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

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.

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.

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

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

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 ring and the spatial frequency

of the spongy layer. C) Predicted reflectance spectra constructed using the

FFT...... 26

3.10 Reflectance spectra of whole feathers for species listed in Table 2 plotted in

CIE 1931 color space according to their corresponding x and y values...... 27

CHAPTER I

AN INVESTIGATION OF PURPLE AND VIOLET FEATHER COLORATION

Introduction

Spectacular displays of color can be found throughout nature in a variety of plants

(Vignolini et al. 2012), animals (Kinoshita et al. 2008), and even some bacteria (Kientz et al. 2013). Birds are a prominent example of a group of animals that have evolved to produce a diverse array of colors in their feathers that completely encapsulate the visible portion of the electromagnetic spectrum (Stoddard and Prum 2011). These colors serve a variety of functions including species recognition, camouflage, and signaling (for review, see Hill and McGraw 2006a). These colors are produced through the selective absorption of light by pigment molecules, coherent scattering of light by organized materials of differing refractive indices (melanin, keratin, and air), or a combination of both pigmentary and structural coloration mechanisms (for review, see Hill and McGraw

2006b).

Previous studies have investigated the mechanisms responsible for producing the variety of colors observed in feathers, and the behaviors or functions associated with them. Non-iridescent rufous-red, brown, grey, and black feathers are among the most commonly observed colors of feathers in many species, and are produced by differing

1 concentrations of eumelanin and phaeomelanin in melanosomes randomly dispersed within the feather (McGraw and Wakamatsu 2004; Li et al. 2012; Yi Liu et al. 2014). In addition to acting as a pigment, the arrangement of melanosomes plays a prominent role in the production of glossy and iridescent feathers (Maia et al. 2010) by the coherent scattering of light due to melanin’s high refractive index of approximately 1.7-1.8

(Stavenga et al. 2015). Melanin is a nearly ubiquitous pigment within Aves and in addition to producing colors that aid in counter shading, species identification, and signaling, the incorporation of melanin has also been correlated with mechanical advantages in feathers. For example, the inclusion of eumelanin has been shown to reduce damage from abrasion (Bonser 1995), reduce colonization (Justyn et al. 2017) and degradation by keratinolytic bacteria (Goldstein et al. 2004).

Non-iridescent long wavelength colors such as red, orange, and yellow are created exclusively by pigments in feathers; primarily carotenoids, psittacofluvins in parrots, and spheniscins in penguins (Thomas et al. 2013). Feathers containing carotenoids have been linked to honest signaling and a birds’ genotypic quality (Hill 1991), immunocompetence

(Nolan et al. 1998), ability to forage successfully (Hill and Montgomerie 1994), and efficiency of cellular respiration, which is linked to several important traits (Hill 2014).

In addition to signaling, carotenoids are thought to provide health benefits by acting as antioxidants (Cornet et al. 2007). In contrast, psittacofulvins deposited into feathers have linked to a reduction in bacterial degradation, similar to eumelanin (Burtt et al. 2010).

However, psittacofulvins have yet to be found circulating in the blood like carotenoids, so they are unlikely to share the antioxidant properties of carotenoids (McGraw and

Nogare 2004). So, although psittacofulvins and carotenoids produce similar colors, their functions likely differ drastically.

Birds rely on structural mechanisms to produce non-iridescent short wavelength colors like blue, green, and violet feathers (Bagnara et al 2007). Recent investigations of non-iridescent structural colors in feathers have become increasingly common to elucidate the mechanisms used to produce these angle independent colors. For example, the color of non-iridescent blue feathers is produced by the coherent scattering of light by quasi-ordered layers of spongy keratin with a basal melanin layer to absorb back- scattered light (Prum 2006; Shawkey et al. 2006).

While pigments and structural colors can produce plumage colors separately, the two are frequently combined to create hues that would otherwise be unavailable to either method independently (Prum and Torres 2003). Non-iridescent green feathers are produced structurally by a combination of yellow pigments and a green-tuned spongy layer with a larger periodicity (D’Alba et al. 2012) or using specialized barbules as is the case in some fruit doves (Dyck 1987). The one known exception to this can be found in turacos that are capable of producing the highly specialized copper containing metalloporphyrin, Turacoverdin, which acts as a green pigment and produces the unique olive-green color seen in the birds’ plumage (Hill and McGraw 2006b).

Brush (1969) originally hypothesized that the described “violet” feather color of the Pompadour Cotinga was caused by a combination of red carotenoids and blue structural color, similar to the combination of mechanisms that produce green colors.

However, this hypothesis was later disproved, and the true origin of the color was shown to be exclusively pigmentary (LaFountain et al. 2010). As far as I know, there are no

3 known examples of a purple feather being produced through a combination of pigmentary and structural mechanisms. Previous studies have since investigated the pigmentary

(Prum et al. 2014; Mendes-Pinto et al. 2012; Berg et al. 2013; LaFountain et al. 2010) and structural (Eliason et al. 2013; Eliason and Shawkey 2012; D’Alba et al. 2012) production mechanisms of purple and violet colored feathers in individual species.

However, purple and violet plumage coloration mechanisms and their potential limitations across all of Aves remains largely undiscussed.

While the majority of plumage colors have been fairly well characterized, and the behaviors associated with them explored, purple and violet-colored plumage remains under-studied and the two colors are even often confused with one another. One issue is that purple and violet are difficult to visibly distinguish from one another without the use of a spectrophotometer. Violet is easily characterized by a single peak in the visible region of the electromagnetic spectrum between 380 nm and 450 nm. However, purple spectra have a large amount of variability created by the combination of reflectance in the violet-blue and the red regions of the visible spectrum. Better understanding the coloration mechanisms that produce these two different colors will allow more accurate assessments of the potential functions associated with the colors and how they may have evolved.

In this study, I investigate 26 different species of purple and violet feathers using light microscopy, UV-vis spectroscopy, confocal Raman spectroscopy, and electron microscopy to elucidate the color production mechanisms. Additionally, I assess the functional and evolutionary implications that these findings may have on feather color production throughout Aves. Lastly, I emphasize the importance of accurately using the

4 correct terminology when examining purple and violet feathers to convey the mechanism of production, and in turn, function of the feathers.

CHAPTER II

MATERIALS AND METHODS

Sampling

I sampled 26 species using UV-vis spectroscopy and microspectrophotometry to determine the color of whole feathers and individual regions of the feather respectively.

Next, I used confocal Raman spectroscopy to detect the presence of any pigments present in the purple or violet regions of the feather. Afterwards, I used a combination of SEM,

TEM, and SAXS to investigation the internal organization of the feather structures.

Lastly, these spectra produced and the images taken were analyzed to determine the contributions that pigments or structural components may have on the observed color of the feathers. Individual feather samples were obtained from a combination of study skins in The University of Akron collections, and opportunistic sampling. A total of 26 species were sampled, including the: purple-breasted cotinga (Cotinga cotinga), shovel-billed kookaburra (Clytoceyx rex), violet-eared waxbill (Uraeginthus granatinus), royal flycatcher (Onychorhynchus coronatus), Ross’s turaco (Musophaga rossae), African pygmy kingfisher (Ispidina picta), white-bellied kingfisher (Corythornis leucogaster), purple swamphen (Porphyrio porphyrio), Southern crowned pigeon (Goura scheepmakeri), vulture guineafowl (Acryllium vulturinum), Knysna turaco (Tauraco

6 corythaix), lilac-breasted roller (Coracias caudatus), purple finch (Haemorhous purpureus), yellow-bibbed fruit dove (Ptilinopus solomonensis), capped conebill

(Conirostrum albifrons), bufflehead (Bucephala albeola), band-tailed pigeon

(Patagioenas fasciata), mallard (Anas platyrhynchos), common starling (Sturnus vulgaris), purple martin (Progne subis), superb fairywren (Malurus cyaneus), scarlet macaw (Ara macao), blue and gold macaw (Ara ararauna), rainbow bee-eater (Merops ornatus), peach-faced lovebird (Agapornis roseicollis), and Costa’s hummingbird

(Calypte costae). These 26 species represent a diverse range of birds from 20 different families, including: Columbidae (3), Cotingidae, Halcyonidae, Estrildidae, Tityridae,

Musophagidae (2), Alcedinidae (2) , Rallidae, Coraciidae, Numididae, Fringillidae,

Thraupidae, Anatidae (2), Sturnidae, Hirundinidae, Maluridae, Meropidae, Psittacidae

(2), Psittaculidae, and Trochilidae. Analysis of barb and barbule morphology and the location of feather coloration of each species were examined using a combination of 10x and 50x light microscopy. The feathers were examined to determine if the purple or violet color, previously quantified using UV-vis spectroscopy, also persists at a microscale within the individual barbs and barbules of the feather.

UV-Vis spectroscopy

I first measured the specular reflectance of each species using UV-visible spectroscopy to determine the color of the feathers. When possible, up to three feathers from the colorful region of the bird were overlain and taped onto black velvet to simulate the arrangement of feathers on actual birds and provide a larger surface area to sample.

When samples were limited in number, a single feather was taped onto a black velvet background. Once prepared, the reflectance of each of the samples was measured following the methods of D’Alba et al., (2012) using an Avantes AvaSpec-2048 spectrometer and an AvaLight-XE pulsed xenon light source, relative to a WS-2 white reflectance standard (Avantes Inc., Boulder, CO, USA). The spectral data were collected at an 80 ° angle of incidence for the light and probe together using AvaSoft software v.7.2.

Using normal specular reflection microspectrophotometry I sampled the small individually colored regions of the feathers. I used a 20/30 PV UV-Visible-NIR microspectrophotomer (CRAIC Technologies Inc.) to obtain the reflectance spectra for each individual region, following the methods of Hsiung et al., (2015). The results were visualized and graphed using OriginPro 8.5.1.

Confocal Raman Spectroscopy

Confocal Raman Spectroscopy was performed as a nondestructive method to detect and characterize pigments present within each of the feather samples based on the change in polarizability of the pigment molecules when they are excited (Thomas et al.,

2014; Hsiung et al. 2017). The peak locations of the spectra collected can be compared to

Raman previously obtained to determine the class of pigment present in feather samples, or compared to data obtained by High-performance liquid chromatography (HPLC) to identify the pigment present (Thomas et al., 2013; Thomas and James 2016). The positions and relative intensities of the different bands give information about which

8 method of spectral tuning may be active in the feather where the concentrations of pigments present may have already been known (LaFountain et al., 2015). However,

Raman spectroscopy is most efficient at detecting pigments with a high number of nonpolar bonds, such as carotenoids, and because of this pigments with a lower number of nonpolar bonds can be overshadowed or completely undetectable using confocal

Raman spectroscopy (Hsiung et al. 2017).

Each of the 26 feather samples were placed onto a glass microscope slide and taped down so that the surface of the feather was as flat as possible. The Raman spectra were then generated using a LabRAM High Resolution Raman microscope (HORIBA

Scientific) which was calibrated using pure silicon. A 532nm green laser source with a spot size of 1.5µm was used with a 1% filter and an exposure time of 5 seconds to prevent burning the sample. For each sample, the spectra were obtained using a 50x objective lens, a 100 µm slit aperture, a 400 µm pinhole, acquisition of x1, and a grating of 1200 lines per mm. The spectra of each sample were recorded in an extended wavenumber range of 300-2500 cm-1. The final spectra generated from each sample were graphed using OriginPro 8.5.1 and the peaks were fit and compared using a Gaussian distribution in Igor Pro 6.36.

Electron microscopy

I performed Field Emission Scanning Electron Microscopy (FESEM) to analyze the internal features of the barbs in three dimensions. During SEM the presence and location of a spongy layer, and the presence and positioning of melanosomes were

9 examined. SEM is also particularly useful for determining the aspect ratio of melanosomes, which is correlated with the color of the feathers, and the ratio of eumelanin to phaeomelanin within the melanosomes (McGraw and Wakamatsu 2004; Li et al. 2012; Yi Liu et al. 2014). Samples for SEM were cut using a razorblade and mounted on an aluminum stub using carbon tape. The samples were sputter coated with gold palladium for 2 minutes using a Polaron E5000 Sputter-Coater (Quorum

Technologies, Laughton, UK), then imaged. The prepared samples were then viewed using a JSM-7401 Field Emission Scanning Electron Microscope (JEOL Solutions for

Innovation) at 7 kV and a 7mm working distance.

I performed Transmission Electron Microscopy (TEM) to observe any micro or nanostructures present in two dimensions within the feathers, so that the size of the features could be more accurately measured. The thickness of the keratin cortex, positioning, morphology, and number of melanosomes were measured due to their previously described roles in color production (Brink and Van der Berg 2004; Maia et al.,

2009; Maia et al., 2010; Eliason et al., 2013). The samples that were examined, were prepared following the methods of Shawkey et al., (2003). Individual barbs were cut from each feather using a razorblade, washed and then dehydrated in 100% ethanol for two 20 minute cycles. Immediately following dehydration, the samples were infiltrated with 15,

50, 70, and 100% Epon, each cycle lasting 24 hours. The samples were then placed into molds with 100% Epon and hardened in an incubator at 60 °C for 16 hours. The blocks were trimmed using a Leica S6 EM-Trim 2 (Leica Microsystems GmbH, Wetzlar,

Germany) and then cut into 100nm thin sections using a Leica UC-6 ultramicrotome

(Leica Microsystems). The samples were then stained with uranyl acetate and lead citrate

10 to increase the contrast between the materials within the sample. The prepared samples were viewed using a JEOL JEM-1230 Transmission Electron Microscope operating at a voltage of 120 kV. Images of spongy layers were taken at 10,000x magnification, and images taken of entire barbules varied from 500 – 1200x magnification to include the whole barbule in the image. The final measurements of the images were made using imageJ software.

SAXS (Small Angle X-ray Scattering)

SAXS was used as a targeted, nondestructive way to sample the length scales of the spongy layer present in the color containing regions of the purple and violet feathers

(Saranathan et al. 2012; Parnell et al. 2015). Measurements were taken by Dr. Andrew

Parnell of The University of Sheffield using the beamline ID02 at the European

Synchotron Radiation Facility (ESRF). The detector used was a Rayonix MX-170HS.

When sampling the feather barbs, x-rays with a wavelength of approximately 1Å, energy of 12.45 keV, a beam size of 20µm by 20µm, and a sample-to-detector distance of

30.6797m was used.

Analyses

Two dimensional Fourier analyses were performed on TEM images when quasi- ordered spongy medullary keratin was found within the feather structure. Fourier transforms reveal both the spatial periodicity of the spongy layers of feathers and provide

11 predicted reflectance of the feather based upon the spacing and refractive index of the air

(1.00) and keratin (1.56). This analysis aids in determining if the structures observed are organized appropriately to produce structural color by coherent scattering of light (Prum et al., 1999; Shawkey et al.., 2003; D’Alba et al., 2012). A section of the TEM image without any melanosomes, cell boundaries, or keratin cortex was chosen and analyzed using the Fourier tool for biological nano-optics (Prum and Torres, 2003).

Using RStudio (RStudio Team 2015) and the package pavo (Maia et al. 2013) I was able to plot where in CIE 1931 color space the individual feathers of each species were located using x and y coordinates.

CHAPTER III

RESULTS

UV-vis Spectroscopy

Using UV-Vis spectroscopy the 26 species sampled were classified as either purple or violet based on the spectra obtained. Purple spectra had a large amount of variation; however, all purple feathers were classified by having some amount of reflectance in both the violet-blue region and red region of the visible spectrum (e.g. costa’s hummingbird; Fig. 3.1A). There was also a substantial amount of variation in the spectra of violet species. Some species showed extremely narrow peaks within the 380-

450nm violet region, while some others had much broader peaks that continued into the blue region of the spectrum (e.g. turaco; Fig 3.1B; to capped conebill; Fig 3.1C).

Microspectrophotometry was performed on feathers where the color visibly differed between the barbs and barbules of the feather, to quantify the color within each of these regions. This confirmed that certain birds like the purple finch and capped conebill were purple at the individual barb level, and not just at the whole feather level

(Fig. 3.2). Other feathers, like the lilac-breasted roller, showed color variation within individual cells along the barb. These cells were sampled along the barb and revealed a color shift from more green cells near the distal tip of a barb, to more violet cells at the basal end (Fig. 3.3).

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.

Figure 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).

Figure 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.

Feather Morphology

Using light microscopy, I found several different morphological differences between species that likely contribute to the color observed. For example, the superb fairywren exhibited flattened barbs while the southern crowned pigeon showed curved barbules containing color only at the tips of them. Additionally, the non-iridescent violet feathers had black barbules compared to those observed in iridescent feathers or non- iridescent purple feathers. When examining the location of color production in the feathers, some species such as the yellow-bibbed fruit dove (Fig. 3.4A) or Costa’s hummingbird (Fig. 3.4B) only had purple coloration in the barbules of the feathers, while other species like the capped conebill, were limited to color production exclusively in their barbs (Fig. 3.4C). Lastly, some species such as the purple-breasted cotinga (Fig.

3.4D) contained purple coloration in both the barbs and barbules of the feather.

Some feathers such as the Southern crowned pigeon and macaw feathers produce purple at the scale of the whole feather or bird, but upon closer inspection, the barbs and barbules were completely different colors. The Southern crown pigeon feathers contain a rufous red colored barb and blue barbules that likely result in the unusual purple coloration of this bird. The purple finch, famous for its’ raspberry colored feathers, has very bright red barbs, but unlike other pigmented birds like the cardinal, the barbules of the feather were dark black. This combination of different colored barbs and barbules to produce a purple color may be much more common than previously expected, and explains the presence of purple colors in zones where colors transition from one color to the next.

Figure 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.

Confocal Raman Spectroscopy

In a majority of the feathers, eumelanin peaks between 1370-1385 cm-1 and 1575-

1580 cm-1 (Peteya et al. 2017) were found when sampling the iridescent purple and violet regions (see Fig. 3.5A). The eumelanin signals indicate that other pigments such as carotenoids, or psittacofulvins were absent from the color containing regions of the feather in any significant concentrations. However, the presence of eumelanin does not rule out the possibility that less Raman active pigments, such as phaeomelanin, may be present in the feather as well. In fact, most melanosomes are thought to have some ratio of both eumelanin and phaeomelanin (McGraw and Wakamatsu 2004; Yi Liu et al.

2014). Interestingly, when performing Raman spectroscopy on the lilac-breasted roller sample the violet and dark blue cells consistently produced eumelanin spectra, but the light blue cells did not. This indicates that birds are capable of selectively depositing melanin within cells along the barbs of their feathers. The lack of melanin in some cells, not absorbing incoherently scattered light, could be the cause for the less saturated green color (e.g Steller’s Jay; Shawkey and Hill 2006). However, it is possible that periodicity changes in the spongy layer between each cell could contribute to the shift in color as well (e.g. budgerigar; D’Alba et al., 2012; Parnell et al. 2015). Several other species (see

Table 1) when sampled produced peaks at 1500–1535 cm-1 1145–1165 cm-1 and 1000–

1010 cm-1 that are consistent with carotenoids (e.g. Purple Finch; see Fig. 3.5B; Hsiung et al. 2017; Thomas et al. 2014). The Knysna turaco and Ross’s turaco revealed the distinctive Raman spectra for turacoverdin (Fig. 3.5C) and turacin (Fig. 3.5D) respectively in their violet colored feathers, and not eumelanin (Thomas et al. 2013).

Figure 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.

Electron Microscopy

TEM images of non-iridescent feathers were determined to only contain eumelanin, revealed the cortex thickness, presence of melanin, spacing between melanosomes, thickness of melanosome layer, and dimensions of a spongy layer in the feather (see Fig. 3.6A). Iridescent feathers, such as the Costa’s humming bird and the bufflehead, revealed an arrangement of melanosomes near the edge of the barbules (Fig.

3.6B; Fig. 3.6C). Carotenoids present in feathers appeared as dark patterns inside (Fig.

3.6D). Additionally, there is an obvious lacking a spongy layer and melanosomes within these feathers. SEM was used to image feathers previously determined to have a spongy layer within the barb of the feather to view the internal structure in three dimensions, and to measure the aspect ratio of any melanosomes present (see Fig. 3.7).

Small Angle X-ray Scattering (SAXS)

Non-iridescent feathers previously determined by Raman spectroscopy to contain eumelanin, and no other pigments, showed peaks characteristic of an organized spongy layer (e.g. Lilac-breasted roller; Fig 3.8A). However, these peaks varied slightly in location and intensity between species which indicates that the size of the spacing and the order of the spongy layer differ between species (Fig. 3.8B). Both noniridescent feathers previously determined to contain carotenoids (Fig. 3.8C), and iridescent feathers (Fig.

3.8D) did not show any peaks indicative of an internal structural, and confirmed the absence of an ordered spongy layer in these feathers.

Figure 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

Figure 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.

Figure 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.

Analyses

The Fourier transforms performed on the TEM images of the spongy layer of the

Southern crown pigeon barbules is isotropic (Fig 3.9A and 3.9B). This confirms that the spongy layers of feathers scatter light equally in all directions, resulting in a non- iridescent color. Additionally, the predicted reflectance spectra produced from this spatial information suggests the color being created by the spongy structure (Fig. 3.9C).

In order to illustrate the wide variation within purple and violet colored feathers, as well as visually representing the breadth of color space that birds are capable of encompassing, I plotted each species in color space to compare and contrast the purple and violet colors produced between species (see Fig. 3.10; Table 3.1). This diagram shows the brilliant blue of a common kingfisher on the far left, and the bright red of both cardinal and red-winged black bird feathers on the far right. In between these two extremes, the violet feathers are found clustered together on the left side of the color space diagram and the purple feathers are spread between the violet and red feathers.

Figure 3.9: A) A magnification of the spongy layer in the barbule of a Southern-crowned pigeon. B) FFT showing an isotropic ring and the spatial frequency of the spongy layer.

C) Predicted reflectance spectra constructed using the FFT.

Figure 3.10: Reflectance spectra of whole feathers for species listed in Table 2 plotted in

CIE 1931 color space according to their corresponding x and y values.

Table 3.1: CIE 1931 Color Space Key

CHAPTER IV

DISCUSSION

In this study, I investigated 26 species of purple and violet-colored birds.

Throughout many previous color studies, the terms purple and violet have been used interchangeably or incorrectly to describe plumage coloration. By using precise terminology to describe these purple and violet feathers, future studies can accurately convey the method of color production and potentially the functions that the colors serve.

Using UV-vis spectroscopy I categorized the 26 species into three different groups: non- iridescent violet, non-iridescent purple and iridescent purple or violet colored species based on their methods of color production. After this initial analysis, I performed a series of other analyses to aid in elucidating the methods of color production in each feather. I used a combination of light microscopy and confocal Raman spectroscopy to determine the region of the feather where the color originated from, and to quantify this color. Next, I used confocal Raman spectroscopy to analyze the pigments present in the barbs and barbules of the feathers. Finally, when possible, Field Emission Scanning

Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM), and Small-

Angle X-ray Scattering (SAXS) were performed to analyze the internal structures present in the feathers. The results of these various analyses for each species are summarized in

Table 4.1.

Table 4.1: Summary of Results for Each Species

Whole Raman of Species Feather Barb Barbule Purple/Violet SAXS of Color Color Color Iridescent Region SEM/TEM Barb Purple-breasted cotinga (Cotinga cotinga) Purple Purple Purple No Carotenoid Central Spongy Layer in Barb and Pigments Peaks Shovel-billed kookaburra (Clytoceyx rex) Violet Violet Black No Eumelanin N/A Peaks Violet-eared waxbill (Uraeginthus granatinus) Violet Violet Black No Eumelanin N/A Peaks Royal flycatcher (Onychorhynchus coronatus) Purple Purple Purple No Eumelanin N/A No Peaks Ross’s turaco (Musophaga rossae) Purple Purple Black No Turacin N/A No Peaks African pygmy kingfisher (Ispidina picta) Violet Violet Black No Eumelanin N/A Peaks White-bellied kingfisher (Corythornis leucogaster) Violet Violet Black No Eumelanin N/A Peaks Purple swamphen (Porphyrio porphyrio) Purple Purple Black No N/A N/A No Peaks Southern crowned pigeon (Goura scheepmakeri) Violet Black Violet No Eumelanin Spongy layer, basal melanin layer in Barbule No Peaks Southern crowned pigeon (Goura scheepmakeri) Purple Orange Violet No Eumelanin N/A No Peaks Vulturine guineafowl (Acryllium vulturinum) Violet Violet Black No Eumelanin N/A Peaks Knysna turaco (Tauraco corythaix) Purple Purple Purple Yes Turacoverdin Pigments only No Peaks Lilac-breasted roller (Coracias caudatus) Violet Violet Black No Eumelanin Spongy layer, basal melanin layer in Barb Peaks Purple finch (Haemorhous purpureus) Purple Purple Black No Carotenoid Pigments only No Peaks Yellow-bibbed fruit dove (Ptilinopus solomonensis) Purple Yellow Purple No Carotenoid N/A No Peaks Capped conebill (Conirostrum albifrons) Violet Violet Black No Eumelanin N/A Peaks Bufflehead (Bucephala albeola) Purple Black Purple Yes Eumelanin Arranged melanosomes No Peaks Band-tailed pigeon (Patagioenas fasciata) Purple Black Purple No N/A N/A No Peaks Mallard (Anas platyrhynchos) Violet Black Violet Yes Eumelanin Arranged melanosomes* No Peaks Common starling (Sturnus vulgaris) Purple Black Purple Yes Eumelanin N/A N/A Purple martin (Progne subis) Violet Black Violet Yes Eumelanin N/A N/A Superb fairywren (Malurus cyaneus) Violet Violet Black No Eumelanin Spongy layer, basal melanin layer in barb N/A Scarlet macaw (Ara macao) Violet Violet Black No Eumelanin N/A Peaks Blue and gold macaw (Ara ararauna) Violet Violet Black No Eumelanin N/A Peaks Rainbow bee-eater (Merops ornatus) Violet Violet Black No Eumelanin N/A Peaks Peach-faced lovebird (Agapornis roseicollis) Violet Violet Black No Eumelanin Spongy layer, basal melanin layer in barb Peaks Costa’s hummingbird (Calypte costae) Purple Black Purple Yes Eumelanin Hollow, arranged melanosomes N/A Northern cardinal (Cardinalis cardinalis) Red Red Red No Carotenoid N/A No Peaks

*Eliason and Shawkey 2012

Non-iridescent violet feathers are characterized by a broad reflectance curve that has a peak in the violet region of the visible spectrum from 380-450 nm. Upon closer inspection, the color from these feathers is generated solely by the barb, and in all cases except the Southern crowned pigeon, the barbules were dark black and lacked any violet color. However, these dark barbules may play a significant role in the overall color production. Pigment analysis of these feathers using confocal Raman spectroscopy revealed eumelanin as the only detectable pigment present in the barbs and barbules of any of these non-iridescent violet colored feathers. Using electron microscopy, I confirmed the presence of a basal layer of melanosomes in these feathers, and also revealed a quasi-ordered spongy layer of keratin and air.My analysis, in agreement with previous studies, has revealed that non-iridescent violet colors observed are produced by a spongy layer in the barbs or barbules containing a smaller spacing in the spongy layer compared to blue feathers and a basal layer of melanosomes (e.g. budgerigar; D’Alba et al. 2012). This use of a spongy layer to produce violet colors, seems to be widespread across Aves in a similar way previously described in blue feathers (Stoddard and Prum

2011).

Non-iridescent purple feathers are identified by a varying amount of reflectance in both the violet-blue and red regions of the visible spectrum that combine to produce the purple color observed. Using confocal Raman spectroscopy, I tested the regions of the feather containing purple color. In some non-iridescent purple feathers the color was limited to only the barbules (e.g. yellow-bibbed fruit dove), however, in others the color was spread uniformly throughout both the barbs and barbules (e.g. purple-breasted cotinga). All Raman spectra from non-iridescent purple feathers shared peaks at 1500–

1535 cm-1 1145–1165 cm-1 and 1000–1010 cm-1, which are characteristic of carotenoids

(Hsiung et al. 2017; Thomas et al. 2014). Previous studies have identified four different carotenoids as the primary contributors to the production of purple feather colors.

Rhodoxanthin is a retro-carotenoid found in the purple feathers of fruit doves

(Columbidae; Berg et al. 2013). Canthaxanthin is a ketocarotenoid found in the white- browed purpletuft is thought to be responsible for the purple plumage coloration observed

(Tityridae; Mendes-Pinto et al. 2012). Cotingin is a methoxy-ketocarotenoid found both alone and in combination with other carotenoids within several species of cotinga that have purple plumage (Cotingidae; LaFountain et al. 2010; Prum et al. 2012). Lastly, cymbirhynchin is the primary carotenoid found within the purple plumage of some species of broadbills (Eurylaimidae; Prum et al. 2014). Electron microscopy confirmed the absence of a spongy layer, or other structural aspect, that may be contributing to the purple color of the feather in a significant way. My data, and the aforementioned studies, appear to suggest that non-iridescent purple feathers are produced exclusively by modified carotenoids through enzymatic conversions of dietary carotenoids.

In this study, I sampled a range of both purple and violet iridescent feathers.

Although the UV-visible spectra for these feathers differ drastically, I grouped them together based on their similar color production mechanisms. For example, the spectra of iridescent feathers are sharp with narrow peaks in comparison to the broad peaks previously observed in non-iridescent violet feathers. Also, iridescence in both purple and violet feathers appears to originate most prominently from the barbules. Confocal Raman spectroscopy revealed eumelanin exclusively in each of the iridescent feathers, except for the Knysna turaco which showed the presence of turacoverdin in the iridescent violet

31 region of the feather. However, this is not surprising due to the feathers’ non-iridescent olive-green appearance under transmitted light, but it is interesting that TEM images show a complete absence of melanosomes. Electron microscopy of all the other iridescent feathers showed a variety of different melanosome arrangements near the surface of the feather that are responsible for striking angle dependent purple and violet colors produced. These findings are also consistent with several previous studies that showed iridescent violet could be produced through a multilayer arrangement of hollow melanosomes (Eliason et al. 2013) or a crystal lattice arrangement (Eliason and Shawkey

2012). Iridescent purple is less common, but in hummingbird feathers is produced by a multilayer arrangement of melanosomes (Greenewalt 1960) or a thin layer interference in rock doves (Yoshioka et al. 2007). Interestingly, the ability to produce certain melanosome arrangements may be as specific or limited to certain groups or families of birds as the enzymatic conversions used to make purple carotenoids.

In addition to detailing the known methods of color production in purple and violet feathers, it is also important to note the apparent production limitations.

Throughout my sampling, and in agreement with previous literature, there is no evidence of a violet pigment in feathers. Modified carotenoids, or any pigments currently known to exist in feathers, do not seem able to create such low wavelength colors without also reflecting light in the red region of the visible spectrum and creating a purple-colored feather instead. Perhaps this is because a pigment with broad band absorption, absorbing all other colors besides violet, would likely result in a similar absorption spectra to melanin and a dark color rather than the intended violet color. The search for a violet

32 pigment in feathers will likely not be found in the form of a modified carotenoid, and if such a pigment exists at all, it would likely be extremely rare like turacoverdin.

In non-iridescent structurally colored feathers the spongy layer of keratin appears to be limited to blue or violet color by default, perhaps limited by the phase separation process that the spongy layer is theorized to be created by. However, the spacing of the spongy layers can be tuned by yellow pigments to produce green (D’Alba et al. 2012). It is then unlikely that a non-iridescent purple spongy layer would be possible in birds without the addition of a pigment to alter the spongy layer. However, there remains no example of a combination of red pigments and blue structural combination in feathers to create such a non-iridescent purple color, as originally hypothesized by Brush (1969). In my study, species such as the southern crowned pigeon that are a unique shade of purple were at first likely suspects for this combination of pigments and structural coloration within the same region of the feather. After investigation, these feathers also have separately colored barbs and barbules that contribute to their unique color. Additionally, purple feathers bridging the gap between blue and red-colored plumage patches, such as the two species of macaws sampled, have structurally colored blue barbs and red pigmented barbules. It appears in both cases that a spongy layer produced the blue structural color, and carotenoids produced the red color, but these mechanisms do not seem to be located within the same region of the feather. The absence of such a combination in species that are clearly capable of producing structural colors and performing the necessary carotenoid conversions to synthesize red carotenoids, is curious. It is especially curious that these types of colorations remain completely separate even when present in the very same feather. This may suggest a potential limitation

33 during feather development that is inhibiting this combination, that isnot affecting the development of green feathers where a combination of spongy layer and carotenoids is possible. Additionally, perhaps it suggests that there is not as strong of a need for such a combination. A diverse range of species can produce non-iridescent purple through several different modified carotenoid combinations. Methods of green color production in feathers appear to be much more limited outside of the combination of yellow pigments and structural colors, with turaco’s and fruit dove’s green production mechanisms being the only current known exceptions. So, either this combination of red pigments and blue structural color to produce purple is not physically possible. Perhaps the perceptual bias of carotenoid based or iridescent purples, or the ease of production, may make these methods more favorable than combining two separate methods of color production.

CHAPTER V

CONCLUSION

In conclusion, making a clear distinction between the two very different colors of purple and violet, and their methods of color production, allows for a better understanding of the roles that these feather colors may be used for. For example, the production of non-iridescent purple feathers can now be invariably linked to carotenoids, non-iridescent violet feathers can be associated with the small spacing of a spongy layer and a basal layer of melanosomes, and iridescent purple and violet feathers can be linked with a particular arrangement of melanosomes in the outer edges of barbules. This knowledge may help to shed light on the many different aspects of feather coloration including the costs of color production and the potential functions known to be associated with these methods of production. I hope this study will reliably allow the mechanism of production, and function of the color to be conveyed just by using the correct phrasing of non-iridescent purple or violet. Future studies should continue to investigate unique examples of purple and violet colorations throughout Aves to discover if there are any exceptions to these three current categories of purple and violet color production in feathers.

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APPENDICES

APPENDIX A

UV-VIS SPECTROSCOPY

Knysna Turaco

APPENDIX B

MICROSPECTROPHOTOMETRY

APPENDIX C

LIGHT MICROSCOPY

Scarlet Macaw

Costa’s Hummingbird

Vulturine Guineafowl

African Pygmy Kingfisher

Band-tailed Pigeon

Bufflehead

Capped Conebill

Shovel-billed kookaburra

Purple-throated Cotinga

Southern Crowned Pigeon

Lilac-breasted Roller

Mallard

Purple Swamphen

Purple Finch

Ross’s Turaco

Royal Flycatcher

Knysna Turaco

Violet-eared Waxbill

White-bellied Kingfisher

Yellow-bibbed Fruit Dove

Peach-faced Lovebird

Superb Fairywren

Rainbow Bee-eater

Northern Cardinal

Purple Martin

Common Starling

APPENDIX D

CONFOCAL RAMAN SPECTROSCOPY

Knysna

APPENDIX E SCANNING ELECTRON MICROSCOPY (SEM)

Peach-faced Lovebird

Superb Fairywren

Lilac-breasted Roller

APPENDIX F

TRANSMISSION ELECTRON MICROSCOPY (TEM)

Southern Crowned Pigeon Costa’s Hummingbird

Knysna Turaco Blue-and-gold Macaw

Bufflehead Purple Finch

Purple-breasted Cotinga Lilac-breasted Roller

APPENDIX G

SMALL ANGLE X-RAY SCATTERING (SAXS)

Lilac-breasted Roller

Knysna Turaco

Royal Flycatcher

Violet-eared Waxbill

Rainbow Bee-eater

Purple-breasted Cotinga

100

Capped Conebill

African Pygmy Kingfisher

101

Blue-and-gold Macaw

Vulturine Guineafowl

102

Peach-faced Lovebird

Purple Finch

103

Shovel-billed Kookaburra

Scarlet Macaw

104

Northern Cardinal

White-bellied Kingfisher

105

Ross’s Turaco

Yellow-bibbed Fruit Dove

106

Band-tailed Pigeon

Bufflehead

107

Purple Swamphen

Southern Crowned Pigeon (Orange Barb)

108

Southern Crowned Pigeon (Blue Barb)

Mallard

109