Impact of pollen resources drift on common bumblebees in NW Europe.

Several bee species are experiencing significant population declines. As bees exclusively rely on pollen for development and survival, such declines could be partly related to changes in their host plant abundance and quality. Here, we investigate whether generalist bumblebee species, with stable population trends over the past years, adapted their diets in response to changes in the distribution and chemical quality of their pollen resources. We selected five common species of bumblebee in NW Europe for which we had a precise description of their pollen diet through two time periods ('prior to 1950' and '2004-2005'). For each species, we assessed whether the shift in their pollen diet was related with the changes in the suitable area of their pollen resources. Concurrently, we evaluated whether the chemical composition of pollen resources changed over time and experimentally tested the impact of new major pollen species on the development of B. terrestris microcolonies. Only one species (i.e. B. lapidarius) significantly included more pollen from resources whose suitable area expanded. This opportunist pattern could partly explain the expansion of B. lapidarius in Europe. Regarding the temporal variation in the chemical composition of the pollen diet, total and essential amino acid contents did not differ significantly between the two time periods while we found significant differences among plant species. This result is driven by the great diversity of resources used by bumblebee species in both periods. Our bioassay revealed that the shift to new major pollen resources allowed microcolonies to develop, bringing new evidence on the opportunist feature of bumblebee in their diets. Overall, this study shows that the response to pollen resource drift varies among closely related pollinators, and a species-rich plant community ensures generalist species to select a nutrient-rich pollen diet.

Nectar foragers contribute to the pollination of buzz-pollinated plant species.

PREMISE OF THE STUDY: Pollination performance may depend on the type of floral resource (pollen or nectar) foraged by visitors. In buzz-pollinated plant species, the poricidal anthers release pollen during active pollen collection that induces flower vibrations. These buzz-pollinated species generally do not produce nectar. Nevertheless, several Ericaceae are buzz-pollinated and produce nectar. We estimated the relative effectiveness of visitors according to the type of resource collected, nectar or pollen (buzzing). METHODS: We compared the relative performance of pollen removal, transport, and deposition (effectiveness) of the main insect visitors on four ericaceous species: three buzz-pollinated species with different pore sizes, Erica tetralix, Vaccinium myrtillus, and V. vitis-idaea; and one non-buzz-pollinated species, Calluna vulgaris. KEY RESULTS: Bumblebees were the main pollinators for the three buzz-pollinated species, whereas hoverflies were the main pollinators for the non-buzz-pollinated generalist C. vulgaris. For the studied plant species, we observed no difference in pollination effectiveness among bumblebee species. Buzzing bumblebees were the most effective visitors for pollination per flower visit for the two Vaccinium species, whereas nectar foragers were the most effective visitors for pollination of E. tetralix. In the case of Vaccinium myrtillus, nectar foragers contributed the most to pollination success because they were more abundant than pollen foragers. CONCLUSIONS: We showed that consideration of the resource collected by visitors and their behavior is necessary to compare their relative performance. The combination of visitation rate and effectiveness per visit reveals that nectar foragers make a substantial contribution to pollination of the buzz-pollinated ericaceous species.

Diversity of the Insect Visitors on Calluna vulgaris (Ericaceae) in Southern France Heathlands.

As part of an ongoing research project on the pollination networks in European heathlands, the objective of this study was to assess the insect visitor guild on Calluna vulgaris (L.) Hull (Ericaceae). We focused the study on a region renowned for its largely well-preserved heathlands, the Cévennes National Park, Southern France. In 2013, flower visitors were observed over 3 d per site, in four heathland sites at mont Lozère. Honeybees (Apis mellifera L.) were the main visitors (62-88% of total visitors). Besides honeybees, a high diversity of visitors was detected with 57 different species identified (42 Diptera and 15 Hymenoptera). Hoverflies (Syrphidae, Diptera) visitors were abundant and diverse, especially individuals belonging to the genera Eristalis and Episyrphus. The reported diversity of visitors was probably due to the preservation of large heathland areas at mont Lozère and to the generalist pollination system of C. vulgaris.

Some bee-pollinated plants provide nutritionally incomplete pollen amino acid resources to their pollinators.

For pollinators such as bees, nectar mainly provides carbohydrates and pollen provides proteins, amino acids, and lipids to cover their nutritional needs. Here, to examine differences in pollinator resources, we compared the amino acid profiles and total amino acid contents of pollen from 32 common entomophilous plants in seven families. Our results showed that the amino acid profiles and contents in pollen samples differed according to the plant family and the chromatography method used, i.e., high-performance liquid chromatography (HPLC) versus ion exchange chromatography (IEX). Pollen from Boraginaceae species had the highest total amino acid contents (361.2-504 µg/mg) whereas pollen from the Malvaceae family had the lowest total amino acid contents (136-243.1 µg/mg). Calculating an amino acid score (AAS) that reflects pollen nutritional quality showed that slightly less than half of the species (19 out of 32) had the maximum nutritional score (AAS = 1) and offered high nutritional quality pollen amino acids for bee pollinators. Though they had high total amino acid contents, the amino acid composition of the studied Boraginaceae species and several members of the Fabaceae was not optimal, as their pollen was deficient in some essential amino acids, resulting in suboptimal amino acid scores (AAS < 0.7). Except for cysteine, the measured amino acid contents were higher using IEX chromatography than using HPLC. IEX chromatography is more robust and is to be preferred over HPLC in future amino acid analyses. Moreover, our observations show that some bee-pollinated species fail to provide complete amino acid resources for their pollinators. Although the implications for pollinator behavior remain to be studied, these deficiencies may force pollinators to forage from different species to obtain all nutritionial requirements.

Climate Change-Induced Stress Reduce Quantity and Alter Composition of Nectar and Pollen From a Bee-Pollinated Species (Borago officinalis, Boraginaceae).

In temperate ecosystems, elevated temperatures, and drought occur especially during spring and summer, which are crucial periods for flowering, pollination, and reproduction of a majority of temperate plants. While many mechanisms may underlie pollinator decline in the wake of climate change, the interactive effects of temperature and water stress on the quantity and quality of floral nectar and pollen resources remain poorly studied. We investigated the impact of temperature rise (+3 and +6°C) and water stress (soil humidity lower than 15%) on the floral resources produced by the bee-pollinated species Borago officinalis. Nectar volume decreased with both temperature rise and water stress (6.1 ± 0.5 µl per flower under control conditions, 0.8 ± 0.1 µl per flower under high temperature and water stress conditions), resulting in a 60% decrease in the total quantity of nectar sugars (mg) produced per flower. Temperature rise but not water stress also induced a 50% decrease in pollen weight per flower but a 65% increase in pollen polypeptide concentration. Both temperature rise and water stress increased the total amino acid concentration and the essential amino acid percentage in nectar but not in pollen. In both pollen and nectar, the relative percentage of the different amino acids were modified under stresses. We discuss these modifications in floral resources in regards to plant-pollinator interactions and consequences on plant pollination success and on insect nutritional needs.

Growing and Flowering in a Changing Climate: Effects of Higher Temperatures and Drought Stress on the Bee-Pollinated Species Impatiens glandulifera Royle.

Drought and higher temperatures caused by climate change are common stress conditions affecting plant growth and development. The reproductive phase is particularly sensitive to stress, but plants also need to allocate their limited resources to produce floral traits and resources to attract pollinators. We investigated the physiological and floral consequences of abiotic stress during the flowering period of Impatiens glandulifera, a bee-pollinated species. Plants were exposed to three temperatures (21, 24, 27 °C) and two watering regimes (well-watered, water stress) for 3 weeks. Not all parameters measured responded in the same manner to drought and/or heat stress. Drought stress induced leaf senescence, decreasing leaf number by 15-30% depending on growth temperature. Drought also reduced photosynthetic output, while temperature rise affected stomatal conductance. The number of flowers produced dropped 40-90% in response to drought stress, while higher temperatures shortened flower life span. Both stresses affected floral traits, but flower resources diminished in response to higher temperatures, with lower nectar volume and pollen protein content. We conclude that increased temperatures and drought stress, which are becoming more frequent with climate change, can negatively affect flowering, even if plants deploy physiological resistance strategies.

Warm Temperatures Reduce Flower Attractiveness and Bumblebee Foraging.

(1) Background: Plants attract pollinators using several visual signals, mainly involving the display, size, shape, and color of flowers. Each signal is relevant for pollinators foraging for floral rewards, pollen, and nectar. Changes in floral signals and rewards can be induced by an increase in temperature, drought, or other abiotic stresses and are expected to increase as global temperatures rise. In this study, we explored how pollinators respond to modified floral signals and rewards following an increase in temperature; (2) Methods: We tested the effects of warmer temperatures on bee-pollinated starflower (Borago officinalis, Boraginaceae) and determined the behavior of one of its main pollinators, the buff-tailed bumblebee (Bombus terrestris). We measured visual floral traits (display and size) and rewards (nectar and pollen) for plants cultivated at 21 °C or 26 °C. We investigated bumblebee behavior by tracking insect visits in a binary choice experiment in an indoor flight arena; (3) Results: Plants cultivated at 26 °C exhibited a smaller floral area (i.e., corolla sizes summed for all flowers per plant, 34.4 ± 2.3 cm2 versus 71.2 ± 2.7 cm2) and a greater flower height (i.e., height of the last inflorescence on the stem, 87 ± 1 cm versus 75 ± 1 cm) compared to plants grown at 21 °C. Nectar production per flower was lower in plants grown at 26 °C than in plants grown at 21 °C (2.67 ± 0.37 µL versus 4.15 ± 0.22 µL), and bumblebees visited flowers from plants grown at 26 °C four times less frequently than they visited those from plants grown at 21 °C; (4) Conclusions: These results show that warmer temperatures affect floral signals and reduce overall floral resources accessible to pollinators. Thus, the global increases in temperature caused by climate change could reduce plant pollination rates and reproductive success by reducing flower visitation.

Multiple introductions boosted genetic diversity in the invasive range of black cherry (Prunus serotina; Rosaceae).

BACKGROUND AND AIMS: Black cherry (Prunus serotina) is a North American tree that is rapidly invading European forests. This species was introduced first as an ornamental plant, then it was massively planted by foresters in many countries, but its origins and the process of invasion remain poorly documented. Based on a genetic survey of both native and invasive ranges, the invasion history of black cherry was investigated by identifying putative source populations and then assessing the importance of multiple introductions on the maintenance of gene diversity. METHODS: Genetic variability and structure of 23 populations from the invasive range and 22 populations from the native range were analysed using eight nuclear microsatellite loci and five chloroplast DNA regions. KEY RESULTS: Chloroplast DNA diversity suggests there were multiple introductions from a single geographic region (the north-eastern United States). A low reduction of genetic diversity was observed in the invasive range for both nuclear and plastid genomes. High propagule pressure including both the size and number of introductions shaped the genetic structure in Europe and boosted genetic diversity. Populations from Denmark, The Netherlands, Belgium and Germany showed high genetic diversity and low differentiation among populations, supporting the hypothesis that numerous introduction events, including multiple individuals and exchanges between sites, have taken place during two centuries of plantation. CONCLUSIONS: This study postulates that the invasive black cherry has originated from east of the Appalachian Mountains (mainly the Allegheny plateau) and its invasiveness in north-western Europe is mainly due to multiple introductions containing high numbers of individuals.

Contribution of Extensive Farming Practices to the Supply of Floral Resources for Pollinators.

Intensification of agricultural practices leads to a loss of floral resources and drives pollinator decline. Extensive agricultural practices are encouraged in Europe and contribute to the preservation of biodiversity. We compared three agricultural landscapes without extensive farming practices with three adjacent landscapes containing organic crops and extensively managed grasslands in Belgium. Nectar resource availability and plant-pollinator interactions were monitored from April to June. Flower density per plant species and plant-pollinator interactions were recorded in different landscape elements. In April, the main nectar resources were provided by linear elements such as hedgerows and forest edges. Nectar production peaked in May, driven by intensive grasslands and mass-flowering crops. Occurrence of extensive grasslands and organic crops significantly alleviated the nectar resource gap observed in June. Our results underscore the importance of maintaining landscape heterogeneity for continuous flower resources and highlight the specific role of extensive grasslands and organic crops in June.

Species-specific responses to combined water stress and increasing temperatures in two bee-pollinated congeners (Echium, Boraginaceae).

Water stress and increasing temperatures are two main constraints faced by plants in the context of climate change. These constraints affect plant physiology and morphology, including phenology, floral traits, and nectar rewards, thus altering plant-pollinator interactions.We compared the abiotic stress responses of two bee-pollinated Boraginaceae species, Echium plantagineum, an annual, and Echium vulgare, a biennial. Plants were grown for 5 weeks during their flowering period under two watering regimes (well-watered and water-stressed) and three temperature regimes (21, 24, 27°C).We measured physiological traits linked to photosynthesis (chlorophyll content, stomatal conductance, and water use efficiency), and vegetative (leaf number and growth rate) and floral (e.g., flower number, phenology, floral morphology, and nectar production) traits.The physiological and morphological traits of both species were affected by the water and temperature stresses, although the effects were greater for the annual species. Both stresses negatively affected floral traits, accelerating flower phenology, decreasing flower size, and, for the annual species, decreasing nectar rewards. In both species, the number of flowers was reduced by 22%-45% under water stress, limiting the total amount of floral rewards.Under water stress and increasing temperatures, which mimic the effects of climate change, floral traits and resources of bee-pollinated species are affected and can lead to disruptions of pollination and reproductive success.

Reproductive biology of the andromonoecious Cucumis melo subsp. agrestis (Cucurbitaceae).

BACKGROUND AND AIMS: Cucumis melo subsp. agrestis (Cucurbitaceae) is cultivated in many African regions for its edible kernels used as a soup thickener. The plant, an annual, andromonoecious, trailing-vine species, is of high social, cultural and economic value for local communities. In order to improve the yield of this crop, the first step and our aim were to elucidate its breeding system. METHODS: Eight experimental pollination treatments were performed during three growing seasons to assess spontaneous selfing, self-compatibility and effects of pollen source (hermaphroditic vs. male flowers). Pollination success was determined by pollen tube growth and reproductive success was assessed by fruit, seed and seedling numbers and characteristics. The pollinator guild was surveyed and the pollination distance determined both by direct observations and by indirect fluorescent dye dispersal. KEY RESULTS: The species is probably pollinated by several Hymenoptera, principally by Hypotrigona para. Pollinator flight distances varied from 25 to 69 cm. No evidence for apomixis or spontaneous self-pollination in the absence of insect visitors was found. The self-fertility index (SFI = 0) indicated a total dependence on pollinators for reproductive success. The effects of hand pollination on fruit set, seed number and seedling fitness differed among years. Pollen tube growth and reproductive success did not differ between self- and cross-pollinations. Accordingly, a high self-compatibility index for the fruit set (SCI = 1.00) and the seed number (SCI = 0.98) and a low inbreeding depression at all developmental stages (cumulative delta = 0.126) suggest a high selfing ability. Finally, pollen origin had no effect on fruit and seed sets. CONCLUSIONS: This andromonoecious species has the potential for a mixed mating system with high dependence on insect-mediated pollination. The selfing rate through geitonogamy should be important.

Morphology of nectaries and biology of nectar production in the distylous species Fagopyrum esculentum.

BACKGROUND AND AIMS: The mechanisms of floral nectar production in buckwheat (Fagopyrum esculentum, Polygonaceae), a distylous pseudo-cereal, have received relatively little attention, prompting an investigation of the factors that regulate this process. The aim was to perform a refined study of the structures that secrete nectar and of the internal and external parameters influencing nectar volumes and sugar concentrations. METHODS: In order to control environmental parameters, plants were cultivated in growth rooms under controlled conditions. The structure of nectaries was studied based on histological sections from flowers and flower buds. Nectar was extracted using glass micropipettes and the sugar concentration was measured with a hand refractometer. Sugar concentration in the phloem sap was measured using the anthrone method. To test the influence of photosynthesis on nectar production, different light and defoliation treatments were applied. KEY RESULTS: Unicellular trichomes were located in the epidermis at the ventral part of eight nectary glands situated on the flower receptacle alternately with stamens. Vascular bundles consisting of both phloem and xylem were identified at the boundary between a multilayered nectary parenchyma and a sub-nectary parenchyma with chloroplasts. A higher volume of nectar in thrum morphs was observed. No other difference was found in morphology or in sugar supply to inflorescences between morphs. Nectar secretion was strongly influenced by plant age and inflorescence position. Nectar volumes were higher in the upper inflorescences and during the flowering peak. Light had a dual role, (1) acting directly on reproductive structures to trigger flower opening, which conditions nectar secretion, and (2) stimulating photosynthetic activity, which regulates nectar accumulation in open flowers. CONCLUSIONS: In buckwheat, nectar is secreted by trichomes and probably proceeds, at least in part, from phloem sap. Nectar secretion is strongly influenced by floral morph type, plant age, inflorescence position and light.

Conservation of hoverflies (Diptera, Syrphidae) requires complementary resources at the landscape and local scales.

Accumulating evidence shows that landscape fragmentation drives the observed worldwide decline in populations of pollinators, particularly in species of Lepidoptera and Hymenoptera. However, Little is known about the effects of landscape fragmentation on hoverfly (Diptera, Syrphidae) communities. Hoverflies provide varied ecosystem services: larvae contribute to waste decomposition (saprophagous species) and pest control (aphidophagous species), and adults pollinate a wide range of flowers.To determine how the diversity and quantity of resources for larvae and adults affect hoverfly abundance and species richness at three spatial scales, we recorded insect visitors of five target plant species in Belgian heathlands, habitats that have decreased considerably due to human activities.Hoverflies represented the most abundant visitors on two plant species, and the second most abundant visitors (after bumblebees) on the other target plant species. A large proportion of hoverflies observed were aphidophagous species associated with coniferous and deciduous forests. Resources for the larvae and floral resources for the adults influenced interactions among hoverflies and plants, but acted at different scales: larval habitat availability (distance to larval habitat) was relevant at the landscape scale, whereas adult resource availability (floral density) was relevant at the plot scale.Hoverfly abundance and species richness decreased with distance to larval habitat but increased with floral density. Moreover, landscape structure and composition had different effects according to hoverfly ecological traits. Landscape composition influenced aphidophagous but not saprophagous hoverflies, in that their abundance and species richness decreased with distance to forests. Maintenance of the interactions between plants and their hoverfly visitors requires complementary resources at both landscape and local scales.

Temperature and water stress affect plant-pollinator interactions in Borago officinalis (Boraginaceae).

Climate change alters the abiotic constraints faced by plants, including increasing temperature and water stress. These changes may affect flower development and production of flower rewards, thus altering plant-pollinator interactions. Here, we investigated the consequences of increased temperature and water stress on plant growth, floral biology, flower-reward production, and insect visitation of a widespread bee-visited species, Borago officinalis. Plants were grown for 5 weeks under three temperature regimes (21, 24, and 27°C) and two watering regimes (well-watered and water-stressed). Plant growth was more affected by temperature rise than water stress, and the reproductive growth was affected by both stresses. Vegetative traits were stimulated at 24°C, but impaired at 27°C. Flower development was mainly affected by water stress, which decreased flower number (15 ± 2 flowers/plant in well-watered plants vs. 8 ± 1 flowers/plant under water stress). Flowers had a reduced corolla surface under temperature rise and water stress (3.8 ± 0.5 cm2 in well-watered plants at 21°C vs. 2.2 ± 0.1 cm2 in water-stressed plants at 27°C). Both constraints reduced flower-reward production. Nectar sugar content decreased from 3.9 ± 0.3 mg/flower in the well-watered plants at 21°C to 1.3 ± 0.4 mg/flower in the water-stressed plants at 27°C. Total pollen quantity was not affected, but pollen viability decreased from 79 ± 4% in the well-watered plants at 21°C to 25 ± 9% in the water-stressed plants at 27°C. Flowers in the well-watered plants at 21°C received at least twice as many bumblebee visits compared with the other treatments. In conclusion, floral modifications induced by abiotic stresses related to climate change affect insect behavior and alter plant-pollinator interactions.

Reproductive traits affect the rescue of valuable and endangered multipurpose tropical trees.

Conservation strategies are urgently needed in Tropical areas for widely used tree species. Increasing numbers of species are threatened by overexploitation and their recovery might be poor due to low reproductive success and poor regeneration rates. One of the first steps in developing any conservation policy should be an assessment of the reproductive biology of species that are threatened by overexploitation. This work aimed to study the flowering biology, pollination and breeding system of V. doniana, a multipurpose threatened African tree, as one step in assessing the development of successful conservation strategies. To this end, we studied (1) traits directly involved in pollinator attraction like flowering phenology, flower numbers and morphology, and floral rewards; (2) abundance, diversity and efficiency of flower visitors; (3) breeding system, through controlled hand-pollination experiments involving exclusion of pollinators and pollen from different sources; and (4) optimal conditions for seed germination. The flowering phenology was asynchronous among inflorescences, trees and sites. The flowers produced a large quantity of pollen and nectar with high sugar content. Flowers attracted diverse and abundant visitors, counting both insects and birds, and efficient pollinators included several Hymenoptera species. We detected no spontaneous self-pollination, indicating a total dependence on pollen vectors. Vitex doniana is self-compatible and no inbreeding depression occurred in the first developmental stages. After extraction of the seed from the fruit, seed germination did not require any particular conditions or pre-treatments and the seeds showed high germination rates. These pollination and breeding characteristics as well as germination potential offer the required conditions to develop successful conservation strategies. Protection, cultivation and integration in agroforestry systems are required to improve the regeneration of the tree.

Multilevel spatial structure impacts on the pollination services of Comarum palustre (Rosaceae).

Habitat destruction and fragmentation accelerate pollinator decline, consequently disrupting ecosystem processes such as pollination. To date, the impacts of multilevel spatial structure on pollination services have rarely been addressed. We focused on the effects of population spatial structure on the pollination services of Comarum palustre at three levels (i.e. within-population, between-populations and landscape). For three years, we investigated 14 Belgian populations, which differed in their within-population flower density, population surface, closure (i.e. proportion of the population edge that consisted of woody elements) and isolation (i.e. percentage of woody area cover within a 500 m radius from the population centre). We tested whether these spatial characteristics impact on pollinator abundance and visitation rate and thus, reproductive success of C. palustre. Insects were observed in 15 randomly-chosen plots in each population. We tested for pollen limitation with supplemental hand-cross pollination. Bumble bees and solitary bees were the major pollinators through all populations. Within populations, plots with high flower densities attracted high numbers of bumble bees and other insects. High bumble bee and solitary bee abundance was observed in populations presenting high proportions of woody edges and in populations within landscapes presenting high proportions of woody areas. Seed set resulting from open pollination varied with bumble bee and solitary bee visitation rate, leading to increased pollen limitation when pollinators were scarce. Since the reproductive success depended on the visitation rate of the main pollinators, which depended on multilevel spatial structure, wetland management plans should pay special attention to favour a mosaic of biotopes, including nesting sites and food resources for insects. This study particularly supports the relevance of a mix wetlands and woody habitats to bees.

The abundance and pollen foraging behaviour of bumble bees in relation to population size of whortleberry (Vaccinium uliginosum).

Habitat fragmentation can have severe effects on plant pollinator interactions, for example changing the foraging behaviour of pollinators. To date, the impact of plant population size on pollen collection by pollinators has not yet been investigated. From 2008 to 2010, we monitored nine bumble bee species (Bombus campestris, Bombus hortorum s.l., Bombus hypnorum, Bombus lapidarius, Bombus pascuorum, Bombus pratorum, Bombus soroensis, Bombus terrestris s.l., Bombus vestalis s.l.) on Vaccinium uliginosum (Ericaceae) in up to nine populations in Belgium ranging in size from 80 m(2) to over 3.1 ha. Bumble bee abundance declined with decreasing plant population size, and especially the proportion of individuals of large bumble bee species diminished in smaller populations. The most remarkable and novel observation was that bumble bees seemed to switch foraging behaviour according to population size: while they collected both pollen and nectar in large populations, they largely neglected pollen collection in small populations. This pattern was due to large bumble bee species, which seem thus to be more likely to suffer from pollen shortages in smaller habitat fragments. Comparing pollen loads of bumble bees we found that fidelity to V. uliginosum pollen did not depend on plant population size but rather on the extent shrub cover and/or openness of the site. Bumble bees collected pollen only from three plant species (V.uliginosum, Sorbus aucuparia and Cytisus scoparius). We also did not discover any pollination limitation of V. uliginosum in small populations. We conclude that habitat fragmentation might not immediately threaten the pollination of V. uliginosum, nevertheless, it provides important nectar and pollen resources for bumble bees and declining populations of this plant could have negative effects for its pollinators. The finding that large bumble bee species abandon pollen collection when plant populations become small is of interest when considering plant and bumble bee conservation.

Development and multiplexing of microsatellite markers in the polyploid perennial herb, Menyanthes trifoliata (Menyanthaceae).

PREMISE OF THE STUDY: We developed microsatellite primers to investigate genetic diversity and population genetic structure of the endangered herb Menyanthes trifoliata. METHODS AND RESULTS: Using the microsatellite-enriched library method, we identified 10 primer pairs in M. trifoliata. The primers amplified nine di- and one tri-nucleotide repeats with 4-13 alleles per locus in two Belgian populations. CONCLUSIONS: The results indicate that these markers offer an appropriate amount of variation to investigate genetic diversity, pollen dispersal (through paternity inference), and other conservation issues.

The evolution of Dactylorhiza (Orchidaceae) allotetraploid complex: insights from nrDNA sequences and cpDNA PCR-RFLP data.

Sequence data from a portion of the external transcribed spacer (ETS) and from the internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were used together with chloroplast DNA PCR-RFLP data to unravel patterns of allotetraploid speciation within the Western European Dactylorhiza polyploid complex. A maximum likelihood tree based on combined ETS and ITS sequences suggests that the Western European Dactylorhiza allotetraploids have evolved by hybridization between four main diploid lineages. Cloned sequences and the topology of the ITS plus ETS tree indicate that the allotetraploid species D. elata, D. brennensis, and D. sphagnicola have originated from the autotetraploid D. maculata together with the diploid D. incarnata, while D. majalis, D. traunsteineri, and D. angustata seem to have evolved by hybridization between the D. fuchsii s.str and D. incarnata lineages. Finally, the diploid D. saccifera lineage seems to have been involved together with the D. incarnata lineage in the formation of the allotetraploid D. praetermissa. The observed congruence between the chloroplast tree and the ITS/ETS tree suggests a directional evolution of the nrDNA after polyploidization in favor of the maternal genome. Considered together with morphological, biogeographical, and ecological evidence, the molecular analysis leads us to recognize four species within the investigated allotetraploid complex, namely D. majalis, D. praetermissa, D. elata, and D. sphagnicola.

Nuclear ribosomal DNA sequence variation and evolution of spotted marsh-orchids (Dactylorhiza maculata group).

Sequences of both internal and external transcribed spacers of nuclear ribosomal DNA were sequenced for four species belonging to the Dactylorhiza maculata group or "spotted marsh-Orchids". These four species are D. fuchsii, D. saccifera, D. foliosa, and D. maculata. Extensive nuclear ribosomal DNA polymorphism was uncovered within the diploid D. fuchsii and the putative autotetraploid D. maculata. Within the phylogenetic trees reconstructed using parsimony and Bayesian analyses, four main lineages (A, B, C, and D) were well supported. While D. saccifera, D. maculata, and D. foliosa were confined to clades B, C, and D, respectively, D. fuchsii accessions were spread over three clades (A, B, and C). Lineage C, which included accessions of the diploid D. fuchsii and the tetraploid D. maculata, was closely related to the lineage of D. foliosa (lineage D), an endemic diploid species from Madeira. Moreover, intra-individual polymorphism was found within accessions of D. maculata, D. fuchsii, and D. saccifera. It is shown that in some instances two lineages, contributed to the observed intra-individual polymorphism (C and A in D. maculata, A and B in D. fuchsii and D. saccifera). Evolutionary scenarios leading to this extensive nuclear ribosomal DNA polymorphism are discussed in the light of results from maternally inherited chloroplast DNA markers and an autopolyploid origin of D. maculata from a D. foliosa-like ancestor is postulated.