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Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs

Conservation Genetics volume 9, pages 653–666 (2008)Cite this article

Abstract

Cryptic species diversity is thought to be common within the class Insecta, posing problems for basic ecological and population genetic studies and conservation management. Within the temperate bumble bee (Bombus spp.) fauna, members of the subgenus Bombus sensu stricto are amongst the most abundant and widespread. However, their species diversity is controversial due to the extreme difficulty or inability morphologically to identify the majority of individuals to species. Our character-based phylogenetic analyses of partial CO1 (700 bp) from 39 individuals spread across their sympatric European ranges provided unequivocal support for five taxa (3–22 diagnostic DNA base pair sites per species). Inclusion of 20 Irish specimens to the dataset revealed ≥2.3% sequence divergence between taxa and ≤1.3% within taxa. We developed a PCR-RFLP based method for unequivocally distinguishing amongst the four cryptic European taxa of this subgenus, B. cryptarum, B. lucorum, B. magnus and B. terrestris, and used it to analyse 391 females of the former three species collected across Ireland, all of which could be unambiguously assigned to species. Bombus lucorum was the most widely distributed and abundant of the cryptarum–lucorum–magnus species complex, comprising 56% of individuals, though it was significantly less abundant at higher altitudes (>200 m) whilst B. cryptarum was relatively more abundant at higher altitudes. Bombus magnus was rarely encountered at urban sites. Both B. lucorum and B. terrestris are nowadays reared commercially for pollination and transported globally. Our RFLP approach to identify native fauna can underpin ecological studies of these important cryptic species as well as the impact of commercial bumble bees on them.

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References

  • Alford DV (1975) Bumblebees. Davis-Poynter, London

    Google Scholar 

  • Banaszak J, Rasmont P (1994) Ocurrence and distribution of the subgenus Bombus Latreille sensu stricto in Poland (Hymenoptera, Apoidea). Pol Pismo Ent 63:337–356

    Google Scholar 

  • Bazin E, Glémin S, Galtier N (2006) Population size does not influence mitochondrial genetic diversity in animals. Science 312:570–572

    PubMed  Article  CAS  Google Scholar 

  • Benton T (2006) Bumblebees. Collins, London

    Google Scholar 

  • Bertsch A (1997a) Abgrenzung der Hummel-Arten Bombus cryptarum und B. lucorum mittels männlicher Labialdrüsen-Sekrete und morphologischer Merkmale (Hymenoptera, Apidae). Entomol Gen 22:129–145

    Google Scholar 

  • Bertsch A (1997b) Wieviele Arten der Untergattung Terrestribombus (Hymenoptera, Apidae) gibt es in Nordhessen; die Abgrenzung von Bombus cryptarum und B. lucorum mittels männlicher Labial-drüsen-Sekrete und morphologischer Merkmale. Marburger Ent Pub 2:1–28

  • Bertsch A, Schweer H, Titze A (2004) Discrimination of the bumblebee species Bombus lucorum, B. crytarum and B. magnus by morphological characters and male labial gland secretions. Beitr Ent 54:365–386

    Google Scholar 

  • Bertsch A, Schweer H, Titze A, Tanaka H (2005) Male labial gland secretions and mitochondrial DNA markers support species status of Bombus cryptarum and B. magnus (Hymenoptera, Apidae). Insectes Soc 52:45–54

    Article  Google Scholar 

  • Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K, Ingram KK, Das I (2007) Cryptic species as a window on diversity and conservation. TREE 22:148–155

    PubMed  Google Scholar 

  • Blaxter ML (2004) The promise of a DNA taxonomy. Philos Trans R Soc Lond B 359:669–679

    Article  CAS  Google Scholar 

  • Cameron SA, Hines HM, Williams PH (2007) A comprehensive phylogeny of the bumble bees (Bombus). Biol J Linn Soc 91:161–188

    Article  Google Scholar 

  • Carvell C, Roy DB, Smart SM, Pywell RF, Preston CD, Goulson D (2006) Declines in forage availability for bumblebees at a national scale. Biol Conserv 132:481–489

    Article  Google Scholar 

  • Caterino MS, Cho S, Sperling FAH (2000) The current state of insect molecular systematics: a thriving Tower of Babel. Annu Rev Entomol 45:1–54

    PubMed  Article  CAS  Google Scholar 

  • Chittka L, Ings TC, Raine NE (2004) Chance and adaptation in the evolution of island bumblebee behaviour. Popul Ecol 46:243–251

    Article  Google Scholar 

  • Cognato A (2006) Standard percent DNA sequence difference for insects does not predict species boundaries. J Econ Entomol 99:1037–1045

    PubMed  CAS  Article  Google Scholar 

  • Colla SR, Otterstatter MC, Gegear RJ, Thomson JD (2006) Plight of the bumble bee: pathogen spillover from commercial to wild populations. Biol Conserv 129:461–467

    Article  Google Scholar 

  • Crozier RH, Crozier C (1993) The mitochondrial genome of the honeybee Apis mellifera: complete sequence and genome organisation. Genetics 133:97–117

    PubMed  CAS  Google Scholar 

  • DeSalle R (2006) Species discovery versus species identification in DNA barcoding efforts: response to Rubinoff. Conserv Biol 20:1545–1547

    PubMed  Article  Google Scholar 

  • DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimitation and DNA barcoding. Philos Trans R Soc 360:1905–1916

    Article  CAS  Google Scholar 

  • Edwards M, Jenner M (2005) Field guide to the bumblebees of Great Britain and Ireland. Ocelli Ltd., England

    Google Scholar 

  • Ellis JS, Knight ME, Goulson D (2005) Delineating species for conservation using mitochondrial sequence data: the taxonomic status of two problematic Bombus species (Hymenoptera: Apidae). J Insect Conserv 9:75–83

    Article  Google Scholar 

  • Ellis JS, Knight TM, Carvell C, Goulson D (2006) Cryptic species identification: a simple diagnostic tool for discrimination between two problematic bumblebee species. Mol Ecol Notes 6:540–542

    Article  CAS  Google Scholar 

  • Feltwell J (2006) Bumblebees. Wildlife Matters, Marlham, UK

    Google Scholar 

  • Fitzpatrick Ú, Murray TE, Byrne A, Paxton RJ, Brown MJF (2006) Regional red data list of Irish bees. In: National parks and wildlife service (Ireland) and environment and heritage service (N. Ireland). Available from: http://www.npws.ie/PublicationsLiterature/RegionalRedLists/file,3658,en.pdf

  • Fitzpatrick Ú, Murray TE, Paxton RJ, Breen J, Cotton D, Santorum V, Brown MJF (2007) Rarity and decline of bumble bees – a test of causes and correlates. Biol Conserv 136:185–194

    Article  Google Scholar 

  • Frankham R, Ballou JD, Briscoe DA (2002) Introduction to conservation genetics. Cambridge University Press

  • Funk DJ, Omland KE (2003) Species-level paraphyly and polyphyly: frequency, causes and consequences, with insights from animal mitochondrial DNA. Annu Rev Ecol Syst 34:397–423

    Article  Google Scholar 

  • Goka K, Okabe K, Yoneda M, Niwa S (2001) Bumblebee commercialization will cause worldwide migration of parasitic mites. Mol Ecol 10:2095–2099

    PubMed  Article  CAS  Google Scholar 

  • Goulson D (2003) Bumblebees: their behaviour and ecology. Oxford University Press, Oxford

    Google Scholar 

  • Goulson D, Hanley ME, Darvill B, Ellis JS, Knight ME (2005) Causes of rarity in bumblebees. Biol Conserv 122:1–8

    Article  Google Scholar 

  • Goulson D, Hanley ME, Darvill B, Ellis JS (2006) Biotope associations and the decline of bumblebees (Bombus spp.). J Insect Conserv 10:95–103

    Article  Google Scholar 

  • Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003a) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B 270:313–321

    Article  CAS  Google Scholar 

  • Hebert PDN, Ratnasingham S, deWaard JR (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond Ser B 270:S96–S99

    Article  CAS  Google Scholar 

  • Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA 101:14812–14817

    PubMed  Article  CAS  Google Scholar 

  • Holehouse KA, Hammond RL, Bourke AFG (2003) Non-lethal sampling of DNA from bumble bees for conservation genetics. Insectes Soc 50:277–285

    Article  Google Scholar 

  • Ings TC, Schikora J, Chittka L (2005) Bumblebees, humble pollinators or assiduous invaders? A population comparison of foraging performance in Bombus terrestris. Oceologia 144:508–516

    Article  Google Scholar 

  • Ings TC, Ward NL, Chittka L (2006) Can commercially imported bumble bees out-compete their native conspecifics? J Appl Ecol 43:940–948

    Article  Google Scholar 

  • IBRA (1980) Atlas of the bumblebees of the British Isles. Institute of Terrestrial Ecology and International Bee Research Association, Cambridge, UK

    Google Scholar 

  • IUCN (2001) IUCN red list categories and criteria, version 3.1. IUCN Species Survival Commission, Gland, Switzerland

    Google Scholar 

  • IUCN (World Conservation Union) (2003) Guidelines for application of IUCN red list criteria at regional levels: version 3.0. IUCN Species Survival Commission. IUCN, Gland, Switzerland

    Google Scholar 

  • IUCN (World Conservation Union) (2006) Guidelines for using the IUCN red list categories and criteria, version 6.1. IUCN Species Survival Commission. IUCN, Gland, Switzerland. Available from http://www.intranet.iucn.org/webfiles/doc/SSC/RedList/RedListGuidelines.pdf

  • Kearns CA, Inouye DW, Waser NM (1998) Endangered mutualisms: the conservation of plant–pollinator interactions. Annu Rev Ecol Syst 29:83–112

    Article  Google Scholar 

  • Kosior A, Celary W, Olejniczak P, Fijal J, Król W, Solarz W, Plonka P (2007) The decline of the bumble bees and cuckoo bees (Hymenoptera: Apidae: Bombini) of Western and Central Europe. Oryx 41:79–88

    Article  Google Scholar 

  • Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

    PubMed  Article  CAS  Google Scholar 

  • Løken A (1973) Studies on Scandinavian bumble bees (Hymenoptera, Apidae). Norsk Entomol Tidsskrift 20:1–218

    Google Scholar 

  • Mallet J (1995) A species definition for the modern synthesis. TREE 10:294–299

    Google Scholar 

  • Mallet J (2007) Species, concepts of. In: Levin SA et al (eds) Encyclopedia of biodiversity, vol 5, 2nd edn. Academic Press, San Diego, CA, pp 427–440

  • Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS Biol 3:2229–2238

    CAS  Google Scholar 

  • Mitsuhata M, Ono M (1996) Hybridization between Japanese and European bumblebees (Bombus spp.). In: Summaries of the 7th international pollination symposium. Lethbridge, Canada

  • Monaghan MT, Balke M, Gregory TR, Vogler AP (2005) DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers. Philos Trans R Soc Lond B 360:1925–1933

    Article  CAS  Google Scholar 

  • Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358

    PubMed  CAS  Google Scholar 

  • Pamilo P, Tengö J, Rasmont P, Pirhonen K, Pekkarinen A, Kaarnama E (1997) Pheromonal and enzyme genetic characteristics of the Bombus lucorum species complex in northern Europe. Entomol Fenn 14:187–194

    Google Scholar 

  • Paxton RJ, Thorén PA, Tengo J, Pamilo P (1996) Mating structure and nestmate relatedness in a communal bee, Andrena jacobi (Hymenoptera, Andrenidae), using microsatellites. Mol Ecol 5:511–519

    PubMed  Article  CAS  Google Scholar 

  • Pedersen BV (2003) European bumblebees (Hymenoptera: Bombini) – phylogenetic relationships inferred from DNA sequences. Insect Syst Evol 33:361–386

    Article  Google Scholar 

  • Prŷs-Jones OE, Corbet SA (1991) Bumblebees. Naturalists’ handbook series 6. Richmond Publishing Co., Slough, UK

    Google Scholar 

  • Rasmont P (1984) Les bourdons du genre Bombus Latreille sensu stricto en Europe occidentale et centrale. Spixiana 7:136–160

    Google Scholar 

  • Rasmont P, Scholl A, de Jonghe R, Obrecht E, Adamski A (1986) Identité et variabilité des mâles de bourdons du genre Bombus Latreille sensu stricto en Europe occidentale et centrale (Hymenoptera, Apidae, Bombinae). Rev Suisse Zool 93:661–682

    Google Scholar 

  • Riddle BR, Hafner DJ, Alexander LF (2000) Phylogeny and systematics of Peromyscus eremicus species group and the historical biogeography of North American warm regional deserts. Mol Phylogenet Evol 17:145–160

    PubMed  Article  CAS  Google Scholar 

  • Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574

    PubMed  Article  CAS  Google Scholar 

  • Rubinoff D (2006) Utility if mitochondrial DNA barcodes in species conservation. Conserv Biol 20:1026–1033

    PubMed  Article  Google Scholar 

  • Savolainen V, Cowan RS, Vogler AP, Roderick GK, Lane R (2005) Towards writing the encyclopedia of life: an introduction to DNA barcoding. Philos Trans R Soc B 360:1805–1811

    Article  CAS  Google Scholar 

  • Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene-sequences and a compilation of conserved polymerase chain-reaction primers. Ann Entomol Soc Am 87:651–701

    CAS  Google Scholar 

  • Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzes. Mol Biol Evol 10:512–526

    PubMed  CAS  Google Scholar 

  • Tanaka H, Roubik DW, Kato M, Liew F, Gunsalam G (2001) Phylogenetic position of Apis nuluensis of northern Borneo and phylogeography of A. cerana as inferred from mitochondrial DNA sequences. Insectes Soc 48:44–51

    Article  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    PubMed  Article  CAS  Google Scholar 

  • Velthuis HHW, van Doorn A (2006) A century of advances in bumblebee domestication and the economic and environmental aspects of its commercialization for pollination. Apidologie 37:421–451

    Article  Google Scholar 

  • von Hagen E (2003) Hummeln: bestimmen, ansiedeln, vermehren, schützen. Fauna-Verlag, Nottuln

    Google Scholar 

  • Walsh PS, Metzger DA, Higuchi R (1991) Chelex-100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10:506–513

    PubMed  CAS  Google Scholar 

  • Wells MM, Henry CS (1998) Songs, reproductive isolation, and speciation in cryptic species of insect: a case study using green lacewings. In: Howard DJ, Berlocher SH (eds) Endless forms: species and speciation. Oxford University Press, Oxford, pp 217–233

    Google Scholar 

  • Westrich P (1989) Die Wildbienen Baden Württembergs, vol 2. Verlag Eugen Ulmer, Stuttgart

    Google Scholar 

  • Williams PH (1986) Environmental change and the distributions of British bumble bees (Bombus Latr.). Bee World 67:50–61

    Google Scholar 

  • Williams PH (1991) The bumble bees of the Kashmir Himalaya (Hymenoptera: Apidae, Bombini). Bull Nat Hist Mus Lond (Entomol) 60:1–204

    Google Scholar 

  • Williams PH (1998) An annotated checklist of bumble bees with an analysis of patterns of description (Hymenoptera: Apidae, Bombini). Bull Nat Hist Mus Lond (Entomol) 67:79–152

    Google Scholar 

  • Williams PH (2000) Are Bombus lucorum and magnus separate species? BWARS Newslett 1:15–17

    Google Scholar 

  • Williams PH (2005) Does specialization explain rarity and decline among British bumblebees? a response to Goulson et al. Biol Conserv 122:33–43

    Article  Google Scholar 

  • Williams PH, Hernandez LM (2000) Distinguishing females of the bumble bees Bombus ruderatus (F.) from Bombus hortorum (L.) in Britain: a preliminary application of quantitative techniques. In: Report to the UK biodiversity action plan bumblebee working group. The Natural History Museum, London

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Acknowledgements

We thank friends and colleagues who helped to collect bumble bees across Ireland: D. Cookson, D. Dominoni, M. Kelly and S. Roos; and Andreas Bertsch for provision of additional samples, discussion and encouragement to engage with the lucorum complex. We also thank Andreas Bertsch, Jim Provan, Alfried Vogler, Paul Williams and an anonymous reviewer for many helpful comments on the manuscript. This work was supported by a grant from the Higher Education Authority of Ireland as part of its North-South Research Programme for Peace and Reconciliation.

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Authors and Affiliations

  1. School of Biological Sciences, Queen’s University Belfast, 97 Lisburn Rd., Belfast, BT9 7BL, UK

    Tomás E. Murray & Robert J. Paxton

  2. Department of Biology, School of Natural Sciences, University of Dublin Trinity College, Dublin 2, Ireland

    Úna Fitzpatrick & Mark J. F. Brown

  3. Crops Research Centre, Teagasc, Oak Park, Carlow, Ireland

    Tomás E. Murray

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  1. Tomás E. Murray
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Correspondence to Tomás E. Murray.

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Murray, T.E., Fitzpatrick, Ú., Brown, M.J.F. et al. Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs. Conserv Genet 9, 653–666 (2008). https://doi.org/10.1007/s10592-007-9394-z

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Keywords

  • Bombus
  • Cytochrome c oxidase subunit 1
  • CO1
  • PCR-RFLP
  • DNA barcode