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© 2006 Nature Publishing Group
Early evolution of the venom system in lizards
and snakes
Bryan G. Fry
1,2
, Nicolas Vidal
3,4
, Janette A. Norman
2
, Freek J. Vonk
5
, Holger Scheib
6,7
, S. F. Ryan Ramjan
1
,
Sanjaya Kuruppu
8
, Kim Fung
9
, S. Blair Hedges
3
, Michael K. Richardson
5
, Wayne. C. Hodgson
8
,
Vera Ignjatovic
10,11
, Robyn Summerhayes
10,11
& Elazar Kochva
12
Among extant reptiles only two lineages are known to have
evolved venom delivery systems, the advanced snakes and helo-
dermatid lizards (Gila Monster and Beaded Lizard)
1
. Evolution of
the venom system is thought to underlie the impressive radiation
of the advanced snakes (2,500 of 3,000 snake species)
2–5
.In
contrast, the lizard venom system is thought to be restricted to
just two species and to have evolved independently from the snake
venom system
1
. Here we report the presence of venom toxins in
two additional lizard lineages (Monitor Lizards and Iguania) and
show that all lineages possessing toxin-secreting oral glands form
a clade, demonstrating a single early origin of the venom system in
lizards and snakes. Construction of gland complementary-DNA
libraries and phylogenetic analysis of transcripts revealed that
nine toxin types are shared between lizards and snakes. Toxino-
logical analyses of venom components from the Lace Monitor
Varanus varius showed potent effects on blood pressure and
clotting ability, bioactivities associated with a rapid loss of con-
sciousness and extensive bleeding in prey. The iguanian lizard
Pogona barbata retains characteristics of the ancestral venom
system, namely serial, lobular non-compound venom-secreting
glands on both the upper and lower jaws, whereas the advanced
snakes and anguimorph lizards (including Monitor Lizards, Gila
Monster and Beaded Lizard) have more derived venom systems
characterized by the loss of the mandibular (lower) or maxillary
(upper) glands. Demonstration that the snakes, iguanians and
anguimorphs form a single clade provides overwhelming support
for a single, early origin of the venom system in lizards and snakes.
These results provide new insights into the evolution of the venom
system in squamate reptiles and open new avenues for biomedical
research and drug design using hitherto unexplored venom
proteins.
In helodermatid lizards, venom is made by a gland on the lower
jaw from which ducts lead onto grooved teeth along the length of the
mandible
1
. In contrast, snake venom is produced by specialized
glands in the upper jaw
1,6–12
and is a shared derived trait of the
advanced snakes
2–5,13
. To investigate the evolution of venomous
function in squamates we first obtained sequence data from five
nuclear protein-coding genes representing major squamate lineages.
Our phylogenetic analyses show that the closest relatives of snakes are
the anguimorph (which include the venomous helodermatids) and
iguanian lizards (Fig. 1, and Supplementary Fig. 1). These results
LETTERS
Figure 1 |Relative glandular development and timing of toxin recruitment
events mapped over the squamate reptile phylogeny. Mucus-secreting
glands are coloured blue; the ancestral form of the protein-secreting gland
(serial, lobular and non-compound) red; the complex, derived form of the
upper snake-venom gland (compound, encapsulated and with a lumen)
fuchsia, and the complex, derived form of the anguimorph mandibular
venom gland (compound, encapsulated and with a lumen) orange. 3FTx,
three-finger toxins; ADAM, a disintegrin and metalloproteinase; CNP-BPP,
C-type natriuretic peptide-bradykinin-potentiating peptide; CVF, cobra
venom factor; NGF, nerve growth factor; VEGF, vascular endothelial growth
factor.
1
Australian Venom Research Unit, Level 8, School of Medicine, University of Melbourne, Parkville, Victoria 3010, Australia.
2
Population and Evolutionary Genetics Unit, Museum
Victoria, GPO Box 666E, Melbourne, Victoria 3001, Australia.
3
Department of Biology and Astrobiology Research Center, 208 Mueller Lab, Pennsylvani a State University,
University Park, Pennsylvania 16802-5301, USA.
4
UMS 602, Taxonomie et collections, Reptiles-Amphibiens, De
´partement Syste
´matique et E
´volution, Muse
´um National
d’Histoire Naturelle, 25 Rue Cuvier, Paris 75005, France.
5
Institute of Biology, Leiden University, Kaiserstraat 63, PO Box 9516, 2300 RA, Leiden, The Netherlands.
6
Department
of Structural Biology and Bioinformatics, University of Geneva and Swiss Institute of Bioinformatics, Centre Me
´dical Universitaire, 1 Rue Michel-Servet, 1211 Geneva 4,
Switzerland.
7
SBC Lab AG, Seebu¨elstrasse 26, 8185 Winkel, Switzerland.
8
Monash Venom Group, Department of Pharmacology, Monash University, Clayton, Victoria 3800,
Australia.
9
Molecular and Health Technologies, CSIRO, 343 Royal Parade, Parkville, Victoria 3010, Austra lia.
10
Department of Pathology, University of Melbourne, Parkville,
Victoria 3010, Australia.
11
Murdoch Children’s Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Austr alia.
12
Department of Zoology,
Tel Aviv University, Tel Aviv 69978, Israel.
Vol 439|2 February 2006|doi:10.1038/nature04328
584
© 2006 Nature Publishing Group
represent a major paradigm shift in the understanding of squamate
evolution. The three lineages were previously considered as part of
a large, unresolved polytomy that also included amphisbaenian,
lacertid and teiioid lizards
14,15
. We subsequently mapped the struc-
ture of protein-secreting oral glands (mandibular and maxillary)
over our revised squamate phylogeny (Fig. 1). The anguimorphs,
iguanians and snakes, which form a well-resolved clade, are shown to
be the only lineages possessing protein-secreting mandibular and/or
maxillary glands. The presence of a protein-secreting gland is
therefore a shared derived trait of this entire clade. The basal
condition of a serial, lobular and non-compound protein-secreting
gland, present in both mandibular and maxillary regions, is retained
in the iguanian lizards (Fig. 2). The restriction of protein-secreting
function to the maxillary (advanced snakes) or mandibular (angu-
imorphs) glands represents highly derived conditions. In the respect-
ive regions, the snakes (maxillary) and anguimorphs (mandibular)
have independently evolved complex, compound venom glands
with encapsulation and lumen formation for the storage of liquid
venom for ready delivery
1,16,17
. Some snakes (for example Natrix) still
express proteins in their serial, lobular, non-compound mandibular
glands, whereas the anguimorphs have lost the maxillary glands
entirely
18
.
To explain the distribution, recruitment
19
and molecular evolu-
tion
20
of venom proteins among them, representatives of the
anguimorph/iguanian/serpent clade, spanning a wide ecological
breadth, were also investigated for the secretion of toxins through
the construction of cDNA libraries. Mandibular gland cDNA
libraries were constructed for the Eastern Bearded Dragon (an
iguanian) and four varanids (anguimorphs); maxillary-gland
cDNA libraries were also constructed for the Eastern Bearded
Dragon. To provide greater coverage of venom evolution, maxillary-
gland cDNA libraries were also constructed from seven advanced
snake families. Transcripts coding for previously characterized lizard
or snake toxin types were identified in all gland cDNA libraries and
we report the presence of venom toxins in lizards (other than
Heloderma). Nine toxin types were recovered from both lizard and
snake cDNA libraries (AVIT, B-type natriuretic peptide (BNP),
CRISP, cobra venom factor, crotamine, cystatin, kallikrein, nerve
growth factor and vespryn), being secreted from mandibular and
maxillary glands. Bayesian phylogenetic analyses
13,19
of these nine
toxin types resulted in the monophyly of each venom toxin to the
Figure 2 |Transverse section of Pogona barbata (Eastern Bearded Dragon)
head to show relative arrangement of glands. Stain: Masson’s trichrome.
Original magnification £40. Abbreviations: d, duct; es, eye socket; ilg,
infralabial gland; lt, lower tooth; mn, mandible; mnivg, mandibular
incipient venom gland; mx, maxillary; mxivg, maxillary incipient venom
gland; pg, palatine gland; slg, supralabial gland; ut, upper tooth.
Figure 3 |Bioactivity of V. varius (Lace Monitor) venom. a, The effect of
different molar concentrations of purified type III PLA
2
toxin DQ139930 on
platelet aggregation. The control was saline. Grey bars, 2
m
M ADP; black
bars, 30
m
M adrenaline; white bars, 5
m
M adrenaline. b, Effect of intravenous
injection of crude venom (1 mg kg
21
) on blood pressure in the anaesthetized
rat. c,d, Effect of crude venom (200
m
gml
21
) on U46619 precontracted
endothelium-intact (c) and endothelium-denuded (d) aortic rings. n¼3;
single traces are shown.
NATURE|Vol 439|2 February 2006 LETTERS
585
© 2006 Nature Publishing Group
exclusion of related non-venom proteins. This pattern strongly
supports single early recruitment events
13
for each toxin type
before the separation of snakes, iguanians and anguimorphs (Sup-
plementary Figs 2–10). An additional toxin, type III phospho-
lipase A
2
(PLA
2
), previously characterized only from Heloderma
venoms
21
(Gila Monster and Beaded Lizard), was identified in
varanid mandibular glands.
The mapping of toxin types, revealed in this study as being secreted
in both mandibular and maxillary glands, over the revised squamate
phylogeny provides additional insights into the evolution of the
reptile venom chemical arsenal (Fig. 1). Most striking is the proposed
complexity of the venom secretions in the common ancestor of
venomous lizards and snakes with nine toxin types present. Seven of
these were previously known only from snake venoms, including one
toxin type (crotamine), sequenced from the Eastern Bearded Dragon
mandibular and maxillary glands, previously characterized only
from rattlesnake venoms. The nine shared venom toxins isolated
all possess previously well-characterized activities, including hypo-
locomotion, hypotension, hypothermia, immunomodulatory
effects, intestinal cramping, myonecrosis, paralysis of peripheral
smooth muscle unregulated activation of the complement cascade,
and hyperalgesia
19
. The type III PLA
2
toxins from Heloderma venoms
have been shown to block platelet aggregation
21
. Some of these toxins
have been shown to have potent systemic effects, such as the profound
hypotension produced by kallikrein and natriuretic toxins
19
leading
to rapid loss of consciousness, or coagulation disorders such as
prolonged bleeding as a consequence of the Heloderma type III PLA
2
toxins
21
. Some toxins exert effects that, although non-lethal, may aid
in the rapid incapacitation of prey items or potential predators, such
as the markedly increased sensitivity to pain (hyperalgesia) and
strong cramping produced by the AVIT toxins
19
.
Varanid venom was revealed by toxinological analyses to be as
complex and potent as previously analysed reptile venoms
5,22
. Liquid
chromatography/mass spectrometry
5
(LC/MS) showed the varanid
secretions to be rich in proteins with molecular masses consistent
with the following toxin types sequenced from varanid cDNA
libraries: natriuretic (2–4 kDa), type III PLA
2
(about15 kDa),
CRISP (23–25 kDa) and kallikrein (23–25 kDa) (Supplementary
Fig. 11). Haematological assays of varanid type III PLA
2
toxin
(DQ139930) purified by reverse-phase high-performance liquid
chromatography
23
showed inhibition of platelet aggregation
(Fig. 3a). Consistent with the same bioactivity as Heloderma type
III PLA
2
(ref. 21) was the preservation of cysteines and cysteine
spacing as well as the conservation of functional residues (Sup-
plementary Fig. 12A). As cDNA sequencing and LC/MS analysis
indicated a high concentration of kallikrein and BNP-type natriuretic
toxins in the varanid secretions, additional assays investigated
hypotension-inducing bioactivity. Intravenous injections of crude
Varanus varius mandibular secretion to anaesthetized rats rapidly
produced a sharp drop in blood pressure (Fig. 3b) and specific
analyses with precontracted rat aortic rings
23
demonstrated the
natriuretic peptide action of relaxation of aortic smooth muscle
(Fig. 3c, d). Consistent with the preserved bioactivity of the varanid
BNP-type natriuretic toxins, sequence analysis and molecular
modelling revealed the retention of residues necessary for natriuretic
activity
23
(Fig. 4, and Supplementary Fig. 10). The varanid forms of
the kallikrein toxins also show conservation of functional residues
and cysteine spacing (Supplementary Fig. 12B). In the CRISP toxins,
the varanid forms all have the loop I doublet (KR) that has been
proposed to be an essential part of the blockage of cyclic-nucleotide-
gated calcium channels (Supplementary Fig. 13). Most varanid
CRISP isoforms also have the loop I motif (EXXF) thought to
contribute to the inhibition of smooth muscle contraction through
the blockage of L-type Ca
2þ
channels (Supplementary Fig. 13). Other
toxin types vary to differing degrees in the relative conservation of
molecular characteristics.
The combined cDNA, LC/MS, molecular modelling and pharma-
cological results are consistent with effects reported for varanid bites,
Figure 4 |Comparative modelling of representative natriuretic peptides.
a, GNP-1 (DQ139927) from V. varius (Lace Monitor); c, TNP-c (P83230)
from Oxyuranus microlepidotus (Inland Taipan); d, DNP (P28374) from
Dendroaspis angusticeps (Eastern Green Mamba); e, Lebetin (Q7LZ09) from
Macrovipera lebetina (Elephant Snake); f, BNP from the rat (P13205) brain
and atria. Blue surface areas indicate positive charges, red areas show
negative charges. Model pairs show the sides of the protein rotated by 1808.
b, GNP in ribbon representation coloured by alignment diversity
29
.
Conserved positions are in blue, brighter colours indicate increasing degree
of variation. Functional residues
23
are CPK (Corey–Pauling–Koltun)
coloured by amino-acid type
29
. Hydrophobic residues are in grey, arginine in
blue. The conserved cysteines are shown as sticks. To minimize confusion,
all sequences are referred to by their SWISS-PROT accession numbers
(http://www.expasy.org/cgi-bin/sprot-search-ful).
LETTERS NATURE|Vol 439|2 February 2006
586
© 2006 Nature Publishing Group
which include severe pain, breathing difficulties, skeletal muscle
weakness and tachycardia
24
. One of the authors (B.G.F.) has also
acted as consultant on three varanid bites by captive bred specimens
(Varanus komodoensis (Komodo Dragon), V. scalaris (Spotted Tree
Monitor) and V. varius (Lace Monitor)), each of which resulted in
rapid swelling (noticeable within minutes), dizziness, localized dis-
ruption of blood clotting and shooting pain extending from the
affected digit up to the elbow, with some symptoms lasting for several
hours. The rapidity and pathology are consistent with bioactive
secretions rather than bacterial infection. In addition, varanid
venom also has been shown to have the ability to rapidly paralyse
small animals such as birds
25
.
As well as providing evidence about the role of bioactive secretions
in the effects produced by varanid bites, the complex and bioactive
secretions present in ‘non-venomous’ lizards forces a fundamental
rethinking of the very concept of ‘non-venomous’ reptile. The
evolution of venomous function is considered to be a key innovation
driving ecological diversification in advanced snakes
2–5
. Our results
indicate that the single origin of venom in squamate reptiles might
also have been a key factor in the adaptive radiation and subsequent
ecological success of lizard lineages such as anguimorphs and
iguanians; the well-supported anguimorph/iguanian/serpent clade
represents about 4,600 out of about 7,900 extant squamate species, or
58% of the total squamate species diversity. There is also palaeonto-
logical evidence for the presence of venom delivery systems in some
Upper Cretaceous anguimorphs and snakes
8,26,27
. Because fossil data
indicate that the clade containing anguimorphs, iguanians and
snakes dates from Late Triassic/Early Jurassic
28
, we infer that the
venomous function arose once in squamate evolution, at about
200 Myr ago. This considerably revises previous estimates of about
100 Myr ago based on the assumed independent origin of venomous
function in snakes and lizards.
Additional work aimed to investigate this special area of reptile
evolution would include investigating the mandibular or maxillary
secretions of all main lineages belonging to the toxin-secreting clade.
Studies of additional transcriptomes may reveal earlier recruitment
of a particular toxin type, such as those currently sequenced only
from snake venoms for example, or may discover previously
unrecognized toxin types. Further pharmacological investigations
may shed more light upon the bioactivities and the relative use in
defence, in prey capture or in predigestion. Because increased
complexity of the venom gland was shown in this study to be linked
to additional toxin recruitment events (Fig. 1), future work should
include an exploration of the relationship between glandular com-
plexity and the relative toxicity of the venom. The new lizard venom
toxins exhibit considerable sequence diversity consistent with the
birth-and-death mode of protein evolution that has given rise to a
wide diversity of bioactivities in snake venoms
20
. These molecules
represent a tremendous hitherto unexplored resource not only for
understanding reptile evolution but also for use in drug design and
development.
METHODS
Toxin molecular evolution. Specimen collection localities: Azemiops feae
(Fea’s Viper), Guizhou, China; Enhydris polylepis (Macleay’s Water Snake),
Darwin, Northern Territory, Australia; Dispholidus typus (Boomslang), Uganda;
Oxyuranus microlepidotus (Inland Taipan), progeny of captive specimens from
Goydnor’s Lagoon, South Australia; Pogona barbata (Eastern Bearded Dragon),
progeny of Brisbane, Queensland, Australia locality captive specimens;
Telescopus dhara (Egyptian Catsnake), Egypt; Trimorphodon biscutatus (Lyre
Snake), Arizona, USA; Liophis poecilogyrus (Gold-bellied Snake), Paraguay;
Leioheterodon madagascariensis (Madagascar Giant Hognosed Snake), Mada-
gascar; Philodryas olfersii (Argentine Racer), Argentina; Rhabdophis tigrinus
(Tiger Keelback), Hunan, China; Varanus acanthurus (Spiny-tailed Monitor),
progeny of captive specimens collected from Newman, Western Australia;
Varanus mitchelli (Mitchell’s Water Monitor), Kununurra, Western Australia;
Varanus panoptes rubidus (Desert Spotted Goanna), Sandstone, Western Aus-
tralia; Varanus varius (Lace Monitor), Mallacoota, Victoria, Australia.
RNA extraction and construction of cDNA library: these steps were under-
taken with the Qiagen RNeasy and Oligotex messenger RNA kits and the Creator
SMART cDNA Library Construction Kit from BD Biosciences. Full details are
available in Supplementary Information.
Histology. Tissue was dissected from animals after killing, then fixed in Bouin’s
fluid and decalcified overnight in acid alcohol (5% HCl in 70% ethanol). The
tissues were dehydrated in graded ethanols, cleared in Histoclear and embedded
in paraffin. Sections were cut to 10
m
m thickness and stained with Masson’s
trichrome (Goldner’s modification), Alcian blue at pH 1.0 and 2.4 alone or
counterstained with haematoxylin–eosin or periodic acid Schiff in accordance
with standard techniques.
Molecular modelling. Three-dimensional models were generated by aligning
target sequences to the 1Q01 template with SPDBV
29
. Sequence-to-structure
alignments were sent to the Swiss-Model server. For the resulting models a van
der Waals surface was calculated in MolMol
30
. Surfaces were coloured by a
‘simplecharge’ potential as calculated in MolMol.
Pharmacology. Male rats were anaesthetized with sodium pentobarbitone (60–
100 mg kg
21
, intraperitoneally). Venom or vehicle (namely 0.1% BSA) was
administered through the jugular-vein cannula. Blood pressure was measured
with a Gould (P23) pressure transducer attached to a carotid artery cannula, and
recorded on a MacLab system.
Platelet aggregometry. Blood samples were collected from normal, healthy adult
volunteers (n¼2) who had not taken any medication during the week before
collection. Whole-blood samples were mixed 9:1 with 0.106 M sodium citrate.
Citrated blood samples were centrifuged for 10min at 100 gat 20 8C. The
supernatant platelet-rich plasma (PRP) was removed and rested at room
temperature for 30 min before assay. Platelet-poor plasma (PPP) for each
volunteer was also prepared (by centrifugation for 20 min at 3,500 g) for platelet
aggregometry. Platelet aggregation was measured with the Aggram platelet
aggregometer and Hemoram software (Helena Laboratories). PRP aliquots
(225
m
l) were incubated in glass cuvettes for 2 min at 378C. Purified Varanus
varius PLA
2
toxin DQ139930 (60, 6 and 0.6
m
M) was added to the PRP and
incubated at 37 8C for 3 min before the addition of agonist (2
m
M ADP, or 5 or
30
m
M adrenaline), to induce platelet aggregation, or sterile saline as a control.
Platelet aggregation was monitored for 10 min. The degree of maximum platelet
aggregation was assessed by measuring the optical transmission of light, zeroed
with the appropriate PPP for each patient sample, through the PRP samples and
compared with the controls (PRP samples assayed without the addition of
toxin).
Squamate reptile phylogeny. Recent studies
14,15
included representatives of all
major squamate lineages and identified a clade comprising the following five
lineages: first, Scincoidea (Scincidae, Xantusiidae and Cordylidae); second,
Teiioidea (Teiidae and Gymnophthalmidae), Lacertidae and Amphisbaenia
(Rhineuridae, Bipedidae, Trogonophidae and Amphisbaenidae); third, Iguania
(Iguanidae, Agamidae and Chamaeleontidae); fourth, Anguimorpha (Varanidae,
Helodermatidae, Anguidae, Shinisaurus and Xenosaurus); and fifth, Serpentes. The
venomous squamates (advanced snakes and helodermatid lizards) are included
in this large clade, whereas two other families of squamates (Gekkonidae and
Dibamidae) are excluded
14,15
. We focused on this clade and sampled five nuclear
protein-coding genes, including two genes (C-mos and RAG1) used in other
studies and three genes (RAG2,R35 and HOXA13) not used previously to clarify
squamate phylogeny. Phylogenies were built with probabilistic approaches
(maximum-likelihood (ML) and bayesian methods of inference). Because
separate analyses showed no significant topological incongruence, we performed
combined analyses, which are considered to be our best estimates of phylogeny.
Scincoidea was used as the outgroup because it was shown to be the most basal
lineage of the clade
14,15
. The bayesian and ML trees obtained were identical and
showed significant support for most nodes (see Supplementary Information). In
particular, a clade that includes Serpentes, Iguania and Anguimorpha was
resolved (bayesian posterior probability 100%; ML bootstrap value 99%). In
turn, we found that the closest relative of this clade is one comprising Teiioidea,
Lacertidae and Amphisbaenia. Full details are available in Supplementary
Information.
Received 13 July; accepted 17 October 2005.
Published online 16 November 2005.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank the following persons and institutions who
helped us or contributed tissue samples used in this study: A. Fry, Alice Springs
Reptile Centre, Australian Reptile Park, M. A. G. de Bakker, R. L. Bezy, B. Branch,
J. Campbell, N. Clemann, C. Clemente, C. Cicero, K. Daoues, A. S. Delmas,
B. Demeter, J. Haberfield, A. Hassanin, Healesville Sanctuary, M. Hird, Louisiana
State University Museum of Zoology, P. Moler, T. Moncuit, P. Moret, National
Museum of Natural History Naturalis Leiden (J. W. Arntzen), T. Pappenfus,
J.-C. Rage, C. Skliris, J. Smith, S. Sweet, Ultimate Reptiles (South Australia),
University of California Museum of Vertebrate Zoology (Berkeley), J. Walker,
R. Waters, J. Weigel and B. Wilson. We also thank A. Webb and T. Purcell for
providing HPLC access; N. Williamson for help with preliminary mass
spectrometry characterization; E. V. Grishin for help in obtaining the references
in Russian; S. Edwards for comments; and T. van Wagner and V. Wexler for
artwork. This work was funded by the Service de Syste
´matique mole
´culaire of
the Muse
´um National d’Histoire Naturelle, Institut de Syste
´matique (N.V.) and
by grants from the Australian Academy of Science (B.G.F.), Australian
Geographic Society (B.G.F.), Australia & Pacific Science Foundation (B.G.F.),
Australian Research Council (B.G.F.), CASS Foundation (B.G.F.), Commonwealth
of Australia Department of Health and Aging (B.G.F.), Ian Potter Foundation
(B.G.F.), International Human Frontiers Science Program Organisation (B.G.F.),
Leiden University (F.J.V., M.K.R.), NASA Astrobiology Institute (S.B.H.), National
Science Foundation (S.B.H.) and University of Melbourne (B.G.F.). We thank the
relevant wildlife departments for granting the scientific permits for field
collection of required specimens.
Author Information The sequences of the cDNA clones have been deposited in
GenBank (accession numbers DQ139877–DQ139931 and DQ184481), as have
the nuclear gene sequences (DQ119594–DQ119641). Reprints and permissions
information is available at npg.nature.com/reprintsandpermissions. The authors
declare no competing financial interests. Correspondence and requests for
materials should be addressed to B.G.F. (bgf@unimelb.edu.au).
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