Pollinator sharing between reproductively isolated genetic lineages of Silene nutans.

High reciprocal pollination specialization leading to pollinator isolation can prevent interspecific pollen transfer and competition for pollinators. Sharing pollinators may induce mating costs, but it may also increase pollination services and pollen dispersal and offer more resources to pollinators, which may be important in case of habitat fragmentation leading to pollination disruption. We estimated pollen dispersal and pollinator isolation or sharing between two reproductively isolated genetic lineages of Silene nutans (Caryophyllaceae), which are rare and occur in parapatry in southern Belgium, forming two edaphic ecotypes. As inter-ecotypic crosses may lead to pollen wastage and inviable progeny, pollinator isolation might have evolved between ecotypes. Silene nutans is mainly pollinated by nocturnal moths, including nursery pollinators, which pollinate and lay their eggs in flowers, and whose caterpillars feed on flowers and seeds. Pollinator assemblages of the two ecotypes are largely unknown and inter-ecotypic pollen flows have never been investigated. Fluorescent powdered dyes were used as pollen analogues to quantify intra- and inter-ecotypic pollen transfers and seeds were germinated to detect chlorotic seedlings resulting from inter-ecotypic pollination. Nocturnal pollinators were observed using infrared cameras on the field, and seed-eating caterpillars were collected and reared to identify nursery pollinator species. No pollinator isolation was found: we detected long-distance (up to 5 km) inter-ecotypic dye transfers and chlorotic seedlings, indicating inter-ecotypic fertilization events. The rare moth Hadena albimacula, a nursery pollinator specialized on S. nutans, was found on both ecotypes, as well as adults visiting flowers (cameras recordings) as seed-eating caterpillars. However, S. nutans populations harbor different abundance and diversity of seed predator communities, including other rare nursery pollinators, suggesting a need for distinct conservation strategies. Our findings demonstrate the efficiency of moths, especially of nursery pollinators, to disperse pollen over long distances in natural landscapes, so to ensure gene flow and population sustainability of the host plant. Seed-predator specificities between the two reproductively isolated genetic lineages of S. nutans, and pollinator sharing instead of pollinator isolation when plants occur in parapatry, suggest that conservation of the host plant is also essential for sustaining (rare) pollinator and seed predator communities.

Does the Phytochemical Diversity of Wild Plants Like the Erythrophleum genus Correlate with Geographical Origin?

Secondary metabolites are essential for plant survival and reproduction. Wild undomesticated and tropical plants are expected to harbor highly diverse metabolomes. We investigated the metabolomic diversity of two morphologically similar trees of tropical Africa, Erythrophleum suaveolens and E. ivorense, known for particular secondary metabolites named the cassaine-type diterpenoids. To assess how the metabolome varies between and within species, we sampled leaves from individuals of different geographic origins but grown from seeds in a common garden in Cameroon. Metabolites were analyzed using reversed phase LC-HRMS(/MS). Data were interpreted by untargeted metabolomics and molecular networks based on MS/MS data. Multivariate analyses enabled us to cluster samples based on species but also on geographic origins. We identified the structures of 28 cassaine-type diterpenoids among which 19 were new, 10 were largely specific to E. ivorense and five to E. suaveolens. Our results showed that the metabolome allows an unequivocal distinction of morphologically-close species, suggesting the potential of metabolite fingerprinting for these species. Plant geographic origin had a significant influence on relative concentrations of metabolites with variations up to eight (suaveolens) and 30 times (ivorense) between origins of the same species. This shows that the metabolome is strongly influenced by the geographical origin of plants (i.e., genetic factors).

Influence of edaphic conditions and nitrogen fertilizers on cadmium and zinc phytoextraction efficiency of Noccaea caerulescens.

The success of cadmium phytoextraction operations with Noccaea caerulescens varies by a factor of 70 between sites of trials. However, soil factors driving the efficiency of cadmium (Cd) and zinc (Zn) phytoextraction are still poorly understood, as are the effects of nitrogen fertilizers. We studied biomass production and Cd and Zn uptake by two contrasting populations of N. caerulescens, Ganges (metallicolous) and Wilwerwiltz (non-metallicolous) grown in pots on a range of 24 field contaminated soils for 20 weeks. The addition of KNO3 and NH4NO3 fertilizers was also tested. Using model averaging of multiple regression models, we show that the major drivers of N. caerulescens growth are physical soil factors such as organic matter and soil bulk density while trace metal accumulation mainly relies on soil Cd and Zn exchangeable concentrations. We confirm the negative effect of soil copper (Cu) on growth, even at exchangeable concentrations below 30 mg kg-1, and therefore on uptake efficiency, while increasing soil lead (Pb) content was related to increased biomass probably due to a protective effect against soil pathogens. Finally, there is a small positive effect of nitrogen fertilization on biomass production only in soils with low initial nitrogen content (under 25 µg g-1 NO3-), while above this value, the positive impact of initial nitrogen content is offset by lower shoot Cd and Zn concentrations. Our data bring substantial information regarding the physico-chemical properties to ensure N. caerulescens growth: a soil bulk density under 1.05 kg/dm3, organic matter above 7% and pH under 7.5. We show that phytoextraction efficiency is maximal for moderate soil contamination in Cd (2-10 mg kg-1) and Zn (300-1000 mg kg-1).

Metabolomics fingerprint of coffee species determined by untargeted-profiling study using LC-HRMS.

Coffee bean extracts are consumed all over the world as beverage and there is a growing interest in coffee leaf extracts as food supplements. The wild diversity in Coffea (Rubiaceae) genus is large and could offer new opportunities and challenges. In the present work, a metabolomics approach was implemented to examine leaf chemical composition of 9 Coffea species grown in the same environmental conditions. Leaves were analyzed by LC-HRMS and a comprehensive statistical workflow was designed. It served for univariate hypothesis testing and multivariate modeling by PCA and partial PLS-DA on the Workflow4Metabolomics infrastructure. The first two axes of PCA and PLS-DA describes more than 40% of variances with good values of explained variances. This strategy permitted to investigate the metabolomics data and their relation with botanic and genetic informations. Finally, the identification of several key metabolites for the discrimination between species was further characterized.

Phytoremediation of urban soils contaminated with trace metals using Noccaea caerulescens: comparing non-metallicolous populations to the metallicolous 'Ganges' in field trials.

Urban soil contamination with trace metals is a major obstacle to the development of urban agriculture as crops grown in urban gardens are prone to accumulate trace metals up to toxic levels for human consumption. Phytoextraction is considered as a potentially cost-effective alternative to conventional methods such as excavation. Field trials of phytoextraction with Noccaea caerulescens were conducted on urban soils contaminated with Cd, Cu, Pb, and Zn (respectively around 2, 150-200, 400-500, and 400-700 µg g-1 of dry soil). Metallicolous (Ganges population) and non-metallicolous (NMET) populations were compared for biomass production and trace metal uptake. Moreover, we tested the effect of compost and fertilizer addition. Maximal biomass of 5 t ha-1 was obtained with NMET populations on some plots. Compared to Ganges- the high Cd-accumulating ecotype from South of France often used in phytoextraction trials- NMET populations have an advantage for biomass production and for Zn accumulation, with an average Zn uptake of 2.5 times higher. The addition of compost seems detrimental due to metal immobilization in the soil with little or no effect on plant growth. In addition to differences between populations, variations of growth and metal accumulation were mostly explained by soil Cd and Zn concentrations and texture. Our field trials confirm the potential of using N. caerulescens for both Cd and Zn remediation of moderately contaminated soils-with uptake values of up to 200 g Cd ha-1 and 47 kg Zn ha-1-and show the interest of selecting the adequate population according to the targeted metal.

Demographic history of the trace metal hyperaccumulator Noccaea caerulescens (J. Presl and C. Presl) F. K. Mey. in Western Europe.

Noccaea caerulescens (Brassicaceae) is a major pseudometallophyte model for the investigation of the genetics and evolution of metal hyperaccumulation in plants. We studied the population genetics and demographic history of this species to advance the understanding of among-population differences in metal hyperaccumulation and tolerance abilities. Sampling of seven to 30 plants was carried out in 62 sites in Western Europe. Genotyping was carried out using a combination of new chloroplast and nuclear neutral markers. A strong genetic structure was detected, allowing the definition of three genetic subunits. Subunits showed a good geographic coherence. Accordingly, distant metallicolous populations generally belonged to distinct subunits. Approximate Bayesian computation analysis of demographic scenarios among subunits further supported a primary isolation of populations from the southern Massif Central prior to last glacial maximum, whereas northern populations may have derived during postglacial recolonization events. Estimated divergence times among subunits were rather recent in comparison with the species history, but certainly before the establishment of anthropogenic metalliferous sites. Our results suggest that the large-scale genetic structure of N. caerulescens populations pre-existed to the local adaptation to metalliferous sites. The population structure of quantitative variation for metal-related adaptive traits must have established independently in isolated gene pools. However, features of the most divergent genetic unit (e.g. extreme levels of Cd accumulation observed in previous studies) question the putative relationships between adaptive evolution of metal-related traits and subunits isolation. Finally, admixture signals among distant metallicolous populations suggest a putative role of human activities in facilitating long-distance genetic exchanges.

Taxonomy of Atlantic Central African orchids 5. A new species of Angraecum sect. Conchoglossum (Orchidaceae, Angraecinae) from Gabon and Cameroon.

Recent field inventories and taxonomic research in Central Africa have resulted in the discovery of many new orchid species. Five specimens of an apparently new Angraecum species were collected in Gabon and Cameroon. They stand out for their hanging habit and short zig-zag stem. Morphology of leaves and habit is somewhat comparable to Angraecum cultriforme and Angraecum stolzii, two species from East Africa. Flowers of the novelty share the general morphology of Angraecum pyriforme from which the new species is distinguished by being smaller and with a different lip-spur ratio. Here we show that these five specimens represent a new species, described here as Angraecum lanceolatum. The distinguishing traits include thin lanceolate leaves, convolute distally, with a rhombic lip shape. Dichotomous key to four Central African species of sect. Conchoglossum and a table of the diagnostic characters of the seven related Continental African Angraecum taxa are included here. A preliminary assessment of the conservation status of Angraecum lanceolatum is provided, using the IUCN Red List Categories and Criteria.

RésuméDes travaux récents d'inventaires et de taxonomie ont abouti à la découverte de nombreuses espèces nouvelles d'orchidées en Afrique centrale. Cinq spécimens n'appartenant à aucune espèce d'Angraecum connue à ce jour ont été récoltés au Gabon et au Cameroun. Ces spécimens se distinguent par un port pendant et une tige courte disposée en zigzag. Leur morphologie foliaire et leur port sont proches de ceuxd'Angraecum cultriforme et d'Angraecum stolzii, deux espèces d'Afrique de l'Est. Les fleurs de cette nouveauté taxonomique présentent la morphologie générale d'Angraecum piriforme, mais sont cependant plus petites et présentent un rapport différent de la longueur du labelle sur celle de l'éperon. Nous montrons ici que ces cinq échantillons représentent une nouvelle espèce, décrite sous le nom d'Angraecum lanceolatum. Les traits distinctifs de ce nouveau taxonsont ses feuilles minces, lancéolées et tordues à l'apex, ainsi que la forme du labelle en losange. Une clé dichotomique des quatre espèces de la section Conchoglossum présentes en Afrique centrale et une table des caractères diagnostiques des six taxons proches d'Angraecum lanceolatum présents en Afrique continentale sont proposées. Le statut de conservation d'Angraecum lanceolatum est évalué selon la méthodologie de la Liste Rouge de l'UICN.

Cost of adaptation to a metalliferous environment for Thlaspi caerulescens: a field reciprocal transplantation approach.

Field reciprocal transplantations of two metallicolous populations (Mpops) and two nonmetallicolous populations (NMpops) of Thlaspi caerulescens were performed here to determine the pattern of local adaptation and to assess the cost of adaptation of Mpops to a metalliferous environment (Menv). The role of herbivores as an important selective pressure in the nonmetalliferous environment (NMenv) was also examined. Growth, survival, fitness, life cycle and herbivore consumption were monitored for each transplant for 2 yr. Local adaptation of Mpops to their own environment was clearly demonstrated, as Mpops consistently outperformed NMpops in Menv. In NMenv, no advantage of NMpops over Mpops was detected. However, the fitness of Mpops was generally lower in NMenv than in Menv. Herbivore consumption appeared to be a significant selective pressure for Mpops in NMenv. An imbalance of selective forces between Menv and NMenv probably explains the greater local adaptation of Mpops. Therefore, colonization of NMenv by Mpops appears possible. Although Mpops were able to survive and reproduce in NMenv, they nevertheless expressed a cost attributable in part to their higher susceptibility to herbivores.

Development of Issoria lathonia (Lepidoptera: Nymphalidae) on zinc-accumulating and nonaccumulating Viola species (Violaceae).

The larvae of Issoria lathonia L. feed in natural conditions on several Viola spp., among which are the zinc-accumulating Viola calaminaria (Gingins) Lej. and the nonmetal-accumulating Viola tricolor L. To examine how I. lathonia caterpillars cope with the naturally high foliar zinc concentration of V. calaminaria, we compared the growth of caterpillars reared on leaves varying in zinc concentration. Larvae were fed in controlled conditions with V. calaminaria and V. tricolor grown on noncontaminated soil (i.e., two low-Zn diets) and with V. calaminaria grown on zinc-enriched soil (i.e., one high-Zn diet). Larvae had a higher growth rate when fed with noncontaminated V. calaminaria compared to zinc-enriched V. calaminaria, suggesting that zinc slows down larval growth. However, larvae consumed more leaves of zinc-enriched V. calaminaria (+45%; estimated from fecal mass) compared with noncontaminated V. calaminaria, suggesting that zinc accumulation would not be advantageous to plants. Caterpillars reared on high-zinc leaves regulate their internal zinc concentration through excretion of highly metal-concentrated feces. When kinetics of growth on both low-zinc diets were compared, it appeared that larval development was faster on noncontaminated V. calaminaria than on V. tricolor. This suggests that more nutrients or less feeding inhibitors in V. calaminaria account for fastest growth. Developmental rates on V. tricolor and on zinc-enriched V. calaminaria were similar, despite the high leaf zinc concentration of the latter species. Together with the abundance of V. calaminaria on calamine soils, this may explain why the largest populations of I. lathonia develop on V. calaminaria in Belgium.

Do metal-rich plants deter herbivores? A field test of the defence hypothesis.

Some plant species growing on metalliferous soils are able to accumulate heavy metals in their shoots up to very high concentrations, but the selective advantage of this behaviour is still unknown. The most popular hypothesis, that metals protect plants against herbivores, has been tested several times in laboratory conditions, with contradictory results. We carried out the first large-scale test of the defence hypothesis in eight natural populations of the model Zn hyperaccumulator Thlaspi caerulescens J. and C. Presl (Brassicaceae). In two climatic regions (temperate, Belgium-Luxembourg, and Mediterranean, southern France), we worked in metalliferous and in normal, uncontaminated environments, with plants spanning a wide range of Zn concentrations. We also examined the importance of glucosinolates (main secondary metabolites of Brassicaceae) as antiherbivore defences. When exposed to natural herbivore populations, T. caerulescens suffered lower herbivory pressures in metal-enriched soils than in normal soils, both in Belgium-Luxembourg and in southern France. The trapping of gastropods shows an overall lower population density in metalliferous compared to normal environments, which suggests that herbivory pressure from gastropods is lower on metalliferous soils. In addition, foliar concentration of glucosinolates was constitutively lower in all populations from metal-enriched soils, suggesting that these have evolved towards lower investment in organic defences in response to lower herbivory pressure. The Zn concentration of plants had a protective role only for Belgian metallicolous plants when transplanted in normal soils of Luxembourg. These results do not support the hypothesis that Zn plays a key role in the protection of T. caerulescens against enemies. In contrast, glucosinolates appear to be directly involved in the defence of this hyperaccumulator against herbivores.

Reaction norms of life history traits in response to zinc in Thlaspi caerulescens from metalliferous and nonmetalliferous sites.

* We examined phenotypic plasticity of fitness components in response to zinc (Zn) in the Zn hyperaccumulator, Thlaspi caerulescens. * Two populations from Zn-enriched soils (M) and two populations from normal soils (NM) were grown in pots at three Zn concentrations (0, 1000 and 8000 mg kg(-1) Zn), for an entire life cycle. Growth, Zn accumulation and fitness components were assessed. * Based on vegetative growth, M and NM populations had similar Zn tolerance at 1000 mg kg(-1) Zn. However, reproductive output was markedly decreased in NM at 1000 and 8000 mg kg(-1) Zn. In M populations, Zn did not affect fitness. However, low Zn status enhanced reproductive output in year 1 compared with year 2 and decreased survival after the first flowering season. * M populations are able to achieve equal fitness across a broad range of Zn concentrations in soil by different combinations of fecundity and longevity. No cost of higher tolerance was demonstrated in M populations. Reproductive traits appeared to be a more sensitive indicator of tolerance than vegetative growth.