A Multigene Approach for Investigating DNA Barcode Lineages in Provisional Cryptic Species of Lepidoptera in Costa Rica
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A multigene approach for investigating DNA Barcode lineages in provisional cryptic species of Lepidoptera in Costa Rica by Claudia Bertrand A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Integrative Biology Guelph, Ontario, Canada © Claudia Bertrand, March, 2012 ABSTRACT A multigene approach for investigating DNA barcode lineages in provisional cryptic species of Lepidoptera in Costa Rica Claudia Bertrand Advisor: University of Guelph, 2012 Professor M. Hajibabaei DNA barcoding has illuminated genetically distinct lineages within what appears to be one morphological species. For example, a large-scale DNA barcode analysis of Lepidoptera in the Área de Conservación Guanacaste has revealed that 8% of the morphospecies show more than one DNA barcode lineage. To assess the evolutionary significance of five of these lineages I conducted further molecular analyses by sequencing mitochondrial cytochrome b, nuclear Elongation Factor 1 subunit α and ribosomal Internal Transcribed Spacer 2, and compare their gene genealogies with the provisional species tree hypothesized by DNA barcode genetic distances. Both mitochondrial and nuclear markers support the existence of three species in Urbanus belli. The lack of corroboration between markers in the four remaining species either suggests that the chosen nuclear markers have not diverged since speciation, or there has been recent hybridization between lineages. In the case of Eacles imperialis, hybridization is strongly suggested. TABLE OF CONTENTS ABSTRACT ..................................................................................................................... I TABLE OF CONTENTS ................................................................................................. III LIST OF TABLES .......................................................................................................... VI LIST OF FIGURES....................................................................................................... VIII ACKNOWLEDGEMENTS ............................................................................................... X LIST OF ABBREVIATIONS .......................................................................................... XII INTRODUCTION ............................................................................................................. 1 1 MULTIGENE ANALYSIS OF INTRASPECIFIC DNA BARCODE LINEAGES OF THREE EXEMPLAR LEPIDOPTERA SPECIES WITH NO APPARENT MORPHOLOGICAL AND ECOLOGICAL DIFFERENCES ............................................ 4 1.1 INTRODUCTION ......................................................................................................... 6 1.2 METHODS .............................................................................................................. 11 1.2.1 Specimens ................................................................................................... 11 1.2.2 Phylogenetic analysis of COI, cytb, EF1α and ITS2 ................................. 11 1.2.3 Next Generation sequencing of ITS2 ......................................................... 13 1.2.4 Non-metric multidimensional scaling (nMDS) .......................................... 15 1.2.5 Wolbachia assay ......................................................................................... 16 1.3 RESULTS ............................................................................................................... 16 1.4 DISCUSSION ........................................................................................................... 18 iii 1.4.1 Incomplete lineage sorting of nuclear loci ................................................ 19 1.4.2 Hybridization upon secondary contact ..................................................... 21 1.4.3 Under-detected pseudogenes .................................................................... 22 1.5 CONCLUSIONS ....................................................................................................... 23 2 MITOCHONDRIAL AND NUCLEAR GENETIC DIVERGENCE UNDERSCORE MORPHOLOGICALLY CRYPTIC SPECIES OF URBANUS BELLI (LEPIDOPTERA: HESPERIIDAE) WITHIN THE ÁREA DE CONSERVACIÓN GUANACASTE ............. 41 2.1 INTRODUCTION ....................................................................................................... 43 2.2 METHODS .............................................................................................................. 46 2.2.1 Specimens ................................................................................................... 46 2.2.2 Molecular analysis ...................................................................................... 46 2.2.3 ITS2 secondary structure and compensatory base change analysis ..... 48 2.3 RESULTS ............................................................................................................... 49 2.3.1 Phylogenetic analysis of COI, cytb and EF1α ........................................... 49 2.3.2 Analysis of ITS2 sequences ....................................................................... 49 2.3.3 ITS2 secondary structure & compensatory base changes ...................... 50 2.3.4 Wolbachia Assays ....................................................................................... 51 2.4 DISCUSSION ........................................................................................................... 51 3 INTROGRESSION THROUGH MALE GENE FLOW BETWEEN CRYPTIC LINEAGES OF THE IMPERIAL MOTH (EACLES IMPERIALIS) IN THE ÁREA DE CONSERVACIÓN GUANACASTE: EVIDENCE FROM MORPHOLOGY, ECOLOGY AND MULTIPLE GENE REGIONS. .............................................................................. 71 iv 3.1 INTRODUCTION ....................................................................................................... 72 3.2 METHODS .............................................................................................................. 75 3.2.1 Sampling ...................................................................................................... 75 3.2.2 Molecular analysis ...................................................................................... 76 3.3 RESULTS ............................................................................................................... 78 3.4 DISCUSSION ........................................................................................................... 79 CONCLUSION ............................................................................................................ 110 REFERENCES ............................................................................................................ 112 APPENDICES ............................................................................................................. 123 APPENDIX A: CHAPTER 1 SUPPLEMENTARY TABLES .................................................... 123 APPENDIX B: CHAPTER 2 SUPPLEMENTARY TABLES AND MATERIALS ........................... 129 Table B.2 Sequence-types of ITS2 sequences from U.belli specimens used in the ITS2 secondary structure and compensatory basepair change analysis. ............................................................................................................................. 145 Nucleotide and secondary structure alignments of ITS2 sequence-types used in the ITS2 secondary structure and compensatory basepair change analysis. ............................................................................................................................. 147 APPENDIX C: CHAPTER 3 SUPPLEMENTARY TABLES .................................................... 170 APPENDIX D: METHODOLOGY SUPPLEMENTARY TABLES AND MATERIAL ......................... 184 Table D.1Description of primers used in this study. ....................................... 184 Macherey-Nagel (MN) Extraction Protocol ...................................................... 185 v LIST OF TABLES Table 1.1 Larval food-plant data for barcode lineages of the three morpho-species used in this study……………………………………………………………………………………..25 Table 1.2 Taxonomic sampling of the butterflies and moths used in this study………...27 Table 1.3 Parameters used in Bayesian analyses of phylogeny for each moth or butterfly species and marker region…………………………………………………………31 Table 1.4 Results of Pyrosequencing ITS2 from three morpho-species of moths and butterfly. ………………………………………………………………………………………..32 Table 2.1 Urbanus belli species complex reared from wild-caught caterpillars in the ACG showing larval food-plant and ecosystems.…………………………………………..58 Table 2.2 Taxonomic sampling of U.belli and gene region success……………………..59 Table 2.3 Parameters used in Bayesian analyses of phylogeny for U.belli for each marker region. …………………………………………………………………………………61 Table 2.4 Results of Pyrosequencing ITS2 from the U. belli complex…………………...62 Table 2.5 CBC matrix showing the number of compensatory base changes within and between each provisional species…………………………………………………………...64 Table 2.6 Wolbachia BLAST results from the wsp and MLST database for the five markers: coxA, hpcA, gatB, fstZ, and fbpA…………………………………………………64 Table 3.1 Eacles imperialisspecimens reared from wild-caught caterpillars in the ACG showing larval food-plant and ecosystems……………….…………………………………85 Table 3.2 Taxonomic sampling and sequencing success………………………………87 vi Table 3.3 Results of Pyrosequencing ITS2 from E.imperialis specimens collected in the ACG…..………………………………………………………………………………..……...96