MOLECULAR PHYLOGENETICS OF THE RIODINIDAE (LEPIDOPTERA) By JONATHAN WALTER SAUNDERS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010 1 © 2010 Jonathan Walter Saunders 2 To my Mom and Dad who fostered my love of the natural world and taught me the importance of education 3 ACKNOWLEDGMENTS I thank the members of my committee for their encouragement and mentoring. And specifically I thank Dr. Tom Emmel for his multifaceted support and positive, uplifting spirit towards me, Dr. Charlie Baer for his generosity and forthrightness, and Dr. Mike Miyamoto for his patience, jokes, and ability to put my mind at ease about graduate school. I thank Mike Perry for all of his help getting me started on this project and Dr. J.D. Turner for sharing with me both his butterflies and his love and knowledge of them. I also thank Dr. Rebecca Kimball for allowing me to use her facilities and answering my questions as well as Dr. Ed Braun, Dr. David Reed, and Julie Allen for being so willing to help me with my many questions. I thank my wife for her loving patience and for her support which allowed me to finish my study. And, I thank God for the ability to study his world full of wonders. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................. 4 LIST OF TABLES............................................................................................................ 7 LIST OF FIGURES.......................................................................................................... 8 LIST OF OBJECTS ......................................................................................................... 9 LIST OF ABBREVIATIONS........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 12 2 MATERIALS AND METHODS ................................................................................ 29 Sequence Data Preparation.................................................................................... 29 Phylogenetic Analyses............................................................................................ 30 3 RESULTS ............................................................................................................... 39 Phylogenetics ......................................................................................................... 39 Myrmecophily.......................................................................................................... 40 4 DISCUSSION ......................................................................................................... 52 Taxonomy ............................................................................................................... 52 Phylogenetics and Gene Trees............................................................................... 56 Myrmecophily.......................................................................................................... 58 3G<2G<1G....................................................................................................... 59 The Hypotheses Are Not Independent ............................................................. 60 The Indirect Method ......................................................................................... 60 Conclusion .............................................................................................................. 62 APPENDIX A COMPARISON OF 3 PARTITIONING SCHEMES FOR THE TOTAL EVIDENCE ANALYSES.......................................................................................... 69 B BAYESIAN TREES WITH POSTERIOR PROBABILITIES..................................... 70 C RANDOM TREES USED TO GENERATE A NULL DISTRIBUTION FOR THE MYRMECOPHILY HYPOTHESES ......................................................................... 74 5 D PROCEDURE FOR SCORING RANDOM TREES WITH A WORKED EXAMPLE............................................................................................................... 75 Procedure ............................................................................................................... 75 Worked Example .................................................................................................... 78 E SCORED RANDOM TREES................................................................................... 89 F RANDOM TREE NULL DISTRIBUTION RESULTS ............................................... 90 REFERENCE LIST........................................................................................................ 92 BIOGRAPHICAL SKETCH............................................................................................ 97 6 LIST OF TABLES Table page 1-1 Taxonomy of riodinid specimens for this study with their respective identification numbers......................................................................................... 22 2-2 Adapted PCR reaction components from (Niklas Wahlberg’s website: http://nymphalidae.utu.fi/Nymphalidae/Molecular.htm). ...................................... 35 2-3 Primers of genes sequenced and their respective sources ................................ 36 2-5 Sequences and accession numbers for three outgroups (denoted by *) and seven riodinids obtained from GenBank (Benson et al. 2006)............................ 38 3-1 Characteristics of the DNA sequence data......................................................... 42 3-2 Likelihood values and AIC values from RAxML likelihood analyses................... 43 3-3 Parsimony score statistics for 1G, 2G, and 3G when mapped onto 100 random trees. ..................................................................................................... 51 4-1 Parsimony scores from mapping gains and losses of myrmecophily onto the Myrmecophily Tree (Figure 4-2). ........................................................................ 65 4-2 Results from comparison of the hypotheses mapped on the Myrmecophily Tree next to the averages and 95% confidence intervals taken from random trees ................................................................................................................... 67 7 LIST OF FIGURES Figure page 1-1 Phylogeny depicting evolutionary relationships of butterfly families adapted from Wahlberg et al. 2005. ................................................................................. 21 1-2 Diagram of caterpillar bodies comparing the auditory, feeding, and chemo- sensory emitting ant organs in three butterfly clades: Nymphidiini/Lemoniini, Eurybini, and Lycaenidae ................................................................................... 25 1-3 Subfamily and tribal relationships inferred from parsimony analysis on morphological data. This phylogeny is adapted from Harvey 1987. ................... 26 1-4 A parsimony phylogeny from Campbell et al. (2000) based on the wingless gene excluding third-position transitions with bootstrap support values less than 50% collapsed into a polytomy. .................................................................. 27 1-5 Two taxon sampling schema represented by A) and B) with the same number of taxa (and in this case, even the same topology). .............................. 28 3-1 RAxML most likely tree for the 3 genes combined and partitioned by gene and by codon position, higher taxa indicated by colored branches..................... 44 3-2 RAxML most likely trees for individual genes, higher taxa indicated by colored branches. ............................................................................................... 45 3-3 Consensuses of bootstrapped combined and individual RAxML trees (bootstraps indicated at nodes) .......................................................................... 46 3-4 50% majority rule consensus tree of the three most likely gene trees. ............... 50 4-1 Morphological phylogenetic hypothesis after Harvey 1987 including his subfamilies and tribes compared to my molecular phylogenetic hypothesis....... 64 4-2 The Myrmecophily Tree..................................................................................... 67 4-4 Adaptation of Campbell’s (1998) molecular phylogeny with myrmecophilous character state indicated by square next to genus. ............................................ 68 D-1 Example of how to mark a phylogeny as in step................................................. 75 D-3 Two interpretations of the evolution of myrmecophily based on the same tree. .......................................................................................................................... 76 D-4 Figure D-4. Further explanation for step 6) and 7).............................................. 77 D-5 Step by step method for scoring a random tree (here it is random tree number 94 which is the most complex................................................................ 78 8 LIST OF OBJECTS Object page C-1 Random Trees.................................................................................................... 74 E-1 Scans of 100 scored random trees..................................................................... 89 9 LIST OF ABBREVIATIONS