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Plant species evolution is driven by many factors that have different roles in space and time. Using different field and laboratory methods, we studied reproductive patterns and their determinants in pure and mixed

Pollinator foraging behavior plays a key role in breeding and therefore affects the evolution of the orchid reproductive strategy. Food-deceptive orchids usually implement a generalized plant pollination strategy and a relatively diverse group of pollinators visit them. Dactylorhiza majalis is a food-deceptive, early-flowering orchid that relies on insect-mediated pollination. This study’s objectives were to identify D. majalis’ pollinators and flower visitors and their foraging behaviors on D. majalis inflorescences. We also assessed the bending movement time to determine the relationship between bending time and the duration of pollinators’ visits. To assess pollination efficiency, we measured the spur length of D. majalis flowers, which is expected to affect the mechanical fit to pollinators/“potential” pollinators. The arthropod fauna were investigated to examine the availability of “potential” pollinators in populations. We identified Apis mellifera as this orchid’s main pollinator and confirmed that few of the flower visitors belonged to Diptera (12 individuals, 9 taxa), Hymenoptera (3 individuals, 3 taxa), or Coleoptera (2 individuals, 2 taxa) in our dataset, which was collected over a 2-year period and includes 360 h of video. The arthropods were collected by a sweep net in D. majalis populations and there were fewer Hymenoptera (2.9–23.2%) and Coleoptera (4.4–23.8%) visitors but more Diptera (23.3–58.6%) visitors. We found that A. mellifera foraged in different ways on D. majalis inflorescences, thereby resulting in cross-pollination and/or geitonogamy; however, the bending time data supported the hypothesis about promoting cross-pollination while decreasing self-pollination, but these data do not exclude the possibility of geitonogamy.

Deceptive plants represent a strategy that promotes outcrossing and protects against facilitated selfing. However, deception does not eliminate the possibility of autonomous self-pollination when pollinators are scarce or absent. Spontaneous autogamy is widespread in Orchidaceae, but the scale, mechanism, time of appearance and effectiveness remain underestimated. Using field and laboratory observations and experiments, for the first time, we documented the possibility of autonomous self-pollination in the deceptive orchid Dactylorhiza fuchsii, which might occur through the previously unspecified mechanism in the tribe Orchideae of caudicle reconfiguration. Self-pollination occurred through the pollinarium twisting to the side and downwards, which was different than caudicle bending forward on the body of pollinators. Caudicle reconfiguration was continuously distributed during anthesis and was common in the studied populations. This mechanism was independent on the flower position in the inflorescence, but was sensitive to pollinator activity. (The frequency of caudicle reconfiguration increased when more pollinaria in the inflorescence were untouched.) This process was effective (self-pollination leading to autogamous fruits and seeds) only when a full caudicle rotation occurred and the pollinium touched the stigma. However, most caudicle reconfigurations were completed before the stigma was reached, resulting in less than 1% of autogamous pollination in the studied populations.

The breeding system in Orchidaceae generates many questions about the selfing potential of its representatives. We investigated the ability of spontaneous autogamy of four orchid species: Cephalanthera rubra and Neottia ovata of the Neottieae tribe and Gymnadenia conopsea and Platanthera bifolia of the Orchideae tribe. These species represent diverse specializations of the gynostemium architecture. The self-compatibility and properties of autogamous seeds were determined in a bagging experiment and seed development analysis. After induced autogamy, a high level of fruiting (80-100%) was noted in all of the four study species. C. rubra, N. ovata, and G. conopsea are completely self-compatible, and P. bifolia is suggested to be partially self-compatible. If autogamy occurred, inbreeding depression and resource limitation on seed development appeared only in the two Orchideae species. Independent of flower specialization, both Neottieae species and P. bifolia were completely allogamous, whereas G. conopsea could be facultatively autogamous.

We carried out translocations of three rare plants that inhabit mineral islands in the marshy Biebrza Valley in order to create alternative populations and facilitate connectivity between existing subpopulations. The species chosen were Iris aphylla and two orchids: Cypripedium calceolus and Cephalanthera rubra. Thirty soil monoliths with vegetative orchid plants or parts of I. aphylla rhizomes were dug out and transplanted to three different sites on new mineral islands (half in 2012 and half in 2013). Prior to translocation, we measured soil moisture and pH and took phytosociological characteristics in 68 potential recipient sites for orchids and 15 for I. aphylla. Then, we monitored the number of shoots for 4 years and retrospectively conducted principal component analysis (PCA) in order to compare the similarity of donor sites and chosen recipient sites. Three years after transplantation we found new C. calceolus populations in good condition, I. aphylla populations in moderate, while C. rubra transplants emerged only in the first and second season. All newly established populations of C. calceolus and I. aphylla survived. In the case of the first species, fruiting was observed in two populations, while a marked increase in shoot number was observed in one population. The most successful C. calceolus translocation site was also the most similar to the donor sites according to PCA.

Background and aims The deception strategies of orchids remain poorly understood, especially in regard to the chemical compounds emitted from their flowers and their interaction with various taxonomic groups of pollinators. We investigated the phylogenetic relationships and compared the variation of floral chemical compounds between food-deceptive Dactylorhiza taxa (D. incarnata var. incarnata and D. incarnata var. ochroleuca, D. fuchsii and D. majalis) from populations in north-eastern Poland. We propose a model of the evolution of deception based on floral chemical signals in this genus. Methods A Bayesian approach based on polymorphic plastid DNA (trnL, trnF and psbC-trnK), internal transcribed spacer (ITS) sequences and flow cytometry data was applied to confirm the taxonomic status of the studied orchids. We also identified and classified the pollinators and flower visitors in each Dactylorhiza population to the taxonomic level and compared our results with literature data. The chemical composition of pentane and diethyl ether extracts from the flowers was analysed by gas chromatography-mass spectrometry. Variation of the floral chemical components was visualized by non-metric multidimensional scaling based on Bray-Curtis dissimilarity. Key results The genetic distinctiveness of D. incarnata, D. fuchsii and D. majalis was confirmed. No hybrids between them were found, but the chloroplast DNA (cpDNA), ITS haplotypes and flow cytometry showed genetic similarity between D. incarnata var. incarnata and D. incarnata var. ochroleuca. We determined that Apis mellifera (Hymenoptera) was the only shared pollinator of these taxa. Strangalia attenuata and Alosterna tabacicolor (Coleoptera) and Volucella pellucens and V. bombylans (Hymenoptera) were observed pollinating D. fuchsii. Visualization of the emission rates of the 61 floral chemical compounds detected from pentane extracts (mainly hydrocarbons and aldehydes) and the 51 from diethyl extracts (with abundant groups of benzenoids and non-aromatic acids) strongly differentiated D. incarnata, D. fuchsii and D. majalis, while those of the two varieties of D. incarnata (var. incarnata and var. ochroleuca) were almost identical. Conclusions While the genetic data clearly supported the distinct lineages of D. incarnata, D. fuchsii and D. majalis, the patterns of emission of their flower chemical compounds were more complex within the series of shared compounds (alkanes and aldehydes) and taxon-specific compounds (benzenoids and esters). Their floral bouquet can influence the sexual, social and feeding behaviour of pollinators in different ways. We observed that the floral chemical compounds attracted both shared and species-specific pollinators to Dactylorhiza, confirming the multidirectional character of floral chemical signals in these food-deceptive taxa. Reduction of species-specific pollination levels in Dactylorhiza orchid taxa may promote hybridization between them.

In the presented study, male and female reproductive success was analyzed in relation to the population size, floral display and pollinators’ availability in natural and anthropogenic populations of the orchid Malaxis monophyllos (L.) Sw. Our results indicated significant differences between all investigated populations in parameters of floral display, including heights and number of flowers per inflorescence, as well the number of flowering individuals and their spatial structure. Additionally, populations differed both in male (pollinia removal) and female (fruit set) reproductive success, but only the fruit set clearly differentiated anthropogenic and natural populations. Despite the average flower number per plant being significantly higher in two of the anthropogenic populations, it was not related to the fruits set, which was significantly lower there. Moreover, our preliminary study concerning the potential pollinators of M. monophyllos showed a higher contribution of flies in natural habitats than in anthropogenic ones. Thus, we can suspect that the main factors influencing the level of female reproductive success in M. monophyllos populations are abundance of effective pollinators, as well as flower visitors, which may have resulted in a different level of pollen discounting in populations. Therefore, further studies concerning breeding system and pollination as important forces that shape demographic processes in M. monophyllos populations are necessary. Our results also indicate that suitable conservation methods in M. monophyllos should always include the preservation of potential pollinators, especially in these new, secondary habitats.

Studies of plant breeding systems are particularly important when they involve facultative processes, which can be overlooked in natural conditions. This especially applies to species that are considered allogamous, due to their pollination syndrome and floral architecture. We examined the potential level and factors enabling or limiting spontaneous autogamy in three populations of the lepidopteran orchid Gymnadenia conopsea. Using a bagging experiment, we noted the stages of the anther thecae and the positions of the pollinaria at the five phenological stages of the flower, as well as the quantity of autogamously set fruits and the number of properly formed seeds. In the studied populations, autogamy represents an accidental character, with a maximum of 3.3% of fruits set spontaneously per analysed sample in a given population, and with seed numbers ranging from 29 to 354. This process is an environmentally dependent co-product of the mechanisms that enable a position appropriate for touching the stigma (bending of the caudicle) and increase male fitness (disintegration of the massulae), preceded by the gradual opening of the anther chambers. Autopollination of G. conopsea may occur in the flowers at various flowering stages (excluding the beginning of anthesis) at each position on the inflorescence.

Polemonium caeruleum (Polemoniaceae) represents a very interesting system of compatibility transition. Studies of its biological and ecological properties in the context of the breeding system of various populations may help to understand the evolutionary mechanism of this process. We investigated some aspects of the breeding system, diversity and foraging behavior of the visitors, and relationship between population properties and fruit set in three populations from NE Poland. We found distinct compatibility systems in two studied populations and showed that if a population is self-compatible (SC), selfing is mediated by insects via geitonogamous pollen transfer. Despite the population properties (compatibility, visitor diversity and activity, population size, density, or floral display), P. caeruleum is not pollen limited and pollinators are highly important as a key factor determining the high reproductive success. Visitor assemblages (including key pollinators, bumblebees, and honey bees) and their foraging behavior on inflorescences vary between the populations, which may influence differences in the breeding system. The self-incompatible population was visited by a more diverse group of insects from Hymenoptera, Diptera, Lepidoptera, Heteroptera, and Coeloptera, which may favor effective cross-pollen transfer, whereas the SC population was pollinated mainly by Apis mellifera, which may promote mixed-mating. Studies on a wider range of P. caeruleum populations are needed to determine selective factors responsible for compatibility transition.

The deficiency of pollinators is indicated as the main factor limiting fruit set in orchids. Nectariferous species are more successful in setting fruits than nectarless species. In the present study, we tested whether pollinator limitation lowers reproductive success in populations of Cypripedium calceolus on environmental islands in the Biebrza National Park, NE Poland. Moreover, we analyzed how population size and structure affect pollination success. Our observations and results of experiments document the low level of fruiting in populations of nectarless C. calceolus (9.2% on average) and strong pollen limitation. Generally, we noted a positive relationship between pollination success and all parameters of population size (measured by both occupied area and number of clumps or shoots) and parameters measuring floral display (number of flowers in population, number of flowers in clumps, spatial structure of flowering shoots). We suggest that information about factors influencing the reproductive success of this endangered species may be useful for planning appropriate conservation actions.

In this work we assessed the genetic diversity of 32 C. calceolus populations from Poland. Mean genetic diversity was moderate (P = 36.4%, A = 1.58, H O = 0.143, F IS = 0.059), and seven geographic regions did not differ significantly in their levels of polymorphism (p > 0.05), although allele frequencies varied greatly. Only four unique alleles were found, at three sites in southern and southeastern Poland. Genetic (P, A) and genotypic diversity parameters (G, G U) were significantly correlated with population size (p

For evolutionary and ecological analyses, genetic diversity at different scales needs to be studied in terms of biological properties, habitat, population size and population history. We surveyed Platanthera bifolia populations from six regions in northeastern Poland to determine the impact of the mating system and population history on genetic diversity. Based on variation at allozyme markers, genetic variation was relatively moderate (P = 22.3%, A = 1.48, H O = 0.083, F IS = −0.015) and similar to other Platanthera species. These parameters varied between populations (P = 13.3%–26.6%, A = 1.26–1.66, H O = 0.055–0.111, F IS = −0.262–0.147). The genetic diversity patterns were shaped by different proportions of facilitated selfing and/or outcrossing, resulting in positive and negative F IS values, respectively. No relationship between inbreeding coefficient and population size, however, and no impact of apomixis on the level of genetic diversity of P. bifolia were found. The relatively low level of genetic differentiation among the investigated regions (F CT = 0.002, P > 0.05) and among populations (F ST = 0.048, P < 0.001), and the lack of a significant relationship between genetic and geographical distance, are discussed in the context of possible scenaria of postglacial expansion.

Factors enabling and limiting self-fertilization were examined in populations of Epipactis palustris, E. helleborine and E. atrorubens located in NE Poland. In these species we found self-compatibility within the same flower (even 100% of fruit set from induced autogamy) and the same inflorescence (reaching 90–100% of fruit set from induced geitonogamy). Facultative autogamy was found only in E. palustris (even 82.5% of fruit set in a bagged inflorescence), due to underdevelopment of the clinandrium and projection of pollinia over the rostellum. Allogamy was predominant in E. helleborine (maximum to 2.8% of fruits set spontaneously) and E. atrorubens (maximum to 3.4%), attributable to a well-developed rostellum and clinandrium and to the position of pollinia. Autonomous selfing takes place during flower wilting, when the viscidium becomes evanescent and the pollinia are fragile. Selfing is enabled mostly by pollinator behaviour, promoting both geitonogamy and autogamy, and influencing mixed-mating.

Enzyme electrophoreses were used to estimate genetic variation in five populations of Epipactis helleborine from two National Parks (Biebrza and Wigry) in northeast Poland. It has been proved that populations from these two regions differed in genetic structure, with the populations belonging to the Biebrza group having a higher level of genetic variation than those from Wigry. The number of polymorphic loci (P) ranged from 18.2% to 40.9% and the mean number of alleles per polymorphic locus (Ap) from 2.25 to 2.80. Although the observed heterozygosity (Ho) was lower than the expected one (He) in each population, the values of Ho and He were 2–3 times lower in the populations from Wigry than in those from Biebrza. Excluding the LUK population with the smallest genotypic diversity, the majority of 345 distinct multilocus genotypes occurred only once, sporadically 2–4 times in each population and their frequency ranged from 0.2% to 3.7%. Moreover, factors shaping genetic structure of E. helleborine and their intensity varied in the populations studied. Reproduction from seeds seems to influence greatly the genetic variation of the populations from the Biebrza National Park, while an assortative mating between related individuals or population size appears to be more important in the case of the populations from the Wigry National Park.