Countergradient variation in the sexual coloration of guppies (Poecilia reticulata): drosopterin synthesis balances carotenoid availability

Trinidad guppies (Poecilia reticulata) are distributed along an environmental gradient in carotenoid availability that limits the carotenoid content of the orange spots of males. The amount of synthetic red pteridines (drosopterins) in the orange spots covaries with the carotenoid content, such that the ratio of the two types of pigments is roughly conserved across streams. Carotenoids and drosopterins have different spectral properties and thus the ratio of the two types of pigments affects the shape of the orange spot reflectance spectrum. Geographic conservation of the carotenoid:drosopterin ratio suggests that males may be under selection to maintain a particular hue. We tested this hypothesis by comparing the pigmentation and coloration of guppies from six streams in the field to that of second-generation descendants of the same populations raised on three dietary carotenoid levels in the laboratory. The results show clearly that the geographic variation in drosopterin production is largely genetic and that the hue of the orange spots is conserved among populations in the field, relative to the laboratory diet groups. This is a countergradient pattern because genetic differences between populations in drosopterin production mask the effect of carotenoid availability on the hue of the orange spots. The potential for countergradient sexual selection to contribute to reproductive isolation between populations is discussed.     

Carotenoid scarcity, synthetic pteridine pigments and the evolution of sexual coloration in guppies (Poecilia reticulata).

Carotenoid-based sexual coloration is the classic example of an honest signal of mate quality. Animals cannot synthesize carotenoid pigments and ultimately depend on dietary sources. Thus, in carotenoid-poor environments, carotenoid coloration may be a direct indicator of foraging ability and an indirect indicator of health and vigour. Carotenoid coloration may also be affected, more directly, by parasites in some species. Carotenoids are not, however, the only conspicuous pigments available to animals. Pteridine pigments, with similar spectral properties, are displayed in the exoskeletons and wings of insects, the irides of birds and the skins of fishes, lizards and amphibians. Unlike carotenoids, pteridines are synthesized de novo by animals. We report that the orange spots that male guppies (Poecilia reticulata) display to females contain red pteridine pigments (drosopterins) in addition to carotenoids. We also examined the relationship between drosopterin production by males and carotenoid availability in the field. The results contrasted sharply with the hypothesis that males use drosopterins to compensate for carotenoid scarcity: males used more, not less, drosopterins in streams with higher carotenoid availability. The positive association between drosopterin use and carotenoid availability could reflect the costs of drosopterin synthesis or it could be a consequence of females preferring a particular pigment ratio or hue. Male guppies appear to use drosopterin pigments in a manner that dilutes, but does not eliminate, the indicator value of carotenoid coloration.     

Relationship between algal-foraging ability and expression of sexually selected traits in male guppies

In the guppy Poecilia reticulata, males exhibit orange spots on their body and tail, and the orange spot patterns are often criteria for female mate choice. The orange spot coloration of males is determined by the intake of algae, a natural source of carotenoids. Therefore, males exhibiting conspicuous orange coloration are considered to possess high algal-foraging ability. In the present study, we examined the influence of algal-foraging ability, measured by algal-searching ability and algal-foraging frequency, on the expression of orange spot patterns and on other sexually selected traits in male guppies. Males exhibiting better performance in terms of both algal-searching ability and algal-foraging frequency grew larger. The size of the orange spots on males also increased with algal-foraging ability. However, neither algal-searching ability nor algal-foraging frequency influenced the coloration of the orange spots. In this experiment, a limited supply of carotenoids possibly prevented the males from completely developing their spots to the intrinsic size. The results of this study suggest that in male guppies under a carotenoid-limited situation, the allocation of carotenoids is directed toward enlargement of the size of the orange spots rather than enhancement of their coloration. Since both the body size and orange spot patterns of males contribute to their sexual attractiveness to females, high algal-foraging ability may enhance their mating success.     

Female mate preference explains countergradient variation in the sexual coloration of guppies (Poecilia reticulata)

We tested the hypothesis that mate choice is responsible for countergradient variation in the sexual coloration of Trinidadian guppies (Poecilia reticulata). The nature of the countergradient pattern is that geographical variation in the carotenoid content of the orange spots of males is counterbalanced by genetic variation in drosopterin production, resulting in a relatively uniform pigment ratio. A female hue preference could produce this pattern, because hue is the axis of colour variation most directly affected by the pigment ratio. To test this hypothesis, we crossed two populations differing in drosopterin production and produced an F(2) generation with variable drosopterin levels. When the carotenoid content of the orange spots was held constant, female guppies preferred males with intermediate drosopterin levels. This shows that females do not simply prefer males with greater orange spot pigment content; instead, the ratio of the pigments also affects male attractiveness. To our knowledge, this is the first direct evidence for a hypothesized agent of countergradient sexual selection.     

Carotenoid limitation of sexual coloration along an environmental gradient in guppies

Carotenoids produce most of the brilliant orange and yellow colours seen in animals, but animals cannot synthesize these pigments and must rely on dietary sources. The idea that carotenoids make good signals because they are a scarce limiting resource was proposed two decades ago and has become the leading hypothesis for the role of carotenoids in animal communication. To our knowledge, until now, however, there has been no direct evidence that carotenoids are a limiting resource in nature. We showed that carotenoid availability in the wild limits the expression of sexual coloration in guppies (Poecilia reticulata), a species in which females prefer males with brighter orange carotenoid-containing spots. Further, the degree of carotenoid limitation varies geographically along a replicated environmental gradient (rainforest canopy cover), which opens new avenues for testing signal evolution theory.     

The Relationship Among Swimming Performance, Courtship Behavior, and Carotenoid Pigmentation of Guppies in Four Rivers of Trinidad

In the laboratory, courtship behavior and carotenoid pigmentation of male guppies are condition-dependent traits, since their expression is affected by physical vigor and environmental factors such as water velocity and diet. Whether these relationships exist in guppies under field conditions is yet to be determined. We compared the swimming performance, courtship behavior, and carotenoid pigmentation of guppies from headwater and downstream localities in four rivers of Trinidad. Swimming performance and courtship behavior of males differed among rivers and between headwater and downstream sites. Guppies from headwater sites swam significantly faster and had higher display rates than those from downstream sites. Mean swimming performance across sites was positively correlated with mean water velocity, but was correlated with the number of orange color spots (carotenoid pigment) in only one river. These results indicate that the courtship behavior of Trinidadian guppies is condition-dependent because the amount of display behavior is positively correlated to swimming performance, a measure of physical endurance. The proximal cause for this condition-dependence may be predator induced variation in microhabitat use by guppies in headwater and downstream locations.     

Colourful male guppies produce faster and more viable sperm

In guppies (Poecilia reticulata) precopulatory sexual selection (via female choice) and post-copulatory selection (via sperm competition) both favour males with relatively high levels of carotenoid (orange) pigmentation, suggesting that colourful males produce more competitive ejaculates. Here we test whether there is a positive association between male orange pigmentation and sperm quality. Our analysis of sperm quality focused on sperm swimming speeds (using CASA: computer-assisted sperm analysis to estimate three parameters of sperm velocity in vitro), sperm viability (proportion of live sperm per stripped ejaculate) and sperm lengths. We found that males with relatively large areas of orange pigmentation had significantly faster and more viable sperm than their less ornamented counterparts, suggesting a possible link between dietary carotenoid intake and sperm quality. By contrast, we found no relationship between sperm length (head length and total sperm length) and male phenotype. These findings, in conjunction with previous work showing that highly ornamented male guppies sire higher quality offspring, suggest that female preference for colourful males and sperm competition work in concert to favour intrinsically higher quality males.     

Effect of parasitic infection on male color pattern and female choice in guppies

We infected male guppies Poecilia reticulata with a naturallyoccurring monogenean parasite, Gyrodactylus turnbulli, in orderto examine effects of parasitism on the expression of colorpatterns and on attractiveness to females. The color of carotenoidspots and ability to attract females were compared between experimentallyinfected fish and a control group of their fullsib brothers,which had identical color patterns and were treated identicallyexcept for actual exposure to parasites. The orange spots ofmales that had been infected for 9 days followed by treatmentwith medication to remove parasites became significandy palerand less saturated. Control males (also treated widi medication)showed no significant changes in their orange spots. Femalesin a divided aquarium choice-apparatus showed no preferencebetween control and infection-treatment males initially, butshowed significant discrimination after the infectiondisinfectiontreatment. Females spent less time near males that had beeninfected and responded to a smaller fraction of their courtshipdisplays relative to control males. There were slight differencesin courting rates of males between treatments. Parasitic infectionappears to reduce the degree of expression of carotenoid colorsin guppies, and females are able to discriminate against recentlyinfected males, probably on the basis of the color change. Byavoiding infected males, females may be able to avoid becominginfected themselves, or they may be able to identify mates basedon "good genes" for parasite resistance diat can be passed onto their offspring.     

Sex-specific effects of carotenoid intake on the immunological response to allografts in guppies (Poecilia reticulata)

Rarely are the evolutionary origins of mate preferences known, but, recently, the preference of female guppies (Poecilia reticulata) for males with carotenoid-based sexual coloration has been linked to a sensory bias that may have originally evolved for detecting carotenoid-rich fruits. If carotenoids enhance the immune systems of these fishes, as has been suggested for other species, this could explain the origin of the attraction to orange fruits as well as the maintenance of the female preference for orange males. We used the classic immunological technique of tissue grafting to assay a component of the immune response of guppies raised on two different dietary levels of carotenoids. Individual scales were transplanted between pairs of unrelated fishes, creating reciprocal allografts. Transplanted scales were scored on a six-point rejection scale every day for 10 days. Five days later, the same pairs of fishes received a second set of allografts and were scored again. Compared with low-carotenoid-diet males, high-carotenoid-diet males mounted a significantly stronger rejection response to the second allograft but not to the first allograft. High-carotenoid-diet females, however, showed no improvement in graft rejection compared with low-carotenoid-diet females. To our knowledge, this is the first experimental evidence for sex-specific effects of carotenoid consumption on the immune system of a species with carotenoid-based sexual coloration. These results are consistent with the hypothesis that the mate preference for carotenoid coloration is maintained by the benefits to females of choosing healthy mates, but they cast doubt on the idea that the benefits of carotenoid consumption, per se, could account for the origin of the preference. The sex-specificity of carotenoid effects on allograft rejection in guppies provides indirect support for the general hypothesis that males pay an immunological cost for sexual ornamentation.     

Differential accumulation and pigmenting ability of dietary carotenoids in colorful finches

Many animals develop bright red, orange, or yellow carotenoid pigmentation that they use to attract mates. Colorful carotenoid pigments are acquired from the diet and are either directly incorporated as integumentary colorants or metabolized into other forms before deposition. Because animals often obtain several different carotenoids from plant and animal food sources, it is possible that these pigments are accumulated at different levels in the body and may play unique roles in shaping the ultimate color expression of individuals. We studied patterns of carotenoid accumulation and integumentary pigmentation in two colorful finch species--the American goldfinch (Carduelis tristis) and the zebra finch (Taeniopygia guttata). Both species acquire two main hydroxycarotenoids, lutein and zeaxanthin, from their seed diet but transform these into a series of metabolites that are used as colorful pigments in the plumage (goldfinches only) and beak (both species). We conducted a series of carotenoid-supplementation experiments to investigate the relative extent to which lutein and zeaxanthin are accumulated in blood and increase carotenoid coloration in feathers and bare parts. First, we supplemented the diets of both species with either lutein or zeaxanthin and measured plasma pigment status, feather carotenoid concentration (goldfinches only), and integumentary color. Zeaxanthin-supplemented males grew more colorful feathers and beaks than lutein-supplemented males, and in goldfinches incorporated a different ratio of carotenoids in feathers (favoring the accumulation of canary xanthophyll B). We also fed goldfinches different concentrations of a standard lutein-zeaxanthin mix and found that at physiologically normal and high concentrations, birds circulated proportionally more zeaxanthin over lutein than occurred in the diet. Collectively, these results demonstrate that zeaxanthin is preferentially accumulated in the body and serves as a more potent substrate for pigmentation than lutein in these finches.     

The effects of carotenoid and food intake on caudal fin regeneration in male guppies

The trade‐offs involved in allocating carotenoid pigments and food to healing and regrowing damaged caudal fin tissue v . other functions were examined in guppies Poecilia reticulata , a species in which females prefer males that display larger amounts of carotenoids in their skin. The guppies were derived from four natural populations in Trinidad that differed in resource availability but not predation intensity. Carotenoids, food and site of origin did not affect either absolute or relative fin regrowth, which suggested that fin regeneration in guppies was not constrained by carotenoid availability. It is possible that carotenoid intake influences fin regeneration in the presence of natural stressors such as predators. There was a significant negative interaction between food level in the laboratory and resource availability in the field: males from low‐resource‐availability sites regrew more fin tissue when raised on the high food level, and males from high‐resource‐availability sites regrew more fin tissue when raised on the low food level. The direction of this interaction runs counter to theoretical expectations.     

Carotenoid limitation and mate preference evolution: a test of the indicator hypothesis in guppies (Poecilia reticulata)

Under the indicator models of mate choice, female preferences evolve to exploit the condition-dependence or "indicator value" of male traits, which in turn may cause these traits to evolve to elaborate extremes. If the indicator value of a male trait changes, the payoff function of the female preference for that trait should change and the preference should evolve to a new optimum. I tested this prediction in the guppy, Poecilia reticulata, a species in which the indicator value of a sexually selected male trait, carotenoid coloration, varies geographically. Carotenoid coloration is thought to be an indicator of foraging ability and health because animals must obtain carotenoid pigments from their diet. The primary dietary source of carotenoids for guppies is unicellular algae, the abundance of which varies among natural streams because of variation in forest canopy cover. Carotenoid availability limits male coloration to a greater extent in streams with greater forest canopy cover. Thus, the indicator value of male coloration covaries positively with canopy cover. To test the indicator model prediction, I measured genetic divergence in the strength of female preferences for carotenoid coloration between high- and low-carotenoid availability streams in each of three river drainages. Second-generation laboratory-born females were given a choice between full-sib males raised on three different dietary levels of carotenoids. For all six populations, male attractiveness (as determined from the responses of females to male courtship displays) increased with dietary carotenoid levels. However, the strength of female preferences differed between populations in the predicted direction in only one of three river drainages. These results fail to support a crucial prediction of the indicator model. More studies taking an interpopulation approach to studying mate preference evolution are needed before the explanatory value of the indicator models can be rigorously assessed.     

The influence of carotenoid acquisition and utilization on the maintenance of species-typical plumage pigmentation in male American goldfinches (Carduelis tristis) and northern cardinals (Cardinalis cardinalis).

Birds display a tremendous variety of carotenoid-based colors in their plumage, but the mechanisms underlying interspecific variability in carotenoid pigmentation remain poorly understood. Because vertebrates cannot synthesize carotenoids de novo, access to pigments in the diet is one proximate factor that may shape species differences in carotenoid-based plumage coloration. However, some birds metabolize ingested carotenoids and deposit pigments that differ in color from their dietary precursors, indicating that metabolic capabilities may also contribute to the diversity of plumage colors we see in nature. In this study, we investigated how the acquisition and utilization of carotenoids influence the maintenance of species-typical plumage pigmentation in male American goldfinches (Carduelis tristis) and northern cardinals (Cardinalis cardinalis). We supplemented the diet of captive goldfinches with red carotenoids to determine whether males, which are typically yellow in color, were capable of growing red plumage. We also deprived cardinals of red dietary pigments to determine whether they could manufacture red carotenoids from yellow precursors to grow species-typical red plumage. We found that American goldfinches were able to deposit novel pigments in their plumage and develop a striking orange appearance. Thus, dietary access to pigments plays a role in determining the degree to which goldfinches express carotenoid-based plumage coloration. We also found that northern cardinals grew pale red feathers in the absence of red dietary pigments, indicating that their ability to metabolize yellow carotenoids in the diet contributes to the bright red plumage that they display.     

Orange Spots as a Visual Cue for Female Mate Choice in the Guppy (Poecilia reticulata)

Previous studies have suggested that orange pigment in the color patterns of male guppies is a cue for female choice. This paper describes a manipulative experiment designed to test this hypothesis. The color patterns perceived by females were manipulated by varying the color of light used to illuminate the experimental aquaria. Orange light dramatically reduces the conspicuousness of orange spots to human observers, and probably also to female guppies. As in previous experiments, female guppies discriminated among males based on differences in the extent of orange pigment, under white, blue, and green light conditions. Under orange light, however, females no longer appeared to discriminate on the basis of orange spots. These results support the hypothesis that orange spots, rather than other correlated characteristics, are a basis for female choice under normal lighting conditions.     

Season-, sex-, and age-specific accumulation of plasma carotenoid pigments in free-ranging white-winged crossbills Loxia leucoptera

Many birds acquire carotenoid pigments from foods and deposit these pigments into feathers and bare‐parts to become sexually attractive, but little work has been done on the interindividual and temporal variability in the types and amounts of carotenoids that free‐ranging individuals have available for use in coloration or other functions (e.g., in immunomodulation). To address this issue, we studied intra‐annual variation in plasma carotenoid profiles of juvenile and adult white‐winged crossbills Loxia leucoptera of both sexes. Adult male crossbills exhibit bright red carotenoid‐based plumage pigmentation, whereas females uniformly display drab yellow feather coloration and juvenile males only occasionally display some orange or pink color. Yellow xanthophylls (e.g., lutein, zeaxanthin) were predominant in plasma of birds from both sexes and age classes throughout the year. Plasma xanthophylls levels tended to be highest in the summer, when crossbills increase seed consumption for breeding as well as supplement their diet with insects. Blood accumulation of three other, less common plasma carotenoids‐β‐cryptoxanthin, rubixanthin, and gazaniaxanthin‐varied in a highly season‐, sex‐, and age‐dependent fashion. These carotenoids were virtually absent in juvenile or adult female plasma at all times of year and were only present in male plasma, at higher concentrations in adults than juveniles, during the period of feather growth (Sept.–Nov.). These pigments have been reported as valuable precursors of the metabolically derived red pigments (e.g., 3‐hydroxy‐echinenone, 4‐oxo‐rubixanthin, and 4‐oxo‐gazaniaxanthin, respectively) that appear in the plumage of male crossbills. These findings suggest that male crossbills either adopt a season‐specific foraging strategy to acquire foods rich in these pigments at the time they are needed to develop red coloration, or have a unique physiological ability to metabolically produce these pigments or absorb them from food during molt, in order to maximize color production.     

Age and experience affect female choice in the guppy (Poecilia reticulata)

Female choices of males, and how these choices are influenced by ecological and social factors, have been studied extensively. However, little is known about the effects of age and breeding experience on female mating decisions. We used video techniques to examine female mate choice in guppies based on the area of carotenoid (orange) pigmentation on the body. Females were presented with paired images of males, one ornamented and the other plain. Visual preference for each male was measured. Age-related changes in the criteria of choice were examined by comparing the responses of the same mature but sexually inexperienced 6-mo-old and 12-mo-old females. Effects of breeding experience on female choice were examined by comparing mate preferences of 12-mo-old female virgins with their preferences after they had mated and produced a brood. Female preferences for ornamented males with large areas of carotenoid pigment changed with age but not with mating experience. Six-month-old virgin females preferred ornamented males, whereas 12-mo-old virgin and postpartum females did not differentiate between males based on orange coloration. The results are discussed in light of life-history theory and have important implications for studies of sexual selection as well as for the design of mate-choice studies.     

The effect of dietary carotenoid access on sexual dichromatism and plumage pigment composition in the American goldfinch

We investigated potential dietary and biochemical bases for carotenoid-based sexual dichromatism in American goldfinches (Carduelis tristis). Captive male and female finches were given access to the same type and amount of carotenoid pigments in the diet during their nuptial molt to assess differences in the degree to which the two sexes incorporated ingested pigments into their plumage. When birds were fed a uniform, plain-seed diet, or one that was supplemented with the red carotenoid canthaxanthin, we found that males grew more colorful plumage than females. HPLC analyses of feather pigments revealed that male finches incorporated a higher concentration of carotenoids into their pigmented feathers than females. Compared to females, males also deposited significantly more canary xanthophyll B into feathers when fed a plain-seed diet and a greater concentration and proportion of canthaxanthin when fed a carotenoid-supplemented diet. These results indicate that sex-specific expression of carotenoid pigmentation in American goldfinches may be affected by the means by which males and females physiologically utilize (e.g. absorb, transport, metabolize, deposit) carotenoid pigments available to them in the diet.     

Female mating preferences for colourful males in a population of guppies subject to high predation

Female guppies Poecilia reticulata descended from the Tacarigua population in Trinidad subject to high predation exhibited strong preferences for males with relatively high levels of carotenoid colouration. The study, which controlled for differences in male courtship, revealed that mate choice in this population is based on the expression of orange pigmentation, irrespective of differences in male motivation.     

Pteridine, not carotenoid, pigments underlie the female-specific orange ornament of striped plateau lizards (Sceloporus virgatus)

Indicator models of sexual selection suggest that signal honesty is maintained via costs of ornament expression. Carotenoid-based visual signals are a well-studied example, as carotenoids may be environmentally limited and impact signaler health. However, not all bright yellow, orange and red ornaments found in vertebrates are carotenoid-based; pteridine pigments may also produce these colors. We examine the contribution of carotenoid and pteridine pigments to the orange reproductive color of female striped plateau lizards (Sceloporus virgatus). This color ornament reliably indicates female mate quality, yet costs maintaining signal honesty are currently unknown. Dietary carotenoid manipulations did not affect orange color, and orange skin differed from surrounding white skin in drosopterin, not carotenoid, content. Further, orange color positively correlated with drosopterin, not carotenoid, concentration. Drosopterin-based female ornaments avoid the direct trade-offs of using carotenoids for ornament production vs egg production, thus may relax counter-selection against color ornament exaggeration in females. Direct experimentation is needed to determine the actual costs of pteridine-based ornaments. Like carotenoids, pteridines influence important biological processes, including immune and antioxidant function; predation and social costs may also be relevant.     

Effect of Dietary Carotenoid Supplementation on Food Intake and Immune Function in a Songbird with no Carotenoid Coloration

Studies of ornamental carotenoid coloration suggest that animals may have evolved specialized mechanisms for maximizing color expression and advertising their potential worth as a mate. For example, when given a choice of foods, many carotenoid‐pigmented fishes and birds select the more colorful, presumably carotenoid‐rich foods, and then accumulate these pigments at high levels in both the integument and systemically, in order to boost their immune system and hence directly advertise their health state with their colors. The majority of animals, however, do not exhibit sexually selected carotenoid coloration, which raises the question of whether they still optimize pigment intake and allocation in ways that boost endogenous accumulation and health. We tested the effect of carotenoid supplementation on food intake, carotenoid accumulation in blood, and both innate and adaptive immunity in male society finches (Lonchura domestica) – a non‐carotenoid‐colored estrildid finch relative of the zebra finch (Taeniopygia guttata; a species in which males do display sexually attractive and health‐revealing carotenoid color). Males fed a carotenoid‐rich diet for 2 wk did not consume more food than control males. Still, consumption of the carotenoid‐rich diet for 2 wk significantly elevated circulating levels of carotenoids in blood in male society finches, yielding the potential for immune enhancement. In fact, carotenoid‐enriched finches performed significantly better than control birds in our assay of constitutive innate immunity (bacterial‐killing activity of whole blood), although not in our test of inducible adaptive immunity (response to a mitogenic challenge with phytohemagglutinin). These results suggest that affinities for carotenoid‐rich foods may be particular to species with sexually selected carotenoid pigmentation, but that, as in humans and other mammals (e.g. mice, rats) without carotenoid color, the immune‐boosting action of carotenoids is conserved regardless of the strength of sexual selection on pigment use.