An Investigation of Purple and Violet Feather Coloration
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AN INVESTIGATION OF PURPLE AND VIOLET FEATHER COLORATION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Nicholas Matthew Justyn August, 2017 AN INVESTIGATION OF PURPLE AND VIOLET FEATHER COLORATION Nicholas Matthew Justyn Thesis Approved: Accepted: Advisor Dean of the College Dr. Todd Blackledge Dr. John Green Co-Advisor Dean of the Graduate School Dr. Matthew Shawkey Dr. Chand Midha Faculty Reader Date Dr. Peter Niewiarowski Department Chair Dr. Stephen Weeks ii ABSTRACT The wide range of feather colors are produced by the pigments deposited within the feather, structural components of the feather interacting with light, or in some cases, a combination of both of these mechanisms. Elucidating the mechanisms of color production is essential to understanding the underlying functions and behaviors that are associated with the colors. However, the mechanisms of purple and violet coloration in feathers have yet to be well-characterized, and in fact, the two colors are often carelessly used interchangeably with one another. Violet feathers can be identified by a single peak in the visible spectrum between 380 nm and 450 nm. However, purple feathers have a much higher amount of variability. This variability is created by differing amounts of reflectance in the violet-blue and red region of the visible spectrum in different species. In this study, I investigated 26 different species of birds with purple or violet plumage, and I attempted to characterize the current known methods of purple and violet feather color production. My data, along with previous studies, strongly suggests that non- iridescent purple feathers are produced exclusively by modified carotenoids, and non- iridescent violet is produced structurally with the inclusion of a spongy keratin layer and a basal melanin layer, similar to blue feathers. Lastly, Iridescent purple and violet colored feathers appear to both be produced structurally using melanin as a medium with a high refractive index to selectively scatter light. Additionally, there appear to be several limitations when producing these two colors. To my knowledge, within feathers there are not currently any examples of a violet pigment, a non-iridescent purple structural color, iii or purple plumage created through the combination of a colorful pigment and non- iridescent structural color. First and foremost, I think that this study will serve as a detailed guide on the current known mechanisms of purple and violet feather color production, and lead to a better understanding of the limitations when producing these two colors. Additionally, by combining both pigmentary and structural color analysis, I hope that this study will encourage more accurate and complete characterizations of animal coloration in the future and the functions that may be associated with the mechanism of color production. iv ACKNOWLEDGEMENTS I would like to thank my committee members Dr. Matthew D. Shawkey, Dr. Todd Blackledge, and Dr. Peter Niewiarowski for their guidance and support throughout the entirety of this project. Additionally, I would like to thank Jennifer A. Peteya, Bor-Kai Hsiung, Ming Xiao, Asritha Nallapaneni, Brani Igic, and Liliana D’Alba for training and assistance during the project. Additional thanks to Dr. Andrew Parnell from The University of Sheffield for providing the SAXS data, Dr. Bojie Wang for instruction on the SEM and TEM, and Dr. Zhorro Nikolov for instruction on the Raman spectrometer. v TABLE OF CONTENTS Page LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES ......................................................................................................... ix CHAPTER I. AN INVESTIGATION OF PURPLE AND VIOLET FEATHER COLORATION .... 1 Introduction ........................................................................................................... 1 II. MATERIALS AND METHODS ................................................................................. 6 Sampling ............................................................................................................... 6 UV-Vis spectroscopy ............................................................................................ 7 Confocal Raman spectroscopy .............................................................................. 8 Electron Microscopy ............................................................................................. 9 Small Angle X-ray Scattering (SAXS) ............................................................... 11 Analyses .............................................................................................................. 11 III. RESULTS ................................................................................................................. 13 UV-Vis spectroscopy ......................................................................................... 13 Feather Morphology............................................................................................ 17 Confocal Raman Spectroscopy ........................................................................... 19 Electron Microscopy ........................................................................................... 21 Small Angle X-ray Scattering (SAXS) ............................................................... 21 Analyses .............................................................................................................. 25 vi IV. DISCUSSION ........................................................................................................... 29 V. CONCLUSION .......................................................................................................... 36 LITERATURE CITED ................................................................................................... 37 APPENDICIES ............................................................................................................... 45 APPENDIX A: UV-VIS SPECTROSCOPY ...................................................... 46 APPENDIX B: MICROSPECTROPHOTOMETRY ......................................... 60 APPENDIX C: LIGHT MICROSCOPY ............................................................ 67 APPENDIX D: CONFOCAL RAMAN SPECTROSCOPY .............................. 80 APPENDIX E: SCANNING ELECTRON MICROSCOPY (SEM) .................. 94 APPENDIX F: TRANSMISSION ELECTRON MICROSCOPY (TEM) ......... 96 APPENDIX G: SMALL ANGLE X-RAY SCATTERING (SAXS) ................. 98 vii LIST OF TABLES Page Table 3.1 CIE 1931 Color Space Key ....................................................................................... 28 4.1 Summary of Results for Each Species ...................................................................... 30 viii LIST OF FIGURES Page Figure 3.1 Measured eflectance spectra exhibiting the variation in violet and purple coloration in A) capped conebill B) Costa’s Hummingbird and C) Knysna turaco feathers. .................................................................................................... 14 3.2 Microspectrophotometry reflectance spectra of the individual purple barbs of both the A) Purple Finch (1, 2, 4, 7) and B) Capped Conebill (1, 2, 3, 4) compared to their black barbules (1, 3, 5, 6, 8) and (5, 6, 7, 8). ......................... 15 3.3 The barb of a lilac-breasted roller feather with A) individual cell boundaries. B) microspectrophotometry sampling of an individual cell. C) Reflectance spectra obtained from cells located in the apical, middle, and lower regions of a single barb running the entire length of the feather. .................................... 16 3.4 Violet or purple color located in the barbules of the A) yellow-bibbed fruit dove. B) Costa’s hummingbird barbs. C) capped conebill. D) Both the barbules and barbs of the purple-breasted cotinga. .................................................................. 18 3.5 Raman spectra of A) eumelanin in barb and barbules of a Bufflehead feather. B) Carotenoids in the barb and eumelanin in the barbules of a purple finch feather. C) Turacoverdin in the barbs and barbules of a Knysna Turaco feather. D) Turacin in the barbs of a Ross’s Turaco feather. .............................. 20 ix 3.6 TEM images of the (A) spongy layer of a southern crowned pigeon barbule with a basal layer of melanosomes. B) Arrangement of elongate hollow melanosomes of a Costa’s hummingbird barbule. C) Arrangement of melanosomes near the outer edge of a Bufflehead barbule D) Carotenoids deposited in the Purple-breasted cotinga barbule (left) and barb (right) of the feather............................................................................................................ 22 3.7 SEM image of a superb fairy wren barb revealing a quasi-ordered, channel- type spongy layer and the shape of the melanosomes that form the basal melanin layer in the center of the barb................................................................ 23 3.8 SAXS showing peaks characteristic of an ordered spongy layer in the barbs of A) lilac-breasted roller and B) capped conebill feathers. An absence of peaks, and therefore structure, is seen in the barbs of C) non-iridescent purple finch and D) iridescent bufflehead feathers. ............................................ 24 3.9 A) A magnification of the spongy layer in the barbule of a Southern- crowned pigeon. B) FFT showing an isotropic