Pollination success in monochromic yellow populations of the rewardless orchid Dactylorhiza sambucina

Dactylorhiza sambucina is a European terrestrial orchid that lacks a pollinator reward. Throughout most of its range, populations contain yellow- and purple-flowering individuals, but in western Germany, monomorphic yellow populations predominate. As elsewhere, bumblebee queens are the most important pollinators in these populations, and mean fruit set over two years was 19%, well within the range reported from dimorphic populations. Multivariate analyses of plant and population traits, including plant height, leaf number, flower number and density on the spikes, flowering population density, and nearest neighbor distance, showed that only individual plant height and population density had a unique positive effect on pollen export; female function was unrelated to height or population density. The positive effects of dense spacing of flowering conspecifics and tall size appear due to greater visual attractiveness. Good visual exposure may also explain that flowers higher up on the spikes, in spite of opening late in the season, had higher male reproductive success than early flowers.     

Floral visual signal increases reproductive success in a sexually deceptive orchid

Sexually deceptive orchids mimic signals emitted by female insects in order to attract mate-searching males. Specific attraction of the targeted pollinator is achieved by sex pheromone mimicry, which constitutes the major attraction channel. In close vicinity of the flower, visual signals may enhance attraction, as was shown recently in the sexually deceptive orchid Ophrys heldreichii. Here, we conducted an in situ manipulation experiment in two populations of O. heldreichii on Crete to investigate whether the presence/absence of the conspicuous pink perianth affects reproductive success in two natural orchid populations. We estimated reproductive success of three treatment groups (with intact, removed and artificial perianth) throughout the flowering period as pollinaria removal (male reproductive success) and massulae deposition (female reproductive success). Reproductive success was significantly increased by the presence of a strong visual signal??the conspicuous perianth??in one study population, however, not in the second, most likely due to the low pollinator abundance in the latter population. This study provides further evidence that the coloured perianth in O. heldreichii is adaptive and thus adds to the olfactory signal to maximise pollinator attraction and reproductive success.     

Enhanced reproductive success revealed key strategy for persistence of devastated populations in Himalayan food‐deceptive orchid,Dactylorhiza hatagirea

Anthropogenic disturbances adversely affect populations of rare and endemic plants, resulting in reduction of their population size and performance. Among different plant groups, deceptive terrestrial orchids are vulnerable and possess greater extinction risks because of rarity in occurrence. To understand the response of food‐deceptive terrestrial orchids to disturbances, we selected Dactylorhiza hatagirea as our representative species, which is endemic to Himalaya, and studied its natural populations. This species is rare for being habitat specific, pollination limited and threatened in its natural habitats. We tested the hypothesis that disturbances lead to reduction in population size and plant performance of food‐deceptive terrestrial orchids. For assessing the impact of disturbance, two contrasting groups, heavily devastated (HD) and lightly devastated (LD), were identified on the basis of frequency and intensity of disturbance (harvesting of plant for tubers) by interviewing local people, medicinal plant extractors and shepherds. HD sites, in comparison to LD sites, were found to have smaller population sizes, but showed an increase in plant growth traits (plant height, specific leaf area, leaf N and specific shoot length). Similarly, plants at HD sites were found to have invested less in inflorescence (inflorescence size, inflorescence length, inflorescence length fraction and flowers per length), but despite that showed higher reproductive success. This was a clear indication of enhanced performance of its populations driven by disturbances. Our findings suggested that food‐deceptive species in small populations tend to reduce the probability of population extinction and have the capability to recover rapidly if conserved in time.     

Confocal scanning laser microscopy, a new technique used in an embryological study of Dactylorhiza maculata (Orchidaceae)

Ovules of Dactylorhiza maculata , fixed in FPA₅₀ and made transparent in Herr's clearing fluid, were investigated with confocal scanning laser microscopy. This new technique makes it possible to obtain thin optical serial sections in perfect alignment without damaging the material. It made possible an interpretation of the development of the embryo sac differing from that based on previous investigations with traditional technique. Using the new technique we found that the young embryo sac generally contains seven nuclei, two of which fuse, and the mature sac is 6-nucleate. The pollen tube does not penetrate any of the synergids when entering the embryo sac. Double fertilization takes place, and all nuclei are still alive at that moment. Four to six endosperm nuclei are formed, but later they degenerate and the growing embryo fills the entire embryo sac.     

Floral display influences male and female reproductive success of the deceptive orchid Phaius delavayi

The influence of floral display on male and female reproductive success is variable without consistent patterns. Few studies concentrate on the integrated influence of the different components of floral display on reproductive fitness at population level. We studied the impact of different components of floral display on reproductive success in the Chinese orchid Phaius delavayi. This deceptive orchid has flowers in which male and female success can be readily assessed in the field by observing rates of pollinarium removal and fruit initiation. The results showed that mean pollinarium removal (52.05?±?2.08%) was higher than fruit set (36.78?±?1.89%), and both male and female reproductive success decreased with increasing clump size in Phaius delavayi. Nevertheless, floral display expressed by clump size and flower position impacted both female and male reproductive success, whereas flowering initiation and duration, and inflorescence size had no effect.     

Differentiation in a tropical deceptive orchid: colour polymorphism and beyond

Colour polymorphism has often been described among individuals of deceptive orchid species, and several studies have investigated reproductive success variations among colour morphs. However, whether colour morphs differed in other traits has received little attention in previous studies. Here, we report the case of a tropical deceptive orchid in Reunion Island that exhibits three different colour varieties. We investigated patterns of trait variation among colour varieties and found significant differences in floral and inflorescence morphology. Interestingly, we found that most populations included only individuals of a single variety, and that strong differences exist among varieties in spatial distribution and flowering phenology. This situation differs from previously reported cases of floral polymorphism in deceptive orchids where colour morphs co-occur and flower in the same populations. The spatio-temporal variation in flowering suggests that colour varieties have independent reproductive ecologies and are adapted to local conditions. We propose that potential variations in pollinator species abundance or diversity and the co-occurrence of nectar-producing species in the community may have driven the adaptation of each variety to its current pollination niche.     

Effects of individual and population parameters on reproductive success in three sexually deceptive orchid species

Reproductive success (RS) in orchids in general, and in non-rewarding species specifically, is extremely low. RS is pollinator and pollination limited in food deceptive orchids, but this has rarely been studied in sexually deceptive orchid species. Here, we tested the effects of several individual (plant height, inflorescence size, nearest neighbour distance and flower position) and population (patch geometry, population density and size) parameters on RS in three sexually deceptive Ophrys (Orchidaceae) species. Inter-specific differences were observed in RS of flowers situated in the upper versus the lower part of the inflorescence, likely due to species-specific pollinator behaviour. For all three species examined, RS increased with increasing plant height, inflorescence size and nearest neighbour distance. RS generally increased with decreasing population density and increasing patch elongation. Given these results, we postulate that pollinator availability, rather than pollinator learning, is the most limiting factor in successful reproduction for sexually deceptive orchids. Our results also suggest that olfactory 'display' ( i.e. versus optical display), in terms of inflorescence size (and co-varying plant height), plays a key role in individual RS of sexually deceptive orchids. In this regard, several hypotheses are suggested and discussed.     

Pollination biology of Luisia curtisii (Orchidaceae): indications of a deceptive system operated by beetles

A population of Luisia curtisii (Orchidaceae: Aeridinae) in northern Thailand was studied with regard to pollination biology. Although a high level of self-compatibility was demonstrated experimentally, the very low natural fruit set (1.4–1.9 %) clearly indicated that the species depends on external agents for pollination. Our observations suggest that L. curtisii is pollinated by beetles, as Lema unicolor (Chrysomelidae) and Clinteria ducalis (Scarabaeidae) were the only flower visitors observed to carry pollinaria of this species. The hypothesis of specialised cantharophily is further supported by 2-methylbutyric acid and caproic acid being striking components of the floral scent. Judging from the lack of nectar and the behaviour of visiting beetles, the pollination system seems to rely on food or brood site deception. Retention of the anther on the pollinarium for some time after pollinarium removal probably reduces the frequency of insect-mediated autogamy and geitonogamy in Luisia curtisii—a possibility that was supported by comparative data on (1) the anther retention time and inflorescence visitation time of Lema unicolor and (2) stigma and anther length in the orchid. Existing reports of specialised beetle pollination in orchids are reviewed, and we conclude that there is accumulating evidence that specialised cantharophily is more common in the Orchidaceae than previously assumed.     

Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia

The Dactylorhiza incarnata/maculata complex (Orchidaceae) was used as a model system to understand genetic differentiation processes in a naturally occurring polyploid complex with much of ongoing diversification and wide distribution in recently glaciated areas in northern Europe. Data were obtained for 12 hypervariable regions in the plastid DNA genome. A total of 166 haplotypes were found in a sample of 1099 plants. Allopolyploid taxa have inherited their plastid genomes from D. maculata s.l. Overall haplotype diversity of the combined group of allopolyploid taxa was comparable to that of maternal D. maculata s.l., but populations of allopolyploids were also more strongly differentiated from each other and contained lower numbers of haplotypes than populations of D. maculata s.l. In addition to haplotypes found in extant D. maculata s.l., the allopolyploids also contained several distinct and widespread haplotypes that were not found in any of the parental lineages. Some of these haplotypes were shared between widespread allopolyploids. Divergent allopolyploids with small distributions did not seem to originate from local polyploidization events, but rather as segregates of already existing allopolyploids. Genetic diversification of allopolyploid Dactylorhiza is the result of repeated polyploid formation, secondary hybridization and introgression between already existing polyploids and extant representatives of parental lineages, hybridization between independently derived polyploid lineages, and phyletic diversification in the group of allopolyploids. Although some polyploid taxa must have evolved after the last glaciation, genetic material from the parental lineages has been transferred continuously for longer periods of time. This combination of processes may explain the taxonomic complexity encountered in Dactylorhiza and other polyploid complexes distributed in previously glaciated parts of Europe.     

利用传粉综合征预测:长瓣兜兰模拟繁殖地欺骗雌性食蚜蝇传粉

在花部进化受传粉者主导这一经典理论影响下,传粉综合征一直被认为是被子植物对应于某些特定传粉者的标志,因此,可以利用传粉综合征来预测传粉者。近年来,这种对应和预测受到多因素影响,以及花部进化理论和传粉者泛化理论的冲击。在本研究中,我们选择长瓣兜兰Paphiopedilum dianthum作为实验材料,来探讨传粉综合征与传粉者的关系以及利用前者预测后者的有效范围。长瓣兜兰具有的一系列特征,同其他依靠假繁殖地欺骗食蚜蝇传粉的兜兰种类相似,包括倒盔状唇瓣,较大的中萼片以及花瓣上的黑色突起和睫状毛,而且长瓣兜兰同这些种类有很近的亲缘关系和相似的生境。根据传粉综合征概念,长瓣兜兰应该是由食蚜蝇传粉,通过模拟繁殖地来欺骗传粉者。为了检验这些预测,我们对长瓣兜兰进行了传粉生物学研究。长瓣兜兰是自交可亲和的物种,不存在自动自交授粉现象,必须依靠昆虫传粉才能结实。结果表明,雌性黑带食蚜蝇Episyrphus balteatus是长瓣兜兰的主要传粉者,并且这种兜兰是通过模拟繁殖地来欺骗食蚜蝇传粉的。根据本实验结果,我们认为传粉综合征只是对应于不同的传粉选择压力而不是简单对应于传粉者种类,而使用传粉综合征预测时还需要考虑生境和演化历史等因素对花部形态的影响,这样才可能得到有效的结果。     

Increase of pollinator attraction by means of a visual signal in the sexually deceptive orchid, Ophrys heldreichii (Orchidaceae)

Orchids of the genus Ophrys are pollinated by males of solitary bees and wasps through sexual deception. Flowers mimic the odor of a receptive female and thus attract males that seek to copulate. Visual stimuli have been assumed so far to play only a minor role in male attraction. We investigated the role of the perigon as a potential visual signal in attracting pollinators in the orchid Ophrys heldreichii and its pollinator, the males of the long-horned bee Tetralonia berlandi (Apidae). In contrast to many other Ophrys species, O. heldreichii exhibits a large and bright pinkish perigon that appears visually conspicuous to a human observer. In a dual choice test we presented two flowers from a single plant and counted visitation rates. We then removed the perigon of one flower and retested the relative attractiveness of both flowers. For 292 male visits in ten trials we found a significant decrease of visitation rate for flowers with the perigon removed. In a second experiment we repeated the dual choice test using photos of the flowers. Males also significantly chose the picture of an intact flower over the picture of a modified flower where the perigon was digitally removed. From our data, we conclude that T. berlandi males respond to and are attracted by the bright pink perigon of the orchid in addition to other stimuli. A bright colorful perigon occurs almost only in the Ophrys holoserica-oestrifera group, a large sub-group of the genus. We hypothesize that this kind of visual signal is adaptive particularly in those Ophrys species where the targeted males patrol resourced-based encounter sites and strongly rely on their visual system while searching for their females.     

Habitat differentiation, hybridization and gene flow patterns in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. (Orchidaceae)

Detailed ecological, morphological and molecular analyses were performed in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. in Scandinavia. Comparisons were made with pure populations of either diploid ssp. fuchsii or tetraploid ssp. maculata. It was shown that mixed populations are the result of secondary contact between ssp. fuchsii and ssp. maculata. No patterns of recent and local autopolyploidization were found. Morphology and nuclear DNA markers (internal transcribed spacers of nuclear ribosomal DNA) showed that diploids and tetraploids from mixed populations have similar levels of differentiation to diploids and tetraploids from pure populations. Vegetation analyses, as well as analyses of environmental variables, revealed that diploid and tetraploid individuals in mixed populations are ecologically well differentiated on a microhabitat level. Diploids and tetraploids in pure populations have wider ecological amplitudes than they do in mixed populations. Triploid hybrids grew in intermediate microhabitats between diploids and tetraploids in the mixed populations. Plastid DNA markers indicated that both diploids and tetraploids may act as the maternal parent. Based on morphology and nuclear markers triploids are more similar to tetraploids than to diploids. There were indications of introgressive gene flow between ploidy levels. Plastid markers indicated that gene flow from diploid to tetraploid level is most common, but nuclear markers suggested that gene flow in opposite direction also may occur. Similar patterns of differentiation and gene flow appeared in localities that represented contrasting biogeographic regions. Disturbance and topography may explain why hybridization was slightly more common and the differentiation patterns somewhat less clear in the Scandinavian mountains than in the coastal lowland. An erratum to this article can be found at     

Systematics of the Dactylorhiza euxina/incarnata/maculata polyploid complex (Orchidaceae) in Turkey: evidence from allozyme data

 Material of Dactylorhiza were sampled from 49 localities in Turkey and investigated for allozyme variation at ten loci (nine enzyme systems). Among diploids, the Anatolian D. osmanica and D. umbrosa were allozymically variable, but not distinct from each other or from D. incarnata. Dactylorhiza saccifera contained the same alleles as the European D. fuchsii. Dactylorhiza iberica and D. euxina were distinct from each other and the other diploids. On basis of allozyme patterns three distinct allotetraploid genotypes were distinguished, and each of them could be treated as a separate species. Dactylorhiza nieschalkiorum is similar to European allotetraploids, and may have arisen from hybridization between D. incarnata s.l. and D. saccifera. Dactylorhiza urvilleana may have arisen from parents related to present-day D. saccifera and D. euxina, but it also contains additional alleles that have not been found in any of the diploids investigated. A third allotetraploid known from four populations in the Ardahan and Kars provinces of north-eastern Turkey combines the allozyme patterns found in material of D. incarnata s.l. from the same area with those from D. euxina. It is here described for the first time as D. armeniaca. Received November 14, 2000 Accepted June 20, 2001     

Pollination biology of Eulophia alta (Orchidaceae) in Amazonia: effects of pollinator composition on reproductive success in different populations

Background and Aims

Spatial variation in pollinator composition and abundance is a well-recognized phenomenon. However, a weakness of many studies claiming specificity of plant–pollinator interactions is that they are often restricted to a single locality. The aim of the present study was to investigate pollinator effectiveness of the different flower visitors to the terrestrial orchid Eulophia alta at three different localities and to analyse whether differences in pollinator abundance and composition effect this plant''s reproductive success.

Methods

Natural pollination was observed in vivo, and manipulative experiments were used to study the pollination biology and breeding system of E. alta at three sites near Manaus, Brazil. To gain a better understanding of the underlying mechanisms of pollinator attraction, nectar composition and secretion patterns were also studied, floral scent composition was analysed and a bioassay was conducted.

Key Results

Flower visitors, pollinator composition, pollinia transfer efficiency of particular pollinator species and natural fruit set differed among the investigated populations of E. alta. Flowers were self-compatible, partially autogamous and effectively pollinated by five bee species (four Centris species and Xylocopa muscaria). Visiting insects appeared to imbibe small amounts of hexose-rich nectar. Nectar sugar content was highest on the third day after flower opening. Floral fragrance analyses revealed 42 compounds, of which monoterpenes and benzenoids predominated. A bioassay using floral parts revealed that only floral tissue from the labellum chamber and labellum tip was attractive to flower visitors.

Conclusions

The data suggest that observed differences in reproductive success in the three populations cannot be explained by absolute abundance of pollinators alone. Due to behavioural patterns such as disturbance of effective pollinators on flowers by male Centris varia bees defending territory, pollinia transfer efficiencies of particular pollinator species also vary between study sites and result in differing reproductive success.     

Effect of pollination success on floral longevity in the orchid Calypso bulbosa (Orchidaceae)

The lifespan of an individual flower is often affected by pollination success. Species differ regarding whether male function (pollen removal), female function (pollen deposition), or both trigger floral senescence. We studied senescence in the singleflowered, deceptive orchid Calypso bulbosa by manipulating the degree of male and female reproductive success. We found that deposition of any amount of pollen resulted in dramatic changes in shape and color within 4 d, whereas unmanipulated flowers and those that had had pollinia removed remained unchanged for 8-11 d after treatment. Selection may favor the reproductive function that is less easily satisfied as the trigger for senescence, because a flower that senesces after accomplishment of this function is likely to have already succeeded at the more easily satisfied one. Deceptive (i.e., rewardless) flowers are more likely to satisfy male than female function since the latter requires that a pollinator be fooled twice, first to pick up pollen and second to deposit it. A survey of naturally pollinated Calypso showed that male function, pollinium removal, was more likely to occur than female function, deposition (95% vs. 66% of visited flowers); thus floral senescence in Calypso is triggered by achievement of the function less likely to succeed. Studies of senescence triggers in species in which female function is more likely to be achieved than male are necessary to further test this hypothesis.     

Molecular phylogenetics of the sexually deceptive orchid genus Ophrys (Orchidaceae) based on nuclear and chloroplast DNA sequences.

We present a phylogenetic analysis of the major lineages of the sexually deceptive orchid genus Ophrys based on nuclear ribosomal (nr) DNA (internal transcribed spacer region) and noncoding chloroplast (cp) DNA (trnL-trnF region) sequences. Sequence divergence within and among major Ophrys lineages was low for both nrDNA and cpDNA sequences. Separate analyses resulted in similar but poorly resolved trees. An incongruence length difference test revealed that nrDNA and cpDNA data sets were not incongruent. A combined analysis resulted in a better-resolved phylogenetic hypothesis of relationships among the major Ophrys lineages. Our data strongly support a division of Ophrys into two groups. These groups do not correspond to the earlier proposed sections Euophrys and Pseudophrys and are thus in conflict with traditional classifications. Our results support a well-resolved monophyletic group that contains the geographically widespread O. bombyliflora, O. speculum, O. tenthredinifera, and the O. fusca-lutea lineage. Relationships in the other group are poorly resolved. Based on our observations that taxa with identical sequences at presumably rapidly evolving loci clearly differ in floral morphology, we hypothesize that the diversity in the genus Ophrys is the result of a recent radiation in this orchid lineage.