Downloaded from rspb.royalsocietypublishing.org on July 16, 2014 The evolution of peafowl and other taxa with ocelli (eyespots): a phylogenomic approach Keping Sun, Kelly A. Meiklejohn, Brant C. Faircloth, Travis C. Glenn, Edward L. Braun and Rebecca T. Kimball Proc. R. Soc. B 2014 281, 20140823, published 16 July 2014 Supplementary data "Data Supplement" http://rspb.royalsocietypublishing.org/content/suppl/2014/07/15/rspb.2014.0823.DC1.h tml References This article cites 34 articles, 10 of which can be accessed free http://rspb.royalsocietypublishing.org/content/281/1790/20140823.full.html#ref-list-1 Subject collections Articles on similar topics can be found in the following collections evolution (1822 articles) taxonomy and systematics (194 articles) Receive free email alerts when new articles cite this article - sign up in the box at the top Email alerting service right-hand corner of the article or click here To subscribe to Proc. R. Soc. B go to: http://rspb.royalsocietypublishing.org/subscriptions Downloaded from rspb.royalsocietypublishing.org on July 16, 2014 The evolution of peafowl and other taxa with ocelli (eyespots): a phylogenomic approach rspb.royalsocietypublishing.org Keping Sun1,2, Kelly A. Meiklejohn1, Brant C. Faircloth3, Travis C. Glenn4, Edward L. Braun1 and Rebecca T. Kimball1 1Department of Biology, University of Florida, Gainesville, FL, USA 2Jilin Key Laboratory of Animal Resource Conservation and Utilization, Northeast Normal University, Changchun, China Research 3Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA, USA 4Department of Environmental Health Science, University of Georgia, Athens, GA, USA Cite this article: Sun K, Meiklejohn KA, BCF, 0000-0002-1943-0217; ELB, 0000-0003-1643-5212; RTK, 0000-0001-5449-5481 Faircloth BC, Glenn TC, Braun EL, Kimball RT. 2014 The evolution of peafowl and other taxa The most striking feature of peafowl (Pavo) is the males’ elaborate train, which with ocelli (eyespots): a phylogenomic exhibits ocelli (ornamental eyespots) that are under sexual selection. Two approach. Proc. R. Soc. B 281: 20140823. additional genera within the Phasianidae (Polyplectron and Argusianus) exhibit http://dx.doi.org/10.1098/rspb.2014.0823 ocelli, but the appearance and location of these ornamental eyespots exhibit substantial variation among these genera, raising the question of whether ocelli are homologous. Within Polyplectron, ocelli are ancestral, suggesting ocelli may have evolved even earlier, prior to the divergence among genera. Received: 4 April 2014 However, it remains unclear whether Pavo, Polyplectron and Argusianus form a monophyletic clade in which ocelli evolved once. We estimated the phylogeny Accepted: 24 June 2014 of the ocellated species using sequences from 1966 ultraconserved elements (UCEs) and three mitochondrial regions. The three ocellated genera did form a strongly supported clade, but each ocellated genus was sister to at least one genus without ocelli. Indeed, Polyplectron and Galloperdix,agenusnotpre- Subject Areas: viously suggested to be related to any ocellated taxon, were sister genera. The evolution, taxonomy and systematics close relationship between taxa with and without ocelli suggests multiple gains or losses. Independent gains, possibly reflecting a pre-existing bias for eye-like structures among females and/or the existence of a simple mutational Keywords: pathway for the origin of ocelli, appears to be the most likely explanation. eyespots, homology, ocelli, sexual selection, ultraconserved elements 1. Introduction Author for correspondence: As no ornaments are more beautiful than the ocelli on the feather of various birds ... Rebecca T. Kimball they deserve to be especially noticed. [1, p. 132] e-mail: [email protected] The wide diversity of colour and ornamental traits in birds has long been of interest to both scientists and non-scientists. Indeed, the ornamental traits of birds were instrumental to Darwin’s development of the theory of sexual selection [1], where he devoted four chapters to the subject. The striking train of male peafowl, comprising elongated tail coverts decorated with ocelli (ornamental eyespots), had a special place within this discussion of avian sexual selection. Assuming gradual evolutionary change, Darwin recognized that there must have been species of birds with successive steps between ‘the magnificent ocelli of [peacocks] and the simpler ocelli or mere coloured spots of other birds’ ([1], p. 137). To gather evidence of their gradual evolution, Darwin studied the striking ocelli present on male peafowls (Pavo sp.) in addition to similar structures found among species of two other genera in the same order (Galliformes) and family (Phasianidae): Polyplectron (peacock-pheasants) and Argusianus Electronic supplementary material is available (argus pheasants). He immediately noted significant variation in the size, at http://dx.doi.org/10.1098/rspb.2014.0823 or number and coloration of ocelli among these genera in addition to observing differences in the distribution of ocelli among taxa—Argusianus have ocelli via http://rspb.royalsocietypublishing.org. located on the wing feathers and Polyplectron have ocelli distributed among different feather types (including tail, tail coverts, flight feathers, wing coverts & 2014 The Author(s) Published by the Royal Society. All rights reserved. Downloaded from rspb.royalsocietypublishing.org on July 16, 2014 and the mantle). Darwin also noted that females either lack 2. Material and methods 2 ocelli (Pavo and Argusianus) or have smaller, less complex rspb.royalsocietypublishing.org and/or less iridescent ocelli (Polyplectron)thanmales.Ithas (a) Supermatrix analyses to identify target taxa since become clear that the ocelli of Pavo species are involved To identify all possible sister taxa of the ocellated taxa, we in sexual selection [2–4] and that male display of ocelli towards consulted Wang et al. [9], who reviewed previous galliform phylo- females during mating [5] also suggests a role for ocelli in sexual genies, and we used available data to construct a mitochondrial selection among Argusianus and Polyplectron. Indeed, males in supermatrix of CYB, ND2 and 12S rRNA sequences from GenBank all of these taxa prominently display ocelli towards females for Galliformes (electronic supplementary material, table S2). We during mating displays [5], often with careful orientation to supplemented those data with mitochondrial sequences collected by different methods, including high-throughput sequencing maximize impact of the ocelli [6]. Darwin postulated that the [17] and PCR amplification from toe pads (see [18] for methods). ocelli of these taxa were homologous (using modern termi- The final matrix included 233 species, corresponding to 83% of nology), suggesting a single origin and then subsequent Galliformes and 91% of Phasianidae (the family that includes the Proc. R. Soc. B modification and elaboration via sexual selection. ocellated taxa). Darwin’s hypothesis predicts that modern, phylogenetic Using this mitochondrial supermatrix, we performed unparti- analyses should strongly support a close relationship among tioned and partitioned (by gene and codon position) maximum- galliforms having ocelli. However, the inferred relationships likelihood (ML) analyses using RAXML v. 7.3.0 [19]. We analysed 281 among these taxa remain controversial. Although there is the mitochondrial DNA using the best model (GTR þ I þ G), strong support for a peafowl clade (Pavo and Afropavo, the identified using the Akaike information criterion (AIC) in MODEL- : 20140823 Congo peafowl) [7–10] and an argus clade (Argusianus and TEST v. 3.06 [20] and assessed support using 1000 bootstrap Rheinardia, the crested argus) [5], the relationships between replicates. We also used Bayesian inference (BI) to assess support, conducting analyses in MRBAYES v. 3.1.2 [21] on the CIPRES Science these two clades and Polyplectron have varied (reviewed in Gateway [22] using the GTR þ I þ G model. We ran the analyses [9]). A few studies unite the peafowl, argus and peacock- for 107 generations using the standard settings (two runs, each pheasants into an ‘ocellated clade’ [5,10], though with little with four heated chains) and we sampled every 1000 generations. support; a larger number of studies unite the peafowl The first 25% of the sampled trees were discarded as burn-in. and argus clades to the exclusion of the peacock-pheasants [9,11–13], whereas others do not unite any of these three clades [14]. The position of Polyplectron is especially variable (b) Ultraconserved element data collection among studies [9], suggesting that large amounts of data We selected 15 species for UCE enrichment using a combination may be necessary to resolve this question. of literature searches and the trees inferred above. These taxa included representatives of the three ocellated genera, putative To further complicate matters, both the peafowl and sister taxa and multiple outgroups. We collected UCE data argus clades contain one genus with ocelli (Pavo and Argusia- using a modification of the Faircloth et al. [23] approach. Briefly, nus) and one genus lacking ocelli (Afropavo and Rheinardia), we prepared Nextera sequencing libraries using the manu- suggesting either a loss in Afropavo and Rheinardia or inde- facturer’s protocol (Illumina Inc., San Diego, CA, USA). We pendent gains of ocelli in Pavo and Argusianus [7]. Ocelli pooled libraries into groups of eight taxa, enriched each library