Abstract
Faced with the decline of pollinators, it is relevant to strengthen our understanding of the whole plant-pollinator web in semi-natural grasslands that serve as refuges for pollinator populations. The aim of this study was to explore the diversity of flower-foraging insects involved in pollen transfer in mountain semi-natural grasslands. Insects actively collecting pollen and/or nectar were caught in spring in six mountain semi-natural grasslands displaying a floristic richness gradient. Individual determinations of insects were made at the finest possible taxonomic scale and pollen loads were removed from the insect body. Using next-generation DNA sequencing, pollens were identified through the ribosomal DNA cistron using the ITS2 database and the ITS plant rDNA cistron sequences from Genbank. A total of 236 flower-foraging insects were collected. Diptera represented 82% of the total catches distantly followed by Hymenoptera (15%) and Apoidea (bees) (11%). Visual observations revealed that Diptera foraged on 16 of the 21 flower species visited by insects. DNA metabarcoding showed that 82% (191) of all of the collected insects were carrying pollen and 44% (104) were carrying two genera of plants or more. Our results demonstrate that Diptera are potential key-pollinators in mountain semi-natural grasslands that cannot be overlooked by the scientific community. However difficulties of taxonomic determination due to severe shortage of experts for Diptera have to be urgently overcome. Further studies on the link between pollen transfer and actual pollination in a global change context are also required. Moreover, our results support the idea that DNA metabarcoding provides accurate information about the plants-insects networks but it also pointed out sensitive issues, especially the necessity to build reliable national barcode databases.
Similar content being viewed by others
References
Al-Shehbaz IA, Mummenhoff K (2011) Stubendorffia and Winklera belong to the expanded Lepidium (Brassicaceae). Edinb J Bot 68(02):165–171
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410
Ankenbrand MJ, Keller A, Wolf M, Schultz J, Förster F (2015) ITS2 Database V: twice as much. Mol Biol Evol 32(11):3030–3032
Bailes EJ, Ollerton J, Pattrick JG, Glover BJ (2015) How can an understanding of plant–pollinator interactions contribute to global food security? Curr Opin Plant Biol 26:72–79. doi:10.1016/j.pbi.2015.06.002
Beattie AJ (1972) The pollination ecology of Viola. 2, Pollen loads of insect-visitors. Watsonia 9:13–25
Biesmeijer JC, Roberts SPM, Reemer M, Ohlemuller R, Edwards M, Peeters T, Schaffers AP, Potts SG, Kleukers R, Thomas CD, Settele J, Kunin WE (2006) Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands. Science 313:351–354
Bosch J, González M, Rodrigo MA, Navarro A D (2009) Plant–pollinator networks: adding the pollinator’s perspective. Ecol Lett 12(5):409–419
Breeze TD, Vaissiere BE, Bommarco R, Petanidou T, Seraphides N, Kozak L, Scheper J, Biesmeijer JC, Kleijn D, Gyldenkaerne S, Moretti M, Holzschuh A, Steffan-Dewenter I, Stout JC, Paertel M, Zobel M, Potts SG (2014) Agricultural policies exacerbate honeybee pollination service supply-demand mismatches across Europe. PLoS ONE 9:e82996. doi:10.1371/journal.pone.0082996
Brittain CA, Vighi M, Bommarco R, Settele J, Potts SG (2010) Impacts of a pesticide on pollinator species richness at different spatial scales. Basic Appl Ecol 11:106–115. doi:10.1016/j.baae.2009.11.007
Carré G, Roche P, Chifflet R, Morison N, Bommarco R, Harrison-Cripps J, Krewenka K, Potts SG, Roberts SPM, Rodet G, Settele J, Steffan-Dewenter I, Szentgyörgyi H, Tscheulin T, Westphal C, Woyciechowski M, Vaissière BE (2009) Landscape context and habitat type as drivers of bee diversity in European annual crops. Agric Ecosyst Environ 133:40–47. doi:10.1016/j.agee.2009.05.001
Chen S, Yao H, Han J, Liu C, Song J, Shi L et al (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5(1):e8613
Dormann CF, Gruber B, Fründ J (2008) Introducing the bipartite package: Analysing Ecological Networks. R News 8(2):8–11
Drescher N, Wallace HM, Katouli M, Massaro CF, Leonhardt SD (2014) Diversity matters: how bees benefit from different resin sources. Oecologia 176:943–953
Ebeling A, Klein AM, Schumacher J, Weisser WW, Tscharntke T (2008) How does plant richness affect pollinator richness and temporal stability of flower visits? Oikos 117:1808–1815. doi:10.1111/j.1600-0706.2008.16819.x
Fluidigm Corporation (2015) Access Array system for Illumina sequencing systems user guide. PN 100–3770 H1. Fluidigm Corporation, South San Francisco, p 162
Free JB (1993) Insect pollination of crops. Academic Press, London
Galimberti A, Mattia FD, Bruni I, Scaccabarozzi D, Sandionigi A, Barbuto M et al (2014) A DNA barcoding approach to characterize pollen collected by honeybees. PLoS ONE 9(10):e109363
Gallai N, Salles J-M, Settele J, Vaissière BE (2009) Economic valuation of the vulnerability of world agriculture confronted with pollinator decline. Ecol Econ 68(3):810–821
Garibaldi LA, Steffan-Dewenter I, Winfree R, Aizen MA, Bommarco R, Cunningham SA, Kremen C, Carvalheiro LG, Harder LD, Afik O et al. (2013) Wild pollinators enhance fruit set of crops regardless of honey bee abundance. Science 339, 1608–1611
Grass I, Albrecht J, Jauker F, Diekötter T, Warzecha D, Wolters V, Farwig N (2016) Much more than bees—wildflower plantings support highly diverse flower-visitor communities from complex to structurally simple agricultural landscapes. Agric Ecosyst Environ 225:45–53. doi:10.1016/j.agee.2016.04.001
Harrison PA, Vandewalle M, Sykes MT, Berry PM, Bugter R, Bello F et al (2010) Identifying and prioritising services in European terrestrial and freshwater ecosystems. Biodivers Conserv 19(10):2791–2821
Hawkins J, Vere N de, Griffith A, Ford CR, Allainguillaume J, Hegarty MJ et al (2015) Using DNA metabarcoding to identify the floral composition of honey: A new tool for investigating honey bee foraging preferences. PLoS ONE 10(8):e0134735
Herrera CM (1987) Components of pollinator ‘quality’: Comparative analysis of a diverse insect assemblage. Oikos 50(1):79
Hicks DM, Ouvrard P, Baldock KCR, Baude M, Goddard MA, Kunin WE et al (2016) Food for pollinators: quantifying the nectar and pollen resources of urban flower meadows. PLoS ONE 11(6):e0158117
Hodkinson ID (2005) Terrestrial insects along elevation gradients: species and community responses to altitude. Biol Rev 80(3):489–513
Hoehn P, Steffan-Dewenter I, Tscharntke T (2010) Relative contribution of agroforestry, rainforest and openland to local and regional bee diversity. Biodivers Conserv 19(8):2189–2200
Keller A, Danner N, Grimmer G, Ankenbrand M, Ohe K, Ohe W et al (2015) Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples. Plant Biol 17(2):558–566
Kleijn D, Winfree R, Bartomeus I, Carvalheiro LG, Henry M, Isaacs R et al (2015) Delivery of crop pollination services is an insufficient argument for wild pollinator conservation. Nat Commun 6:7414
Klein A-M, Vaissière BE, Cane JH, Steffan-Dewenter I, Cunningham SA, Kremen C et al (2007) Importance of pollinators in changing landscapes for world crops. Proc R Soc Lond B Biol Sci 274(1608):303–313
Koetschan C, Hackl T, Müller T, Wolf M, Förster F, Schultz J (2012) ITS2 Database IV: Interactive taxon sampling for internal transcribed spacer 2 based phylogenies. Mol Phylogenet Evol 63(3):585–588
Kohler F, Verhulst J, Knop E, Herzog F, Kleijn D (2007) Indirect effects of grassland extensification schemes on pollinators in two contrasting European countries. Biol Conserv 135(2):302–307
Kraaijeveld K, Weger LA, Ventayol García M, Buermans H, Frank J, Hiemstra PS et al (2015) Efficient and sensitive identification and quantification of airborne pollen using next-generation DNA sequencing. Mol Ecol Resour 15(1):8–16
Kremen C, Williams NM, Thorp RW (2002) Crop pollination from native bees at risk from agricultural intensification. Proc Natl Acad Sci 99(26):16812–16816
Larson BMH, Kevan PG, Inouye DW (2001) Flies and flowers: taxonomic diversity of anthophiles and pollinators. Can Entomol 133(4):439–465
Le Féon V, Schermann-Legionnet A, Delettre Y, Aviron S, Billeter R, Bugter R, Hendrickx F, Burel F, (2010) Intensification of agriculture, landscape composition and wild bee communities: a large scale study in four European countries. Agric Ecosyst Environm 137:143–150. doi:10.1016/j.agee.2010.01.015
Lefebvre V, Fontaine C, Villemant C, Daugeron C (2014) Are empidine dance flies major flower visitors in alpine environments? A case study in the Alps, France. Biol Lett 10(11):20140742
Meier R, Shiyang K, Vaidya G, Ng P (2006) DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success. Syst Biol 55(5):715–728
Memmott J, Godfray HCJ (1993) Parasitoid webs. In: LaSalle J, Gauld ID (eds) Wallingford: CAB International
Menchari Y, Camilleri C, Michel S, Brunel D, Dessaint F, Le Corre V et al (2006) Weed response to herbicides: regional-scale distribution of herbicide resistance alleles in the grass weed Alopecurus myosuroides. New Phytol 171(4):861–874
Meyer S, Reeb C, Bosdeveix R (2004) Botanique: biologie et physiologie végétales. Maloine, Paris, p 461
Michener CD (2000) The bees of the world. In: Michener CD (ed) Johns Hopkins University Press, Baltimore
Ollerton J, Winfree R, Tarrant S (2011) How many flowering plants are pollinated by animals? Oikos 120(3):321–326
Oosterbroek P (2007) The European families of the Diptera—Identification, diagnosis, biology. KNNV Publishing, Boulevard, p 208
Orford KA, Vaughan IP, Memmott J (2015) The forgotten flies: the importance of non-syrphid Diptera as pollinators. Proc R Soc Lond B Biol Sci 282(1805):20142934
Pesson P, Louveaux J (1984) Pollinisation et productions vegetales. Institut National de la Recherche Agronomique, Paris
Pornon A, Escaravage E, Burrus M, Holota H, Khimoun A, Mariette J, Pellizzari C, Iribar A, Etienne R, Taberlet P, Vidal M, Winterton P, Zinger L, Andalo C (2016) Using metabarcoding to reveal and quantify plant-pollinator interactions. Sci Rep 6:27282. doi: 10.1038/srep27282
Praz CJ, Mueller A, Dorn S (2008) Specialized bees fail to develop on non-host pollen: Do plants chemically protect their pollen? Ecology 89:795–804. doi:10.1890/07-0751.1
Rader R, Howlett BG, Cunningham SA, Westcott DA, Newstrom-Lloyd LE, Walker MK et al (2009) Alternative pollinator taxa are equally efficient but not as effective as the honeybee in a mass flowering crop. J Appl Ecol 46(5):1080–1087
Rader R, Bartomeus I, Garibaldi LA, Garratt MPD, Howlett BG, Winfree R, Cunningham SA, Mayfield MM, Arthur AD, Andersson GKS, Bommarco R, Brittain C, Carvalheiro LG, Chacoff NP, Entling MH, Foully B, Freitas BM, Gemmill-Herren B, Ghazoul J, Griffin SR, Gross CL, Herbertsson L, Herzog F, Hipólito J, Jaggar S, Jauker F, Klein AM, Kleijn D, Krishnan S, Lemos CQ, Lindström SAM, Mandelik Y, Monteiro VM, Nelson W, Nilsson L, Pattemore DE, Pereira N, de O, Pisanty, Potts G, Reemer SG, Rundlöf M, Sheffield M, Scheper CS, Schüepp J, Smith C, Stanley HG, Stout DA, Szentgyörgyi JC, Taki H, Vergara H, Viana CH, Woyciechowski BF M (2016) Non-bee insects are important contributors to global crop pollination. PNAS 113:146–151. doi:10.1073/pnas.1517092112
Richardson RT, Lin C-H, Sponsler DB, Quijia JO, Goodell K, Johnson RM (2015a) Application of ITS2 metabarcoding to determine the provenance of pollen collected by honey bees in an agroecosystem. Appl Plant Sci 3(1):1400066
Richardson RT, Lin C-H, Quijia JO, Riusech NS, Goodell K, Johnson RM (2015b) Rank-based characterization of pollen assemblages collected by honey bees using a multi-locus metabarcoding approach. Appl Plant Sci 3(11):1500043
Sanders J (2014) Secrets of wildflowers: a delightful feast of little-known facts, folklore, and history. Rowman & Littlefield, Lanham, p 321
Scheper J, Holzschuh A, Kuussaari M, Potts SG, Rundlöf M, Smith HG et al (2013) Environmental factors driving the effectiveness of European agri-environmental measures in mitigating pollinator loss—a meta-analysis. Ecol Lett 16(7):912–920
Sickel W, Ankenbrand MJ, Grimmer G, Holzschuh A, Härtel S, Lanzen J et al (2015) Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach. BMC Ecol 15(1):1
Skevington JH, Dang PT (2002) Exploring the diversity of flies (Diptera). Biodiversity 3(4):3–27
Steffan-Dewenter I, Tscharntke T (2001) Succession of bee communities on fallows. Ecography 24:83–93. doi:10.1034/j.1600-0587.2001.240110.x
Stork NE, McBroom J, Gely C, Hamilton AJ (2015) New approaches narrow global species estimates for beetles, insects, and terrestrial arthropods. PNAS 112(24):7519–7523
Tscharntke T, Klein AM, Kruess A, Steffan-Dewenter I, Thies C (2005) Landscape perspectives on agricultural intensification and biodiversity—ecosystem service management. Ecol Lett 8(8):857–874
Valentini A, Miquel C, Taberlet P (2010) DNA barcoding for honey biodiversity. Diversity 2(4):610–617
Weiner CN, Werner M, Linsenmair KE, Bluethgen N (2011) Land use intensity in grasslands: changes in biodiversity, species composition and specialisation in flower visitor networks. Basic Appl Ecol 12:292–299. doi:10.1016/j.baae.2010.08.006
Weiner CN, Werner M, Linsenmair KE, Blüthgen N (2014) Land-use impacts on plant–pollinator networks: interaction strength and specialization predict pollinator declines. Ecology 95:466–474. doi:10.1890/13-0436.1
Westphal C, Bommarco R, Carre G, Lamborn E, Morison N, Petanidou T et al (2008) Measuring bee diversity in different European habitats and biogeographical regions. Ecol Monogr 78(4):653–671
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to method and amplifications. Academic Press, San Diego, pp 315–322
Williams NM, Kremen C (2007) Resource distributions among habitats determine solitary bee offspring production in a mosaic landscape. Ecol Appl 17(3):910–921
Willmer P (2011) Pollination and floral ecology. Princeton University Press, Princeton
Wilson E, Sidhu C, Levan K, Holway D (2010) Pollen foraging behaviour of solitary Hawaiian bees revealed through molecular pollen analysis. Mol Ecol 19:4823–4829. doi: 10.1111/j.1365-294X.2010.04849.x
Woodcock TS, Larson BMH, Kevan PG, Inouye DW, Lunau K (2014) Flies and flowers II: floral attractants and rewards. J Pollinat Ecol 12(8):63–94
Acknowledgements
The authors thank David Genoud for insect identifications and ecological expertise, Xavier Lair for Syrphidae identifications, Romain Pradinas from National Botanical Conservatory of Massif Central for help on plant identifications and Michel Frain for help on the botanical surveys. The authors also thank Olivier Troquier from INRA-Herbipôle for valuable help with fieldwork logistics and Yoan Gaudron from INRA-UMRH for the Perl scripts.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Galliot, JN., Brunel, D., Bérard, A. et al. Investigating a flower-insect forager network in a mountain grassland community using pollen DNA barcoding. J Insect Conserv 21, 827–837 (2017). https://doi.org/10.1007/s10841-017-0022-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10841-017-0022-z