The Planalto Woodcreeper (Dendrocolaptes platyrostris) presents “pale” and “dark” plumage variants, which are distributed throughout the Cerrado and Caatinga, and throughout the Atlantic Forest, respectively. To understand the genetic nature of the plumage variation in the species, we partially sequenced the melanocortin-1 receptor (MC1R) gene, which is associated with melanic phenotypes in vertebrates. We found no correlation between variation at MC1R sequences and plumage color in D. platyrostris. Aminoacid sites that were correlated with variation in melanic plumage in other bird species were monomorphic in D. platyrostris. Our results suggested that MC1R seems not to be involved in controlling plumage variation in D. platyrostris.Fil: Corso, Josmael. Universidade Federal do Rio Grande do Sul. Departamento de Genética; BrasilFil: Cabanne, Gustavo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales; ArgentinaFil: Mendonça d'Horta, Fernando. Universidade de Sao Paulo. Instituto de Biociências; BrasilFil: da Silva Loreto, Elgion Lucio. Universidade Federal de Santa Maria. Departamento de Biologia; BrasilFil: Miyaki, Cristina Yumi. Universidade de Sao Paulo. Instituto de Biocièncias; Brasi

Cabanne, Gustavo Sebastián

Corso, Josmael

da Silva Loreto, Elgion Lucio

Mendonça d'Horta, Fernando

Miyaki, Cristina Yumi

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articleRevista Brasileira de Ornitologia, 21(1), 1-4March 2013iNtrODUctiONThe genetic basis of phenotypic diversity within species is of great interest to evolutionary biologists because adaptive evolution depends on selection of genetic variants (Theron et al. 2001). Genetic changes resulting in color and pigmentation variation among closely related taxa might represent important evolutionary events. However, the molecular basis and developmental pathways responsible for phenotypic difference are unknown in most cases (Cheviron et al. 2006). Recently, it has been suggested that the locus encoding the melanocortin-1 receptor (MC1R) may cause color polymorphisms in wild populations (Mundy 2005). This gene encodes the MC1R protein, which is expressed in the melanocytes of developing feathers and hair follicles and plays a critical role in the control of melanin synthesis (Theron et al. 2001, Baião et al. 2007). Point mutations in this locus were associated with color polymorphisms based on melanin and can cause changes from light to dark color all over the body in a variety of taxa (revision in Corso et al. 2012).The planalto woodcreeper (Dendrocolaptes platyrostris) is an endemic bird of the Atlantic forest that inhabits forest enclaves and gallery forests within the Cerrado, Chaco, and Caatinga (Figure 1). As many other woodcreepers, D. platyrostris does not have plumage sexual dimorphism. It has two parapatrically distributed subspecies: D. p. platyrostris – dark plumage morph - individuals are predominantly streaked buff, have blackish crown, dull chestnut wings and tail, and brown underparts, and inhabit the Atlantic Forest domain in southeastern Brazil, eastern Paraguay and extreme northeastern Argentina; D. p. intermedius – pale plumage morph - individuals have browner crown, almost no streaking on mantle, paler and brighter rufous wings and tail, and slightly paler underparts, and inhabit gallery forests of Cerrado / Caatinga domains (Ridgely & Tudor 1994, Willis & Oniki 2001, Cabanne et al. 2011; Figure 1). The study of Cabanne et al. (2011) indicated that plumage type in D. platyrostris (i.e., dark and pale morphs) was not correlated with neutral genetic divergence (mitochondrial DNA) but confirmed that it Plumage variation in the Planalto Woodcreeper (Dendrocolaptes platyrostris) and the                  melanocortin-1 receptor gene (MC1R) Josmael corso1,6, Gustavo Sebastián cabanne2, Fernando Mendonça d’Horta3, Élgion lucio da Silva loreto4       and  cristina Yumi Miyaki51 Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil - josmaelcorso@gmail.com 2 Museo Argentino de Ciencias Naturales, CONICET, Buenos Aires, Argentina - gscabanne@yahoo.com  3 Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil - fmhorta@ig.com.br 4 Departamento de Biologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil - elgion.loreto@pq.cnpq.br5 Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brazil - cymiyaki@ib.usp.br6 Correspondence: Josmael Corso – Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil – CEP:91501-970 - Tel.: 51 3308.6726. E-mail: josmaelcorso@gmail.comReceived on 7 August 2011. Accepted on 6 January 2013.aBStract: The Planalto Woodcreeper (Dendrocolaptes platyrostris) presents “pale” and “dark” plumage variants, which are distributed throughout the Cerrado and Caatinga, and throughout the Atlantic Forest, respectively. To understand the genetic nature of the plumage variation in the species, we partially sequenced the melanocortin-1 receptor (MC1R) gene, which is associated with melanic phenotypes in vertebrates. We found no correlation between variation at MC1R sequences and plumage color in D. platyrostris. Aminoacid sites that were correlated with variation in melanic plumage in other bird species were monomorphic in D. platyrostris. Our results suggested that MC1R seems not to be involved in controlling plumage variation in D. platyrostris.KeY-WOrDS: plumage color polymorphism; melanocortin-1 receptor, Dendrocolaptes 2                                                                                                               Revista Brasileira de Ornitologia, 21(1), 2013                                                                                                                       Revista Brasileira de Ornitologia, 21(1), 2013Plumage variation in the Planalto Woodcreeper (Dendrocolaptes platyrostris) and the melanocortin-1 receptor gene (MC1R) Josmael Corso, Gustavo Sebastián Cabanne, Fernando Mendonça d’Horta, Élgion Lucio da Silva Loreto  and Cristina Yumi Miyakiwas correlated to different types of habitats, with dark and pale morphs occurring respectively in more humid (Atlantic Forest) and dry (Cerrado / Caatinga) habitats. In this study we examined the MC1R sequence variation of D. p platyrostris and D. p. intermedius. Specifically, we looked for fixed non-synonymous differences in MC1R between plumage morphs. Our main question was whether the light and dark phenotypes of this species could be related to changes in the MC1R coding region. Material aND MetHODSWe sampled five adult individuals of D. platyrostris intermedius and five D. p. platyrostris, from localities that are 1600 km apart (Table 1). Total genomic DNA was isolated from blood or muscle samples by a standard phenol/chloroform extraction protocol (Sambrook et al. 2001). A fragment of approximately 500 bp of the avian MC1R gene was amplified by PCR using the following primers: lcorMSHR72 – 5’ AYGCCAGYGAGGGCAACCA 3’ (Cheviron et al. 2006) and MC1RIntRev – 5’ AACATGTGRATGTAGAGCACC 3’. PCR FiGUre 1. Distribution of biomes and of the two subspecies of Dendrocolaptes platyrostris. Modified from Cabanne et al. (2011).                                                                                                               Revista Brasileira de Ornitologia, 21(1), 20133                                                                                                                      Revista Brasileira de Ornitologia, 21(1), 2013Plumage variation in the Planalto Woodcreeper (Dendrocolaptes platyrostris) and the melanocortin-1 receptor gene (MC1R) Josmael Corso, Gustavo Sebastián Cabanne, Fernando Mendonça d’Horta, Élgion Lucio da Silva Loreto  and Cristina Yumi Miyakiconditions were: initial denaturation for 3 min at 94°C, followed by 45 cycles (denaturation at 94°C for 45 s, annealing at 50-60°C for 60 s and extension at 72°C for 90 s) and a final extension at 72°C for 5 min. This MC1R fragment includes main sites previously shown to be associated with melanic phenotypes in birds (Cheviron et al. 2006). Each specimen was submitted to at least two independent amplification and sequencing reactions (using the same primers used in the PCR) to confirm the sequences. Consensus sequences were obtained and deposited in GenBank under the accession numbers: FJ985683–FJ985688, JN224986-JN224989. DNA sequences were aligned and their nucleotides and deduced amino acids were compared to those from bananaquits (Coereba flaveola; GenBank number AF362605 and AF362598) using BIOEDIT v. 7 (Hall, 1999; Table 2). We tested for evidence of selection at MC1R by calculating Tajima’s D statistic using MEGA 5 (Tamura et al.  2011).  taBle 1. Specimens sequenced, sampling localities, sample identification, and voucher identification of tissue samples.taxona locality (habitat) tissue identificationb VoucherscD. p. platyrostrisPinhalão, Paraná (PR). 23°46’S; 50°3’W (Atlantic forest) LGEMA P885 MZUSP 75622Wenceslau Braz, PR. 22°5’S; 48°47’W (Atlantic forest) LGEMA P957 MZUSP 75690Morro Grande State Park, São Paulo (SP). 23°42’S; 46°59’W (Atlantic forest) LGEMA P2480, P2482 -Barreiro Rico, SP. 22°38’S; 48°13’W (Atlantic forest) LGEMA P1696 -D. p. intermedius National Park of Serra das Confusões, Piauí. 9°40’S; 44°8’W (Caatinga)LGEMA P2277, P2278, P2329, P2379, P2418MZUSP 77719, 77720, 77721, 77722 a D. p.= Dendrocolaptes platyrostris.b Samples are deposited at the Laboratório de Genética e Evolução Molecular de Aves (LGEMA), Universidade de São Paulo, São Paulo, Brazil.c Voucher specimens housed at MZUSP: Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil. A dash indicates that a voucher specimen does not exist.taBle 2. MC1R sequences of Dendrocolaptes platyrostris. Sites are numbered based on the alignment with bananaquit (Coereba flaveola) MC1R sequences (see methods). Sites in bold are associated with color polymorphism in other avian taxa (amino acid replacement), all D. platyrostris individuals are monomorphic at these sites. Variable sites in D. platyrostris that resulted in putative aminoacid substitution are underlined. Asterisks show sites that are identical to those of Coereba flaveola. taxa Nucleotide site aminoacid position Plumage 1 1 2 2 5 1 3 6 8 9 1 1 1 1 21 2 0 7 1 6 8 8 5 2 1 5 6 7 03 0 3 4 4         1 7 6 2 7C. flaveola M5 T C A A G A I H V K V Q V V H melanicC. flaveola Y24 * * * G * * * * * E * * * * * yellowD. p. platyrostris P885; P2480; P2482; P1696 * T * G * . * * * E * * * * * darkD. p. platyrostris P957 * T/C * G * . * * * E * * * * * darkD. p. intermedius P2278; P2379; P2418 * T * G A . * * * E * * * I * paleD. p. intermedius P2329 T/A T * G A . * * * E * * * I * paleD. p. intermedius P2277 * T G G * . * R * E * * * * * pale4                                                                                                               Revista Brasileira de Ornitologia, 21(1), 2013Plumage variation in the Planalto Woodcreeper (Dendrocolaptes platyrostris) and the melanocortin-1 receptor gene (MC1R) Josmael Corso, Gustavo Sebastián Cabanne, Fernando Mendonça d’Horta, Élgion Lucio da Silva Loreto  and Cristina Yumi MiyakireSUltSThe alignment matrix presented 525 characters encompassing sites 100 to 624 of the MC1R gene. It had 96–97% nucleotide identity with sequences from other passerine birds such as Coereba flaveola (Theron et al. 2001), Phylloscopus warblers (MacDougall-Shackleton et al. 2003), and Lepidothrix coronata (Cheviron et al. 2006). Two D. platyrostris individuals presented heterozygous sites at positions 113 and 120 (Table 2). There were three variable sites that resulted in non-synonymous substitutions (amino acid positions 38, 68, and 172), which allowed us to identify three polymorphic DNA sites in pale morph birds: c.113 T > A , c.203 A > G, and c.514 G > A (Table 2). These changes could not be correlated with phenotype differences, once those changes maintain the same polarities of original aminoacids. As the number of variable sites observed was very low, our statistical power to detect selection was also low (Tajima’s D: -0.78, not significant at P > 0.10).DiScUSSiONThe results obtained are in contrast with some studies of vertebrate taxa, including birds (reviewed in Mundy 2005), which observed a linkage between MC1R mutations and the appearance of melanic phenotype. In Coereba flaveola, a non-synonymous substitution (E92K) was associated with melanic plumage (Mundy et al. 2004). In the red-footed booby (Sula sula), the white/melanic polymorphism observed was associated to two point substitutions, V85M and H207R (Baião et al. 2007). These three sites were monomorphic in D. platyrostris (Table 2). However, these results should be interpreted with caution, as our sample size is small and it may not have allowed the identification of association between phenotypic and genotypic variations. However, even though we did not completely sequence the MC1R, the segment studied contained the majority of sites previously shown to be correlated with plumage differences in birds (Mundy et al. 2004, Mundy 2005, Cheviron et al. 2006). Therefore, D. platyrostris seems to be another instance of a bird species that does not present any evidence that MC1R is correlated with pigmentation differences as documented previously in Phylloscopus warblers  (MacDougall-Shackleton et al. 2003) and Lepidothrix coronata (Cheviron et al. 2006), the latter belonging to the same Passeriformes suborder (Tyranni) as D. platyrostris. acKNOWleDGMeNtSThis work was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Brazil. We are also grateful to Fabio Raposo and Luis Fabio Silveira (MZUSP), and the members of Laboratório de Biologia Molecular (LabDros) for their help.reFereNceSBaião, P. c.; Schreiber, e. a. & Parker, P. G. 2007. The genetic basis of the plumage polymorphism in red-footed booby (Sula sula): a melanocortin-1 receptor (MC1R) analysis. Journal of Heredity, 98: 287-292.cabanne, G. S.; d’Horta, F. M.; Meyer, D.; Silva, J. M. c. & Miyaki, c. Y. 2011.  Evolution of Dendrocolaptes platyrostris (Aves: Furnariidae) between the South American open vegetation corridor and the Atlantic forest. Biological Journal of the Linnean Society,  103: 801-820. http://www.mendeley.com/research/evolution-dendrocolaptes-platyrostris-aves-furnariidae-between-south-american-open-vegetation-corrid/cheviron, Z. a.; Hackett, S. J. & Brumfield, r. t. 2006. Sequence variation in the coding region of the melanocortin-1 receptor gene (MC1R) is not associated with plumage variation in the blue-crowned manakin (Lepidothrix coronata). Proceedings of the  Royal Society  of  London B, 273: 1613-1618.corso, J.; Gonçalves G. l. &  Freitas t. r. O. 2012. Sequence variation in the melanocortin-1 receptor  (MC1R) pigmentation gene and its role in the cryptic coloration of two South American sand lizards. Genetics and Molecular Biology, 35:  81–87.Hall, t. a. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium  Series, 41:95-98. http://nass.oxfordjournals.org/content/currentMacDougall-Shackleton, e. a.; Blanchard, l. & Gibbs, H. l. 2003. Unmelanized plumage patterns in old world leaf warblers do not correspond to sequence variation at the melanocortin-1 receptor locus (MC1R). Molecular Biology and Evolution, 20: 1675-1681.  Mundy, N. i. 2005. A window on the genetics of evolution: MC1R and plumage coloration in birds. Proceedings of the Royal Society of London B, 272: 1633-1640.Mundy, N. i.; Badcock, N. S.; Hart, t.; Scribner, K.; Janssen, K. & Nadeau, N. J. 2004. Conserved genetic basis of a quantitative plumage trait involved in mate choice. Science, 303: 1870-1873.ridgely, r. S. & tudor, G. 1994. The Birds of South America: The Suboscine Passerines, v. 2. Austin: University of Texas Press.Sambrook, K. J.; russell, D. W. & Sambrook, J. 2001. Molecular Cloning: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M. & Kumar, S. 2011. MEGA 5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28: 2731-2739. http://mbe.oxfordjournals.org/content/28/10/2731.abstractTheron, e.; Hawkins, K.; Bermingham, e.; ricklefs, r. e. & Mundy, N. i. 2001. The molecular basis of avian plumage polymorphism in the wild: a melanocortin-1-receptor point mutation is perfectly associated with the melanic plumage morph of the bananaquit, Coereba flaveola. Current Biology, 11: 550-557.Willis, e. O. & Oniki, Y. 2001. On a nest of the planalto woodcreeper, Dendrocolaptes platyrostris, with taxonomic and conservation notes. Wilson Bulletin, 113: 231-233.Associate Editor: Camila C. Ribas

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Plumage variation in the Planalto Woodcreeper (Dendrocolaptes platyrostris) and the melanocortin-1 receptor gene (MC1R)

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Plumage variation in the Planalto Woodcreeper (Dendrocolaptes platyrostris) and the melanocortin-1 receptor gene (MC1R)

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