Conservation Genetics

, Volume 18, Issue 3, pp 573–584 | Cite as

Microsatellite analysis supports the existence of three cryptic species within the bumble bee Bombus lucorum sensu lato

  • Lorraine McKendrick
  • Jim Provan
  • Úna Fitzpatrick
  • Mark J. F. Brown
  • Tómas E. Murray
  • Eckart Stolle
  • Robert J. Paxton
Research Article


Mitochondrial cytochrome oxidase I (COI) partial sequences are widely used in taxonomy for species identification. Increasingly, these sequence identities are combined with modelling approaches to delineate species. Yet the validity of species delineation based on such DNA ‘barcodes’ is rarely tested and may be called into question by phenomena such as ancestral polymorphisms in DNA sequences, phylogeographic divergence, mitochondrial introgression and hybridization, or distortion of mitochondrial inheritance through such factors as Wolbachia infection. The common and widespread European bumble bee Bombus lucorum s. lato contains three distinct mitochondrial DNA lineages that are assumed to represent three cryptic species, namely Bombus cryptarum, B. lucorum s. str. and B. magnus. To test whether nuclear gene pools of the three putative species were differentiated, we genotyped 304 sympatric members of the lucorum complex (54 B. cryptarum females, 168 B. lucorum s. str. females and 82 B. magnus females, as defined using mtDNA COI haplotypes) from 11 localities spread across the island of Ireland at seven nuclear microsatellite loci. Multilocus genotypes clustered into three discrete groups that largely corresponded to the three mtDNA lineages: B. cryptarum, B. lucorum s. str. and B. magnus. The good fit of mitochondrial haplotype to nuclear (microsatellite) genotypic data supports the view that these three bumble bee taxa are reproductively isolated species, as well as providing a vindication of species identity using so-called DNA barcodes.


DNA barcode Cryptarum Magnus Mitochondrial cytochrome oxidase I Structure software PCoA DAPC Sympatry 



We thank friends and colleagues who helped to collect bumble bees across Ireland: D. Cookson, D. Dominoni, M. Kelly and S. Roos; Andreas Bertsch for use of his photographs, comments on this manuscript and encouragement to engage with the lucorum complex; and Robin Moritz for laboratory and intellectual support. We also thank two anonymous reviewers and editor-in-chief as well as Shalene Jha and Christophe Praz for many insightful comments that helped improve 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. L McKendrick thanks DARD for their financial support (a PhD stipend) and patience.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10592_2017_965_MOESM1_ESM.pdf (949 kb)
Supplementary material 1 (PDF 949 KB)


  1. Ayasse M, Paxton RJ, Tengö J (2001) Mating behavior and chemical communication in the order Hymenoptera. Ann Rev Entomol 46:31–78CrossRefGoogle Scholar
  2. Bergström G, Svensson BG, Appelgren M, Groth I (1981) Complexity of bumble bee marking pheromones: biochemical, ecological and systematic interpretations. In: Hows PE, Clemet J-L (eds) Biosystematics of social insects, vol 19. Academic Press, London, pp 175–183Google Scholar
  3. Berkov A (2002) The impact of redefined species limits in Palame (Coleoptera: Cerambycidae: Lamiinae: Acanthocinini) on assessments of host, seasonal, and stratum specificity. Biol J Linn Soc 76:195–209CrossRefGoogle Scholar
  4. Bertsch A (1997a) Abgrenzung der Hummel-Arten Bombus cryptarum und B. lucorum mittels mänlicher Labialdrüsen-Sekrete und morphologischer Merkmale (Hymenoptera, Apidae). Entomologia Generalis 22:129–145.Google Scholar
  5. 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 Entomologische Publikationen 2:1–28.Google Scholar
  6. Bertsch A, Schweer H, Titze A (2004) Discrimination of the bumblebee species Bombus lucorum, B. cryptarum and B. magnus by morphological characters and male labial gland secretions. Beiträge zur Entomol 54:365–386.Google Scholar
  7. 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.CrossRefGoogle Scholar
  8. Bossert S (2015) Recognition and identification of bumblebee species in the Bombus lucorum-complex (Hymenoptera, Apidae). A review and outlook. Dtsch Entomol Z 62:19–28CrossRefGoogle Scholar
  9. Cameron SA, Lozier JD, Strange JP, Koch JB, Cordes N, Solter LF, Griswold TL (2011) Patterns of widespread decline in North American bumble bees. Proc Natl Acad Sci USA 108:662–667CrossRefPubMedPubMedCentralGoogle Scholar
  10. Carolan JC, Murray TE, Fitzpatrick Ú, Crossley J, Schmidt H, Cederberg B, McNally L, Paxton RJ, Williams PH, Brown MJF (2012) Colour patterns do not diagnose species: quantitative evaluation of a DNA barcoded cryptic bumblebee complex. PLoS ONE 7:e29251CrossRefPubMedPubMedCentralGoogle Scholar
  11. Carstens BC, Pelletier TA, Reid NM, Satler JD (2013) How to fail at species delimitation. Mol Ecol 22:4369–4383CrossRefPubMedGoogle Scholar
  12. Coissac E, Hollingsworth PM, Lavergne S, Taberlet P (2016) From barcodes to genomes: extending the concept of DNA barcoding. Mol Ecol 25:1423–1428CrossRefPubMedGoogle Scholar
  13. Crandall KA, Binindaemonds ORP, Mace GM, Wayne RK (2000) Considering evolutionary processes in conservation biology. Trends Ecol Evol 15:290–295CrossRefPubMedGoogle Scholar
  14. Dieringer D, Schlötterer C (2003) MICROSATELLITE ANALYSER (MSA): a platform independent analysis tool for large microsatellite data sets. Mol Ecol Notes 3:167–169CrossRefGoogle Scholar
  15. Eltz T, Fritzsch F, Pech JR, Zimmermann Y, Ramírez SR, Quezada-Euan JJG, Bembé B (2011) Characterization of the orchid bee Euglossa viridissima (Apidae: Euglossini) and a novel cryptic sibling species, by morphological, chemical, and genetic characters. Zool J Linn Soc 163:1064–1076.CrossRefGoogle Scholar
  16. Fitzpatrick Ú, Murray TE, Byrne A, Paxton RJ, Brown MJF (2006) Regional red list of Irish bees. National Parks and Wildlife Service (Republic of Ireland) and Environment and Heritage Service (Northern Ireland), Dublin, pp 1–38Google Scholar
  17. Fitzpatrick Ú, Murray TE, Paxton RJ, Breen J, Cotton D, Santorum V, Brown MJF (2007) Rarity and decline in bumblebees-a test of causes and correlates in the Irish fauna. Biol Conserv 136:185–194CrossRefGoogle Scholar
  18. Gerth M, Geißler A, Bleidorn C (2011) Wolbachia infections in bees (Anthophila) and possible implications for DNA barcoding. Syst Biodiv 9:319–327CrossRefGoogle Scholar
  19. Gerth M, Röthe J, Bleidorn C (2013a) Tracing horizontal Wolbachia movements among bees (Anthophila): a combined approach using multilocus sequence typing data and host phylogeny. Mol Ecol 22:6149–6162CrossRefPubMedGoogle Scholar
  20. Gerth M, Bleidorn C (2013b) A multilocus sequence typing (MLST) approach to diminish the problems that are associated with DNA barcoding: A reply to Stahlhut et al. (2012). Syst Biodiv 11:1–3CrossRefGoogle Scholar
  21. Gerth M, Gansauge M-T, Weigert A, Bleidorn C (2014) Phylogenomic analyses uncover origin and spread of the Wolbachia pandemic. Nat Commun 5:5117CrossRefPubMedGoogle Scholar
  22. Goulson D (2003) Effects of introduced bees on native ecosystems. Ann Rev Ecol Evol Syst 34:1–26CrossRefGoogle Scholar
  23. Goulson D (2009) Bumblebees. Behaviour, ecology and conservation. 2nd edn. Oxford University Press, OxfordGoogle Scholar
  24. Goulson D, Hanley ME, Darvill B, Ellis JS, Knight ME (2005) Causes of rarity in bumblebees. Biol Conserv 122:1–8CrossRefGoogle Scholar
  25. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–321CrossRefGoogle Scholar
  26. 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 101:14812–14817CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hines HM (2008) Historical biogeography, divergence times, and diversification patterns of bumble bees (Hymenoptera: Apidae: Bombus). Syst Biol 57:58–75CrossRefPubMedGoogle Scholar
  28. Inoue MN, Yokoyama J, Washitani I (2008) Displacement of Japanese native bumblebees by the recently introduced Bombus terrestris (L.) (Hymenoptera: Apidae). J Insect Cons 12:135–146CrossRefGoogle Scholar
  29. Jensen JL, Bohonak AJ, Kelley ST (2005) Isolation by distance, web service. BMC Genet 6:13CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jombart T (2008) adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24:1403–1405CrossRefPubMedGoogle Scholar
  31. Jombart T, Devillard S, Balloux F (2010) Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Genet 11:94CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kanbe Y, Okada I, Yoneda M, Goka K, Tsuchida K (2008) Interspecific mating of the introduced bumblebee Bombus terrestris and the native Japanese bumblebee Bombus hypocrita sapporoensis results in inviable hybrids. Naturwiss 95:1003–1008CrossRefPubMedGoogle Scholar
  33. Kuhlmann M, Else GR, Dawson A, Quicke DLJ (2007) Molecular, biogeographical and phenological evidence for the existence of three western European sibling species in the Colletes succinctus group (Hymenoptera: Apidae). Organisms Divers Evol 7:155–165CrossRefGoogle Scholar
  34. Lozier JD, Zayed A (2016) Bee conservation in the age of genomics. Conserv Genet. doi: 10.1007/s10592-016-0893-7 Google Scholar
  35. Magnacca KN, Brown MJF (2010a) Mitochondrial heteroplasmy and DNA barcoding in Hawaiian Hylaeus (Nesoprosopis) bees (Hymenoptera: Colletidae). BMC Evol Biol 10:174CrossRefPubMedPubMedCentralGoogle Scholar
  36. Magnacca KN, Brown MJF (2010b) Tissue segregation of mitochondrial haplotypes in heteroplasmic Hawaiian bees: implications for DNA barcoding. Mol Ecol Res 10:60–68CrossRefGoogle Scholar
  37. Magnacca KN, Brown MJF (2012) DNA barcoding a regional fauna: Irish solitary bees. Mol. Ecol Res 12:990–998CrossRefGoogle Scholar
  38. Mallet J (1995) A species definition for the Modern Synthesis. Trends Ecol Evol 10:294–299CrossRefPubMedGoogle Scholar
  39. Mallet J (2007) Species, concepts of. In: Levin SA et al, (eds) Encyclopedia of biodiversity, 2nd edn. Academic Press, San Diego, pp 427–440Google Scholar
  40. Meeus I, Brown MJF, De Graaf DC, Smagghe G (2011) Effects of invasive parasites on bumble bee declines. Conserv Biol 25:662–671CrossRefPubMedGoogle Scholar
  41. Murray TE, Fitzpatrick Ú, Brown MJF, Paxton RJ (2008) Cryptic species diversity in a widespread bumble bee complex revealed using mitochondrial DNA RFLPs. Conserv Genet 9:653–666CrossRefGoogle Scholar
  42. Nieto A, Roberts SPM, Kemp J, Rasmont P, Kuhlmann M, García Criado M, Biesmeijer JC, Bogusch, Dathe HH, De la Rúa P, De Meulemeester T, Dehon M, Dewulf A, Ortiz-Sánchez FJ, Lhomme P, Pauly A, Potts SG, Praz C, Quaranta M, Radchenko VG, Scheuchl E, Smit J, Straka J, Terzo M, Tomozii B, Window J, Michez D (2014) European red list of bees. Publication Office of the European Commission, LuxembourgGoogle Scholar
  43. Packer L, Gibbs J, Sheffield CS, Hanner R (2009) DNA barcoding and the mediocrity of morphology. Mol. Ecol Res 9:42–50CrossRefGoogle Scholar
  44. Palsbøll PJ, Berube M, Allendorf FW (2007) Identification of management units using population genetic data. Trends Ecol Evol 22:11–16CrossRefPubMedGoogle Scholar
  45. 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 Fennici 14:187–194.Google Scholar
  46. Paterson HE (1985) The recognition concept of species. In: Vrba ES (ed.) Species and speciation. Transvaal museum Monographs, 4 The Museum, Pretoria, pp 21–29Google Scholar
  47. Paxton RJ, Thoren PA, Tengo J, Estoup A, Pamilo P (1996) Mating structure and nestmate relatedness in a communal bee, Andrena jacobi (Hymenoptera, Andrenidae), using microsatellites. Mol Ecol 5:511–519CrossRefPubMedGoogle Scholar
  48. Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295CrossRefGoogle Scholar
  49. Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus genotype data. Genetics 155:945–959PubMedPubMedCentralGoogle Scholar
  50. R Core Team (2014). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. URL
  51. Rasmont P (1984) Les bourdons du genre Bombus Latreille sensu stricto en Europe occidentale et centrale. Spixiana 7:135–160.Google Scholar
  52. 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 93:661–682CrossRefGoogle Scholar
  53. Rasmont P, Franzén M, Lecocq T, Harpke A, Roberts S, Biesmeijer JC, Castro L, Cederberg B, Dvorak L, Fitzpatrick Ú, Gonseth Y, Haubruge E, Mahé G, Manino A, Michez D, Neumayer J, Ødegaard F, Paukkunen J, Pawlikowski T, Potts S, Reemer M, Settele J, Straka J, Schweiger O (2015) Climatic risk and distribution atlas of European bumblebees. BioRisk 10:1–236CrossRefGoogle Scholar
  54. Raymond M, Rousset F (1995) GenePop (v. 1.2): Population genetic software for exact tests and ecumenicism. J Hered 86:248–249CrossRefGoogle Scholar
  55. Riddle BR, Hafner DJ, Alexander LF, Jaeger JR (2000) Cryptic vicariance in the historical assembly of a Baja California Peninsular Desert biota. Proc Natl Acad Sci USA 97:14438–14443CrossRefPubMedPubMedCentralGoogle Scholar
  56. Ryder OA (1986) Species conservation and systematics: the dilemma of subspecies. Trends Ecol Evol 1:9–10CrossRefGoogle Scholar
  57. Schlötterer C (1998) Microsatellites. In: Hoelzel AR (ed) Molecular Genetic analysis of populations. A practical approach. Oxford University Press, Oxford, pp 237–261Google Scholar
  58. Schmid-Hempel R, Eckhardt M, Goulson D, Heinzmann D, Lange C, Plischuk S, Escudero LR, Salathé R, Scriven JJ, Schmid-Hempel P (2014) The invasion of southern South America by imported bumblebees and associated parasites. J Anim Ecol 83:823–837CrossRefPubMedGoogle Scholar
  59. Schmidt S, Schmid-Egger C, Morinière J, Haszprunar G, Hebert PDN (2015) DNA barcoding largely supports 250 years of classical taxonomy: identifications for Central European bees (Hymenoptera, Apoidea partim). Mol Ecol Res 15:985–1000CrossRefGoogle Scholar
  60. Scriven JJ, Woodall LC, Tinsley MC, Knight ME, Williams PH, Carolan JC, Brown MJF, Goulson D (2015) Revealing the hidden niches of cryptic bumblebees in Great Britain: implications for conservation. Biol Conserv 182:126–133CrossRefGoogle Scholar
  61. Sheffield CS, Hebert PDN, Kevan PG, Packer L (2009) DNA barcoding a regional bee (Hymenoptera: Apoidea) fauna and its potential for ecological studies. Mol. Ecol Res 9:196–207CrossRefGoogle Scholar
  62. Stahlhut JK, Gibbs J, Sheffield CS, Alex Smith M, Packer L (2012) Wolbachia (Rickettsiales) infections and bee (Apoidea) barcoding: a response to Gerth et al. Syst Biodiv 10: 395–401CrossRefGoogle Scholar
  63. Stolle E, Wilfert L, Schmid-Hempel L, Schmid-Hempel P, Kube M, Reinhardt R, Moritz RFA (2011) A second generation genetic map of the bumblebee Bombus terrestris (Linnaeus, 1758) reveals slow genome and chromosome evolution in the Apidae. BMC Genom 12:48CrossRefGoogle Scholar
  64. Tsuchida K, Ito Kondo N, Inoue MN, Goka K (2010) Reproductive disturbance risks to indigenous Japanese bumblebees from introduced Bombus terrestris. Appl Entomol Zool 45:49–58CrossRefGoogle Scholar
  65. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538CrossRefGoogle Scholar
  66. Vesterlund SR, Sorvari J, Vasemägi A (2014) Molecular identification of cryptic bumblebee species from degraded samples using PCR–RFLP approach. Mol Ecol Res 14:122–126CrossRefGoogle 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. Waters J, Darvill B, Lye GC, Goulson D (2011) Niche differentiation of a cryptic bumblebee complex in the Western Isles of Scotland. Insect Conserv Div 4:46–52.CrossRefGoogle Scholar
  70. 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
  71. Williams PH, Cameron SA, Hines HM, Cederberg B, Rasmont P (2008) A simplified subgeneric classification of the bumblebees (genus Bombus). Apidol 39:46–74.CrossRefGoogle Scholar
  72. Williams PH, Brown MJF, Carolan JC, An J, Goulson D, Aytekin AM, Best LR, Byvaltsev AM, Cederberg B, Dawson R, Huang J, Ito M, Monfared A, Raina RH, Schmid-Hempel P, Sheffield CS, Šima P, Xie Z (2012a) Unveiling cryptic species of the bumblebee subgenus Bombus s. str. worldwide with COI barcodes (Hymenoptera: Apidae). Syst Biodiv 10:21–56.CrossRefGoogle Scholar
  73. Williams PH, An J, Brown MJF, Carolan JC, Goulson D, Huang J, Ito M (2012b) Cryptic bumblebee species: consequences for conservation and the trade in greenhouse pollinators. PLoS ONE 7:e32992CrossRefPubMedPubMedCentralGoogle Scholar
  74. Williams PH, Cannings SG, Sheffield CS (2016) Cryptic subarctic diversity: a new bumblebee species from the Yukon and Alaska (Hymenoptera: Apidae). J Nat Hist:1–13. doi:10.1080/00222933.2016.1214294

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Lorraine McKendrick
    • 1
  • Jim Provan
    • 2
  • Úna Fitzpatrick
    • 3
  • Mark J. F. Brown
    • 4
  • Tómas E. Murray
    • 3
  • Eckart Stolle
    • 5
  • Robert J. Paxton
    • 1
    • 6
    • 7
  1. 1.School of Biological SciencesQueen’s University BelfastBelfastUK
  2. 2.Institute of Biological, Environmental and Rural SciencesPenglais, Aberystwyth UniversityAberystwythUK
  3. 3.National Biodiversity Data CentreWaterfordIreland
  4. 4.School of Biological SciencesRoyal Holloway University of LondonSurreyUK
  5. 5.School of Biological and Chemical SciencesQueen Mary University of LondonLondonUK
  6. 6.Institute for BiologyMartin Luther-University Halle-WittenbergHalle (Saale)Germany
  7. 7.German Centre for Integrative Biodiversity Research Halle-Jena-Leipzig (iDiv)LeipzigGermany

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