BUMBLEBEES IN PRIME LANDSCAPES WITH SPECIAL REFERENCE TO THE ARAN ISLAND BUMBLEBEE (HYMENOPTERA: APIDAE) Aislinn Deenihan, B.Sc. (Env. Sci.) A thesis submitted for the degree of Doctor of Philosophy, At the Faculty of Science and Engineering, University of Limerick, Ireland. Supervisors: Prof. John Breen, Department of Life Sciences, University of Limerick. Dr. James Carolan, Department of Biology, National University of Ireland, Maynooth. Submitted to the University of Limerick May 2011 Abstract The Burren region (inclusive of the Aran Islands) in western Ireland is an example of a prime landscapes that hosts internationally rare bumblebee species, such as Bombus muscorum . For the conservation and survival of bumblebees it is important to know nest-site and spring forage plant preferences. Hence nest habitat choices of spring bumblebee queens in the Burren region was investigated by observing their nest-site seeking behaviour. In spring significant nest-site seeking behaviour associations were found for B. sylvarum , with preferences for calcareous grassland habitat and scrub- boundaries. The foraging preferences of bumblebee queens in spring were also recorded with B. sylvarum and B. ruderarius foraging most frequently from Vicia cracca and Lotus corniculatus , respectively. Significant interspecies foraging differences were found between bumblebee species recorded in this study. A melanic colour variety of B. muscorum is found in the Aran Islands, and similar varieties are known from several other islands off the British Isles. Considerable debate has taken place over the last 70 years concerning their taxonomic status. The phylogenetics and genetic differentiation of melanic colour morphs within B. muscorum were examined using DNA barcoding. On dried museum and recently caught alcohol-preserved specimens a novel technique involving a modification of the Qiagen DNeasy PBS DNA extraction protocol for insects was developed to extract DNA from the museum specimens. The CO1 barcoding region, cytochrome B and ITS region were all examined. The results can be used to agrue that melanism in B. muscorum has no underlying phylogenetic significance (e.g. remnants of a Lusitanian distribution or edge of geographic range effect), and the presence of melanic forms on islands is due to convergence. Cumulatively the information gathered through this atypical study of bumblebees in prime landscapes contributes to bumblebee conservation, genetic analysis and taxonomy. More research on insects in prime landscapes is advocated. - i - Declaration This thesis is presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy. “It is entirely my own work, and has not been submitted to any other university or higher institution, or for any academic award in this university. Where use has been made of other people’s work, it has been fully acknowledged and referenced accordingly” _______________________________ Aislinn Deenihan _______________________________ Date - ii - Acknowledgements The financial support of the Irish Research Council for Science, Engineering & Technology (IRCSET) for undertaking this project. My family for their support, and care. Prof. John Breen, Dept. of Life Sciences, University of Limerick (UL), Dr. Mark Brown, School of Biological Sciences, Royal Holloway University of London, Dr. Jim Carolan, National University of Ireland, Maynooth, and Dr. Tomás Murray, Martin-Luther- University Halle-Wittenberg, for guidance, support, and invaluable instruction. Prof. Dave Goulson and Dr. Ben Darvill, Bumblebee Conservation Trust, for supplying samples of bumblebees from Britain. Prof Sean Arkins and Prof James G. Wilson for facilities in the Department of Life Sciences, UL, and Department of Zoology, Trinity College Dublin (TCD). Dr. Nicholas Rudzik, and Sean O’Donovan for help with proof reading and formatting. My colleagues in the University of Limerick in particular Robin Niechoj, Veronica Santorum, Richard O’Hanlon, Maria Cullen, Joan Leahy, Maureen Davoren, Bernadette Norris, Alice Martinon, Dr. Thomas Harrington and Prof. Richard Moles for their advice and support. Klara Golez, Celine Bourdon, Clare McMahon, Michael O’Mahony, Marlene Spaans, Nicola Hogan, Marie T. Conere, Dr. Lisa O’Keeffe, Denis Murphy, Sharon Lucey, Catriona Cunnigham, Joe Colgan, Dr. Rachel Kavanagh, Dr. Frank Cox, Dr. Jennifer Donlan, Eugene O’Regan and Roisin Judge for their advice, support and encouragement. The personnel in the UL Graduate School in particular Marie Beaumont, Anne O’Dwyer and Michael Frain for their kindness and assistance. - iii - Table of Contents ABSTRACT ......................................................................................................I DECLARATION...............................................................................................II ACKNOWLEDGEMENTS ..............................................................................III TABLE OF CONTENTS ................................................................................ IV LIST OF FIGURES ...................................................................................... VIII LIST OF TABLES........................................................................................... X LIST OF ABBREVIATIONS ......................................................................... XII CHAPTER 1 INTRODUCTION ........................................................................0 1.1 Bumblebees in Prime Landscapes...................................................................................................0 1.1.1 Introduction to Bumblebees........................................................................................................1 1.1.2 Bumblebee Classification ...........................................................................................................1 1.1.3 Bumblebee Distribution..............................................................................................................1 1.1.4 Bumblebee Identification............................................................................................................2 1.1.5 Bumblebee Lifecycle ..................................................................................................................4 1.1.6 The Nesting Behaviour of Bumblebees ......................................................................................7 1.1.7 Pollination and Bees ...................................................................................................................9 1.1.8 Bumblebee Foraging...................................................................................................................9 1.1.9 Melanism and Bumblebees.......................................................................................................10 1.1.10 Bumblebees in Ireland ............................................................................................................12 1.1.11 Bombus muscorum ..................................................................................................................13 1.1.12 Bombus lucorum agg. and Bombus terrestris .........................................................................17 1.1.13 Bumblebees in Decline ...........................................................................................................17 1.2 Introduction to Prime Landscapes................................................................................................18 1.2.1 Importance of Prime Landscapes..............................................................................................18 - iv - 1.2.2 Bumblebees in Prime Landscapes ............................................................................................18 1.2.3 The Burren Region ...................................................................................................................19 1.2.4 Prime Habitats within the Burren Region.................................................................................21 1.3 Molecular analysis of insects .........................................................................................................22 1.3.1 Molecular Analysis of Bumblebees ..........................................................................................22 1.4 Introduction to DNA Barcoding....................................................................................................24 1.4.1 Background of DNA Barcoding ...............................................................................................24 1.4.2 Classification of DNA Barcoding Regions...............................................................................25 1.4.3 Utility of DNA Barcoding ........................................................................................................26 1.4.4 The DNA Barcoding Initiative and Repository ........................................................................26 1.4.5 Functionality of DNA Barcoding..............................................................................................27 1.5 Aims .................................................................................................................................................28 1.6 References .......................................................................................................................................28 CHAPTER 2 BUMBLEBEE NEST-SITE SEEKING AND FORAGING IN PRIME LANDSCAPES..................................................................................43