Early Developmental Impacts on Male traits and Female Preference in Zebra Finches (Taeniopygia guttata)

University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School January 2014 Early Developmental Impacts on Male traits and Female Preference in Zebra Finches (Taeniopygia guttata) Martyna Boruta University of South Florida, mb5361@gmail.com Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Biology Commons, and the Social and Behavioral Sciences Commons Scholar Commons Citation Boruta, Martyna, "Early Developmental Impacts on Male traits and Female Preference in Zebra Finches (Taeniopygia guttata)" (2014). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/5189 This Thesis is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact scholarcommons@usf.edu.

Early Developmental Impacts on Male Traits and Female Preference in Zebra Finches (Taeniopygia guttata) by Martyna Boruta A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Integrative Biology with a concentration in Ecology and Evolution College of Arts and Sciences University of South Florida Major Professor: Lynn Martin, Ph.D. Jason Rohr, Ph.D. Peter Stiling, Ph.D. Date of Approval: June 24, 2014 Keywords: early life, female choice, behavior, corticosterone, LPS, infection Copyright 2014, Martyna Boruta

TABLE OF CONTENTS List of Figures... ii Abstract... iii Introduction...1 Methods...5 Zebra finch breeding colony...5 Zebra finch treatments...5 Bill and cheek coloration...6 Female behavioral trials...6 Statistical analyses...8 Male sexually-selected traits...8 Female preference...9 Results...10 Male sexually-selected traits...10 Female preference...11 Discussion...14 Male sexually-selected traits...15 Female preference...16 Conclusions...18 Reference...19 Appendix...23 i

LIST OF FIGURES Figure 1: Predicted effects of early-life treatments on male morphological traits...4 Figure 2: Predicted effects of early life experience on female mate preference...4 Figure 3: Schematic of female choice apparatus based on Sullivan 1994...7 Figure 4: Impact of early-life treatments on male development...10 Figure 5: Impacts of early-life treatments on male coloration...11 Figure 6: Early-life experience of females affected their mate sampling effort...12 Figure 7: Early-life experience affected female mate preference...13 Figure 8: Early-life experience affected female mate preference...13 ii

ABSTRACT Some male sexually selected traits are sensitive to stressors early in life and provide females with information to discriminate among males with different developmental experiences. Moreover, female early life experiences could also impact which males they choose. Females might either choose honest traits indicative of male quality, no matter their own experiences, or they might choose mates to match or compensate for their own experiences. To determine how developmental stressors alter male sexually-selected traits and female preference thereof, I exposed zebra finches (Taeniopygia guttata, ZEFI) to i) lipopolysaccharide (LPS), an immunogenic, Gram-negative bacterial component, ii) corticosterone (CORT), an avian stress steroid, iii) both challenges (CORT/LPS), or iv) none of the above (control vehicles). Finches were exposed during development (12-28 days post-hatch) and male traits (e.g., body size, bill and cheek coloration) and female behaviors (e.g., general activity, male sampling effort, and male preference) were then measured in adulthood. Control males were predicted to express the most elaborate traits followed by LPS, CORT, and then CORT/LPS males. If female preference was generally driven by male quality, control females were predicted to be most selective followed by LPS, CORT, and CORT/LPS females. Alternatively, if female choice was contingent on her own experience, females might choose males with similar (i.e, matching) or distinct (i.e, complementarity) developmental histories. Of the male characteristics measured, only cheek coloration was impacted by treatment early in life; CORT/LPS males had duller, less orange cheeks than controls. For females, overall activity was reduced in CORT/LPS females. iii

More importantly in regards to mate choice, females exhibited a blend of matching and complimentary behavior; females not exposed to LPS or CORT preferred males also not exposed to LPS or CORT. In general, females avoided LPS males no matter their own experience. Altogether, this study suggests that female mate preference is quite sensitive to early-life experiences and driven by a mix of choice of outright male quality and relative complementarity. iv

INTRODUCTION Sexually-selected traits might be honest indicators of mate quality, especially when they are expensive to acquire and maintain[1, 2]. Costs can include physical hindrances (e.g., long tail feathers)[3] or conspicuous behaviors exposing individuals to predators or pathogens (e.g., frog calls)[4, 5]. Good genes for these traits might be inherited from fathers, but their maintenance often requires resources that vary in availability[1, 2]. Many sexually selected traits are sensitive to stressors during development, so some traits could encode a mate s recent as well as developmental experiences[6-8]. Early in development, individuals typically have finite resources to allocate among traits[8]. For example, offspring experiencing nutritional deficiencies might reduce skeletal growth or body mass accretion to allocate resources toward the developing brain, reproductive organs, or sexually-selected traits[9]. Indeed, certain traits are more sensitive to environmental stressors than others; however, not all such sensitivity is necessarily maladaptive. In humans for example, a fetus developing in a poor environment will alter its metabolism to store resources in preparation for a resource-poor environment in adulthood. However, if environmental conditions improve later in life, such an individual will store excess resources, resulting in obesity or diabetes. In other words, the early life environment might be comparatively poor, but the ability of an individual to store resources is unfavorable only if the developmental and adult environments are mismatched[10]. In many contexts, early life predicts conditions during adulthood, so such plastic responses would be adaptive. Subsequently, females may choose males because male traits inform about a mate s 1

past experiences and his potential (or his offspring s potential) to thrive in a particular adult environment[10-12]. Stress hormones are often involved in mediating the effects of early-life adversity on adult phenotypic variation. Such stress hormones can be induced or their regulation altered via sibling competition, food availability, pathogen exposure, pollution or parental effort[7, 11-14]. The hypothalamic-pituitary-adrenal (HPA) axis regulates the major vertebrate stress hormones known as glucocorticoids (GCs)[15]. GCs have extensive and diverse effects on physiology[16-18], morphology and behavior[19]. In both male song sparrows (Melospiza melodia) and zebra finches (Taeniopygia guttata, ZEFI), nestlings with manipulated levels of the main avian GC, corticosterone (CORT), had low song complexity, a trait critical for attracting mates[12, 20, 21]. Further, CORT exposure in early life in ZEFIs was also found to alter the size and structure of the high vocal center of the brain associated with song learning[21]. In addition to their effects on sexually-selected traits, GCs can also have important regulatory effects on the immune system. For example, neonatal rats exposed to bacterial endotoxins, immunogenic molecules that occur on or in some pathogens, had increased sensitivity to a restraint stressor in adulthood[22]. Relevant to mate choice, past pathogen exposure might prime individuals for future encounters with pathogens or permanently alter immune defenses[23]. Collectively, early-life exposure to pathogens (or stressors generally) might have long-term consequences on the ability of an organism to endure infections later in life because immune defenses were altered via changes to the responsiveness of the HPA axis. Mice, for example, exposed perinatally to a Chlamydia infection had low levels of circulating CORT when exposed to a secondary infection compared to mice never exposed to the 2

pathogen[24]. Early-life infection can even affect the attractiveness of males to females. In canaries (Serinus canaria), males infected with malaria (Plasmodium relictum) as juveniles had smaller song repertoires than controls as adults[25]. Although early-life impacts on male sexual traits and female choice thereof are strongly insinuated by prior work, no study (to my knowledge) has considered how female choice is affected by early-life experiences. Females might choose males that experienced infections early in life (e.g., environments with high parasite encounters), no matter their own experience, because such males might cope better with infections better later in life[10]. Alternatively, females might choose males with similar (i.e., matching) or different (i.e., complementarity) early-life experience to their own in order for offspring to be best-suited to environments[26-29]. To address these alternatives, I conducted a study on ZEFI to assess how early life exposure to i) CORT only, ii) LPS only, iii) CORT/LPS, or iv) controls affected male sexually selected traits and female choice in adulthood. I predicted that males experiencing both stressor types would have the least elaborated traits and smallest adult body size (Figure 1). For female preference, I envisioned several possibilities: females would choose the highest-quality (untreated) males no matter their own experience, females exposed to LPS and/or CORT would choose males without such experience, or females would choose males matched to their own experience (Figure 2). I also expected that choosiness might manifest in multiple ways: females experiencing both stressor types might sample the fewest males and choose a mate quickly whereas females experiencing no stressors would sample many males before settling and fixating on a mate. 3

Figure 1. Predicted effects of early-life treatments on male morphological traits. Figure 2. Predicted effects of early life experience on female mate preference. Either (i) females would choose the best (control) males no matter their own experience (left-most area of figure; control males), ii) females would prefer LPS males because their immune systems were primed from prior infection, (green dashed line, an example of complementarity), or (iii) female preference would be contingent on their own early life experience (matching). In case iii) control females were expected to be most selective and CORT/LPS the least (solid lines). Line colors indicate female treatments. 4

METHODS Zebra finch breeding colony Forty adult zebra finches (N=10 females, N=10 males) were acquired from local breeders and housed in four large flight cages (90 x 51 x 51 cm) at the College of Medicine, University of South Florida, Tampa, FL. To stimulate breeding, birds were kept on a 16L:8D cycle and provided with nesting material and nest baskets[30]. All birds had access to standard commercial mixed seeds for songbirds, greens, millet, water, and cuttlebones ad libitum. Once hatched, chicks were marked with non-toxic color markers to indicate hatch order and banded at d 8 post-hatch. Mass (to 0.01 g) and tarsus length (to 0.01 mm) were recorded daily from hatch to d 20, and on d 24 and 28. Zebra finch treatments On d 12, chicks were randomly assigned to one of four treatment groups: i) lipopolysaccharide (N = 45), ii) corticosterone (N = 37), iii) both challenges (N = 44), or iv) none of the above (control; N = 36). Note that because sexes are not dimorphic for weeks posthatch, sexes could not be allocated consistently among treatments. Between 12-15 d post-hatch, individuals were either given an oral of dose of 6.2 ug of CORT dissolved in peanut oil or peanut oil alone (control vehicle). A total of 8.15 ug of CORT was given between 16-28 d to adjust for the size of the developing chick. All oral treatments were given twice a day (12:00h and 17:00h) until d 28 post-hatch[31]. On d 14 post-hatch (17:00h), all chicks were injected s.q. over the 5

breast muscle with either a 0.5mg/kg dose of LPS (from Escherichia coli 055:B55) dissolved in sterile phosphate buffered saline (PBS) or PBS buffer only[31]. Once chicks were nutritionally independent (35-38 d post-hatch), they were separated into mixed treatment, but sex-specific flight cages. Males and females could hear, but not see each other. Fledglings were monitored daily and given food, water, and cuttlebones ad libitum until sexually mature (about three months). Birds that died before mate choice trials were not included in analyses, hence differences in sample sizes in the below analyses. Bill and cheek coloration When males (N=48) were approximately 6-7 months old, bill and cheek patch coloration were scored for the three dimensions of color (hue, brightness (value), and chroma) by comparing to the Munsell color system[32]. Color dimensions were converted into a single continuous color score: 3(15-HUE) + 1.5(6-VALUE) + 0.5(CHROMA-12) On this scale, males with higher scores have redder bills or more orange cheek patches, more saturated traits, and/or brighter traits[32]. All measurements were recorded by the same observer under the same light conditions. Female behavioral trials To test whether females can discriminate among males based on early-life treatment, a four-armed mate choice apparatus was used and female behavior recorded using digital video cameras[33]. The choice apparatus was constructed out of plywood, sealed with polyurethane to facilitate sterilization between trials (Figure 3). Each arm (133 cm x 55 cm x 40 cm) extended 6

from a neutral zone where the female could see all the males at once, but males were unable to see one another. Each arm had a wire mesh divider (placed in the middle of each arm) and four perches (two on each side of the divider)[33]. Males were provided with food and water during trials; females were only given water. Figure 3. Schematic of female choice apparatus based on Sullivan 1994. Thin black lines represent perches and thick black lines represent mesh dividers between male and female birds, depicted here only in the left arm (for clarity). All birds within the choice chamber were in auditory proximity, but males were unable to see each other. Blue arrows indicate male rotation progression between trials. Dimensions: arm length = 133 cm, height of central area = 55 cm; height of arms = 40 cm; perch heights = 20 cm. A subset of males from each of the four treatment groups described above was assigned to one of five quartets (total male N=20). Each quartet had a male representative from each of the treatment groups. Quartets were then presented to a single, unrelated (to males) female (control N=3; LPS N=7; CORT N=5; CORT/LPS N=6) for behavioral trials. Sample sizes were small to i) ensure females were unrelated to males in a quartet, and ii) so no quartet would contain brothers. To query female choice, the following approach was used. Trials began in the dark with one male placed singly and at random into each of the four arms of the apparatus. A 7

single female was then placed in the neutral zone. Birds were given 20 min after lights-on to acclimate to the apparatus. After acclimation, female behavior was video recorded for 15 min. Male birds within a quartet were then rotated among arms either in a clockwise or counter clockwise direction (to reduce directional bias) to obtain four 15-min trials for each male quartet female pairing. A male quartet was not used more than twice in the same day. Female preference was assessed using JWatcher 1.0 software (Los Angeles, USA), and several behaviors were quantified: i) total number of hops (i.e. overall female activity), ii) proportion of time spent in an arm, iii) number of arm entrances, iv) total time spent in an arm in the vicinity of a male, v) total time spent on perch next to male, and vi) total number of perch hops next to male. The behavioral variables used in this study were chosen as proxies for female preference, with increasing proximity to the male serving as an indicator for increased female preference. The former two behaviors characterized male sampling effort whereas the latter three comprised mate preference. These latter three were interpreted as preference because females participate in a courtship dance with males in close proximity[34]. The number of total overall hops was used as a covariate to disentangle general female activity from mate-directed behaviors. All animal care and research was approved by the University of South Florida Institutional Animal Care and Use of Laboratory Animals (IACUC #4349R). Statistical analyses Male sexually-selected traits Differences in mass and tarsus length between male treatment groups from hatch to d 20, and on d 24 and d 28, were analyzed using repeated-measures linear mixed models (LMM) with treatment and sex as fixed effects, individual identity as a random effect, and day 11 mass or 8

tarsus length (i.e., value prior to LPS/CORT treatments) as a covariate to control for size at the time treatment began. Males that survived to d 28 were included in the analysis (N=64); effects of treatment on mass and tarsus length were analyzed separately. To compare male bill and cheek patch coloration, data were analyzed using an ANOVA with treatment as the fixed effect and bill length and width as covariates to adjust for differences in surface area of the bill. Bonferroni post-hoc tests were used to determine pairwise differences between treatments. Female preference Female behavior was analyzed using mixed effects univariate repeated measures ANOVA. Because of small sample sizes (N=21), response variables were averaged across LPSexposed (LPS only and CORT/LPS treatments) or CORT-exposed (control or CORT only treatments) groups. Female treatment, male treatment, and their interaction were fixed effects; female identity was a random effect, and total number of hops was used as a covariate. Total time spent in arm, proportion of time spent in arm, number of arm entrances, total time spent on perch next to male, and number of perch hops next to male were used as dependent variables in separate analyses. Male traits were analyzed with SPSS v21.0 and female behavior data were analyzed with Statistica v12. 9

RESULTS Male sexually-selected traits Body mass in males was not affected by treatment (F 3, 51.1 = 1.177, P = 0.328) or the interaction between treatment and date (F 30, 214.6 = 0.888, P = 0.639). However, date had a significant effect because males were grew (F 10, 214.6 = 42.2, P < 0.001) (Figure 4A). A similar pattern was found for tarsus length in which only date (F 10, 225.7 = 20.176, P < 0.001) but neither treatment nor the interaction of treatment by date affected tarsus length (treatment: F 3, 46.6 = 0.596, P = 0.621; treatment*day: F 30, 226.2 = 1.205, p = 0.223) (Figure 4B). A B Figure 4. Impact of early-life treatments on male development. Treatment did not affect male a) body mass (g) or b) tarsus length (mm). Early-life experience affected cheek coloration (F 3, 44 = 2.913; P = 0.045, Figure 5A). Bonferroni post hoc comparisons indicated that cheek color of control males were significantly brighter than CORT/LPS males (P = 0.045), but not CORT (P = 0.657) or LPS (P = 0.202) males. Bill color was not affected by treatment (F 3, 44 = 0.342, P = 0.795, Figure 5B). 10

A B Figure 5. Impacts of early-life treatments on male coloration. Treatments affected male a) cheek coloration, but not b) bill coloration. Female preference Early-life experience affected female activity (F 5,12 = 36.18, P < 0.001). In terms of female sampling of males, there was significant 3-way interaction for number of arm entrances (MLPS*FLPS*FCORT: F 1,16 = 5.83, P = 0.028), indicating that females receiving either LPS or CORT entered more arms than control females, and that controls entered fewer arms containing LPS-exposed (LPS and CORT/LPS) males (Figure 6). Similar trends were found for the other mate sampling behaviors indicating consistency across response variables (total time and proportion of time spent in each arm (see appendix)). In terms of mate preference, all females, regardless of their early life experience, spent less total and proportional time in arms containing LPS-exposed males (total arm time: F 1,16 = 8.801, P = 0.009; arcsine proportion arm time: F 1, 16 = 7.397, P = 0.015; Figures 7A and 7B). Additionally, females not exposed to LPS spent more time in arms with males that were also unexposed to LPS (MLPS*FLPS: F 1,16 = 6.06, P = 0.025). There was marginally non-significant 3-way interaction for time spent next to a male (MLPS*MCORT*FLPS: F 1,16 = 3.09, P = 0.097; Figure 8) such that unexposed females spent 11

more time next to all males except CORT/LPS males. LPS females showed the opposite reactions, however, spending more time with CORT/LPS males than other options. Perch hops next to males were not affected by treatment (F 1,16 = 2.466, P = 0.135). 4.5 4.0 3.5 Mean number of arm entries 3.0 2.5 2.0 1.5 1.0 0.5 No Male LPS exposure Male LPS exposure 0.0 Female cort: No Yes Female LPS exposure: No Female cort: No Yes Female LPS exposure: Yes Figure 6. Early-life experience of females affected their mate sampling effort. Females that received either LPS or CORT entered on average more arms than control females, and control females entered fewer arms containing LPS-exposed males. Error bars represent +/- SE. 12

210 A 0.50 0.48 B Mean time spent in arm (sec.) 200 190 180 170 160 150 Proportion of time spent in arm (arcsine sqrt) 0.46 0.44 0.42 0.40 0.38 0.36 0.34 140 0.32 130 No Yes 0.30 No Yes Male LPS exposure Male LPS exposure Figure 7. Early-life experience affected female mate preference. All females, regardless of their early life experience, on a) average spent less total time and b) proportional time in arms containing LPS-exposed males. Error bars represent +/- SE. 160 140 Mean time on perch (seconds) 120 100 80 60 40 20 No Female LPS exposure Female LPS exposure 0 Male LPS: No Yes Male cort: No Male LPS: No Yes Male cort: Yes Figure 8. Early-life experience affected female mate preference. Unexposed females spent on average more time next to any male (in sec), regardless of treatment, with the exception of CORT/LPS males which they avoided. LPS females showed the opposite reactions, however, spending more time with CORT/LPS males. Error bars represent +/- SE. 13

DISCUSSION Early-life environments can often induce one genotype to express multiple phenotypes, a process known as developmental plasticity[10, 35]. Such plasticity can produce variation in the traits of potential mates as well as preferences of the choosers, subsequently impacting the evolution of mate choice[35, 36]. To date, developmental plasticity has not yet been well integrated into sexual selection theory, and when it has, it has tended to focus on males only[37]. Classically, females have been predicted to select particular male traits if such traits are honest, condition-dependent and heritable[1, 38, 39]. As a growing literature indicates that early-life adversity can impact male sexually selected traits, female choice should also be impacted by developmental experience[7, 21, 40], but little effort has been made to test this possibility in vertebrates (but see[17, 41, 42]). Females might choose the genuinely best male, no matter their own experience, or they might instead choose a male well-matched to of themselves. Here we found that i) male traits and ii) female choice (sampling effort and preference) were both developmentally plastic. Male cheek coloration was dullest in zebra finches experiencing two stressors in development; no other traits were impacted. Female activity, male sampling effort, and mate preference were also impacted by experience. Females not exposed to either stressor chose a mate quickly, particularly those not exposed to LPS or CORT in development. However, females exposed to LPS or CORT tended to sample more males but be less selective with the exception of CORT/LPS males, whom they avoided. Below I discuss the implications of these findings for our understanding of the evolution of mate choice. 14

Male sexually-selected traits Although most male traits were insensitive to early-life experience, one trait (cheek coloration) was impacted with CORT/LPS males having duller and less orange cheek patches than control males. Exposure to CORT as nestlings has been shown to dampen melanin-based coloration in male barn owls (Tyto alba), a condition-dependent trait that females use to discriminate among males during courtship[43]. Also, although much variation in male sexually selected traits might be mediated by androgens (e.g., testosterone), recent models suggests that CORT may also be an important in how elaborate traits are expressed[44]. GCs and androgens might influence male traits independent of each other[45, 46], or GCs might stabilize variation in certain male traits particularly if high levels of GCs result in a disadvantage[44]. Females select males using more than one trait[47, 48] and because females were able to distinguish among males even though most morphological traits were unaffected, treatments must have affected other traits (i.e., vocalizations, olfactory cues) that were not measured. There is a large body of literature showing that early-life experiences have a profound effect of brain development and song learning[8, 11, 12, 17, 49, 50]. Specifically, early-life stressors are known to reduce the size of brain regions responsible for song learning[50, 51]. Females may have been selecting mates based on song differences and/or females could be cueing in on male activity level, as vigor and coordination are also an important component of zebra finch courtship displays[52]. There is also growing evidence that birds have a developed olfactory system, and certain cues (i.e., preening oils) may be important in conspecific and heterospecific recognition including impacts on sexual behavior[53-55]. 15

As male quality seems quite plastic, females might often have a difficult time selecting the best males if the environment changes (i.e., anthropogenically); sometimes, her historically informed (evolved) choice may be made in the wrong context. Such appears to be the case for plumage coloration in Northern cardinals (Cardinalis cardinalis). In males, red plumage is a condition-dependent trait indicative of high parental care ability, and therefore preferred by females[56, 57]. In urban environments, however, high resource availability (e.g., bird feeders) allows low quality males to misrepresent themselves as good parents[58]. Subsequently, even genuinely low-quality males might persist in population if the conditions females experienced in early-life impose selectivity for such a male type, which may in part resolve the lek paradox. Female preference Female preference was impacted if either females, or the males they sampled, were exposed to LPS early in life. Further, control females sampled the fewest males and/or made their choice of mates quickly. Females, therefore, exhibited a blend of matching and choosing based on absolute quality; in general, females avoided LPS males no matter their own experience. Altogether, this study suggests that female mate preference is quite sensitive to early-life experiences and driven by a mix of choice of outright male quality and phenotypic matching. Assortative mating might explain why females selected males with similar developmental pasts; they may be phenotypically matching their mates to their own developmental experiences[27, 28]. Phenotypic matching, or sexual imprinting, is an example of assortative matching where mate preference is learned early in life, either from the mother, father, or another individual in the population[29, 59]. Evidence exists that ZEFIs match phenotypically, such that 16

high-quality ZEFI pairs and low-quality pairs will match based on song; however, high-quality pairs will breed faster[60]. However, phenotypic matching may not always be advantageous. Paired male and female ZEFIs exposed to early-life stressors had a reduced lifespan compared to pairs where only one sex was exposed to the stressor[31]. In this study, however, female preference was not a result of imprinting because choosing females, their parents did not received treatments. Here, females not exposed to LPS or CORT recognized and chose males with similar developmental backgrounds, unless males were exposed to both stressors. These results support recent models suggesting that females match phenotypically only when mates experienced favorable conditions early in life[61, 62]. Nevertheless, my data are not completely inconsistent with outright choice of males based on male quality. In general, a female should choose males that are a good fit to the current environment[26]. In adult turkeys (Meleagris gallopavo), for example, exposure to pathogens could be energetically costly to females and therefore, infected females should sample fewer males[63]. However, infected females visited more males than control females, but spent less time with each one before initiating copulation[63]. If females recognize their own susceptibility to infection as a result of their developmental history, as adults they select males with genes that would offset or increase offspring survival. Females might therefore select males with different developmental backgrounds than her own to ensure that offspring will be genetically dissimilar[26]. In my study, females, except controls, spent less time in arms next to males, but had more arm entrances suggesting that these females may be sampling more males before making a decision. However, finding high quality males may be difficult, especially if high quality males are rare. Ideally, females would select the highest quality male, but depending on current environmental conditions and past experience, females may be flexible in their 17

selectivity. In threespine stickleback (Gasterosteus spp.), female preference was flexible if the population sex ratio was female-biased and as females become older[64]. If the environment is changing rapidly, female preference, even weak preference, may be more influential in driving population trajectories than previously anticipated, regardless of sexually selected traits[65] and not necessarily if males have the best genes[26]. Conclusion Early-life experiences not only altered mate traits, but also impact female zebra finch behaviors such that mate preference was driven a combination of outright quality and matching based on prior experience. My study provides evidence that developmental history impacts female preference, which may alter population trajectories, particularly in rapidly changing environments[66]. This might have major implications on heritable traits that impact individual physiological and/or immunological responses, thus potentially impacting disease dynamics. 18

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APPENDIX Supplementary Information: A) Univariate repeated measures of variance for number of arm entrances by females. Females receiving either LPS or CORT entered more arms than females receiving neither stressor (controls), and that control females entered fewer arms containing LPS-exposed (LPS and CORT/LPS) males. B) Univariate repeated measures of variance for total time spent in arm and C) proportion of time spent in arm (arcsine square root transformed). All females, regardless of their early life experience, spent less total and proportional time in arms containing LPS-exposed (LPS and CORT/LPS) males. D) Univariate repeated measures of variance of perch time. Unexposed females spent more time next to any male, regardless of treatment, with the exception of CORT/LPS males which they avoided. LPS females showed the opposite reactions, however, spending more time with CORT/LPS males. E) Univariate repeated measures of variance of perch hops. Perch hops next to males were not affected by treatment. * indicates marginal significance, ** indicated statistical significance. A. Arm Entrances Degrees of F-value p-value Source Freedom Intercept 1 2.806 0.113 Overall Female Hops 1 55.039 0.00** Female LPS-exposed 1 0.882 0.362 Female CORT-exposed 1 1.100 0.310 FLPS*FCORT 1 0.610 0.446 Male LPS-exposed 1 5.422 0.033** MLPS*Female Hops 1 9.726 0.006** MLPS*FLPS 1 2.726 0.118 MLPS*FCORT 1 5.852 0.028** MLPS*FLPS*FCORT 1 5.837 0.028** Male CORT-exposed 1 0.529 0.477 MCORT*Female Hops 1 1.791 0.200 MCORT*FLPS 1 4.799 0.044** MCORT*FCORT 1 0.453 0.511 MCORT*FLPS*FCORT 1 2.176 0.160 MLPS*MCORT 1 1.358 0.261 MLPS*MCORT*Female Hops 1 2.649 0.123 MLPS*MCORT*FLPS 1 1.946 0.182 MLPS*MCORT*FCORT 1 0.018 0.894 MLPS*MCORT*FLPS*FCORT 1 0.020 0.890 23

B. Total Time in Arm Degrees of F-value p-value Source Freedom Intercept 1 60.092 0.000** Overall Female Hops 1 0.001 0.974 Female LPS-exposed 1 1.190 0.291 Female CORT-exposed 1 1.227 0.284 FLPS*FCORT 1 0.558 0.466 Male LPS-exposed 1 8.800 0.009** MLPS*Female Hops 1 6.190 0.024** MLPS*FLPS 1 6.065 0.026** MLPS*FCORT 1 0.693 0.417 MLPS*FLPS*FCORT 1 2.170 0.160 Male CORT-exposed 1 2.120 0.165 MCORT*Female Hops 1 1.851 0.193 MCORT*FLPS 1 3.498 0.080 MCORT*FCORT 1 3.185 0.093 MCORT*FLPS*FCORT 1 0.356 0.559 MLPS*MCORT 1 0.339 0.569 MLPS*MCORT*Female Hops 1 0.891 0.359 MLPS*MCORT*FLPS 1 1.413 0.252 MLPS*MCORT*FCORT 1 1.710 0.209 MLPS*MCORT*FLPS*FCORT 1 0.071 0.794 24

C.Proportion Time (Arcsine) Degrees of F-value p-value Source Freedom Intercept 1 63.468 0.00** Overall Female Hops 1 3.579 0.077 Female LPS-exposed 1 0.110 0.744 Female CORT-exposed 1 0.219 0.646 FLPS*FCORT 1 2.014 0.175 Male LPS-exposed 1 7.397 0.015** MLPS*Female Hops 1 5.835 0.028** MLPS*FLPS 1 2.840 0.111 MLPS*FCORT 1 1.371 0.259 MLPS*FLPS*FCORT 1 1.182 0.293 Male CORT-exposed 1 1.266 0.277 MCORT*Female Hops 1 0.765 0.395 MCORT*FLPS 1 3.572 0.077* MCORT*FCORT 1 2.386 0.142 MCORT*FLPS*FCORT 1 1.243 0.281 MLPS*MCORT 1 1.582 0.227 MLPS*MCORT*Female Hops 1 2.317 0.147 MLPS*MCORT*FLPS 1 3.123 0.096 MLPS*MCORT*FCORT 1 0.045 0.835 MLPS*MCORT*FLPS*FCORT 1 0.126 0.727 25

D. Perch Time Degrees of F-value p-value Source Freedom Intercept 1 27.023 0.000** Overall Female Hops 1 1.538 0.233 Female LPS-exposed 1 1.270 0.276 Female CORT-exposed 1 0.013 0.911 FLPS*FCORT 1 0.840 0.373 Male LPS-exposed 1 3.225 0.091 MLPS*Female Hops 1 2.783 0.115 MLPS*FLPS 1 4.305 0.055* MLPS*FCORT 1 0.028 0.870 MLPS*FLPS*FCORT 1 1.674 0.214 Male CORT-exposed 1 2.013 0.175 MCORT*Female Hops 1 1.707 0.210 MCORT*FLPS 1 4.020 0.062* MCORT*FCORT 1 1.499 0.239 MCORT*FLPS*FCORT 1 0.025 0.877 MLPS*MCORT 1 3.603 0.076 MLPS*MCORT*Female Hops 1 2.487 0.134 MLPS*MCORT*FLPS 1 3.098 0.097* MLPS*MCORT*FCORT 1 0.543 0.472 MLPS*MCORT*FLPS*FCORT 1 0.943 0.346 26

E. Perch Hops Degrees of F-value p-value Source Freedom Intercept 1 2.914 0.107 Overall Female Hops 1 19.219 0.000** Female LPS-exposed 1 0.081 0.779 Female CORT-exposed 1 1.327 0.266 FLPS*FCORT 1 2.603 0.126 Male LPS-exposed 1 0.782 0.390 MLPS*Female Hops 1 1.983 0.178 MLPS*FLPS 1 3.913 0.065* MLPS*FCORT 1 0.078 0.784 MLPS*FLPS*FCORT 1 2.466 0.136 Male CORT-exposed 1 0.127 0.726 MCORT*Female Hops 1 0.332 0.573 MCORT*FLPS 1 1.842 0.194 MCORT*FCORT 1 0.798 0.385 MCORT*FLPS*FCORT 1 2.161 0.161 MLPS*MCORT 1 0.130 0.724 MLPS*MCORT*Female Hops 1 0.009 0.926 MLPS*MCORT*FLPS 1 0.763 0.395 MLPS*MCORT*FCORT 1 0.151 0.702 MLPS*MCORT*FLPS*FCORT 1 0.249 0.625 27