Download Adaptive Evolution of 5#HoxD Genes in the

Adaptive Evolution of 5#HoxD Genes in the Origin and Diversification of the
Cetacean Flipper
Zhe Wang,* Lihong Yuan, Stephen J. Rossiter,à Xueguo Zuo,* Binghua Ru,* Hui Zhong,*
Naijian Han,§ Gareth Jones,k Paul D. Jepson,{ and Shuyi Zhang*
*School of Life Science, East China Normal University, Shanghai, China; Guangdong Entomological Institute, Guangzhou, China;
àSchool of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom; §Institute of Zoology,
Chinese Academy of Sciences, Beijing, China; kSchool of Biological Sciences, University of Bristol, Bristol, United Kingdom; and
{Institute of Zoology, Zoological Society of London, Regent’s Park, London, United Kingdom
The homeobox (Hox) genes Hoxd12 and Hoxd13 control digit patterning and limb formation in tetrapods. Both show
strong expression in the limb bud during embryonic development, are highly conserved across vertebrates, and show
mutations that are associated with carpal, metacarpal, and phalangeal deformities. The most dramatic evolutionary
reorganization of the mammalian limb has occurred in cetaceans (whales, dolphins, and porpoises), in which the hind
limbs have been lost and the forelimbs have evolved into paddle-shaped flippers. We reconstructed the phylogeny of
digit patterning in mammals and inferred that digit number has changed twice in the evolution of the cetacean forelimb.
First, the divergence of the early cetaceans from their even-toed relatives coincided with the reacquisition of the
pentadactyl forelimb, whereas the ancestors of tetradactyl baleen whales (Mysticeti) later lost a digit again. To test
whether the evolution of the cetacean forelimb is associated with positive selection or relaxation of Hoxd12 and Hoxd13,
we sequenced these genes in a wide range of mammals. In Hoxd12, we found evidence of Darwinian selection associated
with both episodes of cetacean forelimb reorganization. In Hoxd13, we found a novel expansion of a polyalanine tract in
cetaceans compared with other mammals (17/18 residues vs. 14/15 residues, respectively), lengthening of which has
previously been shown to be linked to synpolydactyly in humans and mice. Both genes also show much greater sequence
variation among cetaceans than across other mammalian lineages. Our results strongly implicate 5#HoxD genes in the
modulation of digit number, web forming, and the high morphological diversity of the cetacean manus.
Introduction
Modifications in the autopod and in the number of digits have occurred recurrently during tetrapod evolution and
are generally viewed as adaptive locomotory responses to
changes in lifestyle and habitat use (Lull 1904; Tabin 1992;
Shubin and Marshall 2000). In mammals, the ancestral amniote manus is typically characterized by five separate digits
(pentadactyly), with each digit consisting of carpals, metacarpals, and phalanges running from proximal to distal
axes. In general, each digit ray consists of one metacarpal
and either two or three phalanges.
Although deviations from this pentadactyl state are
seen in several groups (Lull 1904; Golley and Whitmoyer
1959), major evolutionary reorganization of the manus is
restricted to two clades, Cetartiodactyla (Artiodactyla þ
Cetacea) and Perissodactyla. The ungulate manus shows
several cursorial adaptations for increased speed in a terrestrial environment. In modern perissodactyls, digit loss has
occurred in horses, which are functionally monodactyl
(digit III) with vestigial digits II and IV, and also in tapirs
and rhinoceroses, which possess four and three digits, respectively. Similar reduction in digit size and number
has occurred during the evolution of artiodactyls. Specifically, the reduced first metacarpal of the pentadactyl basal
taxon Diacodexis (Rose 1982) is absent in modern lineages,
which also show a reduction or loss of digits II and V.
Perhaps the most dramatic evolutionary reorganization
of the autopod has occurred in the cetaceans (whales, dolphins, and porpoises), the common ancestor of which diverged from an extinct group of deer-like artiodactyls
Key words: Hoxd12, Hoxd13, adaptive evolution, cetaceans, digits,
flipper.
E-mail: [email protected]; [email protected].
Mol. Biol. Evol. 26(3):613–622. 2009
doi:10.1093/molbev/msn282
Advance Access publication December 10, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
approximately 50 Ma (Thewissen et al. 2007). Changes
in limb morphology associated with the return to water followed three main routes. First, cetacean digits became embedded in skin tissue (an interdigital web), a state also seen
in other mammals (e.g., bats and seals). Second, cetaceans
lost most of their lower limb bones, and some also lost digits in their upper limbs, which evolved into paddle-shaped
flippers (Berta et al. 2006; Thewissen et al. 2006). Specifically, odontocetes (toothed whales) and some mysticetes
(baleen whales) possess a pentadactyl manus, whereas other
mysticetes have lost digit I (Berta et al. 2006; Cooper et al.
2007). Third, cetaceans show considerable variation in their
number of phalanges, with digits I and V typically characterized by few phalanges (ranging from zero to five) and
digits II–IV by as many as 13 in the second digital
ray of some species. Indeed, the carpals of many
cetaceans show clear species-specific polymorphisms,
and phalangeal count can even vary among conspecifics
(Cooper et al. 2007).
In recent years, considerable progress has been made
in our understanding of the molecular basis of digit pattern
control. Many genes expressed in the embryonic limb bud
have been shown to play an important role in autopod formation (Zakany et al. 2004; Tickle 2006). The Hox genes
Hoxd12 (Homeobox D12) and Hoxd13 (Homeobox D13) of
the 5#HoxD cluster both show strong expression in posterior cells of limb buds (Papageorgiou 2007), and comparative studies suggest that their expression (Davis et al.
2007; Freitas et al. 2007), coding sequences, and even noncoding elements (Lee et al. 2006; Lemons and McGinnis
2006) are highly conserved across vertebrates. Much
knowledge has been gained by genetic manipulation or
by screening deformed individuals. For example, Hoxd12
defects in mice are associated with deformities in carpals,
metacarpals, and phalanges (Knezevic et al. 1997), whereas
Hoxd13-defective humans exhibit autopod malformations
614 Wang et al.
characterized by extra digits (polydactyly), short digits
(brachydactyly), and fused digits (syndactyly) (Muragaki
et al. 1996; Zhao et al. 2007). Several studies have also
linked the autosomal-dominant condition of synpolydactyly (polydactyly with syndactyly), in which digits are
webbed and duplicated, to a polyalanine repeat expansion
at the 5#-end of the Hoxd13 protein (Johnson et al. 1998).
Moreover, Hoxd12 and Hoxd13, together with other limbpatterning genes, have been implicated in more severe
forelimb defects (Davis and Capecchi 1996). The Hoxd12
protein directly amplifies the posterior Shh polarizing signal
in a reinforcing positive feedback loop and interacts with
Gli3, both of which also regulate digit number and identity
(Knezevic et al. 1997; Litingtung et al. 2002). More recently, Hoxd12 and Hoxd13 have also been proposed to
modulate digit number (Sheth et al. 2007; Zakany and
Duboule 2007).
Here we use a molecular evolution approach to test
whether key evolutionary transitions in the limbs of mammals have involved the Hox genes Hoxd12 and Hoxd13.
We conduct a wide survey of mammals, including representatives from Cetartiodactyla and Perissodactyla, and apply
codon substitution models to identify lineages and sites under Darwinian selection. Additionally, we examine the
number of repeat units in a polyalanine stretch in Hoxd13
that is implicated in major limb defects in humans and mice.
Materials and Methods
Taxonomic Coverage
Tissue samples held at the School of Life Science, East
China Normal University, Shanghai, China, and the Institute of Zoology, London, United Kingdom, were obtained
for 22 mammal species. These comprised 10 cetaceans (Balaenoptera acutorostrata, Balaenoptera physalus, Megaptera novaeangliae, Physeter macrocephalus, Kogia
breviceps, Ziphius cavirostris, Mesoplodon densirostris,
Phocoena phocoena, Lagenorhynchus albirostris, and
Stenella coeruleoalba), four bats (Rousettus leschenaulti,
Hipposideros armiger, Megaderma lyra, and Taphozous
melanopogon), two artiodactyls (Sus scrofa and Capra hircus), one perissodactyl (Equus caballus), two carnivores
(Felis catus and Arctonyx collaris), one eulipotyphlan (Erinaceus europaeus), one rodent (Petaurista alborufus), and
one lagomorph (Oryctolagus cuniculus).
From GenBank, we obtained Hoxd12 sequences for
three primates(Homo sapiens, NM_021193; Pan troglodytes,
XM_515926; and Papio hamadryas, AC116665), two rodents (Rattus norvegicus, XM_345373; Mus musculus,
NM_008274), one bat (Carollia perspicillata, AY744676),
one carnivore (Canis familiaris, XM_545535), one artiodactyl (Bos taurus, XM_598664), and one perissodactyl (E.
caballus, XM_001500102). Similarly, we obtained Hoxd13
sequences for three primates (H. sapiens, NC_000002; P.
troglodytes, XM_525967; and P. hamadryas, AC116665),
two rodents (R. norvegicus, NM_001105886; M. musculus,
NM_008275), one bat (C. perspicillata, AY744676), and
one carnivore (C. familiaris, AY308818). We also obtained
Ensembl predicted Hoxd12 sequences from published genomes (http://genome.ucsc.edu/and http://www.ensembl
.org) for the primates Macaca mulatta, Callithrix jacchus,
Microcebus murinus, Pongo pygmaeus, and Otolemur
garnettii, and the predicted Hoxd13 sequences for the
artiodactyl B. taurus and the primates C. jacchus and
P. pygmaeus.
Polymerase Chain Reaction (PCR) Amplification and
Sequencing
Genomic DNA was isolated using DNeasy Tissue kits
(Qiagen, Hilden, Germany) and used to amplify the Hoxd12
and Hoxd13 genes via the PCR. For Hoxd12, we designed
one pair of primers (forward: 5#-TTG GAG ATG TGT
GAG CGC AGT CTC TAC AG-3#; reverse: 5#-TTC
ATA CG(A/C) CGG TTC TGG AAC CA-3#) and for
Hoxd13, we designed separate pairs of primers to span
the gene, with an overlap of 75 bp. We refer to these as
primer set 1 (5#-ATG GAC GGG CTG CGG (A/G)C(A/
G) GAC G-3# and 5#-CC(C/T) GAC ACG TCC ATG
TAC TTC TCC AC-3#) and primer set 2 (5#-GCA (A/
G)AA TGC TCT (C/T)AA GTC (G/C)TC GCC-3# and
5#-CCA A(G/C)C TGG (T/A/C)CC ACA TCA (A/
G)GA GAC AGT (A/G)TC-3#), respectively. To verify
the Hoxd13 sequences and extend the overlap, we also amplified a 317-bp sequence using a set of published primers
(5#-CT(G/C) GGC TA(C/T) GGC TAC CAC-3# and 5#TG(G/A) TA(C/G) CCC TCC ATG GAG ATG-3#; Chen
et al. 2005). PCR products for both genes were ligated into
a pMD19-T vector (TaKaRa, Kyoto, Japan), cloned and sequenced using the Big Dye Terminator kit on an ABI 3730
DNA sequencer (Applied Biosystems, Foster City, CA). Finally, the sequences of both genes were edited to remove an
intron and the sections corresponding to the primers, thus
yielding 721 bp (89%) and 965–971 bp (96%) of the protein-coding region of Hoxd12 and Hoxd13, respectively.
GenBank accession numbers of all new sequences are
FJ455455–FJ455496 inclusive.
Ancestral State Reconstruction
To reconstruct the ancestral digit pattern of the forelimb for different mammalian lineages across species
(Murphy et al. 2001; Price et al. 2005; Teeling et al.
2005; O’Leary and Gatesy 2008), we used the parsimony
method in Mesquite 2.01 (Maddison WP and Maddison DR
2007), which minimizes the number of steps between character state changes. We recorded the observed distribution
of digit and phalanx number among extant taxa, and reconstructed the ancestral states at nodes, for six characters: the
total number of digits per manus and the number of phalanges for each of the digits I–V (see fig. 1). Parsimony reconstruction was undertaken assuming both ordered and
unordered models of character change. In the former, the
cost of change from state i to state j is assumed to equal
|i – j| steps, whereas for the latter, one state change is modeled as one step. Ancestral state reconstruction of forelimb
digit number was also undertaken using a maximum likelihood (ML) approach in Mesquite 2.01. However, because
this approach does not support uncertain or polymorphic
character states, it was not used to infer phalanx number.
Adaptive Evolution of 5#HoxD Genes in Cetaceans 615
FIG. 1.—Digit and phalanx number for the forelimb of extant and extinct mammals. Character states of ancestral and extant taxa are shown on the
branches and after the taxon names, respectively. In each case, numbers follow the format: digit number (phalanx number of digit I, digit II, digit III,
digit IV, and digit V). For the extinct taxon Diacodexis, the digit number is five, but the number of phalanges is uncertain. Values above and below the
branches correspond to the results of ordered parsimony and ML reconstructions, respectively. The names of fossil species are framed, and the names of
extant species which were not included in our analyses of molecular evolution are underlined. An asterisk represents the consensus digit pattern of
5(2,3,3,3,3). The four positions in the tree in which digit number is known to have changed are denoted by I, II, III, and IV. Note, in cases where the
number of phalanges is zero, the metacarpal, and thus the digit, can still exist. Lower case letters on the tree illustrate the ancestral branches of the
cetaceans (c) and tetradactyl mysticetes (tm). Upper case letters represent taxonomic groupings as follows: P, Perissodactyla; C, Carnivora; E,
Eulipotyphla; R, Rodentia; and L, Lagomorpha.
We also performed the same methods to reconstruct
the ancestral states of the length of the polyalanine stretch
in Hoxd13. Here, we considered 35 extant species (see
fig. 2), and the single character was the number of alanine
residues.
Molecular Evolution Analyses
To test for evidence of molecular adaptation in
Hoxd12 and Hoxd13, we used the CODEML program in
PAML 4 (Yang 2006, 2007) to derive ML estimates of
the rates of synonymous and nonsynonymous substitutions
(dS and dN, respectively) and the dN/dS ratio (omega, x).
Positive selection is evident where x . 1, neutral evolution
where x 5 1, and purifying selection where x , 1 (Nei and
Kumar 2000). Rates were estimated along specific branches
in a tree, the topology of which was constrained based on
published studies (Murphy et al. 2001; Price et al. 2005;
Teeling et al. 2005; O’Leary and Gatesy 2008) and is shown
as figure 2. For all PAML-based analyses, alignment gaps
were treated as ambiguity characters (setting cleandata 5
0). Branch lengths were estimated simultaneously (iteration
setting method 5 0), and codon frequencies were calculated
from the average nucleotide frequencies at the three codon
positions (setting CodonFreq 5 2 (F3X4)).
We tested for positive selection on the branches ancestral to all cetaceans, to tetradactyl mysticetes, to perissodactyls, and to cetartiodactyls using branch models. Specifically,
a model in which x was fixed across the tree (one-ratio) was
compared with models in which x was allowed to differ between the background and a focal branch (two-ratio model),
or between the background and among two focal branches.
To test whether the x ratio of each focal branch was significantly greater than both the background ratio and one (Yang
1998), we performed likelihood ratio tests (LRTs). The LRT
statistic (2Dl) approximates to a chi-square distribution and
can be used to compare nested likelihood models. Where
we found evidence of a significant change in selection pressure (x) in an ancestral branch, we explored this further with
the free-ratio model, in which x is assumed to be independent
on each branch of the tree.
To identify the sites under positive selection along the
Hox genes, we implemented site models in which x can
vary among sites. However, because positive selection often operates episodically at small numbers of sites and is
thus difficult to detect (Zhang et al. 2005), we also applied
the branch-site model A, in which x can vary among sites
along specific lineages (Yang et al. 2005). Separate branchsite models were implemented with branches c and tm set as
the foreground branches, and model significance was tested
using the LRTs termed branch-site Tests 1 and 2 (Zhang
et al. 2005). In the former, branch-site model A is compared
with two degrees of freedom to a site model (M1a, ‘‘Nearly
Neutral’’) with two site classes: 0 , x0 , 1 and x1 5 1
fixed. In the latter, branch-site model A is compared with
one degree of freedom to an equivalent model in which x2
is fixed at 1. Test 1 is unable to distinguish relaxed constraint from positive selection, whereas test 2 is a direct test
for positive selection on the lineage of interest, but appears
616 Wang et al.
FIG. 2.—Phylogeny of 35 mammals used for evolutionary analysis of Hoxd12 and Hoxd13. The topology is based on Murphy et al. (2001), Price
et al. (2005), and Teeling et al. (2005). Note, for analyses of Hoxd13, the taxon Equus caballus was replaced with Equus asinus, and the sequences of
three primates (Macaca mulatta, Otolemur garnettii, and Microcebus murinus) and the carnivore Canis familiaris were not available from GenBank.
Numbers above the branches refer to the lengths of polyalanine repeats at the 5#-end coding region of Hoxd13 (see Results). Branches c (common
ancestors of cetaceans) and tm (common ancestors of tetradactyl mysticetes) are the major lineages of interest in the analysis of this study. Upper case
letters represent taxonomic groupings as follows: P, Perissodactyla; C, Carnivora; E, Eulipotyphla; R, Rodentia; and L, Lagomorpha.
conservative overall (Zhang et al. 2005). The Bayes Empirical Bayes (BEB) method for identifying positively selected
sites was applied to the results of branch-site model A.
Finally, to visualize variation in x along each gene, we
undertook a sliding window analysis using the software
SWAAP1.0.2 (Pride 2000). We set the window size at
60 bp (20 codons) and the step size at 12 bp (4 codons).
Values of x were estimated following Nei and Gojobori
(1986).
Results
Digit Pattern in the Mammalian Forelimb
Parsimony-based ancestral reconstructions of mammalian forelimb digit pattern yielded broadly similar results
based on ordered and unordered models of character
change, and ancestral digit numbers also agreed with the
results of ML reconstructions (fig. 1). Four major transitions in digit number were detected. Reductions in the number of digits occurred in the ancestral lineages of both the
perissodactyls and cetartiodactyls. Conversely, an increase
in digit number was observed in the ancestral branch of the
cetaceans, whereas a digit was then lost in the ancestral tetradactyl mysticetes. In terms of phalanx number, considerable variation is evident both among ancestral and modern
cetaceans. However, digits I and V typically show reduced
numbers of phalanges, whereas digits II, III, and IV show
increased numbers.
Alanine Repeat Length Polymorphism in Hoxd13
A polyalanine tract at the 5#-end of Hoxd13 was expanded in all the cetacean species studied (fig. 3). The number of alanine repeats ranged from 17 to 18 in modern
cetaceans compared with 14 to 15 in all other mammals.
The nucleotide alignment indicated that two or three alanine
residues have become inserted into the middle of the polyalanine stretch in cetaceans. Ancestral reconstructions of the
number of alanine repeats were identical for both parsimony
(ordered and unordered) and ML models of character change
(fig. 2). We found that two alanines were gained in the
common ancestor of all cetaceans, which was assigned
17 units. Three independent cases of repeat gain were also
detected within the cetaceans, on the branches leading to
Adaptive Evolution of 5#HoxD Genes in Cetaceans 617
FIG. 3.—Alignment of polyalanine repeats tract in Hoxd13. Sequences for 32 mammal species were aligned in ClustalX (Thompson et al. 1997)
and, where indels were present, by eye based on translated amino acid sequences using MEGA 4.0 (Tamura et al. 2007). Short dashes (–) represent
indels and numbers after the sequences indicate the counts of polyalanine residues. Alleles isolated from both a human and mouse exhibiting the
morphological defect synpolydactyly are also included (Muragaki et al. 1996; Johnson et al. 1998). In both cases, there is an insertion of seven alanine
residues into the middle of the polyalanine stretch.
B. acutorostrata, P. phocoena, and K. breviceps. (all possess
18 repeats). Among noncetaceans, all ancestral nodes were
assigned 15 repeat units; however, three species (rabbit, dog,
and badger) were characterized by 14 units and, therefore,
have subsequently lost an alanine.
Molecular Evolution of Hoxd12 and Hoxd13 in
Mammals
Separate one-ratio models undertaken for all mammals, and for cetaceans only, revealed comparable trends
for both genes. For Hoxd12, the x value was 0.105 for
all mammals and 0.320 for cetaceans, whereas for Hoxd13,
the x value was 0.024 for all mammals and 0.063 for cetaceans. Therefore, the ratio of nonsynonymous to synonymous substitutions, indicative of a change in selection
pressure, was approximately three times higher in cetaceans
than in all mammals.
The results of the branch models, with log likelihood
values, parameter estimates, and LRTs for hypothesis testing, are given in table 1. For Hoxd12, separate two-ratio
branch models in which x was allowed to differ from the
estimated background ratio in the ancestral branch of all
618 Wang et al.
Table 1
Log Likelihood Values and Parameter Estimates for Hoxd12 (35 Sequences) under Different Branch Models, with Likelihood
Ratio Tests for Testing Hypotheses (2Dl)
Model
npa
l
x0b
xcb
xtmb
A. One ratio: x0 5 xc 5 xtm
B. Two ratios: x0 5 xtm, xc
C. Two ratios: x0 5 xc, xtm
D. Two ratios: x0, xc 5 xtm
69
70
70
70
4,704.54
4,701.05
4,701.94
4,698.93
0.105
0.103
0.104
0.101
5 x0
0.785
5 x0
1.110
5 x0
5 x0
N
5 xc
E. Three ratios: x0, xc, xtm
F. Two ratios: x0 5 xc, xtm 5 1
G. Two ratios: x0, xc 5 xtm 5 1
H. Three ratios: x0, xc, xtm 5 1
71
69
69
70
4,698.41
4,702.43
4,698.94
4,698.90
0.101
0.104
0.101
0.101
0.785
5 x0
1
0.785
N
1
1
1
a
b
Models Compared
2Dl
P Value
B vs. A
C vs. A
D vs. A
E vs. B
E vs. C
E vs. D
C vs. F
D vs. G
E vs. H
6.98
5.20
11.22
5.28
7.06
1.04
0.98
0.02
0.98
0.0082
0.0226
0.0008
0.0216
0.0079
0.3078
0.3222
0.8875
0.3222
np, number of parameters.
Parameters xc, xtm, and x0 are the x ratios for branches c, tm, and all other branches, respectively (see fig. 2).
cetaceans (branch c in fig. 2), the ancestral branch of the
tetradactyl mysticetes (branch tm in fig. 2), or both together (xc 5 xtm) showed significant model improvement
over a one-ratio model, with higher x values in the foreground branches. The model fit of the former two tworatio models was improved further by the addition of
a third ratio, suggesting that the x also differs between
the branches c and tm. In this three-ratio model, the x values for branch c and the background were 0.785 and 0.101,
respectively, whereas the x value for the branch labeled tm
was ‘‘infinite’’ (i.e., dS 5 0 and dN . 0). A comparison of
the two-ratio model (x0 5 0.101, xc 5 xtm 5 1.110) with
the three-ratio model was not significant (LRT 5 1.04,
P 5 0.3078, df 5 1), suggesting that the two-ratio model,
of which x values of branch c and branch tm were similar
and both higher than one, was not rejected. Two- and
three-ratio models were not found to fit the data better than
equivalent models in which the foreground x ratio was
fixed at one. Therefore, accelerated or adaptive evolution
has occurred in branches c and tm. The same branch
model tests were also performed for Hoxd13; however,
neither accelerated evolution nor positive selection was
detected (data not shown). Additionally, separate tworatio models applied to ancestral branches of the perissodactyls and the cetartiodactyls also showed no significant
improvement over a one-ratio model for either gene (data
not shown).
Site models (M0, M1a, M3, M7, and M8), in which x
was allowed to vary among codons, did not reveal any positively selected sites in the protein-coding regions of
Hoxd12 and Hoxd13 (data not shown). However, positively
selected sites along branches c and tm were identified in
branch-site model A for both genes, the log likelihood values and parameter estimates of which, along with LRT results, are given in supplementary table S1, Supplementary
Material online.
For Hoxd12, we detected seven sites under positive
selection on branch c based on branch-site model A, of
which six had BEB values .0.90 (33S, 60N, 149S,
154E, 155T, and 174Q). The LRT test statistic (2Dl) of Test
1 was 8.84 (P 5 0.012, df 5 2), indicating that model A fit
the data better than did model M1a, and the LRT test statistic of Test 2 was zero (nonsignificant, df 5 1). These results provide evidence of a change in selection pressure, and
reject neutral evolution, on the branch leading to the cetaceans, though it is not possible to reject accelerated evolution due to relaxed selection. On the other hand, for lineage
tm, though two sites were associated with high BEB posterior probabilities (57S with P 5 0.84, 60N with P 5
0.99), the LRT test statistics were not significant for either
Test 1 (2Dl 5 3.98, P 5 0.1367, df 5 2) or Test 2 (2Dl 5
1.00, P 5 0.3173, df 5 1) and so we cannot reject neutral
evolution.
For Hoxd13, a moderately high BEB value was only
found for a single site in each of branch c (74S with P 5
0.62) and tm (76S with P 5 0.75). The results of Tests 1 and
2 were not significant, either for branch c (2Dl 5 0.52, P 5
0.7711, df 5 2; 2Dl 5 0, P 5 1, df 5 1, respectively) or
branch tm (2Dl 5 2.02, P 5 0.3642, df 5 2; 2Dl 5 0, P 5 1,
df 5 1, respectively). Thus, on the basis of these models,
neutral evolution cannot be rejected for this gene.
A free-ratio model, which assumes independent x
values for each branch, was generated for both genes in
the cetaceans only (supplementary fig. S1, Supplementary
Material online). Estimates of x for Hoxd12 ratios were
infinite or greater than one in four branches: the ancestral
branch of the three tetradactyl mysticetes, the ancestral
branch of Megaptera novaeangliae and B. physalus,
the branch leading to B. acutorostrata, and the branch
leading to K. breviceps. For Hoxd13, x values were infinite in the branch leading to Stenella coeruleoalba and the
branch leading to M. densirostris. However, for both
genes, the free-ratio model was not better than the oneratio model (supplementary table S2, Supplementary
Material online).
The results of our sliding window analyses of dN/dS
(x) across the protein-coding regions of Hoxd12 and
Hoxd13 are shown in figure 4. Estimates of x of both
genes are uniformly low, indicative of purifying selection, based on all mammals. In contrast, x estimates
show dramatic variation along the genes in the cetaceans
and exceeds one in two main sections of Hoxd12 and
one section in Hoxd13. However, because these
sections also include the sites identified in the branch-site
model A (see above), this result is unlikely to be statistically significant.
In both data sets, nonsynonymous substitutions were
markedly more concentrated outside of the homeodomain,
Adaptive Evolution of 5#HoxD Genes in Cetaceans 619
FIG. 4.—Sliding window analysis of dN/dS (x). Estimates are based on Nei–Gojobori method (Nei and Gojobori 1986) and given across the
protein-coding regions of Hoxd12 (A) and Hoxd13 (B). In each case, the window was set at 60 bp and step at 12 bp.
in areas that, to our knowledge, are not known or speculated
to be key functional domains.
Discussion
The critical importance of the Hox genes for normal
growth and patterning of the autopod skeleton has been established from screening individuals with morphological
defects (Muragaki et al. 1996; Johnson et al. 1998; Zhao
et al. 2007), as well as via both loss- (Herault et al.
1997; Tarchini et al. 2006) and gain-of-function (Kmita
et al. 2000; Spitz et al. 2005) manipulations. Yet comparatively little consideration has been given to the role of Hox
genes in limb structure and modification over an evolutionary time frame. Consequently, it is generally thought that
these Hox genes are likely to be under strong selective constraint in tetrapods, in spite of several episodes of major
limb reorganization.
Our results suggest that Hoxd12 and Hoxd13 have undergone molecular adaptation in the cetaceans and that these
changes correspond to major rearrangements in the number
of digits. The cetacean fossil record provides one of the most
spectacular examples of evolutionary transition of all mammals, from the small semiaquatic tetrapodal forms of the
lower Eocene (Thewissen et al. 2007) to the large fully
aquatic species of the late Eocene, in which the hind limbs
were already reduced in size and would later disappear altogether (Thewissen et al. 2006). Fossil evidence and ancestral
state reconstruction (Sober 1991; Shimamura et al. 1997;
Nikaido et al. 1999; Price et al. 2005) both suggest that
the ancestors of the cetaceans possessed five digits (Gingerich et al. 2001; Berta et al. 2006) and, therefore, might have
regained a digit after their earlier divergence from the hippopotomids (Nikaido et al. 1999), whose forelimbs have four
digits (Boisserie et al. 2005). An alternative scenario, which
seems more plausible in terms of morphology, but which is
less concordant with the result of our selection tests, is
that digit number in the early cetaceans is a primitive state
and that independent reductions in digit number have occurred in four ancestral artiodactyl lineages, leading to the
camels, the pigs, the common ancestor of cows and goats,
and the hippopotomids.
The broad pentadactyl flipper is thought to be well
adapted for the slow maneuverable swimming necessary
for turning and prey capture in shallow lagoons (Benke
1993). Similarly, the subsequent loss of a digit (I) in some
mysticetes (Cooper et al. 2007) in the late Oligocene
(Fitzgerald 2006; Demere et al. 2008) conferred a more narrow, elongate flipper that is considered well adapted for
swimming at high speeds, which is necessary for filter feeding (Goldbogen et al. 2006; Woodward et al.2006). The results of our analyses of the molecular evolution of Hoxd12
are thus consistent with these adaptive explanations for
transitions in digit number associated with changes in feeding behavior, prey and habitat use, with significantly higher
omega ratios found on the same two branches (ancestral to
the cetaceans and to the tetradactyl mysticetes) than on the
background (see table 1). Although branch model comparisons were unable to reject models in which the omega
value of these branches was fixed at one, models of positive
selection along branches c and tm were also never rejected.
The role of Hoxd12 in the modulation of digit number and
development of the flipper was also suggested by the results
of branch-site models, in which several sites under positive
selection were detected on the branch leading to the cetaceans (see supplementary table S1, Supplementary Material
online). Further research is needed to elucidate the functional significance of the sites in Hoxd12 gene function.
Analyses of the Hoxd13 gene showed no signature of
adaptive change; however, PAML-based analyses did not
take into account the observed length variation in the polyalanine tract at the 5#-end coding region among the cetaceans surveyed. Our ancestral reconstructions revealed
that the addition of two alanine residues occurred in the ancestral branch of the cetaceans and thus coincided with
a gain in digit number and web forming. Abnormally high
numbers of alanine residues (.15) in this stretch in humans
and mice have been linked to morphological deformities
characterized by extra and webbed digits (Muragaki
et al. 1996; Johnson et al. 1998) and, more generally, repeat
length mutations are known to correlate with a range of human conditions or developmental syndromes in humans
(Lavoie et al. 2003; Amiel et al. 2004; Anan et al.
2007), reviewed in Amiel et al. (2004) and Trochet et al.
(2007). Moreover, several of these disease-linked polyalanine domains have also been reported from Hox genes
(Innis et al. 2004). Recently, Fondon and Garner (2004)
proposed that length polymorphisms in amino acid repeat
domains might underpin rapid morphological evolution,
620 Wang et al.
following their discovery that repeat variation in the coding
regions of Alx-4 and Runx-2 were associated with differences in dog limb and skull morphology. Our findings from
Hoxd13 in cetaceans appear to provide support for this
assertion.
As well as changes in digit number, the cetacean manus also shows dramatic interspecific polymorphisms in
the number of carpals and phalanges, and even intraspecific polymorphisms in the number of phalanges (Fedak
and Hall 2004; Berta et al. 2006; Cooper et al. 2007). It
is therefore intriguing that omega estimates (based on
free-ratio models) for both genes were seen to vary markedly among the branches of a tree that included cetaceans
only, and, on average, were much higher in cetaceans than
in other mammals. Sliding windows of omega ratios also
showed considerable fluctuations outside of the homeodomain region of both genes. These adjacent domains have
not been studied individually; however, because both
genes are transcription factors that are typically composed
of a DNA-binding domain and an activator domain, the
area of positively selected sites detected by branch-site
model A and the sliding windows might correspond to
the latter domain and thus influence the efficiency of
DNA binding (Agalioti et al. 2000; Berg et al. 2002).
We thus conclude that the homeodomain is under strong
functional constraint in cetaceans and is not involved in
the observed variation and adaptive change detected in
Hoxd12 and Hoxd13.
In contrast to the cetaceans, no evidence of changes in
selection pressure in 5#HoxD genes was recorded in the
evolution of perissodactyls and artiodactyls, both of which
have also undergone reductions in digit number (Vaughan
et al. 2000). Yet given that diversification of the limb
skeleton can occur via multiple routes (Tanaka et al.
2005; Weatherbee et al. 2006), this lack of parallel signature
is perhaps not surprising. More interesting, however, is how
these Hox genes, which are so highly conserved across vertebrates, including the ungulates, have been able to undergo
such relatively large numbers of changes in the cetaceans.
One clue to this might come from the recent finding that
forelimbs and hind limbs share a common genetic control
path for digit development (Cobb and Duboule 2005). It is
thus plausible that the early loss of the hind limbs in cetaceans reduced the developmental constraint placed on
5#HoxD genes. Therefore, because they no longer controlled
appendicular developments in both limbs (Thewissen et al.
2006), these genes were able to adapt to meet the morphological demands of different cetacean species during their
adaptive radiation.
Previous evidence of the role of Hox genes in evolution has nearly all come from studies of expression (Zakany
et al. 1997). Patterns of expression have been used to assign
digit identity in bird wings (Vargas and Fallon 2005), and
are linked to pelvis loss in the pufferfish Takifugu rubripes
(Tanaka et al. 2005). Expression studies have also suggested that HoxD and Tbx genes were coopted in the evolution of paired fins (Freitas et al. 2006) and that Hox genes
such as those on the HoxA and HoxD clusters were involved
in the subsequent evolution of an ancestral fin-like state into
tetrapod appendages (Zakany and Duboule 2007). Some
studies have examined the molecular evolution of Hox
genes involved in limb organization (Kappen 2000). Lynch
et al. (2006) found that paralog groups of Hox genes
showed signatures of adaptive evolution following duplication of Hox cluster in the early evolution of vertebrates,
whereas work by Amores et al. (2004) suggests that gene
diversification following Hox gene duplication has contributed to the radiation of the teleosts. By comparison, examples of rapid Hox gene evolution in mammals are
uncommon (for an exception, see Wang and Zhang
2007). Our findings, suggesting that limb modification in
the mammals has involved the molecular evolution of
the Hox genes Hoxd12 and Hoxd13, represent an important
addition to this growing body of work and highlights the
potential role that Hox genes are likely to have had in more
recent evolutionary radiations.
Supplementary Material
Supplementary tables S1 and S2 and supplementary
figure S1 are available at Molecular Biology and Evolution
online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
We are very grateful to Rob Deaville for samples of
cetaceans. We also thank Yang Liu for assistance in the laboratory and Annalisa Berta and John Gatesy for helpful
comments on an earlier version of the manuscript. This
work was funded by a grant awarded to S. Zhang under
the Key Construction Program of the National ‘‘985’’ Project and ‘‘211’’ Project, a Royal Society Research Fellowship awarded to S.J.R., and a Darwin grant (14/008)
awarded to G.J. and S.Z.
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Claudia Kappen, Associate Editor
Accepted December 6, 2008