MARIA HEIKKILÄ SYSTEMATIZING MORPHOLOGY: A TOTAL EVIDENCE APPROACH TO DITRYSIAN PHYLOGENETICS (LEPIDOPTERA) Finnish MuseuM oF natural history FaCulty oF BioloGiCal anD enVironMental sCienCes uniVersity oF helsinKi Systematizing morphology: a total evidence approach to ditrysian phylogenetics (Lepidoptera) Maria Heikkilä Finnish Museum of Natural History Zoology Unit University of Helsinki Finland Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Finland Academic Dissertation To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences, University of Helsinki, in the Auditorium C205, Economicum (Arkadiankatu 7), on December 19th 2014 at 12 noon. Helsinki 2014 Supervised by: Senior Curator, Dr. Lauri Kaila, Finnish Museum of Natural History, Zoology Unit, University of Helsinki, Finland. Reviewed by: Dr. Joaquin Baixeras, University of Valencia, Instituto Cavanilles Biodiversidad y Biología Evolutiva, Spain. Prof. Toomas Tammaru, University of Tartu, Estonia. Examined by: Dr. Jurate De Prins, Natural History Museum London, U.K. Custos: Prof. Jouni Laakso University of Helsinki, Finland Author’s address: Finnish Museum of Natural History, Zoology Unit P.O. Box 17, FI-00014, University of Helsinki, Finland [email protected] Cover design: Kaisaleena Halinen Layout: Heidi Viljanen ISBN 978-951-51-0495-3 (paperback) ISBN 978-951-51-0496-0 (PDF) http://ethesis.helsinki.fi Painosalama Oy Turku 2014 My work enraptures but utterly exhausts me… To know that no one before you has seen an organ you are examining, to trace relationships that have occurred to no one before, to immerse yourself in the wondrous crystalline world of the microscope, where silence reigns, circumscribed by its own horizon, a blindingly white arena – all this is so enticing that I cannot describe it. Excerpt from a letter from Vladimir Nabokov (novelist and lepidopterist) to his sister, Elena Sikorski, November 25, 1945. In Selected Letters, p. 58–59. Contents Abstract........................................................................................................6 Summary......................................................................................................7 1. Introduction............................................................................................7 1.1 Phylogenetic systematics..........................................................7 1.2 Morphology and genes..............................................................8 1.3 Lepidoptera: butterflies and moths............................................9 1.4 Ditrysian Lepidoptera and research on their evolutionary history...........................................................................................10 2. Outline and aims of the thesis...............................................................13 3. Material and Methods............................................................................14 3.1 Material...................................................................................14 3.2 Examination of morphology....................................................14 3.3 DNA extraction and sequencing.............................................14 3.4 Phylogenetic analyses.............................................................15 3.5 Divergence time and diversification rate analyses..................15 4. Results and Discussion..........................................................................16 Chapter I: The enigmatic Lypusidae..............................................16 Chapter II: Origin and diversification of the redefined butterflies......................................................................................16 Chapter III: Revised classification for Gelechioidea.....................17 Chapter IV: Morphological insight into the phylogeny of Ditrysia.....................................................................................19 5. Conclusions and future directions.........................................................21 Acknowledgements...................................................................................23 References.................................................................................................25 List of original articles This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I. Heikkilä, M. and Kaila, L. 2010. Reassessment of the enigmatic le- pidopteran family Lypusidae (Lepidoptera: Tineoidea; Gelechioidea). Systematic Entomology. 35 (1): 71-89. II. Heikkilä, M., Kaila, L. Mutanen, M., Peña, C. and Wahlberg, N. 2012. Cretaceous origin and repeated tertiary diversification of the redefined butterflies. Proceedings of the Royal Society B. Biological Sciences. 279: 1093-1099. III. Heikkilä, M., Mutanen, M., Kekkonen, M. and Kaila, L. 2014. Mor- phology reinforces proposed molecular phylogenetic affinities: a re- vised classification for Gelechioidea (Lepidoptera). Cladistics – the in- ternational journal of the Willi Hennig Society. 30 (4): 563-589. IV. Heikkilä, M., Mutanen, M., Wahlberg, N., Sihvonen, P., and Kaila, L. Pinning down rogues? Combining morphological and molecular data in ditrysian Lepidoptera. (manuscript). Table of contributions The following table indicates the major contributions of authors to the original articles and the manuscript. I II III IV Original idea LK MM LK, MM LK, PS Study design LK MH, NW, CP, LK, MM LK, MH LK, PS, MH, MM, NW Morphological data MH, LK MH, LK LK, MH, MK MH, LK, PS Molecular data - MM, NW MM MM, NW Analyses MH, LK NW, CP, MH, MM MH, LK MH, LK, NW, MM Manuscript preparation MH MH, NW, LK, CP, MM MH, LK, MM, MK MH, LK, MM, NW, PS MH=Maria Heikkilä, LK= Lauri Kaila, MK= Mari Kekkonen, MM= Marko Mutanen, CP=Carlos Peña, PS= Pasi Sihvonen, NW= Niklas Wahlberg Chapter I © 2009 The Authors. Journal compilation © 2009 The Royal Entomological Society Chapter II © 2012 The Authors. Journal compilation © The Royal Society Chapter III © Willi Hennig Society 2013 Chapter IV © The Authors Disclaimer This publication is not available for purposes of zoological nomenclature in accordance with the International Code of Zoological Nomenclature, fourth edition, article 8.3. Abstract Lepidoptera (butterflies and moths) are one of the most diverse and species-rich groups of organisms. Currently, the number of described species is close to 160.000, but it is estimated that the true number of species could be as high as half a million (van Nieukerken et al. 2011). Nearly 99% of all Lepidoptera belong to the more advanced subclade Ditrysia (Kristensen & Skalski 1999). The focus of this thesis is the evolutionary history of the enormous ditrysian clade. The relatedness between the 30 ditrysian superfamilies has been a mystery that has only recently begun to unfold. The aim of the thesis is to find new information on the evolutionary relationships between these superfamilies, and within two of the largest of them, Gelechioidea and Papilionoidea, both with over 18.000 described species. Evidence on the evolutionary relationships is sought from both morphological and genetic data. The morphological datasets are based on characters coded from larval, pupal and adult stages, and are thus far the largest in terms of the number of coded characters and of the number of exemplar species. The phylogenetic analysis in chapter I is based solely on morphological character data. In the analyses of chapters II-IV, morphological data are combined to molecular data from eight gene regions sequenced from corresponding species or genera. Morphological characters supporting phylogenetic affinities are discussed in detail. Characters described in the articles of this thesis can be used in the identification of extant and fossil taxa, and in future studies focusing on the evolution of specific morphological traits. Chapter I elucidates the evolutionary affinities of the mysterious genus Lypusa (Lypusidae), the phylogenetic position of which has not been known. It is demonstrated that this genus actually belongs within the megadiverse superfamily Gelechioidea. Chapter II focuses on the phylogenetic relationships between the families of the superfamily Papilionoidea (Papilionidae, Pieridae, Nymphalidae, Lycaenidae, Riodinidae, Hesperiidae and Hedylidae). The results show that the traditional concept of butterflies, i.e. including only the first five above-mentioned families, is paraphyletic. Hesperiidae and Hedylidae are sister-groups and are internested within the rest of the butterflies. A divergence time analysis indicates that this superfamily originated in the Cretaceous and diversified in the Tertiary. Chapter III explores the evolutionary relationships within one of the largest but least studied groups of Lepidoptera, Gelechioidea. The classification of this superfamily has been known to be in the need of revision. Based on the results, a new classification into 16 families is proposed. The aim of chapter IV is to find morphological evidence on the relationships between the ditrysian superfamilies. Several morphological characters claimed to define subgroups of Ditrysia are tested. New characters supporting evolutionary affinities of clades are described and discussed. By combining morphological and sequence data, some taxa that have been unstable in analyses based on either type of data only, find a stable position, e.g. Epipyropidae and Cyclotornidae, Urodoidea + Schreckensteinioidea. 6 Summary organisms in the