J. Avian Biol. 41: 505514, 2010
doi: 10.1111/j.1600-048X.2009.04823.x# 2010 The Authors. J. Compilation # 2010 J. Avian Biol.
Received 2 February 2009, accepted 20 July 2009

The evolution of black plumage from blue in Australian fairy-wrens(Maluridae): genetic and structural evidence
Amy C. Driskell, Richard O. Prum and Stephen Pruett-Jones
A. C. Driskell, Lab of Analytical Biology, National Museum of Natural History, Smithsonian Inst., 4210 Silver Hill Road, Suitland, MD
20746 USA.  R. O. Prum, Dept of Ecology and Evolutionary Biology, and Peabody Museum of Natural History, Yale Univ., Box 208105,
New Haven, CT 06520 USA.  S. Pruett-Jones (pruett-jones@uchicago.edu), Dept of Ecology and Evolution, Univ. of Chicago, 1101 East 57th
Street, Chicago, IL 60546 USA.

Genetic variation in the melanocortin-1 receptor (MC1R) locus is responsible for color variation, particularly melanism,in many groups of vertebrates. Fairy-wrens, Maluridae, are a family of Australian and New Guinean passerines withseveral instances of dramatic shifts in plumage coloration, both intra- and inter-specifically. A number of these color
changes are from bright blue to black plumage. In this study, we examined sequence variation at the MC1R locus in mostgenera and species of fairy-wrens. Our primary focus was subspecies of the white-winged fairy-wren Malurus leucopterus inwhich two subspecies, each endemic to islands off the western Australian coast, are black while the mainland subspecies isblue. We found fourteen variable amino acid residues within M. leucopterus, but at only one position were alleles perfectly
correlated with plumage color. Comparison with other fairy-wren species showed that the blue mainland subspecies, notthe black island subspecies, had a unique genotype. Examination of MC1R protein sequence variation across our sample
of fairy-wrens revealed no correlation between plumage color and sequence in this group. We thus conclude that aminoacid changes in the MC1R locus are not directly responsible for the black plumage of the island subspecies ofM. leucopterus. Our examination of the nanostructure of feathers from both black and blue subspecies of M. leucopterus
and other black and blue fairy-wren species clarifies the evolution of black plumage in this family. Our data indicate thatthe black white-winged fairy-wrens evolved from blue ancestors because vestiges of the nanostructure required for the
production of blue coloration exist within their black feathers. Based on our phylogeographic analysis of M. leucopterus, inwhich the two black subspecies do not appear to be each other’s closest relatives, we infer that there have been two
independent evolutionary transitions from blue to black plumage. A third potential transition from blue to black appears
to have occurred in a sister clade.

Identifying the genetic basis underlying adaptation is acentral goal for evolutionary biologists. Related to this,coloration patterns in vertebrates have been studied extensively. In some instances, intraspecific variation in color has
been shown to be controlled by a simple genetic mechanism. For example, variation at the melanocortin-1 receptor(MC1R) locus is known to affect plumage color in somespecies of birds (Takeuchi et al. 1996, Theron et al. 2001,
Kerje et al. 2003, Ling et al. 2003, Mundy et al. 2003,
2004), coat coloration in some mammals (Majerus andMundy 2003, Nachman et al. 2003), and hair coloration inhumans (Schiöth et al. 1999). The receptor is expressedin the membrane of melanocytes, and is an important
regulator of the synthesis of melanin pigments. Activationof the MC1R increases the production of brown and black
eumelanin pigments (Mundy 2005). In a number of vertebrate species, non-synonymous substitutions in the MC1R
gene are correlated with melanism (Takeuchi et al. 1996,
Theron et al. 2001, Kerje et al. 2003, Ling et al. 2003,
Majerus and Mundy 2003, Mundy et al. 2003, 2004,
Nachman et al. 2003). In many of these cited cases, themechanism by which melanism is triggered is essentiallythe same  mutations in the same region of the MC1R genedisrupt the hormone-recognition site and cause constitutive
activation of the gene (Majerus and Mundy 2003). This
results in the over-production of melanin that eclipses thenormal coat or plumage color.
One of the consistent features of avian species forwhich MC1R has been shown to be important is that themelanistic forms occur within the same population as nonmelanistic forms (Theron et al. 2001, Mundy et al. 2004).
Additionally, the expression of the melanistic phenotype isseldom sex limited  well-known examples exist in which
both males and females are melanistic (Mundy 2005,
Hoekstra 2006), although sex-specific effects of MC1R
variation have been observed in the chicken (Ling et al.
2003). The extent to which MC1R affects coloration varies,
but in general, the entire plumage of an individual ischanged. For example, in the bananaquit Coereba flaveola
all white and yellow colored areas are completely obscured
by melanin (Theron et al. 2001); in the arctic skua
Stercorarius parasiticus the entire plumage of the bird is505

darkened; and in the lesser snow goose Anser caerulescens
caerulescens, although the head of the most melanistic birds
remains white, the rest of the plumage is entirely dark
(Mundy et al. 2004). In both arctic skuas and snow geese,individuals homozygous for the melanism mutation havethe darkest plumage while the plumage of heterozygotes isintermediate (Mundy et al. 2004).
In this paper we examine coloration and MC1R
variation in fairy-wrens (Maluridae), an AustraloPapuan
family of passerines exhibiting a wide range of plumage
patterns. Most species in the family exhibit sexual dichromatism with strikingly colored males and dull colored
females, but important exceptions include the brightly
colored, but essentially monochromatic broad-billed fairywren Malurus grayi in Papua New Guinea, and the grasswrens Amytornis and emu-wrens Stipiturus of Australia(Rowley and Russell 1997) in which sexual dichromatism
is less pronounced than in Malurus. Within the genus
Malurus, geographic variation in plumage can occur infemales (the red-shouldered species group; Schodde andMason 1999) and in males (the blue wren species group;
Schodde and Mason 1999).
One of the most unusual patterns of plumage variation in
fairy-wrens occurs in the white-winged fairy-wren Malurus
leucopterus. The mainland subspecies (M. l. leuconotus),which is distributed over most of the arid regions of theAustralian continent, exhibits no noticeable variation inplumage coloration across this vast range. Males areuniversally bright cobalt blue with white wings, females
are fawn colored, and immature males resemble females. In
contrast, in the two island-endemic subspecies in Western
Australia, M. l. leucopterus on Dirk Hartog Island and M. l.
edouardii on Barrow Island, males are entirely black withwhite wings. The plumages of females and immature malesof the island subspecies are similar to those of the mainland  largely fawn-colored. Based on mitochondrial DNA
evidence, Driskell et al. (2002) demonstrated that: 1) thetwo black-plumaged island subspecies are not sister taxa;
2) M. l. edouard on Barrow Island is the only geneticallydistinct clade; and 3) M. l. leucopterus on Dirk Hartog Islandis genetically indistinguishable from the birds on themainland in Western Australia, in which males are blue
and white (Driskell et al. 2002). These islands are each closer
to the mainland (Dirk Hartog 1 km, Barrow 55 km)
than to each other (600 km) and gene flow between themis unlikely. Thus, either black male plumage evolved
independently, and in parallel in the two island populations,or the mainland subspecies has evolved bright blue plumage
from a M. leucopterus ancestor with black male plumage.
In a study comparing the black Dirk Hartog (M. l.
leucopterus) and mainland blue subspecies (M. l. leuconotus),
Doucet et al. (2004) found five amino acid substitutions in
the MC1R sequence associated with the plumage difference
between these two subspecies. In addition, they concluded
from their examination of the feather nanostructure of these
two subspecies that the ancestor of the black plumaged
island subspecies was blue plumaged (Doucet et al. 2004).
In this paper we examine MC1R variation in all threesubspecies of M. l. leucopterus, the closely related and black
plumaged red-backed fairy-wren (M. melanocephalus ),other species of Malurus, and exemplars of most otherfairy-wren genera (Fig. 1, Appendix 1). Additionally, we
506

examine the nanostructure of feather barbs of the subspeciesof the white-winged fairy-wren, M. melanocephalus, and one
blue-plumaged fairy-wren, the splendid fairy-wren (M.
splendens). As we will demonstrate, patterns of genetic
variation at the MC1R locus within all three subspecies ofwhite-winged fairy-wren as well as among multiple otherspecies of fairy-wrens indicate there is no association
between MC1R variation and plumage variation in fairywrens. However, feather nanostructure does vary in aconsistent manner across the subspecies of white-winged
fairy-wrens, revealing that the black island subspecies have
similar, though not identical, structure and evolved fromblue ancestors.

Methods
Taxon sampling
To examine MC1R variation in Malurus, tissue or blood
samples were obtained from seven of the 11 species in the
Malurus and three of the remaining four genera in
Maluridae. For M. leucopterus, 12 individuals from Barrow
Island (M. l. edouardi ), five individuals from Dirk Hartog
Island (M. l. leucopterus), and 13 individuals from themainland of Australia (M. l. leuconotus) were sampled.
To update our published study of the evolutionary
relationships among the subspecies of M. leucopterus(Driskell et al. 2002), mitochondrial data for five additional
specimens of M. leucopterus edouardi from Barrow Islandand five additional specimens of M. l. leuconotus from
Western Australia were added to the published matrix. In
addition, two M. l. leuconotus specimens had been misidentified as M. l. leucopterus in the previous study and thisis corrected here. Specimens, collection localities, andlending institutions are listed in Appendix 1.
DNA extraction, amplification and sequencing
DNA was extracted from ethanol-preserved tissue or
blood using standard proteinase-k digestion followed by aprotein precipitation method as implemented in the Puregene or Qiagen DNAeasy kits. Multiple primers were
designed for amplification of MC1R in fairy-wrens aspreviously published primers did not amplify reliably.
Two primers were primarily used for amplification: F31 (5?-GCTGGCCCCCCTGCGYCTGCTGC-3?) and R908
(5?-ACCACCTCCCGCAGCGTCCGCC-3?), which produced a 870 bp fragment of the MC1R gene. In a few
instances, the primers MSHR72 and MSHR73 of Mundy
et al. (2003) were also used for either amplification orsequencing. Initially, amplification products were directly
sequenced using the amplification and other internal
primers. However, allelic variation was apparent in a
number of specimens as indicated by the appearance of‘double-peaks’ in resultant electropherograms. In order toaccurately record all MC1R alleles present in the populations, the majority of MC1R data were produced bysequencing cloned PCR products.
A multi-step procedure was used for sequencing clones:
1) PCR amplification from DNA extracts using the primer
pair F31/R908, 2) Gel purification using a 1% low-melt

M. l. edouardi - black body & head, white wings
M. l. leucopterus - black body & head, white wings“Bi-colored”
wrens
M. l. leuconotus - blue body & head, white wings
M. melanocephalus - black belly, head & wings, red backM. alboscapulatus - black body, head & wings, white shoulders“Blue” group: includes M. splendens - nearly entirely blue or purple,
black patches
Other Malurus - various plumages, many with large patches of blue

Figure 1. Relationships among the species and subspecies examined in this study and descriptions of their plumages. Diagram based on
results published in Driskell et al. (2002) and Norman et al. (unpubl.).

agarose gel and excision of the band of interest, 3) Digestionof excised agarose plug using the Gelase enzyme, 4) Cloningof digested amplification products using the TOPO TAcloning kit, 5) Checking 14 clones (on average, two) fromeach specimen by boiling picked colonies for 10 min and
PCR amplification with the primer pair F31/R908, 6)
Clean-up of the amplified product using the ExoSAP-IT
protocol (USB), 7) Double-stranded sequencing using
standard protocols.
Mitochondrial data were produced for the new M.
leucopterus specimens following the procedures of Driskell
et al. (2002).
Sequence alignment and comparison
MC1R sequences were translated using the Se-AL ver. 2.0
program (Rambaut 2001). All apparent base substitutions,in particular non-synonymous substitutions, were verified
by careful examination of the electropherograms. Whenambiguity persisted, the specimen was re-amplified andre-sequenced. No insertions or deletions were observed inany of the protein-coding sequences.
Phylogenetic and phylogeographic analyses of
mtDNA data
The phylogeographic analyses were conducted using
PAUP* ver. 4.0b10 (Swofford 2002). Heuristic searches
under the parsimony criterion were unweighted with 50
multiple random addition sequences and TBR branchswapping limited to 20 min per replicate. An overallmaximum of 5000 trees was saved and strict and majority
rule consensus trees were then calculated using this tree set.
One hundred replicates of bootstrap analysis with TBR
branch-swapping and two random addition sequences per
replicate were used to evaluate nodal support. Branchswapping was again limited to 20 min per replicate. Timelimits were implemented with this data set as the majority
of sequences were identical or nearly so, and almost no wellsupported relationships with M. leucopterus exist. Additionaltime spent branch-swapping almost certainly would not
result in greater resolution of the final tree.
Feather anatomy
We examined the microstructure of feathers of M. splendens,a typical blue plumaged fairy-wren, the three subspecies ofM. leucopterus and M. melanocephalus, a primarily black
plumaged sister species of M. leucopterus (Christidis andSchodde 1997, this study). Contour feathers were washedand prepared for microscopy following the methods outlined in Dyck (1971a). Transmission electron micrographsof thin (100 nm) sections were taken with a Jeol JEM1200 using a Soft-Imaging Megaview II CCD camera(1024 1200 pixels).

Results
MC1R analysis
Fourteen amino acid residues were variable within M.
leucopterus (Table 1). Residue 38 was the only position
correlated with male plumage color in this species 
specimens of both island subspecies had an isoleucine at
this position, while mainland birds had asparagine. However, all other species of fairy-wrens, regardless of plumage
color, and the non-melanistic form of Coereba also had
isoleucine at this position (Theron et al. 2001), and theblack-plumaged M. alboscapulatus individuals had either
isoleucine or valine here. Hence, at residue 38 the blue
mainland M. l. leuconotus, not the black island subspecies,
have a genotype (asparagine) unique among the otherspecies examined. For each of the 14 variable amino acidresidues, the two island subspecies were either homozygous
or heterozygous for the same amino acid as all of the otherspecies, as well as the distantly related bananaquit Coereba.
Furthermore, at these 14 sites the island variant was notpresent in all island individuals sampled, whether homozygous or heterozygous  ruling out the possibility thatthese variants affect plumage color via genetic dominance.
The blue mainland subspecies, M. l. leuconotus, had the507Table 1. Variable amino acid residues in the MC1R sequence of Malurus leucopterus and other fairy-wrens. All residue differences shared by
two or more haplotypes of Malurus leucopterus are shown. Numbers indicate the ratio of haplotypes with each residue, in the cases wheremore than one amino acid was present in a taxon. In this study, 16 haplotypes were identified in M. l. edouardi, 7 in M. l. leucopterus and 19
in M. l. leuconotus. Shaded columns are the residues Doucet et al. (2004) identified as correlated with plumage color. Coereba sequence(GenBank A362594) from Theron et al. 2001.
Amino acid residue
16

28

31

38

111

122

157

166

182

192

248

269

280

296

A

V

I

R

G

F

F

F

A

I

Q

I

R

G

F

F

F

A

Mainland M. l. leuconotus

T

G

N

V/D
6/1
I

I/V
14/2
I

Q

A/T
5/2
A

G/S
14/2
G

I

Dirk Hartog Is M. l. leucopt.

A/T
14/2
A

I

M. alboscapulatus
M. melanocephalus
M. splendens
M. lamberti
M. pulcherrimus
M. cyaneus
Clytomias insignis
Stipiturus mallee
Amytornis striatus
Coereba flaveola y20

A
A
A
A
A
A
A
A
A
A

A
A
A
A
A
A
A
A
A
A

G
G
G
G
G
G
G
G
G
G

I/V
I
I
I
I
I
I
I
I
I

I/V
V
V
V
V
V
V
I
V
V

Q/R
11/8
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q

V/I
16/3
V
I
I
I
I
I
I
I
I
V

R/H
11/8
R/H
R
R
R
R
R
R
R
R
R

G/S
13/6
G
G
G
G
G
G
G
G
G
G

F/L
16/3
F
F
F
F
F
F
F
F
F
F

F/L
17/2
F
F
F
F
F
F
F
F
F
F

F/S
17/2
F
F
F
F
F
F
F
F
F
F

A/P
17/2
A
A
A
A
A
A
A
A
A
A

Barrow Is M. l. edouardi

most divergent amino acid sequence of all those examined 
at nine positions it was either fixed or heterozygous for anamino acid not seen in any of the other taxa. There were no
fixed synonymous differences among the subspecies and apossible cause for the amino acid divergence of M. l.
leuconotus from the other two subspecies is unknown.
Phylogeographic analyses within M. leucopterus
Analysis of the M. leucopterus data set yielded 10 375 equallyparsimonious trees. A strict consensus of these trees showedvery little structure within M. leucopterus  likely due to thereasons of historical demography, which result in very low
levels of sequence divergence within the species (97% of the980 bp of sequence was invariant; Driskell et al. 2002).
A strict consensus tree showed no phylogenetic structure
within M. leucopterus (not shown). However, the majority
rules consensus tree (Fig. 2) retained a monophyletic
M. leucopterus, and three additional clades: A) a monophyletic Barrow Island M. l. edouardi clade (present in 77%of the parsimony trees); B) a monophyletic, but not wellsupported, clade containing all Dirk Hartog specimens; andC) a clade comprised of all Dirk Hartog M. l. leucopterus andall but two of the Western Australian M. l. leuconotus
specimens (present in 100% of the parsimony trees). Thus,
both island subspecies appear as monophyletic groups in the
majority rules consensus tree, albeit neither clade obtains
overwhelming bootstrap support and they are not necessarily
each other’s closest relatives. Bootstrap analysis provided
strong support for only the node subtending the Barrow
Island clade. Sequence divergence among the M. leucopterus
specimens ranged from 00.92% (9 bp different). The
greatest genetic distance observed in the M. leucopterus data
set was between a specimen from Barrow Island and a
specimen from Dirk Hartog Island. Barrow Island specimens had at least 3 and as many as 9 nucleotide differencesfrom all other M. leucopterus specimens.
508

I
I
I
I
I
I
I
I
I
I

Feather anatomy
The morphology of the barbs of blue feathers ofM. splendens (not illustrated) and M. leucopterus leuconotus(Fig. 3A, 4AB) of mainland Australia (including thosefrom Western Australia in close proximity to the island
populations) was very similar. The spongy medullary layerof the feathers of both species was nanostructured in asystem of complex air channels, and is similar to that foundin blue feathers of parrots (Psittaciformes), rollers (Coraciidae), leafbirds (Irenidae), and thrushes (Turdidae) (typef of Dyck 1976; Prum et al. 1999, 2003). The barb rami of
both M. splendens and M. l. leuconotus had 1) ellipticalcross-sections, 2) a thin and uniform cortex completely
devoid of melanosomes, 3) a medullary layer composed oflarge, boxy-shaped cells with extensive spongy keratin
matrices, air-filled vacuoles, and 4) a layer of melanosomes
between the central vacuole and the more superficial spongy
keratin (Fig. 3A, 4AB). The position of melanosomes
within the spongy medullary cells is typical of almost all
structurally colored barb rami (Fig. 4A; Dyck 1971a,
1971b, 1976, Prum et al. 1998, Prum 1999). These
medullary melanosomes function as a light-absorbing layer
below the structurally colored spongy matrix that prevents
the incoherent scattering of white light from the central air
vacuoles (Prum 2006, Shawkey and Hill 2005). Thus, themelanosome layer contributes to the saturation of thestructural blue color. The barbs of M. splendens and M. l.
leuconotus differ only slightly in shape; M. splendens barbs
are narrower and higher than those of M. l. leuconotus.
In contrast, the black feather barbs from the island forms
M. l. edouardi and M. l. leucopterus, and the black-plumaged
red-backed wren (M. melanocephalus ) were substantiallydifferent in morphology from the blue feather barbs ofM. splendens and M. l. leuconotus (Fig. 3BD, 4C, 4E).
Black barbs from all three taxa revealed extensive melanosome deposition in nearly all cells, including the cortex(Fig. 3BD). Furthermore, all three black taxa showed asubstantially thicker cortex with a slight to elongate ridge in

93

77
57
100
100
88
64
78

100
56

100
55

100
100

BIsl 1
BIsl 2
BIsl 8BIsl 5
BIsl 3
BIsl 6
BIsl 7
BIsl 4
BIsl 10
BIsl 11
BIsl 12BIsl 9BIsl 13
DHIsl 1
DHIsl 2
DHIsl 3
DHIsl 4
DHIsl 6
DHIsl 7
DHIsl 5
DHIsl 8
WA 9
WA 10
WA 1
WA 2
WA 3
WA 4
WA 6
WA 8
QLD 3
QLD 4
QLD 5
NSW 3
NSW 4
SA 2
SA 4
SA 3
WA 5
QLD 1
NSW 1
NSW 2
SA 1
WA 7
QLD 2
M. lamberti
M. melano.

Figure 2. Majority-rule consensus of 5000 equally parsimonious trees. Numbers above the nodes indicate the percentage of trees in whichthat node is found. Numbers below the nodes are parsimony bootstrap proportions. Where no bootstrap value is indicated, the node
received B50% bootstrap support. Tree length 193.

the cortex on the upper (or obverse) surface (or margin)of the rami (Fig. 3BD). The barb rami of M. l. edouardi
have the most modest ridge (Fig. 3B), whereas the barb
rami of M. l. leucopterus and M. melanocephalus haveelongate, obverse ridges creating a generally tear drop
shaped ramus in cross-section (Fig. 3CD).
Most interestingly, the barb rami of the two black
subspecies of M. leucopterus and the black M. melanocephalus
showed various evidence of plesiomorphic but vestigial(i.e. secondarily non-functional) spongy medullary keratin(Fig. 4D, 4F). Malurus l. edouardi showed a substantial
spongy keratin matrix that is similar in nanostructure tothat found in blue barbs of blue M. l. leuconotus andM. splendens (Fig. 4D). However, these cells also include a
dense concentration of melanosomes, distributed in thecortex and throughout the medullary keratin (Fig. 4C).
These melanosomes would function to absorb incoming

light before it could interact with the spongy medullary
keratin to produce any structural blue color. Black barb
rami from M. l. leucopterus showed an even more reduced
medullary layer composed of fewer cells with less extensive
spongy keratin within each cell (Fig. 4E). However, these
medullary cells had melanosomes distributed throughout,blocking any direct optical interaction between the rudimentary spongy keratin matrices and incident light. Lastly,
M. melanocephalus exhibited the most rudimentary medullary layer made of fewer cells with an extensive volume of
unstructured ß-keratin (not illustrated).

Discussion
The evolution of color patterns in birds, and vertebrates ingeneral, is complex and involves the interaction of genetics
509

Figure 3. Transmission electron micrographs of the barb rami of white-winged fairy-wrens Malurus leucopterus and red-backed fairy-wren
Malurus melanocephalus (Maluridae). (A) blue, mainland white-winged fairy-wren M. leucopterus leuconotus, (B) black, Barrow Island
white-winged fairy-wren M. leucopterus edouardi, (C) black, Dirk Hartog Island white-winged fairy-wren M. leucopterus leucopterus, (D)black feather from red-backed fairy-wren M. melanocephalus. The cortex (c) differs greatly in thickness and presence of melanization. Scalebars: (A), (C), (D) 10 mm, (B) 5 mm.

Figure 4. Transmission electron micrographs of spongy medullary cells from subspecies of white-winged fairy-wrens Malurus leucopterus.
(A)(B) blue, mainland white-winged fairy-wren M. leucopterus leuconotus, (C)(D) black, Barrow Island white-winged fairy-wren
M. leucopterus edouardi, (E)(F) black, Dirk Hartog Island white-winged fairy-wren M. leucopterus leucopterus. Both blue and blackfeathers have spongy medullary keratin. Blue M. l. leuconotus feathers have melanosomes distributed below the spongy medullary keratin,
whereas black M. l. edouardi M. l. leucopterus feathers have melanosome distributed throughout the spongy medullary keratin. c cortex,
k spongy medullary keratin, m melanosomes. Scale bars: (A), (C) 2 mm, (B) 1 mm, (D), (F) 500 nm, (E) 5 mm.

510

and development with aspects of habitat structure, ecology
and breeding biology. The discovery that one or a very few
mutations in a single gene, the MC1R locus, can have amajor effect on melanism within a number of vertebrate
species has provided a unique opportunity to investigate theinteraction between genes and habitat structure and its
influence on evolution (Majerus and Mundy 2003, Mundy
et al. 2003, 2004, Nachman et al. 2003, Hoekstra 2006).
Although there are instances in which mutations at theMC1R locus clearly underlie melanistic plumage in somebirds (Takeuchi et al. 1996, Theron et al. 2001, Kerje et al.
2003, Ling et al. 2003), other examples are known in which
intra- or interspecific variation in melanistic plumage
is unrelated to genetic variation at the MC1R locus(MacDougall-Shackleton et al. 2003). Additionally, otherstudies clearly reveal the important role that habitat
structure and the light environment play in influencing
evolution of color patterns in birds (Marchetti 1993,
Johnson and Lanyon 2000, Gomez and Théry 2007). In
studies of bananaquit, skua and other birds in which MC1R
appears to influence plumage coloration, the amino acidsubstitutions in MC1R affect the overall quantity of
produced melanin. Such variation cannot entirely explain
the complexity of within-feather pigmentation patterns that
contribute greatly to plumage coloration (Prum andWilliamson 2002).
Our data show that the melanin color variation in malefairy-wrens generally, and the white-winged fairy-wren
specifically, is unrelated to variation at the MC1R locus.
We observed no genotypic variation in MC1R that correlatedwith black plumage in the two island subspecies. At the only
residue (38) at which both island subspecies differed from themainland subspecies, the island subspecies shared this aminoacid with other non-melanistic species examined, and it was
the mainland M. l. leuconotus which possessed the uniquecharacter state. The MC1R locus varied at a number ofamino acid positions, but the most common allele in theblack island populations was the same as that in the majorityof other species examined, be they blue or black and in no
instance was this ‘island’ allele present in every specimen
examined. Although we acknowledge that the genetic
mechanism underlying the plumage variation in whitewinged and other fairy-wrens remains unknown, we contend
it does not appear to be variation at the MC1R locus.
Doucet et al. (2004) found genetic differences in theircomparison of mainland birds and those on Dirk Hartog
Island, but their statement that these differences demonstrate a concordance between MC1R variation and plumage
evolution is not justified based on their two sample
comparison. Any pattern of genetic variation found in a
two-taxon sample would lead to the conclusion that thevariation was associated with differences between the taxa,
but this is not sufficient as a test of the hypothesis ofconcordance. Like a regression based on two data points,
Doucet et al.’s (2004) two population sample was insufficient to make the claim of evolutionary concordance
between genetic and morphological evolution, and ourbroader analysis has falsified their claim.
It is unsurprising that constitutive activation of MC1R,
proposed as the mechanism driving melanism in thebananaquit and many other vertebrates (Mundy et al.
2003, Hoekstra 2006), does not underlie the black plumageof the M. leucopterus subspecies. Not all of the island adultmales’ plumage is obscured by melanin; white wing patches
persist in all subspecies. This seems inconsistent with
continuous activation of MC1R which often completely
obscures plumage and coat patterning by overproduction ofmelanin. In addition, in M. leucopterus leuconotus and M. l.
edouardii only the reproductively mature males have black
plumage  females and immature males retain the samefawn-colored plumage as the mainland blue subspecies.
Clearly, the MC1R locus cannot be consitutively activatedin females and reproductively-immature males, as theirplumage is not melanistic. Color production is a complex
process and involves a very long list of loci other than
MC1R, intricate regulatory mechanisms, and details ofdevelopmental biology (Hoekstra 2006). The biologicaland evolutionary explanation for the appearance of black
plumages within fairy-wrens likely lies somewhere ‘upstream’ of the MC1R locus.
In combination with the phylogeographic results ofDriskell et al. (2002) and those presented here, which show
the two island populations nested within the mainlandsubspecies, our data on feather structure imply thatthe ancestors of both black-plumaged subspecies of M.
leucopterus were structurally blue-plumaged. Doucet et al.
(2004) also reached a similar conclusion based on their
examination of the Dirk Hartog subspecies. The barbules of
black feathers from M. l. leucopterus and M. l. edouardi, andthe closely related black species M. melanocephalus all
showed morphological evidence that these feathers were
derived from the plesiomorphic blue Malurus feather barbs.
However, the different black-plumaged taxa show different
degrees of alteration from the inferred ancestral form.
We believe the differences reflect three separate andconvergent acquisitions of black plumage in these taxa.
Within M. leucopterus, the feathers of M. l. edouardi are themost similar to those of its blue mainland relatives, while
those of M. l. leucopterus are more derived. Moreover, therami of the feather barbules from the two black island
subspecies were different from one another in shape and thetwo taxa exhibit different quantities of spongy medullary
keratin. As the two islands are much closer to the mainland
than to each other, convergent evolution of black plumage
in M. leucopterus seems a reasonable conclusion.
The black feathers of M. melanocephalus differ the mostfrom a typical fairy-wren blue feather; they have completely
lost the spongy medullary nanostructure required for the
production of the structural blue color. If degradation offeather microstructure occurs at a roughly consistent rate,
M. melanocephalus evolved its black feathers much earlier
than either M. leucopterus subspecies. In fact, in light ofrecent phylogenetic information (Fig. 1), it is parsimonious
to assume that black plumage evolved in the ancestor of
M. melanocephalus and M. alboscapulatus  placing the event
still further back in time, before the divergence of the
M. leucopterus subspecies. We predict that examination of
the nanostructure of black feathers of M. alboscapulatus willshow a degree of structural degradation comparable to thatof M. melanocephalus.
We propose the first step in the evolution of blackfeathers from blue in the fairy-wrens is hypermelanizationof the cortex and medullary cells of the feather barbules.
The additional melanin obscures the structural blue color
511

and renders blue feathers black. Once the feathers areblack, pressure to maintain a feather structure specific forthe production of structural blue color is relaxed. Overtime, the ramus of the feather deforms and the spongy
medullary cortex is reduced and simplified. We believe
this progression is more likely than the alternative
hypothesis wherein the ancestor of the entire ‘bi-colored’
clade (Fig. 1) evolved black plumage and the mainland M.
l. leuconotus re-evolved blue. This alternative requires thecomplete re-evolution of the morphology of blue feather
barbules. It would necessitate the evolution of nonfunctioning spongy medullary keratin morphology withina continuous black plumage as well as the subsequentde-melanization of the cortex. It is hard to imagine theselective (or even neutral) process that would cause
this to occur. The evolution of black from blue plumage,as we believe occurred, requires only a greater depositionof melanin and subsequent degradation of feather
nanostructure.
In summary, we find no correlation between sequencevariation at the MC1R locus and variation in blue and black
plumage in white-winged and other fairy-wrens. The
evolution of black plumage in M. leucopterus was not
triggered by a mutation causing constitutive activation of
the MC1R locus, as is proposed for other avian species.
Changes in regulation or developmental biology are more
likely causes. Feather nanostructure in the black subspeciesof M. leucopterus and M. melanocephalus is consistent withinitial evolution of barb hypermelanization, subsequent
derived changes in ramus shape for pigment presentation,and the continuing degradation of spongy medullary
keratin organization. The observed pattern could have
evolved entirely through initially neutral variation in genes
for melanin deposition, followed by a selection on barbmorphology for pigment presentation and release of
selection on spongy matrix morphology. Each of the three
black Malurus taxa has a unique barb morphology andbecause phylogeographic and phylogenetic analyses indicatethat they are not each other’s closest relatives, we propose
at least three instances of evolution from blue to black
plumage in the Maluridae. Exactly why the island subspecies of M. leucopterus are black remains an open
question, but we are certain that it is not the result of
sequence variation at the MC1R locus.
Acknowledgements  This research was supported by the National
Science Foundation (all authors), the Division of Biological
Sciences, University of Chicago (SP-J), the Wettenhall Foundation(SP-J), Western Australian Petroleum (SP-J), Chevron Corporation (SP-J), the Chicago Zoological Society (SP-J) and the Sectionof Evolution and Ecology, Univ. of California Davis (ACD).
We wish to thank the curators at Australian National Museum,
National Museum of Victoria, Western Australian Museum, Burke
Museum, the Univ. of Kansas Natural History Museum, Academyof Natural Sciences, and Leo Joseph and Melanie Rathburn foraccess to specimen samples, the many field and laboratory assistants
that helped us during this work, Trevor Price, Emily Kay and Leo
Joseph for comments on the manuscript, and lastly T. Quinn forelectron microscopy.

512

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513

Appendix 1. Specimens of fairy-wrens examined in this study. SourceMuseum or individual providing specimen; Specimen No. museum
voucher or collection number; Bk. Conserv. Park, SABrookfield Conservation Park, SA; Dryandra SF, WA Dryandra State Forest, WA;
Hattah-Kulkyne NPHattah-Kulkyne National Park, VIC; WA MuseumWestern Australian Museum; ANS, Phil.Academy of Natural
Sciences, Philadelphia; Mus. VictoriaMuseum of Victoria; Burke Mus.University of Washington, Burke Museum; U. KansasUniversityof Kansas, Natural History Museum. Genbank accession numbers for newly generated ATP6 sequences: HM156563-HM156572; ND3
sequences: HM156573-HM756582; MC1R sequences: HM156499-156562.
Taxon

Malurus leucopterus edouardi

M. leucopterus leucopterus

M. leucopterus leuconotus

Malurus alboscapulatus
Malurus melanocephalus
Malurus splendens emmottorum
Malurus cyaneus
Malurus lamberti
Malurus pulcherrimus
Clytomyias insignis
Stipiturus mallee
Amytornis striatus

514

Collection Locality

LatitudeLongitude

SourceBarrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Barrow Island, WA
Dirk Hartog Island, WA
Dirk Hartog Island, WA
Dirk Hartog Island, WA
Dirk Hartog Island, WA
Dirk Hartog Island, WA
Bk. Conserv. Park, SA
Bk. Conserv. Park, SA
Bk. Conserv. Park, SA
Bk. Conserv. Park, SA
Bulloo Downs, QLD
Bulloo Downs, QLD
Ueendoo Creek, WA
Ueendoo Creek, WA
Ueendoo Creek, WA
Ueendoo Creek, WA
Ueendoo Creek, WA
Cape Range, WA
Lancelin, WA
Tetebedi, PNG
Atherton, QLD
Longreach, QLD
Bk. Conserv. Park, SA
Bk. Conserv. Park, SA
Dryandra SF, WA

20847?S20847?S20847?S20847?S20847?S20847?S20847?S20847?S20847?S20847?S20847?S20847?S20847?S25850?S25850?S25850?S25850?S25850?S43821?S43821?S43821?S43821?S
28832?S
28832?S
25803?S
25803?S
25803?S
25803?S
25803?S21855?S
31801?S
9810?S
17816?S24810?S43821?S43821?S32846?S115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
115828?E
113805?E
113805?E
113805?E
113805?E
113805?E
139831?E
139831?E
139831?E
139831?E
142857?E
142857?E
113841?E
113841?E
113841?E
113841?E
113841?E
114804?E
115820?E
148804?E
145828?E
143815?E
139831?E
139831?E
117808?E

Hattah-Kulkyne NP
Hattah-Kulkyne NP

34842?S34842?S

142825?E
142825?E

Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-Jones
WA Museum
M. Rathburn
M. Rathburn
M. Rathburn
M. Rathburn
Pruett-Jones
Pruett-Jones
Pruett-Jones
Pruett-JonesMus. VictoriaMus. Victoria
ANS, Phil.
ANS, Phil.
ANS, Phil.
ANS, Phil.
ANS, Phil.
Mus. Victoria
M. RathburnMus. Victoria
J. Karubian
Burke Mus.
Pruett-Jones
Pruett-Jones
Burke Mus.
U. Kansas
Pruett-Jones
Pruett-Jones

Specimen No.
BW001
BW002
BW003
BW004
BW006
BW007
BW008
BW009
BW011
BW012
BW013
BW015
BW038
A26806
60
81
123
141
WW014
WW020
WW030
WW040
D075
D084
ANSP11230
ANSP11232
ANSP11234
ANSP11236
ANSP11256
MV2512
309
E121
RBW01
SAR7063
SFW91-12
VW104
PLG3004619
MEW1
SGW1