Skua individual collecting sites and genotypes

Collecting sites and genotypes for each individual arctic skua and long-tailed skua. Genotypes are given in terms of haplotype numbers which correspond to numbers given in the haplotype alignment file and Genbank Accession

Skua Haplotype alignments

Alignments of arctic skua and long-tailed skua haplotypes

Museum sample and materials and methods from The genetic basis and enigmatic origin of melanic polymorphism in pomarine skuas (<i>Stercorarius pomarinus</i>)

A key outstanding issue in adaptive evolution is the relationship between the genetics of intraspecific polymorphism and interspecific evolution. Here we show that the pale/dark ventral plumage polymorphism that occurs in both the pomarine skua (<i>Stercorarius pomarinus</i>) and Arctic skua (<i>S. parasiticus</i>) is the result of convergent evolution at the same locus (<i>MC1R</i>), involving some of the same amino acid sites. The dark melanic <i>MC1R</i> allele in the pomarine skua is strongly divergent from the pale <i>MC1R</i> alleles. Whereas the dark allele is closely related to <i>MC1R</i> alleles in three species of great skua (<i>S. skua</i>, <i>S. maccormicki</i>, <i>S. lonnbergi</i>), the pale pomarine skua <i>MC1R</i> alleles present a star-like pattern in an intermediate position on the haplotype network, closer to alleles of the long-tailed skua (<i>S. longicaudus</i>). Variation at other nuclear loci confirms a close relationship between the pomarine skua and the great skuas. The plumage polymorphism in pomarine skuas might have arisen in the common ancestor of pomarine and great skuas, only being retained in pomarine skuas. Alternatively, the pale and melanic <i>MC1R</i> alleles may have evolved independently in different lineages and been brought together in pomarine skuas by hybridization. In this case introgression of a pale <i>MC1R</i> allele into the pomarine skua from another skua lineage is most likely. Our current data do not permit us to distinguish between these hypotheses, and assaying genome-wide variation holds much promise in this regard. Nevertheless, we have uncovered an intriguing example of a functionally important allele within one species that is shared across species

Correspondence between alternatively spliced transcripts of <i>POMC</i> in embryonic and adult Japanese quail, and chicken.

<p>Chicken promoter sites and alternatively spliced transcripts are listed on the left, and the corresponding quail transcript is listed to the right of the transcript. Quail <i>POMC</i> transcripts that have no correspondence with chicken transcripts are listed with the novel promoter site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174714#pone.0174714.ref005" target="_blank">5</a>]. Empty boxes represent non-coding exons and solid boxes denote coding exons. The previously described non-coding exons that were amplified in quail embryonic development are represented with green, whereas red denotes the combination of promoter site and non-coding exons that we were unable to amplify. The promoter sites for T2, T3 and the proximal quail exon are unknown.</p

<i>In situ</i> hybridization results for <i>ASIP</i> and <i>AGRP</i> in quail feather follicles at E8 and E12.

<p>The epidermis is labelled with a small black arrow and feather follicles with a large blue arrow. Ai) <i>ASIP</i> is expressed in the dermal pulp of ventral feather follicles in E8 and E12 embryos, but is not visible on the dorsal surface. Bi) <i>AGRP</i> was not expressed in the feather follicles in early or late development, either ventrally or dorsally. Aii) <i>ASIP</i> expression in a feather follicle with eumelanin on the flank of a Japanese quail embryo at E12. In some feather follicles in the flank or on the wing of E12 embryos <i>ASIP</i> expression was observed with eumelanin pigmentation. Bii). Representative transcription of <i>AGRP</i> in ventral developing feather follicles in E8 Japanese quail embryos. Similar staining was found in feather follicles on the dorsal surface of quail E8, as well as both the ventral and dorsal surface of quail E12, and the epidermis.</p

Patterns of expression of <i>POMC</i>, <i>PC1</i> and <i>PC2</i> in feather follicles at E8 and E12.

<p>The epidermis is labelled with a small black arrow and feather follicles with a large blue arrow. <i>POMC</i> is expressed in the dermal pulp of ventral and dorsal feather follicles in E8 and E12 embryos. Weak expression of <i>PC1</i> was observed in developing feather follicles but did not develop further at E12. There is weak transcription of <i>PC2</i> at E8 on both ventral and dorsal surfaces, but when feather development is more progressed (e.g. E12), <i>PC2</i> is strongly expressed in feather follicles on both the ventral and dorsal surface.</p

Patterns of exon expression for <i>ASIP</i> and <i>AGRP</i> in embryonic quail at developmental stage E8 and E12 determined by RT-PCR.

<p>Patterns of exon expression for <i>ASIP</i> and <i>AGRP</i> in embryonic quail at developmental stage E8 and E12 determined by RT-PCR.</p

RT-PCR of the previously undocumented <i>ASIP</i> alternatively spliced transcript containing novel exon 7 in Japanese quail in three replicates per developmental stage.

<p>The expected amplicon length is 738 base pairs (Table A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174714#pone.0174714.s001" target="_blank">S1 File</a>). The developmental stage is above the gel lanes as well as body surface: V = Ventral, D = Dorsal. N = Negative control.</p

Japanese quail (<i>Coturnix japonica</i>) chick and melanocyte distribution at embryonic stages E8 and E12.

<p>A) The dorsal pigmentation of <i>C</i>. <i>japonica</i> comprises alternating stripes of dark and light feathers that are uniformly pigmented with one type of melanin, as well as feathers that are pigmented with both eumelanin and pheomelanin. Photo by Cowgirl Jules / CC BY 2.0. B) <i>In situ</i> hybridization for <i>SOX10</i> to determine melanocyte distribution in Japanese quail feather follicles during embryogenesis at E8 and E12. Feather follicles are labelled with a large blue arrow and the epidermis with a small black arrow. At E8 and E12 eumelanin is rarely present in ventral feather follicles but is frequently observed in feather follicles on the dorsal surface. Melanocytes are present within the epidermis and feather follicles. Staining of <i>SOX10</i> occurs in areas where individual barbs are developing in feather follicles on both the ventral and the dorsal surface of E8 and E12 quail embryos. The distribution of melanocytes is not restricted to areas pigmented with melanin but also occurs where melanin is absent.</p

Correspondence of <i>ASIP</i> alternatively spliced transcripts between embryonic Japanese quail, adult quail and chicken.

<p>Empty boxes represent non-coding exons and solid boxes denote coding exons (Ce1-3). Green lines and green boxes represent promoter sites and transcripts, respectively, that were successfully amplified in quail embryonic development, whereas red denotes transcripts that were not amplified. Previously reported chicken transcript names begin with “Class” and the exons which they are comprised of begin with “e” and are listed in the middle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174714#pone.0174714.ref004" target="_blank">4</a>]. Previously reported adult quail transcripts begin with “1” and are denoted to the right of the corresponding chicken transcript after the coding exons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174714#pone.0174714.ref004" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174714#pone.0174714.ref021" target="_blank">21</a>]. The names of the novel <i>ASIP</i> transcripts uncovered in this study begin with “Novel” and the exons they are comprised of are represented with blue boxes. The promoter site of the novel transcripts is unknown.</p