BioMed Central
Page 1 of 14
(page number not for citation purposes)
BMC Evolutionary Biology
Open Access
Research article
Inventing an arsenal: adaptive evolution and neofunctionalization of
snake venom phospholipase A2 genes
Vincent J Lynch*
Address: Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, 06511, USA
Email: Vincent J Lynch* - vincent.j.lynch@yale.edu
* Corresponding author
Abstract
Background: Gene duplication followed by functional divergence has long been hypothesized to
be the main source of molecular novelty. Convincing examples of neofunctionalization, however,
remain rare. Snake venom phospholipase A2 genes are members of large multigene families with
many diverse functions, thus they are excellent models to study the emergence of novel functions
after gene duplications.
Results: Here, I show that positive Darwinian selection and neofunctionalization is common in
snake venom phospholipase A2 genes. The pattern of gene duplication and positive selection
indicates that adaptive molecular evolution occurs immediately after duplication events as novel
functions emerge and continues as gene families diversify and are refined. Surprisingly, adaptive
evolution of group-I phospholipases in elapids is also associated with speciation events, suggesting
adaptation of the phospholipase arsenal to novel prey species after niche shifts. Mapping the
location of sites under positive selection onto the crystal structure of phospholipase A2 identified
regions evolving under diversifying selection are located on the molecular surface and are likely
protein-protein interactions sites essential for toxin functions.
Conclusion: These data show that increases in genomic complexity (through gene duplications)
can lead to phenotypic complexity (venom composition) and that positive Darwinian selection is a
common evolutionary force in snake venoms. Finally, regions identified under selection on the
surface of phospholipase A2 enzymes are potential candidate sites for structure based antivenin
design.
Background
Phospholipase A2s (PLA2; EC 3.1.1.4) are esterolytic
enzymes that hydrolyze glycerophospholipids at the sn-2
fatty acyl bond, releasing lysophospholipids and fatty
acids. PLA2s play key roles in various biological processes
in mammals including signal transduction, lipid diges-
tion, host defense and production of eicosanoids and
other lysophospholipid derivates with potent biological
activities [1]. PLA2 enzymes are also the major compo-
nents of snake venoms where they function to immobilize
and rapidly kill prey [1]. PLA2s from elapid venoms
(group-I) are structurally similar to pancreatic secretions
while PLA2s from viper venoms (group-II) are structurally
similar to inflammatory secretions [2]. A third group of
PLA2s (group-III) have been identified from the venom of
bees, jellyfish, scorpions and lizards [2] indicating that
PLA2s have been recruited into a toxic function multiple
times in diverse lineages.
Published: 18 January 2007
BMC Evolutionary Biology 2007, 7:2 doi:10.1186/1471-2148-7-2
Received: 02 October 2006
Accepted: 18 January 2007
This article is available from: http://www.biomedcentral.com/1471-2148/7/2
© 2007 Lynch; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 2 of 14
(page number not for citation purposes)
Snake venom PLA2s are members of large multigene fam-
ilies with diverse pharmacological activities including
neurotoxic, myotoxic, cardiotoxic, anticoagulant and
hemolytic effects [2]. These diverse activities evolved from
an ancestral nontoxic PLA2 by a process of repeated gene
duplication followed by functional divergence. PLA2 tox-
icity is independent of enzymatic activity [3,4] and is
mediated through "pharmacological sites" on the protein
surface that directly interact with ligands on the cell mem-
brane [5]. Thus, the surface of PLA2s forms a scaffold for
adaptive modification that has been used to generate a
diverse array of pharmacological effects through a process
of neofunctionalization (the generation of new protein
functions that were not the primary function of the ances-
tral protein).
Previous studies of PLA2 genes identified accelerated evo-
lution of group-I genes from Naja naja [6] and group-II
genes from Trimeresurus [7] and Vipera [8] consistent with
positive Darwinian selection, however these studies
focused on one or two species, included relatively few
genes and used methods that lack power to detect episodic
adaptive evolution. A larger study of group-I and -II genes
found that amino acid substitutions were correlated with
surface accessibility [9], suggesting that modifications of
surface residues and positive selection play important
roles in generating toxin diversity. To further explore this
possibility I compiled an extensive dataset of full length
snake venom PLA2 genes from public databases, inferred
the gene trees for these toxins and tested for episodes of
positive Darwinian selection coincident with the origin of
novel pharmacological effects and recurrent diversifying
selection on specific sites. In addition, I used a larger
amino acids dataset to test if conclusions drawn from the
smaller nucleotide dataset were robust to phylogenetic
inference.
The results indicate that adaptive evolution is common in
snake venom PLA2 genes and is associated with the evolu-
tion of new toxin functions and speciation events, dem-
onstrating that molecular adaptation has played a
pervasive role in the evolution of snakes and their venom
arsenal. This analysis has identified the mutational path-
way leading from non-toxic to highly toxic PLA2 enzymes,
reconstructing the processes of mutation and adaptation.
Finally, increases in genomic complexity gained through
gene duplications has promoted the evolution an increas-
ingly complex phenotype (venom composition), provid-
ing a link between molecular, phenotypic and organismal
evolution.
Results and discussion
Gene duplication and speciation history
To study the molecular evolution of snake venom PLA2
genes, I compiled a dataset of 83 group-I and 90 group-II
genes from public databases and inferred the evolutionary
history of these genes using Bayesian phylogenetics [10].
The molecular phylogeny inferred for group-I PLA2
enzymes (Figs. 1 and 3) indicates that genes group with
higher-order snake phylogeny and are divided into two
sister clades containing marine and Australian species (the
"Hydrophiids") and African, American and Asian species
(the "Elapids"). This division is similar to the results of
earlier phylogenetic studies of group-I PLA2 genes [11]
and likely reflects a deep divergence between these
groups. Within these two major clades, subclades contain
genes from closely related taxa that have similar pharma-
cological effects, suggesting functional diversification
occurred after speciation. In contrast, group-II genes clus-
ter by pharmacological effect with little species distinction
(Figs. 2 and 4), indicating that functional divergence arose
before the divergence of these species.
Even though branch support for the group-I and group-II
nucleotide trees was high in this analysis, nucleotide data
are only about a third of the gene sequences that are avail-
able, the majority are amino acid data (and thus not suit-
able for codon-based selection analysis discussed later).
To test if the topology of nucleotide-based gene trees was
sensitive to taxon sampling I inferring group-I and group-
II phylogenies using larger amino acids datasets (245
group-I and 271 group-II genes, respectively). Although
the trees inferred from amino acid data (Figs. 3 and 4) had
lower support for recent lineages than the nucleotide data,
perhaps because protein sequences have not accumulated
enough phylogenetically informative substitutions to
accurately reconstruct recent branching orders, the deeper
nodes were well supported and the overall topology was
congruent between amino acid and nucleotide data indi-
cating that inferences based on the nucleotide datasets are
reliable.
Origin and elaboration of toxic genes
Snake venom PLA2s have diverse pharmacological activi-
ties including neurotoxic, myotoxic, cardiotoxic, anticoag-
ulant and hemolytic effects [2], which must have
originated after they diverged from nontoxic ancestors.
Although uncertainty in the tree topology and the richness
of toxin functions makes assessment of specific ancestral
and derived functions difficult for all lineages, it is clear
from these phylogenies that many novel functions have
originated in PLA2 genes after gene duplications. For
example, three nontoxic group-I PLA2 genes isolated from
Laticuadata semifasciata pancreas [12] branch near the base
of the Elapinae group in the nucleotide tree, but are the
most basal snake group-I genes in the amino acids tree
(Figs. 1 and 3). These pancreatic genes have been sug-
gested to be intermediates between nontoxic and toxic
enzymes [12], suggesting that duplication of an ancestral
nontoxic gene originally expressed in the pancreas was
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 3 of 14
(page number not for citation purposes)
Molecular phylogeny of group-I phospholipase A2 genesFigure 1
Molecular phylogeny of group-I phospholipase A2 genes. (A) Bayesian phylogeny, branch lengths are given as number of substi-
tutions per codon. (B) Evolution of group-I genes. Numbers above the branches are Bayesian posterior probability values (BP)
followed by the dN/dS ratio (ω) or the number of nonsynonymous substitutions (N) if no synonymous substitutions (S)
occurred along that branch (BP | ω or N/S). Branches in red were inferred to be under positive selection. Genes are labeled
according the species they were identified from followed by the GenBank GI number for that gene. Pharmacological effects and
higher order classifications are given to the right of clades. Pan, nontoxic pancreatic isoforms. AtC, anticoagulent. Ctx, cardio-
toxic. PrC, procoagulent.
L.laticaudata.17129631
L.laticaudata.17129629
L.laticaudata.64047
L.laticaudata.17129627
L.laticaudata.17129625
L.colubrina.17129637
L.colubrina.17129639
L.colubrina.17129633
L.semifasciata.9453877
L.semifasciata.9453907
L.semifasciata.9453879
L.semifasciata.9453881
L.semifasciata.9453909
L.semifasciata.17129623
L.semifasciata.9049438
L.semifasciata.17129621
L.semifasciata.9453913
A.superbus.5924334
A.laevis.62401
N.scutatus.64105
A.superbus.27802488
A.superbus.5924322
A.superbus.5924330
A.superbus.5924320
A.superbus.5924324
A.superbus.5924326
A.superbus.5924328
A.superbus.5924332
A.superbus.5924336
N.scutatus.64109
A.superbus.5924342
A.superbus.5924346
A.superbus.5924348
A.superbus.5924344
A.superbus.5924340
A.superbus.5924338
L.hardwickii.18000322
L.hardwickii.18026637
A.superbus.5923907
A.superbus.5923909
L.semifasciata.21734659
L.semifasciata.21734661
L.semifasciata.21734657
O.hannah.10442707
O.hannah.10863759
O.hannah.10863761
N.naja.804797
N.sputatrix.8953898
N.naja.804793
N.naja.804795
N.sputatrix.8953900
N.naja.395191
N.naja.456270
N.kaouthia.4115522
N.kaouthia.4115520
M.corallinus.24496494
B.caeruleus.10121879
B.multicinctus.62501
B.fasciatus.14423357
B.fasciatus.14423359
B.caeruleus.19526594
B.multicinctus.24412702
B.multicinctus.62505
B.multicinctus.1000449
B.multicinctus.19069523
B.multicinctus.24412704
B.multicinctus.24412706
B.multicinctus.6523108
B.multicinctus.6523110
B.candidus.24459191
B.multicinctus.62473
B.multicinctus.24412700
B.multicinctus.24412708
B.multicinctus.10129662
B.candidus.24459193
B.multicinctus.6523112
B.candidus.24459189
B.caeruleus.19526596
B.candidus.29422776
P.textilis.5230717
P.textilis.19067870
P.textilis.5230715
P.textilis.19067868
100| (14/0)
100| 1.76
100| 2.11
85| 2.65
100| 2.04
100|(16/ 0) 1.19
1.03
(22/0)
96| 2.29
60| 1. 77
99|(21/0)
97| 3.20
99| 1.58
4.67
68| (7/0)
95| 1.02
1.39
100| 1.24
100| 5.50
100| 3.91
1.83
100| 1. 29
91| (11/0)
99| 1.26
2.16
3.62
100| 3.34
79| 2.89
84| 1.28
62|(6/0)
1.29
1.10
2.19
100|(14/0)
100| 1. 63
100| 2.33
1.81
(4/0)
85| 5.12
100| 4. 06
100| 3. 06
2.9
(5/0)
91| 9.06
55| 1. 35
1.67
65| 1. 29
64| (6/0)
(12/0)
94| 1.07
1.23
95| 3.49
100| 1. 30
95
53 94
75
94
100
90
93
53
98
89
94 92
59
85
100
93
68
72
84
100 95
100
62
100
72
76
97
82
100
L.laticaudata.17129631
L.laticaudata.17129629
L.laticaudata.64047
L.laticaudata.17129627
L.laticaudata.17129625
L.colubrina.17129637
L.colubrina.17129639
L.colubrina.17129633
L.semifasciata.9453877
L.semifasciata.9453907
L.semifasciata.9453879
L.semifasciata.9453881
L.semifasciata.9453909
L.semifasciata.17129623
L.semifasciata.9049438
L.semifasciata.17129621
L.semifasciata.9453913
A.superbus.5924334 A.laevis.62401
N.scutatus.64105
A.superbus.27802488
A.superbus.5924322
A.superbus.5924330
A.superbus.5924320
A.superbus.5924324
A.superbus.5924326
A.superbus.5924328
A.superbus.5924332
A.superbus.5924336
N.scutatus.64109
A.superbus.5924342
A.superbus.5924346
A.superbus.5924348
A.superbus.5924344
A.superbus.5924340
A.superbus.5924338
L.hardwickii.18000322
L.hardwickii.18026637
A.superbus.5923907
A.superbus.5923909
L.semifasciata.21734659
L.semifasciata.21734661
L.semifasciata.21734657
O.hannah.10442707
O.hannah.10863759
O.hannah.10863761
N.naja.804797
N.sputatrix.8953898
N.naja.804793
N.naja.804795
N.sputatrix.8953900
N.naja.395191
N.naja.456270
N.kaouthia.4115522
N.kaouthia.4115520
M.corallinus.24496494
B.caeruleus.10121879
B.multicinctus.62501
B.fasciatus.14423357
B.fasciatus.14423359
B.caeruleus.19526594
B.multicinctus.24412702
B.multicinctus.62505
B.multicinctus.1000449
B.multicinctus.19069523
B.multicinctus.24412704
B.multicinctus.24412706
B.multicinctus.6523108
B.multicinctus.6523110
B.candidus.24459191
B.multicinctus.62473
B.multicinctus.24412700
B.multicinctus.24412708
B.multicinctus.10129662
B.candidus.24459193
B.multicinctus.6523112
B.candidus.24459189
B.caeruleus.19526596
B.candidus.29422776
P.textilis.5230717
P.textilis.19067870
P.textilis.5230715
P.textilis.19067868
A
B
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 4 of 14
(page number not for citation purposes)
Molecular phylogeny of group-II (B) phospholipase A2 genesFigure 2
Molecular phylogeny of group-II (B) phospholipase A2 genes. (A) Bayesian phylogeny, branch lengths are given as number of
substitutions per codon. (B) Evolution of group-II genes. Numbers above the branches are Bayesian posterior probability val-
ues (BP) followed by the dN/dS ratio (ω) or the number of nonsynonymous substitutions (N) if no synonymous substitutions (S)
occurred along that branch (BP | ω or N/S). Branches in red were inferred to be under positive selection. Genes are labeled
according the species they were identified from followed by the GenBank GI number for that gene. Pharmacological effects and
higher order classifications are given to the right of clades. AtC, anticoagulent. Chp/Chaperone, neurotoxin chaperone. Ntx,
neurotoxic.
T.mucrosquamatus.7636221
Ca.rhodostoma.6073845
Ca.rhodostoma.6073844
T.jerdonii.20977210
E.ocellatus.25992664
V.ammodytes.871759
V.ammodytes.64448
V.ammodytes.296137
V.ammodytes.64441
V.ammodytes.2231121
V.ammodytes.64444
T.flavoviridis.28268776
T.flavoviridis.28268778
T.mucrosquamatus.895913
P.mucrosquamatus.15420984
D.acutus.1177378
C.durissusCtB2.62696
C.durissusCtB.62692
C.scutulatusMtB.451317
B.jararacussu.475923
B.jararacussu.28194388
T.mucrosquamatus.1177690
D.acutus.1177376
D.acutus.1177379
Ca.rhodostoma.6073834
Ca.rhodostoma.6073832
Ca.rhodostoma.6073846
Ca.rhodostoma.6073847
T.flavoviridis.222954
T.flavoviridis.222952
B.asper.6492259
B.neuwiedi.7673018
B.jararacussu.28194117
B.moojeni.7673016
B.schlegelii.17432517
C.godmani.17432519
C.atro.17224434
A.contortri.809484
T.gramineus.992956
T.gramineus.994789
T.gramineus.994787
D.acutus.17224438
A.acutus.2760480
T.okinavensis.1469806
T.flavoviridis.21698859
T.flavoviridis.15799260
T.flavoviridis.222956
G.halys.2460024
G.halys.2460026
T.mucrosquamatus.517489
T.flavoviridis.222958
T.flavoviridis.222960
T.flavoviridis.15799262
T.flavoviridis.15799264
T.okinavensis.1469804
T.gramineus.992955
G.halys.2460034
C.durissusCtA.62685
C.scutulatusMtA.451315
B.pictus.9652396
C.viridis.28893825
C.viridis.28893821
C.viridis.29692355
C.atro.17224436
C.viridis.29692349
C.viridis.29692351
C.viridis.29692353
C.viridis.28893823
Ca.rhodostoma.6073842
Ca.rhodostoma.6073838
Ca.rhodostoma.6073840
Ca.rhodostoma.6073836
D.acutus.1177377
B.jararacussu.25140376
B.insularis.20069136
A.piscivorus.263959
G.halys.2460030
G.halys.2460028
E.coloratus.13936544
E.carinatus.25992660
E.pyramidum.25992662
E.coloratus.13936542
V.palaestinae.1575330
V.aspis.22797864
V.russelli.64454
V.russelli.30142139
V.ammodytes.5702035
V.ammodytes.6967297
V.russelli.64452
V.aspis.22797866
78
88
62| (10/0)
62| 3.97
100| 1.54 100
78| (11/0 )
100
94 100| 2.17 51| (3/0) 100
100| 1.59
97
98| 2.32 76 100 100
100
99| (5/0)
100| 1.21
100
100| 3. 85
100| 1.68
100| 1.4
100 74| (5/0) 89
100| 1.5
91
83
100| 1.26 100
59| (6/0) 100
100
100
94| 2.35
71 100| 1.65 92
100| 1.99
100
100
92| 2.38
88| 2.71
100 100| (8/0) 100
100 51 87
100
100
67| 1.22
71| (9/0)
93
88
100| (10/0)
100 100| 1.02
93| 1.43 94
100
100| 1. 03
100| 1. 29
100 100
65
99| (23/0)
98
97| 2.48
100| 3.67
100|1. 62
2.44
2.44
B
T.mucrosquamatus.7636221
Ca.rhodostoma.6073845
Ca.rhodostoma.6073844
T.jerdonii.20977210
E.ocellatus.25992664
V.ammodytes.871759
V.ammodytes.64448
V.ammodytes.296137
V.ammodytes.64441
V.ammodytes.2231121
V.ammodytes.64444
T.flavoviridis.28268776
T.flavoviridis.28268778
T.mucrosquamatus.895913
P.mucrosquamatus.15420984
D.acutus.1177378
C.durissusCtB2.62696
C.durissusCtB.62692
C.scutulatusMtB.451317
B.jararacussu.475923
B.jararacussu.28194388
T.mucrosquamatus.1177690
D.acutus.1177376
D.acutus.1177379
Ca.rhodostoma.6073834 Ca.rhodostoma.6073832
Ca.rhodostoma.6073846
Ca.rhodostoma.6073847
T.flavoviridis.222954
T.flavoviridis.222952
B.asper.6492259
B.neuwiedi.7673018
B.jararacussu.28194117
B.moojeni.7673016 B.schlegelii.17432517
C.godmani.17432519
C.atro.17224434
A.contortri.809484
T.gramineus.992956
T.gramineus.994789
T.gramineus.994787
D.acutus.17224438
A.acutus.2760480
T.okinavensis.1469806
T.flavoviridis.21698859
T.flavoviridis.15799260
T.flavoviridis.222956
G.halys.2460024
G.halys.2460026 T.mucrosquamatus.517489
T.flavoviridis.222958
T.flavoviridis.222960
T.flavoviridis.15799262
T.flavoviridis.15799264
T.okinavensis.1469804
T.gramineus.992955
G.halys.2460034 C.durissusCtA.62685
C.scutulatusMtA.451315
B.pictus.9652396
C.viridis.28893825
C.viridis.28893821
C.viridis.29692355
C.atro.17224436
C.viridis.29692349
C.viridis.29692351
C.viridis.29692353
C.viridis.28893823
Ca.rhodostoma.6073842
Ca.rhodostoma.6073838
Ca.rhodostoma.6073840
Ca.rhodostoma.6073836
D.acutus.1177377
B.jararacussu.25140376
B.insularis.20069136 A.piscivorus.263959
G.halys.2460030
G.halys.2460028
E.coloratus.13936544
E.carinatus.25992660
E.pyramidum.25992662
E.coloratus.13936542
V.palaestinae.1575330
V.aspis.22797864
V.russelli.64454
V.russelli.30142139
V.ammodytes.5702035
V.ammodytes.6967297
V.russelli.64452
V.aspis.22797866
A
B
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 5 of 14
(page number not for citation purposes)
followed by recruitment into the venom gland and the
emergence of toxic functions. Also within group-I, a clade
containing neurotoxins from Laticuadata has emerged
from antiplatelet enzymes; the nested position of this
clade indicates that the neurotoxic effect of these enzymes
is derived from more ancestral antiplatelet enzymes. The
origin of group-II toxins that target muscle is also associ-
ated with a gene duplication event. Group-II myotoxins
share a unique amino acid substitution at residue 49
(aspartate to lysine) that abolishes enzymatic activity
[13,14]. Thus, Lys49-myotoxins evolved a novel nonhy-
drolytic mechanism to induce membrane damage [15,16]
Molecular phylogeny of group-I phospholipase A2 genesFigure 3
Molecular phylogeny of group-I phospholipase A2 genes. Amino acid dataset with Bayesian posterior probabilities shown along
branches.
T.carinatus 71066772
T.carinatus 71066770
T.carinatus 71066768
T.carinatus 71066764
T.carinatus71066766
N.scutatus 999320
N.scutatus 71066760
T.carinatus 71066774
N.scutatus 71066762
O.scutellatus 66475090
O.scutellatus 66475092
O.scutellatus 71066730
O.scutellatus 913014
O.scutellatus71066726
O.microlepidotus71066742
O.microlepidotus 71066736
O.microlepidotus 71066738
O.microlepidotus 71066740
O.scutellatus 71066728
O.scutellatusalphataipoxin2pre
O.scutellatus 129413
P.textilis400716
P.textilis 265534
O.scutellatus 71066718
O.scutellatus 67194
O.scutellatus66475082
O.microlepidotus 71066732
P.textilistextilotoxinC 710667
P.textilis 71066750
P.textilis 71066752
P.textilis 265535
Pn.textilis 265533
L.laticaudata17129632
L.laticaudata 17129630
L.laticaudata 17129628
L.laticaudata 17129626
L.laticaudata 64048
L.semifasciata 25453166
L.semifasciata 25453169
L.semifasciata 25453168
L.semifasciata 25453167
L.semifasciata 25453170
L.semifasciata 129439
L.semifasciata 129450
L.semifasciata 17129624
L.semifasciata 25453165
L.semifasciata 9049439
A.superbus 5924341
A.superbus 5924343
A.superbus 5924345
A.superbus 5924349
A.superbus 5924347
A.superbus 5924339
N.scutatus 129434
N.scutatus 1097976
N.scutatus 71066754
N.scutatus 71066756
N.scutatus 129445
E.schistos 129509
Lapemishardwickii 18026640
L.hardwickii18000323
L.colubrina 17129640
L.colubrina 17129634
L.colubrina 17129636
L.colubrina 17129638
L.colubrina85961
L.colubrina 129428
Pe.porphyriacus71066800
Pe.porphyriacus 71066790
P.australis 129447
P.australis 129477
P.australis 71066778
Pe.australis 71066782
P.australis 71066780
P.australis71066784
Pe.porphyriacus 71066792
Pe.australis 129458
P.australis 129415
Pe.australis 71066776
Pe.australis 129471
Pe.australis129452
P.australis 129397
P.australis 129454
Pe.australis 71066788
P.porphyriacus 129465
P.porphyriacus 71066798
A.antarcticus 3183115
A.antarcticus 3183114
P.porphyriacus 129459
P.porphyriacus 71066794
P.australis129474
Pe.australis 71066786
P.australis 129476
P.porphyriacus71066796
A.eydouxii 49472978
A.eydouxii49472970
A.eydouxii49472990
A.eydouxii 49472982
A.eydouxii 49472966
A.eydouxii 49472984
A.eydouxii 49472976
A.eydouxii49472980
A.eydouxii 49472992
A.eydouxii 49472964
A.eydouxii49472988
A.eydouxii 49472986
Aipysuruslaevis 62402
A.superbus5924335
A.superbus 5924337
N.scutatus 64106
N.scutatus241602
A.superbus 5924333
N.scutatus 71066758
N.scutatus 64110
N.scutatus67183
A.superbus5924323
A.superbus 27802489
A.superbus 5924325
A.superbus5924331
A.superbus 5924329
A.superbus 5924327
Lapemishardwickii18026638
P.textilis 19067871
P.textilis 19067869
O.scutellatus71066720
O.scutellatus 913013
O.scutellatus 71066724
O.scutellatus71066722
O.microlepidotus 71066734
O.scutellatus66
475086
O.scutellatus129446
A.superbus 5923910
A.superbus 5923908
P.textilis2
65536
P.textilisextilotoxinDchain 71
P.textilis 71066746
M.Ikaheka 61679797
M.Ikaheka 55669537
M.Ikaheka 55669544
O.hannah 10863760
O.Hannah 34809960
O.hannah 10863762
B.fasciatus 129494
N.kaouthia 2144440
N.kaouthia 67172
N.sputatrix8
953899
N.sputatrix 25453163
N.sputatrix 8953901
N.sputatrix 25453162
N.nigricollis 129444
N.mossambica 129443
N.pallida129515
N.mossambica 129410
N.mossambica 129433
Aspidelapsscutatu 129422
N.melanoleuca 129432
N.melanoleuca 129409
N.melanoleuca 129442
N.kaouthia 24638468
N.naja395192
N.kaouthia 24638469
N.naja 558355
N.atra 443188
N.naja 64104
N.naja 129514
N.oxiana 129411
N.sagittifera37
700489
N.Sagittifera 46015732
N.Sagittifera3
1615585
N.sagittifera 38017492
N.sagittifera 48425162
N.sagittifera 71042391
N.Sagittifera37926586
N.Sagittifera31
615586
N.sagittifera 33357328
N.sagittifera 38324522
N.sagittifera 38565478
N.Sagittifera 46015731
N.Sagittifera 31615584
N.sagittifera58197905
N.sagittifera66
360669
N.naja 31615941
N.sagittifera 58197903
N.sagittifera 66360668
N.naja 31615940
Hemachatushaemachatus129403
M.nigrocinctus 3914267
M.nigrocinctus3914266
M.corallinus 24496495
B.candidus 29422777
B.flaviceps31745051
B.flaviceps 31745049
B.multicinctus 6562011
B.multicinctus 6562013
B.multicinctus 1359511
B.multicinctus62506
B.multicinctus 10129663
B.multicinctus 6523113
B.candidus 34368939
B.multicinctus 6562009
B.multicinctus 6523111
B.multicinctus 62474
B.multicinctus350799
B.candidus 40363689
B.caeruleus 19526597
B.caeruleus19526595
B.multicinctus 24412709
B.multicinctus 1431755
B.multicinctus 6523109
B.multicinctus 6562022
B.multicinctus 6522953
B.multicinctus 24412705
B.multicinctus 19069524
B.multicinctus 23396780
B.candidus 38016965
B.candidus2
4459190
B.candidus 38016969
B.candidus 24459192
B.multicinctus 24412703
B.flaviceps31745057
B.flaviceps 31745055
B.caeruleus 10121880
B.Caeruleus 13096371
B.caeruleus14278702
B.caeruleus38374048
B.Caeruleus 52696003
B.caeruleus 42563725
B.caeruleus 49259299
B.caeruleus 38374050
B.caeruleus 33356923
B.multicinctus62502
B.multicinctus 350460
B.fasciatus1
29455
B.fasciatus 129462
B.fasciatus 14423358
B.fasciatus 129453
B.fasciatus 14423360
B.fasciatus 129497
B.fasciatus 263083
B.candidus 40363695
O.hannah 10442708
L.semifasciata 25453145
L.semifasciata 25453146
Human
Sus
100
71
81
100
63
84
99
76
80
87
88
99
100
98
100
100
100
54
75
79
98
100
100
94
100
83
100
99
100
99
100
51
79
92
93
83
100
100
100
89
81
60
65
99
100
100
100
100
100
98
71
98
100
99
97
100
87
100
62
99
100
99
96
97
100
64
88
100
92
89
100
76
92
100
100
100
100
100
100
100
77
99
94
97
100
100
91
100
64
100
94
100
100
100
100
86
100
100
50
100
93
99
100
100
80
100
99
96
77
100
98
100
100
100
100
55
52
81
61
82
100
100
70
100
100
96
84
59
82
63
8
9
73
50
83
92
100
100
66
60
83
78
76
75
100
100
99
99
92
58
100
61
100
75
100
100
100
100
100
84
100
100
100
100
92
81
68
62
83
99
99
100
95
100
68
64
83
91
100
100
100
100
97
100
94
100
100
100
100
100
100
98
94
100
100
100
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 6 of 14
(page number not for citation purposes)
after duplication of an ancestral Asp49-PLA2 that was not
myotoxic.
I used maximum likelihood models of coding-sequence
evolution [17,18] to test the hypothesis that functional
diversification of snake venom PLA2 genes is driven by
positive Darwinian selection. This method determines the
strength and direction of selection by estimating the non-
synonymous-to-synonymous substitution rate (dN/dS =
ω), with ω = 1, <1, and >1 indicating neutral evolution,
purifying selection and directional selection, respectively.
The branch-specific one-ratio model is the simplest, esti-
mating the same ω for all branches in the phylogeny. The
estimate of ω for group-I genes under this model, 1.28, is
an average over all codons and lineages, highlighting the
dominant role of positive selection on elapid venom
Molecular phylogeny of group-II phospholipase A2 genesFigure 4
Molecular phylogeny of group-II phospholipase A2 genes. Amino acid dataset with Bayesian posterior probabilities shown along
branches
V.a.aspis50874338
V.a.aspis50874398
V.a.aspis50874354
V.a.atra50874366
V.a.zinnikeri33187134
V.a.aspis50874396
V.a.aspis50874342
V.b.berus50874326
V.a.aspis50874344
V.b.berus50874330
V.a.atra50874368
V.a.aspis50874262
V.a.aspis50874348
V.a.zinnikeri50874266
V.a.atra50874362
V.a.aspis50874260
V.b.berus50874324
V.a.atra50874356
V.a.atra50874360
V.a.zinnikeri50874320
V.b.berus50874332
V.a.aspis50874350
V.a.aspis50874288
V.a.aspis50874290
V.a.aspis50874292
V.a.aspis50874388
V.a.montandoni50874384
V.a.ammodytes50874374
V.a.33187132
V.a.ruffoi50874254
V.a.aspis50874304
V.a.aspis50874302
V.b.berus50874310
V.b.berus33187136
Viperaursinii50874334
V.a.ruffoi50874252
V.a.aspis50874294
V.palaestinae3885850
E.macmahoni237894
E.coloratus13936543
Viperapalaestinae3885848
V.a.atra50874480
V.a.aspis50874476
V.a.atra50874482
V.a.aspis50874472
V.a.ruffoi50874458
V.a.aspis50874444
V.b.berus50874416
V.b.berus33187126
V.a.aspis50874474
V.a.aspis50874442
V.a.aspis50874430
V.a.aspis50874448
V.a.aspis50874452
V.a.aspis50874402
V.a.aspis50874438
V.a.aspis50874436
V.b.berus50874464
V.a.aspis50874410
V.a.aspis50874440
V.a.ammodytes50874486
V.a.ammodytes50874488
V.a.aspis50874432
V.a.aspis50874424
V.a.ammodytes50874490
V.a.montandoni50874510
V.a.montandoni50874508
V.a.montandoni50874506
V.a.meridionalis50874496
V.a.meridionalis50874502
V.a.meridionalis50874500
V.ursinii50874466
E.macmahoni237893
V.r.siamensis31790290
D.r.russellii87130854
B.schlegelii87130858
D.r.russellii87130860
VipoxinAchain2851544
VipoxinChainA16974940
V.a.meridionalis48425249
VipoxinChainA2914536
V.a.1408314
V.a.aspis50874228
V.a.zinnikeri50874248
V.a.zinnikeri50874246
V.a.zinnikeri33187114
V.a.aspis50874232
V.a.zinnikeri50874238
P.fieldi1345182
Daboiarusselii64455
V.r.siamensis30142140
E.pyramidumleakeyi25992663
B.arietans62547945
E.carinatussochureki25992661
E.coloratus13936545
E.carinatus30983918
E.Carinatus40889259
V.a.aspis33187140
V.a.aspis50878167
VipoxinChainB129419
VipoxinChainB2914537
V.a.zinnikeri50878199
V.a.1408315
V.a.zinnikeri50878179
V.a.zinnikeri33187118
V.a.zinnikeri50878195
V.nikolskii91199942
V.a.aspis50878159
V.a.zinnikeri50878197
V.a.zinnikeri50878169
V.a.zinnikeri50878187
V.a.zinnikeri50878193
V.a.zinnikeri50878173
V.nikolskii91199940
P.fieldi1345181
D.russelii64453
V.r.siamensis30909285
D.r.russellii87130856
V.b.berus33187120
Caudoxin129502
T.stejnegeri37785863
T.puniceus38230139
T.puniceus38230135
T.borneensis38230145
T.gramineus263545
Ovophisokinavensis1769398
T.gramineus430834
T.gramineus1644274
T.gramineus430833
T.stejnegeri37785859
G.shedaoensis38146948
B.erythromelas86450426
B.pictus9652397
C.durissus62686
C.scutulatusscutulatus451316
S.c.tergeminus45934758
C.v.viridis28893822
V.a.montandoni28893826
C.v.viridis28893824
C.v.viridis29692350
C.atrox17224437
C.v.viridis29692354
C.v.viridis29692352
C.v.viridis29692356
T.stejnegeri37785865
C.rhodostoma6073837
C.rhodostoma6073839
C.rhodostoma6073841
C.rhodostoma6073843
S.c.tergeminus45934756
D.acutus90265326
D.Acutus18158787
A.piscivorus300394
T.stejnegeri37785861
T.stejnegeri37785867
A.Halys28948590
B.Jararacussu93278529
B.Jararacussu37928232
B.jararacussu25140377
B.insularis20069137
T.flavoviridis15799265
T.flavoviridis222959
P.elegans84578889
P.mucrosquamatus517490
A.piscivorus300395
A.piscivorus263960
B.Pirajai16974919
PiratoxinIII17865540
B.jararacussu51890398
B.toxinI17433157
B.jararacussu265051
B.Pirajai10835912
PiratoxinII17368328
PiratoxinI17433154
B.neuwiedipauloensis7673019
B.jararacussu28194118
MyotoxinI17368325
MyotoxinII1171973
MjTXII17865560
B.moojeni7673017
B.asper6492260
B.atrox40888878
MyotoxinII3122600
MyotoxinII17433156
C.godmani17432520
B.schlegelii17432518
AGKPI
A.Contortrix460157
A.contortrixlaticinct
C.atrox17224435
BPI
T.flavoviridis222955
C.rhodostoma6073835
P.smucrosquamatus83
P.elegans84578893
P.elegans84578891
T.stejnegeri37785831
T.gramineus1644275
T.stejnegeri37785833
T.stejnegeri37785829
T.stejnegeri37785857
T.puniceus38230133
T.puniceus38230131
T.borneensis38230143
D.acutus17224439
D.acutus2760481
C.rhodostoma6073833
B.jararacussu28194389
MyotoxinI129400
T.flavoviridis28268779
T.flavoviridis28268777
P.mucrosquamatus15420985
D.acutus90265327
S.m.streckeri38230121
S.c.tergeminus38230127
C.v.viridis33337339
C.godmani38230123
B.schlegelii38230125
C.durissus62693
S.c.tergeminus38230129
C.durissus62697
Agkistrotoxin129437
A.HalysPallas4389403
A.HalysPallas5821835
T.jerdonii20977211
P.mucrosquamatus7636222
T.borneensis38230147
T.puniceus38230137
T.stejnegeri37785869
V.a.129481
V.a.242334
V.a.ammodytes50874542
V.a.ammodytes2231122
V.a.ammodytes50874556
V.a.ruffoi50874536
V.a.ammodytes50874560
V.a.montandoni50878139
V.a.montandoni50878137
V.a.montandoni50878133
V.a.montandoni50878135
V.a.montandoni50874568
V.a.ammodytes50874562
V.b.berus50874522
V.b.berus50874526
V.b.berus50874530
V.b.berus50874524
Viperaberusberus50874532
V.b.berus50874528
V.b.berus50874538
V.a.aspis50878149
V.a.871760
V.a.aspis50878145
V.a.296138
V.a.aspis50878151
V.a.1334641
V.a.64442
V.a.aspis50878153
V.a.aspis50878143
DRusselliiPulchella31615
D.r.Pulchella48425253
E.ocellatus25992665
B.arietans62547939
T.stejnegeri37785827
T.stejnegeri37785825
P.smucrosquamatus77
A.HalysPallas6980609
A.h.Pallas22218627
G.halys2460025
T.flavoviridis21698860
human
62
64
90
88
74
94
96
94
97
60
58
84
79
56
96
98
75
82
84
91
74
98
88
84
98
98
99
96
99
99
99
99
96
96
96
95
65
84
98
98
90
91
98
98
95
98
97
98
98
98
98
73
98
99
100
92
74
89
93
89
98
98
71
99
99
99
99
99
98
98
99
99
99
99
99
98
99
98
99
54
54
61
52
70
78
51
98
76
98
77
99
74
92
99
98
69
71
90
95
91
98
98
97
99
98
98
81
54
70
99
98
96
98
98
97
98
98
94
99
99
99
98
74
86
94
55
63
99
99
99
99
99
76
98
99
97
98
84
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 7 of 14
(page number not for citation purposes)
phospholipases. The estimate of ω for group-II genes
under the one-ratio model, 0.686, indicates that group-II
genes are generally under purifying selection, however,
this estimate is higher than reported from most genes.
The one-ratio model can only detect adaptive evolution
when the majority of amino acids and lineages under
study have been under positive selection (such as in
group-I genes). If adaptive evolution is primarily episodic,
then short episodes of positive selection, which are fol-
lowed by long periods of purifying selection, will not be
detected. To test for episodes of positive selection in
group-I and group-II gene lineages, I used a free-ratios
model that estimates separate dN/dS ratios for all lineages
in the tree. These models fit the data significantly better
than either the one-ratio model or a constrained one-ratio
model with ω forced to be 1 (group-I genes) or a free-ratio
model with lineages previously identified with ω >1 con-
strained to be 1 (group-II genes), indicating that episodes
of directional selection are common in snake venom PLA2
evolution with nearly 32% and 21% of group-I and -II
gene lineages, respectively, having been under directional
selection (Figs. 1 and 2). Moreover, there are several
branches with extremely high ω values, including two
group-I and three group-II branches with ω >3, one group-
I branch with ω >5 and one group-I branch with ω = 9.06
(Figs. 1 and 2).
Ohno's model [19] of post-duplication divergence pre-
dicts an increase in the nonsynonymous substitution rate
following duplication as positive Darwinian selection
drives the fixation of mutations that confer new or modi-
fied functions on gene duplicates. To test for accelerated
evolution after duplication I used smaller datasets for
which speciation and duplication events could be unam-
biguously assigned to each branch and a two-ratios model
that estimated different ω parameters for post-duplication
(PD) and post-speciation (PS) branches. Surprisingly, in
group-I genes PD and PS branches have nearly identical ω
values (ωPD = 1.12, ωPS = 1.22), indicating that positive
selection is associated with both gene duplication and
speciation. In contrast to group-I genes, group-II gene PD
branches evolve much faster than PS branches (ωPD = 1.4,
ωPS = 0.63), consistent with the classical model of neo-
functionalization.
Here, neofunctionalization is defined as the emergence of
a new toxic effect from an ancestral enzyme that did not
posses that effect as its main toxin function (for example,
neurotoxic Laticuadata genes and Lys49-myotoxins dis-
cussed above). Strikingly, positive selection occurred in
the stem-lineage of 67% (4/6) of group-I functional
groups and 88% (7/8) of group-II functional groups (Figs.
1 and 2) indicating that positive selection played a perva-
sive role in the origin of novel toxin functions during the
diversification of vipers and elapids and their venoms. It
also suggest lineages which can be targeted for ancestral
sequence reconstructions for characterization of ancestral
toxin functions to compare extant functions to.
The importance of gene duplication to the evolution of
species-specific traits is relatively unknown, but duplica-
tions resulting in species-specific adaptations have been
demonstrated for some genes [20,21]. The unexpectedly
high group-I ωPS may be the result of enzyme adaptation
to new prey preference after speciation. Indeed, the three
semi-aquatic kraits (Laticaudata sp.) prey primarily on
moray and conger eels and assorted fishes [22,23] while
Australian copperheads (Austrelaps) prey on frogs and liz-
ards [24]. In the Elapinae group, the king cobra (Ophi-
ophagus hannah) and kraits (Bungarus sp.) feed almost
exclusively on snakes and other reptiles [25], while the
true cobras (Naja sp.) and the Eastern brown snake (Pseu-
donaja textilis) feed on small mammals, amphibians and
birds [25]. This pattern suggest a scenario where dietary
shifts after speciation runs the PLA2 gene repertoire
through a "selective sieve"; those genes which are no
longer effective in subduing new prey species are lost,
while genes that are still effective adapt to the new prey
type and subsequently diversify.
A limitation of the lineage-specific models of protein evo-
lution utilized above is that they can only detect direc-
tional selection when the average ω over all amino acids
in the protein is >1. Thus, lineage-specific models have
limited ability to detect short episodes of directional selec-
tion that affect only a few amino acids or amino acids
under recurrent diversifying selection. Site-specific mod-
els [26] account for rate variation among sites and are
powerful tools for detecting diversifying selection. I used
three pairs of site-specific models to test for recurrent,
diversifying, selection: M0 (one ratio) and M3 (Discrete),
M1 (Neutral) and M2 (Selection), and M7 (Beta) and M8
(Beta & ω). Parameter estimates under models M2, M3
and M8, which allow for sites with ω >1, identified that up
to 65% of sites in group-I genes and 27% of sites in group-
II genes are under positive selection (Tables 1 and 2). This
is strong evidence that diversification of snake venom
PLA2 genes is driven by recurrent positive selection and
suggest that venomous snakes are caught in a co-evolu-
tionary arms race with prey as prey evolve resistance to the
current venom arsenal and snakes evolve ever more toxic
venoms.
The three dimensional structure of PLA2 enzymes are
extremely conserved, obscuring the mechanisms that pro-
duce such a wide spectrum of pharmacological effects. To
investigate how functional diversity is generated in PLA2
enzymes, I mapped sites that were identified as being
under diversifying selection on to the crystal structure of
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 8 of 14
(page number not for citation purposes)
group-I and -II phospholipases (Fig. 5). The vast majority
of amino acids under diversifying selection occur outside
of the α-helicical central scaffold and in regions of the
protein that form connecting loops, however, the scaffold
of group-II proteins is more conserved than group-I pro-
teins. Functionally important residues, including cysteins
that participate in disulfide bonds, the catalytic triad, the
calcium-binding site and the hydrophobic channel have
dN/dS ratios near zero indicating these regions are under
strong structural and functional constraint. In contrast,
there are several clusters of amino acids on the molecular
surface under intense diversifying selection in both group-
I and -II proteins (Fig. 5). These rapidly evolving regions
are similar to sites known to produce toxic effects in PLA2
Table 2: Maximum Likelihood Parameter Estimates for Group-II PLA2 Genes.
Model ᐍω0Parameters Sig Positive Sites
Lineage-specific
M0: one ratio... -11770.60 0.686 = ω0
M0: one ratio-2 -4303.29 0.694 = ω0
PD-PS.............. -4300.30 ωPD = 1.41, ωPS = 0.63 P = 0.014
Free ratios....... -11580.19 see Figure 1 P << 0.001
Free ratios-2.... -11591.23 1ω0 constrained to 1 P << 0.01
Site-specific
M1: neutral....... -11547.79 0.876 p0 = 0.124, ω0 = 0
p1 = 0.876, ω2 = 1
M2: selection... -11266.10 1.64 p0 = 0.124, ω0 = 0 P << 0.001 30 (PP ≥ 0.99)
p1 = 0.605, ω2 = 1 2 (0.95 ≤ PP < 0.99)
p2 = 0.271, ω2 = 3.81 9 (PP < 0.95)
M3: discrete..... -11096.30 0.971 p0 = 0.346, ω0 = 0.078 P << 0.001 21 (PP ≥ 0.99)
p1 = 0.42, ω2 = 0.83 5 (0.95 ≤ PP < 0.99)
p2 = 0.234, ω2 = 2.55 8 (PP < 0.95)
M7: beta........... -11179.33 0.532 p = 0.332, q = 0.293
M8: beta&ω...... -11073.18 0.899 p0 = 0.81, p = 0.352, q = 0.348 P << 0.001 15 (PP ≥ 0.99)
p1 = 0.19, ω = 2.58 7 (0.95 ≤ PP < 0.99)
6 (PP < 0.95)
L is the log likelihood of the model; ω0 is the estimate of the dN/dS ratio under the model (given as a weighted average for the site-specific models);
Sig. is the significance of the model when compared to it's neutral partner under the χ2-distribution; Positive sites gives the number of sites
Table 1: Maximum Likelihood Parameter Estimates for Group-I PLA2 Genes.
Model ᐍω0Parameters Sig. Positive Sites
Lineage-specific
M0: one ratio... -9102.48 1.28 = ω0P < 0.01
M0: one ratio-C -9106.69 1ω0 constrained to 1
M0: one ratio-2 -5153.65 1.16 = ω0
PD-PS............. -5153.55 ωPD = 1.22, ωPS = 1.12 n.s.
Free ratio......... -8980.48 see Figure 1 P << 0.001
Site-specific
M1: neutral....... -8909.37 0.852 p0 = 0.148, ω0 = 0
p1 = 0.582, ω2 = 1
M2: selection... -8600.95 2.11 p0 = 0.145, ω0 = 0 P << 0.001 36 (PP ≥ 0.99)
p1 = 0.5, ω2 = 1 4 (0.95 ≤ PP < 0.99)
p2 = 0.355, ω2 = 4.53 10 (PP < 0.95)
M3: discrete..... -8540.48 1.69 p0 = 0.343, ω0 = 0.115 P << 0.001 74 (PP ≥ 0.99)
p1 = 0.435, ω2 = 1.43 7 (0.95 ≤ PP < 0.99)
p2 = 0.222, ω2 = 4.63 4 (PP < 0.95)
M7: beta........... -8725.96 0.594 p = 0.266, q= 0.182
M8: beta&ω...... -8544.24 1.4 p0 = 0.70, p = 0.276, q = 0.228 P << 0.001 28 (PP ≥ 0.99)
p1 = 0.297, ω = 3.41 2 (0.95 ≤ PP < 0.99)
9 (PP < 0.95)
L is the log likelihood of the model; ω0 is the estimate of the dN/dS ratio under the model (given as a weighted average for the site-specific models);
Sig. is the significance of the model when compared to it's neutral partner under the χ2-distribution; Positive sites gives the number of sites that fall
within a particular Baysean posterior probability value (PP) of being in the site class ω >1. n.s., not significant.
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 9 of 14
(page number not for citation purposes)
enzymes [27-29], highly suggesting that regions under
positive selection on the proteins surface are responsible
for generating toxic functions.
Although positive selection is often associated with the
origin of novel toxin functions (such as antiplatelet, neu-
rotoxic and procoagulent toxins), there are several line-
ages in which new functions emerge without a significant
increase in the nonsynonymous substitution rate (other
neurotoxins and cardiotoxins). This is not unexpected
since it has long been known that relatively few amino
acid changes can have drastic effects on protein functions
[ref], suggesting some lineages may have substitutions
that contribute to the origins of novel functions but that
might have been missed in the lineage and site-specific
analyses above. To further clarify the pattern of amino
acid replacement that promotes functional changes, I
mapped amino acid changes inferred from ancestral
sequence reconstructions for select group-I (Figs. 6 and 7)
and group-II genes (Fig. 8) onto the crystal structure of
these proteins. Clearly, replacements are nearly evenly dis-
tributed on protein surface in both group-I and -II genes,
but there are several regions that are devoid of amino
acids changes including residues in and around the active
site and patches of conserved residues on the "back" of the
proteins. These regions also contain residues in the slow-
est evolving site-class from the site-specific analysis. Taken
together, these patterns suggest that while only a few
changes on the surface are needed to evolve a new func-
tion, there are regions under strong structural/functional
constraints that limit divergence such as the hydrophobic
core and patches of conservation on the surface. Given
this, it is interesting to note that several basal clades in the
group-II genes with uncharacterized pharmacological
effects have strong evidence of selection and many amino
acid replacements that map to the surface (Figs. 2 and 8)
suggesting that they may have evolved novel, if as of yet
unidentified, functions.
Conclusion
The molecular evolution of group-I and group-II PLA2
genes, such as the birth and death like and "selective
sieve" processes of gene duplication, divergence and loss
are similar to evolution of other snake venom proteins,
The structure of group-I (A-C) and group-II (D-F) phospholipase A2 proteinsFigure 5
The structure of group-I (A-C) and group-II (D-F) phospholipase A2 proteins. The structures are represented by ribbons in A
and D with disulfide bonds and catalytic residues shown as sticks and as molecular surfaces rendered in 3D in B, C, E and F.
Residues are colored coded according to their approximate posterior mean ω (scale shown between rows) calculated under
model M3 (discrete). B and E are in the same orientation as A and D, respectively, while C and F are rotated 180° about a hor-
izontal axis through the molecule.
DF
1.00>3
E
ABC
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 10 of 14
(page number not for citation purposes)
Ancestral sequence mapping for hydrophiinae group genesFigure 6
Ancestral sequence mapping for hydrophiinae group genes. The upper left panel shows the generalized phylogeny while each
additional panel shows the cumulative amino acid changes that occurred for that lineage. Amino acids in panels A-E are colored
by the lineage they changed in. For example, amino acid changes that occurred in the stem-lineage of neurotoxic genes (lineage
C, panel C) are colored X; amino acid changes that occurred in ancestral lineages of neutrotoxins are colored Y and Z. In each
panel the top two structures are shown with the molecular surface and the bottom structures are ribbons. Structures on left
and right are rotated about a central axis 180°. Cystienes are shown in yellow
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 11 of 14
(page number not for citation purposes)
Ancestral sequence mapping for elapinae group genesFigure 7
Ancestral sequence mapping for elapinae group genes. Organization follows Figure 6.
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 12 of 14
(page number not for citation purposes)
Ancestral sequence mapping for group-II genesFigure 8
Ancestral sequence mapping for group-II genes. Organization follows Figure 6. Antipla., antiplatelet. Chp., chaperone. UnChar.,
genes with uncharacterized pharmacological effects.
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 13 of 14
(page number not for citation purposes)
particularly the elapid three-finger toxins [30]. Indeed,
there is even evidence for species-specific toxin adaptation
to prey type within the three-finger toxins and mainte-
nance of a well-ordered tertiary structure [30] similar to
that seen in PLA2 genes, suggesting that this mode of
molecular evolution may be common in venom genes.
Kini and Evans [5] have proposed that 'target sites' on the
surface of prey cells are recognized by 'pharmacological
sites' on PLA2 enzymes. These protein-protein interactions
determine PLA2 specificity by having complementary
charges, hydrophobicities, and Van der Waals contact sur-
faces. This model, combined with the analyses above, sug-
gest that entirely new functions originate after duplication
through substitutions in pharmacological sites that alter
binding specificities. Although most substitutions will
likely disrupt binding specificity for the current target site,
a few may create new interaction sites leading to the emer-
gence of novel functions.
The extraordinary level of positive selection acting on
snake venom phospholipase A2 genes indicates that adap-
tive molecular evolution plays an important role in the
emergence of these novel functions, continues as func-
tions are diversified and refined, and may contribute to
niche differentiation after speciation. Interestingly, map-
ping sites under positive selection onto the structure of
PLA2 enzymes has identified regions that are attractive
candidates for structure-based drug design. These data
also demonstrate that increases in genomic complexity
gained through gene duplications has lead to an increase
in phenotypic complexity (venom composition) and
likely the ability of venomous snakes to adapt to new prey
types.
Methods
Sequence alignment and phylogenetic reconstruction
PLA2 genes were obtained from public database (GenBank
GI's for each gene are shown in Figs. 1 and 2 for nucleo-
tide data and in Figs. 3 and 4 for amino acid data). Partial
sequences, sequences with insertions-deletions that
caused reading frame shifts and sequences with premature
stop codons were excluded from analysis as likely pseudo-
genes in the nucleotide analysis. Group-I and -II protein
sequences were aligned in Clustal W [31] and adjusted by
eye using Se-Al. Bayesian phylogenetic analyses were per-
formed using MrBayes v3.0 [10]. Tree searches were run
using four Markov chains for 3,000,000 generations sav-
ing every 100th tree and a codon-based GTR+Γ+I model of
sequence evolution or a JTT+Γ+I model for amino acid
data. Models are nucleotide and amino acid data were
selected using ModelGenerator. After stationarity, the
final 15000 trees were used to build a consensus tree. Each
analysis was performed three times to ensure convergence
of tree topologies.
Tests for selection and ancestral sequence reconstructions
I used codon-based maximum likelihood models imple-
mented in CODEML in the PAML package of programs
(version 3.14) [32] to test for lineages under positive
selection using the one-ratio and free-ratios models; this
package of programs was also used for ancestral sequence
reconstructions. To test for differences in post-duplication
(PD) and post-speciation (PS) branches I used a smaller
dataset of group-I and -II genes that included at least five
representatives of each species/pharmacological group
and for which gene duplication and speciation events
could accurately be assigned for each branch. Twice the
log likelihood difference between models, 2Δᐍ = 2(ᐍ1-ᐍ0),
is compared to a χ2-distribution with the degrees of free-
dom equal to the number of parameter differences
between the models to test whether the alternative model
(free-ratio or PD-PS) fits the data significantly better than
the null model (one-ratio). If a lineage has a dN/dS > 1 and
the likelihood ratio test is significant, than the neutral
model of evolution is violated and positive selection is
suggested. To explicitly test for positive selection I used
constrained models that fixed ω at 1.
I used three pairs of site-specific models [33] to identify
specific amino acids under diversifying selection: M0 and
M3 (discrete), M1 (neutral) and M2 (selection), and M7
(beta) and M8 (beta & ω). Model M0 estimates a single ω
parameter for all sites and branches, while model M3 (dis-
crete) estimates three independent ω parameters and the
proportion of sites belonging to each ω-class directly from
the data. Model M1 (neutral) assumes two classes of sites
in the protein with the proportion of conserved sites (ω =
0) and neutral sites (ω = 1) estimated from the data, while
model M2 (selection) adds a third site class with an addi-
tional ω estimated as a free parameter allowing for sites
with ω > 1. Model M7 (beta) uses a beta distribution B(p,
q) with ω restricted to the interval (0,1) while Model M8
(beta & ω) adds a site class with the ω and the proportion
of sites with that ω estimated from the data, allowing for
sites with ω > 1. Twice the log likelihood difference
between the models is compared to the χ2 distribution
and tests for variation in ω among sites. After maximum
likelihood parameter estimates are calculated, the Bayes
theorem is used to calculate the posterior probability of
belonging to a site class, when a site is identified with ω >
1 than positive selection is indicated. Sites with posterior
probabilities of > 0.5 are reported here. The approximate
posterior mean ω for each site from model M3 with two
site classes were mapped onto the crystal structures of
group-I (PDB ID: 1A3D) and -II (PDB ID: 1OZ6) PLA2
proteins using ICM-Browser (available from http://
www.molsoft.com) or Chimera. The three-dimensonal
space filling structures were generated with Deep View –
spdbv v3.7 [34].
Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
BMC Evolutionary Biology 2007, 7:2 http://www.biomedcentral.com/1471-2148/7/2
Page 14 of 14
(page number not for citation purposes)
Authors' contributions
VJL designed the study, carried out the statistical analyses
and drafted the manuscript. All authors have read and
approved the final manuscript.
Acknowledgements
I thank G. P. Wagner for helpful comments and discussion of the manu-
script and Z. Yang for answering questions regarding the use of PAML. I also
thank M. J. McCarron for careful reading of the manuscript and the anony-
mous reviewers for their comments. Support for this research was pro-
vided by departmental graduate student research funds provided to VJL.
References
1. Dennis EA: Phospholipases. In The Enzymes Volume 16. 3rd edition.
Edited by: Boyer PD. New York, Academic Press; 1983.
2. Kini RM: Venom Phospholipase A2 enzymes: Structure, Func-
tion and Mechanism. Chichester, Wiley; 1997:511.
3. Kini RM, Evans HJ: The role of enzymatic activity in inhibition
of the extrinsic tenase complex by phospholipase A2 isoen-
zymes from Naja nigricollis venom. Toxicon 1995,
33:1585-1590.
4. Rufini S Cesaroni, M.P., Balestro, N., Luly, P.: The proliferative
effects of ammodytin L from the venom of Vipera ammo-
dytes on 208F rat fibroblasts in culture. Biochemical Journal
1996, 320:318-326.
5. Kini RM, Evans HJ: A model to explain the pharmacological
effects of snake venom phospholipases A2. Toxicon 1989,
27:613-635.
6. Chuman Y, Nobuhisa I, Ogawa T, Deshimaru M, Chijiwa T, Tan NH,
Fukumaki Y, Shimohigashi Y, Ducancel F, Boulain JC: Regional and
accelerated molecular evolution in group I snake venom
gland phospholipase A2 isozymes. Toxicon 2000, 38:449-462.
7. Nakashima K, Nobuhisa I, Deshimaru M, Nakai M, Ogawa T, Shimoh-
igashi Y, Fukumaki Y, Hattori M, Sakaki Y, Hattori S, Ohno M: Accel-
erated Evolution in the Protein-Coding Regions is Universal
in Crotalinae Snake Venom Gland Phospholipase A2 Iso-
zyme Genes. PNAS 1995, 92:5605-5609.
8. Kordis D, Bdolah A, Gubensek F: Positive Darwinian Selection
inVipera palaestinaePhospholipase A2Genes Is Unexpect-
edly Limited to the Third Exon. Biochemical and Biophysical
Research Communications 1998, 251:613-619.
9. Kini RM Chan, Y.M.: Accelerated evolution and molecular sur-
face of venom phospholipase A2 enzymes. Journal of Molecular
Evolution 1999, 48:125-132.
10. Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic
inference under mixed models. Bioinformatics 2003,
19:1572-1574.
11. Slowinski JB, Knight A, Rooney AP: Inferring Species Trees from
Gene Trees: A Phylogenetic Analysis of the Elapidae (Ser-
pentes) Based on the Amino Acid Sequences of Venom Pro-
teins. Molecular Phylogenetics and Evolution 1997, 8:349-362.
12. Fujimi TJ, Kariya Y, Tsuchiya T, Tamiya T: Nucleotide sequence of
phospholipase A2 gene expressed in snake pancreas reveals
the molecular evolution of toxic phospholipase A2 genes.
Gene 2002, 292:225-231.
13. Maraganore JM, Heinrikson RL: The lysine-49 phospholipase A2
from the venom of Agkistrodon piscivorus piscivorus. Rela-
tion of structure and function to other phospholipases A2
[published erratum appears in J Biol Chem 1993 Mar
15;268(8):6064]. J Biol Chem 1986, 261:4797-4804.
14. van den Bergh CJ Slotboom, A.J., Verheij, H.M., de Haas, G.H.,: The
role of aspartic acid-49 in the active site of phospholipase A2.
A site-specific mutagenesis study of porcine pancreatic phos-
pholipase A2 and the rationale of the enzymatic activity of
[lysine49]phospholipase A2 from Agkistrodon piscivorus pis-
civorus' venom. European Journal of Biochemistry 1998,
176:353-357.
15. Diaz C Gutierez, J.M., Lomonte, B., Gene, J.A.: The effect of myo-
toxins isolated from Bothrops snake venoms on multilamel-
lar liposomes: relationship to phospholipase A2,
anticoagulant and myotoxic activities. Biochim Biophys Acta
1991, 1070:455-460.
16. Rufini S Cesaroni, P., Desideri, A., Farias, R., Gubensek, F., Gutierrez,
J.M., Luly, P., Massoud, R., Morero, R., Pedersen, J.Z.: Calcium ion
independent membrane leakage induced by phospholipase-
like myotoxins. Biochemistry 1992, 31:12424-12430.
17. Goldman N, Yang Z: A codon-based model of nucleotide sub-
stitution for protein-coding DNA sequences. Mol Biol Evol
1994, 11:725-736.
18. Yang Z: Likelihood ratio tests for detecting positive selection
and application to primate lysozyme evolution. Mol Biol Evol
1998, 15:568-573.
19. Ohno S: Evolution by Gene Duplication. Berlin, Springer; 1970.
20. Zhang J , Zhang, Y., Rosenberg, H.F: Adaptive evolution of a dupli-
cated pancreatic ribonuclease gene in a leaf-eating monkey.
Nature Genetics 2002, 30:411-415.
21. Riehle MM, Bennett AF, Long AD: Genetic architecture of ther-
mal adaptation in Escherichia coli. PNAS 2001, 98:525-530.
22. Shine R Reed, R. N., Shelty, S., Cogger, H. G.: Relationships
between sexual dimophism and niche partitioning within a
clade of sea snakes (Laticaudinae). Oecologia 2002, 133:45-53.
23. Su Y Fong, S.-C., Tu, M.-C.: Food Habits of the Sea Snake, Lati-
cauda semifasciata. Zoological Studies 2005, 44:403-408.
24. Shine R: Habitats, diets, and sympatry in snakes: a study from
Australia. Canadian Journal of Zoology 1977, 55:1118-1128.
25. Oriov N Ananjeva, N., Ryabov, S., Rao, D.-Q.: Venomous snakes
of southern China. Reptilia 1992, 31:22-29.
26. Yang Z Bielawski, J. P.: Statistical Methods for detecting molec-
ular adaptation. Trends in Ecology and Evoltuion 2000,
15:1994-1997.
27. Kini RM: Excitement ahead: structure, function and mecha-
nism of snake venom phospholipase A2 enzymes. Toxicon
2003, 42:827-840.
28. Chioato L, Ward RJ: Mapping structural determinants of bio-
logical activities in snake venom phospholipases A2 by
sequence analysis and site directed mutagenesis. Toxicon
2003, 42:869-883.
29. Soares AM, Giglio JR: Chemical modifications of phospholi-
pases A2 from snake venoms: effects on catalytic and phar-
macological properties. Toxicon 2003, 42:855-868.
30. Fry BG WW Kini RM, Brusic V, Khan A, Venkataraman D, Rooney
AP.: Molecular evolution and phylogeny of elapid snake
venom three-finger toxins. Journal of Molecular Evolution 2003,
57:110-129.
31. Higgins D Thompson, J., Gibson, T., Thompson, J.D., Higgins, D.G.,
Gibson, T.J.: CLUSTAL W: improving the sensitivity of pro-
gressivemultiple sequence alignment through sequence
weighting,position-specific gap penalties and weight matrix
choice. Nucleic Acids Research 1994,