類胡蘿蔔素呈色及黑色素呈色的特徵在紅嘴黑鵯的研究

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(1)國立台灣師範大學生命科學系博士論文. 類胡蘿蔔素呈色及黑色素呈色的特徵在紅嘴黑鵯的研究 Studies of carotenoid-based and melanin-based characteristics in Himalayan black bulbul (Hypsipetes leucocephalus regerrimus). 研究生 : 洪心怡 Hsin-Yi Hung 指導教授: 李壽先博士 Shou-Hsien Li. 中華民國 104 年八月.

(2) Table of Contents Table of contents. 1. List of Tables. 2. List of Figures. 3. 致謝. 4. Abstract. 7. 中文摘要. 9. Chapter One. General introduction. 11. Chapter Two. Sexual dichromatism cryptic to the human eye and the. 23. quality of museum specimens in the Himalayan black bulbul (H. leucocephalus nigerrimus, Pycnonotidae) Chapter Three. Negative effects of molting on carotenoid-based. 52. characteristics and individual’s physical condition in the Himalayan black bulbul (H. leucocephalus nigerrimus) Chapter Four. Negative correlation between melanin-based plumage. 73. coloration and oxidative stress in Himalayan black bulbul (H. leucocephalus nigerrimu) Chapter Five. Carotenoid-based traits alone might not be the sexually. 90. selected cues for female Himalayan black bulbuls (H. leucocephalus nigerrimu) Chapter Six. General conclusions and perspectives. Supplementary. 105 115. materials. 1.

(3) List of Tables Chapter Two Table 2.1. ΔS between two sexes for live birds and skin. 44. specimens in different parts Table 2.2. Post-hoc test (Student’s t) of total brightness and. 45. chromauv between live birds and skin specimens after two-way ANOVA test (S2.2 Table) Table 2.3. Variabilities (Standard Deviation) of brightness in the. 47. same sex among different parts Chapter Three Table 3.1. Multiple regressions of whether molting could affect 70 individuals’ physical conditions in Caro(-) group and Caro(+) group respectively. Chapter Four Table 4.1. Two-way ANOVA tests of brightness in eight body 87 parts. Table 4.2. Multiple regressions of brightness in different body 88 parts in 2010 and 0211. Chapter Six Table 6.1. The summary of my results. 2. 114.

(4) List of Figures Chapter Two Figure 2.1. Spectra of ten characteristics in two sexes. 48. Figure 2.2. The colorimetric variables in carotenoid-based. 49. bill and tarsi between sexes in live birds Figure 2.3. The colorimetric variables in melanin-based. 51. characteristics between sexes in live bird Chapter Three Figure 3.1. Correlations between bill coloration and. 72. individual’s physical indicators: (A) swelling (B) Log (H/L ratio) Chapter Four Figure 4.1. The Log (H/L ratio) of two sexes in different. 89. years Chapter Five Figure 5.1. Plain view of the mate choice apparatus. 3. 104.

(5) 致謝漫長的研究路程，終於走到最後了。在這過程中，如果沒有許多人的幫忙，這本論文無法完成。非常感謝我的指導教授，李壽先老師，這個論文的方向是他建議我的。一路上，他盡心盡力幫我進行研究，除了經費上的支持，同時也隨時提供新的研究方向與方法，促使我想得更深更遠。同時老師也不吝於鼓勵與協助我出國參與國際會議，或是進行研究，老師也積極讓我與他國外認識的學者進行接觸，讓我有更開闊的視野。而到了口試以及修改論文的階段，李老師更是費盡心力，幫我修改論文及口試幻燈片；口試前兩天刮颱風，老師也辛苦的到學校幫我準備口試。可以說，這本論文如果沒有他，就無法完成了。真的非常謝謝老師一路上的幫忙，以及對我的所有教誨。謝謝所有在師大族群遺傳多樣性實驗室的學弟、學妹、學姊們，他們每一個人都幫助、支持過我，讓我能順利完成實驗。葉佳芬、林容千、洪貫捷、褚瑞華及楊愷樂都幫助過我處理帶回來的紅嘴黑鵯，幫我抽血及實驗記錄，有時甚至需要幫我照顧他們。新進來的學妹賴郁婷與游艾雲在口試前兩天還在學校幫助我練習。在實驗室的日子，因為有他們而變得更有趣，也更難讓人離開。. 4.

(6) 感謝李佩珍老師借我他們實驗室精良的顯微鏡，也謝謝羅諠憶教我使用儀器，讓我能順利完成實驗。謝謝台中科博館的姚秋如學姊與集集特生中心的姚正得學長，他們讓我借用該單位的紅嘴黑鵯標本，讓我能與活體進行比對。謝謝師大的許鈺鸚老師，她提供了我很多生物統計與動物行為實驗的建議，讓我能更正確且有效率地進行實驗與分析。謝謝中興大學獸醫系的曾秋隆老師，他花費自己的時間，多次耐心教我辨識不同的白血球細胞，同時也給我他配製的細胞染色劑，讓我能完成換羽試驗的部分。謝謝洪志銘博士常替我修改論文，同時也給我寫作上非常多的建議。也要謝謝幫我照顧紅嘴黑鵯的所有人，其中最辛苦的是我母親伊玉珍，我的實驗有兩年的時間將鳥養在家裡，當我不在的時候，完全是我母親幫忙餵養換水，讓我能繼續實驗。謝謝李明忠學長協助幫忙申請及整理系上動物房的空間，讓我能將紅嘴黑鵯飼養在更適合的環境。謝謝系上學弟、學妹們，洪梓容、林文琪、吳相余、鄭元誠、林佩璇、張鈞睿、金宣安及何懿洲，都曾經幫我照顧鳥，分擔了我非常多的日常工作，而能專心在實驗上。謝謝 Alan Watson 及 Martin Craig 幫我的論文進行英文編寫，讓論文能更流暢，也更為有條理，容易閱讀。 5.

(7) 謝謝國立自然科學博物館及特有生物研究保育中心提供紅嘴黑鵯標本。感謝綠世界、鳳凰谷鳥園、新光兆豐農場提供良好的場地，收容我實驗完後的紅嘴黑鵯。感謝台北市立動物園在實驗中進行的所有任何幫助。要謝謝我的家人，他們忍受我長久以來因為進行研究而對家裡造成的不便。謝謝我爸媽及阿姨忍受了長時間與數十隻活鳥相處，同時替我照顧牠們。最後，要特別謝謝我的先生，陳恆毅，在我念書期間，他一肩擔負家中的經濟責任，同時對我追求自己的理想實踐沒有任何意見，一直支持我，他也忍受多年單獨生活的日子．真的非常謝謝他的體諒。. 最後，還是一句. 謝謝大家. ！！！！！. 6.

(8) Abstract Integumentary colorations are essential signals in avian communication. Among all the various color types, carotenoid-based (expressed in yellow, orange or red) and melanin-based (expressed in black, grey or brown) colors are the most common and important traits in birds. Studies have shown that these traits are informative and serve vital functions, such as cues for choosing mates or assessing opponents.. Traditionally, in avian. communication, carotenoid-based colors are thought to be sexually selected traits and melanin-based colors signal social status. However, the results of various studies indicate these traits serve several functions in different avian species. It suggests that the evolution of both traits is more complex than we used to think. Nevertheless, most relevant studies have focused on the role in sexual dichromatic species, which might be suggested to be under higher pressure of sexual selection. There is a gap of how these traits evolved and what their functions are in less sexually dichromatic species, which might be under different selection regimes from sexually dichromatic ones. In order to understand the imperatives of coloration in avian communication completely, studies to characterize variations and functionality of both traits on less sexually dichromatic species are needed. I chose to study Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus), which are sexually monomorphic to human vision and contain a carotenoid-based bill and tarsi and melanin-based plumage to analyze the possible roles of both types of traits. My results showed that both carotenoid- and several melanin-based parts were sexually dichromatic in avian vision. Furthermore, I found that the 7.

(9) expression of a carotenoid-based bill and melanin-based breast and scapular colorations were correlated with individuals’ physical conditions, including immunocompetence and oxidative stress levels. The results of the female-preference test on carotenoid-based traits showed that the red bared parts alone may not be the cues for mate choice for female black bulbuls. My dissertation provides clear evidence that both carotenoid- and melanin-based traits should be informative cues reflecting bearers’ physical condition; it suggests that these traits may play a role in the signaling of Himalayan black bulbuls, but the functions of the traits need to be further investigated.. Keywords: carotenoid-based trait, Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus), melanin-based trait, quality cue, sexual selection. 8.

(10) 中文摘要鳥類體表顏色多樣性極大，這些顏色大部分具有功能性，對於鳥類溝通非常重要。在各種體表顏色中，由類胡蘿蔔素呈色 (呈現黃、橘或紅色)以及黑色素呈色（黑、灰或棕色）的特徵是最普遍也最重要的特徵。這類特徵通常能傳遞個體的訊息，並且具有重要的功能，例如個體可據此選擇配偶或評估對手。傳統上，類胡蘿蔔素呈色的特徵，被普遍認為是個體選擇配偶的依據，而黑色素呈色的特徵，則被認為與社會互動有關。但是這種觀念已被質疑，因為隨著越來越多的鳥種被研究，發現這兩種特徵在不同種類，可能功能不同，顯示這兩種特徵在鳥類的演化機制，可能比目前所知的更為複雜。除此之外，大部分的相關研究都在探討這兩類特徵在性擇上扮演的角色，所以多是以兩性有明顯型態差異的物種為研究標的（這類物種通常可能受到較高的性擇壓力），較少以在兩性型態差異較小的物種為主題，而這些物種可能受到與兩性型態差異大的物種不同的選汰壓力。為了能更完整地了解顏色在鳥類溝通上的演化機制，需要研究這兩類特徵在兩性型態差異較小的物種上扮演的角色。本研究選擇紅嘴黑鵯 (Hypsipetes. leucocephalus nigerrimus )為研究物種,他們具有類胡蘿蔔素呈色的喙部、跗蹠與黑色素呈色的羽毛，對人眼來說是雌雄單型性的鳥種。我的結果顯示，在鳥類視覺上，這兩類特徵在兩性間是有差異的。而類胡蘿 9.

(11) 蔔素呈色的喙部與黑色素呈色的胸部與肩部羽毛的顏色表現，也與個體的生理狀況（包括個體的免疫能力與受到的氧化壓力）有關。雌性偏好試驗的結果顯示，單獨類胡蘿蔔素呈色的部位，可能不是雌性黑鵯選擇配偶的依據。我的論文提出清楚的證據，顯示類胡蘿蔔素呈色以及黑色素呈色的特徵，都能顯示紅嘴黑鵯個體的生理狀況，因此對紅嘴黑鵯而言，這些特徵可能具有重要的功能。但是要知道這些特徵在這個物種真正的功能，還需要之後更詳細的研究。. 關鍵字：紅嘴黑鵯、類胡蘿蔔素、黑色素、性擇、鳥類通訊. 10. 格式化: 置中.

(12) Chapter One General introduction. Birds show highly diverse colorations, and even the color variations among species within the same genus can be drastic. How such colorations evolved has always been intriguing to evolutionists. Although some evidence suggests that traits such as white color or plumage patterns (spots, stripes and bars) are selectively neutral and the consequence of developmental constraints (Price and Pavelka 1996, Tickell 2003), most color characteristics in avian species are believed to be adaptive (Hill and McGraw 2006) and serve imperative functions, such as communication signaling. This could include facilitating individual, kin or species recognition (Mayr 1972); enhancing. or survivorship. through concealing or deceiving to predators (Bortolotti 2006); or promoting appropriate mate choice or social interactions (Bortolotti 2006, Hill 2006). Sometimes, coloration may serve multiple purposes in one single species (Hill and McGraw 2006).. Communication theory Most research on bird coloration has focused on color as potential signals of quality – cues that communicate information about aspects of the bearer’s relative phenotypicand genetic constitution, or other abilities (Dale 2006). According to Stevens’ (2013) definition, signals are any act or structure that influences the behavior of other organisms, also known 11.

(13) as receivers, and which evolved specifically because of that effect. He defines cue as an incidental source of information that may influence the behavior of a receiver despite not having evolved under selection for that purpose. Communication theory suggests that signalers would use coloration providing information to receivers (Stevens 2013).. Qualities revealed from informative coloration The broad aspects of “quality” reflected in the color of the bearer include genetic qualities and an individual’s condition. Genetic qualities revealed from the coloration include whether the individuals have good genes and/or better genetic compatibility (Neff and Pitcher 2005). An individual’s condition is revealed from its coloration including physical conditions; social status; and ability to forage, care for young and defend territory (Dale 2006). The information obtained from signalers could be used by receivers to assess the qualities and make choices about potential mates and/ or opponents. In my study, I focused on whether the coloration traits may reflect an individual’s physical condition, namely immunocompetence and oxidative stress levels. Among all kinds of colorations, carotenoid- and melanin-based traits are the most widely studied and discussed (Hill and McGraw 2006), particularly in birds.. Evolution of carotenoid-based ornaments Carotenoids, mainly lutein and zeaxanthin, are responsible for red, yellow or orange coloration in vertebrates. Carotenoids also function as natural antioxidants that remove harmful free radicals, which are produced 12.

(14) through normal cellular metabolism or induced by environmental stressors (Chew 1996). Through antioxidative activities, carotenoids can increase the number of lymphocytes or the ability of anti-pathogens and hence, can directly or indirectly regulate multiple somatic processes, such as the functioning of the immune system and molting. (Chew and Park. 2004, McGraw 2006a). However, an individual’s use of carotenoids in the diet is likely limited. Carotenoids cannot be synthesized de novo by animals themselves but rather must be ingested from their diet (Brush 1990) and the ability to utilize carotenoids in food is constrained by genotypes and physiological conditions (AlonsoAlvarez et al. 2004, Hill 1991, Latscha 1990, Olson and Owens 1998). The process of different somatic demands competing for limited carotenoids could result in trade-offs of carotenoids allocation among different demands, possibly making the expression of carotenoid-based ornaments correlate with an individual’s physical condition. In avian species, males with carotenoid-rich traits usually have higher immunocompetence (e.g., Blount and Matheson 2006, Chew and Park 2004, Faivre et al. 2003), possess higher quality territories (e.g., Bostrom and Ritchison 2006, Casagrande et al. 2006, Reudink et al. 2009), provide better parental care (e.g., Pike et al. 2007, Senar et al. 2002), or have better mobility (e.g., Blount and Matheson 2006) than those with carotenoid-poor traits. Based on the indicator hypothesis, which suggests that females would use honestly informative characteristics to choose mates (Andersson and Simmons 2006), carotenoid-based ornaments are suggested as sexually selected (mate-choice) signals in avian species. Studies have shown that 13.

(15) males with carotenoid-richer ornaments are preferred by females (e.g. Hill 1991) and that carotenoid-based plumage is positively correlated with the intensity of sexual selection (Badyaev and Hill 2000, Gray 1996). However, the concept of carotenoid-based ornaments having mainly evolved through sexual selection has been overemphasized. In some avian species, carotenoid-based ornaments have been found to be functional only as quality cues reflecting an individual’s physical condition or parental ability but not as the sexually selected traits (e.g. Pryke et al. 2001), or the main cue of individual identification (Dale 2000). The idea that carotenoid-based ornaments are costly to produce has also been challenged. Some studies suggest that carotenoids are rich in the environment and if over-indigested, may even toxic to animals (Olson and Owens 1998). Data also show that the variabilities of carotenoid-based ornaments may be under the control of genes (Walsh et al. 2012). Therefore, more studies are required to test how carotenoid-based traits serve as a cue for mate choice.. Evolution of melanin-based traits Melanins include eumelanin and pheomelanin and cause black, brown, gray, chestnut, or buff coloration in vertebrates (McGraw 2006b). Besides producing pigmentation, the process of melannogenesis reduces the production of free radicals; therefore, melanins are also powerful antioxidants and could affect multiple somatic functions in a way similar to carotenoids (McGraw 2006b). Studies on avian species show that individuals with darker coloration or larger patch sizes of melanin-based 14.

(16) traits are usually more aggressive (e.g. Da Silva et al. 2013, Mennill et al. 2003), possess larger territories (Jawor and Breitwisch 2003), and have better immunocompetence (e.g., Jacquin et al. 2011) than species with lighter coloration or smaller patch size. Unlike carotenoids, melanins can be synthesized within the body from amino acids, which are generally not considered to be limited (Fox 1976, Jawor and Breitwisch 2003). The expression of melanin-based traits is thought to be under the strong control of genes (McGraw 2006b). Hence, melanin-based traits are less costly to be used as signals than carotenoids (Badyaev and Hill 2000). It is conventionally believed that melanin-based ornaments are less likely to be used by females in mate choice and are more likely to be involved in male–male competition (Jawor and Breitwisch 2003, Senar 1999). However, more studies show that melanin-based ornaments could also serve as sexually selected traits in avian species such as Eurasian penduline tits (Remiz pendulinus, Kingma et al. 2008) and yellowthroats (Geothlypis trichas, Tarof et al. 2005). In addition, the debate about whether or not the level of melanism is correlated with an individual’s condition has been raised; empirical studies on the alpine swifts (Apus melba, Bize et al. 2006), house sparrows (Passer domesticus) and brown-headed cowbirds (Molothrus ater, McGraw et al. 2002) indicate that environmental nutrition and body condition do not significantly influence the expression of melanin-based traits; but studies of barn owls (Tyto alba, Roulin et al. 2008) and Eurasian kestrels (Falco tinnunculus, Fargallo et al. 2007) showed that melanin-based ornaments were affected by the abundance of nutrition in the habitat. Such controversies indicate the necessity to test if melanin-based ornaments could reflect an 15.

(17) individual’s physical quality in a diverse range of species, so that the evolutionary mechanism of such traits can be understood better.. Himalayan black bulbuls Although there are ample studies about the functions of carotenoid- or melanin-based traits, most of them were conducted in sexually dichromatic species. The studies in less sexually dichromatic species, which may be under less sexual selection compared to sexually dichromatic ones, are relatively rare. In order to understand how impetrative both traits are in avian communication completely, the gap of studying sexually monomorphic species are needed. In this dissertation, I examined the roles of both carotenoid- and melanin-based traits in a sexually monomorphic avian species, Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus). Himalayan black bulbuls are widely distributed in Taiwan’s broad-leaf forests at elevations ranging from 100 m to 1500 m. The black bulbul provides both types of pigment-based traits to be measured; it is covered by an entirely black plumage (melanin-based) and its bill and tarsi are both in red (carotenoid-based).. Questions in my dissertation In my dissertation, I explored four questions: (1) Are the carotenoid- and melanin-based characteristics potential cues for sexual selection in black bulbuls? I tested this by examining an indicator of the level of sexual selection, i.e., the degree of sexual dichromatism, from the avian vision (Chapter Two) 16.

(18) (2) Are the carotenoid-based characteristics quality cues that could reflect an individual’s physical condition (including immunocompetence and level of oxidative stress) under the stress of molting? Through this experiment, I examined the effects of molting, which is a poorly studied life-history trait, on the allocation of carotenoids between an individual’s physical condition and the expression of ornamented traits simultaneously (Chapter Three) (3) Are the melanin-based plumages quality cues in black bulbuls? If so, a correlation between the coloration of melanin-based plumage and an individual’s physical condition (oxidative stress) should be observed (Chapter Four) (4) Can female black bulbuls use carotenoid-based ornaments (the red bill and tarsi) as sexually selected cues? If so, females black bulbuls would show preferences for redder (carotenoid-richer) males (Chapter Five).. References AlonsoAlvarez C, Bertrand S, Devevey G, Gaillard M, Prost J, Faivre B, Sorci G (2004) An experimental test of the dosedependent effect of carotenoids and immune activation on sexual signals and antioxidant activity. Am Nat 164:651-659. Andersson M, Simmons L W (2006) Sexual selection and mate choice. Trends Ecol Evol 21:296-302. Badyaev A V, Hill G E (2000) Evolution of sexual dichromatism: contribution of carotenoidversus melaninbased coloration. Bio J 17.

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(22) integrated framework for good genes and compatible genes. Mol Ecol 14:19-38. Olson V A, Owens I P F (1998) Costly sexual signals: are carotenoids rare, risky or required? Trends Ecol Evol 13:510-514. Pike T W, Blount J D, Lindstrom J, Metcalfe N B (2007) Dietary carotenoid availability influences a male's ability to provide parental care. Behav Ecol 18:1100-1105. Pryke S R, Andersson S, Lawes M J (2001) Sexual selection of multiple handicaps in the redcollared widowbird: female choice of tail length but not carotenoid display. Evolution 55:1452-1463. Reudink M W, Studds C E, Marra P P, Kurt Kyser T, Ratcliffe L M (2009) Plumage brightness predicts non breeding season territory quality in a long distance migratory songbird, the American redstart Setophaga ruticilla. J Avian Biol 40:34-41. Roulin A, Gasparini J, Bize P, Ritschard M, Richner H (2008) Melanin-based colorations signal strategies to cope with poor and rich environments. Behav Ecol Sociobiol 62:507-519. Senar J C, Figuerola J, Pascual J (2002) Brighter yellow blue tits make better parents. Proc R Soc Lond B Biol Sci 269:257-261. Stevens M (2013) Sensory ecology, behaviour, and evolution. OUP Oxford, United kingdom. Tarof S A, Dunn P O, Whittingham L A (2005) Dual functions of a melanin-based ornament in the common yellowthroat. Proc R Soc Lond B Biol Sci 272:1121-1127. Walsh N, Dale J, McGraw K, Pointer M, Mundy N (2012) Candidate 21.

(23) genes for carotenoid coloration in vertebrates and their expression profiles in the carotenoid-containing plumage and bill of a wild bird. Proc R Soc Lond B Biol Sci 279:58-66.. 22.

(24) Chapter Two. Sexual dichromatism cryptic to the human eye and the quality of museum specimens in the Himalayan black bulbul (Hypsipetes leucocephalus nigerrimus, Pycnonotidae). 23.

(25) Abstract Sexual dichromatism is an important proxy for the intensity of sexual selection, yet related studies in birds based on museum specimens or conspicuous visual traits in live animals may have led to an underestimation of the intensity and complexity of sexual selection. Using the Himalayan black bulbul (Hypsipetes leucocephalus nigerrimus) which is sexually monomorphic to the human eye, I investigated the extent of overall bodily sexual dichromatism. I measured the reflectanceboth within human visual perceptive range and in the ultra-violet rangeof two carotenoid-based parts and eight dull, melanin-based parts for each individual live bird or museum skin sampled. I found that males had redder beaks, brighter tarsi and darker plumage than females. These are perceptible to the bird according to a model of color discrimination thresholds, suggesting the existence of multiple cryptic sexually selected traits within the species. I also found significant degradation of the color in skin specimens compared with that in live birds, indicating that sexual dichromatism could be underestimated by using skin specimens alone.. Keywords: carotenoid-based characteristics, cryptic sexual dichromatism, melanin-based plumage, Himalayan black bulbul (Hypsipetes leucocephalus nigerrimus), sexual dichromatism. 24.

(26) Introduction One of the most robust and widely used indexes of the intensity of sexual selection in birds is sexual dichromatism, in which the male is typically brighter or more colorful than the female or has more distinguishing features (e.g., Owens and Hartley 1998, Seddon et al. 2013). Although intersexual differentiation in mating behavior, habitat preference or predator avoidance can also promote the evolution of coloration, sexual dichromatism is considered to be driven mainly by female preference or male-male competition (Andersson 1994) and to function in sexual recognition, individual quality assessment and sexual attraction (Dale 2006). Still, several pitfalls in studies of sexual dichromatism may have led to an overall underestimation of sexual selection in birds. Most significantly, these studies have mainly focused on conspicuous differences (e.g., Bortolotti et al. 1996, Eaton 2005, Gray 1996) in the range 400 to 700nm perceptible to human vision (Cuthill et al. 1999, Neitz and Jacobs 1986). However, birds have a wider visual sensory range (300 to 700nm), and can detect intersexual differences in ultra-violet (UV; 300 to 400nm, Chen et al. 1984). Therefore, UV coloration could be also used as the signal (e.g., Alonso-Alvarez et al. 2004, Siefferman and Hill 2005) or target (Bennett et al. 1997) for mate choice. With the aid of spectrometers, several avian species presumed monochromatic have been found to have dichromatic UV coloration (e.g., Igic et al. 2010, Mays Jr et al. 2004), but more studies are needed to evaluate the prevalence of UV-dichromatism. Also underrepresented in avian sexual dichromatism studies is the examination of melanin-based coloration, which appears dull to humans 25.

(27) but may still carry signals of individual quality to birds given their superior vision. Melanin-based characteristics have been found to be associated with individual qualities such as social rank, aggressive behavior and immunocompetence and should be no less important targets for sexual selection than carotenoid-based characteristics (reviewed in McGraw 2006b, Kingma et al. 2008, Tarof et al. 2005). Moreover, melanin deposition appears to be controlled by genes and not easily affected by environmental factors such as diet (Fox 1976, Buckley 1989), whereas the expression of carotenoids depends on nutritional status and foraging ability (Hill 1992, Nolan et al. 1998, Thompson et al. 1997). The sexual selection pressures on these traits might differ from those on other types of trait existing in the same organism. However, little attention has been paid to the relative contribution of the two pigment-based colorations within the same species. The use of museum skin specimens in studies of avian coloration could also lead to the underestimation of sexual dichromatism. The concern that specimens’ feather color might fade over time has been noted in several studies: the color degradation might be species dependent and also determined by when the specimen was collected (Doucet and Hill 2009, McNett et al. 2005, Pohland and Mullen 2006). It has been shown that color fading is significant for museum skin specimens collected more than 50 years previously (Pohland and Mullen 2006). But the level of color degradation of newly collected museum specimens has been controversial (Doucet and Hill 2009, McNett et al. 2005, Pohland and Mullen 2006). In this study, I used a spectrometer to study sexual dichromatism in a 26.

(28) passerine, the Himalayan black bulbul (H. leucocephalus nigerrimus), which is sexually monomorphic to the human eye: both sexes are entirely covered by black plumage with a grey patch on their wings (both melanin-based) and a red beak and tarsus (both carotenoid-based (McGraw 2006a). I tested whether intersexual plumage-color differences would be perceptible to the bulbul itself with the Vorobyev-Osorio color discrimination model which is based on the avian tetrahedral color space (Eaton 2005, Stoddard and Prum 2008). I show that sexual dichromatism does exist in the Himalayan black bulbul, providing insights into the potential functional roles of melanin-based and carotenoid-based characteristics in the species. I also found significant fading of museum skin specimens less than five years old. This raises concerns about the use of recently collected skin specimens for study of avian cryptic sexual dichromatism.. Materials and methods Study Species The Himalayan black bulbul (H. leucocephalus nigerrimus) is a widely distributed species inhabiting broadleaf evergreen and mixed deciduous forests, groves, clearings and edges. A total of 112 live individuals were bought from the pet-shop (San Xing Bird Shop, Taipei, 25.034398,121.504444) during the non-breeding seasons of 2008, 2009 and 2011. The birds were all captured from the southern mountain areas in Taiwan. A blood sample was taken from each bulbul for molecular sex typing before proceeding to color quantification. I also examined 37 specimens from the archives of Taiwan’s National Museum of Natural 27.

(29) Science (female: 5, male: 11) and Endemic Species Research Institute (female: 7, male: 14), all of which had been collected within the previous 15 years.. Molecular Sex Typing Gross DNA was extracted from blood samples with traditional proteinase K digestion followed by LiCl extraction (modified from the procedure of Gemmell and Akiyama 1996). Extracted DNA was resuspended in ddH2O and stored at −20°C. Less than 100 ng of genomic DNA was added to 12.5 μL of PCR (polymerase chain reaction) mix containing 0.5 mM of each of the dNTPs, 0.3 μM of each PCR primer (2550F / 2718R, Fridolfsson and Ellegren 1999), 10 mM Tric-HCL, 50 mM KCL, 1.5 mM of MgCl2 and 0.4 U of Taq DNA polymerase (Amersham Biosciences). The PCR profile was 94°C for 3 min, followed by 40 cycles of 95°C for 20 s, 46°C for 30 s and 72°C for 40 s, finished at 72°C for 2 min. The PCR reactions were carried out in iCyclers (Bio-Rad, Hercules, CA, USA). After PCR reactions, I conducted electrophoresis with 1.2% agarose gel to determine the sex. In total, 55 male and 57 female live bulbuls were identified.. Color Measurement For each individual, the reflectance of ten body regions including two carotenoid-based parts- the beak and tarsus- and eight melanin-based parts- the forehead, nape, back, breast, belly, tail, remige and scapular feathers- were measured by an USB2000 spectrometer (Ocean Optics) with a HL2000 deuterium-halogen light source (Ocean Optics). A 28.

(30) R600-7-UV/125F probe (Ocean Optics) was held perpendicular to the surface of the feathers with a cylindrical cap at the end to standardize measuring distance (5 mm) and to shield ambient light. To calculate relative reflectance, a white standard (Labsphere) was used. To collect the dark reference, the light source was capped by a black plastic plate. Each part was measured three times to calculate repeatability (repeatability > 90% (Lessells and Boag 1987). Due to the obvious fading of carotenoid-based coloration, I did not score the coloration of beak and tarsus in skin specimens. I measured the coloration after checking that there was no obvious stain or abrasion on the surface in order to reduce errors of diminished light reflectance.. Color Quantification I used a combination of colorimetric variables to quantify coloration. These included hue, total brightness and chroma (Montgomerie 2006). Hue was calculated for beak and tarsus by finding the wavelength of the mean of maximum and minimum reflectance values in the wavelength range 550 to 700nm. Total brightness was calculated for all parts by averaging the reflectance from 300 to 700 nm. Two kinds of chroma were calculated. One was chromaRED, calculated as the proportion of reflectance from 550 to 700 nm in the total brightness for beak and tarsus. The other was chromaUV, calculated as the proportion of reflectance from 300 to 400 nm on the total brightness for all parts.. Color Discrimination To distinguish between the hue, brightness and chroma of the two sexes, I 29.

(31) used two-way ANOVA to compare the male and female average measurements by considering the cofactor of different examining year. I also used two-way ANOVA to test whether the date of sample collection (in years) had any significant effect on these colorimetric variables. I also compared the same colorimetric variables between skin specimens and the live birds to examine the extent of color by using two-way ANOVA. Because hue, brightness and chroma were compared for each of two carotenoid-based and eight melanin-based body parts, I applied a Bonferroni adjustment for multiple comparisons which reduced the p value from 0.05 to 0.025 in red parts and to 0.00625 in black plumage. I calculated the variability (standard deviation) of brightness within each sex to test whether the divergence of color differences within the females was different from that within the males at different parts. I also used two-way ANOVA to test whether sexually dichromatic parts had higher variability than non-sexually dichromatic parts. In addition, considering the different spectral sensitivity of the four avian cone types, I mapped the spectra onto Goldsmith’s tetrahedral color space system (Goldsmith 1990) that has recently been recommended for analyzing avian coloration (Eaton 2005, Stoddard and Prum 2008, Stoddard and Prum 2011). I converted the spectrum measured into points within a tetrahedron in which the vertices correspond to exclusive stimulation of the ultraviolet (UV)-, blue (B)-, green (G)- and red (R)-sensitive cones in the avian eye. The quantum catch of each receptor is as follows: Qi = ∫λRi(λ)S(λ)I(λ)dλ, where λ denotes wavelength, Ri(λ) is the spectral sensitivity of cone cell 30.

(32) type i (i from 1 to 4 represent the four cone cells, UVS or VS, SWS, MWS and LWS respectively), S(λ) is the reflectance spectrum of a given feather patch, I (λ) is the irradiance spectrum entering the eye and integration is over the entire avian visual range(300-700 nm). The program Tetracolorspace (Stoddard and Prum 2008) was used for spectrum conversion, and I chose the average spectral sensitivity curves of UVS-type retinas (Endler and Mielke JR 2005) as the candidate avian vision in this study. After calculating the Qi, I calculated discriminability of color for each pair of average males and females in different body patches using the Vorobyev-Osorio color discrimination model (Vorobyev and Osorio 1998, Vorobyev et al. 1998). The model calculates a distance in avian color space (ΔS) defined by the quantum catch of each receptor type (i.e., cone cell) in the avian retina (Eaton 2005). To calculate ΔS, I used the following formula: (ΔS)2 = [(ω1ω2)2(Δf4-Δf3)2 + (ω1ω3)2(Δf4-Δf2)2 + (ω1ω4)2(Δf3-Δf2)2 + (ω2ω3)2(Δf4-Δf1)2 + (ω2ω4)2(Δf3-Δf1)2 + (ω3ω4) 2 (Δf2-Δf1)2] / [(ω1ω2ω3)2 + (ω1ω2ω4)2 + (ω1ω3ω4)2 + (ω2ω3ω4)2] where ωi is the constant noise-to-signal ratio (Weber fraction) for receptor type i, which is based here on empirical estimates from the Pekin robin (Leiothrix lutea, ω4=0.05, following the ratio of the numbers of cones (UV: S: M: L= 1:2:2:4). fi is proportional to the natural logarithm of the respective receptor quantum catches, which are normalized against an adapting background (equation 2 and 3 of (Vorobyev et al. 1998)). Δfi is the difference between the signals in receptor i between the stimuli (two colors). When ΔS is below a threshold value 1, colors are assumed to be indistinguishable. 31.

(33) Results The average spectrums of two sexes were similar in shape but different in total reflectance (Fig. 2.1). In the carotenoid-based beak and tarsus, the spectrums show two peaks at wavelengths ranging from 300 to 400 nm and 600 to 700 nm, which are the reflectance ranges of UV light and carotenoid feathers respectively. Conversely, the spectrums for the melanin-based parts were almost flat but with a slight rise in the UV section. Among the colorimetric variables, the hue and the total brightness were different between sexes in two places. The carotenoid-based beaks of males had higher hues than those of females (male 590.25±0.62 nm, female 587.95±0.71nm; least square mean ± SE; Fig. 2.2., two-way ANOVA, p = 0.016). At the melanin-based belly, males had lower total brightness than females (male 4.30±0.21%, female: 5.16±0.20%, p=0.004, Fig.2.3). As for skin specimens, all parts were the same in the two sexes (S 2.1 Table). Applying the Vorobyev-Osorio color discrimination model, more parts were found to be significantly dichromatic in live male and female birds, including the carotenoid-based beak and tarsus and the melanin-based belly, remige and tail (Table 2.1). In museum skin specimens, the belly and scapular- in addition to breast- were also found to be sexually dichromatic. As such, different subsets of body parts were found to be sexually dichromatic in live birds and museum skin specimens (Table 2.1). Color comparisons between live birds and skin specimens showed 32.

(34) coloration fading in several parts. Live birds had higher brightness in breast and scapular but lower brightness in the tail (Table2.2 a, S2.2 Table); they also had higher chromaUV in every part (Table2.2 b, S2.2 Table b). Although the sampled skin specimens were all collected less than 20 years previously, an effect of specimens’ preserved years was found in the scapular: older specimens showed significantly lower brightness than more recent ones (specimens 15-10 years old 4.52±0.99%, specimens 10-5 years old 5.13±0.53%, specimens less than 5 years old 7.21±0.69%; F =3.70, p=0.042, S2.2 Table), although the difference is not statistically significant after a Bonferroni correction to account for multiple comparisons. Color variabilities of brightness within females were the same with those within males in all parts (Table 2.3). Additionally, the variabilities of brightness of sexually dichromatic parts (carotenoid-based beak, tarsus and melanin-based belly, remige and tail) were larger than those of sexually monochromatic ones (Table 2.3, variances of sexual dichromatic traits 3.67±0.62, variances of non-sexual dichromatic traits 1.06±0.50, Two-way ANOVA with cofactor sex, F=10.663, p=0.005).. Discussion I have shown significant sexual dichromatism in both carotenoid-based and melanin-based body regions of the Himalayan black bulbul, including in reflectance and spectral shape. Males’ redder beak, brighter tarsus and darker plumage were significantly different enough for birds to distinguish between them and females, which could provide an insight 33.

(35) into this species’ mating behavior. I also show that color degradation could lead to different results on sexually dichromatism in skin specimens and live birds. The Himalayan black bulbul and most pycnonotids (part of Pycnonotidae), are dull to humans and listed as monomorphic (Fishpool and Tobias 2005), but my results suggest that the extent of their sexual dichromatism could be underestimated; it is significant but not very large (Table 1; ΔS of Black bird (Turdus merula): 5.56-9.21; ΔS of Black cap (Sylvia atricapilla): 1.48-16.9; ΔS of Greenfinch (Carduelis chloris): 2.26-8.10 (Delhey and Peters 2008), which may be indicative of mild sexual selection. Like most pycnonotids, the Himalayan black bulbul is socially monogamous and provides biparental care (Fishpool and Tobias 2005), personal observation). Dunn et. al. (2010) analyzed more than 1000 species of birds and found lower sexual dimorphism in species with monogamous than with polygynous or lekking mating systems where variance in male mating success is thought to be lower. Nevertheless, other aspects of Himalayan black bulbul and related species’ reproductive biology may contribute to sexual dichromatism; these include the genetic mating system and the parental investments of each sex, which need to be investigated further. The Himalayan black bulbul’s sexually dichromatic characteristics could be function-signaling and therefore the objects of sexual selection. In a study of six avian species, Delhey and Peters (2008) found that most function-signaling patches were sexually dichromatic. Sexually dichromatic traits have been proved to function in quality signaling (e.g., Hill 1996, Walker et al. 2013) and agonistic interactions in several avian 34.

(36) species (e.g., Alonso-Alvarez et al. 2004, Bright and Waas 2002, Préault et al. 2002), and are often the object of female choice. Moreover, brightness varied more in the sexually dichromatic parts of the Himalayan black bulbul (beak, tarsus, belly, remige and tail) than in sexually monochromatic ones, consistent with theoretical and empirical expectations (Andersson 1994, Delhey and Peters 2008). Where males are subject to female mate choice, their sexually selected traits are usually more variable than females’ (Andersson 1994, Darwin 1872). The similar variability that I found in both female and black bulbuls’ sexually dichromatic traits suggests that mate choice might be mutual in this species. Whereas studies of sexual selection have mostly focused on female choice and male–male competition, data increasingly shows that males can be choosy and benefit from mating females whose reproductive potential is high (reviewed in Edward and Chapman 2011, e.g., Amundsen and Forsgren 2001, Griggio et al. 2005, Jones et al. 2001). Kokko and Johnstone (2002) suggested that high species-specific and high sex-specific mate-encounter rates, high cost of breeding (parental investment), low cost of mate searching and highly variable quality of the opposite sex could promote the evolution of choosiness and that the primary determinant of sex roles in mate choice is parental investment. According to this hypothesis, the sex for which the cost of breeding (mortality during signaling and caring) is the larger should evolve to be choosy. The reproductive biology of Himalayan black bulbuls is unclear, but research on pycnonotids suggests comparable parental care loads between the sexes, and the breeding success is generally low (8.3-15%, Balakrishnan 2010, Fishpool and Tobias 2005) while the rate of predation 35.

(37) is high. As such, high cost of breeding and comparable parental care load between the sexes might promote mutual selection in pycnonotids. My data also suggest the involvement of multiple Himalayan black bulbul ornaments in sexual selection - as both carotenoid-based and melanin-based characteristics were found to be sexually dichromatic. Studies have shown females choosing mates based on multiple sexual ornaments (Chaine and Lyon 2008, Doucet and Montgomerie 2003); multiple ornaments provide females with different kinds of information in different stages of mate choice (Borgia 1995), or function as redundant signals to improve the accuracy of mate assessment (Johnstone 1994, Moller and Pomiankowski 1993). Different sets of sexually dichromatic parts were found in live birds and museum skin specimens, and significant degradation of colorwhether pigment-based or structural- was found in skin specimens, some of which had been preserved for less than 5 years. These results suggest that the use of skins in avian coloration study may be error-prone, contradicting the previous finding that melanin- and carotenoid-based skins colors remain the same for at least 50 years after preservation (Armenta et al. 2008). Conversely, my results corroborate the conclusion drawn in a study comparing live and skin long-tailed manakins that significant differences in colorimetric variables were attributable to the age of specimens (Doucet and Hill 2009). They also agree with another study that found UV color degradation in preserved skin specimens of some 300 bird species throughout Europe and the USA (Pohland and Mullen 2006). There are many possible reasons for color degradation, including the preservation process, preservation agents, specimen 36.

(38) preparation, contamination or simply age (reviewed in (Doucet and Hill 2009). Given that museum skin specimens are widely used in studies of avian coloration (e.g., Bridge et al. 2008, Kennedy 2010), I suggest that skin specimen coloration should be pre-tested against live birds; measurements obtained from skin samples should be corrected for age and/or condition of preservation, and the results should be interpreted with greater caution.. References Alonso-Alvarez C, Doutrelant C, Sorci G (2004) Ultraviolet reflectance affects male-male interactions in the blue tit (Parus caeruleus ultramarinus). Behav Ecol 15:805-809. Amundsen T, Forsgren E (2001) Male mate choice selects for female coloration in a fish. P Natl Acad Sci USA 98:13155-13160. Andersson M B (1994) Sexual selection. Princeton University Press, NJ. Armenta J K, Dunn P O, Whittingham L A (2008) Effects of specimen age on plumage color. Auk 125:803-808. Balakrishnan P (2010) Reproductive biology of the square-tailed Black Bulbul Hypsipetes ganeesa in the Western Ghats, India. Indian Birds 5:134-138. Bennett A T, Cuthill I C, Partridge J C, Lunau K (1997) Ultraviolet plumage colors predict mate preferences in starlings. P Natl Acad Sci USA 94:8618-8621. Borgia G (1995) Complex male display and female choice in the spotted bowerbird: specialized functions for different bower decorations. 37.

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(45) Table 2.1 ΔS between two sexes for live birds and skin specimens in different parts. Parts. Live birds. Beak. 3.15. -. Tarsus. 2.28. -. Back. 0.28. 0.88. Belly. 0.64. 1.49. Nape. 0.08. 0.61. Breast. 0.09. 1.14. Forehead. 0.28. 0.81. Remige. 1.19. 0.99. Scapular. 0.66. 1.12. Tail. 3.81. 0.52. Skin specimens. ΔS >1 is in bold and italics.. 44.

(46) Table 2.2. Post-hoc test (Student’s t) of total brightness and chromaUV between live birds and skin specimens after two-way ANOVA test (S2.2 Table). (a) Total brightness Parts. Item. Mean* ± SE. Breast. Live. 3.49 ± 0.08. Skin. 2.87 ± 0.17. Scapular Live. 6.61 ± 0.19. Skin. 5.36 ± 0.38. Live. 4.23 ± 0.10. Skin. 5.22 ± 0.20. Tail. *Least Square Mean, unit=%. 45. Lower CL. Upper CL. Difference. Difference. 0.24. 1.00. 0.41. 2.11. -1.44. -0.55.

(47) (b) ChromaUV. Parts. Item. Mean* ± SE. Back. Live. 24.13 ± 0.19. Skin. 21.70 ± 0.39. Live. 23.13 ± 0.18. Skin. 21.70 ± 0.38. Live. 24.03 ± 0.23. Skin. 20.83 ± 0.45. Live. 24.10 ± 0.20. Skin. 21.09 ± 0.42. Forehead Live. 23.39 ± 0.24. Skin. 20.63 ± 0.48. Live. 22.99 ± 0.17. Skin. 22.13 ± 0.36. Live. 23.95 ± 0.16. Skin. 22.30 ± 0.32. Live. 25.79 ± 0.16. Skin. 24.65 ± 0.33. Belly. Nape. Breast. Remige. Scapula. Tail. *Least Square Mean, unit=%. 46. Lower CL. Upper CL. Difference. Difference. 1.57. 3.29. 0.60. 2.26. 2.19. 4.20. 2.09. 3.93. 1.71. 3.81. 0.07. 1.64. 0.95. 2.36. 0.41. 1.87.

(48) Table 2.3. Variabilities (Standard Deviation) of brightness in the same sex among different parts. Parts. Female. Male. Beak. 6.59. 6.50. Tarsus. 5.49. 5.52. Back. 0.68. 0.62. Belly. 1.43. 1.44. Nape. 0.89. 0.53. Breast. 1.01. 0.94. Forehead. 0.64. 0.60. Remige. 1.71. 1.45. Scapular feather. 1.99. 1.98. Tail. 0.95. 1.14. Chi-square test. P < 0.05 is in bold and italics.. 47.

(49) 35. 40. Beak. Tarsus 30. 30 25 20. 20 15. 10 10 5 7. 0 4.0. Belly. Back 3.5. 6. 3.0. 5. 2.5. 4. 2.0 4.5. 3 3.0. Reflectance (%). Breast. Forehead. 4.0. 2.5. 3.5. 2.0. 3.0. 1.5. 2.5 4.0. 1.0 14. Neck. Remige. 3.5. 12. 3.0 10 2.5 8. 2.0 1.5 8.0. 6 7. Tail. Scapular 7.5. 6. 7.0 5 6.5 4 6.0 3. 5.5. 2. 5.0 300. 400. 500. 600. 300. 700. 400. 500. 600. 700. Wavelength (nm). Fig. 2.1. Spectra of ten characteristics in two sexes. Dotted lines indicate spectra of males and solid lines indicate females. 48.

(50) Tarsus. Sex: F0.05,2=5.95, p=0.016 Year: F0.05,2=52.18, p<0.0001 Sex*Year: F0.05,2=0.20, p=0.82. Sex: F0.05,2=0.73, p=0.40 Year: F0.05,2=12.40, p<0.0001 Sex*Year: F0.05,2=0.37, p=0.69. Sex: F0.05,2=0.34, p=0.56 Year: F0.05,2=120.78, p<0.0001 Sex*Year: F0.05,2=0.57, p=0.57. Sex: F0.05,2=0.18, p=0.67 Year: F0.05,2=28.43, p<0.0001 Sex*Year: F0.05,2=2.42, p=0.09. Sex: F0.05,2=1.13, p=0.29 Year: F0.05,2=18.40, p<0.0001 Sex*Year: F0.05,2=0.64, p=0.53. Sex: F0.05,2=0.52, p=0.47 Year: F0.05,2=4.69, p<0.0112 Sex*Year: F0.05,2=3.790, p=0.03. Sex: F0.05,2=1.61, p=0.21 Year: F0.05,2=8.28, p=0.0005 Sex*Year: F0.05,2=09220, p=0.40. Sex: F0.05,2=1.38, p=0.24 Year: F0.05,2=13.03, p<0.0001 Sex*Year: F0.05,2=3.55, p=0.03. Chroma UV (%). Chroma RED (%). Brightness (%). Hue (wavelength,nm). Beak. 49.

(51) Fig. 2.2. The colorimetric variables in carotenoid-based bill and tarsi between sexes in live birds. The hollow dots indicate males and the solid dots indicate females. Two-way ANOVA test, factor “Year” included three categories: 2008, 2009 and 2011, factor “Sex” included two categories: female and male.. 50.

(52) Brightness (%) Beak. Chroma UV (%). Belly. Beak. Belly. *. Sex: F0.05,2=1.01, p=0.32 Year: F0.05,2=6.13, p=0.003. Nape. Sex: F0.05,2=0.00, p=0.99 Year: F0.05,2=0.19, p=0.83. Forehead. Sex: F0.05,2=0.09, p=0.77 Year: F0.05,2=0.70, p=0.50. Scapular. Sex: F0.05,2=0.12, p=0.73 Year: F0.05,2=55.84, p<0.001. Sex: F0.05,*2=8.67, p=0.004 Year: F0.05,2=10.07, p<0.001. Sex: F0.05,2=1.11, p=0.30 Year: F0.05,2=5.46, p=0.006. Breast. Nape. Sex: F0.05,2=0.76, p=0.83 Year: F0.05,2=14.43, p<0.001. Sex: F0.05,2=0.37 p=0.54 Year: F0.05,2=1.69, p=0.19. Remige. Forehead. Sex: F0.05,2=2.05, p=0.16 Year: F0.05,2=3.18, p=0.046. Sex: F0.05,2=0.00 p=0.99 Year: F0.05,2=1.37, p=0.26. Tail. Scapular. Sex: F0.05,2=0.59, p=0.45 Year: F0.05,2=1.26, p=0.29. Sex: F0.05,2=0.06, p=0.80 Year: F0.05,2=13.48, p<0.001. Sex: F0.05,2=3.72 p=0.06 Year: F0.05,2=10.75, p<0.001. Breast. Sex: F0.05,2=0.15 p=0.70 Year: F0.05,2=10.89, p<0.001. Remige. Sex: F0.05,2=0.49, p=0.49 Year: F0.05,2=62.34, p<0.001. Tail. Sex: F0.05,2=1.63, p=0.21 Year: F0.05,2=1.82, p=0.17. Fig. 2.3. The colorimetric variables in melanin-based characteristics between sexes in live birds. The hollow dots indicate males, and the solid dots indicate females. Two-way ANOVA test factor “Year” included three categories: 2008, 2009 and 2011, factor “Sex” included two categories: female and male. The result of interaction between two factors in each colorimetric variable was not listed because of the insignificant effect of it. The asterisk indicates the significant effect of the factor (adjusted p < 0.005).. 51.

(53) Chapter Three. Negative effects of molting on carotenoid-based characteristics and individuals’ physical condition in the Himalayan black bulbul (Hypsipetes leucocephalus nigerrimus). 52.

(54) Abstract Traits that compete limited internal resources against other somatic needs could lead to trade-offs of resource allocations between them; their expression could therefore be correlate. Because the amount of carotenoids available for use is limited and carotenoids can be used in many physical functions, trade-offs arising among carotenoid-based ornaments and other physical demands. Molting is energetically and nutritionally demanding, but the effects of molting on major somatic needs are vague and contentious. In this study, I aimed to investigate whether carotenoid-based ornaments are quality cues that could reflect individuals’ physical condition following severe physical stress, namely molting induced by artificial plucking in Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus). I conducted a 2×2 grouping design experiment involving plucking and carotenoid supplementation. My data showed that bill redness was positively correlated with an individual’s immunocompetence and negatively correlated with oxidative stress level it experienced, but such correlations were not found in the carotenoid-supplemented group. My results suggest that the bill is a quality cue in black bulbuls, and additional carotenoids would reduce the negative effect of molting on both decorative traits and physical condition. Keywords: carotenoid-based ornaments, Heterocyte/liphocyte ratio, Himalayan black bulbul (Hypsipetes leucocephalus nigerrimus), molting, quality cues, PHA test.. 53.

(55) Introduction Competition for limited internal resources among different functions could lead to life history trait trade-offs, which may occur between physiological traits expressed either during the same stage or different stages of the life cycle (Zera and Harshman 2001). Signal traits, which could reflect individuals’ physical condition or agonistic abilities, are usually costly to their bearers because these traits share resources with other physically demanding ones (e.g., Spencer et al. 2003). Consequently, trade-offs between variable life-history traits and those that could serve as signal traits are to be expected. Among different types of signal traits, carotenoid-based ornaments are the most studied till date. Besides their ability to producing striking colors (i.e. yellow, orange and red, Fox and Vevers 1960, Latscha 1990), their powerful antioxidant properties also make carotenoids crucial to several life-history traits like breeding (McGraw et al. 2005, Surai 2002), migration (Alan et al. 2013, Metzger and Bairlein 2011) and molting (e.g., McGraw et al. 2006). Nevertheless, animals can only acquire carotenoids from food, and the ability of an animal to utilize the carotenoids in food depends on its genotypes and physiological condition (Alonso-Alvarez et al. 2004, Olson and Owens 1998). It has been suggested that the quantities of carotenoids available to individuals are limited, although carotenoids may be abundant in the natural environments (Olson and Owens 1998). Due to the limited availability of carotenoids, individuals may have to trade-off carotenoids among different life history traits (Alonso-Alvarez et al. 2004). For instance, females may have to balance the benefits of carotenoids with those investing in offspring quality, or of impairing their 54.

(56) own antioxidant damage during reproduction (Bertrand et al. 2006, Biard et al. 2005). In migratory birds, early-arriving males, who usually have a longer reproductive season and greater reproductive success (e.g., Klomp 1970, Perrins 1970), may be strong enough to allocate carotenoids into coloration rather than use them as antioxidants during migration (Ninni et al. 2004). In summary, the appropriate allocation of carotenoids among life traits and physiological functions (i.e., decorative plumage and antioxidation) are necessary in order to maximize individual fitness (e.g. Bertrand et al. 2006, Biard et al. 2005, Faivre et al. 2003, Nordeide et al. 2008). Molting is energetically and nutritionally demanding (Jenni and Winkler 1994, Klaassen 1995, Kuenzel 2003, Lindstrom et al. 1993) and would decrease the accumulation of circulating plasma carotenoids (Barbosa et al. 2013, Del Val et al. 2014, Del Val et al. 2013). However, to the best of my knowledge, studies that addressing whether molting can affect the expression of carotenoid-based characteristics have been rare. The only relevant study showed that molting speed constrains the expression of yellow throat in the rock sparrows (Petronia petronia, (Serra et al. 2007). Meanwhile, the effect of molting on the expression of carotenoids on the bared ornaments like bill, tarsus, or dewlap is also unknown. It is suggested that the expression of carotenoid-based bared parts could reflect an individual’s physical condition faster than that of plumage (e.g. Ardia et al. 2010, Faivre et al. 2003). The effects of molting on major somatic functions are vague and contentious. With immunocompetence, an indicator of the individual’s immune response, molting has either positive (Sanz et al. 2004) or negative (Martin 2005, 55.

(57) Sanz et al. 2004) correlations, or none at all (Pap et al. 2008). Although it is suggested that oxidative stress is higher during or after molting due to the depletion of plasma carotenoids, the effects of molting on an individual’s oxidative stress level has not been tested directly (Del Val et al. 2013). To the best of my knowledge, only one study has found that individuals’ oxidative stress levels did not change by natural molting in great tits (Parus major, Vaugoyeau et al. 2015). Knowledge of the effect of carotenoid abundance on molting is lacking; therefore, I would like to know whether the effect of molting can be diminished by carotenoid supplementation, because carotenoids are powerful antioxidants. In this study, I aim to investigate whether carotenoid-based ornaments are the quality cues that reflect individuals’ physical condition after a severe physical stress. I used Himalayan black bulbuls (Hypsipetes leucocephalus nigerrimus), which have carotenoid-based bills and tarsi, as the study species to examine whether the expression of carotenoid-based characteristics and individuals’ physical condition (both immunocompetence and oxidative stress level) could be affected under the stress of molting. Traditionally, the study of molting effects in birds involves the natural induction of molting, but this may be inappropriate under the current study conditions. Because both natural molting and carotenoid allocation can be influenced by hormones, such as testosterone, thyroxine, or corticosterone (Cherel et al. 1988, Rehder et al. 1986), it would be difficult to control the confounding effects of hormones when conducting experiments based on natural molting. I therefore plucked feathers directly from study animals to initiate molting artificially. A 2×2 grouping design was utilized within the carotenoid supplementary group, 56.

(58) tail and secondary feathers were pulled off on half of the birds, while the other half of them served as a control group with their feathers intact. I predicted that molting would have negative effects on the physical condition and cause trade-off of carotenoids allocation in black bulbuls; therefore, I should have observed a significant trade-off between decorative traits and individuals’ physical condition in the molting group. I also predicted that the effects of molting could be diminished when the carotenoids were in abundant supply.. Materials and methods Captive setting and sampling Thirty-one Himalayan black bulbuls were purchased from a pet-shop in 2010. They were individually housed in cages with opaque covers between them, so that the birds could have acoustic contact but not visual perception of others. They were placed in a room at constant temperature (27℃) and humidity (80%). Blood (150 μL) was collected from each bird using a heparinized capillary tube. Blood samples were stored in a −20°C freezer for molecular sex-typing. In addition, a drop of blood, approximately 5μL, was put on a slide to assay the oxidative stress level.. Molecular sex typing Genomic DNA was extracted from blood samples with traditional proteinase K digestion followed by LiCl extraction (Gemmell and Akiyama 1996). The polymerase chain reactions (PCRs) program used for molecular sex typing (Fridolfsson and Ellegren 1999) was the same as 57.

(59) that described in Hung et al. (2015 in revision).. Experimental procedures Experimental design Thirty-one birds were used in this experiment. After sex typing, individuals of each sex were randomly and equally assigned to four treatment groups: carotenoid-supplemented and plucked [Caro(+), plucked, F = 4, M = 4], carotenoid-supplemented and unplucked [Caro(+), unplucked, F = 2, M = 4], carotenoid-unsupplemented and pluckded [Caro(-), plucked, F = 3, M = 4], or control [Caro(-), unplucked, F = 5, M = 5]. In the plucked group, individuals’ tails and secondary feathers had been removed. Carotenoid supplementation was initiated on February, 10, 2010, a month prior to the molting experiment. The molting experiment was initiated on the March, 8, 2010, this day also served as the baseline for the immune challenge experiment. During the experimental period, only commercial feed formulated with or without supplementary carotenoids was fed. Oxidative stress testing was conducted on two dates: the baseline day, on which tails and secondary feathers were removed and the testing date, on which the tails and secondary feathers of individuals in the experimental group had grown to half of their average length (approximately three weeks later, around 5 to 5.5 cm for tail feathers and 1.5 to 2.0 cm for secondary feathers). The immune challenge was conducted only on the testing day. Carotenoid-supplementation experiment Each individual was fed 40 grams of commercial bird food every day. In the carotenoid-supplemented groups, the additional canthaxanthin (0.9 58.

(60) mg/40 g, Orpharma, Belgium) and lutein (0.32 mg/40g, Orpharma, Belgium) were added to the commercial bird foods. Physiological conditions Immune challenge: Phytohaemagglutinin assay (PHA assay) The PHA assay was conducted on May, 26, 2010. PHA solution (80μl of 2.5ng/μL, Sigma L-1668, Sigma Chemical Co., St. Louis, Mo, USA) was injected into individuals, according to the method of Smits et al. (1999). A pressure-sensitive caliper (TecLock Inc., Japan) was used to measure the thickness of the right wing web before injection to the nearest 0.01 mm. Twenty-four hours after the injection, the level of swelling was measured on the wing web of each individual to determine the degree of immune response (Smits et al. 1999). Individuals with larger swollen wing webs were considered to have stronger immune responses. Oxidative stress test: Heterocyte to lymphocyte ratio (H/L ratio) I calculated the ratio of lymphocytes (L) to heterocytes (H) in a total of 100 leukocytes as a measure of oxidative stress for each bulbul individual, according to the method of Vleck et al. (2000). The detailed procedures were the same as that described by Hung et al. (2015 in revision). A higher H/L ratio indicates that the individual were under a higher level of oxidative stress (Gross and Siegel 1983). The assay of oxidative stress was conducted on the baseline and testing dates. Molting speed measurements To test whether carotenoid supplementation affected molting speed, I measured the regrowth lengths of tail and secondary feathers on the testing date in the plucked groups, then divided by 21 days to determine each individual’s molting speed. 59.