Plumage polymorphism and variation in the melanocortin-1 receptor gene in the Fuscous Flycatcher, Cnemotriccus fuscatus (Wied, 1831)

We investigated the possible mechanisms behind the variation plumage color of the Fuscous Flycatcher, Cnemotriccus fuscatus, by sequencing the melanocortin-1 receptor (MC1R) gene, which has been associated with the variation in plumage coloration in birds. C. fuscatus is widely distributed in South America and includes seven subspecies, which diff er in their plumage coloration. Here we tested the hypothesis that the variation in the MC1R gene explains the plumage polymorphism found in C. fuscatus. We sequenced the MC1R gene in six subspecies, representing two groups: group 1 (yellow morph), with three subspecies, C. f. duidae, C. f. fumosus, and C. f. fuscatus, and group 2 (white morph), with the remaining subspecies, C. f. bimaculatus, C. f. beniensis, and C. f. fuscatior. Th e only variation we found among the C. fuscatus sequences were six non-synonymous substitutions from 22 variable sites, none of which were associated systematically with either plumage morph. Th e result of the neutrality test indicated that the polymorphism of the MC1R gene is not suggestive of signifi cant selection pressure. We conclude that variation in plumage coloration in C. fuscatus does not appear to be determined by the MC1R gene, and that it may be related to other loci or under the infl uence of environmental factors. KEY-WORDS: birds, MC1R gene, mutation, pigmentation, Tyrannidae. the diff erentiation of the plumage in avian species, due to the association between mutations in this gene and the phenotypic variation found in a number of diff erent groups of wild birds (Johnson et al. 2012, Ran et al. 2016). For example, single non-synonymous mutations in the MC1R gene were associated with plumage polymorphisms in the bananaquit (Coereba fl aveola) and the chestnut-bellied monarch, Monarcha castaneiventris (Th eron et al. 2001, Uy et al. 2009). Studies in birds have also shown that the MC1R gene controls the amount of both eumelanin (brown/black) and pheomelanin (red/ yellow) produced (Takeuchi et al. 1996, Wen et al. 2015). In particular, García-Borrón et al. (2005) showed that the yellow (pheomelanin) phenotype is produced by recessive MC1R extension (e) alleles. In this context, we investigated the variation in coloration found among the subspecies of the Fuscous Flycatcher, Cnemotriccus fuscatus, a monotypic genus widely distributed in South America (Fig. 1). Th ere are seven C. fuscatus subspecies, which are diff erentiated not only on the basis of their morphological characters, but also their vocalizations and ecology (Fitzpatrick et al. 2004). Th ese subspecies can be divided into two groups, Plumage polymorphism in Fuscous Flycatcher, Cnemotriccus fuscatus Vieira et al. 252 Revista Brasileira de Ornitologia 26(4): 2018 Species distribution


INTRODUCTION
Th e variation in plumage coloration has been studied from ecological, evolutionary and genetic perspectives (Hoekstra & Price 2004, Mundy 2005, Uy et al. 2016. Such diversity has been related to visual communication, and may have evolved in response to the evolution of the avian visual system (Osorio & Vorobyev 2008), although there is also some evidence that changes in plumage coloration may be a response to varying pressures in diff erent types of habitat (Gomez & Th éry 2004, McNaught & Owens 2002. Many questions remain unresolved, however, on the evolution of plumage coloration and its relation to speciation in birds (Stoddard & Prum 2011, Seddon et al. 2013, such as the mechanisms that mediate the change in coloration between juveniles and adults (Galván & Jorge 2015), and the factors determining changes in coloration despite the considerable energetic costs of this process (Legagneux et al. 2012, Mercadante & Hill 2014. Previous studies (e.g., Robbins et al. 1993, Vidal et al. 2010, Johnson et al. 2012 have suggested that the melanocortin-1 receptor (MC1R) gene may be involved in the diff erentiation of the plumage in avian species, due to the association between mutations in this gene and the phenotypic variation found in a number of diff erent groups of wild birds (Johnson et al. 2012, Ran et al. 2016. For example, single non-synonymous mutations in the MC1R gene were associated with plumage polymorphisms in the bananaquit (Coereba fl aveola) and the chestnut-bellied monarch, Monarcha castaneiventris (Th eron et al. 2001, Uy et al. 2009). Studies in birds have also shown that the MC1R gene controls the amount of both eumelanin (brown/black) and pheomelanin (red/ yellow) produced (Takeuchi et al. 1996, Wen et al. 2015. In particular, García-Borrón et al. (2005) showed that the yellow (pheomelanin) phenotype is produced by recessive MC1R extension (e) alleles.
In this context, we investigated the variation in coloration found among the subspecies of the Fuscous Flycatcher, Cnemotriccus fuscatus, a monotypic genus widely distributed in South America (Fig. 1). Th ere are seven C. fuscatus subspecies, which are diff erentiated not only on the basis of their morphological characters, but also their vocalizations and ecology (Fitzpatrick et al. 2004). Th ese subspecies can be divided into two groups, based primarily on the coloration of the belly, which is either white or yellow. Th ese fl ycatchers can be found in a variety of habitats, including fl uvial islands, rainforest, dry forests, riparian habitats, and lowland and secondary forests (Rasmussen & Collar 2002). It is thus important to understand which factors may infl uence the variation in the coloration of plumage found among the diff erent subspecies of the Fuscous Flycatcher (Farnsworth & Lebbin 2017). In particular, if a relationship can be found between genotype and phenotype, it might represent evidence of the role of natural selection in the fi xation of subspecifi c coloration patterns (Hewitt 1988, Chunco et al. 2007).
Here we investigated the possible mechanisms that determine diff erentiation in plumage amongst the subspecies of C. fuscatus. Specifi cally, we tested whether non-synonymous mutations in the sequence of the melanocortin-1 receptor (MC1R) gene were associated systematically with variation in plumage coloration amongst the six subspecies, and whether these mutations are suff ering selection pressures.
Total DNA was isolated from the muscle tissue using the Wizard © Genomic DNA purifi cation kit (Promega), following the manufacturer's instructions. To obtain a partial sequence of the MC1R gene, we amplifi ed the samples by PCR using the primers described by Cheviron et al. (2006): lcorMSHR9 (5' -CTG GCT CCG GAA GGC RTA GAT -3') and lcorMSHR72 (5' -AYG CCA GYG AGG GCA ACC A -3'). Th e PCR conditions were the same as those used by Cheviron et al. (2006), and the PCR products were sequenced by Sanger's didesoxiterminal method (Sanger et al. 1977), using an ABI 3500 automatic sequencer.
Th e DNA sequences were aligned and their nucleotides were compared to those from the bananaquit, Coereba fl aveola (GenBank access numbers AF362598 and AF362601) and Gallus gallus (AB201631) using Bioedit   samples (Table 2), including all six subspecies. Th ese variable sites of the MC1R locus determined six nonsynonymous mutations for the codifi cation of amino acids, A8G, S9R, S10N, S89N, V226I, and L240I (Table 3). None of these sites were associated with the coloration patterns of either the two groups or any of the subspecies. Tajima's D was not signifi cant (-1.603, P > 0.05), indicating that the variation found in the study locus in C. fuscatus is neutral, with a signal of recent demographic expansion, against the constant demographic model. All the sequences generated in the present study were deposited in GenBank (www. ncbi.nlm.nih.gov) under access numbers MK102986 through MK103006 (Table 3).

DISCUSSION
Our study of Cnemotriccus fuscatus indicates that there is no clear association between the plumage polymorphism found in this species and mutations of the MC1R gene.
As in previous studies of bird species such as Phylloscopus toutinegras (MacDougall-Shackleton et al. 2003), Lepidothrix coronata (Cheviron et al. 2006), Dendrocolaptes platyrostris (Corso et al. 2013), Philomachus pugnax v. 7.2.5, to confi rm the position of amplifi ed fragment. We then assessed if aminoacid sequence presented stop codons and indels, which could indicate pseudogenes. Th e potential association of variable sites with the plumage morphotype of each species was confi rmed by visual inspection. We calculated Tajima's D (Tajima 1989) in DnaSP (version 3.51, Rozas et al. 2003) to verify whether the MC1R gene was under selection pressure in the two groups.

Cfu008 -C. f. fumosus G C A G A C C C G C G C C T C C C/G T C G C C/A C Yellow
In addition to genetics, the variation found in the coloration of C. fuscatus may be related to environmental factors, given the diversity of habitats occupied by the species (Fig. 1). Uy et al. (2009), for example, found that natural selection may favor distinct coloration in diff erent habitats based on the existence of several population patterns, with habitats dominated by short-wavelength light (e.g., shaded woodland) favoring darker birds, and habitats rich in long-wavelength light (e.g., forest clearings with direct sunlight) favor lighter-colored species.
Furthermore, the studied part of the gene MC1R includes all the main sites that were showed in previous research with plumage polymorphism of birds (Mundy 2005, Cheviron et al. 2006). Overall, our results reinforce the conclusion that understanding the evolution of plumage coloration in C. fuscatus with varying patterns of eumelanin/pheomelanin pigmentation requires a more profound investigation of the genes in the melanocortin pathway and their potential variation, as well as other loci and environmental factors. Unlike many other bird species (see e.g., Cheviron et al. 2006, Corso et al. 2013, Farrell et al. 2014, Luna et al. 2018, the variation in the plumage coloration of C. fuscatus does not appear to be related to mutations of the MC1R gene.