in I. sinensis var. sinensis.