Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Current Biology 23, 1–10, June 17, 2013 ª2013 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.cub.2013.04.062
Article
Developmental Basis of Phallus Reduction
during Bird Evolution
Ana M. Herrera,1,4 Simone G. Shuster,2,4 Claire L. Perriton,3,4
and Martin J. Cohn1,2,3,*
1Howard Hughes Medical Institute, Department of Molecular
Genetics and Microbiology, and the Genetics Institute
2Department of Biology
University of Florida, Gainesville, FL 32610, USA
3School of Animal and Microbial Sciences, University of
Reading, Reading RG6 6AJ, UK
Summary
Background: One of the most puzzling events in evolution is
the reduction and loss of the phallus in birds. All birds reproduce by internal fertilization, but only w3% of birds have
retained a phallus capable of intromission. A number of
hypotheses have been proposed for the evolutionary mechanisms that drove phallus reduction; however, the underlying
developmental mechanisms are unknown.
Results: We investigated genital development in two sister
clades of birds, Galliformes (land fowl), most of which lack
an intromittent phallus, and Anseriformes (waterfowl), which
have well developed phalluses; and in two outgroups, Paleognathae (emus) and Crocodilia (alligators). Galliform embryos
undergo cryptic development of a genital tubercle, the precursor of the phallus, but this later undergoes apoptosis, leading
to regression of the tubercle. At the molecular level, a derived
pattern of Bmp4 expression was identified in chick (a galliform)
genital tubercles. Inhibition of Bmp signaling in chick genitalia
rescues cells from apoptosis and prevents phallus regression,
whereas activation of Bmp signaling in duck (an anseriform)
genitalia induces a galliform-like pattern of apoptosis. Thus,
distal Bmp activity is necessary and sufficient to induce
apoptosis in Galloanserae genital tubercles.
Conclusions: Our results indicate that evolutionary reduction
of the intromittent phallus in galliform birds occurred not by
disruption of outgrowth signals but by de novo activation of
cell death by Bmp4 in the genital tubercle. These findings,
together with discoveries implicating Bmps in evolution of
beak shape, feathers, and toothlessness, suggest that modulation of Bmp gene regulation played a major role in the evolution of avian morphology.
Introduction
Few morphological characters in the animal kingdom rival the
diversity, complexity, and evolvability of male external genitalia [1]. Among animals with internal fertilization, genital
morphology evolves rapidly, and the size, shape, and anatomical details of the external genitalia can show dramatic variation, even between closely related species [2]. The basis of
this diversity has been the subject of debate, and a number
of evolutionary mechanisms have been proposed to account
for the rapid divergence of genital form [1–9].
4These
authors contributed equally to this work
*Correspondence: [email protected]
Some of the most striking examples of the evolutionary
diversification of genital form and function are found in birds
[10–18]. All birds reproduce by internal fertilization, yet only
w3% of extant birds possess a phallus capable of intromission [19]. Indeed, external genitalia are reduced or absent in
nearly 10,000 species of birds. These evolutionary changes
have resulted in phallus morphologies that can be classified
operationally into three general categories: (1) an intromittent
phallus that is capable of insertion during copulation (e.g.,
ducks), (2) a nonintromittent phallus that is reduced but not
lost (e.g., chickens), and (3) absence of the phallus that
involves a complete loss of a phallic protuberance (e.g., neoaves). Recent progress in phylogenomics has begun to clarify
the phylogenetic relationships among birds [20], and evolutionary reductions of the intromittent phallus can be mapped
to a relatively small number of nodes in the avian phylogeny;
for example, after their divergence from Anseriformes (e.g.,
ducks, swans, geese, and allies), Galliformes (e.g., chickens,
quails, pheasants, and megapodes) evolved a highly reduced,
nonintromittent phallic rudiment. Even more extreme reduction occurred near the base of Neoaves, the largest clade of
birds, which have no remnant of the phallus [21, 22].
Although most galliforms underwent extreme reduction of
the external genitalia [21, 22], members of their sister clade,
the anseriforms, retained an intromittent phallus, and in
some species these elongated coiled organs can exceed the
length of the body [23]. These morphological changes led to
changes in copulation strategies in different lineages. For
example, male birds that lack a prominent intromittent organ
(IO) transfer sperm by apposing the cloaca with that of the
female, in a maneuver known as the ‘‘cloacal kiss,’’ which
requires cooperation of the female [10, 11]. By contrast, males
with an IO can manipulate females and even forcibly copulate
with unwilling females, a behavior that is well documented in
waterfowl [10, 13, 24].
While selective pressures for elaborate phallus morphologies are obvious, it is less clear how selection could favor
reduction or loss of an IO. A number of hypotheses have
been proposed for the adaptive significance of genital reduction, although the precise nature of the evolutionary mechanisms is unclear due to the paucity of ecological or experimental data [10, 11, 19, 21, 22]. One of the most influential
hypotheses for genital diversity, the ‘‘lock and key’’ hypothesis, argues that rapid evolution of genitalia facilitated speciation through breeding incompatibility (i.e., only males and
females of the same species can fit their genitalia together)
[7]. Although anatomical barriers to hybridization can in theory
lead to reproductive isolation, experimental studies have failed
to support the idea that strict mechanical incompatibility has
been a driving force in the evolution of genital form [2, 6, 7].
Alternatively, Mayr proposed that genital morphology
undergoes frequent changes due to pleiotropic effects [9]. It
is unclear why neutral changes resulting from pleiotropy would
affect genitalia disproportionately [2], although studies of the
genetic control of genital development have identified a multitude of developmental mechanisms (including cis-acting DNA
regulatory elements) that are shared by other organ systems
[25–28], which raises the possibility that modulating gene
Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Current Biology Vol 23 No 12
2
expression in structures such as limbs could have collateral
effects on the genitalia [29] A third hypothesis argues that
diversification of external genitalia has been driven by sexual
selection acting on variation that affects sperm competition,
sensory features of genital morphology, female choice, and
sexually antagonistic coevolution (i.e., males and females
evolve adaptations and counteradaptations to gain control
over copulation and fertilization) [2, 6, 7, 12, 30].
At a developmental level, little is known about the molecular
genetic mechanisms of external genital evolution. Recent
progress in the developmental biology of mammalian external
genitalia has led to the identification of genetic pathways that
control outgrowth and patterning of the penis [29, 31–42].
These discoveries, together with independent advances in
avian phylogenetics, reproductive ecology, and comparative
morphology [12, 20, 21, 43], create a new opportunity to
investigate the developmental genetic basis of genital evolution in birds. Here we report that the same mechanisms that
regulate mammalian penis development also operate in bird
external genitalia, and that the proliferative cues that direct
genital outgrowth are conserved in chick (a galliform) and
duck (an anseriform) embryos despite striking differences in
their adult genital morphologies. Although early development
of the genital tubercle (the precursor of the phallus) is
conserved in chicks and ducks, we find that chicks have a
unique domain of cell death distally. This domain of cell death
is associated with ectopic activation of the gene encoding
bone morphogenetic protein 4 (Bmp4), which is known to
induce apoptosis in other organs. Experimental inactivation
of Bmp signaling in chick embryos blocks apoptosis and prevents regression of the genital tubercle. Furthermore, ectopic
application of Bmp protein to the distal tip of duck genital
tubercles leads to a chick-like pattern of apoptosis in ducks.
We then extended our study to the most basal group of birds,
the Paleognathae, and to the sister group to the birds, the
Crocodilia, both of which have intromittent phalluses. The
results show that ducks retained the plesiomorphic pattern
of Bmp4 expression and cell death, whereas chickens
evolved a novel domain of Bmp-mediated cell death that
causes regression of the phallus before hatching. Taken
together, our results suggest that reduction of the intromittent
phallus in galliform birds evolved not by disruption of
outgrowth signals but by de novo activation of cell death in
the developing genital tubercle.
Results
Chicks and Ducks Initiate Early Outgrowth of Genital
Tubercles, but Chick Tubercles Arrest and Regress
Reanalysis of avian external genital morphology in light of
recent genomic studies of bird interrelationships suggests
that reduction of the phallus likely occurred in stem galliforms
after their divergence from anseriforms (Figure 1A). Comparison of male genital morphology in two anseriforms, domestic graylag goose (Anser anser) and Pekin duck (Anas
platyrhynchos), and in two galliforms, domestic chicken
(Gallus gallus domesticus) and Old World quail (Coturnix
coturnix), reveals striking differences (Figure 1B). Geese and
ducks have elongated, coiled phalluses with dermal spines,
whereas chickens and quails have only rudimentary phallic
swellings on the anterior margin of the cloacal wall (Figure 1B).
Although highly reduced, chicken and quail phallic swellings
retain proximodistal and dorsoventral polarity and a ventral
sulcus, suggesting that early genital patterning may be
conserved in galliform embryos. To determine when these
morphological differences arise, we used scanning electron
microscopy to investigate genital development in a staged
[44] series of chick and duck embryos. External genital development begins at the same embryonic stage (stage 26) in
chicks and ducks (Figures 1C and 1D). Despite the internal
position of the phallus within the cloaca in adult birds, embryonic development of the genital tubercle occurs externally and
resembles the mouse genital tubercle at early stages (Figures
1C–1H) [32] Development of the phallus begins with the initiation of paired genital swellings between the anterior and posterior cloacal swellings (Figures 1C and 1D). By stage 28, the
paired swellings have merged to form a single genital tubercle
in both chicks and ducks (Figures 1E–1H). Outgrowth of the
tubercle continues until stage 35 in chicks, when development
arrests, but in ducks the phallus continues to grow and extend
from the cloaca (Figures 1I–1L). In chicks at stage 45, the rudimentary phallus is visible only as a bulge on the inferior margin
of the cloacal collar (Figure 1M). Ducks at the same stage have
a pronounced phallus that begins to form the characteristic
coiled pattern (Figure 1N). Thus, initiation and early outgrowth
of the genital tubercle occurs in both chicks and ducks, but in
chickens, outgrowth arrests and the tubercle then regresses
within the cloaca.
Mechanisms of Genital Tubercle Outgrowth Are Retained
in Chicks
The precocious arrest of chick phallus development
suggested that the molecular mechanisms that regulate
genital tubercle outgrowth are disrupted. In mice, sustained
outgrowth of the genital tubercle requires Sonic hedgehog
(Shh) and Hoxa13/d13 [28, 32–34, 38, 41, 45]. Given that loss
of Shh or Hoxa13 and Hoxd13 function in mouse embryos
results in arrest of genital tubercle outgrowth, which mimics
the situation in chicks, we tested whether these pathways
are disrupted in chick genital tubercles. Unexpectedly, strong
expression patterns of Shh, Hoxa13, and Hoxd13 are well
conserved in chick and duck genitalia, despite the failure of
chick genital development to progress beyond the early bud
stage (Figure 2). Shh is expressed along the ventral sulcus in
chick and duck at stage 29 and persists through stage 35
(Figures 2A–2D). To determine whether mesenchymal cells
respond to Shh, we examined Ptch1 and Ptch2, transcriptional
targets of Hedgehog [47]. Both genes show strong expression
in mesenchyme on either side of the sulcus as late as stage 35
(Figures 2E–2L), indicating activation of the Hedgehog
pathway. Hoxd13 and Hoxa13 are expressed throughout the
genital tubercles of both chick and duck embryos at stage 29
(Figures 2M and 2N, 2Q, and 2R), and expression weakens in
both species by stage 35 (Figures 2O and 2P, 2S, and 2T),
Thus, despite the extreme reduction of the galliform phallus,
the mechanisms known to direct early outgrowth of the genital
tubercle have been retained in chicks.
The similar expression patterns of outgrowth-promoting
genes in chick and duck genitalia led us to investigate whether
the precocious arrest of genital development in chicks
involves a failure of genital tubercle cells to respond to proliferative cues. Quantitative analysis of cell proliferation in chick
and duck genital tubercle mesenchyme revealed no significant
differences (p = 0.6) in their mitotic indices, despite the
obvious differences in outgrowth at stage 35 (see Figure S1
available online). These results indicate that chick genital
mesenchyme exhibits a normal response to the growthpromoting genes described above.
Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Evolutionary Reduction of the Penis in Birds
3
Figure 1. Phylogenetic, Adult, and Embryological Comparison of External Genitalia in Birds
(A) Phylogenetic distribution of intromittent phalluses in birds. + indicates present; 2 indicates absent or reduced in the majority of species in the clade.
*Most tinamous have an intromittent phallus, but an exception has been reported for one species [21]. Phylogeny is from [20]. Branch colors are as follows:
purple, Paleognathae; green, Anseriformes; cyan, Galliformes; orange, Neoaves; black, Crocodilia (root).
(B) Anatomical preparations of adult male external genitalia in two anseriforms (goose and duck) and two galliforms (chicken and quail). Arrows point to the
reduced phalluses in galliform species. Scale bar represents 1 cm.
(C–N) Scanning electron microscopy of chick and duck external genital development. Anterior is to the left in (C)–(F) and to the top in (G)–(N). Chick and duck
embryos initiate formation of paired genital swellings (C and D), which form a single tubercle during early outgrowth (E and H). Outgrowth arrests and the
genital tubercle regresses in chick embryos (I, K, and M), but genital development continues in duck embryos (J, L, and N). Hamburger-Hamilton (HH) stage
numbers are shown above. Abbreviations: gs, genital swellings; gt, genital tubercle; acs, anterior cloacal swelling; pcs, posterior cloacal swelling.
Increased Apoptosis Is a Synapomorphic Character
of Galliform Genital Tubercles
Appendage outgrowth requires a balance between cell proliferation and cell death, and truncation can result from either
decreased proliferation or increased cell death [33]. To determine whether increased apoptosis could underlie the arrest of
chick genital development, we compared patterns of cell
death in the genital tubercles of galliform, anseriform, paleognath, and crocodilian embryos. LysoTracker Red staining
shows that chicks and ducks have similar patterns of
apoptosis at stage 29, with labeled cells along the sulcus in
both species (Figure S2), in a pattern similar to that reported
for E11.5 mice [32]. Comparison at stage 36, however,
revealed marked differences: chick genital tubercles showed
widespread apoptosis throughout the distal mesenchyme
and along the sulcus, whereas duck genitalia had few labeled
cells in the sulcus or distal region of the tubercle (Figures 3A
and 3C). To determine whether the differences in apoptosis
between chick and duck genital tubercles are conserved features of Galliformes and Anseriformes, respectively, we examined additional members of each clade. Quail genital tubercles
at stage 29 showed an apoptotic pattern similar to that seen in
chick, whereas goose genitalia at this stage showed an
apoptotic pattern resembling that of duck (Figure S2). By
stage 36, quail genital tubercles showed extensive apoptosis
throughout the distal mesenchyme and sulcus, whereas goose
genital tubercles exhibited very low levels only along the
sulcus (Figures 3B and 3D). These results indicate that the
galliforms examined here (chicken and quail) undergo a late
wave of apoptosis in the distal genital tubercle, which coincides temporally with the arrest of outgrowth and onset of
regression.
If the evolutionary reduction of galliform external genitalia
was driven by increased apoptosis, then the pattern of cell
death observed in chick and quail should be a synapomorphic
(shared derived) character, whereas the duck and goose
pattern should be plesiomorphic (ancestral). We tested this
hypothesis by extending our study to the most basal clade
of birds, the Palaeognathae, and to the sister group to birds,
the Crocodilia. Analysis of the emu, a paleognath, showed
that there is low-level cell death along the sulcus at stage 36,
as seen in ducks and geese (Figure 3E). In comparably staged
alligator embryos, the genital tubercles show even fewer
apoptotic cells than in emus, ducks, and geese (Figure 3F).
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(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Current Biology Vol 23 No 12
4
Figure 2. Comparative Analysis of Gene Expression in Developing External
Genitalia of Chick and Duck Embryos
Whole-mount RNA in situ hybridizations of chick and duck genital tubercles
at stages 29 and 35 showing expression patterns of genes that promote
mouse external genital development [28, 29, 32, 34, 45, 46]. Genital tubercles are shown in ventral view with distal toward the top. Gene names are
shown at top; species and Hamburger-Hamilton (HH) stage are shown at
left.
Thus, the wave of cell death in the distal genital tubercle
appears to be a synapomorphy of galliforms.
Chicken Genital Tubercles Have a Derived Pattern of Bmp4
Expression
The finding that regression of galliform genital tubercles is
associated with apoptosis, rather than loss of an outgrowth
signal, raised new questions about the molecular mechanism
responsible for the distal cell death. Bone morphogenetic proteins (Bmps) have been shown to induce apoptosis in a number of developmental contexts, including the external genitalia
of mice [31, 48–50]. To determine whether endogenous differences in Bmp activity could underlie the differences in
apoptosis between galliform and anseriform genitalia, we
compared expression of Bmp2, Bmp4, and Bmp7 (ligands
for BmpR1a, which controls apoptosis of the mouse genital
tubercle [31]) in chick and duck embryos. Chicks and ducks
showed different patterns of Bmp4 and Bmp7 expression at
stage 29 (Figures 3G–3P), before differences in phallus development can be detected (Figures 1I and 1J). In chicks, strong
expression of Bmp4 was detected along the entire proximodistal axis of the genital tubercle (Figure 3G), whereas in
duck genitalia, Bmp4 was expressed in the cloaca and at the
base of the genital tubercle, but not at the distal tip (Figure 3H).
Bmp7 also showed strong expression along the chick genital
tubercle at stage 29, particularly at the distal tip, but comparatively little expression was seen in duck genitalia (Figures 3I
and 3J).
At stage 35, Bmp4 continued to be expressed distally in
chicks only (Figures 3G0 and 3H0 ). By this stage, Bmp7 and
Bmp2 expression had diminished in both chick and ducks
(Figures 3I0 –3L0 ). To quantify levels of Bmp4 expression in
chick and duck genital tubercles, we used quantitative realtime PCR (qRT-PCR) and found that chick genitalia have
significantly higher levels of Bmp4 mRNA at stage 29
(>3-fold higher) and stage 35 (>2-fold higher; Figure 3Q). The
sustained expression of Bmp4 in chick genitalia led us to ask
whether Msx1 and Msx2, downstream effectors of Bmp
signaling [48], show differential activity in chick and duck
genital tubercles. At stages 29 and 35, Msx1 and Msx2 were
expressed distally in chick genitalia (Figures 3M, 3M0 , 3O,
and 3O0 ), where the region of apoptosis occurs (Figure 3A),
but neither gene could be detected in duck genitalia (Figures
3N, 3N0 , 3P, and 3P0 ). These findings show that distal activation
of the Bmp4 pathway coincides spatially and temporally with
the wave of cell death that occurs in galliform external
genitalia.
The results described above raised the possibility that galliforms evolved a new domain of Bmp4 (and apoptosis) in their
distal genital tubercles, but comparison of duck and chick
genital development could not exclude the possibility that
distal Bmp4 expression is a primitive character that was lost
in anseriforms. Therefore, we examined Bmp4 expression in
the emu, a basal bird with a well-developed phallus that
does not undergo widespread distal apoptosis (Figure 3E). In
emu embryos at stage 29, Bmp4 showed a duck-like pattern
of expression: Bmp4 expression was detected in the cloacal
collar and interdigitally in the hindlimbs, but not at the distal
tip of the genital tubercle (Figures 3R and 3S). Taken together
with our phylogenetic survey of apoptosis across archosaur
(avian + crocodilian) external genitalia (Figures 3A–3F), these
results indicate that Bmp4 expression and apoptosis in the
distal genital tubercle evolved in the galliform lineage after
its divergence from the anseriforms.
Inhibition of Bmp Signaling Rescues Apoptosis and
Prevents Regression of Chick Genital Tubercles
Having established the relative timing of the evolutionary
acquisition of distal Bmp4 expression, we went on to perform
functional experiments to test the hypothesis that cell death in
galliform external genitalia is caused by Bmp signaling. If this
hypothesis is correct, then antagonism of Bmp activity should
rescue cells from apoptosis. We applied beads soaked in
Noggin protein (0.275 mg/ml), which binds Bmps and blocks
their interaction with Bmp receptors [51], to one side of chick
genital tubercles at stage 26. Noggin beads resulted in local
inhibition of apoptosis within 24 hr, whereas the contralateral
side showed no change in apoptosis (Figure 4A). Chick tubercles receiving control beads (soaked in PBS) showed no
change in apoptosis on either side (Figure 4B). To confirm
the specificity of Noggin treatments, we monitored expression
of the Bmp target gene Msx1. On the Noggin-treated side of
the genital tubercle, Msx1 was downregulated within 24 hr
(the contralateral side, by contrast, continued to show strong
expression of Msx1), indicating that Bmp signaling had been
inactivated (Figure 4C). PBS control beads did not affect
Msx1 expression (Figure 4D).
Interestingly, the distal tip of the genital tubercle appeared
to extend further on the Noggin-treated side than on the
contralateral side (Figure 4C), suggesting that increased cell
survival resulted in sustained outgrowth. We therefore quantified proximodistal outgrowth of the bead side versus the
contralateral side 24 hr after implantation of either a Noggin
bead or PBS control bead. The difference between the bead
side and contralateral side was w6.5 times greater in
Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
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Evolutionary Reduction of the Penis in Birds
5
Figure 3. Apoptosis and Bmp Signaling Are Increased in Galliform Genital Tubercles
(A–F) LysoTracker Red staining shows widespread apoptosis in galliform (chick and quail) genital tubercles, whereas anseriform (duck and goose), emu, and
alligator genitalia at comparable stages show limited apoptosis mostly along the sulcus. Phylogenetic relationships [20] are shown at top.
(G–P) Bmp4, Bmp7, Bmp2, Msx1, and Msx2 expression in chicks and ducks at stage 29 (G–P) and 35 (G0 –P0 ). Bmp4 (G and H), Bmp7 (I and J), Msx1 (M and N),
and Msx2 (O and P) show distal expression in chick but not duck genital tubercles at stage 29 (G–J). Distal expression domains of Bmp4 (G0 and H0 ), Msx1
(M0 and N0 ), and Msx2 (O0 and P0 ) persist in chick genital tubercles through stage 35. Bmp2 shows no significant difference at either stage (K, L, K0 , and L0 ).
(Q) qRT-PCR shows that Bmp4 expression is significantly higher in genital tubercles (GTs) of chicks relative to ducks at stage 29 (p = 0.002) and stage 35
(p = 0.0003). Error bars show standard error of the mean.
(R) In the emu genital tubercle, Bmp4 expression is absent from the distal tip. Expression is limited to the base of the tubercle and the cloacal collar, as in
ducks (compare with H).
(S) Internal control of emu hindlimb showing interdigital expression of Bmp4.
Noggin-treated tubercles (100.8 mm) than in control tubercles
(15.6 mm; p = 0.01; Figure 4E). Thus, antagonism of Bmp
signaling in the chick genital tubercle resulted in significantly
increased outgrowth. These results indicate that Bmp function
is required for apoptosis and the arrest of outgrowth in chick
genital tubercles.
The results described above led us to ask whether, during
the evolution of Galloanserae, activation of Bmp signaling
could have been sufficient to induce apoptosis in a genital
tubercle fated to form an intromittent phallus. We tested this
hypothesis by applying beads loaded with Bmp2 protein
(a paralog of Bmp4 that also signals through BmpR1a [52]) to
the genital tubercles of duck embryos at stage 26, the stage
when Bmp is required for apoptosis in chicks. Eight hours after
application of a Bmp2 bead, duck genital tubercles showed
ectopic apoptosis on the treated side of the tubercle, whereas
application of control beads had no effect on cell death (Figure S3). Taken together, these results show that distal Bmp
activity is both necessary and sufficient for apoptosis and
regression of the phallus in Galloanserae. The findings presented here suggest that a novel domain of Bmp4 arose in
the distal genital tubercle during the evolution of galliforms,
and this led to apoptosis-meditated regression of the phallus
(Figure 4F).
Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Current Biology Vol 23 No 12
6
Figure 4. Bmp Signaling Causes Apoptosis and Inhibition of Outgrowth in Chick Genitalia
(A–D) Beads were loaded with either Noggin protein (A and C) or PBS (B and D) and implanted into the left side (right in photos) of stage 29 chick genital
tubercles.
(A) Noggin bead (arrow) decreases apoptosis (red cells are LysoTracker positive) on the treated side of tubercles. Contralateral side shows widespread
apoptosis.
(B) PBS bead has no effect on either side.
(C) Msx1 is downregulated on the side of the tubercle with Noggin bead (circle), but expression is maintained on the contralateral side. Note that the Noggintreated side shows more outgrowth.
(D) Msx1 is expressed in a normal pattern on both sides of the tubercle receiving PBS bead (circle).
(E) Comparison of outgrowth in treated versus contralateral sides in Noggin-treated (green) and PBS control-treated (red) genital tubercles 24 hr after bead
implantation. Noggin beads induced a mean increase of 100.8 mm over the contralateral side, which is significantly greater than the difference between PBS
control and contralateral sides (15.8 mm; p = 0.01).
(F) Developmental model for evolutionary loss of the intromittent phallus in galliforms. Black circles indicate presence of an intromittent phallus; open circle
with diagonal line indicates loss of intromittent phallus; asterisk indicates all galliforms with the exception of cracids, which have a fully intromittent phallus.
Discussion
During the evolution of birds, an intromittent phallus was
reduced or lost in multiple lineages, including at least once
in galliforms and again in the common ancestor of neoaves
[19, 21, 22]. Consequently, w97% of extant bird species lack
an intromittent phallus [10, 11, 22]. Our comparative study of
external genital development in birds shows conservation of
the signals that promote initiation and early outgrowth of the
genital tubercle across galliform, anseriform, and paleognath
lineages, but our data from chick embryos suggest that galliforms are unique in their expression of Bmp4 in the distal genital tubercle. Experimental manipulations of Bmp signaling in
chick and duck embryos show that this derived domain of
Bmp activity is necessary and sufficient for the wave of distal
apoptosis that causes regression of the genital tubercle in
galliforms. The results presented above suggest that a new
region of Bmp4 expression arose in the galliform lineage after
its divergence from anseriforms, and that this resulted in a
domain of apoptosis that arrests genital outgrowth and causes
regression of the phallus.
Evolution of Phallus Reduction
The marked divergence of genital morphology between
anseriforms and galliforms raises questions about the selective pressures that led to phallic reduction. Loss of intromittent
organs is an evolutionary paradox: How can reduction of a
structure that facilitates internal fertilization have a positive
effect on reproductive fitness? It is unlikely that reduction of
the phallus resulted in improved delivery of sperm. Darwin first
proposed that variable characters with no apparent adaptive
value were not subject to natural selection, but that these traits
Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Evolutionary Reduction of the Penis in Birds
7
could be stabilized in a population if they confer an advantage
‘‘over other individuals of the same sex and species, in exclusive relation to reproduction’’ [53]. Copulation with males lacking an intromittent phallus requires female cooperation, such
as presentation of the cloaca and eversion of the vagina [10,
11]. Accordingly, by selecting males with reduced or nonintromittent phalluses, females can control paternity, which is
consistent with the idea that female choice played a role in
reduction and loss of the phallus [10, 11, 14, 19, 22, 43].
An alternative to the sexual selection hypothesis is that
reduction of phallus size may have been a secondary or pleiotropic effect of developmental changes to other characters [9].
The molecular mechanisms of appendage development have
been deeply conserved during animal evolution, and the
gene networks that regulate development of external genitalia
also control formation of a wide range of other structures,
including limbs, gut, nervous system, muscle, and integumentary appendages such as feathers and scales. Moreover, tissue-specific expression of a number of genes expressed in
the genital tubercle, including Bmp4, Msx2, and Hoxd13, has
been shown to be controlled by enhancers that also direct
transcription in other regions of the embryo. In mouse, an
evolutionarily conserved enhancer located more than 45 kb
50 to the Bmp4 promoter can drive expression in the genital
tubercle, digit tips, dorsal root ganglia, and whisker hairs
[25]. Similarly, Msx2 expression in the genital tubercle ectoderm and in the apical ectodermal ridge of the limb buds is
regulated by a shared cis-regulatory element [27], as is
Hoxd13 expression in the digits and the genital tubercle [54].
In the latter example, the length of the digits and the penis
scale together in response to changes in the quantity of
Hoxd13 expression [28]. Therefore, modulation of gene regulation in other organ systems has the potential to have pleiotropic effects on gene expression in the genital tubercle (and
vice versa). However, even if reduction of the intromittent
phallus arose due to pleiotropic effects of Bmp4 regulatory
evolution in another cell population and therefore was not
the primary target of selection, persistence of a reduced
phallus in galliforms would have required stabilizing selection.
Thus, we propose that regression of the intromittent phallus
in galliforms arose due to acquisition of a novel domain of
Bmp4 expression in the distal region of the genital tubercle,
and that stabilization of this domain may have been due to
selection for males with small or nonintromittent phalluses.
A Developmental Mechanism for Reduction of the Phallus
in Galliform Birds
Outgrowth of appendages generally requires induction and
maintenance of cell proliferation as well as attenuation of
apoptosis in order to keep cell death below the rate of cell
proliferation. Regionalized regulation of cell death plays an
important role in morphological sculpting of appendages,
influencing processes and traits such as digit differentiation,
joint cavitation, removal of interdigital webbing, urethra formation, and limb and genital length. Activation of apoptosis by
Bmp signaling has been implicated in a number of evolutionary
reductions, such as loss of teeth in birds [55] and reduction of
forewings in soldier ants [56]. Reciprocally, inhibition of the
apoptotic activity of Bmps through upregulation of Bmp
antagonists (e.g., Gremlin) and/or survival cues (e.g., Fgf8)
has been implicated in other evolutionary innovations, such
as retention of interdigital webbing in bat wings. Our analysis
of signals known to promote outgrowth of the mouse genital
tubercle, including Shh and Hoxd13/a13, revealed surprising
conservation between galliforms and anseriforms, suggesting
that galliforms have not lost the mechanisms for external genital development. Indeed, quantification of cell division in chick
and duck genital tubercles showed that there is no significant
difference in cell proliferation. This is consistent with our previous report that, in the mouse, Shh promotes outgrowth of
the genital tubercle by regulating cell-cycle kinetics [33]. By
contrast, galliforms have a derived pattern of apoptosis at
the distal tip of the genital tubercle, and this coincides with a
domain of Bmp4 expression not detected in anseriforms or
paleognaths. These results indicate that the distal expression
of Bmp4 and the resulting increase in apoptosis in the distal
region of the genital tubercle are a synapomorphic characteristic of galliforms.
The ability of the Bmp antagonist Noggin to prevent cell
death and to sustain outgrowth of galliform genital tubercles
provides a causal link between the distal domain of Bmp
signaling and apoptotic regression of the phallus. Furthermore,
our finding that a distal wave of apoptosis can be induced by
experimental activation of Bmp signaling at the distal tip of
duck genital tubercles demonstrates that Bmp activity alone
is sufficient to induce cell death and regression of a genital
tubercle that otherwise is fated to form an elongated phallus.
Thus, Bmp signaling in the distal genital tubercle of Galloanserae is both necessary and sufficient to trigger regression of
the phallus during embryonic development. These findings
are consistent with studies of Bmp function in mouse external
genital development. Deletion of Bmpr1A results in reduced
cell death in the mouse genital tubercle, which leads to hyperplasia of the penis [31]. Moreover, Noggin knockout mice have
excessive Bmp signaling, which causes reduction of the penis
[31]. Taken together, our findings support the hypothesis that
evolutionary reduction of an intromittent phallus in galliform
birds resulted from the acquisition of a new distal domain of
Bmp activity in the developing genital tubercle.
Interestingly, cracids are an exception to the galliform rule in
that they have a fully intromittent phallus (although not as pronounced as in anseriforms [21]). In light of current phylogeny
[20], cracids may have re-evolved an intromittent phallus
(Figure 1A), which would suggest that galliforms have retained
the competence to redevelop a phallus from a rudimentary
genital organ. Our findings that (1) the molecular machinery
for genital tubercle outgrowth has not been disrupted in
chicks, (2) regression of the phallus is caused by Bmpmediated apoptosis, and (3) antagonism of Bmp signaling is
sufficient to block apoptosis and rescue outgrowth raise the
possibility that modulating Bmp activity may have been
sufficient for reemergence of an intromittent phallus.
Whether the same developmental mechanism that we
identified in galliforms also played a role in the independent
reductions of the phallus in neoaves remains to be tested.
Comparative studies of birds have the potential to identify
the developmental mechanisms responsible for a number of
other major morphological transitions, such as how genital
outgrowth is sustained for such an extensive period in waterfowl, how coiling of the penis and vagina is regulated, what
determines the direction of the coiling, and how the microanatomy of penile spines and other dermal ornamentation is
specified in birds.
Bmp Regulation and the Evolution of Bird Morphology
Modulation of Bmp expression has been implicated in evolution of at least four morphological innovations in birds:
feathers [57], toothlessness [55], beak shape [58, 59], and,
Please cite this article in press as: Herrera et al., Developmental Basis of Phallus Reduction during Bird Evolution, Current Biology
(2013), http://dx.doi.org/10.1016/j.cub.2013.04.062
Current Biology Vol 23 No 12
8
now, phallic reduction. It is interesting that the phallus is one of
several avian structures, including teeth and the most anterior
and posterior digits, that undergo partial development during
embryogenesis and then regress. It is possible that this also
reflects gene regulatory elements that are shared among multiple organ systems. If transcription of a gene in a developing
organ is regulated by an enhancer that also regulates gene
expression in the progenitors of an organ that has been lost
during evolution, then that early phase of expression (and
perhaps an early phase of development) could be maintained
in both cell populations. Such conservation of gene regulatory
mechanisms may underlie the occasional reemergence of
structures that have been lost during evolution.
Experimental Procedures
Embryo Collection
Fertilized chicken, quail, duck, goose, and emu eggs were obtained from
commercial suppliers, and alligator eggs were obtained from the Rockefeller National Wildlife Refuge. Chicken, quail, goose, and duck eggs were
incubated in a humidified incubator at 38 C, and emu eggs were incubated
at 36.4 C. Stages of development were determined using the HamburgerHamilton staging series for comparison of multiple anatomical landmarks
[44]. Alligator eggs were incubated at 33 C to induce male development.
Scanning Electron Microscopy
Genital tubercles were dissected in PBS, fixed in 1% glutaraldehyde in PBS,
and stored at 4 C. After postfixation in 1% osmium tetroxide, specimens
were dehydrated through a graded ethanol series and placed in acetone.
Specimens were then critical-point dried, mounted on metal studs, sputter
coated with gold particles, and imaged on a Hitachi S4000 scanning electron microscope.
RNA Isolation, cDNA Synthesis, and RT-PCR
Genital tubercles were dissected from staged embryos and stored in
RNAlater. Tissue samples were collected from each embryo for genomic
DNA extraction and genotyping using sex-specific primers [60]. Male
tubercles were pooled and RNA was extracted using an RNeasy Mini Kit
(QIAGEN). RNA quantity and purity were determined using a NanoDrop
ND-1000. For each pooled sample, cDNA was synthesized from 2 mg of total
RNA according to manufacturer’s specifications (Bio-Rad). Total RT-PCR
reactions (10 ml) were performed using SYBR Green qPCR Master
Mix (Bio-Rad). The primers used for RT-PCR were as follows: Bmp4
(F 50 -AACTCCACCAACCACGCCATC-30 , R 50 -AGCACCACCTTGTCATACT
CAT-30 ), chick b-actin (F 50 -TGATGGACTCTGGTGATGGTGTTAC-30 , R 50 CTCTCGGCTGTGGTGGTGAAG-30 ), duck b-actin (F 50 -GCCTCCCTGTC
CACCTTCC-30 , R 50 -GCTGCTGATACCTTCACCATTCC-30 ). Relative level
of expression was calculated using the DDCt method.
In Situ Hybridization
Embryos harvested for in situ hybridization were fixed in 4% paraformaldehyde (PFA) at 4 C overnight and dehydrated in a graded methanol series.
Whole-mount RNA in situ hybridization was performed as described by
Nieto et al. [61] with the following modifications: Proteinase K concentration
was increased to 70 mg/ml, following the protocol of Laufer et al. [62]; KTBT
buffer contained 1% Triton X-100; alkaline phosphatase buffer (NTMT)
contained 0.1% Tween-20; and dimethylformamide (10%) was added to
NBT-BCIP staining solution.
Detection of Apoptotic Cells
Apoptotic cells were localized using LysoTracker Red (Molecular Probes/
Invitrogen). Embryos were rinsed briefly in PBS and incubated in
LysoTracker Red (1:200) in PBS at 37 C for 30 min and then washed briefly
in PBS before fixation in 4% PFA. Samples were rinsed in methanol to
remove background, viewed and photographed under epifluorescence,
and then photographed again in the same plane of focus with bright-field
illumination.
Bead Implantation
Heparin acrylic beads (Sigma) were size selected (125–250 mm) and soaked
in either Xenopus Noggin protein (a gift from Richard Harland, University of
California, Berkeley) at a concentration of 0.275 mg/ml, Bmp2 protein at a
concentration of 1 mg/ml (a gift from Vicki Rosen, Harvard School of Dental
Medicine), or PBS (control) for a minimum of 1 hr. Beads were implanted
under the surface ectoderm on one side of the distal tubercle via an incision
made with a tungsten needle. Duck genitalia were manipulated in ovo and
collected for LysoTracker staining after 8 hr. Chick genital tubercles were
explanted to organ culture in Dulbecco’s modified Eagle’s medium with
L-glutamine (Invitrogen) with 10% fetal calf serum and 13 Antibiotic-Antimycotic (Gibco/Invitrogen) for 24 hr and then fixed in 4% PFA at 4 C for
further analysis. Proximodistal outgrowth of the genital tubercle was
measured using NIH ImageJ.
Analysis of Cell Proliferation
Chick and duck embryos were incubated to stage 35. Embryos (n = 7 for
each species) were then harvested, fixed in 4% PFA, and processed for histology. Coronal sections (16 mm) were stained with mouse anti-PCNA antibody (Abcam) at a concentration of 1:1,000 and developed using an ABC
Kit Elite (Vector Labs) according to the manufacturer’s instructions. Cell
counts were made by positioning a counting box (6,670 3 8,940 mm) at
the distal tip of the genital tubercle, immediately under the distal ectoderm.
Three sections were counted for each embryo, one at the midpoint of the
tubercle (the longest proximal-to-distal section) and one on either side
(anterior and posterior) of the midpoint. The three sections were used to
calculate a mean for each embryo. Significance was determined by ANOVA.
Supplemental Information
Supplemental Information includes three figures and can be found with this
article online at http://dx.doi.org/10.1016/j.cub.2013.04.062.
Acknowledgments
We thank Rebecca Kimball, Ehab Abouheif, Brian Harfe, and members of
our laboratory for discussions; Patricia Brennan and two other anonymous
reviewers for critical comments on the manuscript; Richard Harland and
Vicki Rosen for generous gifts of Noggin and Bmp2 protein, respectively;
Patsy Fitch-Hand, Ruth Elsey, and Mark and Cheryl Wagner for eggs and
cadavers; and Krista McCoy for assistance with quantitative analysis. This
research was supported by a grant from the National Science Foundation
(IOS-0843590).
Received: March 13, 2013
Revised: April 18, 2013
Accepted: April 23, 2013
Published: June 6, 2013
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