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The transcriptome data are archived at NCBI’s Gene Expression
Omnibus database (series record GSE56278: www.ncbi.nlm.nih.
gov/geo/query/ acc.cgi?acc=GSE56278). Funding was provided
by The Gordon and Betty Moore Foundation through grant
GBMF2629, the Schmidt Ocean Institute, and a fellowship
from the NSF to N. T.K. We thank M. van Oppen, I. Baums,
M. De Salvo, L. Bay, M. Morikawa, and O. Hoegh-Guldberg for
comments on earlier versions of the manuscript. We also thank
the U.S. National Park of American Samoa for permission to work
on Ofu reefs and C. Caruso for logistical and research help.
Materials and Methods
Figs. S1 and S2
Tables S1 to S4
27 January 2014; accepted 1 April 2014
Published online 24 April 2014;
Ancient DNA reveals elephant birds
and kiwi are sister taxa and clarifies
ratite bird evolution
Kieren J. Mitchell,1 Bastien Llamas,1 Julien Soubrier,1 Nicolas J. Rawlence,1*
Trevor H. Worthy,2 Jamie Wood,3 Michael S. Y. Lee,1,4 Alan Cooper1†
The evolution of the ratite birds has been widely attributed to vicariant speciation,
driven by the Cretaceous breakup of the supercontinent Gondwana. The early isolation of
Africa and Madagascar implies that the ostrich and extinct Madagascan elephant birds
(Aepyornithidae) should be the oldest ratite lineages. We sequenced the mitochondrial
genomes of two elephant birds and performed phylogenetic analyses, which revealed
that these birds are the closest relatives of the New Zealand kiwi and are distant from
the basal ratite lineage of ostriches. This unexpected result strongly contradicts
continental vicariance and instead supports flighted dispersal in all major ratite lineages.
We suggest that convergence toward gigantism and flightlessness was facilitated
by early Tertiary expansion into the diurnal herbivory niche after the extinction of
Despite extensive studies, the evolutionary history of the giant flightless ratite birds of the Southern Hemisphere landmasses and the related flighted tinamous of South America has remained a major unresolved
question. The ratites and tinamous, termed
“palaeognaths” due to their shared basal palate
structure, form the sister taxon to all other living
birds (neognaths). The living ratites are one of
the few bird groups composed largely of giant
terrestrial herbivores and include: the emu and
cassowary in Australia and New Guinea, the
kiwi in New Zealand, the ostrich in Africa, and
the rhea in South America. In addition, two re-
cently extinct groups included the largest birds
known: the moa from New Zealand (height up
to 2 to 3 m, 250 kg in weight) (1) and elephant
birds from Madagascar (2 to 3 m in height, up
to 275 kg in weight) (2, 3). Ratites have been
believed to have originated through vicariant
speciation driven by the continental breakup of
the supercontinent Gondwana on the basis of
congruence between the sequence of continental
rifting and the presumed order of lineage diver-
gence and distribution of ratites (4, 5).
New Zealand is the only landmass to have sup-
ported two major ratite lineages: the giant her-
bivorous moa and the chicken-sized, nocturnal,
omnivorous kiwi. Morphological phylogenetic
analyses initially suggested that these two groups
were each other’s closest relatives (6, 7), presum-
ably diverging after the isolation of an ancestral
form following the separation of New Zealand
and Australia in the late Cretaceous ~80 to 60
million years ago (Ma) (8). However, subsequent
studies suggest that kiwi are more closely related
to the Australasian emu and cassowaries (9, 10),
whereas the closest living relatives of the giant
moa are the flighted South American tinamous
(11–14). The latter relationship was completely un-
expected on morphological grounds and sug-
gests a more complex evolutionary history than
predicted by a model of strict vicariant specia-
tion. By rendering ratites paraphyletic, the rela-
tionship between the moa and tinamous also
strongly suggests that gigantism and flightless-
ness have evolved multiple times among palae-
ognaths (12, 13).
Perhaps the most enigmatic of the modern
palaeognaths are the recently extinct giant Madagascan elephant birds. Africa and Madagascar
were the first continental fragments to rift from
the supercontinent Gondwana, separating from
the other continents (and each other) completely
during the Early Cretaceous (~130 to 100 Ma)
(15). Consequently, the continental vicariance
model predicts that elephant birds and ostriches should be the basal palaeognath lineages (16). Most molecular analyses recover the
ostrich in a basal position, consistent with a vicariant model. However, the phylogenetic position of the elephant birds remains unresolved,
as cladistic studies of ratite morphology are sensitive to character choice and may be confounded
by convergence (17), whereas DNA studies have
been hampered by the generally poor molecular
preservation of elephant bird remains (18).
We used hybridization enrichment with in-solution RNA arrays of palaeognath mitochondrial genome sequences and high-throughput
sequencing to sequence near-complete mitochondrial genomes from both elephant bird genera:
Aepyornis and Mullerornis. Phylogenetic analyses
placed the two taxa, Aepyornis hildebrandti
(15,547 base pairs) and Mullerornis agilis (15,731
base pairs), unequivocally as the sister taxa to
the kiwi (Fig. 1 and fig. S1). This result was consistently retrieved, regardless of phylogenetic
method or taxon sampling, and was strongly supported by topological tests (19). To our knowledge, no previous study has suggested this
relationship, probably because of the disparate
morphology, ecology, and distribution of the two
groups. Elephant birds were herbivorous, almost
certainly diurnal, and among the largest birds
1Australian Centre for Ancient DNA, School of Earth and
Environmental Sciences, University of Adelaide, North
Terrace Campus, South Australia 5005, Australia. 2School of
Biological Sciences, Flinders University, South Australia
5001, Australia. 3Landcare Research, Post Office Box 40,
Lincoln 7640, New Zealand. 4South Australian Museum,
North Terrace, South Australia 5000, Australia.
*Present address: Allan Wilson Centre for Molecular Ecology and
Evolution, Department of Zoology, University of Otago, Dunedin,
New Zealand. †Corresponding author. E-mail: alan.cooper@