THE EFFECT OF MALE PLUMAGE COLORATION ON PARENTAL EFFORT IN EASTERN BLUEBIRDS (Sialia sialis) by Maria Sara Costantini A thesis submited to the Graduate Faculty of Auburn University in partial fulfilment of the requirements for the Degree of Master of Science Auburn, Alabama August 3, 2013 Copyright 2013 by Maria Sara Costantini Approved by Jack Feminela, Chair, Profesor of Biological Sciences Geoffrey Hil, Profesor of Biological Sciences Wendy Hood, Asistant Profesor of Biological Sciences Haruka Wada, Asistant Profesor of Biological Sciences i THESIS ABSTRACT In iteroparous species, there is tradeoff betwen current and future reproductive investment. Parents that invest more in current offspring wil provide resources that give those offspring beter chances of survival and reproductive succes. If the current brood is of poor quality, parents may invest les and save energy for other proceses, as wel as future reproduction, maximizing lifetime reproductive succes. The diferential alocation hypothesis predicts that individuals wil adjust their parental investment based on the quality of their mate. Females wil invest more in offspring of atractive males than unatractive males. In response, atractive males wil invest les in current reproduction and alocate more time and resources to other proceses. In the field, I tested the diferential alocation hypothesis in the socialy monogamous Eastern Bluebird (Sialia sialis). I experimentaly manipulated the brightnes of the structural blue coloration of Eastern Bluebirds, which is a known condition-dependent trait in the species. The brightnes of male birds was either enhanced using a violet marker or reduced using a black marker. I recorded nest visitation rates prior to and after marker application to test the efects of treatment on both female and male visitation rates. I then tested the efect of treatment on offspring growth rates. I predicted that 1) females mated to brighter males would visit the nest more than females mated to duller males, 2) brighter males would visit the nest les than duller males, and 3) offspring of brighter males would grow at a faster rate than offspring of duller males. I found a ii trend suggesting that females of brighter males provision offspring at a higher rate than females mated to duller males, but there was no significant diference betwen males of each group. Tarsus growth rate of chicks betwen the groups did not difer, but chicks of the bright treatment gained mas at a faster rate than chicks of the dull treatment. The significant efect of treatment on chick body mas supported the trend demonstrated in female provisioning. I suggest that diferential alocation by female bluebirds has the potential to create a selective presure on male plumage brightnes through enhanced offspring growth rates. iii ACKNOWLEDGEMENTS There are several people that I owe a great deal of gratitude to for helping me stay calm and on tract during this proces. I am beyond lucky to have been able to work under both Geoffrey Hil and Wendy Hood. Both of who were incredibly patient and provided countles insights and suggestions and have helped me as I start my path to becoming a scientist. I would also like to thank Haruka Wada for al her help, especialy for doing so while out of the country. I am grateful to Bob Montgomerie for alowing me to use his spectral procesing program CLR (v1.05). Al my lab-mates were crucial in perfecting my seminar talk and I appreciate their suggestions tremendously. I could not have made it through my field season without the help of undergraduates in the Hood lab, especialy A.J. Pate. Finaly, I would like to thank my parents and family for being confident in me, even when I could not be confident in myself. iv iv Style manual or journal used: Auk Computer Software used: Microsoft Word v TABLE OF CONTENTS ABSTRACT..................................................................................................................................ii ACKNOWLEDGEMENTS.........................................................................................................iv LIST OF FIGURES.....................................................................................................................vi CHAPTER 1: THE EFECT OF MALE BRIGHTNES ON PARENTAL NEST VISITATION RATE............................................................................................................................................1 Introduction .....................................................................................................................1 Methods ............................................................................................................................4 Results ..............................................................................................................................7 Discussion.........................................................................................................................8 CHAPTER 2: CAN ALTERED MALE PHENOTYPE AFECT OFSPRING GROWTH RATES?......................................................................................................................................16 Introduction ...................................................................................................................16 Methods ..........................................................................................................................20 Results ............................................................................................................................22 Discussion.......................................................................................................................23 LITERATURE CITED .............................................................................................................29 vi LIST OF FIGURES CHAPTER 1 Figure 1. A timeline of nesting sequence and procedures in the study ........................................7 Figure 2. Reflectance spectra of male plumage coloration showing the efect of treatment by permanent markers on rump feathers ...........................................................................................9 Figure 3. Change in the rate at which female Eastern Bluebirds visited nestlings following experimental manipulation of the color of the male at days 7 and 10 .......................................11 Figure 4. Change in the rate at which male Eastern Bluebirds visited nestlings following experimental manipulation of the color of the male at days 7 and 10........................................21 CHAPTER 2 Figure 1. A timeline of Eastern Bluebird nesting sequence and procedures in the study .........41 Figure 2. Change in clutch tarsus length of Eastern Bluebird chicks from day 5 ......................48 Figure 3. Change in mean body mas of Eastern Bluebird chicks from day 5 ..........................50 1 THE EFFECT OF MALE BRIGHTNESS ON PARENTAL NEST VISITATION RATE INTRODUCTION Indicator models of sexual selection suggest that elaborate ornaments in animals evolve to signal phenotypic or genetic quality (Zahavi 1975; Andersson 1994). Such signals can be used in mate choice or aid in intrasexual competition. In species with biparental care, these signals can indicate the quality of a variety of characteristics that could benefit potential mates, such as genetic quality, parental ability, physiological condition and resistance to stresors, parasite resistance, and competitive ability (Hil 2011). Honest signaling can evolve as a consequence of indirect or direct benefits of mate choice (Andersson 1994). Indirect benefit models suggest that there is a positive genetic link betwen an ornament and fitnes (Kokko et al. 2002) and that heritably of the sexualy selected trait is high (Kirkpatrick and Barton 1997). In contrast, direct benefits of sexual selection are more important when heritability of the trait is low (Kokko 1998). In birds, the most important direct benefits are typicaly the level of parental care (Heywood 1989). The Good Parent Hypothesis, suggests that sexualy selected ornaments signal parental quality (Hoelzer 1989). Under this hypothesis, individuals choose mates based on traits that signal potential parental quality. Several studies of bird coloration have supported the Good Parent Hypothesis (Senar et al. 2002; Maguire and Safran 2010). Males with more brightly colored feathers have been shown to provision offspring more frequently, acquire limited nest sites more succesfully, and produce heavier offspring (Sieferman and Hil 2003; Sieferman and Hill 2005a; Grindstaf et al. 2012). Another hypothesis addresing parental care, specificaly in species with biparental care is the diferential alocation hypothesis. This hypothesis predicts 2 that individuals adjust their own parental investment based on the quality of their mate; mates of higher quality individuals are expected to provide more parental care than mates of les quality individuals, regardles of that individual?s contributions (Burley 1986). A second part of the hypothesis states that higher quality mates wil reduce parental investment to save energy for future reproductive atempts, as wel as increase its chance of survival, thus leading to directional selection on ornamental traits (Burley 1986). Parents are constantly faced with decisions related to the trade-off betwen current and future reproduction (Trivers 1972). Reproduction, as wel as behaviors asociated with parental investment, are a great energetic burden that afects the condition and ultimately the survival of individuals (Trivers 1972). Sieferman and Hil (2005) supported this hypothesis when they observed that male Eastern bluebirds (Sialia sialis) trade future ornamentation for current reproductive investment. When broods were experimentaly reduced, males invested les in parental care and the reduction had a significant positive efect plumage brightnes the following year. The diferential alocation hypothesis directly addreses Trivers (1972) description of the trade-off betwen current and future reproductive atempts. Eforts to increase the fitnes of current offspring are expected to increase costs asociated with parents? own future survival and reproduction. Therefore, it is in the parent?s best interest to adjust parental investment to the apparent value of the current offspring in each breeding atempt. In avian species, diferential alocation can occur at various breeding stages. For example, females can alter their investment in the quantity or quality of eggs produced (Johnsen et al. 2005), or through diferential parental efort (Burley 1988; Johnsen et al. 2005; Limbourg et al. 2013). If the current mate is perceived as of higher quality and the probability that future mates are of equal or higher quality is low, then current offspring wil have a higher value than 3 the expected value of future offspring. Parental care is costly (Clutton-Brock 1991), so selective investment must be balanced by a benefit (Moller and Thornhil 1998), such as more atractive and higher quality offspring. I experimentaly tested the diferential alocation hypothesis in the field on a breeding population of Eastern bluebirds in Alabama. The Eastern bluebird is a socialy monogamous, sexualy dimorphic species that exhibits biparental care (Gowaty and Plisner 1998). Males display a structural UV-blue coloration on the rump, back, head, wings, and tail. Females display the same, but duller plumage patern. The UV-blue plumage is produced through modification of the nanostructure of feather barbs (Shawkey et al. 2005; Shawkey et al. 2006). A feather barb has an outer keratin cortex, beneath which is a spongy layer of air spaces and keratin rods. The precise size and arrangement of those nanostructures alows for coherent scatering of light that alows short wavelengths of light, blue and violet colors, to be reflected and amplified, while longer wavelengths of light to get absorbed by melanin under the spongy layer. The blue plumage brightnes of bluebirds has been shown to correlate with individual condition in both males and females (Sieferman and Hil 2005b; Sieferman and Hill 2005a; Sieferman et al. 2005; Grindstaf et al. 2012). Individual condition, particularly that asociated with nutrition, is believed to afect structural coloration due to deficiencies in the precision of the formation of feather nanostructure (Hil 2006a). Structural coloration has also been shown to be a positive indicator of offspring provisioning (Keyser and Hil 2000; Sieferman and Hil 2003; Limbourg et al. 2004; Johnsen et al. 2005; Grindstaf et al. 2012). Many previous studies have tested the diferential alocation hypothesis by altering male phenotypic traits asociated with quality, often by using artificial traits (Burley 1988; Swaddle 1996) or by manipulating outside the natural variation of an ornament (de Lope and Moller 1993; 4 Limbourg et al. 2004). A beter approach is to manipulate the trait within the range of natural variation of male atractivenes (Hil 2006b), as in a study of male blue tits (Cyanistes caeruleus) (Johnsen et al. 2005). I altered male Eastern bluebird plumage coloration using either a black marker to decrease the brightnes of the feathers making them appear duller or a violet marker to increase the brightnes of the feathers and the perceived quality of the individual. It has previously been demonstrated that these methods can alter plumage color within its range of natural variation (Liu et al. 2007). I then quantified the change in rate of parental visitations for both treatment groups. In acordance with the diferential alocation hypothesis, I predicted that (1) females paired to males with enhanced blue plumage brightnes would have a higher nestling visitation rate than females paired with experimentaly reduced blue plumage brightnes, and also that, (2) males with enhanced blue plumage brightnes would have a lower nestling visitation rate than males with reduced blue plumage brightnes. METHODS Study Species.- The Eastern bluebird is a medium-sized songbird that breeds throughout eastern North America (Gowaty and Plisner 1998). Eastern Bluebirds are cavity-nesters and adults practice biparental care of offspring. Adult males have briliant blue plumage on their heads, backs, rumps, tails, and wings, while their upper breast feathers are a warm orange-brown color and belies white. This blue coloration has a spectral reflectance peak at approximately 400 nm and is known to reflect UV and blue wavelengths equaly (Sieferman and Hil 2003). Adult female bluebirds are paler and blue-gray on top with dull blue wing and tail feathers and pale orange breasts. 5 Study Site and General Field Procedures.- The study was conducted in early April through July of 2012. Two groups of nestboxes were monitored (approximately 11.3 km apart) in Auburn, Le County, Alabama. Bluebirds at these locations have been banded since 1999. Any adult birds that were not previously banded were banded upon capture with a U.S. Fish and Wildlife aluminum band and a unique set of 3 color bands. Al pairs of bluebirds included in the study were rearing their second clutch that year. The first day of hatching for a nestbox was clasified as day 1. I caught adult males atending chicks on day 5 post-hatching for color manipulation. Males were mist neted betwen the 1000 and 1200 h, and a second atempt was made at 1700 and 1900 h, as necesary. Nest visitation rate was recorded 3 hr on days 7 and 10 starting at sunrise (Se Fig. 1 for timeline). I used Eastern Bluebird playback cals and a male bluebird museum skin to atract males to the net. I carefully plucked 7-10 rump feathers from each bird. Al feathers were placed in sealed envelopes for future spectrometry analysis. Previous studies have demonstrated that permanent markers can be used to experimentaly enhance or reduce structural plumage brightnes within the natural range of variation, while not eliminating UV reflectance (Balentine and Hil 2003; Liu et al. 2007). Plumage brightnes has been shown to be condition dependent in the Eastern Bluebird, while hue and chroma have not, therefore brightnes is what was manipulated in this experiment (Sieferman and Hill 2005a). Because structural coloration is produced by feather nanostructure, rather than pigments, feathers colored with markers stil looked blue to a human observer (Liu et al. 2007). I used non-toxic, permanent markers (Sharpie ? permanent marker) to either decrease brightnes of the natural plumage with a black marker or increase brightnes with a violet marker. I carefully colored al feathers on the head, back, rump, tail, and wings of each bird. To confirm that reflectance spectra of the manipulated feathers reflected natural variation in color 6 within the population (as shown by Liu et al. 2007), feathers were collected from male birds outside the study for post-manipulation samples. After color application, the bird was placed in a brown paper bag 1-2 minutes to alow the color to dry, and then birds were released. Nestboxes were asigned to a treatment group randomly for each location. Visitation Rate.- All nestboxes where a male was succesfully captured and color manipulated were monitored for parental visitation. Birds were first monitored with cameras on day 5, prior to color manipulation to determine initial visitation rate. On days 7 and 10, activity at nestboxes was recorded with a Reconyx game camera for 3 hr, starting at sunrise. Pictures were taken at a rate of one per second and thus, making it unlikely that visitations were mised. The sex of the parents feding the chick was determined based on characteristic plumage. If I could not consistently determine the diference betwen the male and female of a particular box, that box was not used in the study. Parental visitation rate was quantified rather than feding rate because I could not be confident the male or female brought food to the nest with each visit. A visitation atempt was counted when the adult inserted its full head into the nestbox. The treatment group that each male belonged to was blind to the observer quantifying visitations at the nest avoid biases. The number of visitations was recorded for males and females on day 5 prior to treatment, and then 2 days and 5 days post-treatment, which was equal to day 7 and 10 post-hatching. Relative visitation rate was quantified relative to the visitation rate prior to the color manipulation, as the number of visits on day 7 divided by the number of visits on day 5, and number of visits on day 10 over the number of visits on day 5 Spectrometry Analysis.- The rump feathers collected from the adult males were taped in an overlapping patern to recreate a colored patch and then along with the tail feathers taped onto black construction paper. Plumage reflectance was measured using an Ocean Optics S2000 7 spectrometer and deuterium tungsten halogen light source (Dunedin, Florida, USA) and a micron fiber-optic probe with a rubber cap (to exclude ambient light) held at a 90? angle to the feather surface. I took five measurements for each feather sample and averaged them to represent the UV-blue coloration of each male. The program CLR (v1.05) was used for spectral analysis. I produced spectral curves for normal and manipulated feathers to show how the manipulation afected the percent reflectance. CLR calculates mean brightnes as the average reflectance in the 300-700 nm range. Statistical Analysis.- Al analyses were performed with JMP 10.0.2 (SAS Institute, Cary, NC) and p-value of < 0.05 was considered statisticaly significant. To determine if the treatment groups had equal variance, clutch size, pre-treatment nest visitation rate, and male coloration pre- treatment were compared betwen groups using a two-sample t-test. . A t test was also used to show that there was a treatment efect on brightnes of plumage from the markers. Controlling for the efects of repeated data within individual (day 7 and 10), a repeated measures analysis of variance (ANOVA) was used to compare the relative visitation rate betwen treatment groups at day 7 and day 10. Nest visitation rates were transformed prior to analysis. RESULTS Clutch Size and Pre-Treatment Variables.- There was no diference in clutch size betwen treatments (t= 0, df= 17.6, P= 1.0). The number of times males (t= 0.63, df= 13, P= 0.537) and females (t= 1.45, df= 12.79, P= 0.173) visited the nest did not vary betwen groups prior to treatment application. Pre-treatment male plumage brightnes on day 5 did not vary betwen groups (t= -0.92, df= 6.05, P=0.395). 8 Spectrometry.- Both treatments produced spectral curves characteristic of blue (Fig. 2). Mean brightnes for the feathers of al groups fel within the natural range of variation as reported by Liu et al. (2007). The bright treatment had a mean brightnes significantly higher than the dull treatment (t= -4.79, df= 5.03, P= 0.005). Female Visitation Rate.- The overal model testing for the efect of treatment, days post- manipulation, and individual ID on female relative visitation rate bordered on significance (overal: F 15, 8 =2.93, P=0.065), with females paired to more brightly colored males increasing their number of visitations relatively more than females paired to dull colored males (treatment efect: partial F 1 = 15.08 P= 0.005) (Fig. 3). Relative provisioning rate did not vary betwen day 7 and 10 (repeatability efect: partial F 1 = 4.97, P= 0.056) and visitation rate did not vary betwen individual females (ID efect: partial F 13 = 1.67, P= 0.238). Male Visitation Rate.- The overal model testing for the efects of treatment, days post- manipulation, and individual on male relative visitation rate was significant (overal: F 15,8 = 4.18, P= 0.024), but this efect was driven by a significant variation in visitation rate betwen individual males (ID efect: partial F 13 = 4.10, p= 0.026). Male plumage brightnes (treatment efect: partial F 1 = 3.63, P= 0.093) and relative provisioning rate betwen day 7 and 10 (repeatability efect: partial F 1 = 0.06, P= 0.807) was not significant (Fig. 4). DISCUSSION Burley?s (1986) diferential alocation hypothesis predicts that because there is a trade-off betwen current and future reproduction, mates of higher quality individuals wil invest more in the current offspring than mates of lower quality individuals. The results of this study are suggestive that diferential alocation may exist in Eastern Bluebirds. There was a trend 9 suggesting that females mated to brighter males increase their visitation rate to a greater degree as chicks age that females mated to duller males, suggesting that female may interpret their mates as in beter or worse condition, respectively. Similar results were found in other studies testing the diferential alocation hypothesis. In blue tits, Johnsen et al. (2005) found male color typicaly does not impact female provisioning rates, with the exception of first time breeders (1- year olds) that did display diferential alocation. In a separate study, Limbourg et al. (2004) showed that blue tit females decreased their feding rates when males displayed reduced coloration. Visitation rate was used as a measure of parental efort because it could not be reliably determined whether parents carried food upon entering the nestbox. Nevertheles, the behaviors displayed at each visit were suggestive of feder and thus, feding and nest visitation should be strongly correlated. As nestlings age and grow they require greater quantities of food, thus parental visitation rate to increase through nestling development. Although the change in visitation rate betwen day 5 and 7 and 5 and 10 average greater than 1 across al treatment groups, there was no diference in provisioning rates betwen days 7 and 10. It is possible that no low sample size contributed to lack of diference betwen these two time points. Under the diferential alocation hypothesis, mate quality is used as a tool for making decisions betwen current and future reproduction. Females may be more likely to expres positive diferent alocation earlier in a breeding season, or earlier in a lifetime, when there is a higher possibility of more breeding atempts. It is in best interest for individuals to maximize their investment in the best quality mates. This may explain why only young females significantly expresed positive diferential alocation in Limbourg et al. 2004. Unfortunately, the sample size was not large enough in this study to test for this efect. 10 In Burley?s (1986) original presentation of the diferential alocation hypothesis, it is stated that individuals may increase their parental investment to either obtain or maintain their high quality mates. Another interpretation for the trend of more maternal investment displayed by females when mated to high quality mates is that they are trying to maintain their mate for future reproduction. If females mated to brighter males display increased parental efort, then males are expected to decrease their own parental investment in response to save energy for future reproduction or extra-pair mating atempts. Moller and Thornhil (1998) showed that diferential alocation strategies are asociated with higher frequencies of extra-pair paternity, and it has been specificaly demonstrated in the closely related Mountain Bluebird (Sialia currucoides) that brighter blue males sired more offspring both with their own mate and extra- pair mates than duller males (Balenger et al. 2009). It is possible that higher quality males have the chance to breed with higher quality females in subsequent breeding atempts. Then, if a female recognizes that her mate is in good condition and can provide benefits to her offspring, she may increase her parental investment to retain her mate for future reproduction. Although males cannot judge their own altered phenotype, it is most likely that they receive cues from the female about their phenotypic condition (Johnsen et al. 2005). These cues could be transferred through brief interactions made with their mate during the nestling period or could be cues from the increased parental efort by the females. Burley (1986) predicted that high quality mates would decrease their parental investment as compensation for the increase parental investment in their mates. She later found support for this claim in zebra finches (Taeniopygia guttata) where the male?s phenotypic was manipulated with color bands (Burley 1988). ?Atractive? males decreased their parental investment as females increased their parental investment. A similar result was observed in the study done by Johnsen et al. (2005) on blue tits. 11 UV reduced (les atractive) males fed at a higher rate than UV enhanced (more atractive) males. My study demonstrated that male Eastern Bluebirds that were experimentaly made brighter showed no diference in nest visitation from males that were dulled, which does not support the second prediction made by the diferential alocation hypothesis. This finding agrees with a similar study done on blue tits where they manipulated male atractivenes by eliminating UV reflectance in a group of males (Limbourg et al. 2004). However, it disagrees with what has been observed in other studies of diferential alocation on birds. Diferences betwen this study and other studies may be an efect of treatment time period; males may respond diferently based on the time of the nestling stage that manipulation occurs. This study demonstrates support for primary predictions of the diferential alocation hypothesis; individuals paired to brighter mates, have higher parental investment than individuals paired to duller mates. Female bluebirds showed a trend for a greater change in nest visitation rate when paired with brighter males. However, my observations did not support the second asumption made by the diferential alocation hypothesis that high quality mates wil have reduced parental investment to save for future reproduction. Females appear to be constantly acesing the condition of their mate to make decisions regarding the tradeoff betwen current and future reproduction. The level of parental efort expresed by female bluebirds has the ability to create directional selection presures on male structural coloration. 12 Figure 1. A timeline of nesting sequence and procedures in the study. Numbers in timeline represent days from hatching of eggs. 13 Figure 2. Reflectance spectra of male plumage coloration showing the efect of treatment by permanent markers on rump feathers. The think black line represents the natural male plumage coloration before treatment. The gray line represents the brightnes-enhanced treatment by violet markers. The thick black line represents the spectral curve of the brightnes-reduced treatment by black markers. 14 0" 2" 4" 6" Day$7$ Day$10$ Ch a n g e$i n $N es t $Vi si t a 2o n $fr o m$ Pr e8t r ea t men t $ Bright$ Dull$ Figure 3. Change in the rate at which female Eastern Bluebirds visited nestlings following experimental manipulation of the color of the male at days 7 and 10. Dark columns represent females paired with males of enhanced plumage brightnes. Light columns indicate females paired with males of reduced plumage brightnes. Visitation rate is presented relative to the rate of visitation before manipulation on day 5. 15 0" 0.5" 1" 1.5" 2" Day$7$ Day$10$ Ch a n g e$i n $N es t $Vi si t a 2o n $fr o m$ Pr e8t r ea t men t $ Bright$ Dull$ Figure 4. Change in the rate at which male Eastern Bluebirds visited nestlings following experimental manipulation of the color of the male at days 7 and 10. Dark columns represent males with enhanced plumage brightnes. Light columns indicate males with reduced plumage brightnes. Visitation rate is presented relative to the rate of visitation before manipulation on day 5. 16 CAN ALTERED MALE PHENOTYPE AFECT OFSPRING ROWTH RATES? INTRODUCTION In species with repeated reproduction, a tradeoff many occur betwen current and future reproductive efort (Trivers 1972). This conflict arises because, while parental investment is beneficial for viable offspring, it presents a substantial cost in terms of reduced survival or fecundity to the parent (Clutton-Brock 1991). Species with biparental care deal with an added element to the conflict, because there is often a negotiation betwen both sexes over the level of investment (Hinde 2006). In birds and other species with ornamental displays, the relative level of parental investment is often correlated with the quality of ornamental traits (e.g. refs). Theses traits can be both exaggerated and costly and may even adversely afect survival (Darwin 1871). Ornamental traits are typicaly asociated with honest signals of genetic quality (Zahavi 1975). But Hoelzer (1989) suggested by the Good Parent Hypothesis that ornamental traits could be sexualy selected as indicators of potential parental ability. In species where parental ability is correlated with the execution of a trait, it is in the best interest of one individual to mate with another with a higher quality display that wil not only provide more parental care, but wil pas along genes asociated with a higher quality display. The ability to acquire good nesting sites, provide nest defense, and fed nestlings and mates are examples of direct benefits provided by good parents (Heywood 1989; Hoelzer 1989). Traits asociated with parental ability are often used in negotiations betwen parents in relative level of care (Hinde 2006), as wel as in tradeoffs betwen current and future breeding atempts (Burley 1986). If mate selection is driven by selection for parenting ability, as suggested by good parent hypothesis, then it is also probably that relative parental efort may be adjusted based on the 17 quality of that mate (Burley 1986). The diferential alocation hypothesis, as described in chapter 1, asumes that: 1) the potential for mates with high quality ornaments to produce higher quality offspring must be greater than mates with low quality ornaments, 2) resource investment by the choosing sex must be costly, creating a tradeoff betwen investment and condition, and 3) the resource must be beneficial to the offspring of the current reproductive atempt (Sheldon 2000). Most often it is the female that is diferentialy alocating parental investment based on the atractivenes of the male. It is asumed that females of most species have a finite, renewable amount of resources to use for reproduction (Harris and Uler 2009). Thus, by the diferential alocation hypothesis, females are expected to invest more in the most viable offspring. Females that invest more in a current reproductive atempt wil be at a selective disadvantage unles their investment is balanced, or preferably outweighed, by a benefit (Moller and Thornhil 1998). By investing more in the offspring of mates with that indicate high genetic and phenotypic quality, a female wil further increases the potential quality of the offspring. Females wil obtain an indirect fitnes benefit because their offspring should experience enhanced ornament quality ultimately resulting in a greater long-term fitnes payoff (Moller and Thornhil 1998). Several studies across diferent taxa have demonstrated support for the diferential alocation hypothesis (reviewed in Sheldon 2000). Most of these studies have focused on the direct benefits asociated with sexual selection, such as parental efort, nest defense, and clutch size. However, few studies testing diferential alocation have focused on the reproductive output and succes of offspring (Sheldon 2000). For the cost of diferentialy alocating resources to current offspring to be beneficial, enhanced offspring viability must exist (Burley 1986). Reproduction and parental care are costly endeavors, and those that invest more usualy suffer decreases in survival and condition in following years (Clutton-Brock 1991). If females invest 18 more in the current brood, without experiencing increased reproductive succes, than they are at a selective disadvantage (Moller and Thornhil 1998). Therefore, there is a need to not only to study parental investment as it relates to the diferential alocation hypothesis, but also to se if parental investment influences indirect genetic efects for the offspring. As a consequence to the diferential alocation hypothesis, the higher investment in offspring of atractive mates should lead to increased offspring condition (Limbourg et al. 2004). Not only direct genetic efects, but also indirect genetic efects, of parental quality influence offspring viability (Clutton-Brock 1991). Indirect genetic efects can occur whenever individuals provide non-genetic resources to offspring (Wolf et al. 1997). Wolf et al. (1997) also refers to these types of efects as ?inherited environments?, because while they are efects due to the environment in the offspring generation, the phenotypes of the parental generation that produced the environmental efects are heritable. Thus, if an individual diferentialy alocates resources based on mate atractivenes or quality, it can create a link betwen mate atractivenes and offspring condition (Harris and Uler 2009). Without an increase in offspring condition and survival, a parent?s efort is in vain. The Eastern Bluebird (Sialia sialias) is a wel-studied organism for its plumage coloration in relation to aspects of sexual selection and parental care. It is a socialy monogamous, sexualy dimorphic species with biparental care of offspring (Gowaty and Plisner 1998). Males display a briliant ultraviolet (UV) blue structural coloration on the rump, back, head, wings, and tail. Females display a similar, but duller blue-grey patern. The UV-blue structural coloration is due to changes in the nanostructure of the feathers (Shawkey et al. 2006). The nanostructure, which afects the quality of the structural coloration, is thought to be greatly afected by factors asociated with individual condition (Hil 2006). The blue plumage of Eastern 19 Bluebirds is considered an ornamental trait that has experimentaly been shown to correlate with factors asociated with condition in both sexes (Sieferman and Hill 2005a; Sieferman and Hil 2005b; Sieferman et al. 2005; Grindstaf et al. 2012). Structural coloration is also known to be asociated with parental ability in many species, including in Eastern Bluebirds (Keyser and Hil 2000; Sieferman and Hil 2003; Limbourg et al. 2004; Johnsen et al. 2005; Grindstaf et al. 2012). It has previously been demonstrated that more colorful bluebirds produce heavier offspring (Sieferman and Hil 2003; Grindstaf et al. 2012). Female bluebirds may use the blue plumage in males as an indicator of the value of potential offspring (Limbourg et al. 2004), and diferentialy alocate more resources to brighter blue males. If unatractive males receive decreased provisioning from females, their offspring wil experience hindered development of sexualy atractive traits, as wel as decreased body size (Limbourg et al. 2004). Smal body size in offspring is asociated with adverse efects on survival post fledging and competitive ability later in life (Garnet 1981; Alatalo and Lundberg 1986). In this study I experimentaly manipulated male Eastern Bluebird atractivenes by altering blue plumage brightnes. Natural variation of structural plumage is due to coherent scatering of light from the spongy layer of feather barbs (Shawkey et al. 2006). I was able to change male coloration to the extremes of the blue spectrum sen in nature using markers (protocol from Liu et al. 2007). Males were either made more atractive using a blue marker, or les atractive using a black marker. I tested whether females observed the change in phenotype, and invested more in ?higher quality? males as sen by altered offspring condition. Ofspring condition was quantified as the change in clutch growth rate. In Eastern Bluebirds, females that invest more in current reproduction are les likely to survive to the following year (Sieferman and Hil 2008). If the current mate is considered atractive, females are expected to provide more 20 care and produce offspring of beter quality, trading off their own survival and future reproductive suces. I expect offspring of experimentaly enhanced male color to be of beter condition than offspring of experimentaly reduced male color in Eastern Bluebirds. METHODS Study Site and General Field Procedures.- As described in chapter 1, monitoring of bluebird nesting began in early April and continued through July of 2012. Al data collected in this study was limited to the second clutch of each bluebird pairs. The day that the first egg of a clutch hatched was considered day 1. The to color manipulation was applied to males on day 5, as described in chapter 1. Following color manipulation, the chicks were removed from the nest and body mas and tarsus length was recorded for al chicks. Chick morphological measurements were recorded again on days 7 and 10 betwen the 1000 and 1200 hr (Se Fig. 1 for timeline of study) body mas was measured to the nearest 0.01 g using a digital balance. Tarsus length was measured to the nearest 0.01 mm using calipers. Wing chord and bil length were also measured, but were not used in analyses. Color Manipulation.- It has been demonstrated that for Eastern Bluebirds plumage brightnes, and not chroma or hue, is a condition-dependent trait (Sieferman and Hill 2005a), therefore brightnes was the component of structural plumage that was manipulated in this study. Permanent markers have been shown to be able to enhance or reduce structural plumage brightnes with the natural range of variation, while not eliminating UV reflectance (Balentine and Hil 2003; Liu et al. 2007). Because structural coloration is produced by feather nanostructure, rather than pigments, feathers colored with markers stil looked blue to a human observer and color measurements stil fal within the range of natural variation (Liu et al. 2007). I used non-toxic, permanent markers (Sharpie ? permanent marker) to either decrease with a 21 black marker or increase with a violet marker the brightnes of male natural plumage. Upon capture, I carefully colored al feathers on the head, back, rump, tail, and wings of each bird. Once color was applied, I placed the bird in a brown paper bag for 1-2 minutes to alow the color to dry, and then the birds were released near the nest. Nestboxes were asigned to treatment groups randomly for each location. Manipulated feathers were collected from male birds 2-3 days post-manipulation from separate nestboxes that were not a part of the study to confirm that reflectance spectra reflected natural variation in color within population, as shown previously by Liu et al. (2007). Chick Morphological Measurements.- Pre-treatment morphological measurements were taken for al chicks in a brood on day 5, and post-treatment measurements were taken on days 7 and 10 morphological measurements were taken again betwen the hours of 1000 and 1200. Mas was measured to the nearest 0.01 g using a digital balance. Tarsus length was measured to the nearest 0.01 mm using calipers. Wing chord and bil length were also measured, but were not used in analyses. Spectrometry Analysis.- The rump feathers collected from the adult males were taped in an overlapping patern to recreate a colored patch and then along with the tail feathers taped onto black construction paper. Plumage reflectance was measured using an Ocean Optics S2000 spectrometer and deuterium tungsten halogen light source (Dunedin, Florida, USA) and a micron fiber-optic probe with a rubber cap (to exclude ambient light) held at a 90? angle to the feather surface. I took five measurements for each feather sample and averaged them to represent the UV-blue coloration of each male. I used the program developed by Montgomerie (2008) (CLR v1.05) to analyze reflectance data. Percent reflectance data was used to create a spectral curve for natural and 22 manipulated feathers. Because brightnes is known to be the trait asociated with condition dependence in the species, it was the standard descriptor of reflectance that I used. Mean brightnes is the average reflectance in the 300-700 nm range. Analysis was done on both rump and tail feathers, but both feathers displayed a similar patern so al data reported is indicative of rump feathers. Statistical Analysis.- Al analyses were performed with JMP 10.0.2 (SAS Institute) and a p-value < 0.05 was considered statisticaly significant. A two-sample t test was used to confirm that there was not a significant diference in clutch size, pre-treatment chick tarsus length, pre- treatment chick mas, and male coloration pre-treatment betwen groups. A t test was also used to demonstrate if the markers had an efect on brightnes betwen groups. A repeated measures analysis of variance (ANOVA) was used to examine the efect of color manipulation on growth of chick tarsus length and chick mas. ID number was included as a covariate to control for repeated sampling events within individual (day 7 and 10). Chick age and the interaction betwen days and color manipulation were also included in the model. Tarsus length and chick mas were log transformed prior to analysis. RESULTS Clutch Size and Pre-Treatment Variables.- There was no diference in clutch size betwen treatments (t= 0, df= 17.6, P= 1.0). Pre-treatment male plumage brightnes on day 5 did not vary betwen groups (t= -0.92, df= 6.05, P=0.395). Average clutch tarsus length (t= 1.17, df= 19.9, P= 0.255) and mas (t= 0.98, df= 19.9, P= 0.338) did not vary prior to treatment. Color Analysis.- The manipulated brightnes of the two treatment groups was shown to be significantly diferent from one another (t=-4.78, df= 5.03, p= 0.005), indicating that the permanent marker treatments efectively changed the plumage coloration of male Eastern 23 Bluebirds. The mean brightnes for both treatment groups fel within the natural range of plumage brightnes reported by Liu et al. (2007). The treatments appeared to afect plumage brightnes but did not significantly alter the shape of the spectral curve (Fig. 2). Change in Tarsus Length The overal model testing for the efects of treatment, age, and individual on relative chick tarsus growth for a clutch was significant (overal: F 22,15 = 6.15, P= 0.0004), but this efect was driven by a significant variation in tarsus growth betwen individual clutches (clutch ID efect: partial F 20 = 3.10, p= 0.015 and the growth in the length of the tarsus betwen days 7 and 10 (age efect: partial F 1 = 49.6, P< 0.0001). Clutch tarsus length betwen treatments (treatment efect: partial F 1 = 2.19, P= 0.160) was not significantly diferent (Fig. 2). Change in Body Mass The overal model testing for the efect of treatment, days post-manipulation, and individual ID on change in body mas change was significant (overal: F 22,15 = 4.86, P=0.0014), with clutches of brightly colored males having a faster rate of mas change than clutches of dull colored males (treatment efect: partial F 1 = 4.71, P= 0.046) (Fig. 3). Change in clutch mas varied betwen days 7 and 10 significantly (age: partial F 1 = 56.7, P< 0.0001). Clutch mas change did not vary betwen clutches (clutch ID efect: partial F 20 = 1.75, P= 0.135). DISCUSSION In this study, I tested the efect of male ornamentation on offspring growth rate by experimentaly manipulating the plumage coloration of males. I found no diference in rate of tarsus growth betwen clutches of treatments. However, I did find a significant efect of treatment on mas change. There was a significant diference in plumage coloration due to manipulation betwen the bright and dull treatments. I previously demonstrated that females in 24 pairs where the male was experimentaly brightened showed a trend for visiting the nest at a higher rate than females paired to males with reduced brightnes (Chapter 1). Interestingly, even over just a period of a few days, this trend in females appears to have an efect on nestling growth. Nestling body size is geneticaly determined, but can be influenced by environmental effects (Senar et al. 2003). It has been demonstrated that brighter male and female plumage in Eastern Bluebirds is asociated with heavier offspring or offspring in beter condition (Sieferman and Hil 2003; Sieferman and Hill 2005a; Grindstaf et al. 2012). In a study using blue tits (Cyanistes caeruleus) where male phenotype was experimentaly reduced offspring had lower growth rates (Limbourg et al. 2004). However, previous studies looking at the efect of male phenotype on offspring fitnes have found no efect (Sheldon 2000; Johnsen et al. 2005; Balenger et al. 2007). Diferences betwen studies could be species-specific or come from diferent time points when measurements were taken. Increased investment in offspring of atractive mates should lead to increased offspring condition (Burley 1986; Sheldon 2000). In my first chapter I found that Eastern Bluebird females mated to brighter males showed a trend towards a higher relative provisioning rate than females mated to duller males. The only way for the trade-of proposed by Trivers (1972) betwen current and future reproduction to be beneficial for the parent is for the costs of reproduction to be outweighed by the benefits of enhanced offspring viability. It was experimentaly demonstrated in a study of Drospholia melanogaster that the direct cost of diferentialy alocating more resources to offspring of atractive mates was outweighed by the indirect benefits of offspring quality (Head et al. 2005). Small body size of nestlings is known to have adverse efects on survival post fledging and competitive ability later in life (Garnet 1981; Alatalo and Lundberg 1986). Decreased provisioning is suggested to also hamper development 25 of sexualy atractive traits (Ohlson et al. 2002; Blount et al. 2003). To demonstrate the possible benefit of increased reproductive efort for offspring of atractive mates it would have been useful to test for future survival, sexual atractivenes, and reproductive succes of the offspring. A beter measure of reproductive succes and offspring quality may be necesary to beter understand the benefit of enhanced parental efort related to atractive mates. However, it is interesting that a change in male phenotype has the ability to efect offspring condition through female behavior over such a short span of time. 26 Figure 1. A timeline of Eastern Bluebird nesting sequence and procedures done in the study. Numbers indicate days from the hatching of eggs. 27 0" 0. 5" 1" 1. 5" 2" Da y $7 $ Da y $1 0 $ Ch a n g e$i n $T a r su s$Len g t h $fr o m$ Pr e8T r ea t men t $ Br ig h t$ Du ll$ Figure 2. Change in clutch tarsus length of Eastern Bluebird chicks from day 5. The dark columns represent pairs where the male?s plumage brightnes was experimentaly enhanced. 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