A Limited Role for Gene Duplications in the Evolution of
Platypus Venom
Emily S. W. Wong,*,1 Anthony T. Papenfuss,2 Camilla M. Whittington,1 Wesley C. Warren,3 and
Katherine Belov*,1
1
Faculty of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia
Bioinformatics Division, The Walter and Eliza Hall Institute for Medical Research, Parkville, Victoria, Australia
3
The Genome Center, Washington University School of Medicine
*Corresponding author: E-mail: [email protected]; [email protected].
Associate editor: Willie Swanson
2
Abstract
Key words: gene duplications, venom, platypus, evolution.
Introduction
Platypuses are venomous mammals. Male platypuses possess spurs on their hind legs that deliver venom during the
breeding season. In humans, envenomation causes both excruciating immediate and long-lasting pain that does not
respond to traditional painkillers such as morphine. Other
symptoms of envenomation include edema, a drop in blood
pressure, and blood coagulation (Martin and Tidswell 1895;
Fenner et al. 1992). Proteomic studies in the 1990s revealed
that platypus venom contains at least 19 different peptide
components (de Plater et al. 1995) of which only three
fractions were characterized: C-type natriuretic peptides,
defensin-like peptides, and nerve growth factors. More recently, a molecular transcriptomic study of an in-season
venom gland led to the identification of proteases, spider
venom alpha-latrotoxin-like peptides, cysteine-rich secretory proteins, cytolytic toxin-like peptides, and venom proteins with homology to stonefish stonustoxin (Whittington
et al. 2010).
Similar venom toxin gene families are found in a diverse
range of venomous animals including shrews, snakes and
other reptiles, amphibians, fish, mollusks, insects, cnidarians,
and echinoderms. These toxins have evolved in a convergent manner in different lineages independently (Fry et al.
2009). Protein scaffolds that are recruited into venom peptides include chitinase, cystatin, defensin, hyaluronidase,
Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1,
phospholipase A2, sphingomyelinase D, SPRY, and cysteine-rich secretory proteins including the AVIT family
(Fry et al. 2009).
Gene duplications appear to play an important role in
the recruitment and diversification of toxin genes (Kordis
and Gubensek 2000; Reza et al. 2006; Lynch 2007; Juárez
et al. 2008). The evolutionary fate of duplicated genes
was first developed by Ohno (1970). He proposed that immediately following a duplication event, one gene copy is
expected to maintain the ‘‘ancestral’’ role, whereas the other
copy is free to evolve new function (neofunctionalization).
It is now accepted that duplicated genes can be also maintained through the sharing of ancestral gene function (subfunctionalization) and that selective pressures can play
a role in the preservation of a gene duplicate (Bergthorsson
et al. 2007). Across frog species, caeruleins, which are skin
toxin decapeptides, have arisen by independent duplications
(Roelants et al. 2010). Venom serine proteases in the North
© Crown copyright 2011.
Mol. Biol. Evol. 29(1):167–177. 2012 doi:10.1093/molbev/msr180
Advance Access publication August 3, 2011
167
Research article
Gene duplication followed by adaptive selection is believed to be the primary driver of venom evolution. However, to date,
no studies have evaluated the importance of gene duplications for venom evolution using a genomic approach. The
availability of a sequenced genome and a venom gland transcriptome for the enigmatic platypus provides a unique
opportunity to explore the role that gene duplication plays in venom evolution. Here, we identify gene duplication events
and correlate them with expressed transcripts in an in-season venom gland. Gene duplicates (1,508) were identified. These
duplicated pairs (421), including genes that have undergone multiple rounds of gene duplications, were expressed in the
venom gland. The majority of these genes are involved in metabolism and protein synthesis not toxin functions. Twelve
secretory genes including serine proteases, metalloproteinases, and protease inhibitors likely to produce symptoms of
envenomation such as vasodilation and pain were detected. Only 16 of 107 platypus genes with high similarity to known
toxins evolved through gene duplication. Platypus venom C-type natriuretic peptides and nerve growth factor do not
possess lineage-specific gene duplicates. Extensive duplications, believed to increase the potency of toxic content and
promote toxin diversification, were not found. This is the first study to take a genome-wide approach in order to examine
the impact of gene duplication on venom evolution. Our findings support the idea that adaptive selection acts on gene
duplicates to drive the independent evolution and functional diversification of similar venom genes in venomous species.
However, gene duplications alone do not explain the ‘‘venome’’ of the platypus. Other mechanisms, such as alternative
splicing and mutation, may be important in venom innovation.
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Wong et al. · doi:10.1093/molbev/msr180
American shrew have evolved adaptively through amino
acid changes to regulatory loops surrounding the active site,
also following gene duplications (Aminetzach et al. 2009).
Further examples of gene duplications in venom include
contoxins in the cone snail (Duda and Palumbi 1999),
a multitude of gene families including phospholipase A2
(Gutiérrez and Lomonte 1995), serine proteases (Kini
2005), serine protease inhibitors (Zupunski et al. 2003),
C-type lectins (Ogawa et al. 2005), coagulation factor V
(Minh Le et al. 2005; Reza et al. 2006), and three finger toxins (Fry et al. 2003), across a range of snake species and
cysteine-enriched toxins in scorpions (Zhijian et al. 2006).
Venom toxins across animal taxa are members of some
of the fastest duplicating gene families in humans: serine
proteases, defensins, and natural killer cell (NKC) receptor
genes (lectins) (Bailey et al. 2002). In humans, these genes
are involved in pathogen defense. In platypus, antimicrobial
peptides have given rise through tandem gene duplications
to venom peptides (Whittington et al. 2008). The rapid duplication rate in immune genes, driven by intense competition between host and pathogen, is likely to be favorable
toward the initial creation of new traits, such as venom
function. This is then followed by subsequent duplications
that are likely to reinforce the venom system. Venom genes,
like immune genes, are also likely to face strong adaptive
pressures. In most venomous animals, such pressure is
brought about through predator–prey interactions where
venom is used to immobilize prey and defend against predators and/or circumvent prey resistance (Poran et al. 1987;
Daltry et al. 1996; Biardi et al. 2006; Calvete et al. 2009).
However, in platypus, this adaptive pressure is most likely
exerted by intraspecific competition, to assert dominance
over other males during the breeding season (Grant 2007).
Prior studies on the selective constraints of gene duplicates in venom have focused on the venomous snakes and
the cone snail. In snakes, venom gene families such as phospholipase A2, serine protease, C-type lectin-like proteins,
metalloproteinases, and serine proteinase inhibitors appear
to have evolved through neofunctionalization under strong
selective constraints (Kordis and Gubensek 2000; Lynch
2007; Juárez et al. 2008; Aminetzach et al. 2009; Casewell,
Wagstaff, Harrison, Renjifo, et al. 2011). In the venomous
gastropod genus Conus, a number of contoxin families
block sodium and calcium channels and neural receptors
in prey species and have diversified their gene function
through strong positive selection following extensive duplications (Duda and Palumbi 1999). In addition to toxin peptides that have evolved through gene duplications, genes
involved in the biosynthesis of toxins, such as the sxt gene
cluster involved in the production of cyanobacteria neurotoxins, have also undergone duplications followed by adaptive evolution (Murray et al. 2011).
Although previous studies suggest that duplicated genes
are the major driver of venom evolution, the direct impact
of gene duplications on the evolution of venom has not
been characterized. This is primarily due to difficulties in
assigning gene duplicates in the absence of a sequenced
genome. Given that the platypus is the only venomous
168
animal with a sequenced genome (Warren et al. 2008)
and venom gland transcriptome (Whittington et al.
2010), we have taken a novel genome-wide approach to
assess how duplications have contributed to venom function. To place the role of gene duplication in the context of
platypus venom innovation, here, we identify lineagespecific gene duplicates in the platypus genome that are
also expressed in an in-season venom gland. Our results
suggest that gene duplications do not appear to be the major driver of toxin evolution in the platypus.
Materials and Methods
Identification of Platypus-Specific Gene
Duplications
We used homology data from Ensembl Compara Release 61
to identify all platypus genes that are duplicated in the
monotreme lineage including genes that are derived from
multiple lineage-specific duplications (Vilella et al. 2009;
Flicek et al. 2010). All cDNA and protein sequences from
monotreme gene duplicates were extracted. We took into
consideration the fact that gene loss in other lineages will
lead to incorrect inferences of monotreme duplications. A
phylogenetic tree may appear to show a species-specific
duplication when, in fact, the topology is the result of
multiple gene losses in other species (Casewell, Wagstaff,
Harrison, and Wüster 2011). However, due to the large
number of species in Ensembl, we expect this type of error
to be negligible. A list of nonredundant duplicated gene pairs
containing monotreme-specific duplication events was
constructed (supplementary file 1, Supplementary Material
online). Each pair contained at least one gene expressed in
the venom gland. We also constructed expansion groups
comprising two or more genes where at least one gene
is expressed in the venom gland. To confirm Ensembl gene
predictions in the absence of expression in the venom
gland, we checked for the expression of these genes in other
tissue transcriptomes.
Identification of Gene Duplicates Expressed in the
Venom Gland
A normalized cDNA library from a breeding season platypus venom gland was sequenced using the Illumina platform, as described in Whittington et al. 2010. The raw data
set comprised 19,069,168 reads of 36 nucleotides in length.
These reads were mapped against the platypus genome assembly (5.0) using MAQ (Li), and the number of reads
matching each Ensembl platypus gene was tallied and used
as digital expression values (see Whittington et al. 2010).
Multimapped reads were treated in a uniform random
manner to increase transcriptomic coverage and decrease
false negatives in the detection of expressed duplicates. Only monotreme genes with read counts above 100 were used
for further analysis. Protein searches were conducted using
BLASTP (Altschul et al. 1997) against Tox-Prot, a UniProtKB/
Swiss-Prot database of animal toxins (Release 2010_10) (Jungo and Bairoch 2005) with an E value cutoff of 1 105.
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Identification of Secreted Gene Duplicates
Table 1. Number of Monotreme-Specific Duplications.
To date, all known toxin peptides are secretory proteins
(Fry et al. 2009). We used SignalP 3.0 hidden Markov models
(Bendtsen et al. 2004) and Gene Ontology (GO) (Ashburner
et al. 2000) terms assigned by basic local alignment search
tool (BLAST) to predict the cellular localization of expressed
protein sequences. Disulfide cross-linking, common to
secreted proteins, enhances stable tertiary structures (Mamathambika and Bardwell 2008). To confirm that our predictions are secreted, we compared the ratio of cysteine
residues with other amino acid residues in secretory and
nonsecretory peptides. Functional annotations were inferred by sequence similarity to human proteins. However,
we note that as these genes are the result of gene duplication, it is possible that completely novel function has
evolved.
Genes
Gene pairs
Number of duplication events
Pairs of duplicates
PAML
Test for Adaptive Selection
We used PAML (Yang 2007) to obtain dn/ds values for each
pair of duplicates under the pairwise M0 model. Since an
averaged omega ratio (M0) over the entire length of a gene
is not a sensitive measure of positive selection, which may
only occur on a small subset of sites (Anisimova et al. 2001),
we tested branches of venom gland expressed genes for
adaptive selection using the branch-site test, ‘‘test 2.’’
We note that both these approaches give conservative estimations of positive selection. Branch lengths taken from
M0 model runs were used as initial branch lengths in the
branch-site test. Pairs where branch lengths were greater
than five nucleotide substitutions per codon were removed
from further analysis. Alignments were constructed using
PRANK and a codon substitution matrix (Löytynoja and
Goldman 2008). Two test 2 runs were conducted. Sequences showing convergence of likelihood values with a difference of ,0.001 were kept. Alignments were carefully
checked to ensure gene duplicates were not incorrectly assigned due to short contig lengths. The chi-square distribution with one degree of freedom was used to evaluate
the likelihood ratio test statistic for positive selection.
FDR was used to adjust P values for multiple testing (Hochberg and Benjamini 1990). We checked for significant (P ,
0.05) cases of gene conversions using GENECONV (Sawyer
1999).
Phylogenetic Trees
Evolutionary relationships of protease protein sequences
were constructed using MEGA5 (Tamura et al. 2007).
The bootstrapped (500 replicates) trees were inferred using
the neighbor-joining (NJ) method with evolutionary distances computed using the Poisson correction method. Ambiguous positions were removed for each sequence pair.
Results
Identification of Duplicated Gene Groups
Fifty-six percent (10,676,863 reads) of the raw reads were
aligned to the platypus genome. We identified 378 pairs of
monotreme-specific duplicates, where at least one gene
3,741
1,508
421
378
159 pairs; 46 groups
NOTE.—‘‘Genes’’ denote all genes that are duplicated in the monotreme lineage.
‘‘Gene pairs’’ represent the number of duplications that have resulted in two
Ensembl genes. The ‘‘number of gene duplication events’’ denotes genes where
one or both duplicates are expressed. ‘‘Pairs of duplicates’’ consist of expressed
genes pair only. ‘‘PAML’’ duplicates include only genes that meet strict criteria;
these groups were subsequently used for selection tests.
copy (.100 Illumina reads) was one of the 5,410 protein-coding genes expressed in the platypus venom gland.
Of these, 327 showed venom gland expression in only one
duplicated copy, which is indicative of venom-specific
function evolution following gene duplication. A summary
of the number of monotreme-specific duplicates is shown
in table 1.
Gene family expansions were also identified. Forty-two
gene duplication events leading to a monophyletic clade
containing three or more genes were identified. In each
case, a gene duplication occurred within an internal, or ancestral, node. In the majority of cases, venom gland expression was not detected for all members of a clade. After
removing genes on short contigs, we found that the maximum number of genes expressed in the venom gland in
any monotreme-specific clade was three. We found evidence of expression of 34% of gene duplicates that were
not found in in-season venom gland in other tissue transcriptomes.
Duplicated Venom Genes
The vast majority of genes expressed in the venom gland
were not significantly similar to any known toxin proteins.
Only 107 genes (from 5,410 genes) matched existing toxin
and venom proteins from the Tox-Prot database (E value ,
1 105). Of these, only 16 genes were monotreme lineage-specific duplicates (supplementary file 2, Supplementary
Material online). Only two genes of these possessed signal
peptides (ENSOANG00000001418, ENSOANG00000012443).
To explore the possibility that unique toxin genes exist
in the platypus, it is necessary to distinguish likely toxin
peptides from genes with other functional roles that are
also expressed in the venom gland. We focused on expressed genes that have duplicated in the monotreme lineage and had features of secretory peptides, as we would
expect that these are more likely to have toxin function
(Fry et al. 2009). Forty-two putative secretory genes were
identified using a predictive algorithm implemented in SignalP and GO term analysis based on similarity to human
secretory proteins (table 2). GO terms were used due to the
fragmentation of the platypus assembly, and we recognize
that some secreted genes may have truncated 5# termini,
resulting in missing signal motifs and nondetection using
SignalP.
We expected that the cysteine contents of putative secretory genes would be higher than average compared with
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Table 2. List of Duplicated Putative Platypus Venom Genes that Are Expressed in an In-Season Venom Gland and Are Likely to be Secreted.
Ensembl ID
Protein degradation
ENSOANG00000001418
ENSOANG00000001214
ENSOANG00000002285
ENSOANG00000013866
ENSOANG00000013077
ENSOANG00000008022
ENSOANG00000005148
Immunity
ENSOANG00000002927
ENSOANG00000002926
ENSOANG00000006911
ENSOANG00000001407
ENSOANG00000001539
ENSOANG00000021638
ENSOANG00000003125
ENSOANG00000007061
ENSOANG00000010729
ENSOANG00000003004
ENSOANG00000005314
ENSOANG00000014191
Cell regulation
ENSOANG00000015762
ENSOANG00000008500
Transport
ENSOANG00000012538
ENSOANG00000011884
ENSOANG00000007590
ENSOANG00000022698
ENSOANG00000008168
ENSOANG00000004183
Metabolism
ENSOANG00000006512
ENSOANG00000006519
ENSOANG00000010188
ENSOANG00000001438
ENSOANG00000000453
ENSOANG00000009571
Protein folding
ENSOANG00000001063
ENSOANG00000000957
RNA processing
ENSOANG00000005365
ENSOANG00000000815
Cell adhesion
ENSOANG00000015748
Other physiological function
ENSOANG00000012443
ENSOANG00000010405
ENSOANG00000011883
ENSOANG00000004710
Symbol
Description
Signal Peptide
CMA1
ERAP1
CUL9
TPSG1
TPSG1
ADAMDEC1
MBTPS1
Chymase 1, mast cell
ERAP1
Cullin 9
Tryptase gamma 1
Tryptase gamma 1
Disintegrin and metalloproteinase domain-like protein
Membrane-bound transcription factor peptidase, site 1
Yes
Yes
Yes
Yes
Trun
Yes
Yes
CCL3
CCL3L1
CXCL2
CFH
B2M
MR1
CD94
CLEC2L
TRDC
HV303
HV303
A2M
Chemokine (C-C motif) ligand 3
Chemokine (C-C motif) ligand 3-like 1
Chemokine (C-X-C motif) ligand 2
Complement factor H
B2M
Major histocompatibility complex, class I related
Killer cell lectin-like receptor subfamily
Killer cell lectin-like receptor subfamily
T-cell receptor delta chain C region
Immunoglobulin heavy chain V region
Immunoglobulin heavy chain V region
Alpha-2-macroglobulin
Yes
Yes
Yes
Trun
Yes
Yesa
Yes
Trun
Yes
Yes
Yes
Trun
AGGF1
NENF
Angiogenic factor with G patch and FHA domains 1
Neuron-derived neurotrophic factor
Trun
Yes
TRPV6
S28A2
S28A2
ACRV1
SLC2A5
YIF1A
TRP cation channel, subfamily V, member 6
Sodium/nucleoside cotransporter 2
Sodium/nucleoside cotransporter 2
Acrosomal vesicle protein 1
Solute carrier family 2
yip1 interacting factor homolog A (Saccharomyces cerevisiae)
Yes
Yes
Yes
Yes
Yes
Yes
BTN2A1
PIGG
MOXD1
HHIPL2
FUT3
CES1
Butyrophilin, subfamily 2, member A1
Phosphatidylinositol glycan anchor biosynthesis, class G
Monooxygenase, DBH-like 1
Hedgehog interacting protein-like 2
Galactoside 3(4)-L-fucosyltransferase
Carboxylesterase 1 (monocyte/macrophage serine esterase 1)
Yes
Yes
Yes
Yes
Yes
Yes
PPIL4
TOR3A
Peptidylprolyl isomerase (cyclophilin)-like 4
Torsin family 3, member A
Yes
Yes
INTS10
RBM28
Integrator complex subunit 10
RNA binding motif protein 28
Yes
Yes
ICAM3
Intercellular adhesion molecule 3
Yes
BTN1A1
EN DOD1
Butyrophilin, subfamily 1, member A1
Endonuclease domain containing 1
Serpin peptidase inhibitor, clade A
(alpha-1 antiproteinase, antitrypsin), member 3
Microfibril-associated glycoprotein 3
Yes
Yes
SERPINA3
MFAP3
Trun
Yes
NOTE.—Gene name and gene descriptions reflect the most similar human gene. ‘‘Trun’’ denotes truncated 5# end.
a
Curated gene prediction. Gene pairs in italics denote duplication pairs where both copies are expressed and possess signal peptides.
intracellular genes due to disulfide bonding which provide
structural stability (Clark 2005). The average ratios of cysteine versus noncysteine residues for secreted and nonsecreted genes were 1:52 (standard error [SE] 5 7) and 1:77
(SE 5 4), respectively.
Twelve of these genes share similarity with known
venom peptides in other species. These included five genes
involved in protein degradation (three serine proteases and
two metalloproteinase), two protease inhibitors, two C-type
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lectin genes, an isomerase, and one SPRY domain-containing
gene similar to snake Ohanin protein.
Adaptive Selection
Twenty of 159 pairs of duplicates (;12%) show evidence of
positive selection (FDR , 0.05) as identified using the
branch-site model implemented in PAML (test 2) (table 3).
To eliminate errors in our results, we took a series of steps
to ensure accurate inference of gene duplication and
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Table 3. Venom Gland-Expressed Monotreme-Specific Duplicate Pairs under Positive Selection.
Ensembl Gene ID
ENSOANG00000004421
Ensembl Protein ID
ENSOANP00000007003
ENSOANG00000011604
ENSOANG00000004530
ENSOANP00000018383
ENSOANP00000007182
ENSOANG00000003557
ENSOANG00000007061
ENSOANG00000006435
ENSOANG00000006437
ENSOANG00000021791
ENSOANG00000012329
ENSOANG00000019992
ENSOANG00000010184
ENSOANP00000005638
ENSOANP00000011236
ENSOANP00000010269
ENSOANP00000010273
ENSOANP00000027772
ENSOANP00000019509
ENSOANP00000025376
ENSOANP00000016146
ENSOANG00000012538
ENSOANG00000020293
ENSOANG00000009915
ENSOANG00000004696
ENSOANG00000008958
ENSOANG00000007394
ENSOANG00000010340
ENSOANG00000007030
ENSOANG00000019734
ENSOANP00000019830
ENSOANP00000025784
ENSOANP00000015714
ENSOANP00000007439
ENSOANP00000014266
ENSOANP00000011768
ENSOANP00000016404
ENSOANP00000011196
ENSOANP00000025024
Ensembl Family Description
ATP-binding cassette subfamily B
member P glycoprotein
Golgin subfamily A member 1
Disintegrin and metalloproteinase domain
containing precursor ADAM
Ring finger 112 zinc finger 179
C-type lectin domain familya
Interferon-induced GTP binding
Interferon-induced GTP binding
Coiled-coil domaina
Presplicing factor SLU7
Ig domain familya
Interferon induced with tetratricopeptide
repeats IFIT
TRP cation channel subfamily V
Unknown
Ig gamma chain C region
2-Hydroxyacyl coA lyase 1
Polyadenylation factor subunit 2
Methylthioribulose 1 phosphate dehydratase
Ig domain familya
ATP sythase subunit D
Aldo-keto reductase familya
P Value
FDR
2.04 3 1026
8.73 3 1026
3.67 3 1024
7.86 3 1024
1.83 3.25 3
7.92 1.19 3
1.19 3
1.38 3
1.92 3
2.26 3
105
1025
105
1024
1024
1024
1024
1024
1.10 1.46 3
2.85 3.07 3
3.07 3
3.09 3
3.46 3
3.69 3
103
1023
103
1023
1023
1023
1023
1023
3
3
3
3
3
3
3
3
3
3
1024
1024
1024
1023
1023
1023
1023
1023
1023
1023
8.13
8.13
8.96
1.35
2.39
2.39
2.49
2.69
4.11
4.76
3
3
3
3
3
3
3
3
3
3
1023
1023
1023
1022
1022
1022
1022
1022
1022
1022
5.69
5.87
6.97
1.12
2.19
2.26
2.49
2.84
4.58
5.56
NOTE.—Genes in bold are functionally related to known toxins.
a
A self-annotated gene family where Ensembl descriptions were unavailable.
accurate alignment. We filtered pairs based on gene lengths,
sequence divergence, and likelihood of past gene conversions in order to eliminate factors that can lead to incorrect
results. First, we removed gene duplicates that did not show
overlap in multiple alignments with other species. We then
aligned sequences using PRANK incorporating a codon substitution model. This produces alignments with a lower
number of false positive results compared with other multiple aligners (Fletcher and Yang 2010). Due to the fragmentation of the platypus genome assembly, many genes are
truncated. This could lead to incorrect assignment of gene
duplicate, where one gene, split over two or more contigs, is
considered to be two or more genes and treated as gene
duplicates. Truncated genes can also lead to gene prediction
errors, which may elevate the level of positive selection. To
overcome these issues, we only kept putative gene duplicates that overlapped by 60 bp or more when aligned. Short
uninformative alignments were removed from the selection
analysis. Highly divergent sequences can lead to spuriously
high levels of positive selection due to alignment errors, different codon usage, and different nucleotide composition
(Fletcher and Yang 2010). To address this, we filtered gene
pairs based on tree branch lengths. We further removed all
gene pairs that showed signs of gene conversion, as recombination events can also lead to elevated rates of adaptive
selection (Anisimova et al. 2003).
We also performed selection tests on duplication groups
(.2 genes) that are likely to have toxin roles. We identified
strong positive selection acting on the lineage of an expressed
protease gene (P ,, 0.001; ENSOANG00000002931) belonging to a group of four serine protease inhibitors that have
expanded in the monotreme lineage. All genes contained
the Kunitz-type domain, which has been identified in a wide
range of venomous species, including snake, wasp, scorpion,
sea anemones, and coral (Fry et al. 2009). The branch of an
expressed C-type lectin gene (ENSOANG00000019797), within
a monotreme group containing six genes, was also under
positive selection P , 0.05.
Discussion
Our results show that only a small proportion of putative
venom genes are duplicated with a smaller subset displaying
evidence of adaptive evolution. Here, we discuss the role of
gene duplications in platypus venom evolution in secreted
proteases, protease inhibitors, C-type lectins, ion channel
proteins, isomerases, and beta-2-microglobulin (B2M).
Proteases
Whittington et al. (2010) identified 14 serine proteases in
the platypus genome. Only three of the 14 appear to result
from lineage-specific gene duplications, including two
genes which derive from a lineage-specific clade-expansion
of five genes that share similarity with human trypase (fig. 1)
and one gene that is most similar to human chymase. A
platypus peptidase with high similarity to a metalloprotease, endoplasmic reticulum aminopeptidase 1 (ERAP1)
was also identified. Both ERAP1 and chymase are involved
in the regulation of blood pressure in humans. The physiological function of ERAP1 in humans suggests that it may
cause hypotension by cleaving angiotensin II (Hattori et al.
2000). Hence, it is possible that this gene duplicate may
contribute to the hypotensive effect of platypus envenomation (Martin and Tidswell 1895). On the other hand,
chymase, a member of the peptidase S1 family, cleaves inactive angiotensin I into angiotensin II, causing an increase
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MBE
FIG. 1. Evolutionary relationship of serine protease protein sequences. There were 366 positions in the final data set. Bootstrap values greater
than 70% are shown. Tree is drawn to scale based on the number of amino acid substitutions per site. Platypus sequences are in bold;
a diamond denotes genes expressed in the platypus venom gland. Snake venom genes are denoted by a triangle. Labels contain the gene
accession (if no Ensembl accession is available the UniProt one is used).
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in blood pressure (Wu et al. 2005) which is not consistent
with the physiological effect of platypus envenomation. It is
possible that this hypertensive effect may be neutralized by
the expression of ten serine protease inhibitors, which are
found in venom (Whittington et al. 2010). The presence of
both hypertensive and hypotensive proteins may aid in the
regulation of blood pressure inside the venom gland. Additionally, proteases typically possess a wide range of functions, including smooth muscle contraction, inflammation,
blood clotting, degradation of extracellular matrix, and
pain (Wu et al. 2005). Therefore, it is possible that the gene
duplicate was not selected for its role in hypertension but
rather for other functional properties. In support of this,
a platypus chymase-like duplicate also shares high levels
of homology to a toxin from short-trailed shrew (BLAST;
Accession: Q76B45; E value 5 5 1031), which has kallikrein-like activity and dilatory effects on blood vessel walls
(Kita et al. 2004).
Homology of platypus peptidases to chymase and trypase suggests that platypus venom serine proteases arose
from different ancestral genes to snake peptidases, which
are derived from tissue kallikrein (fig. 1) (Deshimaru et al.
1996). Venom peptidases have also independently evolved
in shrew, reptiles, and insects (Fry et al. 2009).
Protease Inhibitors
Two pairs of serine protease inhibitors have resulted from
lineage-specific gene duplications: SERPINA3 and A2M.
Both genes are associated with acute phase response in humans. Serpins belong to a superfamily of protease inhibitors
that are found in gene clusters and evolve through a series
of tandem gene duplications and control coagulation pathways and immune processes (van Gent et al. 2003). SERPINA3 inhibits chymase, which has also experienced a gene
duplication in monotremes, suggestive of coevolution of
the receptor and ligand pair in venom function. Serpin inhibition of chymase leads to in a drop in blood pressure
(Rubin et al. 1990), consistent with observations in rabbits
that have been injected with platypus venom. The platypus
gene duplicate may also have a protective function: In wasp
venom, serpins are believed to protect host tissues from
toxin effects generated by serine proteases (Colinet et al.
2009). A2M is also a protease inhibitor that is capable of
eliminating proteolytic effects of several crotalid snake venoms (Kress and Catanese 1981). A2M is homologous to C3,
a thiolester-containing protein of the vertebrate complement and homolog of cobra venom factor (Sottrup-Jensen
et al. 1985), suggesting a convergent role of A2M-like domains in venom. This is the first time, to our knowledge,
that two A2M genes have been identified in the venom
component of any species. We hypothesize that it functions in a protective and regulatory capacity.
Ion Channel Protein
A duplication of an ion channel gene similar to TRPV6 was
identified. This member of the transient receptor potential
(TRP) ion channel family plays a role in neurotransmission,
muscle contraction, and exocytosis using calcium as
MBE
a secondary messenger in humans (Clapham et al. 2001).
Given its expression in the venom gland, this receptor
may function in the secretion of venom.
Isomerase
We identified a secreted gene duplicate belonging to the
peptidylprolyl cis-trans isomerase (PPIase) family. PPIases
catalyze the cis-trans isomerization of prolines. Alterations
in the folding of proteins can lead to novel structures and
toxin function. This is supported by the discovery of
a platypus venom L–D-isomerase, which may protect toxins from protease degradation and increase toxin potency
(Torres et al. 2006; Torres et al. 2007). In addition to cellular signaling (Luban et al. 1993) and transcriptional regulation (Rycyzyn and Clevenger 2002), PPIases in cone
snail have been documented to accelerate the folding
of disulfide-rich toxins containing prolines by acting as
chaperons in protein folding (Safavi-Hemami et al.
2010). The PPIase may possess a similar role in platypus
venom.
Beta-2-Microglobulin
An expressed B2M duplicate was indentified. It is the third
most highly expressed gene in the Illumina data set, higher
than any of the characterized venom peptides. Expression
of B2M in venom or venom tissues in other venomous species has not been described, but its high expression level
and lineage-specific duplication warrants further investigation. Due to the absence of reports of B2M in the venom of
other species, we checked whether its high level of expression in platypus was due to the presence of noncoding
RNA. We mapped reads back to the genome and confirmed that its expression is indeed due to the proteincoding gene itself. High levels of B2M in plasma cause
destructive osteoarthropathies in humans, including joint
pain and swelling (Drüeke 1999). These symptoms are a result of B2M forming nonselective voltage independent
ion channels or pores in phospholipid bilayer membranes
(Hirakura and Kagan 2001). Formation of such channels
could compromise membrane potential and allow toxic extracellular components, such as calcium, into the tissue of
an envenomated victim, while causing the loss of crucial
ions including potassium and magnesium (Hirakura and
Kagan 2001). It may also cause cell death through the destruction of intracellular structures and leakage of lysosomal enzymes (Hirakura and Kagan 2001).
C-Type Lectins
Two of 12 C-type lectin lineage-specific duplicates were expressed in the venom gland over our threshold of 100 reads.
In snakes, over 80 C-type lectin venom peptides affecting
hemostatsis and thrombosis have been identified (Ogawa
et al. 2005). Lectins have also been found in the venoms of
caterpillars, blood-sucking insects, and stonefish (Fry et al.
2009). Interestingly, the platypus has experienced a massive
expansion of C-type lectin domain genes (Wong et al.
2009). Due to their similarity to NKC receptors, it was
thought that these genes primarily have immunological
173
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Wong et al. · doi:10.1093/molbev/msr180
function, but evidence of high expression in venom gland
warrants further exploration of a venom function.
Evidence of Adaptive Selection
We do not expect that all genes under positive selection will
have venom-related roles. Rather, the majority appear to be
associated with ubiquitous cellular processes such as transcription and cell metabolism. Among the 20 pairs of gene
duplicates that showed evidence of positive selection, two
pairs belonged to gene families with a known venom function (table 2): a disintegrin and metalloproteinase (ADAM)
family protein (ENSOANP00000007182) and a C-type lectin
protein (ENSOANP00000011236). Genes from the ADAM
gene family are closely related to snake venom metalloproteinases (SVMPs), which have been shown to evolve
through positive selection (Casewell, Wagstaff, Harrison,
Renjifo, et al. 2011). SVMPs can be cleaved into disintegrin
peptides with potent inhibition of platelet aggregation activity but can also have hemorrhagic activities (Jia et al.
1996; Silva et al. 2003; Modesto et al. 2005). Based on
the coagulatory properties of platypus venom, we suggest
that the platypus peptide likely acts in a procoagulatory
capacity. We did not identify a predicted signal peptide
in the platypus ADAM peptide using SignalP. This is not
unexpected, as it is thought that certain venom metalloproteinases possess an internal signal peptide in the form
of a hydrophobic segment which only acts as a signal peptide in the mature peptide following proteolysis of the precursor (Kini 1995). Platypus C-type lectin genes have
previously been shown to evolve under positive selection
(Wong et al. 2009). However, this is the first time that positively selected C-type lectins have been identified in the
venom gland. In snakes, C-type lectins have also evolved
adaptively and have acquired numerous hemostatic and
thrombotic functions (Ogawa et al. 2005). We expect these
platypus lectins to possess coagulatory and hypotensive
roles due to the observed symptoms of envenomation.
We also identified that the TRP channel receptor gene, ENSOANP00000019830, possibly involved in venom secretion,
is also under adaptive selection.
In total, we found that approximately 12% of all platypus
venom-expressed lineage-specific duplicates showed evidence of positive selection. This is likely to be an underestimate for several reasons. There is a limited time period
postduplication when positive selection can be detected,
and signatures of positive selection can be obscured by purifying selection in older gene duplicates (Han et al. 2009).
In addition, positive selection likely acts on a small number
of amino acids for a short period of time after duplication
and therefore is most detectable in younger duplicates
(Zhang 2003). Positive selection is also difficult to detect
in recently diverged genes. The chi-square test, used to test
the significance of the model of positive selection, also gives
conservative results for short and highly similar data sets
(Anisimova et al. 2001). Moreover, it is generally difficult
to detect positive selection between gene pairs due to
the small numbers of sites available for comparison, resulting in little discriminatory power. Despite this, our result is
174
comparable to the amount of positive selection in lineagespecific gene duplicates in four eutherian lineages: human,
macaque, rat, and mouse (Han et al. 2009). There, the authors only examined ‘‘young’’ duplicates, those with ds , 1,
enriching their data set for genes that show evidence of
adaptive selection (Lynch and Conery 2000; Kondrashov
et al. 2002). In comparison, approximately 8% of our duplicates possessed a ds value greater than 1.
Limited Role of Gene Duplications
We did not find instances where all gene members of a large
multigene family expansion were expressed in the venom
gland. We identified expanded lineage-specific clades for
ADAM peptides, serine proteases, Kunitz-type serine proteases inhibitors, and C-type lectin genes, but only some
and not all members of the expanded clade were expressed
in the venom gland. This suggests multiple recruitment
events from the same gene family without further duplications that increase toxin copies. Extensive gene duplications can lead to increase in toxin expression, thereby
increasing the efficiency and potency of the venom (Fry
et al. 2009). Multiple gene copies can increase toxin concentration and hasten venom replacement. Cone snail toxins use this pattern of evolution; where between 50 and 100
thousand individual peptides have been identified across
a number of species (Bingham et al. 2010). There may
be several reasons why we did not observe expansions
of multivenom copies in platypus. Rapid venom replenishment may not be required in the platypus, as only small
quantities of venom are released (70 ll) and the full venom
gland capacity is not fully delivered by the animal when it
spurs (Whittington et al. 2009). Additionally, the role of
platypus venom is believed to be for asserting dominance
over conspecifics in the context of mating rather than to
kill or immobilize prey, as in cone snails and snakes. These
functional differences may account for differences in
strength and direction of selective pressure on venom genes
as it is likely that there is an increased fitness cost in producing lethal venom as opposed to a less potent venom
that incapacitates their mating competitors. However,
we note that lowly expressed genes, incorrect phylogenetic assignments or inaccurate gene predictions may
have affected our ability to detect multiple toxin copies
in the platypus.
Most platypus venom genes do not appear to have
arisen via gene duplication in the monotreme lineage. Only
16 of 107 putative venom genes identified through similarity to known toxin genes are lineage-specific duplicates. If
we assume convergent evolution of venom function, only
15% of platypus toxins have evolved through gene duplications. Consistent with this, nine of 12 serine proteases
identified by Whittington et al. (2010) have one-to-one orthologs with other species; in addition, platypus venom
protein fractions, C-type natriuretic peptides (ENSOANG00000012322) (de Plater et al. 1998) and nerve growth
factor (most likely to be ENSOANG00000004523) (de Plater
et al. 1995) identified previously via proteomic studies do
not appear to have duplicated in the platypus lineage.
Gene Duplications and Platypus Venom Evolution · doi:10.1093/molbev/msr180
We suggest that changes other than gene duplications
play an important part in the recruitment of toxin genes.
There are 12 known splice variants of platypus venom Ctype natriuretic peptide (Kita et al. 2009). Snake venom
C-type natriuretic peptides also undergo extensive molecular processing forming a large number of isoforms from
a single precursor mRNA (Higuchi et al. 1999). This extensive cleavage pattern has also been identified in viper sarafotoxins (Ducancel et al. 1993). Alternatively, spliced
variants have been identified in other venom components
including snake disintegrins (Scarborough et al. 1993) and
Factor X (St. Pierre et al. 2005). Splice variants are considered to be a major contributor to protein diversity (Modrek
and Lee 2002; Keren et al. 2010). Human genes (92–94%)
undergo alternative splicing and are responsible for the 4fold increase in the number of proteins synthesized compared with protein-coding genes (Wang et al. 2008; Keren
et al. 2010). Protein isoforms derived from alternatively
spliced genes can have distinct or even antagonistic functions (Das et al. 2002; Zhang and Mount 2009) and may
possess different tissue specificities (Wang et al. 2008).
Gene mutations can also increase the functional diversity
of a gene, through changes in either the regulatory or the
protein-coding regions. In snakes, CRISP and kallikrein
venom genes are believed to have evolved through mutational modifications (Fry 2005).
It is also possible that we have underestimated the number of putative venom genes that have undergone duplication. This could be due to several factors. First, limited
gene divergence or short gene length could lead to difficulties identifying duplicates. For example, previously gene
trees were not constructed for defensin-like venom peptides (Whittington et al. 2008) due to their high degree of
divergence. Second, although a large number of proteincoding genes were predicted in the platypus assembly
(17,951), it is possible that some venom gene duplicates
were missed. Third, the fragmented assembly is likely to
have led to truncations in some gene predictions and
hence failure to call some duplications.
Gene duplications are only partially responsible for the
adaptation of platypus venom. The availability of whole genome sequence has allowed us to detect gene duplicates
that have led to venom function. Multispecies comparisons
have allowed us to distinguish between duplicates that
stem from speciation events, where venom orthologs exist
in nonvenomous species and true lineage-specific innovations. Comparative studies of other venomous animals
will provide insights on the likely variable role of gene duplication in venom evolution. We anticipate that genomic
strategies will play an increasingly useful role in the understanding of venom evolution as whole genome sequences
from other venomous animals become available.
Conclusion
Our genomic and transcriptomic data analysis shows that
duplications of proteases, their inhibitors, and C-type lectins
play a role in the evolution of platypus venom. However,
MBE
extensive venom gene duplications were not identified
and key venom genes do not appear to have arisen through
gene duplications in the platypus lineage. This points to
other evolutionary mechanisms being important in platypus venom evolution. Our study highlights the usefulness
of genomic data in understanding the evolution of novel
gene functions.
Supplementary Material
Supplementary files 1 and 2 are available at Molecular Biology
and Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
This work was funded by the University of Sydney and the
Australian Research Council. KB is supported by an ARC
Future Fellowship.
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