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Like males of many bird species, male House Finches (Carpodacus mexicanus) have patches of feathers with ornamental coloration that are due to carotenoid pigments. Within populations, male House Finches vary in expression of ornamental coloration from pale yellow to bright red, which previous research suggested was the result of variation in types and amounts of carotenoid pigments deposited in feathers.

CAROTENOID PIGMENTS ARE responsible for the bright red, orange, and yellow coloration of plumage. Birds obtain those carotenoids exclusively through their diet. No animal has been shown unequivocally to be capable of in vivo synthesis of carotenoids (Goodwin 1984, 1986; Schiedt 1990). In birds, dietary carotenoids may either be deposited directly into feathers or chemically changed from ingested forms prior to pigment deposition, typically by addition or elimination of oxygen groups to one or both end rings of the molecule (Davies 1985, Goodwin 1986, Tyzckowski and Hamilton 1986a, b; Brush 1990, Schiedt 1990).

The House Finch (Carpodacus mexicanus) is a sexually dichromatic passerine bird species in which males display bright, carotenoid-based patches of color on their crowns, throats, breasts, and rumps, and male House Finches vary in expression of that ornamental coloration from a bright red to a dull yellow (Michener and Michener 1931, Hill 1990, 1993a). The carotenoid pigments responsible for colorful plumage in the House Finch and the pigmentary basis for variation among males in expression of that coloration were first studied by Brush and Power (1976). They attributed plumage color variation to differences in constituent carotenoids in feathers. Red birds had the most complex assemblage of pigments, consisting of beta-carotene, a group of unidentified mixed xanthophylls, orange isocryptoxanthin, and red echinenone; orange birds had the same subset of carotenoids without echinenone; and yellow birds lacked both echinenone and isocryptoxanthin. Recent analyses of several congeneric finch species of the Palearctic Carduelinae done by Stradi et al. (1995a, b; 1996, 1997), using new analytical techniques, revealed a more complex pattern that differed substantially from that described by Brush and Power (1976).

The proximate basis of variation in carotenoid-based plumage coloration in House Finches is of interest beyond improved understanding of the physiological control of avian pigmentation. Plumage redness in House Finches has been shown to be a primary criterion used by females in choosing mates (Hill 1990, 1991, 1994a). In addition, plumage brightness in male House Finches is correlated with overwinter survival (Hill 1991), nutritional condition during molt (Hill and Montgomerie 1994), parasite load (Thompson et al. 1997, Brawner et al. 2000), and provisioning of females during incubation (Hill 1991). It has been proposed that male plumage brightness is an honest signal of male condition, because carotenoids may be scarce resources in the environment and carotenoid-based color displays may be costly to produce (Hill 1994b, 1996a, 2002). A thorough understanding of the signal content of carotenoid-based ornamental displays can only be achieved, however, through an understanding of the proximate control of variation among males in expression of these displays (Hill 1992, 1996a, 2002).

Feeding experiments conducted with captive House Finches have demonstrated that variation among males in plumage hue and saturation is dependent upon carotenoid access during molt (Brush and Power 1976, Hill 1992, 1993a). When males are held in flight cages and fed a standardized diet, variation in ornamental plumage coloration is minimized (Hill 1992, 1993a). Moreover, after molting in captivity under conditions of standardized carotenoid access, males from populations that are typically drab in coloration grow ornamental plumage coloration that is indistinguishable from that grown by males from populations that are typically bright in coloration (Hill 1993a). However, the degree to which access to dietary carotenoids affects expression of carotenoidbased plumage coloration in the wild remains controversial (Hill 1994c, 2002; Hudon 1994a, Thompson et al. 1997, Inouye 1999). Birds are capable of endogenous modification of ingested carotenoids prior to deposition into target tissues (Fox et al. 1969, Davies 1985, Schiedt et al. 1985, Tyczkowski and Hamilton 1986b, c, d; Brush 1990, Hencken 1992). Therefore, mechanisms involved in the digestion, absorption, transport, modification, and deposition of dietary carotenoids may contribute to plumage color variation (Hill 1999, 2002). Furthermore, viral, bacterial, and coccidial infections may have a significant effect on expression of ornamental plumage coloration by male House Finches (Thompson et al. 1997, Nolan et al. 1998, Hill and Brawner 1998, Brawner et al. 2000).

There is substantial variation in expression of ornamental plumage coloration in House Finches not just among males within populations, but also among populations and subspecies (Moore 1939, Hill 1993a). There are approximately 15 subspecies of House Finches in North America (Moore 1939, Hill 1996b), each of which has had a unique evolutionary history for thousands of years (Moore 1939). Each subspecies is characterized by specific plumage traits, some of which involve carotenoid coloration (Moore 1939, Hill 1996b). Two subspecies are studied in this paper: C. m. frontalis, originally native to coastal California but now introduced to the Hawaiian Islands and the eastern United States and Canada, and C. m. griscomi, found in a relatively small region of southern Mexico. Male House Finches from the frontalis population have much more extensive ventral carotenoid pigmentation (larger patch size) than those from the griscomi population (Moore 1939, Hill 1993a), but some adult male griscomi have more intense red coloration than any male frontalis (Hill 1993a).

In contrast to the plasticity of expression of the color (hue, brightness, and saturation) of carotenoid-based plumage coloration, differences between frontalis and griscomi males in expression of size of ventral patches of ornamental coloration reflect fixed genetic differences between populations. When they are fed a lowcarotenoid diet, both frontalis and griscomi males grow drab yellow plumage; when they are fed a red-carotenoid-supplemented diet, they grow bright red feathers (Hill 1993a). Regardless of diet treatment and plumage color, griscomi males always display a small patch of ornamental color, and frontalis males always display a relatively large patch of color (Hill 1993a). Moreover, hybrid males produced by crossing a griscomi female with a frontalis male showed a patch size intermediate to the parent types (Hill 1993a). Thus, there are some fixed genetic differences between subspecies at least in distribution of carotenoid pigments in the plumage. Whether there are also differences among subspecies in types and amounts of carotenoid pigments that color feathers has not previously been investigated.

In this study, we identified and quantified the carotenoids responsible for male House Finch plumage coloration in the subspecies frontalis and griscomi. Our objectives were, first, to elucidate the pigmentary basis for extreme variation in expression of ornamental plumage coloration among males observed within age classes and subspecies. To do that, we investigated how types and amounts of carotenoid pigments in feathers affected both hue (redness) and saturation of plumage coloration among individual males. Alhough we studied males in only two subspecies, a wide range of hues, saturations and patch sizes of ornamental plumage color has been recorded in frontalis and griscomi (Hill 1993a). Second, within both subspecies, we compared carotenoid pigments of hatch-year versus older males to analyze extent to which feather pigmenation was affected by age. Finally, to investigate the proximate basis for differences between subspecies in carotenoid display, we compared their patch sizes and mean hues, total carotenoid abundances, and carotenoid composition of their feathers.

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METHODS

DISCUSSION

Pigmentary basis for individual variation in plumage coloration.-For male House Finches of the subspecies C. m. frontalis and C. m. griscomi, the hue of ornamental plumage depended primarily on proportion of red (4-keto-carotenoids) versus yellow pigments deposited in feathers. Relationship between proportion of red pigments and plumage redness was strong and significant for hatch-year and adult frontalis males and for hatch-year griscomi males, but there was no relationship between plumage redness and proportion of red pigments in adult griscomi males. It seems likely that proportion of red pigments was not correlated with plumage redness in adult griscomi males, because there was little detectable variation in hue. That is, all adult males were bright red with a hue of 9 or 10. Lack of variation in plumage hue among adult griscomi males would also explain weak relationships between hue and virtually all component carotenoids (Table 2), the weak negative relationships between plumage redness and concentration of several red pigments, and the weak positive relationships between plumage redness and concentration of several yellow pigments (Table 2).

Contributions of specific carotenoids to variation in plumage hue were different for the various subspecies and age classes of males. 3Hydroxy-echinenone was the most abundant red pigment in the plumage of males from both subspecies and age classes, but its concentration was not always the best predictor of plumage redness. Likewise, lutein was the most abundant yellow pigment in the plumage of both subspecies and age classes that we sampled, but its contribution to plumage redness varied among groups. Total carotenoid concentration in feathers also had a weak but significant effect on plumage hue.

Our observations are similar to those reported for Common (Carduelis flammea) and Hoary (C. hornemanni) redpolls, in which hue differences among individuals were attributed to the concentrations of both lutein (a yellow carotenoid) and echinenone (a red carotenoid) as well as relative concentration of lutein-individuals with higher levels of lutein appeared more orange (Troy and Brush 1983). Similarly, the color polymorphism exhibited by the Sooty-capped Bush Tanager (Chlorospingus pileatus) is due to differing concentrations of lutein in the feathers (Johnson and Brush 1972). 3-Hydroxy-echinenone has also been found to be the primary carotenoid pigment responsible for red plumage color in several other Carpodacus finches, for example, C. roseus and C. rubricilloides (Stradi et al. 1995a, b; 1997), as well as the plumages of Pine Grosbeak (Pinicola eunucleator; Stradi et al. 1996).

The same basic relationship between redness of integumentary display and proportion of red carotenoid pigments has also been observed in fish. In the stickleback (Gasterosteus aculeatus), red males had primarily the red carotenoid pigment astaxanthin in their skin whereas yellow males had primarily yellow pigments tunaxanthin and lutein in their skin (Wedekind et al. 1998).

The color saturation (intensity) of ornamental plumage appeared to depend primarily on concentration of carotenoids in feathers, but relationship between saturation and carotenoid concentration was different for frontalis and griscomi males. Among frontalis males, total concentration of red pigments was the best predictor of plumage color saturation, but among griscomi males, total concentration of yellow pigments was the best predictor of plumage intensity. Most griscomi males had abundant red pigments in their plumage, and variation in concentration of red pigments apparently had little influence on plumage color saturation. On the other hand, abundance of yellow pigments was variable, and that variation in yellow-pigment concentration appeared to determine plumage intensity in griscomi males. Conversely, most frontalis males had abundant yellow pigments but variable concentrations of red pigments. In that subspecies, the concentration of red pigments determined plumage color saturation. Overall, the relationship between color saturation and pigment concentration was not as strong as expected. However, color saturation was difficult to assess by the visual scoring methods used in this study. Hence, error in scoring saturation may have obscured patterns. Future studies that quantify plumage saturation with a spectrophotometer may better resolve the issue of what pigment properties determine plumage saturation.

Age effects.-We found significant differences in plumage color and ventral patch size between adult and hatch-year males in griscomi but not frontalis. The smaller patches of color and drabber plumage of hatch-year griscomi males is not surprising, because males from that subspecies have been shown to have a distinctive and female-like first-year plumage (Hill 1996b). In frontalis populations, hatchyear males have generally been observed to be less colorful than adults (Michener and Michener 1931, Gill and Lanyon 1965, Hill 1992, 1993b), but hatch-year and adult males exhibit the same range of colors. What was surprising was that, despite delayed plumage maturation, adult and hatch-year griscomi did not differ significantly in pigment composition of their feathers. Clearly, many hatch-year griscomi males had the same carotenoid composition in their feathers as adult males (Fig. 6), but others had more and higher concentrations of yellow pigments. In contrast, despite their similarity in appearance, adult and hatch-year frontalis finches had significantly different pigment compositions, suggesting that different carotenoid combinations may result in the same plumage coloration.

Differences in the pigment composition of the feathers of adult and hatch-year frontalis males may reflect differences between those age groups in physiological mechanisms involved in feather pigmentation. Higher levels of zeaxanthin and lutein and the lower levels of adonirubin and astaxanthin in hatch-year compared to adult frontalis males suggest that hatch-year males may have a greater tendency to deposit unmodified dietary carotenoids directly into the feathers. Thus, ability to convert dietary precursors into 4-keto-carotenoids may increase with age. Similar results have been documented for female Red-winged Blackbirds (Agelaius phoeniceus) in which epaulet color changed from yellow in juveniles to orange in adults (Miskimen 1980). Such changes could be regulated by sex hormones (Stoehr and Hill 2001), such that ability to convert dietary carotenoids into the redder 4-keto-carotenoids is enhanced by onset of sexual maturity. The agerelated differences in pigment composition may also be due to differences between adult and hatch-year birds in access to dietary carotenoids, levels of parasite infection, immunocompetence, or general nutrition during molt.

Implications for potential pathways of carotenoid metabolism.-Birds are known to be capable of metabolically altering ingested carotenoids (Fox et al. 1969, Schiedt et al. 1985, Tyczkowski and Hamilton 1986a, b, c, d; Brush 1990, Schiedt 1990). Much of the transformation and modification of dietary carotenoids occurs by the introduction of oxo- or hydroxy-groups into the main beta-ionone ring (Schiedt 1990), by the alteration of end rings, for example, a Pinto an E-ring (Davies 1985), or both. Stradi et al. (1996) indicated that many cardueline finches are capable of converting carotenoids by this pathway, for example, converting dietary zeaxanthin into astaxanthin that is deposited into feathers.

Results from the present study suggest that House Finches are also capable of the addition of a keto group at the C-4 position, C-4' position, or both, thus explaining presence of red 4keto-carotenoids in the feathers. Keto groups at the C-4(') position functionally extend the chain of conjugated double bonds in the carotenoid molecule, causing a bathochromic shift, that is, shift of hue towards red (Hudon 1994b). Thus, higher levels of 4-keto-carotenoids may intensify redness of feathers, accounting for the observation that redder House Finches had higher proportions of 4-keto-carotenoids in their plumages.

Many vertebrates have the ability to convert beta-into E-end rings, which shortens the chain of conjugated double bonds and alters carotenoid color to bright yellow (Matsuno et al. 1985, Miki et al. 1985). In birds, that may be done to produce "canary xanthophylls" (Stradi et al. 1995a, b; 1997). House Finches may also be capable of those conversions, as demonstrated by presence of the yellow carotenoids, E,E-caroten3,3'-dione and 3'-hydroxy-E,E-caroten-3-one, in feathers. Those pigments may be produced from dietary sources of lutein or zeaxanthin because House Finch diets probably do not contain E,E-carotenoids (Inouye 1999). Many birds have been shown to oxidize hydroxy- into ketogroups at the C-3(') position (Stradi et al. 1996), and that mechanism may account for the occurrence of 3'-dehydro-lutein in House Finch feathers, converted from dietary lutein.

Subspecies comparisons.-We found substantial differences in amounts and kinds of carotenoids in both hatch-year and adult males from the two subspecies. Those differences were large and consistent enough that we were able to use carotenoid concentrations to classify correctly to subspecies all adult males and 96% of hatch-year males on the basis of discriminant function analysis. In frontalis males, the yellow 3'-hydroxy-E,E-caroten-3-one and E,E-caroten3,3'-dione were more likely to be found in plumage than the other component carotenoids. Those yellow carotenoids are most likely derived from dietary sources of lutein or zeaxanthin. On the other hand, the 4-keto-carotenoids, echinenone, 4-oxo-rubixanthin, canthaxanthin, adonirubin, and astaxanthin, in the plumage occurred in a greater percentage of the population of griscomi males. With the exception of astaxanthin, those carotenoids are probably not lutein- or zeaxanthin-derived. Those results indicate (a) that there may be differences between the two subspecies in levels of dietary lutein and zeaxanthin, and (b) that griscomi males demonstrate increased capacities for adding keto- functions at C-4('), whereas frontalis males show increased capacities for converting beta- into E-end rings.

Carotenoid-based coloration as an honest signal.-In this paper, we describe the pigmentary basis for variation in color expression among male House Finches. The data, however, provide no direct information about what causes some males to have more red pigments in their feathers than other males. Diet may play a role in determining plumage coloration in males (Hill 1992). Male House Finches must either ingest red pigments (which are rare in the diet; Inouye 1999) or specific precursors to the red pigments that are ultimately deposited in the feathers. However, the role of dietary access to carotenoid pigments in determining variation in expression of plumage coloration among wild birds remains controversial (Hill 1994c, 1999; Hudon 1994a, Thompson et al. 1997, Inouye 1999). To convert precursors in their diet to red pigments in their feathers, male House Finches must establish and maintain complex physiological systems for carotenoid absorption, transport, and deposition (Allen 1987, Erdman et al. 1993, Parker 1996, Furr and Clark 1997, Hill 2002). Those systems require energy (Hill 1996a, 2002), although whether or not the energy required for carotenoid utilization is great enough to constrain expression of ornamental plumage coloration is unknown (Hill 1996a, Inouye 1999). There is evidence that nutritionally stressed House Finches produce less red plumage than birds that are not stressed even when they have access to the same carotenoid pigments (Hill 2000). Furthermore, parasites potentially play a large role in determining expression of carotenoids; coccidia may inhibit intestinal absorption of carotenoids and have been shown to decrease plumage redness upon molt in male House Finches (Hill and Brawner 1998, Brawner et al. 2000). Other diseases that affect the overall health of finches, including pox and Mycoplasma galliceptum, cause males to grow a less red plumage (Thompson et al. 1997, Nolan et al. 1998, Brawner et al. 2000). Carefully controlled studies will be required to determine the relative contribution of those various factors to the expression of ornamental plumage coloration in wild House Finches.

The differences in plumage coloration and plumage pigment composition that we observed between age classes, between subspecies, and among males within an age class and subspecies may be the result of any of the factors listed above acting alone or in combination. The different mix of carotenoid pigments in the plumages of hatch-year versus adult frontalis House Finches suggests that hatch-year males may utilize dietary carotenoid pigments differently than adult males, and such age-specific carotenoid utilization has potentially important implications for signal content of ornamental plumage coloration in this species (Hill 1990, 1994a, 2002). In addition, differences in the carotenoid composition of feathers of males from the two subspecies that we sampled suggest that those subspecies utilize different metabolic pathways in modifying dietary pigments. Whether those differences in pigment physiology evolved under sexual selection for brighter color display (Hill 1994c) and what the difference means for signal honesty in those two subspecies can perhaps be answered through a comparative study looking at pigment composition of other subspecies as well as the evolutionary relationships of those taxa.

ACKNOWLEDGMENTS

We extend special thanks to the individuals and families who provided logistical support and hospitality during collecting trips, particularly Fran Mewaldt, Jorge Serrano, Sr., Jorge Serrano, Jr., the Serrano family, and Mike Rigney and the staff at Coyote Creek Riparian Station. Jim Dale assisted with the field work in California. Birds were collected under a joint U.S. Fish and Wildlife and California Fish and Game Scientific Collecting Permit in California and a scientific collecting permit from La Secretaria de Relaciones Exteriores, Mexico. This study was approved by the Chancellor's Animal Research Committee at University of California Los Angeles and the Animal Care Committee at Queen's University. Funding for this research was provided in part by the American Museum of Natural History (Frank M. Chapman Memorial Fund), American Ornithologists' Union, Los Angeles Audubon Society, and Sigma Xi grants to C.I., and Natural Sciences and Engineering Research Council of Canada (NSERC) research and equipment grants to R.M. G.H. was supported during field work by an NSERC International Postdoctoral Fellowship and during write-up by NSF grant IBN9722172. Hoffman-La Roche, Basel, Switzerland generously donated carotenoid standards used in this study. Kevin McGraw, Jocelyn Hudon, David Chapman, Ken Nagy, and two anonymous reviewers provided comments on the manuscript.