microanatomy and evolution of the nanostructures responsible … · 2018. 2. 5. · trogons and...

ORIGINAL ARTICLE

Microanatomy and evolution of the nanostructuresresponsible for iridescent coloration in Trogoniformes (Aves)

Esther Quintero & Alejandro Espinosa de los Monteros

Received: 2 February 2011 /Accepted: 16 June 2011 /Published online: 24 June 2011# Gesellschaft für Biologische Systematik 2011

Abstract One of the most outstanding features of the orderTrogoniformes is the presence of iridescent plumage, whichis widely distributed throughout the group except in thespecies of the Asian genus Harpactes. Previous studiesindicated that the iridescence-producing nanostructuresvary in form and array throughout the order. Thus, thepresent study aimed at reconstructing the evolutionaryhistory of those nanostructures in a phylogenetic context.The results show some clear tendencies in the evolution ofiridescence-producing nanostructures throughout the order.

Keywords Trogoniformes . Coloration . Iridescence .

Iridescence production . Character evolution . Transmissionelectron microscopy

Introduction

Trogons and quetzals (Aves: Trogoniformes: Trogonidae)form part of a relatively small, pantropical order of 39recognized species of frugivorous birds (Sibley and Monroe1990). Almost all species within the order are cavity-nesting birds (Collar 2001). Some species use previouslyexisting cavities, whereas others excavate in a variety of

soft rotting trees, termite mounds, or in the nests of othersocial insects (Brightsmith 2005). Both sexes participate innest excavation, egg incubation, and parental care.

In both male and female trogons, coloration comes fromcarotenoid and melanin pigmentation, as well as frommodifications in feather microstructure (Johnsgard 2000). Itis this latter mechanism that gives trogons one of their mostoutstanding features, iridescence, which is widely distributedamong the order, with the exception of the species in theAsian genus Harpactes.

Iridescence is a form of structural coloration in which theperceived color changes according to the angle of observa-tion or illumination. This phenomenon is produced byinteraction of light with nanostructures that, as pointed outby Prum et al. (1999b), “are similar in size to thewavelengths of visible light.” Color-producing nanostruc-tures are widely distributed among birds, other animals, andeven plants (Lee et al. 2000), and they are ancient, as isevidenced by the many fossils of the Burgess Shale (MiddleCambrian, British Columbia, 515 Mya; Parker 2000) thatexhibit this kind of coloration. Moreover, color-producingnanostructures have been found in fossil feathers of theMessel Oil Shale (Middle Eocene, Germany; Vinther et al.2010), which suggests that structural coloration has been afeature present among birds for a long time. Within Aves,there is a great diversity of these nanostructures (see Durrer1977, 1986), but most share a similar structural organiza-tion and thus a similar physical mechanism to producecolor (for a detailed explanation, see Prum 2006; Prum etal. 1999b).

Durrer and Villiger (1966) conducted an electronmicroscopy study of the iridescence-producing nanostruc-tures in seven species belonging to five genera ofTrogoniformes (Pharomachrus, Trogon, Priotelus, Apalo-derma, and Harpactes). Their results showed that these

E. Quintero :A. Espinosa de los MonterosDepartamento de Biología Evolutiva, Instituto de Ecología A.C.,Carretera antigua a Coatepec 351,Xalapa, Veracruz 91070, Mexico

Present Address:E. Quintero (*)Department of Ornithology,American Museum of Natural History,Central Park West at 79th Street,New York, NY 10024, USAe-mail: [email protected]

Org Divers Evol (2011) 11:237–248DOI 10.1007/s13127-011-0049-z

nanostructures are arrays of melanin granules and airvacuoles suspended in the barbule keratin (a furtherdetailed description of the variation in these structures canbe found in Durrer 1977). Similar iridescence-producingstructures have been reported from several orders andfamilies of birds, including ducks, swifts, hummingbirds,pigeons, turacos, kingfishers, and several Galliformes andPasseriformes (Durrer 1977, 1986; Dyck 1971, 1976, 1978,1987; Fox 1976; Lucas and Stettenheim 1972). In trogons,however, these structures vary in form and array within theorder. These results suggest the Trogoniformes are a goodmodel for studying the evolution of these nanostructures.However, as no phylogenetic hypothesis was available atthe time of Durrer’s and Villiger’s (1966) study, it was notpossible to make any further inference about the way thesenanostructures might have evolved within the group, as theuse of phylogenies is the only way to understand characterevolution in an historical context (Losos 1996).

In the study presented here, our objective was toreconstruct the evolutionary history of the iridescence-producing nanostructures within trogons. In order to dothis, we (1) conducted a transmission electron microscopystudy of the iridescent feathers of the trogons, (2) inferredthe phylogeny of the group using mitochondrial and nuclearsequence data, and (3) mapped the morphology and arrayof the iridescence-producing nanostructures onto the mo-lecular phylogeny.

Material and methods

Transmission electron microscopy study of the feathers

A transmission electron microscope (TEM; JEOL JEM 100CXII) was used to obtain images of the iridescence-producing nanostructures, using one feather each from themantle region in 24 species of Trogoniformes, and in twospecies of Coliiformes. Feathers were obtained from theOrnithology Collection of the American Museum ofNatural History (AMNH; Table 1).

To prepare the feathers for electron microscopy, amodified version of the standard protocol by Bozzola andRussell (1992) was used. First, fragments 1 mm in lengthfrom the barbules of each of the feathers were obtained andthoroughly cleaned with common soap and tap water toremove any remaining oils. Samples were then dehydratedusing a graded ethanol series (70%, 80%, 96%, 100%,100%, 100%; one hour per change), followed by twochanges of propylene oxide to complete the dehydrationprocess. Next, samples were infiltrated with resin (Araldite6005 Kit; Spi-Chem) and propylene oxide at the followingconcentrations: 1:2 for two hours at 60°C, 1:1 for 24 h atroom temperature, and 2:1 for 24 h at room temperature.

Finally, samples were embedded in fresh pure resin, usingflat embedding molds (Ted Pella), and were left topolymerize for 24 h at 60°C.

Resin blocks containing the feathers were cut into thinsections using a crystal knife and an ultramicrotome(Ultracut E, Reichert-Jung) to obtain sections of about600Å thickness. These sections were placed on 3-mm 300-mesh nickel grids (Electron Microscopy Sciences), andstained with a 5% uranyl acetate (Spi-Chem) alcoholicsolution, and a 0.4% lead citrate (Spi-Chem) aqueoussolution for ten and five minutes, respectively.

Photographs of the nanostructures were obtained foreach of the samples at 8000× magnification, and analyzedusing the software Image Pro Plus, version 3.0 (MediaCybernetics). For each sample, the following characterswere measured (see Fig. 1): melanin granule (referred to asmicrotube from here on) area (n=50 per feather); airchamber area (i.e. area within the melanin microtube) (n=50 per feather); and microtube diameter (n=50 per feather).Melanin area within each microtube was determined bysubtracting the air chamber area from the total microtubearea; from this result, the percentage of melanin for eachmicrotube was calculated. In order to compare the amountsof microtubes among the different species, the totalnumbers of microtubes within six 1.25 μ2 quadrants werecounted in the interior, exterior, and matrix (medulla) ofeach barbule.

DNA sequence data

Deducing the phylogenetic relationships of trogons andquetzals has been the focus of several molecular studies(DaCosta and Klicka 2008; Espinosa de los Monteros 1998,2000; Hosner et al. 2010; Johansson 1988; Johansson andEricson 2003, 2005; Moyle 2005; Sorenson et al. 2003).Therefore, DNA sequences from several genes are availablefrom GenBank (Table 2), from which sequences for 24species of trogons representing 62% of all recognizedspecies (Sibley and Monroe 1990) were obtained. Follow-ing the conclusions presented by Espinosa de los Monteros(2000), three species of mousebirds (Coliiformes: Coliuscolius Linné; C. striatus J. F. Gmelin; and C. leucocephalusReichenow), and three species of Cuculiiformes (Centropussinensis Stephens; Geococcyx velox Wagner; and Cuculusfugax Horsfield) were used as outgroups, the latter as amore distant outgroup lineage. [The original publicationsfor the species names involved here are: Gmelin 1789;Horsfield 1821; von Linné 1766; Reichenow 1879; Stephens1815; and Wagner 1836.]

Sequences from three mitochondrial and four nucleargenes were used to infer the molecular phylogeny. Mostcytochrome b (1143 bp) and 12S ribosomal RNA (1016 bp)sequences were obtained from Espinosa de los Monteros

238 E. Quintero, A. Espinosa de los Monteros

(1998). Sequences for the NADH dehydrogenase subunit 2(NADH 2; 1042 bp) and for the recombination activatingprotein 1 (RAG1; 2873 bp) were taken from Moyle (2005);those for the glyceraldehyde-3-phosphate dehydrogenaseintron 11 (GAPDH; 358 bp) and the myoglobin intron 2(734 bp) were taken from Johansson and Ericson (2003,2005), whereas sequences for the β-fibrinogen intron 7

(939 bp) were taken from Fain and Houde (2004). Finally,the mitochondrial genes cytochrome b and 12S weresequenced for this study for Pharomachrus mocinno,Trogon citreolus, T. melanocephalus, and T. massena. Themethods used for mtDNA extraction, amplification, andsequencing have been detailed in Espinosa de los Monteros(1998). Initial alignment of the sequences was performedusing the program Clustal X (Thompson et al. 1997),followed by manual depuration.

Phylogenetic analyses

Phylogenetic hypotheses concerning the concatenated dataset were derived via maximum parsimony (MP), maximumlikelihood (ML), and Bayesian Inference (BI). The MP andML analyses were performed using PAUP* (Swofford2002), the BI ones with MrBayes 3.0 (Huelsenbeck andRonquist 2001; Ronquist and Huelsenbeck 2003). MPsearches were aided by PAUPRat (Sikes and Lewis 2001),which implements the Parsimony Ratchet (Nixon 1999).Twenty repetitions of the Ratchet were performed with 10%

Keratinematrix

Barbule edge

Microtube

Melanin wall

Air chamber1µ

Fig. 1 General anatomy of feather barbules in Trogoniformes,showing the iridescence-producing nanostructures

Table 1 Data on voucher specimens from which feathers were obtained

Species Original publication AMNH Catalog # Collecting locality Collecting date

Harpactes oreskios Temminck and Laugier (1823) 648322 Malaysia, Sarawak, Kelabit plateau 26 December 1947

Harpactes ardens Temminck and Laugier (1826) 782353 Philippines, Polillo Island March 1959

Harpactes diardii Temminck and Laugier (1832) 633707 Malaysia, Malacca 1 February 1885

Priotelus temnurus Temminck and Laugier (1825) 96241 Cuba, Holguin, Santiago ?

Apaloderma narina Stephens (1815) 764203 DR Congo, falls of Lwiro River 28 January 1954

Apaloderma vittatum Shelley (1882) 799954 Kenya, Meru Forest 11 February 1944

Pharomachrus antisianus d’Orbigny and Lafresnaye (1837) 478108 Venezuela, Mérida ?

Pharomachrus auriceps Gould (1842) 167900 Colombia, Cauca 6 January 1911

Pharomachrus pavoninus von Spix (1824) 270845 Venezuela, Mt. Dursa 18 January 1929

Pharomachrus mocinno de La Llave (1832) 172647 Honduras, Mt. San Jacinto ?

Euptilotis neoxenus Gould (1835–1838) 99502 Mexico, Chihuahua, Pachaco 15 August 1905

Trogon citreolus Gould (1835–1838) 815339 Mexico, Oaxaca, Pto Escondido 16 May 1965

Trogon melanocephalus Gould (1835–1838) 813361 Guatemala, Sta Rosa, La Avellana 23 April 1975

Trogon massena Gould (1835–1838) 448275 Mexico, Oaxaca, Montebello 29 March 1962

Trogon curucui von Linné (1766) 58647 Brazil, Mato Grosso 10 June 1885

Trogon violaceus Gmelin (1789) 186715 Panama, Vranguas, Santiago 11 July 1924

Trogon viridis von Linné (1766) 231198 Peru, Amazonas, Pto Indiana 19 July 1926

Trogon comptus Zimmer (1948) 786910 Colombia, Chocó, Rio Bajaya 13 March 1965

Trogon melanurus Swainson (1838) 769867 Ecuador, Chone Morai 4 January 1913

Trogon elegans Gould (1834) 802790 Mexico, Sonora, Alamos 22 July 1971

Trogon rufus Gmelin (1789) 769867 Uruguay, Arroyo 9 May 1958

Trogon collaris Vieillot (1817) 820909 Peru, Rio Llullapichis 24 June 1964

Trogon mexicanus Swainson (1827) 799147 Mexico, Sinaloa, Palmito 22 March 1964

Trogon personatus Gould (1842) 180174 Ecuador, Raeza 30 September 1923

Colius castaneus Verreaux and Verreaux (1855) 633897 Angola, Benquella 5 July 1904

Colius macrurus von Linné (1766) 214577 Sudan, Zaidab Babor 15 August 1924

Evolution of the iridescent coloration in Trogoniformes 239

of the characters perturbed, and with 200 iterations, assuggested by Sikes and Lewis (2001). Character trans-formations were considered as unordered, the tree bisectionreconnection algorithm (TBR) was used to perform branchswapping, and outgroups were not constrained to bemonophyletic. Retention and consistency indexes werecomputed to evaluate the level of homoplasy in the mostparsimonious tree, and 500 full-heuristic bootstrap repli-cations were performed to evaluate relative strength for thenodes in each phylogenetic hypothesis (Felsenstein 1985).For these bootstrap replicates, input order bias wasminimized by random addition of taxa, nucleotide trans-formations were considered as unordered, and branchswapping was performed by the tree bisection reconnection(TBR) algorithm. For the ML search, the best-fit model forthe complete data matrix as determined using Modeltest3.04 (Posada and Crandall 1998) corresponded to the GTR+Γ+invariant sites model. The likelihood settings usedwere: gamma distribution shape=0.4492; proportion ofinvariable sites=0.4575; base frequencies (A=0.3188, G=0.2869, C=0.1816, T=0.2127); rate matrix (1.6295,8.0770, 1.7310, 0.5030, 20.6357, 1.0000); and molecularclock not enforced. The starting branch lengths were

obtained using the Rogers-Swofford approximation methodas implemented in PAUP* (Swofford 2002). Clade supportwas inferred by 200 full-heuristic bootstrap replications(Felsenstein 1985), using random addition of taxa, unor-dered nucleotide transformations, and TBR as the branch-swapping algorithm. Finally, the BI analysis was generatedusing MrBayes 3.0 (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003), using the Markov chainMonte Carlo with Metropolis-Hasting algorithm. The MPtree was used as a starting point for the searches. Each genefragment was declared as an individual partition, and therespective best-fit models for each set of sequences, asfound through Modeltest, were GTR+Γ+invariant sites forcytochrome b; HKY+Γ for the fibrinogen; K80+Γ formyoglobin; TVM+Γ+invariant sites for NADH2; HKY forGAPDH; and TrN+Γ+invariant sites for RAG1 and 12S.The search ran for five million generations, with four parallelchains, and sampling every 100th generation. Resulting –ln Lscores were plotted against generation time to assess when thevalues had reached an equilibrium. Sample points prior to theasymptote were discarded as ‘burn-in’. The majority-ruleconsensus tree after burn-in was used to calculate the posteriorprobabilities for each node.

Table 2 GenBank accession numbers for sequences from trogon species

Species Cytochrome b(1143 bp)

12S rRNA(1016 bp)

RAG1(2873 bp)

NADH2(1042 bp)

Myoglobin(734 bp)

NAPDH(358 bp)

β-fibrinogen(939 bp)

Total bp

Harpactes oreskios U89199 U89239 AY625238 AY625209 AY165827 AY600484 AY600469 8105

Harpactes ardens U94796 U94810 AY625239 AY625210 AY600499 AY600485 AY600470 8105

Harpactes diardii U94797 U94811 AY625243 AY625214 AY600500 AY600486 AY600471 8105

Priotelus temnurus U89202 U89237 AY625245 AY625216 AY600501 AY600487 AY600472 8105

Apaloderma narina U94798 U94812 – AY625223 AY600502 AY600488 AY600473 5232

Apaloderma vittatum U89200 U89234 AY625251 AY625222 AY600503 AY600489 AY600474 8105

Pharomachrus antisianus U89204 U89235 AY625247 AY625218 – – – 6074

Pharomachrus auriceps U94799 U94813 AY625248 AY625219 AY600504 AY600490 AY600475 8105

Pharomachrus pavoninus U94800 U94814 AY625249 AY625220 AY600505 AY600491 AY600476 8105

Pharomachrus mocinno EF622094 EF622093 – – – – – 2159

Euptilotis neoxenus U89203 U89236 AY625250 AY625221 – AY600496 – 6432

Trogon citreolus EF622090 EF622089 – – – – – 2159

Trogon melanocephalus EF622088 EF622087 AY625237 AY625208 – – – 6074

Trogon massena EF622092 EF622091 AY625228 AY625199 – – – 6074

Trogon curucui U94801 U94815 AY625233 AY625204 AY600506 AY600492 AY600477 8105

Trogon violaceus U94802 U94816 AY625234 AY625205 AY600507 AY600493 AY600478 8105

Trogon viridis U94803 U94817 AY625236 AY625207 – – AY695161 7013

Trogon comptus U94804 U94818 AY625230 AY625201 – – – 6074

Trogon melanurus U94805 U94819 AY625229 AY625200 AY165828 AY600494 AY600479 8105

Trogon elegans U94806 U94820 AY625232 AY625203 – – – 6074

Trogon rufus U94807 U94821 AY625231 AY625202 – – – 6074

Trogon collaris U94808 U94822 AY625225 AY625196 AY600508 AY600495 AY600480 8105

Trogon mexicanus U94809 U94823 AY625226 AY625197 AY600509 – AY600481 7747

Trogon personatus U89201 U89238 AY625227 AY625198 – – – 6074


Results

Molecular phylogeny

Taxon sampling differed among the studies from which DNAsequences were compiled; therefore the resulting data matrixis missing some data. The amount of sequence data presentper species ranged from 2159 to 8105 bp. GenBank accessionnumbers for all trogon specimens used in this study are listedin Table 2; the resultant trees can be accessed at TreeBASE(http://purl.org/phylo/treebase/phylows/study/TB2:S11397).

All three analyses recovered the same basic phylogeneticrelationships among species in the Trogoniformes (Fig. 2).MP recovered two equally parsimonious trees of 6773 steps(strict consensus presented in Fig. 2a), whereas the MLanalysis recovered a single tree with a maximum-likelihoodscore of –ln L 41975.1 (Fig. 2b), which is topologicallyidentical to the one recovered by BI. In general, theresulting relationships obtained for the Trogonidae con-curred with the previous hypotheses presented by Espinosade los Monteros (1998), Johansson and Ericson (2005), andOrnelas et al. (2009). Four main clades were recovered inthe analysis: African trogons, Asian trogons, Quetzals, andthe remaining New World trogons (Fig. 2). All generawithin the order were recovered as monophyletic groups.The Asiatic Harpactes lineage formed the sister clade to theNew World trogons, while the African Apaloderma was themost basal genus and sister to the former two genera

combined. Within the New World clade, quetzals (Phar-omachrus spp.) + Euptilotis neoxenus were located at thebase as sister to the other two genera (Priotelus andTrogon). The core of the phylogeny was formed by thegenus Trogon. This genus was divided in three subclades(Fig. 2): white-breasted trogons (T. rufus / T. collaris), red-breasted trogons (T. comptus / T. massena), and yellow-breasted trogons (T. violaceus / T. citreolus). Even thoughthere were several short internodes (Fig. 2b), the mono-phyly of the main lineages was well supported by both,bootstrap and Bayesian posterior probability values.

It is important to highlight that sequences for some taxacame from individuals of different populations. Thus, inorder to compile the matrix for the analyses, we assumedthat the recognized species of Trogoniformes are mono-phyletic, an assumption supported by the results ofEspinosa de los Monteros (1998). Although the dissimilarorigins of some sequences might be a potential source ofnoise for phylogenetic reconstruction (due to, e.g., mixtureof different phylogroups, fusion of non-sister lineages), wenonetheless consider that the cladograms presented here arerobust hypotheses supported by the current taxonomicknowledge and the available data.

Transmission electron microscopy study of the feathers

Results from the measurements can be found in Table 3.Harpactes oreskios, H. ardens, and H. diardii were

a) b)

100100

100

100

100

100

100

100

100100

100

100

10099

75

93

85

8470

89

77

100/100

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96/100

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89/94100/100

100/10098/100

66/10097/100

100/100100/100

95/100

83/100

100/10099/100

100/100

100/100

Outgroup Outgroup0.05

African

trogonsA

sian trogons

Quetzals

New

World trogons

Fig. 2 Phylogenetic hypotheses derived from combined analyses ofthe molecular data set. a Strict consensus tree from two equallyparsimonious trees (length 6773, consistency index 0.455, retention

index 0.544); numbers above branches are bootstrap support values. bMaximum likelihood phylogram (−ln L 41975.083); numbers abovebranches: bootstrap support / Bayesian posterior credibility values


http://purl.org/phylo/treebase/phylows/study/TB2:S11397

excluded from this table, as they did not show iridescence,hence lacked nanostructures comparable to those in theother genera.

Our microscopy results gave the following evidence. (1)Coliiformes, used as an outgroup in this part of the study,do not exhibit iridescence. Therefore, within the context ofthis taxon sample, iridescence-producing nanostructures area local evolutionary innovation for the Trogoniformes(Fig. 3). (2) Given the present data, the reconstruction ofthe evolutionary scenario for iridescence within Trogoni-formes is equivocal, as the included species of Harpactes(H. oreskios, H. ardens, H. diardii) do not present this typeof coloration. This means that iridescence has either beenlost at least once within the family, or that it has beengained twice independently. (3) There is a decrease, fromthe base to the tip of the phylogeny, in the followingmeasurements: microtube area (from 0.179 μ2 to 0.036 μ2),microtube diameter (from 0.610 μ to 0.253 μ), air chamberarea (from 0.032 μ2 to 0.009 μ2), area occupied by melanin(from 0.147 μ2 to 0.027 μ2), and percentage of melaninwithin each microtube (from 85% to 61%). (4) There is anincrease in the amount of microtubes from the base to thetip of the phylogeny, and a tendency to find them in a morecompact arrangement across the barbule (Fig. 3).

Within trogons, the basal Apaloderma has the biggestmicrotubes (Fig. 3). These are less dense, and dispersedthroughout the barbule (Fig. 4a, b). The three species of

Harpactes used in the present study show pigmentarycoloration exclusively. Feather coloration in these birds isproduced by melanin and carotenoid granules embeddedwithin the barbule’s medulla, and there is no evidence forthe existence of iridescent microtubes (Fig. 4c–e). In theNew World clade there are two main tendencies: oneexhibited by the Pharomachrus + Euptilotis clade, the otherby the Priotelus + Trogon clade. The eared quetzal (E.neoxenus), the most basal taxon in the former clade (Fig. 3),has microtubes that resemble those found in Pharomachrus(Fig. 4f). In all species of Pharomachrus (Fig. 4g–j) themicrotubes have fused to produce multi-chambered struc-tures, and there is a tendency towards reduction in area andthe amount of melanin present within each microtube.Except for P. antisianus (Fig. 4h), all species show themulti-chambered structures ordered in a concentric mannerthroughout the barbule. Although there are fewer micro-tubes in Pharomachrus than in all other species of NewWorld trogons, this reduction has no observable effect onthe intensity of their iridescence. Rather, species ofPharomachrus are the most brilliant in the whole family,as a result of the interaction between light and the differentoptical densities of the multi-chambered nanostructures (seereview by Durrer 1977). An alternative tendency wasobserved for the evolution of nanostructures in the remain-ing clade within the New World trogons (Priotelus +Trogon). In Priotelus (Fig. 4k), the density of microtubes is

Species Ta Td Ca Ma M% Ti Te Tm

Apaloderma narina 0.179 0.610 0.032 0.147 83 14 4 14

Apaloderma vittatum 0.141 0.587 0.020 0.120 85 10 6 12

Euptilotis neoxenus 0.111 0.525 0.013 0.097 87 22 22 22

Pharomachrus antisianus 0.074 0.344 0.016 0.058 77 17 17 17

Pharomachrus auriceps 0.019 0.508 0.022 0.106 83 18 18 18

Pharomachrus pavoninus 0.126 0.506 0.037 0.088 70 14 14 14

Pharomachrus mocinno 0.065 0.398 0.010 0.048 74 24 24 24

Priotelus temnurus 0.096 0.412 0.018 0.077 80 21 20 12

Trogon citreolus 0.054 0.286 0.012 0.042 77 53 22 33

Trogon melanocephalus 0.054 0.329 0.019 0.034 68 51 21 8

Trogon massena 0.062 0.330 0.023 0.038 63 41 43 17

Trogon curucui 0.040 0.289 0.013 0.026 65 34 22 32

Trogon violaceus 0.061 0.343 0.019 0.042 68 29 19 28

Trogon viridis 0.036 0.253 0.009 0.027 75 45 19 24

Trogon comptus 0.050 0.310 0.011 0.038 77 53 42 18

Trogon melanurus 0.072 0.440 0.017 0.055 76 34 35 18

Trogon elegans 0.070 0.406 0.026 0.043 61 30 20 9

Trogon rufus 0.051 0.298 0.016 0.034 67 17 22 18

Trogon collaris 0.052 0.035 0.010 0.040 78 68 49 11

Trogon mexicanus 0.073 0.423 0.027 0.046 62 29 25 21

Trogon personatus 0.060 0.361 0.014 0.045 76 42 38 13

Table 3 Results of measure-ments of the color-producingnanostructures in Trogoni-formes, averaged per speciesand character; for explanations,refer to text

Ta = microtube area (μ2 ); Td =microtube diameter (μ); Ca = airchamber area (μ2 ); Ma = mela-nin area (μ2 ); M% = percentageof melanin; Ti = number ofmicrotubes per quadrant in inte-rior part of barbule; Te = numberof microtubes per quadrant inexterior part of barbule; Tm =number of microtubes per quad-rant in medulla of barbule


lower than that found in Trogon, and the microtubes arebigger and more dispersed in the barbule. In Trogon, apartfrom the copious individual microtubes, these are neatly

distributed between the barbule edge and the keratin layer,in compact layers, with only few of them dispersed alongthe medulla (Fig. 5).

1 µ

0.05 substitutions/site

Apaloderma narina

Apaloderma vittatum

Harpactes ardens

Harpactes diardii

Harpactes oreskios

Euptilotis neoxenus

Pharomachrus mocinno

Pharomachrus antisianus

Pharomachrus pavoninus

Pharomachrus auriceps

Priotelus temnurus

Trogon curucui

Trogon melanocephalus

Trogon citreolus

Trogon viridis

Trogon violaceus

Trogon massena

Trogon melanurus

Trogon comptus

Trogon curucui

Trogon collaris

Trogon personatus

Trogon mexicanus

Trogon elegans

Trogon rufus

Fig. 3 Global scenario for anatomical tendencies in the evolution of the nanostructures responsible for iridescent coloration in Trogoniformes


aa b c

d e f

g h i

j k l

1µ

Fig. 4 Iridescence-producing nanostructures at 8000× magnification.a Apaloderma vittatum. b Apaloderma narina. c Harpactes oreskios.d Harpactes diardii. e Harpactes ardens. f Euptilotis neoxenus. g

Pharomachrus mocinno. h Pharomachrus antisianus. i Pharomachruspavoninus. j Pharomachrus auriceps. k Priotelus temnurus. l Trogonrufus


a b c

d e f

g h i

j k l

1µ

Fig. 5 Iridescence-producing nanostructures at 8000× magnification in species of Trogon. a T. elegans. b T. mexicanus. c T. personatus. d T.collaris. e T. comptus. f T. melanurus. g T. massena. h T. violaceus. i T. curucui. j T. viridis. k T. citreolus. l T. melanocephalus


Discussion

Iridescence in Trogoniformes and other groups might havemultiple functions (e.g. intra-specific recognition, inter-specific signaling, sexual selection, mimetic patterns, etc.).Our work cannot address the advantages or specificfunctions of trogon coloration; instead, we focus thisdiscussion on the observed anatomical patterns.

As we have presented, iridescence-producing nanostruc-tures vary among trogoniform genera. Shawkey et al.(2006) suggest that simple rearrangements in morphologymay be responsible for the diversity found in the nano-structures that produce iridescence, as well as other types ofstructural coloration. Further research into the developmen-tal paths of these nanostructures could provide us withanswers as to why some groups, such as the Trogoniformesand some families of Galliformes and Passeriformes, showsuch diversity of iridescence-producing nanostructureswithin them, whereas others, e.g. Anatidae, Trochilidae,and Nectariniidae, present no corresponding internal vari-ation (Durrer 1977).

Among iridescent trogons, the species in Apalodermaare the least iridescent, whereas quetzals are the mostiridescent. The multi-chambered structures observed in thegenera Euptilotis and Pharomachrus may have evolved bythe fusion of individual tubes, as those found in the sistergroup, which includes Trogon and Priotelus (Fig. 3). Thenotion that these layered, multi-chambered structures mayhave derived from individual tubes was also suggested byDurrer (1977). This author drew a relationship between thetypes of nanostructures that each genus possesses with thedegree of iridescence produced. He proposed that themultiple layers of regularly spaced multi-chambered ultra-structures found in Pharomachrus maximize light interfer-ence, resulting in the most intense iridescence found amongbirds (Durrer 1977). Because quetzals have also developedcrests, as well as elongated wing and upper-tail coverts, itwould be worth exploring further whether iridescence hasevolved in a sexual-selection context, and/or whether itpossesses any other implications (see, for instance, Endler1992, 1993).

Additional work is needed to clarify the status ofiridescence within the Asian trogons. Currently, thesetrogons are divided in two genera: Apalharpactes andHarpactes. The two species within Apalharpactes are theonly Asian trogons with metallic tones in their plumage. Wewere unable to obtain tissue samples for any of the twospecies of Apalharpactes. In a recent analysis, Hosner et al.(2010) concluded that Apalharpactes is more closelyrelated to the African trogons than to the Asian genusHarpactes. However, those authors’ study only included A.rienwarrdtii. If indeed Apalharpactes is more closelyrelated to the African genus Apaloderma than to the Asian

Harpactes, this would mean that iridescence is primitive forthe Trogoniformes, and has been lost in Harpactes.Moreover, it is worth noting that in our study, feathersfrom the non-iridescent species of Harpactes do not show auniform distribution of melanin pigments throughout thekeratin matrix as in other non-iridescent birds. Rather, thesemelanin pigments are distributed in well-defined clustersresembling the general aspect of ancestral microtubes(Fig. 3). It is possible that such clusters evolved after theloss of the keratin wall that kept the coherence of themicrotubes. Shawkey et al. (2006) present examples ofreversals from iridescence to non-iridescent colorationwithin grackles and allies (Icteridae), arguing that only afew steps of morphological modification are needed to gofrom one state to the other. Although the nanostructures thatproduce iridescence in the Icteridae are different from thosein Trogoniformes (Durrer 1977, 1986), it is nonethelesspossible that simple rearrangements of these nanostructureswithin a group have considerable implications to theresulting coloration.

Further studies of the optical properties of the arrange-ments produced by the diversity of nanostructures in thisfamily (as in Prum 2002; Prum et al. 1994, 1999a, b, 2003;Prum and Torres 2003; Shawkey et al. 2006) could give usfurther insight into the way in which iridescence, as aphysical phenomenon, has evolved within this group alongwith the evolution of the iridescence-producing nano-structures, and how brighter forms are a result of thesechanges.

Acknowledgements The authors wish to gratefully acknowledge thegenerosity of Fernando García-Hernández and his lab team, especiallyVerónica Anaya and Jesús Espinosa, who trained the first author andprovided all the necessary help and material for the electronmicroscopy study. Feather samples were obtained from the ornithol-ogy collection of the American Museum of Natural History thanks tothe generosity and help of Joel Cracraft, George Barrowclough, andPaul Sweet. Blanca Hernández and Adolfo Navarro from the Museode Zoología Alfonso L. Herrera, UNAM, provided us with sometissues for the molecular analysis. Finally, Blanca Hernández, AdolfoNavarro, Francisco Ornelas, Fanny Rebón, Joel Cracraft, OlafBininda-Emonds, and two anonymous reviewers all read and gavevaluable comments to an earlier version of this paper. This work waspartly funded by a CONABIO research grant to A. Espinosa de losMonteros (FB612/R174/98), and by a B.Sc. fellowship from theInstituto de Ecología to E. Quintero.

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