Molecular Phylogenetics of the Superfamily Curculionoidea (Insecta: Coleoptera) Conrad Paulus Dias Trafford Gillett A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of East Anglia Norwich, Norfolk, England March 2014 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there-from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. 1 Molecular Phylogenetics of the Superfamily Curculionoidea (Insecta: Coleoptera) Conrad Paulus Dias Trafford Gillett March 2014 Thesis abstract This thesis examines higher-level evolutionary history within the superfamily Curculionoidea, the most speciose family-level taxon, which includes beetles commonly known as weevils. This is achieved using a phylogenetic approach incorporating the largest datamatrix yet employed for weevil molecular systematics, and includes an investigation into the prospect of obtaining short phylogenetically informative amplicons from archival museum specimens. Newly obtained DNA sequence data is analysed from a variety of mitochondrial and nuclear loci, including 92 mitogenomes assembled through the approach of next-generation sequencing of pooled genomic DNA. The resulting trees are used to test previous morphological- and molecular-based hypotheses of weevil relationships and classification schemes. Mitogenomic-derived trees reveal topologies that are highly congruent with previous molecular studies, but that conflict with some morphological hypotheses. Strong nodal support strengthens inferences into the relationships amongst most weevil families and suggests that the largest family, the Curculionidae, is monophyletic, if the subfamily Platypodinae is excluded. Division of the Curculionidae into two large clades is well supported and the wood-boring habit adopted by three subfamilies is shown to have arisen multiple times, contradicting most morphological analyses. i Addition of several nuclear loci to the mitogenomic data is found to provide little additional value, in terms of improving nodal bootstrap support. A suggestion is made that future efforts to enhance understanding of relationships should focus on improving taxon sampling. Statistical tests of an augmented dataset, derived from public database sequences for single mitochondrial genes, wherein multiple tribes and subfamilies within the broad-nosed weevils are constrained as monophyletic, indicate that three entimine tribes, as currently defined, are each not consistent with the hypothesis for their monophyly. Incongruences between molecular data and classical morphological taxonomy are suggestive that the current weevil classification system is misleading if used to interpret species richness, geographic distributions or ecological traits within currently recognised lineages. ii Supervisory Information University of East Anglia Primary supervisor: Dr. Brent C. Emerson Island Ecology and Evolution Research Group, Instituto de Productos Naturales y Agrobiología (IPNA-CSIC), C/Astrofísico Francisco Sánchez 3, La Laguna, Tenerife (Canary Islands, 38206), Spain Secondary supervisor: Dr. David S. Richardson School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk NR4 7TJ United Kingdom The Natural History Museum, London Prof. Alfried P. Vogler Department of Life Sciences, The Natural History Museum, Cromwell Road, South Kensington, London SW7 5BD United Kingdom Dr. Christopher H. Lyal Department of Life Sciences, The Natural History Museum, Cromwell Road, South Kensington, London SW7 5BD United Kingdom iii List of Contents Thesis abstract ............................................................................................................................................. i Supervisory Information ....................................................................................................................... iii List of Contents .......................................................................................................................................... iv List of Tables, Figures and Appendices ......................................................................................... viii Dedication................................................................................................................................................... xii Acknowledgements ............................................................................................................................... xiii Chapter 1 From Linnæus 1758 to next-generation sequencing 2014: an introductory review of curculionoid higher-level phylogeny and molecular systematics ...................... 1 1.1 Weevil diversity and biology ..................................................................................................... 2 1.2 Weevil classification and phylogeny to date ....................................................................... 5 1.3 Molecular markers employed in weevil systematics ................................................... 12 1.3.1 Summary of loci used in weevil higher-level phylogeny reconstruction ............ 14 1.3.2 Mitochondrial genes ................................................................................................................. 14 1.3.3 Nuclear genes .............................................................................................................................. 17 1.3.4 New approaches ......................................................................................................................... 21 1.4 Thesis aims and structure ....................................................................................................... 24 1.5 References ..................................................................................................................................... 26 Chapter 2 From mini-amplicons to mitochondrial genomes – developing molecular techniques for reconstructing curculionoid phylogeny .......................................................... 30 2.1 Abstract .......................................................................................................................................... 31 2.2 Introduction .................................................................................................................................. 32 2.3 General materials and methods ............................................................................................ 34 2.3.1 Taxon sampling .......................................................................................................................... 34 2.3.2 DNA extraction for standard PCR ....................................................................................... 37 2.4 Molecular markers ..................................................................................................................... 38 2.4.1 Mitochondrial markers ........................................................................................................... 38 iv 2.4.1.3 16S small subunit ribosomal RNA (rrnL) .................................................................... 42 2.4.2 Nuclear markers ........................................................................................................................ 43 2.4.3 Short phylogenetically informative amplicons (SPIAs) ............................................ 46 2.4.4 Mitochondrial genomes .......................................................................................................... 57 2.5 Discussion ...................................................................................................................................... 63 2.6 References ..................................................................................................................................... 68 2.7 Appendices .................................................................................................................................... 71 Chapter 3 Bulk de novo mitogenome assembly from pooled total DNA elucidates the phylogeny of weevils (Coleoptera: Curculionoidea) ................................................................ 80 3.1 Abstract .......................................................................................................................................... 81 3.2 Introduction .................................................................................................................................. 82 3.3 Materials and methods ............................................................................................................. 85 3.3.1 Taxon sampling, DNA extraction and quantification ................................................ 85 3.3.2 ‘Bait’ sequence PCR ................................................................................................................... 86 3.3.3 Sample pooling and sequencing .......................................................................................... 86 3.3.4 Mitogenomic assembly pipeline .......................................................................................... 87 3.3.5 Identification of mitogenomic assemblies using ‘bait’ sequences......................... 90 3.3.6 tRNA gene order ........................................................................................................................