Introduction

Intraspecific variation in individual colouration is widespread across the animal kingdom [1,2,3]. This naturally occurring variation reflects underlying differences in the extent and pattern of pigment deposition, which in turn usually have a genetic basis. For example, the melanocortin-1 receptor (MC1R), which plays a central role in the deposition of black to brown eumelanin and the yellow to reddish-brown phaeomelanin, has been associated with plumage polymorphism in multiple bird species including, for example, bananaquits, snow geese, arctic skuas, red-footed boobies and chickens [1–4] as well as with hair colouration in humans and other mammals [5–7].

Despite many of the genes involved in pigmentation having no known pleiotropic effects, colour morphs of several species show significant differences in fitness-relevant life history traits. Notable exceptions are the melanocortin receptor genes which are known, in conjunction with the pro-opiomelanocortin (POMC) gene, to influence not only plumage pigmentation but also behaviour, immunity and stress responses [8]. For example, in barn and tawny owls, plumage colouration has been linked to fitness via differences in immune responsiveness [9–12]. Similarly, in common buzzards, three colour morphs termed 'light', 'intermediate' and 'dark' appear to be maintained by contrasting selection pressures on the extreme morphs, with darker individuals being less aggressive and more dispersive but more susceptible to ectoparasites, whereas light individuals are more aggressive, disperse later and carry more endoparasites [13–15]. Consequently, intermediately melanised birds tend to experience the best of both worlds and have the greatest overall lifetime reproductive success [13, 16].

Another potential mechanism by which melanin-based colouration may affect fitness involves differences in energy homeostasis [17–19]. Studies of several vertebrate species have shown that colouration can affect the regulation of food intake and energy expenditure, resulting in variation in adult body size [8, 18]. Typically, highly melanised individuals tend to be both larger and heavier than individuals with lighter colouration (reviewed by [8]).

Numerous songbird species are plumage polymorphic [20], making this group well suited to exploring the fitness consequences of plumage polymorphism. Within the Estrildid finches, for example, Gouldian finches possess different plumage morphs that are stable in the wild [21] and which differ in their fitness [22, 23]. The zebra finch (Taeniopygia guttata) provides another potential avian model system for exploring the effects of plumage colouration on morphological and fitness variation. This Estrildid finch has distinctive melanin based plumage ornaments that differ in the extent to which eumelanin and phaeomelanin are present [24]. Furthermore, variation in plumage colouration occurs naturally as well as extensively in domesticated populations in the form of different colour morphs, of which over 30 have been documented [25]. Although the colour morphs of parents and other conspecifics play a role in mate preferences and song development [26–28], little is known in general about the broader behavioural and fitness consequences of these plumage polymorphisms. Several studies have uncovered effects of colour morph on nest preferences [29] and it is also known that white zebra finches have a partially impaired visual system [30, 31], but no effects of morph have been found on cognitive skills [32].

Although zebra finches are in many respects ideally suited to exploring the fitness consequences of plumage polymorphism, they also exemplify an issue that is common to many association studies–that of the potentially confounding effects of cryptic population structure. It has long been recognised in studies of humans that associations between genotypes and phenotypes can be confounded by population stratification, which can lead to either false positives or false negatives [33]. Various approaches have been proposed to deal with this problem [34] but these typically rely on the use of very large panels of unlinked genetic markers, which are unavailable for most non-human study systems. Consequently, the majority of studies of colour polymorphisms in birds and other organisms have not been able to control for differences in the genetic backgrounds of individuals [8]. While it remains unclear how important this could be in wild populations, many of which do not appear to be strongly structured, population stratification may be more prevalent in domesticated populations, especially where multiple stocks are present.

An example of this is provided by a recent study of zebra finches that looked for sequence differences in the MC1R gene between two plumage morphs that differ maximally with regard to the extent of melanin-based coloration, termed 'wild-type' and 'white' respectively [35]. Wild-type birds (Fig 1) are greyish in colour with males carrying additional secondary sexual ornamentation in the form of orange cheek patches, a black throat, black breast stripes and maroon-coloured flanks [25, 36]. By contrast, the white morph (Fig 1), which has an autosomal recessive mode of inheritance [25], is leucistic and completely lacks both phaeomelanin and eumelanin. Sequencing of the MC1R gene uncovered a highly significant association with morph, but this was confounded by strong underlying population structure as revealed by microsatellites. After crossing the two plumage morphs over two generations and rearing the F₂ birds to adulthood (for details of the breeding scheme, see Fig 1), the original association between MC1R genotype and colouration was lost [35].

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Fig 1. Overview of the controlled breeding experiment.

a) F₀ birds, b) F₁ birds were backcrossed with white birds, resulting in c) F₂ backcrossed birds, d) the F₂ birds were paired with a partner of the same plumage morph. Photos are taken from [35, 66].

https://doi.org/10.1371/journal.pone.0188582.g001

A further interesting observation is that the white zebra finch colour morph only occurs sporadically in the wild [25]. So far, it is unclear whether this is simply a reflection of low underlying allele frequencies, or whether white birds could be under stronger selective pressures than their wild-type counterparts. Overall, the consequences of plumage colour variation in zebra finches on physiology, behaviour and fitness have been rarely addressed in experimental studies, as pointed out by a recent review [37]. Consequently, in this study, we compared morphological and life-history traits between wild-type and white zebra finches using F₂ birds from the experiment described above to control for background genetic differences. Specifically, we reared the F₂ birds to adulthood, collected morphological measurements from them and then randomly paired birds of the same plumage morph and gave them the opportunity to breed. Given that the white morph is rare in the wild [25] and that darker individuals of many vertebrates are often in better body condition than lighter ones [8] we hypothesized that wild-type birds should have a selective advantage over white birds. Specifically, we expected to find that wild-type birds would be superior to white birds in key fitness-related traits such as body condition and reproductive investment.

Methods

Results

Body condition variation among the plumage morphs

Body condition, expressed as the ratio of body mass to tarsus length, differed significantly among the three plumage morphs and was also significantly higher in females relative to males (Fig 2; LME_condition: factor plumage morph F_2,61 = 7.66, p = 0.001, factor sex F_1,61 = 20.06, p < 0.0001, R² of the model = 0.63). Post hoc pairwise comparisons revealed significant differences between white and wild-type birds (p = 0.012) as well as between wild-type and intermediate birds (p = 0.002), whereas white and intermediate birds were not significantly different (p = 0.16) from one another.

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Fig 2. Body conditions of the three plumage morphs of the F₂ birds prior to breeding.

Shown are the means ± SE of each plumage morph. The three groups differ significantly from each other and the post hoc comparisons reveal differences between wild-type and intermediate birds (p = 0.002, indicated by “**”) and wild-type and white birds (p = 0.012, indicated by “*”). White and intermediated coloured birds did not differ from each other in body condition (p = 0.16, indicated by “ns”). See the texts for details of the tests.

https://doi.org/10.1371/journal.pone.0188582.g002

Effects of plumage morph on reproductive traits

The majority of breeding pairs (27 out of 30) laid at least one egg during the experiment. Latency to lay the first egg varied significantly among the three plumage morphs (LM_{latency to first egg}: factor morph F_2,24 = 3.98, p = 0.03, R² = 0.25; Fig 3A). Post hoc comparisons indicated that white and intermediate birds started laying eggs around the same time (p = 0.71; Fig 3A) and that both of these morphs began laying significantly earlier than wild-type birds (both p<0.024; Fig 3A).

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Fig 3. Reproductive parameters of the F₂ breeding pairs belonging to one of the three plumage morphs.

Shown are means ± SE for a) latency until pairs laid the first egg (in days); and b) relative egg mass (calculated as mean egg mass / female body mass). Latency until pairs laid the first egg was significantly different between the three plumage morphs and the post hoc comparisons revealed that wild-type pairs had higher latencies than both white and intermediate birds (both p<0.024, as indicated by “*”). Whereas latencies of intermediate and white pairs did not differ (p = 0.71, indicated by “ns”). The relative egg mass also differed between the three morphs and the post hoc test indicated that white birds had a higher relative egg mass than intermediate (p = 0.03, indicated by “*”) and wild-type birds (p = 0.005, indicated by “**”). Intermediate and wild-type birds did not differ (p = 0.63, indicated by “ns”). See text for details.

https://doi.org/10.1371/journal.pone.0188582.g003

The total number of eggs produced did not differ significantly by morph (LM_{number of eggs}: factor plumage morph F_2,27 = 2.04, p = 0.15, R² = 0.13, Power = 0.37). However, female investment in terms of mean relative egg mass varied significantly (Fig 3B; LM_{mean rel. egg mass}: factor plumage morph F_2,24 = 5.55, p = 0.01, R² = 0.32; Fig 3B). Post hoc comparisons showed that white pairs had higher values than intermediate (p = 0.03; Fig 3B) and wild-type (p = 0.005; Fig 3B) pairs but the relative egg masses of intermediate and wild-type birds did not differ significantly (p = 0.63; Fig 3B). The total effort in egg mass production did not vary significantly by plumage morph (LM_{total effort in egg mass}: factor plumage morph F_2,24 = 2.13, p = 0.14, R² = 0.15, Power = 0.43). Similarly, plumage morph was unrelated to hatchling mass (LM_{mean hatchling mass}: plumage morph F_2,8 = 0.16, p = 0.86, R² = 0.04, Power = 0.08).

Discussion

Using zebra finches of different plumage morphs from a controlled breeding experiment, we showed that plumage colouration is associated with differences in adult body condition and reproductive traits. Birds of the white morph exhibited shorter latencies to start reproduction and tended to invest more into their eggs than wild-type birds after controlling for maternal body mass. By contrast, the morphs did not differ significantly with respect to either the total number or biomass of offspring, although our experiment had limited statistical power in respect of these particular traits. Taken at face value, our findings suggest that wild-type and white birds may be exploiting different reproductive strategies that under controlled laboratory conditions do not appear to result in a net difference in reproductive fitness. However, larger sample sizes in the laboratory as well as confirmatory work in the wild would be required to reach more confident conclusions in respect of the ultimate fitness outcomes.

Our study was partly motivated by the observation that, although both wild-type and white zebra finches are present in the wild, white animals are quite rare [36], although they have been maintained in captive populations through selective breeding [35, 38]. While frequency differences in the wild could simply be a reflection of the underlying allele frequencies, in which case the white allele should be relatively rare, another possibility is that white birds might be subjected to stronger natural selection under natural conditions. For example, highly conspicuous phenotypes like bright white colouration might be associated with increased predation pressure in the wild, as has been observed in several other species [53–55]. Alternatively, as shown for example in guppies, rare phenotypes could potentially accrue fitness advantages depending on the prevailing population structure [56]. This has been advocated in predators as the apostatic selection hypothesis [57] where a rare morph might not be as readily recognised as either a predator or prey due to its rarity, resulting in negative frequency-dependent selection.

In a previous study, we showed that laboratory strains of wild-type and white zebra finches differ significantly from one another in their genetic background as measured using microsatellites, which reflect their long history of selective breeding in isolation [35]. As population structure can be a problem for association studies [33, 34, 58, 59], we therefore used a controlled breeding design to reduce the risk of obtaining artefactual associations that reflect differences in the genetic background rather than the trait in question (i.e. plumage colouration). Having done so, we found a number of significant associations between plumage colouration and morphological and life-history traits. It remains unclear whether these associations could be directly caused by the gene(s) responsible for colouration, or whether genes in linkage disequilibrium with them could be involved. Direct effects are plausible as POMC, for example, is expressed in multiple tissues and can have downstream influences on diverse processes ranging from stress responsiveness, food intake, energy expenditure and sexual activity [8]. However, given the unusual recombination landscape of zebra finches, in which almost a third of the genome typically segregates in only four independent blocks [60], possible effects of linked genes cannot be discounted.

One of our main findings was that wild-type and white birds differed significantly in body condition. White birds on average had lower body condition in adulthood than darker birds, consistent with findings in other vertebrate species [8]. However, white zebra finches appeared to compensate for these differences during reproduction by laying earlier and investing a significantly greater proportion of their total body mass into eggs. This increased investment would be expected to be beneficial in the short term, as egg mass correlates positively with hatchling mass in zebra finches, and heavier chicks in turn have a greater likelihood of surviving into adulthood [43]. Compensation in reproductive traits has been shown in other contexts in zebra finches. For example, females paired with an attractive partner invest more into their eggs [61], whereas breeding pairs that have experienced harsh conditions during early life tend to have a shorter latency to reproduce [62]. Flexibility in these traits would thus appear to be adaptive, at least in the short term, although compensatory investment may well carry costs later in life as one would expect there to be a trade-off between reproductive and somatic investment [63–65].

The potential costs arising from compensation in important fitness-related traits are worthy of further study. We focused on birds during their first breeding attempt, which seems reasonable as zebra finches are opportunistic breeders [25] and may not always have multiple opportunities to reproduce. However, as studies of other bird species have uncovered links between lifetime breeding success and plumage colouration [16], an obvious next step would be to investigate the effects of colour morph over the entire lifespan in order to test whether the patterns we have uncovered remain stable throughout adulthood or whether they diminish over time. Such a study would also be long enough to test for differences between the morphs in rates of senescence and longevity.

During our experiment, compensatory investment on the part of white zebra finches appeared to result in similar levels of overall reproductive investment as for wild-type birds, despite the latter being in better body condition. If this is indeed the case, our findings would contrast with several studies of wild vertebrate populations (e.g. owls, buzzards or Gouldian finches [9–11, 13, 23]) showing that colour morphs often differ markedly in respect of key fitness traits and ultimately reproductive success. We can think of two main possible explanations for such a discrepancy. First, the results of post hoc power tests corresponding to our non-significant statistical analyses revealed generally rather limited power, particularly for the effect of plumage morph on hatchling mass. This suggests that some of our tests lacked the statistical power to detect anything but very strong effects, an issue that could potentially be remedied in future studies by further increasing sample sizes. Second, our experiment was carried out under controlled laboratory conditions where high quality food was provided ad libitum and the birds were not exposed to predators. Under more natural conditions, any fitness differentials should be amplified, a possibility that could be tested by replicating our study in semi-natural enclosures and / or by manipulating food availability. Finally, there is also the question of how representative our study may be of the situation in the wild, as our crossing design will have altered the genetic background of the colour polymorphism(s) responsible for the white phenotype. To evaluate this, a confirmatory study would be required in the wild, although this may prove difficult due to the natural rarity of white birds.

To summarise, our study uncovered a number of differences between wild-type and white zebra finches after having controlled for the potentially confounding effects of population structure through a controlled breeding design. Specifically, white birds were significantly lighter and had lower body condition than wild-type birds, yet they reproduced earlier and laid heavier eggs relative to their own body mass. The net result was no significant difference in total reproductive output, although our study was underpowered to detect anything but very large differences in total egg mass production and total hatchling mass. Although it appears that differences in morphological traits between wild-type and white birds might be compensated for during reproduction in captivity, it may be more difficult for white birds to compensate for their lower body condition under natural conditions, which could potentially help to explain the low frequency of white birds in the wild. Furthermore, due to their conspicuous phenotype, white birds may also suffer from greater predation under natural conditions.

Supporting information

Acknowledgments

We thank the animal caretakers from the Department of Animal Behaviour, Bielefeld University. Two anonymous reviewers provided helpful comments to an earlier version of the manuscript. We acknowledge support for the Article Processing Charge from the Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.