Harshman et al., Phylogenomic evidence for multiple losses of flight in ratite birds (2008).pdf

Phylogenomic evidence for multiple losses of flight

in ratite birds

John Harshman a,b,c , Edward L. Braun c,d,e , Michael J. Braun c,f,g , Christopher J. Huddleston f , Rauri C. K. Bowie a,h,i ,

Jena L. Chojnowski d , Shannon J. Hackett a , Kin-Lan Han d,f,g , Rebecca T. Kimball d , Ben D. Marks j , Kathleen J. Miglia k ,

William S. Moore k , Sushma Reddy a , Frederick H. Sheldon j , David W. Steadman l , Scott J. Steppan m , Christopher C. Witt j,n ,

and Tamaki Yuri d,f

a Zoology Department, Field Museum of Natural History, 1400 South Lakeshore Drive, Chicago, IL 60605; b 4869 Pepperwood Way, San Jose, CA 95124;

d Department of Zoology, University of Florida, Gainesville, FL 32611; f Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian

Institution, 4210 Silver Hill Road, Suitland, MD 20746; g Behavior, Ecology, Evolution, and Systematics Program, University of Maryland, College Park, MD

20742; h Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720; i Department of Science and

Technology/National Resource Foundation Centre of Excellence at the Percy FitzPatrick Institute, Department of Botany and Zoology, Stellenbosch

University, Matieland 7602, South Africa; j Museum of Natural Science, 119 Foster Hall, Louisiana State University, Baton Rouge, LA 70803; k Department of

Biological Sciences, Wayne State University, 5047 Gullen Mall, Detroit, MI 48202; l Florida Museum of Natural History, University of Florida, Gainesville, FL

32611; m Department of Biological Science, Florida State University, Tallahassee, FL 32306; and n Department of Biology and Museum of Southwestern

Biology, University of New Mexico, Albuquerque, NM 87131

Edited by Morris Goodman, Wayne State University School of Medicine, Detroit, MI, and approved July 17, 2008 (received for review April 2, 2008)

Ratites (ostriches, emus, rheas, cassowaries, and kiwis) are large,

ﬂightless birds that have long fascinated biologists. Their current

distribution on isolated southern land masses is believed to reﬂect

the breakup of the paleocontinent of Gondwana. The prevailing

view is that ratites are monophyletic, with the ﬂighted tinamous

as their sister group, suggesting a single loss of ﬂight in the

common ancestry of ratites. However, phylogenetic analyses of 20

unlinked nuclear genes reveal a genome-wide signal that unequiv-

ocally places tinamous within ratites, making ratites polyphyletic

and suggesting multiple losses of ﬂight. Phenomena that can

mislead phylogenetic analyses, including long branch attraction,

base compositional bias, discordance between gene trees and

species trees, and sequence alignment errors, have been elimi-

nated as explanations for this result. Themost plausible hypothesis

requires at least three losses of ﬂight and explains the many

morphological and behavioral similarities among ratites by parallel

or convergent evolution. Finally, this phylogeny demands funda-

mental reconsideration of proposals that relate ratite evolution to

continental drift.

them for f light’’ (5). Ratite monophyly was debated throughout

much of the last century, with Mayr and Amadon (6) stating in

1951 that the ‘‘present consensus is that the main groups of these

birds are of independent origin.’’ DeBeer provided a develop-

mental explanation for the similarities among ratites when he

interpreted the paleognathous palate and other features of

extant ratites as neotenic (7). Paleognath monophyly was ques-

tioned as late as the 1980s (8), but it has been confirmed by many

recent morphological and molecular studies (9–13).

Most recent studies have also strongly supported ratite mono-

phyly (9–12, 14), suggesting a single loss of f light in their

common ancestor. This puzzled biogeographers for more than a

century, because ratites would be unable to achieve their current

distribution on southern land masses if their common ancestor

was f lightless. Continental drift provided a compelling solution.

No longer was it necessary to imagine giant f lightless birds

crossing vast oceans; they could have rafted to their current

distributions on fragments of the Earth’s crust (15). Although

the proposed phyletic branching patterns for ratites do not

correspond perfectly to the order of separation of land masses

during the breakup of Gondwana, the convenient serendipity of

continental drift as a mechanistic explanation for ratite distri-

bution proved irresistible (10, 11, 14, 16), and it stands today as

a textbook example of vicariance biogeography (17, 18).

Despite the current consensus, some continue to question

ratite monophyly (13, 19) and the role of Gondwana in ratite

distribution (20). It has long been recognized that adaptation to

a f lightless, cursorial lifestyle can result in morphological con-

vergence or parallelism, especially in the postcranial skeleton (1,

19, 21). Such convergent adaptations might mislead phylogenetic

inference based on morphology. In fact, one study based solely

convergence

ﬂightlessness

Paleognath

homoplasy

vicariance biogeography

and Neognathae (1, 2), a classification based originally on

bony palate structure (3, 4). Palaeognathae also is traditionally

divided into two groups, the f lightless ratites (defined by absence

of a keel on the sternum) and the volant tinamous. Although they

include fewer than 1% of extant avian species, paleognaths have

long been viewed as central to understanding the early evolution

of birds. The many morphological and behavioral similarities of

ratites suggest common ancestry, but some have proposed that

they instead ref lect convergent adaptation to a f lightless, cur-

sorial lifestyle. The distribution of ratites is also remarkable;

ostriches live in Africa, rheas in South America, emus and

cassowaries in Australasia, kiwis and moas (now extinct) in New

Zealand, and elephant birds (also now extinct) in Madagascar.

How did these f lightless birds get to these far-f lung southern

landmasses?

Paleognath relationships have been controversial since the

earliest days of evolutionary biology. When Huxley defined the

paleognathous palate he also stated that extant ratites ‘‘are but

waifs and strays of what was once a very large and important

group’’ (3). Nevertheless, the notion that ratites have indepen-

dent origins arose around the same time, when Owen suggested

that they have closer affinities to various volant groups while

being united by the ‘‘arrested development of wings unfitting

Author contributions: J.H., E.L.B., M.J.B., S.J.H., R.T.K., W.S.M., F.H.S., and D.W.S. designed

research; J.H., E.L.B., M.J.B., C.J.H., R.C.K.B., J.L.C., S.J.H., K.-L.H., R.T.K., B.D.M., K.J.M., S.R.,

S.J.S., C.C.W., and T.Y. performed research; J.H., E.L.B., M.J.B., and C.J.H. analyzed data; and

J.H., E.L.B., and M.J.B. wrote the paper.

The authors declare no conﬂict of interest.

This article is a PNAS Direct Submission.

Data deposition: NewDNA sequences are deposited inGenBank (accession nos. EU805776–

EU805796, and EU822937). Alignments and trees have been deposited in TreeBase (study

accession no. S2138).

c J.H., E.L.B., and M.J.B. contributed equally to this work.

e To whom correspondence should be addressed at: Department of Zoology, P.O. Box

118525, University of Florida, Gainesville, FL 32611. E-mail: ebraun68@uﬂ.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/

0803242105/DCSupplemental .

13462–13467

PNAS

September 9, 2008

vol. 105

no. 36

www.pnas.org

cgi

doi

10.1073

pnas.0803242105

L iving birds are divided into two major groups, Palaeognathae

Fig. 1. Phylogenetic analyses of a 20-gene, 24-kb nuclear DNA dataset

strongly supporting ratite polyphyly. All analyses used Anas , Gallus , Buteo ,

and Ciconia as outgroups. Branches forwhich all supportmeasureswere 100%

or 1.0 are indicated with an asterisk; support for ratite polyphyly is high-

lighted. Topology obtained by using both partitioned (by locus) and unpar-

titioned ML and Bayesian analyses. Branch lengths reﬂect the unpartitioned

ML analysis. Support measures are partitioned RAxML bootstrap ( Upper Left ),

unpartitioned ML bootstrap ( Upper Right ), unpartitioned Bayesian posterior

probability ( Lower Left ), and partitioned Bayesian posterior probability

( Lower Right ).

Fig. 2. Phylogenetic analyses including crocodilian outgroups and two

passerine birds ( Corvus and Smithornis ) strongly support the conventional

position of the avian root and ratite polyphyly. Analyses were conducted by

using all sequences that could be aligned between crocodilians and birds

(4,668 bp). Support measures are unpartitioned ML bootstrap ( Upper Left ),

MP bootstrap (U pper Right ), unpartitioned Bayesian posterior probability

( Lower Left ), and partitioned Bayesian posterior probability ( Lower Right ).

Branch lengths shown reﬂect the unpartitioned ML analysis. Branches for

which all support measures were 100% or 1.0 are indicated with an asterisk;

the branch with no support values had 50% bootstrap support and 0.5

Bayesian posterior probability in all analyses. MP and ML analyses conducted

after Y coding produced similar results (not shown).

on cranial characters, where convergence may be less likely,

suggested that ratites are not monophyletic (13). These mor-

phological results are provocative in light of recent sophisticated

analyses of mitochondrial DNA (mtDNA) that show equivocal

support for ratite monophyly (22, 23). Given the profound

implications of this group for Gondwanan biogeography and the

evolution of f lightlessness, determining the true phylogeny of

ratites is a key question in avian systematics.

Phylogenomic studies, which combine data frommany genetic

loci sampled to represent the genome, are proving useful in

resolving difficult phylogenetic problems (24, 25). We assembled

a dataset of 20 nuclear loci widely dispersed in the avian genome

[ supporting information (SI) Table S1 ] to examine ratite mono-

phyly. It contains

30% protein-coding and 70% noncoding

sequence ( Table S1 ) , taking advantage of the phylogenetic signal

in archosaur noncoding sequences (26–28). The dataset com-

prises 18 taxa, including all extant ratite genera, four tinamou

genera, and eight outgroup taxa ( Table S2 ) . Analyses of this

dataset support a phylogeny in which paleognaths are mono-

phyletic but ratites are not.

specific analyses. The critical branch was strongly supported in

analyses using purine/pyrimidine (RY) coding ( Fig. S1 A ) , only

protein-coding exons ( Fig. S3 ) and different data partitioning

schemes (see SI Methods and Fig. S1 B ) . Furthermore, our

conclusions do not ref lect the specific set of outgroups used;

analyses including crocodilians (Fig. 2) and/or up to 150 addi-

tional neognaths, representing all major living avian lineages,

also support ratite polyphyly (25, 26, 30).

Separate analyses of individual loci show that 19 of 20 support

paleognath monophyly in all analytical approaches (data not

shown). The one that does not, BDNF , has a severe base

compositional bias (see below). The great majority of single-

locus trees (17 for ML and 15 for MP) support the ostrich as the

sister group of all other paleognaths (Table 1). The probability

of 15 or more of 20 independent gene trees agreeing by chance

is extremely low ( P

Results and Discussion

Nuclear DNA Sequences Strongly Support Ratite Polyphyly. All phy-

logenetic analyses of our 20-locus dataset revealed strong sup-

port for the ostrich as the sister group of all other paleognaths,

placing the volant tinamous within ratites and making ratites

polyphyletic. o When all loci were combined, the branch uniting

tinamous with all ratites except the ostrich received 100%

bootstrap support in maximum-likelihood (ML) analyses (Fig.

1), whether analyses were partitioned by locus (each locus

assigned its own best-fit evolutionary model) or unpartitioned (a

single model for all loci). Maximum-parsimony (MP) analyses

produced similar results ( Fig. S1 A ) . The critical branch uniting

non-ostrich paleognaths had a posterior probability of 1.00 in

both partitioned and unpartitioned Bayesian analyses (Fig. 1)

and highly significant support ( P

10 9 ; binomial test using equiprobable

trees null model). A number of loci that fail to support ratite

polyphyly in individual analyses show hidden support (31, 32) in

combined analyses ( Table S3 ) . Thus, the phylogenetic signal is

widespread in the nuclear genome and any attempt to explain

ratite polyphyly as an artifact must invoke a genome-wide

systematic bias.

0.001) in the Shimodaira–

Hasegawa (SH) test (see SI Methods and Fig. S2 C ) .

Support for ratite polyphyly is robust to the assumptions of

Rare Genomic Changes also Support Ratite Polyphyly. Beyond the

strong signal present in nucleotide substitutions, three insertion/

deletion events (indels) provide additional information regard-

ing ratite phylogeny. Rare genomic changes like indels are

thought to be valuable phylogenetic markers that may be free

from a number of caveats that apply to nucleotide substitutions

(33). The ostrich shares the ancestral character state with

neognaths for two indels, an 8-bp deletion in ALDOB (Fig. 3)

and a 9-bp insertion in MYC ( Fig. S4 ) . Tinamous share the

derived character state with all other ratites, so both indels

support the optimal tree found here. A single 1-bp indel in CLTC

( Fig. S5 ) is found in ratites (including ostrich) but not tinamous,

thus conf licting with the other indels and analyses of nucleotide

substitutions. Vertebrate indels consistently exhibit less ho-

moplasy than nucleotide substitutions (26, 34), and an exami-

o A group is polyphyletic if its deﬁning characters are convergent (29). Ratites have long

been deﬁned by the absence of a keel on their sternum (e.g., ref. 3), a character related

to ﬂightlessness. Our analyses (see below) indicate the common ancestor of ratites was

likely capable of ﬂight and thus had a keeled sternum and was not a ratite.

Harshman et al.

PNAS

September 9, 2008

vol. 105

no. 36

13463

Table 1. Bootstrap support for the crucial branch uniting

a non-ostrich paleognath clade from ML and MP analyses

of individual loci

Gene

Support

Conﬂict

Support

Conﬂict

ALDOB

64 —

—

BDNF

— 57*

—

CLTC

42 —

—

CLTCL1 †

68 —

—

CRYAA

66 —

—

EEF2

99 —

—

EGR1

77 —

—

FGB

— 42

—

GH1

71 —

—

HMGN2

81 —

—

IRF2

60 —

—

97 —

—

MUSK

95 —

—

MYC †

85 —

—

NGF

78 —

—

Fig. 3. An 8-bp deletion in ALDOB supports ratite polyphyly. ( A ) Alignment

of the region around the informative deletion in ALDOB (positions 3213–

3220). The ostrich shares its character state (

NTF3

83 —

—

PCBD1

92 —

—

8 bp) with neognaths, whereas

tinamous share the character state of all other ratites (

RHO

78 —

—

8 bp). ( B ) The

distribution of character states can be mapped as a single deletion on the

optimal topology found in this study. ( C ) The distribution requires at least two

steps on the traditional topology (one possible reconstruction shown).

TGFB2 †

— 52

—

TPM1

47 —

—

Number of loci

17 ‡

15 §

*ML analysis of BDNF does not support paleognath monophyly, instead

placing the tinamous within neognaths.

† These genes have multiple MP trees, some of which support and some of

which conﬂict with ratite polyphyly.

‡ Binomial test, P

10 15 (pure birth model) or 3

10 12 (equiprobable

tional convergence, we would expect a tree derived from base-

compositional information alone to have tinamous nested within

ratites. When we clustered taxa using base-compositional dis-

tances, however, tinamous fell within ratites for few loci ( Table

S5 ) . Instead, the most common grouping (10 of 20 loci) clustered

ratites, a signal expected to reinforce the conventional topology.

Furthermore, ratite polyphyly was supported both by analyses

conducted after RY coding ( Fig. S1 A ) , which increases histor-

ical signal relative to compositional bias (37, 41), and ML

analyses using a model allowing base compositional change

( Table S6 and Fig. S1 B ) . Base compositional convergence

therefore cannot explain our results.

The one locus that failed to support paleognath monophyly

was BDNF , which places tinamous within neognaths in ML

analyses. However, BDNF has the greatest base-compositional

variation and longest tinamou branch of all loci studied ( Table

S4 ) . ML analyses of BDNF after RY coding do not place

tinamous within neognaths (data not shown), suggesting that the

anomalous results for that locus are artifactual.

Another potential problem occurs when individual gene trees

differ from the species tree. Under some conditions, stochastic

lineage sorting can produce a bias toward gene trees more

symmetrical than the species tree (38), and the gene tree signal

can predominate when many loci are analyzed (42). However,

this is unlikely to occur unless the relevant branches are short

relative to the coalescence time for the genes examined. The

expected coalescence time for nuclear genes is twice the effective

population size in generations. Because the relevant branch

length and the ancestral paleognath population size are un-

known, we assessed the potential impact of lineage sorting

empirically. The gene-tree bias is weak (42), so it is unlikely to

produce the consistency of single-gene trees we observed (Table

1). The aggregate probability of all trees with non-ostrich

paleognath monophyly is 1/10, given the pure birth model for

gene trees (42), so the probability of finding this result in 15 or

more of 20 single-gene analyses is very small ( P

model).

§ Binomial test, P

10 12 (pure birth model) or 2

10 9 (equiprobable

model).

nation of avian FGB intron 7 indels (34) revealed that 1-bp indels

are more than twice as likely to exhibit homoplasy as longer

(

Known Phylogenetic Artifacts Do Not Explain Ratite Polyphyly. Sev-

eral distinct systematic biases that can mislead phylogenetic

analysis have been characterized (35–38), and we determined

whether any of these artifacts could explain ratite polyphyly.

Inconsistent phylogenetic estimation due to long-branch at-

traction (35) is a concern in phylogenomics. Superficially, it

might appear problematic for paleognath phylogeny because

tinamous have a high rate of molecular evolution ( Table S4 ) .

Although the branch leading to tinamous is long (e.g., Fig. 1), it

is not united with the other long branch on the tree, which leads

to the outgroup. In fact, long-branch attraction would be ex-

pected to favor the conventional hypothesis of ratite monophyly

rather than the surprising alternative of ratite polyphyly that we

observed. We confirmed this expectation in two ways. First, a

parametric bootstrap (Swofford–Olson–Waddell–Hillis,

SOWH) test (39) rejected a null hypothesis of ratite monophyly

( P

10 12 ).

This calculation is conservative; it ref lects the minimum number

of loci supporting the relevant branch in single-gene MP anal-

yses. More loci support non-ostrich paleognath monophyly using

13464

www.pnas.org

cgi

doi

10.1073

pnas.0803242105

Harshman et al.

5-bp) indels. Thus, the likelihood of homoplasy in the 1-bp

indel is much higher than the combined likelihood of homoplasy

in the two longer indels that support ratite polyphyly.

0.001). Second, a series of 1,000 MP analyses in which the

outgroup was replaced with a random sequence of similar base

composition, simulating the longest possible branch (40), all

rooted the tree within tinamous or along the branch leading to

them. This confirmed our expectations and rejected long-branch

attraction as an explanation for ratite polyphyly ( P 0.001).

Convergence in base composition can also create artifactual

relationships (23). If the observed topology ref lects composi-

ML (Table 1 and Table S6 ) , and all loci that fail to support this

group in single-gene ML analyses have positive partitioned

hidden branch support in combined analyses ( Table S3 ) .

Phylogenetic analyses can be inf luenced by alignment (43),

although our alignment protocol was conservative, and we

excluded ambiguously aligned sites. We further tested the pos-

sibility of an alignment artifact by performing two analyses using

only length-conservative regions: first, those regions that could

be reliably aligned with distantly related crocodilian sequences

(Fig. 2) and second, protein-coding exon sequences ( Fig. S3 ) .

Both analyses strongly supported the non-ostrich paleognath

clade, like the full data. In sum, our analyses indicate that the

signal evident in the 20-locus dataset does not ref lect any known

phylogenetic artifact.

branch leading to the outgroup is likely (as described above).

Thus, we do not consider mtDNA to be in strong conf lict with

ratite polyphyly; indeed, the ambivalent signal evident in recent

analyses (22, 23) suggests more sophisticated analyses of mtDNA

may ultimately support polyphyly.

Several morphological studies strongly support ratite mono-

phyly (9, 12) in conf lict with our nuclear genetic data. Simply

collecting more data or combining molecular and morphological

data are unlikely to resolve the impasse generated by these

incongruent signals (53). Both signals cannot represent evolu-

tionary relationships; at least one must be nonphylogenetic.

Cranial morphology offers a potential solution, because two

studies using cranial characters (13, 19) agree with our data.

There is no reason to expect convergence in the cranial char-

acters of tinamous and any particular subset of ratites. However,

convergence is common in the postcranial skeletons of other

f lightless birds (54), and morphological convergence certainly

can mislead phylogenetic analysis (55). Flightlessness is expected

to result in reduction or loss of the sternal keel; reduction in size,

complexity, and number of wing bones; increase in size of leg

bones; and nonaerodynamic changes in plumage structure. Be-

cause the volant ancestors of each ratite lineage may have been

morphologically similar, parallel evolution could have produced

some ratite traits identical in state but not by descent.

Monophyly of Australasian Ratites, Placement of Tinamous, and the

Root of the Avian Tree. Other relationships strongly supported by

our results include monophyly of rheas, tinamous, and an emu–

cassowary clade (e.g., Fig. 1), in agreement with previous studies.

Monophyly of extant Australasian ratites (kiwis, emus, and cas-

sowaries) is also strongly supported (Fig. 1, Fig. S1 ) , in agreement

with previous molecular analyses (9–11, 14) but contrary to some

morphological analyses (9, 12). Kiwis may be difficult to place in

morphological phylogenies because of the striking differences

between kiwis and other ratites in life history, behavior, and size

(44). The molecular support for nesting kiwis within ratites is

especially interesting given suggestions that kiwis were derived

directly from volant paleognaths (45).

Although placement of tinamous within the ratites is strongly

supported, the sister group of tinamous is unclear. ML and

Bayesian analyses place tinamous sister to Australasian ratites

(Fig. 1, Fig. S1 B ) , whereas MP and RY-coded ML analyses place

tinamous sister to rheas ( Fig. S1 A ) . The alternative trees do not

differ significantly when the SH test is used ( Fig. S2 ) , suggesting

that the topology is sensitive to the model of sequence evolution

applied. The second alternative is most parsimonious from a

biogeographic standpoint, because both tinamous and rheas are

exclusively Neotropical.

Our large nuclear dataset allowed us to address a controversy

regarding the root of the avian tree and examine the impact of

the root on our conclusions. Although the traditional view is that

the root lies between paleognath and neognath clades (12, 27,

46), early analyses of mtDNA placed the root either between

passerines and all other birds (47) or within passerines (48, 49),

contradicting neognath monophyly. More sophisticated analyses

(e.g., RY-coding) of mtDNA data strongly support the tradi-

tional rooting (22, 23, 50, 51), unlike the early analyses. Some

morphological studies also suggest nonmonophyly of paleog-

naths (8, 45). Given these questions, we analyzed our dataset

using several different methods and confirmed that the position

of the root lies between paleognath and neognath clades (Fig. 2).

Evolution of Flightlessness. Any topology that nests the volant

tinamous within the f lightless ratites requires either multiple

losses of f light or a loss of f light in the ancestral paleognath and

a regain in tinamous. Although loss and regain is more parsi-

monious if both transitions are equally probable, multiple losses

of f light are more likely. Flight has been lost in members of 18

extant bird families, many more times in extinct groups, and

hundreds of times in the family Rallidae alone (21, 54, 56). Thus,

the loss of f light is much more probable than gain. Given the

position of tinamous in either optimal tree based on the com-

plete dataset (Fig. 1, Fig. S1 A ) , f light must have been lost

independently at least three times, in ostriches, rheas, and

Australasian ratites. A scenario in which tinamous regained

f light would be even more interesting, but there are no examples

of avian lineages that have lost and regained f light.

Biogeographic Implications. There have been many proposals

relating divergences among living ratites to the breakup of

Gondwana (10, 11, 14, 16), but all assume ratite monophyly. Our

phylogeny actually fits a strictly vicariant hypothesis better than

previous phylogenies. Africa was the first piece of Gondwana to

separate (16), and the African ostriches are the first clade to

diverge (e.g., Fig. 1). Nevertheless, no proposed phylogeny, ours

included, can be explained entirely by the order of separation of

Gondwanan fragments.

Multiple losses of f light, with the implication of greater

dispersal capability for ancestral paleognaths, make a strictly

vicariant model less compelling. The existence of volant pale-

ognaths in the Paleogene of Europe and North America also

suggests that dispersal must be considered (45, 57). Dispersal of

ratites is further suggested by phylogenies in which the extinct

moas of New Zealand are not sister to the extant kiwis (2, 10, 11),

as would be predicted by strict vicariance. Thus, fossil data

confirm that simple vicariant models can be rejected. It may be

possible to distinguish among evolutionary scenarios with time-

calibrated trees, but the large differences in evolutionary rates

evident in our analyses (e.g., Fig. 1) combined with the paucity

of good fossil calibration points for paleognaths render this task

difficult.

Contrasts with Previous Studies. The strong support for ratite

polyphyly in our study raises an important question: Why have

most modern analyses supported ratite monophyly? We cannot

fully answer this question, but we can offer plausible hypotheses.

A study based on a single, short nuclear intron (52) showed

limited support for ratite monophyly, probably ref lecting limited

power. DNA–DNA hybridization (14) required extrapolation of

distances far beyond the useful range of the method. Early

studies using mtDNA strongly supported ratite monophyly (9–

11), but recent analyses using more sophisticated methods

revealed that support for a ratite clade is weak (22, 23). Avian

mtDNA evolves rapidly and exhibits high among-site rate vari-

ation (10, 11), making analyses sensitive to the evolutionary

model and taxon sample used (23). Furthermore, tinamous have

a much higher mtDNA evolutionary rate than other paleognaths

(11), so long-branch attraction uniting tinamous with the long

Conclusions

Exhaustive analyses of DNA sequence data from 20 unlinked

nuclear genes provide strong evidence that ratites are polyphyl-

Harshman et al.

PNAS

September 9, 2008

vol. 105

no. 36

13465

etic. We have discovered a robust genome-wide signal that is not

associated with any known phylogenetic artifact. We believe this

phylogeny resolves a debate on ratite origins that began in the

time of Huxley and Owen (3–5). Our phylogeny implies that the

numerous striking similarities associated with f lightlessness (1)

had independent origins in various ratite lineages. Thus, the

f lightless ratites are living evidence of parallel evolutionary

trajectories from f lighted ancestors. The possibility that multi-

ple, unique developmental genetic pathways underlie the ratite

form should be tested in light of this new phylogenetic hypoth-

esis. Finally, our phylogeny removes the need to postulate

vicariance by continental drift to explain ratite distribution.

Although that theory seemed to represent a consilience between

evolutionary biology and geology, it was never completely

consistent either with any published phylogeny or the existence

of paleognath fossils in the Northern hemisphere (45, 57).

Perhaps the impact of our phylogeny should be viewed as yet

another example of the phenomenon that Huxley called ‘‘the

great tragedy of science—the slaying of a beautiful theory by an

ugly fact.’’

used the best-ﬁt model for each locus ( Table S8 ) and nhPhyML analyses used

the Galtier and Gouy (62) model, which allows the GC content to vary across

the tree. In these analyses, support was assessed by using 1,000 resampling of

estimated log-likelihoods (RELL) (63) bootstrap replicates.

Bayesian Markov chain Monte Carlo analyses were performed by using

MrBayes 3.1.1 (64). We ran four chains for 10 million generations, sampling

every 500 generations and discarding the ﬁrst 500 trees, with other run

parameters set at defaults. Partitioned Bayesian analyses used linked branch

lengths.

The relationship between the probability that a clade is correct and boot-

strap support is complex (65, 66), but the bootstrap is conservative undermany

circumstances. In contrast, Bayesian posterior probabilities can overestimate

the probability that a clade is correct (e.g., 66). We also assessed branch

(Bremer) support as well as hidden support and conﬂict for speciﬁc branches

(31, 32).

Tests of Topologies, Base Composition, and Individual Gene Trees. We used two

topology tests appropriate for comparisons of trees that were not speciﬁed a

priori (67) to examine the position of the ostrich. The SH test (68) was

performed by using the plausible set of 315 distinct trees ( Fig. S2 ) because the

test requires the inclusion of all trees that can be entertained as the true

topology (67). The parametric bootstrap (SOWH) test (39) compares the

difference in optimality scores for the empirical data on the optimal tree and

a null hypothesis tree to a distribution generated by simulation. The null

hypothesis topology was the most likely one with ratite monophyly (tree 19a

of Fig. S2 ) ; 1,000 simulated datasets were analyzed by using MP and ML ( SI

Methods ) .

Base-compositional clusteringwas performed by usingminimumevolution

in PAUP* with a matrix of Euclidean distances between base-composition

vectors for each taxon (see SI Methods ) . Topologies examined were limited to

the plausible set ( Fig. S2 ) .

Individual gene trees may differ from the species tree. We tested whether

the observed number of gene trees showing monophyly of non-ostrich pale-

ognaths was unexpected given the null hypothesis of a completely polyto-

mous species tree. We used binomial tests and two appropriatemodels of tree

probability to assess this possibility ( SI Methods ) .

Methods

Sequencing and Alignment of Nuclear Loci. We ampliﬁed and sequenced 20

nuclear loci (see SI Methods for detailed methods). Sequences (accession

numbers listed in Table S7 ) were aligned manually (see SI Methods ) ; ambig-

uously aligned regions and sparsely sampled sites (those not present in at least

four birds and three paleognaths) were excluded from analyses.

Phylogenetic Analyses. Four nonpasserine neognaths were used as outgroups

in most analyses of the 14 taxon, 23,902-bp dataset (e.g., Fig. 1). Crocodilians

and passerines were included only in analyses designed to test the position of

the root of the avian tree using the subset of sequences for which crocodilian

and avian sequences could be aligned (4,668 bp; Fig. 2).

OptimalMP andML trees usingunpartitioneddatawere identiﬁedby using

PAUP* 4.0b10 (58). MP analyses used branch-and-bound searches with equally

weighted characters and assessed support using 1,000 bootstrap replicates.

The appropriate nucleotide substitutionmodel ( Table S8 ) for ML analyses was

determined by using the Akaike information criterion and Modeltest 3.6 (59)

or the models appropriate for RY-coded data (see SI Methods ) . ML analyses

used heuristic searches with 10 random addition sequence replicates and tree

bisection and reconnection (TBR) branch swapping; support was assessed by

using 100 bootstrap replicates.

ThreeML analyses with the data partitioned by locus were conducted. First,

a partitioned RAxML (60) analysis, using the general time-reversible (GTR)

Identification of Informative Indels. To identify low-homoplasy indels, all gaps

in a 19-gene, 171-taxon dataset (25) were coded by using the simple gap

codingmethod (69). These indels weremapped by usingMP on theML tree for

the 171-taxon dataset (25) and the same tree rearranged so ratites were

monophyletic. Indels mapping unambiguously on the branch of interest with

a consistency index

model with distinct parameter values for each partition and linked branch

length parameters, was conducted and support assessed by using 100 boot-

strap replicates. The other two analyses used PAUP* and nhPhyML (61),

allowing use of a more diverse set of nucleotide substitution models. Branch

lengths were unlinked andmonophyly of well established clades was assumed

a priori , focusing on a ‘‘plausible set’’ of 315 trees (105 arrangements of the

ﬁve major paleognath groups and three arrangements within tinamous; Fig.

S2 ) . The log likelihoods of all partitions for each treewere summed after being

calculated using PAUP* ( Table S9 ) or nhPhyML (not shown). PAUP* analyses

ACKNOWLEDGMENTS. We thank D. L. Swofford, P. O. Lewis, and D. J. Zwickl

for advice on phylogenetic analyses; D. J. Levey, K. Braun, and anonymous

reviewers for helpful comments; and A. B. Thistle for a technical edit of the

manuscript. For the loan of samples, we thank the Field Museum of Natural

History, the University of Kansas, Louisiana State University, the United States

National Museumof Natural History, L. D. Densmore, and the collectors ( Table

S2 ) . This work was supported by the U.S. National Science Foundation Assem-

bling the Tree of Life Program Grants DEB-0228675 (to S.J.H.); DEB-0228682

(to R.T.K., E.L.B., and D.W.S.); DEB-0228688 (to F.H.S.); and DEB-0228617 (to

W.S.M.).

1. Davies SJJF (2002) Ratites and Tinamous: Tinamidae, Rheidae, Dromaiidae, Casuari-

idae, Apterygidae, Struthionidae (Oxford Univ Press, Oxford).

2. Harshman J (2007) in Reproductive Biology and Phylogeny of Birds , ed Jamieson BGM

(Science Publishers, Enﬁeld, NH), pp 1–35.

3. Huxley TH (1867) On the classiﬁcation of birds; and on the taxonomic value of the

modiﬁcations of certain of the cranial bones observable in that class. Proc Zool Soc

London 145:415–472.

4. Pycraft WP (1900) Part II. On the morphology and phylogeny of the Palaeognathae

(Ratitae and Crypturi) and Neognathae (Carinatae). Trans Zool Soc London 15:149–

290.

5. Owen R (1866) The Anatomy of Vertebrates, Vol. 2: Birds and Mammals (Longmans,

Green, London).

6. Mayr E, Amadon D (1951) A classiﬁcation of recent birds. Am Mus Novit 1496:

1–42.

7. DeBeer G (1956) The evolution of ratites. Bull Br Mus (Nat Hist) Zool 4:57–76.

8. Olson SL (1985) The fossil record of birds. Avian Biol VIII:79–238.

9. Lee K, Feinstein J, Cracraft J (1997) in Avian Molecular Evolution and Systematics ,ed

Mindell DP (Academic, San Diego), pp 173–211.

10. Cooper A, et al. (2001) Completemitochondrial genome sequences of twoextinctmoas

clarify ratite evolution. Nature 409:704–707.

11. Haddrath O, Baker AJ (2001) Complete mitochondrial DNA genome sequences of

extinct birds: Ratite phylogenetics and the vicariance biogeography hypothesis. Proc R

Soc London Ser B 268:939–945.

12. Livezey BC, Zusi RL (2007) Higher-order phylogeny of modern birds (Theropoda, Aves:

Neornithes) based on comparative anatomy. II. Analysis and discussion. Zool J Linn Soc

149:1–95.

13. Elzanowski A (1995) Cretaceous birds and avian phylogeny. Cour Forsch Inst Senck

181:37–53.

14. Sibley CG, Ahlquist JA (1990) Phylogeny and Classiﬁcation of Birds (Yale Univ Press,

New Haven, CT).

15. Cracraft J (1973) Continental drift, palaeoclimatology, and the evolution and bioge-

ography of birds. J Zool 169:455–545.

16. Cracraft J (2001) Avian evolution, Gondwana biogeography and the Cretaceous-

Tertiary mass extinction event. Proc R Soc London Ser B 268:459–469.

17. Futuyma DJ (2005) Evolution (Sinauer, Sunderland, MA).

18. Gill F (2007) Ornithology (Freeman, New York).

19. Bock WJ, B¨ hler P (1990) in Proceedings of the International Centennial Meeting of

the Deutsche Ornithologen-Gesellschaft , eds van den Elzen R, Schuchmann K-L,

Schmidt-Koenig K (Verlag der Deutschen Ornithologen-Gesellschaft, Bonn, Germany),

pp 31–36.

13466

www.pnas.org

cgi

doi

10.1073

pnas.0803242105

Harshman et al.

0.5 were examined further, and ambiguously aligned

regionswere removed. CLTCL1 was not included in the 171-taxon analysis (25),

so it was examined independently by using a similar methodology.

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika: