Red-winged blackbirds Agelaius phoeniceus use carotenoid and melanin pigments to color their epaulets

Over the past three decades, the red‐winged blackbird Agelaius phoeniceus has served as a model species for studies of sexual selection and the evolution of ornamental traits. Particular attention has been paid to the role of the colorful red‐and‐yellow epaulets that are striking in males but reduced in females and juveniles. It has been assumed that carotenoid pigments bestow the brilliant red and yellow colors on epaulet feathers, but this has never been tested biochemically. Here, we use high‐performance liquid chromatography (HPLC) to describe the pigments present in these colorful feathers. Two red ketocarotenoids (astaxanthin and canthaxanthin) are responsible for the bright red hue of epaulets. Two yellow dietary precursors pigments (lutein and zeaxanthin) are also present in moderately high concentrations in red feathers. After extracting carotenoids, however, red feathers remained deep brown in color. HPLC tests show that melanin pigments (primarily eumelanin) are also found in the red‐pigmented barbules of epaulet feathers, at an approximately equal concentration to carotenoids. This appears to be an uncommon feature of carotenoid‐based ornamental plumage in birds, as was shown by comparable analyses of melanin in the yellow feathers of male American goldfinches Carduelis tristis and the red feathers of northern cardinals Cardinalis cardinalis, in which we detected virtually no melanins. Furthermore, the yellow bordering feathers of male epaulets are devoid of carotenoids (except when tinged with a carotenoid‐derived pink coloration on occasion) and instead are comprised of a high concentration of primarily phaeomelanin pigments. The dual pigment composition of red epaulet feathers and the melanin‐only basis for yellow coloration may have important implications for the honesty‐reinforcing mechanisms underlying ornamental epaulets in red‐winged blackbirds, and shed light on the difficulties researchers have had to date in characterizing the signaling function of this trait. As in several other birds, the melanic nature of feathers may explain why epaulets are used largely to settle aggressive contests rather than to attract mates.     

Polygyny and extrapair fertilizations in eastern red-winged blackbirds (Agelaius phoeniceus)

Parentage of nestling red-winged blackbirds (Agelaius phoeniceus)from an eastern population was determinedusing DNA fingerprintingtechniques. Of 235 nestlings surveyed, 58 had fingerprints excludingthemale, but none excluded the female tending the nest. Data onpairing status during the female's fertilizable period was availablefor 232 offspring; 55 (25%of 1988 nestlings, 23% of 1989 nestlings)of those were sired through extrapair copulations. Of these55 offspring, 33 could be assigned to nearby territory holders;16 of the remaining nestlings may have been sired by nearbymales that were not captured. During both years, 44% of territorialmales had more than one female nesting simultaneously on theirterritory. The number of extrapair fertilizations gained bymales increased significandy with harem size in 1 year. Paternity(die proportion of nesdings on the territory sired by die territoryholder) showed a positive but nonsignificant increase widi haremsize in bodi years. There was no apparent cost in paternityfor males guarding two or more fertilizable females at the sametime. The broods of females that were fertilizable at die sametime anodier female was setding on die same territory tendedto have a greater proportion of extrapair fertilizations (0.37)than did die broods of odier females within harems (0.15). Establishedfertilizable females were chased significantly more by die territoryowner and by extrapair males when a new female was setding.There were no associations between a male's paternity or successat gaining extrapair fertilizations and his age or color-bandcombination. Overall, extrapair fertilizations had litde effecton die relationship between fledgling success and harem sizeand appeared to have a minimal impact on die overall intensityof sexual selection on males.     

Sound-pressure Levels and Song Preferences in Female Red-winged Blackbirds (Agelaius phoeniceus) (Aves,Emberizidae)

Female red-winged blackbirds (Agelaius phoeniceus) were tested for response to songs of male red-winged blackbirds differing in sound-pressure level (SPL) of playback. Subjects gave significantly more copulation-solicitation displays in response to playback of songs at 85 dB SPL than to playback of the same songs at 72 dB SPL. The strength of the preference, as judged by the ratio of response to high and low SPL playback (1.15:1), was lower than that of preferences for high SPL shown by insects and anurans. Female red-winged blackbirds responded preferentially to a conspecific song relative to a heterospecific imitation, even when the imitation was presented at an SPL 13 dB higher than that of the conspecific song. By contrast, female redwings did not maintain a preference for multiple conspecific song types over single types when the single song types were presented at the higher SPL. These results are compatible with Klump & Gerhardt's (1987) suggestion that the intensity independence of female preferences varies with the relative benefit females obtain from each preference.     

Premigratory Changes in the Hypothalamo-Hypophysial Neurosecretory System in the Red-winged Blackbird (Agelaius phoeniceus)*

Abstract

• 1 The hypothalamo-hypophysial neurosecretory system in the Red-winged Blackbird (Agelaius phoeniceus) has been studied with special reference to changes during the period prior to fall migration.
• 2 In September the supraoptic and paraventricular cells were found to be considerably larger with substantial amounts of aldehyde fuchsin positive neurosecretory material (NSM) accumulating in the perikaryon. Large amounts of the NSM were found also in the anterior median eminence and pars nervosa.
• 3 Towards migration time (November), the cells in the hypothalamic nuclei contained very little NSM. A marked decline in the amount of NSM was also seen in the anterior median eminence and probably in the pars nervosa too.
• 4 The release of NSM toward migration time has been discussed as an important event in the sequence of events that control and regulate migratory activity.

     

Hitchhiking and recombination in birds: evidence from Mhc-linked and unlinked loci in Red-winged Blackbirds (Agelaius phoeniceus)

Hitchhiking phenomena and genetic recombination have important consequences for a variety of fields for which birds are model species, yet we know virtually nothing about naturally occurring rates of recombination or the extent of linkage disequilibrium in birds. We took advantage of a previously sequenced cosmid clone from Red-winged Blackbirds (Agelaius phoeniceus) bearing a highly polymorphic Mhc class II gene, Agph-DABI, to measure the extent of linkage disequilibrium across approximately 40 kb of genomic DNA and to determine whether non-coding nucleotide diversity was elevated as a result of physical proximity to a target of balancing selection. Application of coalescent theory predicts that the hitchhiking effect is enhanced by the larger effective population size of blackbirds compared with humans, despite the presumably higher rates of recombination in birds. We surveyed sequence polymorphism at three Mhc-linked loci occurring 1.5-40 kb away from Agph-DAB1 and found that nucleotide diversity was indistinguishable from that found at three presumably unlinked, non-coding introns (beta-actin intron 2, beta-fibrinogen intron 7 and rhodopsin intron 2). Linkage disequilibrium as measured by Lewontin's D' was found only across a few hundred base pairs within any given locus, and was not detectable among any Mhc-linked loci. Estimated rates of the per site recombination rate p derived from three different analytical methods suggest that the amounts of recombination in blackbirds are up to two orders of magnitude higher than in humans, a discrepancy that cannot be explained entirely by the higher effective population size of blackbirds relative to humans. In addition, the ratio of the number of estimated recombination events per mutation frequently exceeds 1, as in Drosophila, again much higher than estimates in humans. Although the confidence limits of the blackbird estimates themselves span an order of magnitude, these data suggest that in blackbirds the hitchhiking effect for this region is negligible and may imply that the per site per individual recombination rate is high, resembling those of Drosophila more than those of humans.     

Secondary sexual traits, parasites, and polygyny in red-winged blackbirds, Agelaius phoeniceus

Data from a 3-year study of red-winged blackbirds (Agelaiusphoeniceus) were used to test the hypothesis that parasites(in this case, haematozoa) reduce male fitness and cause diminishedexpression of secondary sexual traits, which, in turn, are usedby females to select parasite-free males as mates. There wasno evidence indicating a fitness cost to being parasitized becauseparasitized males were as likely as unparasitized males to acquirea territory and to survive from one year to the next. Similarly,parasitized and unparasitized females did not differ with regardto how early they started nesting, how many eggs they laid,or their year-to-year survival. Secondary sexual traits, particularlyintrasexual aggression, did reliably (>80%) reveal the parasitestatus of males. Plumage and morphological traits also alloweddiscrimination of parasitized and unparasitized females. However,apparent mating patterns were unrelated to either the males'or the females' parasite status. Only if genetic analyses revealthat unparasitized males actually realize higher productivesuccess will these results potentially provide support for theparasite hypothesis of sexual selection.     

Habituation,Recovery and the Similarity of Song Types within Repertoires in Red-winged Blackbirds (Agelaius phoeniceus) (Aves,Emberizidae)

Male red-winged blackbirds (Agelaius phoeniceus) respond to playback of conspecific song on their territories with the song spread, a graded aggressive display in which males extend their wings to expose their red epaulets while singing. We show that the intensity of song spread display declines with repeated presentation of one song type, and recovers when song types are switched. Recovery is greater for switches between song types that are acoustically dissimilar than for switches between song types that are acoustically similar. Recovery is no different for switches between two song types taken from the repertoires of different males than for switches between song types recorded from the same male. Analysis of acoustic features also indicates that song types recorded from different males are not more dissimilar than are song types from the same male. Our results do not support the idea that repertoires of red-winged blackbirds are composed of similar song types in order to facilitate individual recognition. Rather, repertoires may be constructed of dissimilar song types, so as to help maintain the response of listeners despite habituation.     

Microsatellite Variation in Red-Winged Blackbirds (Agelaius phoeniceus)

Territorial male red-winged blackbirds from five locations in the United States and Canada were genotyped using a suite of six microsatellite loci. Each population possessed unique alleles, but numbers of alleles per locus (range = 7.3-8.8) and expected multilocus heterozygosities (range = 0.76-0.80) were similar in all populations. Significant overall allele frequency differences were detected between some population pairs, and some pairwise Fst values were significant (but small). However, Fst among populations, although significant, was also small (0.009). Despite revealing low levels of population structure, the high multilocus polymorphism indicates these loci will be valuable in the genetic analysis of behavior and reproductive strategies in this species.     

Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.     

Aggressive and Pair-bond Maintenance Songs of Female Red-winged Blackbirds (Agelaius pboeniceus)

Female red-winged blackbirds sing two distinct song types. Field observations and playback experiments indicate that Type 1 female song is sung chiefly in the presence of mates and probably functions in pair-bond maintenance, while Type 2 song is sung chiefly in the presence of female conspecifics, and appears to be aggressive in nature.     

Egg crypsis and clutch survival in three species of blackbirds (Icteridae)

For most open-nesting passerine birds, the primary function of egg pigmentation is crypsis. Interspecific variation for egg coloration is quite high, even among species that nest in the same locale. This variation might be maintained by selection to blend into species-specific nest cups, which vary in pattern because they are constructed from different materials. To test this hypothesis we photographed 30 clutches of Brewer's blackbirds ( Euphagus cyanocephalus ), 26 clutches of red-winged blackbirds ( Agelaius phoeniceus ), and 24 clutches of yellow-headed blackbirds ( Xanthocephalus xanthocephalus ), and quantified crypsis in terms of pattern (PD crypsis), brightness (MB crypsis) and brightness disparity (BD crypsis). The species were equally cryptic in terms of pattern crypsis, but differed significantly ( P < 0.01) in MB and BD crypsis. Intraspecific variation in pattern crypsis was surprisingly high. Mean clutch pattern disparity was highly correlated with mean background pattern disparity ( r = 0.999); this suggests selection for disparity matching, rather than for crypsis. We also monitored 25 clutches (7 Brewer's, 9 red-winged, and 9 yellow-headed blackbirds) for a 9-day period to determine if survival was related to crypsis. For Brewer's and yellow-headed blackbirds, successful nests were no more cryptic than failed nests. For red-winged blackbirds, there was a tendency ( P = 0.1) for less cryptic clutches to have higher survival; in addition, successful red-winged blackbird clutches had significantly ( P < 0.05) higher egg pattern disparity and background pattern disparity. Red-winged blackbird clutches are not concealed by overhanging vegetation, and high pattern disparity may enable them to blend into a large-scale heterogeneous background.     

Plasma LH and androgen levels in the red-winged blackbird (Agelaius phoeniceus) treated with a potent GnRH analogue.

1. Low doses of GnRH-A (0.01-0.10-1.0 micrograms) given during the annual testes growth period did not clearly affect plasma LH and androgen levels 10 min following the injection. 2. The first injection of high doses of GnRH-A (2.0-10.0-20.0 micrograms) markedly increased plasma LH and androgen levels measured 10 min following the injection. The increase in plasma LH level was dose-dependent and the maximal LH level was obtained with 10.0 micrograms of GnRH-A. 3. Impairment of the LH response to GnRH-A was assessed by comparing the first and the fourteenth injection of high doses of GnRH-A. Evidences of pituitary gland desensitization are reported since plasma LH levels were reduced following the fourteenth injection in all groups. 4. Plasma androgen levels following high doses of GnRH-A were not clearly affected in red-winged blackbirds.     

Ontogeny of catabolic and morphological properties of skeletal muscle of the red-winged blackbird (Agelaius phoeniceus)

Thermogenic capabilities of red-winged blackbirds improve markedly during their 10-12-day nestling period, especially between day 5 and day 8. The time course of improvements may be determined by the maturation of skeletal muscles involved in shivering thermogenesis, particularly the pectoralis muscles. To test this hypothesis, morphological and biochemical changes in pectoral and leg muscles were measured in young and adult blackbirds. Both muscles grew disproportionately relative to body mass. The pectoralis consisted entirely of fast-twitch fibers, predominantly fast oxidative glycolytic. In contrast, the gastrocnemius muscle consisted of a mixture of slow and fast fibers (predominantly fast glycolytic). Although fiber composition was constant, both cross-sectional area and density of fibers increased with age in both muscles. Catabolic capacities of the pectoralis increased significantly (approximately 7-8-fold) throughout the nestling period, most abruptly after day 3 (citrate synthase, CS) or day 4 (3-hydroxacyl-CoA-dehydrogenase, HOAD). Myofibrillar ATPase activities in the pectoralis were initially low, but increased after day 5. Further increases in CS and myofibrillar ATPase activities occurred in the pectoralis after fledging. CS and HOAD activities in the leg were much lower, but myofibrillar ATPase activities were remarkably similar in the two muscles, differing only in adults. These results are consistent with the hypothesis that the development of endothermy is dependent on the morphological and biochemical maturation of skeletal muscles important in thermogenesis.     

Responses to tones of single cells in nucleus magnocellularis and nucleus angularis of the redwing blackbird (Agelaius phoeniceus)

Summary Responses to tones were recorded from single cells in the brainstem auditory nuclei n. magnocellularis and n. angularis of the redwing blackbird (Agelaius phoeniceus). Whereas 30% of the cells in angularis had rates of spontaneous activity less than 40/s, only 5% of those in magnocellularis had rates this low. Two types of response maps are found in n. angularis. Type III maps have a single excitatory area with regions of inhibition on one or both sides. Type IV maps have a region of excitation at sound levels near threshold at the best frequency of the cell; at higher levels the best frequency is inhibitory; there can be broad bands of inhibition or interleaved bands of inhibition and excitation at higher levels. Cells in magnocellularis show only Type III response maps with generally weaker inhibitory sidebands than those in angularis. Responses to excitatory tone bursts in magnocellularis are primary-like in that they show a rapid increase in discharge rate at tone onset followed by a decrease in rate of 30 to 70% over the next 50 ms. On the other hand, a number of response types are seen in angularis, including primary-like, pauser, and onset. These results for magnocellularis and angularis are compared with results from their presumed mammalian homologues, the anteroventral cochlear nucleus, and posteroventral and dorsal cochlear nuclei, respectively. Finally, the relationship between response patterns in these avian brainstem nuclei and those seen in higher auditory centers is considered.This work was supported by a grant from the National Institute of Neurological and Communicative Diseases and Stroke. J. Sinnott is a National Institutes of Health Postdoctoral Fellow. M.B. Sachs is a National Institutes of Health Research Career Development Awardee. Mrs. Phyllis Taylor worked out many of the histologic procedures and handled all of the histology for the project. Orlando Asuncion was responsible for the care of the birds used in this study. We wish to gratefully acknowledge the cooperation of the Patuxent Wildlife Center, Laurel, Maryland, and in particular Brooke Meanly.     

Fitness cost of incubation in great tits (Parus major) is related to clutch size

Life-history theory predicts that parents produce the number of offspring that maximizes their fitness. In birds, natural selection on parental decisions regarding clutch size may act during egg laying, incubation or nestling phase. To study the fitness consequences of clutch size during the incubation phase, we manipulated the clutch sizes during this phase only in three breeding seasons and measured the fitness consequences on the short and the long term. Clutch enlargement did not affect the offspring fitness of the manipulated first clutches, but fledging probability of the subsequent clutch in the same season was reduced. Parents incubating enlarged first clutches provided adequate care for the offspring of their first clutches during the nestling phase, but paid the price when caring for the offspring of their second clutch. Parents that incubated enlarged first clutches had lower local survival in the 2 years when the population had a relatively high production of second clutches, but not in the third year when there was a very low production of second clutches. During these 2 years, the costs of incubation were strong enough to change positive selection, as established by brood size manipulations in this study population, into stabilizing selection through the negative effect of incubation on parental fitness.