DISENTANGLING THE PHENOTYPIC VARIATION AND POLLINATION BIOLOGY OF THE CYCLOCEPHALA SEXPUNCTATA SPECIES COMPLEX (COLEOPTERA: SCARABAEIDAE: DYNASTINAE) A Thesis by Matthew Robert Moore Bachelor of Science, University of Nebraska-Lincoln, 2009 Submitted to the Department of Biological Sciences and the faculty of the Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Master of Science July 2011 © Copyright 2011 by Matthew Robert Moore All Rights Reserved DISENTANGLING THE PHENOTYPIC VARIATION AND POLLINATION BIOLOGY OF THE CYCLOCEPHALA SEXPUNCTATA SPECIES COMPLEX (COLEOPTERA: SCARABAEIDAE: DYNASTINAE) The following faculty members have examined the final copy of this thesis for form and content, and recommend that it be accepted in partial fulfillment of the requirement for the degree of Master of Science with a major in Biological Sciences. ________________________ Mary Jameson, Committee Chair ________________________ Bin Shuai, Committee Member ________________________ Gregory Houseman, Committee Member ________________________ Peer Moore-Jansen, Committee Member iii DEDICATION To my parents and my dearest friends iv "The most beautiful thing we can experience is the mysterious. It is the source of all true art and all science. He to whom this emotion is a stranger, who can no longer pause to wonder and stand rapt in awe, is as good as dead: his eyes are closed." – Albert Einstein v ACKNOWLEDMENTS I would like to thank my academic advisor, Mary Jameson, whose years of guidance, patience and enthusiasm have so positively influenced my development as a scientist and person. I would like to thank Brett Ratcliffe and Matt Paulsen of the University of Nebraska State Museum for their generous help with this project. Several others deserve credit for helping me along the way: Dr. Maxi Polihronakis, Dr. Bin Shuai and Dr. Karen Brown-Sullivan. Lastly, I am very grateful to the researchers at the Biofinity Project (http://biofinity.unl.edu) for their funding of this work. vi ABSTRACT Researching cryptic biodiversity is an integrative process that uses a “total evidence” approach to identify population-level evolutionary lineages (species). Cryptic species of aroids have been discovered but the existence of cryptic pollinator species has not been addressed. The highly polymorphic scarab beetle, Cyclocephala sexpunctata, is a hypothesized pollinator of two cryptic aroid species. This research integrates detailed morphological data, spatial and distribution data, mitochondrial CO1 sequence data and host plant associations to test the hypothesis that cryptic species of Cyclocephala are visiting aroid flowers. Nine morphologically similar Cyclocephala species were included to address identification problems among similar species. A new country record was found for C. pan (Honduras). A female paratype specimen of C. letiranti was determined to be a female C. sexpunctata raising the possibility that there are no female type specimens of C. letiranti. Four unique male paramere forms (morphotypes) were found in C. sexpunctata and the allied species C. brevis. These paramere forms were associated with four female morphotypes that have a diagnostic form of the ventral surface of the epipleural pillow. The ventral form of the female epipleural pillow is described here for the first time and is a new character for the genus Cyclocephala. Detailed elevational and distribution data indicate that the morphotypes of C. sexpunctata and C. brevis are rarely collected together at specific localities. A checklist of cyclocephaline floral associations was compiled. Examination of voucher specimens and published floral associations indicate that the morphotypes described here visit different species of flowers within their hypothesized elevational range. Mitochondrial CO1 data demonstrate that C. sexpunctata is polyphyletic but the monophyly of C. brevis could not be addressed. The combination of these datasets indicates that the morphotypes described here are cryptic species though their taxonomy remains unresolved due to large numbers of synonyms. vii TABLE OF CONTENTS Chapter Page 1. INTRODUCTION 1 1.1 Introduction 1 1.2 Questions and Significance 2 2. BACKGROUND 4 2.1 The genus Cyclocephala 4 2.2 Variation in Species-level Diagnostic Characters 5 2.3 Variation and Diagnosability of C. sexpunctata, C. brevis and Morphologically Allied Species 5 3. MATERIALS AND METHODS 8 3.1 Taxa Selection 8 3.2 Morphological and Distribution Data 8 3.3 Scoring Maculae Phenotypes 10 3.4 Molecular Methods 11 3.4.1 DNA Extraction 11 3.4.2 Mitochondrial CO1 Amplification 11 3.4.3 Purification, Sequencing and Contig Assembly 12 3.4.4 Alignment and Parsimony Analysis 13 3.5 Checklist of Floral Associations for the Cyclocephalini 13 4. RESULTS AND DISCUSSION 16 4.1 Taxa Selection and Specimen Acquisition 16 4.1.1 Taxa Selection 16 4.1.2 Specimen Acquisition 17 4.1.3 Discussion 18 4.2 The Morphology and Distribution of Morphotypes 21 4.2.1 Morphotype 1 22 4.2.2 Morphotype 2 29 4.2.3 Morphotype 3 36 4.2.4 Morphotype 4 45 4.2.5 Sympatry of the Morphotypes 51 4.2.6 Discussion 51 4.3 Maculae Phenotypes 54 4.3.1 Morphotype 1 55 4.3.2 Morphotype 2 57 4.3.3 Morphotype 3 60 4.3.4 Morphotype 4 63 4.3.5 Discussion 65 4.4 Molecular Relationships 66 4.4.1 Discussion 68 4.5 Checklist of Floral Associations for the Cyclocephalini 70 4.5.1 Discussion 80 5. CONCLUSIONS 83 viii TABLE OF CONTENTS (continued) Chapter Page REFERENCES 87 APPENDICES 101 1. Molecular Voucher Specimen and Sequence Data 102 2. Mitochondrial CO1 Alignment 113 3. Checklist of Floral Associations for the Cyclocephalini 121 ix LIST OF TABLES Table Page 1. Taxonomic history of C. sexpunctata. 7 2. Taxonomic history of C. brevis. 7 3. Primers for CO1 amplification. 12 4. CO1 sequences from NCBI. 13 5. Cyclocephala species of the C. sexpunctata species group examined during this study. 16 6. Private and institutional collections providing loaned material. 17 7. Elevational distribution of morphotype 1. 27 8. Elevational distribution of morphotype 2. 34 9. Elevational distribution of morphotype 3. 43 10. Elevational distribution of morphotype 4. 50 11. Localities of sympatry between morphotypes. 51 12. Cyclocephaline synonyms reported in floral association literature. 71 13. Unresolved cyclocephaline names reported in floral association literature. 71 14. Plant synonyms reported in floral association literature. 71 15. Manuscript names and unresolved plant names reported in floral association literature. 73 16. Generic level summary of cyclocephaline floral associations based on most specific data. 73 17. Host plant data for the C. sexpunctata species complex. 78 x LIST OF FIGURES Figure Page 1. Dorsal maculae of C. sexpunctata. 10 2. Ventral view of female epipleural flange of C. letiranti from Costa Rica, Puntarenas (Monte Verde Forest Reserve). 20 3. Lateral view of female epipleural flange of C. letiranti from Costa Rica, Puntarenas (Monte Verde Forest Reserve). 20 4. Dorsal habitus of morphotype 1 female and male.. 23 5. Caudal view of parameres of a Morphotype 1 male from El Salvador, Ahuachapán (Parque Nacional El Imposible, Cancha San Benito). 23 6. Lateral view of parameres of a Morphotype 1 male from El Salvador, Ahuachapán (Parque Nacional El Imposible, Cancha San Benito). 23 7. Caudal view of parameres of a morphotype 1 male from Mexico, Chiapas (Finca Irlanda). 24 8. Lateral view of parameres of a morphotype 1 male from Mexico, Chiapas (Finca Irlanda). 24 9. Ventral view of female epipleural flange of morphotype 1 from Honduras, Yoro (Sinai, 5.3 km NW of Sn. Fco. Campo). 24 10. Ventral view of female epipleural flange of morphotype 1 from Honduras, Yoro (Pico Pijol National Park). 25 11. Lateral view of female epipleural flange of morphotype 1 from Honduras, Yoro (Sinai, 5.3 km NW of Sn. Fco. Campo). 25 12. Ventral form of female epipleural pillow of morphotype 1 from Honduras, Yoro (Sinai, 5.3 km NW of Sn. Fco. Campo). 26 13. The distribution of morphotype 1 in Mexico, Guatemala, Honduras and El Salvador. 27 14. Dorsal habitus of morphotype 2 female and male. 30 15. Caudal view of parameres of a morphotype 2 male from Costa Rica, Guanacaste (Rio San Lorenzo, R. F. Cord., Tenorio). 30 16. lateral view of parameres of a morphotype 2 male from Costa Rica, Guanacaste (Rio San Lorenzo, R. F. Cord., Tenorio). 30 17. Caudal view of parameres of a morphotype 2 male from Costa Rica, Puntarenas (Est. La Casona Res. Biol. Monteverde). 31 xi LIST OF FIGURES (continued) Figure Page 18. Lateral view of parameres of a morphotype 2 male from Costa Rica, Puntarenas (Est. La Casona Res. Biol. Monteverde). 31 19. Ventral view of female epipleural flange of morphotype 2 from Costa Rica, Puntarenas (Monteverde Forest Reserve). 31 20. Ventral view of female epipleural flange of morphotype 2 from Panama, Chiriquí (Santa Clara). 32 21. Lateral view of female epipleural flange of morphotype 2 from Panama, Chiriquí (Finca La Suiza, 5.3 km N. Los Planes). 32 22. Ventral form of female epipleural pillow of morphotype 2 from Costa Rica, Puntarenas (Monteverde Forest Reserve). 33 23. The distribution of morphotype 2 in Costa Rica and Panama. 34 24. Dorsal habitus of morphotype 3 female and male. 37 25. Caudal view of parameres of a morphotype 3 male from Ecuador, Pichincha (Maquipucuna For. Res., 50 km NW Quito). 37 26. Lateral view of parameres of a morphotype 3 male from Ecuador, Pichincha (Maquipucuna For. Res., 50 km NW Quito). 37 27. Caudal view of parameres of a morphotype 3 male from Nicaragua, Jinotega (Cerro Kilambé, Camp 6-Las Torres). 38 28. Lateral view of parameres of a morphotype 3 male from Nicaragua, Jinotega (Cerro Kilambé, Camp 6-Las Torres). 38 29. Ventral view of female epipleural flange morphotype 3 from Costa Rica, Guanacaste (Est. Pitilla, 9 km S Santa Cecilia, P.N. Guanacaste). 38 30.