The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town MOLECULAR PHYLOGENETICS AND THE EVOLUTION OF HIGH-FREQUENCY ECHOLOCATION IN HORSESHOE BATS (GENUS RHINOLOPHUS) SAMANTHA STOFFBERG Town Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY in the DepartmentCape of Zoology UNIVERSITY OFof CAPE TOWN August 2007 Supervised by: UniversityAssociate Professor David S. Jacobs Department of Zoology, University of Cape Town Co-supervised by: Associate Professor Conrad A. MaUhee Evolutionary Genomics Group, University of Stellenbosch DECLARATION I, Samantha Stoffberg, hereby declare that the work contained in this thesis is the result of my own research and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. The text does not exceed 80,000 words and no part has been submitted in the past, or is being submitted, to any other university in fulfilment of a degree. Signature: ..... ~....... Date: ..I.~ .. I~\~.~.-?~. ?-0??- University of Cape Town 11 ABSTRACT Horseshoe bats (genus Rhinolophus) belong to the Old World family Rhinolophidae. They are high-duty cycle bats and many species use echolocation calls dominated by high frequencies (above 60 kHz). Much is known about how they use their echolocation calls, but very little is known about why these bats use echolocation calls of such high frequencies, or what has caused the divergence in echolocation call frequency between rhinolophid species. I test five hypotheses that may explain the evolution and divergence of high frequencies in the horseshoe bats: (1) The Allotonic Frequency Hypothesis - echolocation frequencies outside of moth hearing range (allotonic frequencies) have evolved in response to moth hearing; (2) The Allometry Hypothesis - high­ frequency echolocation calls are simply a function of body size; (3) The Acoustic Adaptation Hypothesis - selection pressures linked toTown habitat structure have shaped the evolution of high-frequency echolocation calls; (4) The Foraging Habitat Hypothesis - foraging style and habitat of a bat should correspond to echolocation call frequency and wing Capedesign; and (5) The Acoustic Communication Hypothesis - echolocationof frequencies evolved under selection pressure which eliminated overlap among sympatric species of rhinolophids, within the context of effective communication. To explore the evolution and divergence of high frequencies, a robust phylogeny is required. To date no robust phylogenetic hypothesis has been erected for the family, and in manyUniversity instances South African species have been excluded from partial phylogenies. To develop a robust phylogeny for the majority of the rhinolophids and in particular for the South African Rhinolophus species, I used a molecular supermatrix approach which included mitochondrial cytochrome band three nuclear introns (TG, THY and PRKC1). The resultant robust phylogenetic hypothesis allowed me to investigate the geographical centre of origin for the Rhinolophidae and to estimate dates of divergence using a relaxed Bayesian 11l clock. The phylogeny for the family, multivariate analyses on echolocation and morphological data, together with the inclusion of habitat data, were used to test the predictions made by the above hypotheses. Acceptance of the Allotonic Frequency Hypothesis requires two independent predictions to be validated. Numerous studies support the first prediction that the proportion of tympanate insects will be highest in the diets of bats whose echolocation calls are dominated by frequencies outside the hearing range of moths. Here I tested the second prediction of the Allotonic frequency Hypothesis, that within any family of bats, species using allotonic frequencies should be phylogenetically more derived. I found no support for the second prediction of the Allotonic Frequency Hypothesis, viz that bats using allotonic frequencies should be derived rather than ancestral if high frequencies in this genus have evolved in response to the selection pressure imposed by moth hearing. Rather, high frequencies are the ancestral condition. Rhinolophids evolvedTown in forests, probably within Asia, and the high frequencies that characterize this genus are an adaptation to the cluttered habitats in which they arose and still occur today. Cape An allometric relationship exists betweenof body size and echolocation frequency within the Rhinolophidae: however, body size alone cannot explain the evolution of high frequencies in this family. There is a complete overlap in body sizes between the Rhinolophidae and other families, such as the Vespertilionidae, which use lower echolocation frequencies. Among the rhinolophids echolocation frequency has stronger allometric relationships with morphological characters directly associatedUniversity with sound production, emission, and reception. This contrast strongly suggests that selection has acted directly on echolocation rather than on body size. Furthermore, many species echolocate at frequencies much higher than predicted by body size allometry. These deviations towards higher frequencies cannot be explained by the Acoustic Adaptation Hypothesis. For present-day species, differences in habitat IV structure and climatic variables cannot explain differences in echolocation call frequency, nor can they explain why some species use echolocation calls of much higher frequency than would be predicted by the allometric relationship between call frequency and body size. Both low-frequency and high-frequency species occupy syntopic habitats (the same habitat within the same geographical range) and, because of this, habitat alone cannot explain the higher frequencies used by this family of bats. Limited support was found for the Foraging Habitat hypothesis, with some species that deviated from the body size/echolocation frequency relationship also deviating from the body size/wing design relationship. However no relationship exists between wing loading and peak echolocation frequency for the South African rhinolophids, nor for the global set of horseshoe bats. Thus, the evolution of frequencies higher than predicted by allometry are not due to selection acting simultaneously on wing morphology and call frequency. Town Echolocation call frequencies of South African rhinolophids do, however, support predictions of the Acoustic Communication Hypothesis and also show support for the hypothesis that the high frequencies usedCape by many rhinolophid species have evolved to enable efficient conspecificof communication. Thus, evolutionary divergence of echolocation frequency among the Rhinolophidae was driven by the partitioning of sonar frequency bands for effective communication. University v ACKNOWLEDGEMENTS I am greatly indebted to my supervisor David S. Jacobs for introducing me to the world of bats and for the wealth of knowledge he has passed onto me during my studies at UCT. This thesis would not have been possible without the input of an outstanding molecular biologist, namely Conrad A. Matthee, and without a huge amount of information exchange in the field by my great friend, companion and supporter Corrie Schoeman. Without the support of these people, this thesis would never have come to fruition. I thank, in no particular order: Daniel Ribeiro, Geeta Eick, Pieter Malan, Ernest Seamark, and Maryalice Walker for their assistance in the field. I also thank the staff and students of the Evolutionary Genomics Group at the University of Stellenbosch for laboratory and analytical advice. I am grateful to the numerous people who have donated tissue samples, without whose generous spirit this work would not have been accomplished: Gabor Csorba, Manuel Ruedi, Teresa Kearny (National Flagship Institute) and ErnestTown Seamark, Bill Stanely (Field Museum of Chicago), Jim Dines (Natural History Museum of Los Angeles County), Christian Deitz, and Yoram Yom-Tov. lain Mackie must be especially thanked for the echolocation data he provided in addition to the tissue.Cape To Gerhard Groenewald and the 4x4 Eco-Challenge team ... thank you! of A special thanks to my family for their continued loving support, encouragement and for always trying to understand. To Phil, thank you for all your love, patience and reassurance, especially during the last crazy months - I love you completely! Finally, this thesis is dedicated to my gran who has always believed in me and who has taken an interest in my research from the start. Gran - I love you and am forever grateful for your continued loveUniversity and support. I also thank the following referees for their valuable comments: Robert Barclay, Emma Teeling and Gareth Jones. This research was funded by the National Research Foundation and the University of Cape Town Research Associateship Award. VI TABLE OF CONTENTS Title page Declaration ii Abstract iii Acknowledgements vi Table of contents vii Chapter 1: The evolution of high-frequency echolocation in horseshoe bats: an introduction to the hypotheses 1 Chapter 2: A molecular phylogeny of the Rhinolophidae 19 Chapter 3: Evolution of echolocation in the genus Rhinolophus:Town a test of the Allotonic Frequency Hypothesis 68 Chapter 4: Alternative explanations for the Capeevolution