Faculteit Bio-ingenieurswetenschappen Academiejaar 2015 – 2016 Microsatellites as a molecular tool to assess population genetics in declining Brazilian bumblebee and stingless bee species Laura Golsteyn Promotoren: Prof. dr. ir. Guy Smagghe & dr. Ivan Meeus Tutor: dr. Kevin Maebe Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen: Landbouwkunde Preface To bee or not to be… That’s the question. This definitely accounted for me during this master thesis! But it is also an important issue for the rest of the world. For that reason I was really determined to do a study concerning bees and their conservation. That is how I ended up in the bumble-bee lab of Professor Smagghe and Ivan Meeus. I would like to thank my promotors for the opportunity to conduct my thesis at their research group. Without Kevin I would never have accomplished this thesis, he taught me a lot and guided me throughout the whole process. Furthermore, I thank my Brazilian supervisors Betina and Patricia for welcoming me so warmly in Brazil and offering their helping hand both personally and scientifically during my stay there. Finally, I am very grateful to be surrounded by such great people as my family and friends, they supported and encouraged me through the whole journey. Thank you! 1 Contents Preface ..................................................................................................................................................... 1 List of abbreviations ................................................................................................................................ 4 Abstract ................................................................................................................................................... 5 Samenvatting ........................................................................................................................................... 7 Introduction ............................................................................................................................................. 9 1. Literature study ............................................................................................................................. 11 1.1 Bumblebee species of Southern Brazil .................................................................................. 11 1.2 Bumblebee decline: general .................................................................................................. 14 1.2.1 Introduction ................................................................................................................... 14 1.2.2 The role of genetics ....................................................................................................... 14 1.2.3 Previous research .......................................................................................................... 15 1.3 Bumblebee decline: the case of Southern Brazil ................................................................... 15 1.3.1 Introduction ................................................................................................................... 15 1.3.2 Local extinction of Bombus bellicosus ........................................................................... 16 1.4 Stingless bees and the genus Melipona ................................................................................ 17 1.4.1 Introduction ................................................................................................................... 17 1.4.2 Stingless bee reproduction ............................................................................................ 18 1.4.3 Stingless bee pollination ................................................................................................ 18 1.4.4 Melipona species of Southern Brazil ............................................................................. 19 1.5 Meliponiculture and its genetic consequences ..................................................................... 20 1.5.1 Introduction ................................................................................................................... 20 1.5.2 Melipona decline ........................................................................................................... 20 1.5.3 Meliponiculture for conservation.................................................................................. 21 1.5.4 Genetic consequences of artificial maintenance of Melipona colonies ........................ 22 1.6 Microsatellites as a molecular tool for population genetic research .................................... 23 1.6.1 Microsatellites ............................................................................................................... 23 1.6.2 Population genetic parameters ..................................................................................... 28 2. Material and methods ................................................................................................................... 30 2.1 Collection of samples ............................................................................................................ 30 2.2 DNA-extraction, primer selection, PCR, capillary electrophoresis ........................................ 32 2.2.1 DNA-extraction .............................................................................................................. 32 2.2.2 Selection MS primers ..................................................................................................... 32 2 2.2.3 Amplification & visualization ......................................................................................... 33 2.2.4 Genotyping .................................................................................................................... 34 2.3 Data analysis .......................................................................................................................... 35 2.3.1 Genetic diversity ............................................................................................................ 37 2.3.2 Population structure ...................................................................................................... 37 3. Results ........................................................................................................................................... 38 3.1 Melipona ................................................................................................................................ 38 3.1.1 Primer selection ............................................................................................................. 38 3.1.2 Dataset preparation ...................................................................................................... 39 3.2 Bombus .................................................................................................................................. 40 3.2.1 Dataset preparation ...................................................................................................... 40 3.2.2 Estimation of parameters for genetic diversity and inbreeding ................................... 43 3.2.3 Estimation of population structure ............................................................................... 51 4. Discussion ...................................................................................................................................... 53 4.1 Collection of Brazilian wild bees ............................................................................................ 53 4.2 Initialisation Melipona project .............................................................................................. 54 4.3 Genetic parameters of bumblebees in Rio Grande do Sul .................................................... 55 5. Conclusion & future perspectives ................................................................................................. 58 References ............................................................................................................................................. 59 Supplementary data .............................................................................................................................. 71 3 List of abbreviations AFLP Amplified Fragment Length Polymorphism AR Allelic richness bp Base pairs Dest Jost’ D DNA Deoxyribonucleic acid F1 First filial generation F2 Second filial generation FIS Inbreeding coefficient FST Genetic differentiation : gentic structure values HE Expected Heterozygosity HO Observed Heterozygosity HWE Hardy-Weinberg Equilibrium MS Microsatellite n Number Ne Effective population size nt Nucleotide p Probability PCR Polymerase Chain Reaction RNA Ribonucleic acid SE Standard Error SSR Simple Sequence Repeat Ta Annealing temperature Taq Thermus aquaticus DNA polymerase VNTR Variable Number Tandem Repeats 4 Abstract Worldwide there is a decline in pollinators going on. Also in South-Brazil, the bumblebee species Bombus bellicosus has been reported to be extinct in the most north-eastern range of its distribution. In literature, several drivers are described to explain the phenomenon of pollinator decline. Also genetic factors can play a role, as low genetic variability can make bees more vulnerable to changes in the environment. In order to understand whether low genetic diversity was historically inherent to the endangered species B. bellicosus, or was caused by its recent decline, its population genetics