Conservation Genetics

, Volume 9, Issue 3, pp 653–666 | Cite as

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

  • Tomás E. Murray
  • Úna Fitzpatrick
  • Mark J. F. Brown
  • Robert J. Paxton
Research 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.

Keywords

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

Notes

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.

References

  1. Alford DV (1975) Bumblebees. Davis-Poynter, LondonGoogle Scholar
  2. Banaszak J, Rasmont P (1994) Ocurrence and distribution of the subgenus Bombus Latreille sensu stricto in Poland (Hymenoptera, Apoidea). Pol Pismo Ent 63:337–356Google Scholar
  3. Bazin E, Glémin S, Galtier N (2006) Population size does not influence mitochondrial genetic diversity in animals. Science 312:570–572PubMedCrossRefGoogle Scholar
  4. Benton T (2006) Bumblebees. Collins, LondonGoogle Scholar
  5. Bertsch A (1997a) Abgrenzung der Hummel-Arten Bombus cryptarum und B. lucorum mittels männlicher Labialdrüsen-Sekrete und morphologischer Merkmale (Hymenoptera, Apidae). Entomol Gen 22:129–145Google Scholar
  6. 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 2:1–28Google Scholar
  7. 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 54:365–386Google Scholar
  8. 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 52:45–54CrossRefGoogle Scholar
  9. 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 22:148–155PubMedGoogle Scholar
  10. Blaxter ML (2004) The promise of a DNA taxonomy. Philos Trans R Soc Lond B 359:669–679CrossRefGoogle Scholar
  11. Cameron SA, Hines HM, Williams PH (2007) A comprehensive phylogeny of the bumble bees (Bombus). Biol J Linn Soc 91:161–188CrossRefGoogle Scholar
  12. 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 132:481–489CrossRefGoogle Scholar
  13. Caterino MS, Cho S, Sperling FAH (2000) The current state of insect molecular systematics: a thriving Tower of Babel. Annu Rev Entomol 45:1–54PubMedCrossRefGoogle Scholar
  14. Chittka L, Ings TC, Raine NE (2004) Chance and adaptation in the evolution of island bumblebee behaviour. Popul Ecol 46:243–251CrossRefGoogle Scholar
  15. Cognato A (2006) Standard percent DNA sequence difference for insects does not predict species boundaries. J Econ Entomol 99:1037–1045PubMedCrossRefGoogle Scholar
  16. Colla SR, Otterstatter MC, Gegear RJ, Thomson JD (2006) Plight of the bumble bee: pathogen spillover from commercial to wild populations. Biol Conserv 129:461–467CrossRefGoogle Scholar
  17. Crozier RH, Crozier C (1993) The mitochondrial genome of the honeybee Apis mellifera: complete sequence and genome organisation. Genetics 133:97–117PubMedGoogle Scholar
  18. DeSalle R (2006) Species discovery versus species identification in DNA barcoding efforts: response to Rubinoff. Conserv Biol 20:1545–1547PubMedCrossRefGoogle Scholar
  19. DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimitation and DNA barcoding. Philos Trans R Soc 360:1905–1916CrossRefGoogle Scholar
  20. Edwards M, Jenner M (2005) Field guide to the bumblebees of Great Britain and Ireland. Ocelli Ltd., EnglandGoogle Scholar
  21. 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 9:75–83CrossRefGoogle Scholar
  22. 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 6:540–542CrossRefGoogle Scholar
  23. Feltwell J (2006) Bumblebees. Wildlife Matters, Marlham, UKGoogle Scholar
  24. 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
  25. 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 136:185–194CrossRefGoogle Scholar
  26. Frankham R, Ballou JD, Briscoe DA (2002) Introduction to conservation genetics. Cambridge University PressGoogle Scholar
  27. Funk DJ, Omland KE (2003) Species-level paraphyly and polyphyly: frequency, causes and consequences, with insights from animal mitochondrial DNA. Annu Rev Ecol Syst 34:397–423CrossRefGoogle Scholar
  28. Goka K, Okabe K, Yoneda M, Niwa S (2001) Bumblebee commercialization will cause worldwide migration of parasitic mites. Mol Ecol 10:2095–2099PubMedCrossRefGoogle Scholar
  29. Goulson D (2003) Bumblebees: their behaviour and ecology. Oxford University Press, OxfordGoogle Scholar
  30. Goulson D, Hanley ME, Darvill B, Ellis JS, Knight ME (2005) Causes of rarity in bumblebees. Biol Conserv 122:1–8CrossRefGoogle Scholar
  31. Goulson D, Hanley ME, Darvill B, Ellis JS (2006) Biotope associations and the decline of bumblebees (Bombus spp.). J Insect Conserv 10:95–103CrossRefGoogle Scholar
  32. Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003a) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B 270:313–321CrossRefGoogle Scholar
  33. 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–S99CrossRefGoogle Scholar
  34. 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 101:14812–14817PubMedCrossRefGoogle Scholar
  35. Holehouse KA, Hammond RL, Bourke AFG (2003) Non-lethal sampling of DNA from bumble bees for conservation genetics. Insectes Soc 50:277–285CrossRefGoogle Scholar
  36. Ings TC, Schikora J, Chittka L (2005) Bumblebees, humble pollinators or assiduous invaders? A population comparison of foraging performance in Bombus terrestris. Oceologia 144:508–516CrossRefGoogle Scholar
  37. Ings TC, Ward NL, Chittka L (2006) Can commercially imported bumble bees out-compete their native conspecifics? J Appl Ecol 43:940–948CrossRefGoogle Scholar
  38. IBRA (1980) Atlas of the bumblebees of the British Isles. Institute of Terrestrial Ecology and International Bee Research Association, Cambridge, UKGoogle Scholar
  39. IUCN (2001) IUCN red list categories and criteria, version 3.1. IUCN Species Survival Commission, Gland, SwitzerlandGoogle Scholar
  40. 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, SwitzerlandGoogle Scholar
  41. 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
  42. Kearns CA, Inouye DW, Waser NM (1998) Endangered mutualisms: the conservation of plant–pollinator interactions. Annu Rev Ecol Syst 29:83–112CrossRefGoogle Scholar
  43. 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 41:79–88CrossRefGoogle Scholar
  44. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  45. Løken A (1973) Studies on Scandinavian bumble bees (Hymenoptera, Apidae). Norsk Entomol Tidsskrift 20:1–218Google Scholar
  46. Mallet J (1995) A species definition for the modern synthesis. TREE 10:294–299Google Scholar
  47. 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 427–440Google Scholar
  48. Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS Biol 3:2229–2238Google Scholar
  49. Mitsuhata M, Ono M (1996) Hybridization between Japanese and European bumblebees (Bombus spp.). In: Summaries of the 7th international pollination symposium. Lethbridge, CanadaGoogle Scholar
  50. 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 360:1925–1933CrossRefGoogle Scholar
  51. Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358PubMedGoogle Scholar
  52. 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 14:187–194Google Scholar
  53. 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 5:511–519PubMedCrossRefGoogle Scholar
  54. Pedersen BV (2003) European bumblebees (Hymenoptera: Bombini) – phylogenetic relationships inferred from DNA sequences. Insect Syst Evol 33:361–386CrossRefGoogle Scholar
  55. Prŷs-Jones OE, Corbet SA (1991) Bumblebees. Naturalists’ handbook series 6. Richmond Publishing Co., Slough, UKGoogle Scholar
  56. Rasmont P (1984) Les bourdons du genre Bombus Latreille sensu stricto en Europe occidentale et centrale. Spixiana 7:136–160Google Scholar
  57. 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 93:661–682Google Scholar
  58. 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 17:145–160PubMedCrossRefGoogle Scholar
  59. Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  60. Rubinoff D (2006) Utility if mitochondrial DNA barcodes in species conservation. Conserv Biol 20:1026–1033PubMedCrossRefGoogle Scholar
  61. 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 360:1805–1811CrossRefGoogle Scholar
  62. 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 87:651–701Google Scholar
  63. 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 10:512–526PubMedGoogle Scholar
  64. 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 48:44–51CrossRefGoogle Scholar
  65. 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 22:4673–4680PubMedCrossRefGoogle Scholar
  66. 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 37:421–451CrossRefGoogle Scholar
  67. von Hagen E (2003) Hummeln: bestimmen, ansiedeln, vermehren, schützen. Fauna-Verlag, NottulnGoogle Scholar
  68. 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 10:506–513PubMedGoogle Scholar
  69. 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 217–233Google Scholar
  70. Westrich P (1989) Die Wildbienen Baden Württembergs, vol 2. Verlag Eugen Ulmer, StuttgartGoogle Scholar
  71. Williams PH (1986) Environmental change and the distributions of British bumble bees (Bombus Latr.). Bee World 67:50–61Google Scholar
  72. Williams PH (1991) The bumble bees of the Kashmir Himalaya (Hymenoptera: Apidae, Bombini). Bull Nat Hist Mus Lond (Entomol) 60:1–204Google Scholar
  73. 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) 67:79–152Google Scholar
  74. Williams PH (2000) Are Bombus lucorum and magnus separate species? BWARS Newslett 1:15–17Google Scholar
  75. Williams PH (2005) Does specialization explain rarity and decline among British bumblebees? a response to Goulson et al. Biol Conserv 122:33–43CrossRefGoogle Scholar
  76. 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, LondonGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Tomás E. Murray
    • 1
    • 3
  • Úna Fitzpatrick
    • 2
  • Mark J. F. Brown
    • 2
  • Robert J. Paxton
    • 1
  1. 1.School of Biological SciencesQueen’s University BelfastBelfastUK
  2. 2.Department of Biology, School of Natural SciencesUniversity of Dublin Trinity CollegeDublin 2Ireland
  3. 3.Crops Research CentreCarlowIreland

Personalised recommendations